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Contribution of Methanotrophic Groundwater and Rhizosphere Bacteria to Phytoremediation

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PAGE 1

1 C O N T R I B U T I O N O F M E T H A N O T R O P H I C G R O U N D W A T E R A N D R H I Z O S P H E R E B A C T E R I A T O P H Y T O R E M E D I A T I O N B y A D R I A N A P A C H E C O A D I S S E R T A T I O N P R E S E N T E D T O T H E G R A D U A T E S C H O O L O F T H E U N I V E R S I T Y O F F L O R I D A I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y U N I V E R S I T Y O F F L O R I D A 2006

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2 C opyr i ght 2006 by A dr i a na P a c he c o

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3 T hi s di s s e r t a t i on i s de di c a t e d t o m y pa r e nt s

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4 A C K N O W L E D G M E N T S I t ha nk D r G a br i e l B i t t on D r R obi n L B r i gm on D r D ona l d L R oc kw oo d, D r W i l l i a n R W i s e a nd D r A nge l a S L i ndne r f or t he i r t i m e a nd a s s i s t a nc e of f e r e d a s m y a dvi s or y c om m i t t e e I e s pe c i a l l y t ha nk D r A nge l a S L i ndne r m y a dvi s or p r of e s s or f or he r m e nt or i ng he r pa t i e nc e a nd e s pe c i a l l y, f or s h a r i ng he r e nt hus i a s m t ow a r ds t e a c hi ng a nd s c i e nt i f i c r e s e a r c h t ha t w i l l a l w a ys be a s our c e of i ns pi r a t i o n. A l s o, I w oul d l i ke t o t ha nk D r J ud I s e br a nds a nd D r M a de l i ne R a s c he f or t he i r a bs ol ut e s uppor t I a l s o t ha nk our r e s e a r c h gr oup f or a l l t he i r s uppor t ; J e s s i c a S t r a t e C ha nc e L a ude r da l e a nd C a l l i e W hi t f i e l d. F i na l l y I t ha nk m y f a m i l y a nd f r i e nds f or t he i r unc ondi t i ona l s uppor t w i t hou t w hi c h none of t hi s w oul d ha ve be e n pos s i bl e

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5 T A B L E O F C O N T E N T S pa ge A C K N O W L E D G M E N T S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 L I S T O F T A B L E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 L I S T O F F I G U R E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 A B S T R A C T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 C H A P T E R 1 I N T R O D U C T I O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 S i gni f i c a nc e of t he S t udy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 L i t e r a t ur e R e vi e w . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 C hl or i na t e d C om pounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 P hyt or e m e di a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 T he R hi z os phe r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 M onot e r pe ne s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 M e t ha not r ophi c B a c t e r i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 E c ol ogy a nd ha bi t a t s o f m e t ha not r ophs . . . . . . . . . . . . . . . . . . . . . . . . . . 34 E nvi r onm e nt a l f a c t or s a f f e c t i ng m e t ha not r ophs . . . . . . . . . . . . . . . . . . . . 36 M e t ha not r ophs a nd c hl or i na t e d c om pounds . . . . . . . . . . . . . . . . . . . . . . . 37 M e t ha not r ophs a nd pl a nt s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 P hyl oge ne t i c s of m e t ha not r ophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 M ol e c ul a r a na l ys i s of m e t ha not r ophs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 M e t hods U s e d t o A s s e s s R hi z ode gr a da t i on P ot e nt i a l i n P hyt o r e m e di a t i on . . . 44 C ul t ur e de pe nde nt t e c hni que s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 C ul t ur e i nde pe nde nt t e c hni que s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 S t udy H ypot he s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 S t udy O bj e c t i ve s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 B r oa d O bj e c t i ve s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 S pe c i f i c O bj e c t i ve s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 2 M E T H Y L O C Y ST I S A L D R I C H I I S P N O V A N O V E L M E T H A N O T R O P H I S O L A T E D F R O M A G R O U N D W A T E R A Q U I F E R . . . . . . . . . . . . . . . . . . . . . . 57 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

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6 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 3 E F F E C T S O F A L P H A P I N E N E A N D T R I C H L O R O E T H Y L E N E O N O X I D A T I O N P O T E N T I A L S O F M E T H A N O T R O P H I C B A C T E R I A . . . . . . . . . 78 I nt r o duc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 R e s ul t s a nd D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 4 S T A B L E I S O T O P E P R O B I N G F O R C H A R A C T E R I Z A T I O N O F M E T H A N O T R O P H I C B A C T E R I A I N T H E R H I Z O S P H E R E O F P H Y T O R E M E D I A T I N G P L A N T S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 S i t e D e s c r i pt i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 S a m pl i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 S t a bl e I s ot ope P r ob i ng ( S I P ) S oi l M i c r oc os m s . . . . . . . . . . . . . . . . . . . . . . . . 92 D e na t ur i ng G r a di e nt G e l E l e c t r opho r e s i s A na l ys i s ( D G G E ) S e que nc i ng, a nd P hyl oge ne t i c A na l ys i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 S t a t i s t i c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 S I P P r ot oc ol I m pl e m e nt a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 M e t ha not r oph A c t i vi t y a nd C om pos i t i on i n t he T C E S i t e . . . . . . . . . . . . . . . . 98 M e t ha not r oph A c t i vi t y a nd C om pos i t i on i n t he P C E S i t e . . . . . . . . . . . . . . . 101 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 5 C H A R A C T E R I Z A T I O N O F R H I Z O S P H E R E M E T H A N O T R O P H I C B A C T E R I A I N T C E P H Y T O R E M E D I A T I O N : I M P A C T O F T H E D E S I G N . . . 111 I nt r oduc t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 M a t e r i a l s a nd M e t hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 S i t e D e s c r i pt i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 S a m pl i ng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 S oi l C ha r a c t e r i z a t i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 M i c r obi a l C ount s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 C ha r a c t e r i z a t i on of E n r i c hm e nt s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 S t a bl e I s ot ope P r ob i ng ( S I P ) S oi l M i c r oc os m s . . . . . . . . . . . . . . . . . . . . . . . 120 P hyl oge ne t i c A na l ys i s of E n r i c hm e nt s a nd S I P M i c r oc os m s . . . . . . . . . . . . . 121 S t a t i s t i c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 R e s ul t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 D e s c r i pt i on of S i t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 M i c r obi a l C ount s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 R oot B i om a s s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 E nr i c hm e nt s A c t i vi t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 P hyl oge ne t i c s of E n r i c hm e nt s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 9

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7 S I P S o i l M i c r oc os m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 P r i nc i pa l C om pone nt A na l ys i s ( P C A ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 D i s c us s i on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 M i c r obi a l A bunda nc e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 A c t i vi t y a nd P h yl oge ne t i c s of E n r i c hm e nt s . . . . . . . . . . . . . . . . . . . . . . . . . . 136 A c t i vi t y a nd P hyl oge ne t i c s of S I P S oi l M i c r oc os m s . . . . . . . . . . . . . . . . . . . 139 P r i nc i pa l C om pone nt A na l ys i s ( P C A ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 6 C O N C L U S I O N S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 A P P E N D I X A D D I T I O N A L T A B L E S A N D F I G U R E S . . . . . . . . . . . . . . . . . . . . . . 157 L I S T O F R E F E R E N C E S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 B I O G R A P H I C A L S K E T C H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

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8 L I S T O F T A B L E S T a bl e pa ge 1 1. P hys i c a l a nd c he m i c a l p r ope r t i e s of T C E a nd P C E . . . . . . . . . . . . . . . . . . . . . . . . 53 1 2. P hys i c a l a nd c he m i c a l p r ope r t i e s of pi ne ne . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 1 3. C ha r a c t e r i s t i c s of di f f e r e nt m e t ha not r oph t yp e s . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2 1. P he not ypi c c ha r a c t e r i s t i c s di f f e r e nt i a t i ng S t r a i n C S C 1 f r om M e t hy l os i nus t r i c hos por i um M e t hy l oc y s t i s e c hi noi de s a nd M e t hy l oc y s t i s par v u s . . . . . . . . . . 75 5 1. G e ne r a l c ha r a c t e r i s t i c s of t he di f f e r e nt phy t or e m e di a t i on pl ot s a t t he S R S a nd L a S a l l e s i t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 5 2. S um m a r y of phyl oge ne t i c a s s i gnm e nt s ( B L A S T s e a r c h) o f t he pm oA ge ne s e que nc e s of t he a c t i ve m e t ha not r oph popul a t i ons ( 1 3 C D N A f r a c t i on) f r om t he S I P s oi l m i c r oc os m s i n e a c h phyt or e m e di a t i on pl ot . . . . . . . . . . . . . . . . . . . . . . 150 A 1. S oi l c ha r a c t e r i s t i c s of t he S R S a nd L a S a l l e ph yt or e m e di a t i on pl ot s f r om h i gh c ont a m i na nt r e gi ons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 A 2. A na l ys i s of va r i a nc e r e s ul t s ( P va l ue s ) f or t he e f f e c t o f t i m e a nd de pt h on r hi z os phe r e ( R H ) a nd r hi z opl a ne ( R P ) m i c r obi a l a bunda nc e of t he L a S a l l e phyt or e m e di a t i on pl ot s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

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9 L I S T O F F I G U R E S F i gur e pa ge 1 1. S c he m a t i c of pr oc e s s e s i n a phyt or e m e di a t i on s ys t e m . . . . . . . . . . . . . . . . . . . . . 53 1 2. C 1 m e t a bol i s m by m e t ha not r ophs a nd m e t hyl ot r ophs a s de s c r i be d by W a c ke t t ( 1995) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 1 3. O xi da t i on o f T C E by a e r obi c m e t ha not r oph d e gr a da t i on ( A ) a nd a na e r obi c r e duc t i ve de ha l oge na t i on ( B ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2 1. 16 S r R N A phyl oge ny of S t r a i n C S C 1 a nd r e l a t e d M e t hy l os i nus a nd M e t hy l oc y s t i s s pe c i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 2 2. F unc t i ona l ge ne s phyl oge ni e s of S t r a i n C S C 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 2 3. T r a ns m i s s i on e l e c t r on m i c r os c opy phot ogr a p hs of S t r a i n C S C 1 a nd M e t hy l oc y s t i s e c hi noi de s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 2 4. E l e c t r on m i c r os c ope c yt oc he m i s t r y o f t he S l a ye r of S t r a i n C S C 1. . . . . . . . . . . . . 77 3 1. N o r m a l i z e d r a t e o f oxyge n upt a ke by t he r e pr e s e nt a t i ve m e t ha not r ophs i n t he pr e s e nc e of va r yi ng c onc e nt r a t i ons o f T C E ( ) a nd ( R ) pi ne ne . . . . . . . . . . . . 86 3 2. C ha nge i n t he no r m a l i z e d oxyge n upt a ke r a t e by r e pr e s e nt a t i ve m e t ha not r ophs obs e r ve d i n t he pr e s e nc e of 20 ppm T C E a t va r yi n g c onc e nt r a t i ons of ( R ) pi ne ne . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 4 1. E qui l i br i um c e nt r i f uga t i on of i s ot opi c a l l y l a b e l e d D N A i n C s C l de ns i t y gr a di e nt c ol um ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 4 2. I ni t i a l 1 3 C H 4 de pl e t i on r a t e s ( ba r s ) obs e r ve d i n S I P m i c r oc os m s a f t e r t he t hr e e s a m pl i ng pe r i ods a t t he T C E S i t e ( A ) a nd P C E S i t e ( B ) . . . . . . . . . . . . . . . . . . . 108 4 3. D G G E ge l s of pm o A P C R p r oduc t s de r i ve d f r om t he 1 3 C D N A f r a c t i on of S I P m i c r oc os m s a t t he T C E S i t e ( A C ) a nd P C E S i t e ( D ) . . . . . . . . . . . . . . . . . . . . 109 4 4. N e i ghbor j oi ni ng phy l oge ne t i c t r e e o f pm oA s e que nc e s de r i ve d f r om t he 1 3 C D N A f r a c t i on o f 1 3 C H 4 S I P m i c r oc os m s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

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10 5 1. L oc a t i on of phyt or e m e di a t i on s i t e s a nd di a g r a m of s a m pl i ng a r e a s a t t he ( A ) S a va nna h R i ve r S i t e ( S R S ) S C a nd ( B ) L a S a l l e I L . . . . . . . . . . . . . . . . . . . . 144 5 2. M i c r obi a l c ount s pe r t r e e t ype f r om t he di f f e r e nt phyt or e m e di a t i on pl o t s a t S R S a nd L a S a l l e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 5 3. O xyge n upt a ke r a t e s of e nr i c hm e nt s f r om di f f e r e nt t r e e t ype s i n t he p r e s e nc e of C H 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 5 4. F r e que nc y o f phyl um a f f i l i a t i ons pe r t r e e t yp e of t he N M S w i t h C u e nr i c hm e nt c om pone nt s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 5 5. P hyl oge ne t i c a na l ys i s of t he S R S S I P s oi l m i c r oc os m s . . . . . . . . . . . . . . . . . . . . 149 5 6. P r i nc i pa l c om pone nt a na l ys i s ( P C A ) of c ul t ur e de pe nde nt a nd c ul t ur e i nde pe nde nt m e a s ur e m e nt s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 A 1. D G G E ge l o f P C R a m pl i f i e d pa r t i a l pm oA f r a gm e nt s of di f f e r e nt m e t ha not r oph t ype s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 A 2. E f f e c t of de pt h on oxyge n upt a ke r a t e s o f N M S w i t h C u e n r i c hm e nt s . . . . . . . . . 160 A 3. D G G E ge l s of 16S r D N A pa r t i a l s e que nc e s f r om N M S w i t h C u e nr i c hm e nt s . . . 161 A 4. P hyl oge ne t i c t r e e o f 16 S r D N A pa r t i a l s e que n c e s f r om N M S w i t h C u e nr i c hm e nt s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

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11 A bs t r a c t of D i s s e r t a t i on P r e s e nt e d t o t he G r a dua t e S c hool of t he U ni ve r s i t y of F l or i da i n P a r t i a l F u l f i l l m e nt o f t he R e qui r e m e nt s f or t he D e g r e e of D oc t o r of P hi l os o phy C O N T R I B U T I O N O F M E T H A N O T R O P H I C G R O U N D W A T E R A N D R H I Z O S P H E R E B A C T E R I A T O P H Y T O R E M E D I A T I O N B y A dr i a na P a c he c o A ugus t 2006 C ha i r : A nge l a S L i ndne r M a j or D e pa r t m e nt : E nvi r on m e nt a l E ngi ne e r i ng S c i e nc e s T r i c hl or o e t hyl e ne ( T C E ) a w i de l y us e d s ol ve nt a n d ubi qui t ous c ont a m i na nt i s e f f e c t i ve l y r e m ove d f r om s oi l a nd g r oundw a t e r by t he us e of pl a nt s ( phyt or e m e di a t i on) R a pi d r e m ova l ha s be e n r e por t e d a t t he r oot z one ( r hi z os phe r e ) w he r e m e t ha not r ophs ( m e t ha ne oxi di z i ng ba c t e r i a ) c a pa bl e of c o oxi di z i ng T C E a r e pr e s e nt T he obj e c t i ve o f t he s t udy w a s t o de t e r m i ne by t he de ve l opm e nt o f a n a de qua t e pr ot oc ol how pl a nt t ype s ys t e m de s i gn, a nd e nvi r onm e nt a l c ondi t i ons p r e s e nt a t t w o phyt or e m e di a t i on s i t e s i m pa c t s m e t ha not r oph s bi ode gr a da t i on po t e nt i a l T o de ve l op c ha r a c t e r i z a t i on m e t hods phe not ypi c a nd ge not ypi c a na l ys e s of a n unc ha r a c t e r i z e d m e t ha not r oph, S t r a i n C S C 1, i s ol a t e d f r om a n unc ont a m i na t e d gr oundw a t e r a qui f e r w e r e pe r f o r m e d. F i e l d s i t e s r e pr e s e nt e d a n e n gi ne e r e d s ys t e m w i t h pop l a r a nd w i l l ow t r e e s a nd a na t u r a l l obl ol l y pi ne r e gr ow t h a r e a L a bor a t or y s t udi e s w e r e c onduc t e d t o a s s e s s t he a bi l i t y of m e t ha not r ophs t o oxi di z e pi ne e xuda t e s ( m onot e r p e ne s ) a nd i t s e f f e c t s on T C E ox i da t i on. F i e l d s a m pl e s w e r e a na l yz e d by c ul t ur e de pe nde nt m i c r obi a l c ount s a nd e nr i c hm e nt s a nd

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12 c ul t ur e i nde pe nde nt s t a bl e i s ot ope pr obi ng ( S I P ) m i c r oc os m s a nd m ol e c ul a r m e t hods S t r a i n C S C 1 pos s e s s e d a uni que s pi ny S l a ye r a nd w a s s how n t o be a nove l s t r a i n of t he ge nus M e t hy l oc y s t i s a nd w a s na m e d M e t hy l oc y s t i s al dr i c hi i s p. nov C ha r a c t e r i z a t i on m e t hods de ve l ope d w i t h S t r a i n C S C 1 w e r e s uc c e s s f ul l y a ppl i e d t o phyt or e m e di a t i on f i e l d s a m pl e s a nd i s ol a t e s D i f f e r e nt t ype s of m e t ha not r ophs w e r e c a pa bl e of oxi di z i ng m onot e r pe ne s ( pi ne ne ) a nd, i n t he pr e s e nc e of T C E ; a nt a goni s t i c a nd s yne r gi s t i c r e s pons e s w e r e obs e r ve d de pe ndi ng on m e t ha not r o ph t ype R hi z os phe r e s a m pl e s a na l yz e d by c ul t ur e de pe nde nt m e t hods c onf i r m e d t he pr e s e nc e of m e t ha not r ophs a t bot h s i t e s ; how e ve r e nr i c hm e nt s w e r e bi a s e d t ow a r ds t ype I I m e t ha not r ophs a nd di d not c or r e s pond w i t h t he a c t i ve popul a t i ons A c t i ve po pul a t i ons w e r e m or e di ve r s e a nd a bunda nt i n t he pl a nt e d s a m pl e s a nd s t r ongl y i nf l u e nc e d by t he de s i gn, e s pe c i a l l y t he us e of pl a nt i ng m a t e r i a l t ha t r e s ul t e d i n a dom i na nc e o f t he r m ot ol e r a nt m e t ha not r ophs V a r i a bl e r e s ul t s be t w e e n t he e ngi ne e r e d a nd na t u r a l s e t t i ngs hi ghl i ght t he i m por t a nc e o f m e a s ur i ng oxi da t i on pot e nt i a l s a nd di ve r s i t y of r hi z os phe r e m e t ha not r ophs a t a ny phyt or e m e di a t i on s i t e e s pe c i a l l y i f m onot e r pe ne r e l e a s i ng pl a nt s a r e c ont e m pl a t e d f or us e A l s o, s t udy of a c t i ve popul a t i ons w a s s how n t o be t he m os t a c c ur a t e c ha r a c t e r i z a t i on m e t hod. P hyl oge ne t i c a na l ys i s c o m bi ne d w i t h S I P m i c r oc os m s of f e r s pow e r f ul a na l yt i c a l t ool s t ha t c a n ul t i m a t e l y a i d p r a c t i t i one r s i n opt i m i z i ng phyt or e m e di a t i on f o r m o r e e f f e c t i ve t r e a t m e nt

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13 C H A P T E R 1 I N T R O D U C T I O N C hl or i na t e d s ol ve nt s s uc h a s t r i c hl or oe t hyl e ne ( T C E ) a r e a m a j o r s our c e of gr oundw a t e r a nd s oi l po l l ut i on t hr oughout t he U ni t e d S t a t e s I t i s w e l l know n t ha t s oi l m i c r oor ga ni s m s i n t he p r e s e nc e a nd a bs e nc e of ox yge n a r e c a pa bl e of de gr a di ng t he s e c om pounds ( B a r r i o L a ge e t a l 1986 ; F ox e t a l 1 990; H a ns on a nd H a ns on, 1996) T C E c a n be m e t a bol i z e d t o vi nyl c hl or i de a po t e nt c a r c i noge n, i f oxyge n i s not pr e s e nt ( B a r r i o L a ge e t a l 1986 ; E ns l e y, 1991) T he r e f or e t hi s pa t hw a y o f de gr a da t i on i s unde s i r a bl e gi ve n c ondi t i ons w he r e vi nyl c hl or i de c a n a c c um ul a t e w i t h no f u r t he r br e a kdow n. I n c ont r a s t m e t ha ne oxi di z i ng ba c t e r i a ( m e t ha not r ophs ) a e r obi c m i c r oor ga ni s m s know n f or t he i r bi or e m e di a t i on po t e nt i a l a nd pr e va l e nc e i n t he e nvi r onm e nt c a n c o m e t a bol i z e T C E t o C O 2 a t hi ghe r r a t e s t ha n ot he r m i c r oo r ga ni s m s ( W i l s on a nd W i l s on, 1985; L i t t l e e t a l 1988; F ox e t a l 1990) H ow e ve r t he pa t hw a y of T C E de gr a da t i on unde r a e r obi c c ondi t i ons i s not w i t hout r i s k o f f or m i ng t oxi c i nt e r m e di a t e s i nc l udi ng c hl o r a l hydr a t e di c hl o r oa c e t a t e a nd e t h yl e ne gl yc ol ( O l de nhui s e t a l 1989; A l va r e z C ohe n a nd M c C a r t y, 1991a ; S t a c pool e e t a l 1998; L a s h e t a l 2000) R e c e nt l y, t he pos s i bi l i t y of us i ng ve ge t a t i on t o e nh a nc e de gr a da t i on of o r ga ni c c ont a m i na nt s i n s oi l s ys t e m s ( phyt or e m e di a t i on) h a s r e c e i ve d a t t e nt i on a s a n a t t r a c t i ve l ow c os t a l t e r na t i ve t o t he t r a di t i ona l e ngi ne e r i ng a ppr oa c he s of s oi l e xc a va t i on a nd i nc i ne r a t i on, a i r s t r i ppi ng a nd pum p a nd t r e a t ( E P A 2000b; E P A 2001; M c C ut c he on a nd S c hnoor 2003) W he n pl a nt s a r e us e d f o r t h i s pur pos e a s e r i e s of m e c ha ni s m s a r e

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14 i nvol ve d, i nc l udi ng phyt o vol a t i l i z a t i on, a c c um ul a t i on, de g r a da t i on, s t a bi l i z a t i on, a nd r hi z ode gr a da t i on. H ow e ve r t he r ol e a nd c ont r i but i on of e a c h o f t he s e pr oc e s s e s t o t he ove r a l l r e m e di a t i on s ys t e m ha s not be e n a c c ur a t e l y c ha r a c t e r i z e d ( O r c ha r d e t a l 2000b; S ha ng e t a l 2003 ) I t i s w e l l know n t ha t t he m i c r oe nvi r onm e nt s ur r ou ndi ng t he r oo t z one of pl a nt s ( r hi z os phe r e ) i s c ha r a c t e r i z e d by hi ghe r num be r s o f ba c t e r i a a nd i nc r e a s e d m i c r obi a l a c t i vi t y ( C ur l a nd T r ue l ove 1986 ) T he r e f or e r hi z os phe r e m e t a bol i s m c a n s i gni f i c a nt l y c ont r i but e or gove r n t he r e m e di a l po t e nt i a l of ve ge t a t i on. P i l ot s t udi e s of phyt or e m e di a t i on s ys t e m s a r e be i ng t e s t e d i n t he f i e l d i n a r e a s c ont a m i na t e d w i t h c hl or i na t e d c om pounds T he t w o s i t e s s t udi e d i n t hi s pr oj e c t a r e t he S a va nna h R i ve r S i t e ( S R S ) i n A i ke n, S out h C a r ol i na a nd t he f o r m e r L a S a l l e E l e c t r i c a l U t i l i t i e s i n L a S a l l e I l l i noi s bot h i nvol vi ng a c t i ve phyt or e m e di a t i on of T C E a nd P C E ( B r i gm on e t a l 2001; E P A 2002; L a nge 2004 ) R e s e a r c h c onduc t e d a t t he S R S ha s de m ons t r a t e d t ha t T C E de gr a da t i on oc c ur s f a s t e r i n t he r h i z os phe r e of t r e e s ( W a l t on a nd A nde r s on, 1990; A nde r s on a nd W a l t on, 1995; B r i gm on e t a l 2001) T hus ve ge t a t i on m a y be us e d t o a c t i ve l y pr om ot e m i c r obi a l r e s t or a t i on of c ont a m i n a t e d s oi l s a nd e nha nc e d ha z a r dous c ont a m i na nt bi ode gr a da t i on. I n t he s e s i t e s s e ve r a l t r e e s pe c i e s a r e be i ng s t udi e d f or t he i r phyt o r e m e di a t i on pot e nt i a l L obl ol l y pi ne ( P i nus t ae da ) i s be i ng t e s t e d a s a pr om i s i ng s pe c i e s t ha t i s c a pa bl e of up t o 90% T C E r e m ova l f r om s oi l s a nd gr oundw a t e r a t t he S R S ( B r i gm on e t a l 2001 ) T hi s s pe c i e s i s c ha r a c t e r i z e d by t he p r o duc t i on of s i gni f i c a nt qua nt i t i e s of oi l e xt r a c t s c om pos e d m a i nl y of m onot e r pe ne s ( A m a r a l e t a l 1998; S a vi t h i r y e t a l 1998; P hi l l i ps e t a l 1999) T he r e f o r e t he s e c om pounds m a y i nf l ue nc e t he m i c r obi a l p r oc e s s e s

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15 oc c ur r i ng i n t he r hi z os phe r e e i t he r a s r oot e xuda t e s or l e a c ha t e f r om t h e de c a yi ng f ol i a ge i n t he s ur f a c e s oi l l a ye r s T he i m por t a nc e a nd oc c ur r e nc e of t he s e i nt e r a c t i ons on m i c r obi a l m e t a bol i s m ha ve be e n a dd r e s s e d pr e vi ous l y i n s e ve r a l s t udi e s ( W hi t e 1986; M i s r a e t a l 1996; A m a r a l a nd K now l e s 1997; W a r d e t a l 1997) C onf l i c t i ng f i ndi ngs on t he r ol e of pl a nt e xuda t e s on m i c r obi a l na t ur a l p r oc e s s e s i n t he uppe r s oi l l a ye r s s uc h a s ni t r i f i c a t i on a nd m e t ha ne c ons um pt i on, ha ve c l a i m e d i n hi bi t i on a s w e l l a s s t i m ul a t i on by t he s e c om pounds ( W hi t e 1986; M i s r a e t a l 19 96; A m a r a l a nd K now l e s 1997; W a r d e t a l 1997) I n pl a nt e d s oi l s a t phyt o r e m e di a t i on s i t e s t he r e i s no e vi de nc e t ha t c onne c t s t he i nc r e a s e d m i c r obi a l m i ne r a l i z a t i on of c ont a m i n a nt s w i t h t he pr e s e nc e of pl a nt c om pounds T he l a c k of unde r s t a ndi ng of t he po t e nt i a l r ol e s t ha t r hi z os phe r e ba c t e r i a c a n a s s um e i n t he ove r a l l r e m ova l of c ont a m i na nt s i s h i nde r e d by t he i na bi l i t y t o di r e c t l y a s s e s s t he a c t i vi t y a nd di ve r s i t y o f m i c r oo r ga ni s m s i n s i t u us i ng t r a di t i ona l c ul t ur e de pe nde nt m e t hods ( F r y 2004; S m a l l a 2004) T h e r e c e nt de ve l opm e nt of c ul t ur e i nde pe nde nt m e t hods t ha t i nvol ve s oi l m i c r o c os m s a nd l a be l e d s ubs t r a t e s c om bi ne d w i t h m ol e c ul a r t e c hni que s ha ve e na bl e d s c i e nt i s t s t o m or e e f f e c t i ve l y t e s t i n s i t u c ondi t i ons a nd, m or e i m por t a nt l y a c c ur a t e l y i de nt i f y a nd c ha r a c t e r i z e a c t i ve m i c r obi a l popul a t i ons ( R a da j e w s ki e t a l 2000 ) T he m a i n pu r pos e of t h i s s t udy i s t o de t e r m i ne by t he de ve l opm e nt of a n a de qua t e pr ot oc ol how pl a nt t ype s ys t e m de s i gn, a nd e nvi r onm e nt a l c ondi t i ons pr e s e nt a t t w o phyt or e m e di a t i on s i t e s i m pa c t t he po t e nt i a l a bi l i t y of m e t ha not r ophi c ba c t e r i a t o a c hi e ve bi ode gr a da t i on of c hl o r i na t e d s ol ve nt s i n c ont a m i n a t e d r hi z os phe r e s oi l s a nd gr oundw a t e r T he c ha r a c t e r i z a t i on of t hi s m e c ha ni s m a nd t he pr ovi s i on of a n a de qua t e

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16 t e c hni que s pe c i f i c t o t he r hi z os phe r e c a n ul t i m a t e l y l e a d t o m o r e e f f i c i e nt phyt or e m e di a t i on f o r m o r e e f f e c t i ve e nvi r onm e nt a l r e s t or a t i on. S i gn i f i c an c e o f t h e S t u d y A ny s ubs t a nc e t ha t pos e s a s i gni f i c a nt t hr e a t t o hu m a n he a l t h a nd t he e nvi r onm e nt de s e r ve s pr i or i t i z e d a t t e nt i on not onl y t o t he s t udy of i t s t oxi c ol ogy pr of i l e but a l s o t o i t s e nvi r onm e nt a l f a t e a nd de ve l opm e nt of r e m e di a t i o n t e c hnol ogi e s T he U S E P A ha s c l a s s i f i e d c hl or i na t e d s ol ve nt s T C E a nd P C E a s p r i or i t y pol l ut a nt s on t he ba s i s of i t s w i de s pr e a d c ont a m i na t i on i n gr oundw a t e r i t s pos s i bl e c a r c i noge ni c na t ur e a nd i t s pot e nt i a l t o be bi ol ogi c a l l y c onve r t e d t o t he m or e pot e nt c a r c i noge n vi nyl c hl or i de unde r a na e r obi c c ondi t i ons T he r e f o r e t he m a j or i t y of t h e N a t i ona l P r i o r i t y L i s t ( N P L ) s i t e s a r e de a l i ng w i t h t hi s t ype of c ont a m i na t i on G r oundw a t e r a nd s oi l c ont a m i na t i on by c hl o r i na t e d s ol ve nt s pr e s e nt s uni que c ha l l e nge s t o r e m e di a t i on t e c hnol ogi e s D ue t o t h e c he m i c a l a nd phys i c a l pr ope r t i e s o f t he s e c om pounds s m a l l a m ount s of t he s e s ol ve nt s c a n c ont a m i na t e a l a r ge vol um e of gr oundw a t e r T he r e m e di a t i on o f c ont a m i na t e d s o i l us ua l l y i nvol ve s e xc a va t i on a nd di s pos a l of t he i m pa c t e d m e di a H ow e ve r i f t he c ont a m i na nt ha s r e a c he d t he gr oundw a t e r t he r i s k t o t he publ i c t he r e m e di a l c o s t a nd t he a m ount of t i m e r e qui r e d t o r e m ove t he c ont a m i na nt s c a n i nc r e a s e s ubs t a nt i a l l y ( C he r e m i s i nof f 2001) A l t e r na t i ve l y, i n s i t u r e m e di a t i on t e c hnol ogi e s s uc h a s bi or e m e di a t i on a nd phyt or e m e di a t i on a r e be i ng t e s t e d a t s e ve r a l N P L f i e l d s i t e s O ne e xa m pl e i s t he S R S w he r e t he pot e nt i a l f or phyt or e m e di a t i on o f c hl o r i na t e d s ol ve nt s ha s be e n de m ons t r a t e d w i t h l obl ol l y pi ne s c a pa bl e of up t o 90 % T C E r e m ova l i n s oi l a nd gr oundw a t e r ( W a l t on a nd A nde r s on, 1990; A nde r s on a nd W a l t on, 1995) T he s e r e s ul t s a r e e nc our a gi ng f o r t he a ppl i c a t i on of m o r e s us t a i na bl e r e m e di a t i on t e c hno l ogi e s t ha t do not r e qui r e l a r ge

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17 a m ount s of i nput s a nd pr om ot e t he a ppl i c a t i on o f b i ol ogi c a l s ys t e m s a l r e a dy i n na t ur e t o e nvi r onm e nt a l pr obl e m s c r e a t e d by hum a ns I n or de r t o be t t e r unde r s t a nd a nd m oni t or t he s e bi o l ogi c a l pr oc e s s e s oc c ur r i ng d ur i ng phyt or e m e di a t i on t h i s s t udy c om bi ne d a l a bor a t or y a nd f i e l d a pp r oa c h. T he f oc us of t he s t udy i s on t he pot e nt i a l c ont r i b ut i on o f m e t ha not r ophi c ba c t e r i a c a pa bl e of c o oxi di z i ng c hl or i na t e d c om pounds t o t he r hi z ode gr a da t i on m e c ha ni s m i n phyt or e m e d i a t i on s ys t e m s A ddi t i ona l l y t he pr oj e c t c onc e nt r a t e s e f f or t s on m e t hod de ve l opm e nt s pe c i f i c t o t he r hi z os phe r e e nvi r onm e nt a nd c om pa r e s t r a di t i ona l l y us e d m e t hodol ogi e s w i t h m or e r e c e nt c ul t ur e i nde pe nde nt m e t hods L a bor a t or y ba s e d s t udi e s i nvol ve d c ha r a c t e r i z a t i on of i s ol a t e d pur e a nd m i x e d c ul t ur e s i nc l udi ng a gr oundw a t e r i s ol a t e S t r a i n C S C 1, w hi c h s e r ve d a s a m e a ns t o d e ve l op phe not ypi c a nd ge not ypi c pr ot oc ol s us e d i n t he phyt o r e m e di a t i on po r t i on o f t hi s s t udy. I n a ddi t i on, t he r ol e of pl a nt e xuda t e s a nd t he i r i m pa c t on T C E bi ode gr a da t i on w a s de t e r m i ne d w i t h r e p r e s e nt a t i ve m e t ha not r ophs i n t he l a bor a t or y. F i e l d ba s e d s t udi e s i nvol ve d c ha r a c t e r i z a t i on of t he m e t ha not r ophi c c om m uni t y i n r hi z os phe r e s a m pl e s f r om t w o c ur r e nt T C E a nd P C E c ont a m i na t e d s i t e s unde r goi ng phyt or e m e di a t i on w i t h di f f e r e nt t r e e t ype s L i t e r at u r e R e vi e w C h l or i n at e d C om p ou n d s T he w i de s pr e a d us e of c hl o r i na t e d hydr oc a r bons a s s ol ve nt s a nd de gr e a s e r s i n t he m e t a l a nd dr y c l e a ni ng i ndus t r i e s a nd t he i r i ndi s c r i m i na t e di s pos a l ha ve r e s ul t e d i n a s i gni f i c a nt a dve r s e e f f e c t on t he e nvi r onm e nt T r i c hl or oe t hyl e ne ( T C E ) a nd t e t r a c hl or oe t hyl e ne ( P C E ) pr i m a r y c hl or i na t e d s ol ve nt s f ound a t ha z a r dous w a s t e s i t e s oc c upi e d t he 16 t h a nd 31 s t pos i t i on, r e s pe c t i ve l y, i n t he P r i or i t y L i s t o f t he U n i t e d S t a t e s C om pr e he ns i ve E nvi r onm e nt a l R e s pons e C om pe ns a t i on, a nd L i a bi l i t y A c t ( C E R C L A )

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18 know n a s S upe r f und ( A T S D R 2006) T he pol l ut a nt s r a nk a c c or di ng t o t he i r pr e s e nc e a t N a t i ona l P r i or i t y L i s t f a c i l i t i e s pos s i bl e c a r c i noge ni c na t ur e a nd pot e nt i a l t o be c onve r t e d t o m o r e t oxi c byp r oduc t s a s vi nyl c hl or i de t ha t oc c upi e s t he 4 t h pos i t i on i n t he C E R C L A P r i or i t y L i s t ( V oge l a nd M c C a r t y, 1 985) A r e c e nt r e por t f r om t he N a t i ona l A c a de m i e s of S c i e nc e s C om m i t t e e on H um a n H e a l t h R i s ks of T r i c hl or oe t hyl e ne c onc l ude d t ha t t he e vi de nc e of T C E s c a r c i noge ni c r i s k ha s i nc r e a s e d s i nc e 2001 ( N A S 2006) C on s e que nt l y, t he s e c om pounds a r e he a vi l y r e gul a t e d by f e de r a l a nd s t a t e s t a nda r ds T he S a f e D r i nk i ng W a t e r A c t r e gul a t e s t he na t i ona l m a xi m um c ont a m i na nt l e ve l ( M C L ) o f T C E a nd P C E i n dr i nki ng w a t e r a t 5 ppb, w i t h a z e r o m a xi m um c ont a m i na nt l e ve l goa l ( M C L G ) ( E P A 2000a ) T he f a t e o f T C E a nd P C E r e l e a s e d i nt o t he e nvi r on m e nt t hr ough a va r i e t y of w a s t e s t r e a m s w i l l be di c t a t e d by t he i r phys i c a l a nd c he m i c a l pr ope r t i e s ( T a bl e 1 1 ) B e c a us e t he i r de ns i t i e s a r e gr e a t e r t ha n 1 g m l 1 P C E a nd T C E a r e c ons i de r e d de ns e nona que ous pha s e l i qui ds ( D N A P L s ) T he s e c om pounds c ons i de r e d vol a t i l e o r ga ni c c om pounds ( V O C s ) be c a us e of t he i r hi gh va l ue s of va por pr e s s ur e ( 74 18. 5 m m H g) a nd H e nr y s l a w c ons t a nt s ( 0 011 0. 0 18 a t m m 3 m ol 1 ) w he n r e l e a s e d i nt o t he a t m os phe r e or s ur f a c e w a t e r a nd s oi l w i l l vol a t i l i z e i nt o t he a t m os phe r e I n t he a t m os phe r e bot h c om pounds a r e s ubj e c t e d t o phot ooxi da t i on w i t h a ha l f l i f e of a c oupl e of m ont hs t o da ys A l s o, t he y bot h w oul d be p r e di c t e d t o r e a c h t he g r oundw a t e r gi ve n t he i r l ow pa r t i t i on i ng c oe f f i c i e nt va l ue s ( l og K o c a nd l og K o w o f 2 3) hi gh s pe c i f i c g r a vi t y ( > 1) a nd r e s ul t i ng l ow t e nde nc i e s t o a ds or b t o s e di m e nt s or s oi l s a nd t o bi oc onc e nt r a t e i n a ni m a l s a nd pl a nt s N e ve r t he l e s s s pe c i f i c s i t e c ondi t i ons s uc h a s or ga ni c s oi l c ont e nt c a n r e a di l y c ont r i but e t o t r a ns i e nt s or pt i on of T C E ( B r i gm on e t a l 1998 ; S he r e m a t a e t a l 2000) P C E m a y

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19 m ov e s l ow e r t ha n T C E i n s oi l i n f i l t r a t i on pr oc e s s e s be c a us e of i t s l ow e r w a t e r s ol ubi l i t y ( 150 m g l 1 ) c om pa r e d t o T C E ( 1 366 m g l 1 ) ( A T S D R 1999) A s a r e s ul t of t he i r c he m i c a l a nd phys i c a l c ha r a c t e r i s t i c s gr oundw a t e r c ont a m i na t i on by c hl or i na t e d c om pound s pr e s e nt s s e ve r a l c ha l l e nge s f or r e m e di a t i on. W he n T C E a nd P C E r e a c h t he gr oundw a t e r t he y a r e a nt i c i pa t e d t o s i nk de e pe r i nt o t he s ubs ur f a c e unt i l t he y r e a c h a l e s s pe r m e a bl e s t r a t um ( c onf i ni ng l a ye r ) I n t hi s l a ye r t he y w i l l s pr e a d out o r e s c a pe t hr ou gh f r a c t ur e s of t he r oc k or c l a y ( K ue pe r a nd M c W hor t e r 1991) T he r e f or e r e m e di a t i on i s m or e di f f i c ul t t ha n s pi l l s of l i gh t N A P L s ( L N A P L s ) s uc h a s ga s ol i ne f ue l s t ha t f l oa t ne a r t he s ur f a c e of t he w a t e r t a bl e a s a c om pa c t m a s s a nd do not a c t a s a s l ow r e l e a s i ng, c ont i nuous s our c e o f pol l ut i on ( C he r e m i s i nof f 2001 ) C hl or i na t e d c om pounds i n t he e nvi r onm e nt a r e pr o ne t o m i c r obi a l de gr a da t i on; how e ve r t he r a t e a nd e xt e nt of ox i da t i on i n t he pr e s e nc e of oxyge n i s i nve r s e l y r e l a t e d t o t he c hl or i ne t o c a r bon r a t i o ( H a ns on a nd H a ns on, 1996) H i ghl y c hl or i na t e d hy dr oc a r bons s uc h a s P C E a r e no t de gr a de d a e r o bi c a l l y. P C E i s r e duc t i ve l y de ha l oge na t e d unde r a na e r obi c c ondi t i ons ( U c hi ya m a e t a l 1989; B ow m a n e t a l 1993b) I n a e r obi c e nvi r onm e nt s T C E a nd t he m e t a bol i t e s of t he r e duc t i ve de ha l oge na t i on of P C E a nd T C E s uc h a s di c hl or oe t hyl e ne ( D C E ) a nd vi nyl c hl or i de ( V C ) a r e c om e t a bol i c a l l y oxi di z e d t o C O 2 by ba c t e r i a t ha t p os s e s s oxyge na s e e nz ym e s S om e of t he s e e nz ym e s a r e t he m e t ha ne m onooxyge na s e ( M M O ) of m e t ha not r ophs t ol ue ne di oxyge na s e ( T D O ) of P s e udom onas put i da F 1, t o l ue ne 2 m onooxyge na s e s ( T M O ) of B ur k hol de r i a c e pac i a G 4, pr opa ne m ono xyge na s e of M y c obac t e r i um v ac c ae J O B 5, phe nol hydr oxyl a s e ( P H ) of A l c al i ge ne s e ut r ophus J M P 134 a nd B ur k hol de r i a c e pac i a

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20 G 4, a l ke ne m onooxyge na s e ( A M O ) of A l c al i ge ne s de ni t r i f i c ans s pp. a m m on i a m onooxyge na s e of N i t r os om onas e ur opae a a nd i s opr opyl be nz e ne di oxyge na s e ( I P B ) o f P s e udom onas s p J R 1 ( A r c i e r o e t a l 1989; W a c k e t t e t a l 1989 ; E w e r s e t a l 1990 ; F ol s om e t a l 1990; F ox e t a l 1990; D a br oc k e t a l 1992; K i m e t a l 1996; S m i t h e t a l 1997) F r o m t hi s c om e t a bol i c pr oc e s s m i c r oor ga n i s m s do not ga i n e ne r gy or c a r bon o f t he oxi di z e d pol l ut a nt T he r e f o r e a n e xt e r na l s our c e of c a r bon ( e l e c t r on dono r ) m us t be pr e s e nt a pa r t f r o m oxyge n t ha t s e r ve s a s t he e l e c t r on a c c e pt or i n t he r e a c t i on. I n a na e r obi c c ondi t i ons pa t hw a ys of de gr a da t i on o c c ur vi a t he p r oc e s s of de ha l or e s pi r a t i on c a t a l yz e d by t he r e duc t i ve de ha l oge na s e e nz ym e T he c hl o r i na t e d c om pound f unc t i ons a s t he e l e c t r on a c c e pt or a nd, c om m onl y, hyd r oge n a s t he e l e c t r on do nor T he onl y know n m i c r oor ga ni s m t ha t pe r f or m s r e duc t i ve de c hl or i na t i on o f T C E a nd P C E t o c om pl e t i on i s D e hal oc oc c oi de s e t he noge ne s s t r a i n 195 ( M a ym o G a t e l l e t a l 1999) O t he r a na e r obe s a nd f a c ul t a t i ve a na e r obe s s uc h a s s ul f a t e r e duc e r s a nd m e t ha noge ns de gr a de T C E a nd P C E i nc om pl e t e l y t o c i s D C E a nd V C ( H ol l i ge r e t a l 1998) D e s ul f ur om onas c hl or oe t he ni c a a s ul f ur r e duc i ng ba c t e r i um ut i l i z e s pyr uva t e or a c e t a t e a s t he e l e c t r on donor a nd de gr a de s P C E o r T C E t o c i s D C E ( K r um hol z 1997) P h yt or e m e d i at i on P hyt or e m e di a t i on, t he us e of pl a nt s t o r e m e di a t e c ont a m i na t e d s i t e s t a ke s a dva nt a ge of t he a bi l i t y of pl a nt s t o e xt r a c t s e que s t e r or de gr a de pol l ut a nt s by t he m e c ha ni s m s of phyt o e xt r a c t i on, vol a t i l i z a t i on, d e gr a da t i on, s t a bi l i z a t i on a nd r hi z ode gr a da t i on ( F i g 1 1 ) T he l a s t m e c ha ni s m i s of s pe c i a l i nt e r e s t be c a us e i t i nvol ve s pl a nt m i c r obe i nt e r a c t i ons oc c ur r i ng i n t he r oot s y s t e m ( r hi z os phe r e ) T he r ol e of r hi z os phe r e m i c r oor ga ni s m s i n t he ove r a l l b r e a kdow n a nd r e m ova l o f pol l u t a nt s i s i nf l ue nc e d by t he t ype of c ont a m i na nt a nd p l a nt s p e c i e s ut i l i z e d. R hi z ode gr a da t i on ha s

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21 be e n r e por t e d a s t he m a i n pr oc e s s i n or ga ni c pol l ut a nt r e m e di a t i on o f t o l ue ne phe nol a nd T C E ( N a r a ya na n e t a l 1999) T he us e of p l a nt s r e pr e s e nt s a n a l t e r na t i ve t e c hnol ogy t o t r a di t i ona l w a s t e m a na ge m e nt pr a c t i c e s s uc h a s i nc i ne r a t i on, e xc a v a t i on a nd l a ndf i l l i ng, a nd pum p a nd t r e a t s ys t e m s T he e f f e c t i ve ne s s of phyt or e m e di a t i on ha s be e n de m ons t r a t e d i n a w i de r a nge of a ppl i c a t i ons s uc h a s he r bi c i de s pe t r ol e u m hydr oc a r bons m e t a l s r a di onuc l i de s l e a c ha t e s f r om l a ndf i l l s a nd s e w a ge s nut r i e nt s pe nt a c hl or ophe nol pol yc yc l i c a r om a t i c hydr oc a r bons a nd c hl o r i na t e d s ol ve nt s P hyt or e m e di a t i on of f e r s m u l t i pl e a dva nt a ge s i nc l udi ng be i ng a l ow c os t i n s i t u t e c hnol og y t ha t i s e nvi r onm e nt f r i e ndl y a nd publ i c l y a c c e pt e d. M os t i m por t a nt l y t he r e i s no ne e d t o di s t ur b t he s i t e a nd a f t e r t he t r e a t m e nt t he s oi l i s l e f t f e r t i l e f o r f u r t he r us e H ow e ve r s o m e l i m i t a t i ons a nd c onc e r ns di c t a t e t he pot e nt i a l a ppl i c a t i ons of t h i s t e c hnol ogy, i nc l udi ng t he t i m e ne c e s s a r y f or a c c e pt a bl e e f f e c t s t o t a ke pl a c e t he l i m i t e d de pt h o f t he r oot s ys t e m t he s e ns i t i vi t y of pl a nt s a nd m i c r obe s t ow a r ds t he c ont a m i na nt t he s e a s ona l va r i a bi l i t y i n t he r a t e of t r e a t m e nt a nd t he pot e nt i a l of c ont a m i na nt bi oa c c um ul a t i on or t r a ns por t i nt o t he f ood c ha i n N e ve r t he l e s s s om e of t he s e l i m i t a t i ons c a n be ove r c om e by s e l e c t i ng t he a pp r opr i a t e pl a nt s pe c i e s or by c om bi ni ng ot he r t e c hnol ogi e s s uc h a s pum pi ng a nd i r r i ga t i ng t he t r e e s w i t h t he de e pe r c ont a m i na t e d gr oundw a t e r ( E P A 2000b; M c C ut c he on a nd S c hnoor 2003) P hyt or e m e di a t i on e f f i c i e nc y i s s t i l l l i m i t e d by a l a c k of know l e dge o f m a ny ba s i c pl a nt pr oc e s s e s a nd i nt e r a c t i ons w i t h ot he r o r ga ni s m s s uc h a s ba c t e r i a a nd f ungi P ol l ut a nt de gr a da t i on by ba c t e r i a a nd f ungi ha ve b e e n s t udi e d e xt e ns i ve l y a nd, e ve n t hough pl a nt s c a n a l s o e xpr e s s s i m i l a r m e t a bol i c p a t hw a ys i t i s onl y r e c e nt l y t ha t e f f o r t s

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22 ha ve be e n c onc e nt r a t e d t ow a r ds unde r s t a ndi ng t he pl a nt s ys t e m M os t e nz ym e s i nvol ve d i n or ga ni c xe nobi ot i c de gr a da t i on, s uc h a s c yt oc hr om e P 450 oxi da s e s pe r oxi da s e s a nd gl ut a t hi one S t r a ns f e r a s e a r e know n t o be p r e s e nt i n bot h m i c r oor ga ni s m s a n d pl a nt s ( S a nde r m a nn, 1994 ; S ha ng e t a l 2003 ; C ha udhr y e t a l 2005) P hyt or e m e di a t i on of c hl or i na t e d s ol ve nt s f r om g r o undw a t e r a nd s oi l ha ve r e por t e d up t o 90% c ont a m i na nt r e m ova l by t he us e of di f f e r e nt pl a nt s pe c i e s ( W a l t on a nd A nde r s on, 1990; N e w m a n e t a l 1999; B r i gm on e t a l 2001; N e vi us e t a l 2004 ) H ow e ve r w he n a s s e s s i ng t h e r e s pons i bl e m e c ha ni s m s of c ont a m i na nt r e m ova l s t udi e s a r e not c ons i s t e nt T he m a i n c ont r a di c t i on i n phyt or e m e di a t i on of c hl or i na t e d s ol ve nt s r e ga r ds t he m a gni t ude o f pl a nt upt a ke phy t ovol a t i l i z a t i on, a nd r hi z ode gr a da t i on ( O r c ha r d e t a l 2000a ) S e ve r a l s t udi e s ha ve r e po r t e d t ha t T C E di s a ppe a r a nc e i n pl a nt e d s ys t e m s i s m a i nl y due t o pl a nt up t a ke f ol l ow e d by phyt ovol a t i l i z a t i on a nd di f f us i on t hr ough t he s t e m a nd/ o r m e t a bol i s m by t he p l a nt ( S c hr ol l e t a l 1994; A nde r s on a nd W a l t o n, 1995; N e w m a n e t a l 1997 ; B ur ke n a nd S c hnoor 1998 ) O n t he c ont r a r y ot he r s t udi e s ha ve obs e r ve d T C E de gr a da t i on oc c ur r i ng m a i nl y a s a r e s ul t o f r h i z os phe r e m i c r obi a l m e t a bol i s m ( W a l t on a nd A nde r s on, 199 0; A nde r s on e t a l 1993; S c hna be l e t a l 1997 ; O r c ha r d e t a l 2000a ) C ont r a di c t o r y r e s ul t s m a y be t he ou t c om e of e xpe r i m e nt a l a r t i f a c t s c a us e d by hi gh e xpos ur e t o T C E c onc e nt r a t i ons us e of c o s ol ve nt s t he s hor t dur a t i on of m a ny s t udi e s a nd pl a nt s t r e s s or i gi na t e d by t he us e of s t a t i c c ha m be r s t o a s s e s s a m a s s ba l a nc e of t he s ys t e m A ddi t i ona l l y, pr obl e m s e xi s t i n t he s e pa r a t i on of t he a bove a nd be l ow gr ound c om pa r t m e nt s s e l e c t i on of a de qua t e c ont r ol s a nd l a c k of m e t hods t o c or r e l a t e be nc h s c a l e s t udi e s t o t he f i e l d ( O r c ha r d e t a l 2000b; O r c ha r d e t a l 2000a )

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23 P opl a r a nd w i l l ow t r e e s a r e t he pr e f e r r e d pl a nt s pe c i e s i n t e m pe r a t e c l i m a t e s f or T C E a nd P C E phyt o r e m e di a t i on. T he y ha ve a l s o be e n us e d f or t he r e m e di a t i on o f he a vy m e t a l s s a l t s pe s t i c i de s e xpl os i ve s r a di onuc l i de s hydr oc a r bons a nd l a ndf i l l l e a c ha t e s ( I s e br a nds a nd K a r nos ky, 2001 ) V a l ua bl e popl a r a nd w i l l ow c ha r a c t e r i s t i c s t ha t m a ke t he m i de a l f o r t hi s a ppl i c a t i on a r e t ha t t he y a r e f a s t gr ow i ng, e a s i l y pr opa ga t e d, t ol e r a nt t o hi gh l e ve l s of c ont a m i na nt s ( < 550 ppm T C E ) r e s i s t a nt t o s a t ur a t e d c ondi t i ons a nd t he y a r e phr e a t ophyt e s ( de e p r oot e d pl a nt w he r e w a t e r upt a ke i s m a i nl y f r om t he gr oundw a t e r ) ( I s e br a nds a nd K a r nos ky, 2001 ; P i l o n S m i t s 2005 ) I n pa r t i c ul a r w i l l ow s ha ve be e n f ound t o c ons i s t e nt l y ut i l i z e gr oundw a t e r s our c e s e ve n dur i ng pe r i ods of r a i nf a l l ( S nyde r a nd W i l l i a m s 2000 ) A ddi t i ona l l y, popl a r a nd w i l l ow t r e e s pos s e s s s pe c i a l i z e d r oot ve s s e l s ( a e r e nc hym a ) t ha t m a y c o m pr i s e up t o 60% o f t he i nt r a c e l l ul a r vol um e a nd m e di a t e oxyge n d i f f us i on de e pe r i nt o t he s oi l pr of i l e ( C ha udhr y e t a l 2005 ) I t ha s be e n hypot he s i z e d t ha t w i l l ow t r e e s m a y c on t a i n a hi ghe r c onc e nt r a t i on of oxi da t i ve e nz ym e s W he n popl a r a nd w i l l ow t r e e s w e r e dos e d w i t h P C E onl y by pr oduc t s of de gr a da t i on w e r e f ound i n w i l l ow a nd no T C E w a s de t e c t e d, a s i t w a s c om m onl y f ound i n popl a r t i s s ue a nd i t s r h i z os phe r e ( N z e ngung a nd J e f f e r s 2001) T he l a r ge s ur f a c e a r e a a nd po r ous w ood of popl a r t r e e s a l l ow s w a t e r t r a ns por t t hr ough t he e nt i r e c r os s s e c t i on of t he s t e m w hi c h c a n r e s ul t i n 3 m ye a r 1 gr ow t h unde r opt i m a l c ondi t i ons ( L a ndm e ye r 2001) T r a ns pi r a t i on r a t e s c a n i nc r e a s e f r om 19 t o 200 1000 L o f w a t e r da y 1 i n young t o m a t u r e t r e e s ( N e w m a n e t a l 1997; P i l on S m i t s 2005) T he s e hi gh t r a ns pi r a t i on r a t e s c a n e xt r a c t e nough w a t e r t o de pr e s s t he w a t e r t a bl e l oc a l l y, i nduc i ng f l ow t ow a r d t he t r e e s a nd, c ons e que n t l y, c ont a i ni ng t he c ont a m i na nt pl u m e ( hydr a ul i c c ont r ol ) A ddi t i ona l l y, popl a r t r e e s pos s e s s e ndophyt i c ba c t e r i a i nc l udi ng

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24 m e t ha not r ophs t ha t l i ve s ym bi ot i c a l l y w i t h i n t he p l a nt S om e o f t he s e ba c t e r i a i s ol a t e d f r om pl a nt s a r e know n f or t he i r bi o r e m e d i a t i on pot e nt i a l i nc l udi ng m e m be r s of P s e udom onas s p. E nt e r obac t e r C l os t r i di um s pe c i e s a nd m e t hyl ot r oph s pe c i e s s uc h a s M e t hy l obac t e r i um popul i s p. nov ( B r i gm on e t a l 1999; V a n A ke n e t a l 2004a ; V a n A ke n e t a l 2004b) T he e f f e c t i ve ne s s of c hl or i na t e d s ol ve nt phyt or e m e di a t i on by popl a r a nd w i l l ow t r e e s i s s t r ongl y i nf l ue nc e d by t he c hoi c e o f ge not y pe s ( c l one s ) C ons i de r a t i on o f t he a de qua t e c l one i s a n e s s e nt i a l s e l e c t i on c r i t e r i on, a s t he c hoi c e m us t be c om pa t i bl e w i t h t he i nt e nde d us e t he s i t e c ha r a c t e r i s t i c s ( s oi l t ype m i c r oc l i m a t e pe s t s a nd di s e a s e s ) a nd w i t h t he l oc a l op i ni on c onc e r ni ng us e of na t i ve v e r s us e xot i c t r e e s ( I s e br a nds a nd K a r nos ky, 2001) O t he r po t e nt i a l t r e e s pe c i e s t ha t ha ve be e n s t udi e d f or c hl or i na t e d s ol ve nt phyt or e m e di a t i on a r e c oni f e r s i n pa r t i c ul a r t he l ob l ol l y pi ne ( A nde r s on a nd W a l t on, 1995; P uns hon e t a l 2002; B r i g m on e t a l 2003) I n a s t udy w he r e pi ne w i l l ow a nd popl a r t r e e s w e r e c om pa r e d f or t he i r T C E phyt or e m e di a t i on pot e nt i a l unde gr a d e d T C E w a s f ound pr i m a r i l y i n t he va s c ul a r s ys t e m a nd l e a ve s of pi ne w he r e a s pl a nt m e t a bol i t e s of T C E w e r e f ound w i t hi n t he l e a f t i s s ue of popl a r a nd w i l l ow t r e e s s ugge s t i ng pl a nt de gr a da t i on pot e nt i a l of t he s e t ype of t r e e s ( P uns hon e t a l 2002 ) M e a n w hi l e f or pi ne s i t ha s be e n pos t ul a t e d t ha t r hi z ode gr a da t i on i s t he m a i n phyt or e m e di a t i on m e c ha ni s m ( A nde r s on a nd W a l t on, 1995) T h e R h i z os p h e r e T he r hi z os phe r e i s t he r oot z one unde r t he i nf l ue nc e of t he pl a nt ( C ur l a nd T r ue l ove 1986) T h i s z one i s c ons t a nt l y e nr i c he d w i t h a va r i e t y of p l a nt de r i ve d c om pounds a nd, a s a r e s ul t h i ghe r m i c r obi a l de ns i t i e s ( 5 20 t i m e s ) a nd r a t e s of a c t i vi t y

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25 ( 2 3 o r de r s ) oc c ur r e d i n t hi s a r e a c om pa r e d t o non ve ge t a t e d s oi l ( W a l t on e t a l 1994) I n r hi z os phe r e s t udi e s t he r hi z opl a ne i s de f i ne d a s t he s oi l a dhe r e d t o t he r oot s t he r hi z os phe r e a s t he s oi l unde r t he i nf l ue nc e of t he pl a nt a nd t he r h i z os phe r e e f f e c t ( R / S r a t i o ) a s t he r a t i o be t w e e n t he a bunda nc e of m i c r obi a l popul a t i ons i n t he r hi z os phe r e t o t ha t i n bul k s oi l H ow e ve r t he e f f e c t of t h e pl a n t i s not on l y t r a ns l a t e d i n hi ghe r a bunda nc e but a l s o i n hi ghe r a c t i vi t y. T he r e f or e w he n pl a nt s a r e pr e s e nt s e l e c t i ve e nr i c hm e nt of popu l a t i ons m a y or m a y no t t r a ns l a t e t o hi ghe r R / S r a t i os a l t hough hi ghe r de gr a da t i on a c t i vi t y i s obs e r ve d ( H a by a nd C r ow l e y, 1996) U p t o 10 40 % o f t he a s s i m i l a t e d c a r bon m a y be e x ude d by pl a nt s i nt o t he r hi z os ph e r e ( r hi z ode pos i t i on) i n t he f or m of c om p ounds t ha t a r e r e a di l y ut i l i z e d by m i c r oor ga ni s m s ( W hi pps a nd L ync h, 1983 ) P l a nt e xuda t e s i nc l ude s uga r s a m i no a c i ds or ga ni c a c i ds nuc l e ot i de s f l a vonone s phe nol i c c o m pounds t e r pe ne s a nd c e r t a i n e nz ym e s T he r a t e of e xuda t i on de pe nds on t he a g e of t he pl a nt s oi l nut r i e nt a va i l a bi l i t y pr e s e nc e of c ont a m i na nt s a nd s e a s ona l i t y. T he s e c om pounds ha ve be e n s how n t o be r e l e a s e d i nt o t he r hi z os phe r e i n gr e a t e r a m ount s a t t he e nd of t he gr ow i ng s e a s on dur i ng l e a f s e ne s c e nc e ( H e gde a nd F l e t c he r 1996) D ur i ng t hi s pe r i od a bout 58% o f t he pr oduc e d f i ne r oot bi om a s s di e s ( r oot t u r nove r ) a n d, a s a r e s ul t a n i nc r e a s e of up t o 2 f ol d i n phe nol i c c om pounds ha s be e n obs e r ve d a t t he r hi z os phe r e ( L e i gh e t a l 2002) T he s e c om pounds a r e know n t o s t i m ul a t e pol yc hl o r i na t e d bi phe nyl ( P C B ) bi ode gr a da t i on ( D onne l l y e t a l 1994) A pa r t f r om t he va r i e t y of c a r bon s our c e s t he r hi z os phe r e pr ovi de s s t e a dy r e dox c ondi t i ons a nd i de a l a t t a c hm e nt s i t e s f or ba c t e r i a l pr ol i f e r a t i on ( C ur l a nd T r ue l ove 1986; S hi m e t a l 2000 )

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26 P l a nt s be ne f i t f r om t he pr e s e nc e of r hi z os phe r e m i c r oor ga ni s m s be c a us e t he y c a n i nc r e a s e nut r i e nt a va i l a bi l i t y t h r ough bi os ur f a c t a nt pr oduc t i on ( s ol ubi l i z e s s oi l bound nut r i e nt s ) a nd N 2 f i xa t i on p r oduc e hor m one s t ha t pr om ot e pl a nt g r ow t h, s uppr e s s de l e t e r i ous m i c r oor ga ni s m s by t he pr oduc t i on o f a nt i bi ot i c s a nd de gr a de phyt ot oxi c s oi l c on t a m i na nt s ( S m a l l a e t a l 2001) T hus t he r e i s a l s o c ons i de r a bl e i nt e r e s t i n c ha r a c t e r i z i ng t he s t r uc t ur e a nd f unc t i on of r hi z os phe r e m i c r obi a l c om m un i t i e s f or t he a dva nt a ge ous e f f e c t s t o pl a nt s P hyt or e m e di a t i on m a y e xpl o i t t he be ne f i c i a l e f f e c t of m ode r a t e pl a nt s t r e s s ( B a r oc s i e t a l 2003; C ha udhr y e t a l 2005) C e r t a i n l e ve l s of nut r i e nt a nd w a t e r de f i c i e nc i e s a nd c he m i c a l t oxi c i t y m a y i nduc e s t r e s s a da pt a t i on, r oot p r ol i f e r a t i on a nd e xuda t i on, a nd e nha nc e r oot ha i r de ns i t y. F or e xa m pl e P or K de f i c i e nc y i s know n t o s t i m ul a t e e xuda t i on of o r ga ni c a c i ds a nd c e r t a i n e nz ym e s M e a nw hi l e F e o r Z n de f i c i e nc y i nduc e s t he pr oduc t i on o f m e t a l c he l a t or s ( phyt os i de r ophor e s ) ( C ha udhr y e t a l 2005 ) P l a nt t ol e r a nc e t o he a vy m e t a l s w a s e n ha nc e d w he n a s ynt he t i c c he l a t e ( e t hyl e ne di a m i nt e t r a a c e t i c a c i d, E D T A ) w hi c h r a pi dl y i nc r e a s e s m e t a l bi oa va i l a bi l i t y, w a s a ppl i e d i n s e ve r a l l ow dos e s a voi di ng pl a nt de t r i m e nt a l e f f e c t s a nd s e c ur i ng t i m e f or pl a nt a da pt a t i on ( B l a yl oc k e t a l 1997; B a r oc s i e t a l 2003 ) W i t hi n t he di ve r s i t y of r hi z os phe r e m i c r oo r ga ni s m s t he r e a r e s t r a i ns c a pa bl e of de gr a di ng xe nobi ot i c c om pounds ( C ur l a nd T r ue l o ve 1986; W a l t on a nd A nde r s on, 1990; W a l t on e t a l 1994; A nde r s on a nd W a l t on 1995; B r i gm on e t a l 1999) T he di ve r s i t y of he t e r ot r oph m i c r oo r ga ni s m s m a y e nha nc e s t e pw i s e t r a ns f or m a t i on o f c ont a m i na nt s by m i c r obi a l c ons or t i um a nd/ o r p r ovi de a n e nvi r onm e nt t ha t i s f a vor a bl e f o r ge ne t i c e xc ha nge a nd ge ne r e a r r a nge m e nt s of t he de gr a da t i ve t r a i t s T he pr e s e nc e of s t r uc t u r a l

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27 a na l ogs t o c ont a m i na nt s i n r oot e xuda t e s c e l l w a l l c om pone nt s a nd l ys a t e s a s w e l l a s s e c onda r y pr oduc t s of de gr a da t i on of t he s e m a t e r i a l s m i ght f or t ui t ous l y s e l e c t f or m i c r obe s t ha t m e t a bol i z e ( a c c om pa ni e d by e ne r gy ga i n) or c om e t a bol i z e ( i nvol vi ng no e ne r gy ga i n) xe nobi ot i c s T e r pe ne s ( s e c onda r y pl a nt m e t a bol i t e s ) a nd P C B s pl a nt a na l ogs ( phe nol i c c om pounds ) ha ve be e n r e po r t e d t o pl a y a n i m por t a nt r ol e i n a c t i va t i ng or t r a ns f or m i ng s pe c i f i c ba c t e r i a l ha bi t a t s by i nduc i ng bi phe nyl di oxyge na s e i n P C B de gr a di ng ba c t e r i a a nd i nc r e a s e popul a t i ons of t hi s de gr a de r s by up t o 100 f ol d ( D onne l l y e t a l 1994; F l e t c he r a nd H e gde 1995; H a by a nd C r ow l e y, 1996) T he r hi z os phe r e pr ov i de s s t a bl e s our c e s of oxyge n a nd m e t ha ne t ha t c a n s uppor t t he a c t i vi t y of m e t ha ne oxi di z i ng ba c t e r i a ( m e t ha n ot r ophs ) know n t o c om e t a bol i c a l l y oxi di z e T C E a t hi ghe r r a t e s t ha n ot he r ba c t e r i a ( L i t t l e e t a l 1988 ; F ox e t a l 1990; B r i gm on e t a l 1999) I t ha s be e n de m ons t r a t e d t h a t T C E de gr a da t i on oc c ur s f a s t e r i n t he r hi z os phe r e of pl a nt s ( W a l t on a nd A nde r s on, 1990 ; A nde r s on a nd W a l t on, 1995) w he r e t he pr e s e nc e a nd de ns i t y of m e t ha not r oph s ha s be e n s how n t o pl a y a n i m por t a nt r ol e i n T C E de gr a da t i on ( B r i gm on e t a l 1999) M i c r obi a l de gr a da t i on of c ont a m i na nt s i s us ua l l y n ot dr i ve n by e ne r gy ne e ds but by a ne c e s s i t y t o r e duc e t oxi c i t y f or w hi c h m i c r ob e s m a y e xpe r i e nc e a n e ne r gy de f i c i t T he r e f or e t he p r oc e s s m a y be a s s i s t e d a nd dr i ve n by t he a bunda nt e ne r gy a va i l a bl e i n t he r hi z os phe r e e n vi r onm e nt a s r oot e xuda t e s a nd a c c um ul a t e d pl a nt bi om a s s T he t ype of c om pounds t he s pe c i e s of pl a nt a nd t he de gr e e of c ont a m i na t i on m a y ha ve t he pot e nt i a l t o e xe r t p r e s s ur e a nd t hus s e l e c t f or s pe c i a l i z e d de gr a di ng ba c t e r i a l popul a t i ons C ons e que nt l y, r hi z os phe r e m i c r obi a l popul a t i ons m a y c ha nge c ons i de r a bl y w i t h t i m e i n r e s pons e t o t he t ype a nd de g r e e of c ont a m i na t i on ( F l e t c he r a nd H e gde 1995; H e r na nde z

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28 e t a l 1997; B r i gm on e t a l 1999; K oz dr oj a nd va n E l s a s 200 0) H ow e ve r t he r e i s a l a c k of i n f or m a t i on on s pe c i f i c pl a nt c ha r a c t e r i s t i c s t ha t pr om ot e m i c r obi a l de g r a da t i on of or ga ni c po l l ut a nt s ( C ha udhr y e t a l 2005 ) A not he r une x pl or e d a r e a of r hi z os phe r e m i c r oe nvi r onm e nt s i s t he i nt e r a c t i on of pl a nt a nd m i c r obi a l popul a t i ons w i t h m yc or r hi z a e M yc or r hi z a e s ym bi ot i c r oot f ungi pl a y a n i m por t a nt r ol e i n pl a nt e s t a bl i s hm e nt a nd s ur vi va l S om e of t he s e s ym bi ot i c a s s oc i a t i on s a r e s pe c i f i c t o pl a nt s pe c i e s s uc h a s w i t h l obl ol l y pi ne t r e e s us e d i n phyt or e m e di a t i on. I n t he s e pi ne s hi ghe r de ns i t i e s of m e t ha not r ophi c ba c t e r i a w e r e obs e r ve d t o be a s s oc i a t e d w i t h t he f ung i ( B r i gm on e t a l 1999) T he r e f o r e m yc or r hi z a e m a y c ont r i but e s i gni f i c a nt l y t o t he r e m e di a t i on pot e nt i a l of s e ve r a l pl a nt s pe c i e s T he f ungi pr ovi de s t he pl a nt a nd r h i z os phe r e ba c t e r i a p r ot e c t i on a ga i ns t dr ought a nd t oxi c pol l ut a nt s be c a us e of t he phys i c a l ba r r i e r c r e a t e d b y t he i r e xt e ns i ve hypha e ne t w or k A l s o, t hi s ne t w or k c a n i nc r e a s e t he s ur f a c e a r e a ov e r w hi c h t he p l a nt s a nd a s s oc i a t e d m i c r oor ga ni s m s e xpl or e f o r w a t e r nut r i e nt s a nd p ol l ut a nt upt a ke A ddi t i ona l l y m yc or r hi z a e i s know n f or t he e xt r a c t i on of he a vy m e t a l s a nd de gr a da t i on of or ga ni c pol l ut a nt s f r om s oi l i nc l udi ng 2 4 D a t r a z i ne a nd P C B s ( D onne l l y a nd F l e t c he r 1995 ; M e ha r g a nd C a i r ne y, 2000; C ha udhr y e t a l 2005) R hi z os phe r e e nha nc e d m i c r obi a l de gr a da t i on pr oc e s s e s a r e poor l y unde r s t ood a nd c e r t a i nl y va r y a c c or di ng t o s oi l c ondi t i ons pl a nt s pe c i e s a nd t ype of c ont a m i na nt ( H a by a nd C r ow l e y, 1996 ) I t i s r e l e va nt t o s t udy t he s e pr oc e s s e s a s t he y ha ve t he pot e nt i a l t o c om pl e t e l y m i ne r a l i z e c ont a m i na nt s A s a r e s ul t c ont a m i na nt s a r e not t r a ns p or t e d i nt o t he pl a nt r e duc i ng t he pos s i bi l i t y of pa s s i ng t he t o xi c c om pound i nt o ot he r o r ga ni s m s i n t he f ood c ha i n a nd t he r e l e a s e of pot e nt i a l l y ha r m f ul pol l ut a nt s i nt o t he a t m os phe r e T hi s

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29 s c e na r i o m a y r e pr e s e nt t he i de a l i n s i t u r e m e di a t i o n s ys t e m w h e r e t he r ol e o f t he p l a nt i s t o s uppor t a nd s t i m ul a t e m i c r oor ga ni s m s c a pa bl e o f c ont a m i na nt de gr a da t i on M on ot e r p e n e s M or e t ha n 70 hyd r oc a r bons i nc l udi ng i s opr e ne m ono a nd s e s qui t e r pe ne s a nd a s ubs t a nt i a l num be r of oxyge na t e d o r ga ni c s a r e t he pr e dom i na nt c he m i c a l s pe c i e s e m i t t e d by ve ge t a t i on ( B e nj a m i n e t a l 1996) M onot e r pe ne s a nd i s opr e ne a r e t he m a j o r na t ur a l vol a t i l e or ga ni c c om pounds ( V O C s ) a nd a nd pi ne ne a r e t he r e pr e s e nt a t i ve m onot e r pe ne s ( K i m 2001) M onot e r pe ne s a r e t he s i m pl e s t c ons t i t ue nt s of t he pl a nt e s s e nt i a l oi l s a nd t he m a j or non m e t ha ne hydr oc a r bon e m i t t e d t o t he a t m os phe r e ( 4. 8 X 10 1 4 g ye a r 1 ) w hi c h c o nt r i but e s t o t he f o r m a t i on o f t r opos phe r i c oz one ( Z i m m e r m a n e t a l 1978 ) E m i s s i ons f r om c oni f e r f o r e s t s a r e pr e dom i na n t l y m onot e r pe ne s ( A m a r a l a nd K now l e s 1998; S a vi t hi r y e t a l 1998) F or P i nus t ae da ( l obl ol l y pi ne ) m onot e r pe ne e m i s s i ons a r e 5. 1 g g l e a f dw 1 h 1 w i t h gr e a t e r t h a n 60% r e pr e s e nt e d by pi ne ne ( B e nj a m i n e t a l 1996; K i m 2001) O n t he c ont r a r y, b r oa d l e a f s pe c i e s s uc h a s P opul us de l t oi de s ( popl a r ) a nd Sal i x ni gr a ( w i l l ow ) a r e a m ong t he hi gh i s opr e ne e m i t t i ng s pe c i e s ( G e r on e t a l 2001) w i t h 37 0 a nd 25. 2 g l e a f dw 1 h 1 r e s pe c t i ve l y, w i t h no de t e c t e d m onot e r pe ne e m i s s i ons ( L a m b e t a l 1985) A pa r t f r om t r e e e m i s s i ons m onot e r pe ne s c a n be r e l e a s e d i nt o t he e nvi r onm e nt f r om di s c ha r ge e f f l ue nt s of t he pul p m a nuf a c t ur i n g i ndus t r y, a s m onot e r pe ne s a r e t he pr e dom i na nt c om pone nt o f t u r pe nt i ne ( K l e i nhe i nz e t a l 1999) M onot e r pe ne s a r e a l s o be i ng us e d i n t he f ood, pe r f um e pha r m a c e ut i c a l i n dus t r i e s a nd, r e c e nt l y, a t a l a r ge r s c a l e i n a n e f f o r t t o s ubs t i t ut e f o r c hl or o f l uor oc a r bons a nd ha l oge na t e d s ol ve nt s ( A m a r a l e t a l

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30 1998) T he r e f or e t he i r e nvi r onm e nt a l f a t e a nd i n t e r a c t i ons w i t h ot he r s ubs t a nc e s a r e of i m por t a nc e T he s e c om pounds pos s e s s a br oa d r a nge of f unc t i o ns i n na t ur e f r o m e c ol ogi c a l i nt e r a c t i ons t ha t e xt e nd f r om a l l e l opa t hy a ge nt s ( a nt i m i c r obi a l s a nd f ungi c i de s ) t o pol l i na t or a t t r a c t a nt s ( T ooke r e t a l 2002) P ot e nt i a l s our c e s of m onot e r pe ne s i n s oi l s i nc l ude l e a c ha t e f r om l e a f l i t t e r a nd c a nopy l e a ve s r oot e xud a t i on a nd de pos i t i on f r om t he a t m os phe r e T he r ol e of m onot e r pe ne s a nd t he i r e f f e c t on s oi l m i c r obi a l c om m un i t i e s i s c om pl e x a nd ha s not be e n f ul l y e l uc i da t e d. I t i s know n t ha t c e r t a i n m i c r obi a l e nz ym e s a r e s t i m ul a t e d by t he pr e s e nc e of m onot e r pe ne s a nd t ha t s e ve r a l m i c r oo r ga ni s m s i nc l udi ng P s e udom onas s p. A l c al i ge ne s x y l os ox i dans a nd B ac i l l us s p. c a n us e t he s e c om pounds a s c a r bon a nd e ne r gy s our c e s ( V okou e t a l 1984; M i s r a e t a l 1996; V okou a nd L i ot i r i 1999; Y oo e t a l 2001 ) A l s o, i t ha s be e n r e por t e d t ha t m onot e r pe n e s f i r s t i nt r oduc e d a s de c a yi ng pl a nt m a t e r i a l or e xuda t e s of m onot e r pe ne r e l e a s i ng pl a nt s m a y e nha nc e bi ot r a ns f or m a t i on of P C B s ( H e r na nde z e t a l 1997 ) H ow e ve r t he r e a r e a l s o r e por t s of i nhi bi t i on of d i f f e r e nt m i c r obi a l pr oc e s s e s by t he s e c om pounds ( V okou e t a l 1984; W hi t e 1986 ; W a r d e t a l 1997 ) N i t r oge n m i ne r a l i z a t i on a nd ni t r i f i c a t i on i s i nhi bi t e d i n t he pr e s e nc e of m onot e r pe ne s but t he pr e c i s e m ode of a c t i on ha s not ye t be e n e l uc i da t e d ( W hi t e 1986 ; W hi t e 1988; W hi t e 1994 ) W hi t e ( 1988) pr opo s e d t ha t m onot e r pe ne s hi nde r ni t r i f i c a t i on by i nhi bi t i ng t he e nz ym a t i c a c t i vi t y of a m m oni um m onooxyge na s e ( A M O ) t he f i r s t e nz ym e i n t he a m m oni a oxi da t i on pa t hw a y, a nd t ha t t he de gr e e of i nhi bi t i on w a s de t e r m i ne d by t he s t r uc t ur e o f t he c om pound T he s e r e s ul t s ha ve pr ovoke d ot he r s t udi e s on t he e f f e c t of m onot e r pe ne s on m e t ha ne oxi da t i o n be c a us e of t he s i m i l a r i t y be t w e e n t he

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31 m onooxyge na s e e nz ym e s of t he s e t w o gr oups of b a c t e r i a ( A m a r a l a nd K now l e s 1997; A m a r a l e t a l 1998 ) T he s pe c ul a t i on i s t ha t m ono t e r pe ne s i nhi bi t M M O s i m i l a r l y t o A M O r e s ul t i ng i n t he i nhi bi t i on of C H 4 upt a ke T he a ut hor s a l s o s uppor t t hi s hypo t he s i s by m e nt i oni ng t ha t m e t ha not r ophs a r e not t ha t c om m onl y f ound i n t he s ur f a c e l a ye r s of f or e s t s oi l s w he r e m ono t e r pe ne c onc e nt r a t i ons a r e t he hi ghe s t ( A m a r a l a nd K now l e s 1997; A m a r a l e t a l 1998) H ow e ve r m e t ha not r o phs i n s oi l s ur f a c e s oxi di z e d a t m os phe r i c C H 4 ( a t c onc e nt r a t i ons o f 1 7 ppm ) a nd t he i r i s ol a t i on ha s pr ove n t o be di f f i c ul t be c a us e of t he c om pe t i t i ve a dva nt a ge o f l ow a f f i ni t y m e t ha not r ophs i n ge ne r a l l y us e d l a bor a t or y c ondi t i ons a t hi gh C H 4 c onc e nt r a t i ons ( us ua l l y 20% ( v/ v) C H 4 ) T o da t e hi gh a f f i ni t y m e t ha not r ophs ha ve be e n phyl oge ne t i c a l l y i de nt i f i e d but not i s ol a t e d ( H ol m e s e t a l 1999; J e ns e n e t a l 2000) C ons e que n t l y, t he l ow a bunda nc e of m e t ha not r ophs i n s oi l s ur f a c e s m a y be t he r e s ul t of i na de qua t e c ul t i va t i on t e c hni que s A l pha pi ne ne one of t he m os t a bunda nt m onot e r p e ne s e xi s t s pr e dom i na nt l y i n N or t h A m e r i c a a s t he r i ght e na nt i om e r ( + ) p i ne ne ( S a vi t hi r y e t a l 1998) T hi s c om pound i s a bi c yc l i c a l ke ne c om pos e d of t w o i s opr e ne uni t s C 5 H 8 W he n r e l e a s e d i nt o t he e nvi r onm e nt t he f a t e of t hi s m onot e r pe ne i s di c t a t e d by i t s phys i c a l a nd c he m i c a l pr ope r t i e s ( T a bl e 1 2 ) A l pha pi ne ne r e l e a s e d i nt o t he a t m os phe r e e xi s t s s ol e l y a s a va por t ha t c a n be de gr a de d by t he r e a c t i on w i t h ph ot oc he m i c a l l y pr oduc e d hydr oxyl r a di c a l s ( ha l f l i f e = 4 h ) oz one ( ha l f l i f e = 40 m i n) a nd ni t r a t e r a di c a l s a nd i n a n i ght t i m e r e a c t i on ( ha l f l i f e = 6 m i n) I n s oi l s pi ne ne s ho w s l ow m obi l i t y be c a us e i t a ds or bs t o s oi l pa r t i c l e s ( K o c of 1 200 a nd l og K o w o f 4 83) H ow e ve r i n m oi s t s oi l s ur f a c e s vol a t i l i z a t i on i s e xpe c t e d t o be a n i m po r t a nt pr oc e s s ba s e d on i t s H e nr y s l a w c ons t a nt ( 0. 107 a t m m 3 m ol 1 ) I n w a t e r pi ne ne w i l l a ds or b t o s us pe nde d s ol i ds a nd s e di m e nt s

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32 a nd e xhi bi t a hi gh pot e nt i a l f or bi oc onc e nt r a t i on i n a qua t i c or ga ni s m s ( bi oc onc e nt r a t i on f a c t or of 2, 800 ) ( H S D B 1999) B i ode gr a da t i on o f pi ne ne oc c ur s i n s oi l s w he r e a s a va r i e t y of ba c t e r i a ( P s e udom ona s s p. A l c al i ge ne s x y l os ox i dans B ac i l l us s p. ) a nd f ungi ( C l ados por i um s p. ) pa r t i a l l y de gr a de t hi s c om poun d i n bot h a e r obi c a nd a na e r obi c c ondi t i ons ( H a r de r a nd P r obi a n 1995; M i s r a e t a l 1996; M i s r a a nd P a vl o s t a t hi s 1997; K l e i nhe i nz e t a l 1999; P a vl os t a t hi s a nd M i s r a 19 99; Y oo e t a l 2001) M e t h an ot r op h i c B ac t e r i a M e t ha not r ophs be l ong t o t he phys i ol ogi c a l g r oup of m e t hyl ot r ophs M e t hyl ot r ophs a r e a e r obi c m i c r oo r ga ni s m s t ha t ut i l i z e a s t he i r s ol e s our c e of c a r bon a nd e ne r gy r e duc e d c a r bon s ubs t r a t e s w i t h no C C bonds ( C 1 c om poun ds ) a nd a s s i m i l a t e c a r bon vi a f or m a l de hyde ( F i g. 1 2 ) ( H a ns on a nd H a ns on, 199 6) M e t ha not r ophs a r e c ons i de r e d obl i ga t e m e t hyl ot r ophs be c a us e t he y onl y gr ow on C 1 c om pounds i nc l ud i ng m e t ha ne a nd m e t ha nol ( L i ds t r o m 2001) H ow e ve r r e c e nt l y, a ne w s pe c i e s w a s de s c r i be d w i t h t he c a pa bi l i t y of f a c ul t a t i ve gr ow t h on m u l t i c a r bon c o m pounds M e t hy l oc e l l a s i l v e s t r i s B L 2 ( T he i s e n e t a l 2005 ) T he a bi l i t y t o g r ow on C H 4 i s a l m os t e xc l us i ve t o m e t ha not r ophs e xc e pt f o r a gr a m pos i t i ve m e t hyl ot r oph of t he ge nus M y c obac t e r i um ( R e e d a nd D uga n, 1987 ) M e t ha not r ophs pos s e s s c om pl e x i nt r a c yt opl a s m i c m e m br a ne s ys t e m s w hi c h a ppe a r t o be i nvol ve d i n C H 4 upt a ke T he c onf i gur a t i on of t he s e m e m br a ne s a pa r t f r om ot he r c ha r a c t e r i s t i c s s e pa r a t e s m e t ha not r ophs i nt o t w o gr oups T hos e t ha t pos s e s s m e m b r a ne s a s bundl e s of di s ks s t a c ke d t hr oughout t he c e nt e r o f t he c e l l ( t ype I ) a nd t hos e w i t h m e m br a ne s a r r a nge d a s r i ngs a t t he pe r i phe r y of t h e c e l l ( t ype I I ) ( T a bl e 1 3 ) O t he r c ha r a c t e r i s t i c s t ha t c or r e l a t e t o t he t ype c l a s s i f i c a t i on i nc l ude D N A G C c ont e nt pa t hw a ys of C a s s i m i l a t i on, r os e t t e f or m a t i on t ype s o f c ys t s a nd a bi l i t y t o f i x N 2 A s m a l l nu m be r

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33 of m e t ha not r ophs f r om t he ge nus M e t hy l oc oc c us p os s e s s c ha r a c t e r i s t i c s of bot h gr oups ; t he r e f or e t he y ha ve be e n c l a s s i f i e d i nt o t he t ype X c a t e gor y ( H a ns on a nd H a ns on, 1996; G r a ha m e t a l 2002 ) T he e xi s t e nc e of t h i s t ype X gr oupi ng i s how e ve r a poi n t of de ba t e w i t hi n t he C 1 r e s e a r c h c om m uni t y. T he m e c ha ni s m by w hi c h m e t ha not r ophs oxi di z e C H 4 t o m e t ha nol a nd c om e t a bol i z e ( t he m i c r oo r ga ni s m ga i ns no c a r bon or e ne r gy f r om t he s ubs t r a t e i t oxi di z e s a s pr e vi ous l y de f i ne d) m a ny ot he r c om po unds i nc l udi ng c hl or i na t e d s ol ve nt s i s f a c i l i t a t e d by t he e nz ym e m e t ha ne m onooxyge na s e ( M M O ) uni que t o m e t ha not r ophs M M O e xi s t s i n t he s ol ubl e ( s M M O ) or pa r t i c ul a t e ( pM M O ) f or m de pe ndi ng on t he bi oa va i l a bi l i t y of c oppe r i n t he e nvi r onm e nt pM M O i s a C u a nd F e c ont a i ni ng e nz ym e bound t o t he i nt r a c yt opl a s m i c m e m br a ne ( N guye n e t a l 1994; L i e be r m a n a nd R os e nz w e i g, 2004) w he r e a s s M M O w i t h a uni qu e di i r on s i t e a t i t s c a t a l yt i c c e nt e r i s l oc a t e d i n t he c yt opl a s m ( L i ps c om b, 1994; K opp a nd L i ppa r d 2002 ) A l t hough bot h f or m s of M M O e xhi bi t a l a c k o f s ubs t r a t e s pe c i f i c i t y, t he s ol ubl e f or m ha s be e n s how n t o di s pl a y a br oa de r r a nge i nc l udi ng a l ka ne s a l ke ne s a nd a r o m a t i c c om pounds s M M O i s a m ong t he m os t nons pe c i f i c e nz ym e s know n t o da t e a nd e xhi bi t hi gh s ubs t r a t e t ur nove r r a t e s T he r e f or e s M M O i s m or e s ui t a bl e f o r t he d e gr a da t i on of a w i de r va r i e t y of c ont a m i na nt s H ow e ve r s M M O i s onl y s ynt he s i z e d by t ype I I a nd X m e t ha not r ophs i n e nvi r onm e nt s w i t h C u c onc e nt r a t i ons l e s s t ha n 50 nM ( < 0. 89 1 m ol C u g 1 dw c e l l s ) ( O l de nhui s e t a l 1989; H a ns on a nd H a ns on, 1996 ) T he oxyge n a nd m e t ha ne l e ve l s a l s o i nf l ue nc e t he e xpr e s s i on of e i t he r f or m of M M O I n e nvi r onm e nt s w i t h a bunda nt oxyge n a n d l i m i t i ng c onc e nt r a t i ons of C H 4 m e t ha not r ophs e xpr e s s pM M O r e ga r dl e s s of w he t he r C u i s l i m i t i ng. O n t he c ont r a r y i n

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34 oxyge n l i m i t e d e nvi r onm e nt s w i t h hi gh C H 4 c onc e nt r a t i ons t he e xp r e s s i on of e i t he r e nz ym e i s di c t a t e d s ol e l y by t he C u a va i l a bi l i t y. A t l ow C u c onc e nt r a t i ons a nd hi gh c e l l de ns i t i e s s M M O i s e xpr e s s e d. C e l l s t ha t e xpr e s s pM M O ha ve hi ghe r gr ow t h yi e l ds a nd gr e a t e r a f f i ni t y f o r C H 4 be c a us e pM M O e m pl oys a n a bunda nt hi gh e ne r gy e l e c t r on dono r f or C H 4 oxi da t i on. M e a nw hi l e s M M O pos s e s s e s a hi gh e ne r gy de m a nd be c a u s e of t he i nvol ve m e nt of N A D H + H + a s a n e l e c t r on donor t h a t c a t a l yz e s t hi s r e a c t i on ( H a ns on a nd H a ns on, 1996; S ul l i va n e t a l 1998) E c ol ogy an d h ab i t at s o f m e t h an ot r op h s M e t ha not r ophs ha ve be e n w i de l y s t udi e d f or t he i r r ol e i n t he c a r bon c yc l e T he y i nt e r c e pt a nd oxi di z e C H 4 t ha t e s c a pe s f r om a na e r obi c e nvi r onm e nt s t hus p r e ve nt i ng l a r ge qua nt i t i e s f r o m e s c a pi ng i nt o t he a t m os phe r e ( H a ns on a nd H a ns on, 1996) T hus m e t ha not r ophs a r e c ons i de r e d t he p r i nc i pa l bi ol ogi c a l s i nk of a t m os phe r i c C H 4 by r e gul a t i ng t he a m ount of C H 4 pr e s e nt i n t he a t m os phe r e a nd, c ons e que nt l y, de c r e a s i ng t he i m pa c t t ha t C H 4 23 t i m e s m or e pot e nt t ha n C O 2 ha s on gl oba l w a r m i ng ( H ought on e t a l 2001) M e t ha not r ophs a r e w i de s pr e a d i n na t ur e f ound i n a ny e nvi r onm e nt w he r e C H 4 a nd oxyge n a r e pr e s e nt U nde r f l oode d c ondi t i ons s uc h a s r i c e pa ddi e s w e t l a nd s oi l s s w a m ps a nd bogs t he y a r e r e s t r i c t e d t o t he s oi l s ur f a c e l a ye r s a nd t o t he r h i z os phe r e of pl a nt s w he r e t he y i nt e r c e pt t he C H 4 be i ng pr oduc e d ne a r by unde r a na e r obi c c ondi t i ons M e a nw hi l e i n upl a nd s oi l s non f l oode d ha bi t a t s s uc h a s f or e s t g r a s s l a nds a nd a r a bl e l a nd, m e t ha not r ophs a r e f ound i n t he t op s oi l l a ye r s w he r e t he y oxi di z e a t m os phe r i c C H 4 ( H a ns on a nd H a ns on, 1996; H o r z e t a l 2002) A l s o, i n t he s e ha bi t a t s t he y a r e f ound de e pe r i n t he s oi l pr of i l e s t r a t i f i e d i n a na r r ow ba n d a t t he oxi c a noxi c i nt e r f a c e w he r e c onc e nt r a t i ons of C H 4 a nd oxyge n a r e t he hi ghe s t M e t ha not r ophs ha ve be e n i s ol a t e d

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35 f r om m a r i ne f r e s hw a t e r a nd t e r r e s t r i a l ha bi t a t s u nde r c ondi t i ons of hi gh a nd l ow pH a nd t e m pe r a t ur e s up t o 55C T he y e x i s t a s s ym bi ont s w i t h i nve r t e br a t e s a nd p l a nt s H ow e ve r l i t t l e a t t e nt i on ha s be e n pa i d t o s ym bi ot i c r e l a t i ons hi ps be t w e e n pl a nt s a nd m e t ha not r ophs e ve n t hough t he f i r s t m e t ha not r op h i s ol a t e d w a s f r om t he l e a ve s of a m a c r ophyt e i n 1906 by S hnge n ( H a ns on a nd H a n s on, 1996) I t i s w e l l know n t ha t di f f e r e nt t ype s of m e t ha not r o phs a da pt be t t e r t o di f f e r e nt e nvi r onm e nt a l c ondi t i ons M e t ha ne oxyge n, a nd ni t r oge n c onc e nt r a t i ons a r e t he pr i m a r y de t e r m i na nt s of t he t ype of m e t ha not r oph pr e s e nt i n a n e nvi r onm e nt T ype I m e t ha not r ophs out c om pe t e t ype I I s pe c i e s a t l ow C H 4 c onc e nt r a t i ons ( < 2 ppm v i n s oi l s ) w he r e a s gr ow t h of t ype I I m e t ha not r ophs i s f a vor e d unde r l ow oxyge n ( < 0 2 ppm i n de e p w a t e r s ) a nd hi gh C H 4 c ondi t i ons ( > 1 000 ppm v i n s e di m e nt s ) ( H a ns on a nd H a n s on, 1996) H ow e ve r be c a us e of ha bi t a t he t e r oge ne i t y or di f f e r e nc e s i n e xpe r i m e nt a l t e c hni que s us e d, a c ons i s t e nt pa t t e r n c onc e r ni ng t h e c om pe t i t i ve dom i na nc e of c e r t a i n t ype s of m e t ha not r ophs ha s be e n di f f i c ul t t o di s c e r n. T ype I I m e t ha not r ophs ha ve be e n r e por t e d t o be dom i na nt i n s oi l s ; how e ve r a n a bun da nc e of t ype I or of bot h t ype s ha s a l s o be e n r e por t e d i n t he s oi l e nvi r onm e nt ( V e c he r s ka ya e t a l 1993; B r us s e a u e t a l 1994; S undh e t a l 1995; H a ns on a nd H a ns on, 199 6; S e ghe r s e t a l 2005) O n t he c ont r a r y, t ype I m e t ha not r ophs a ppe a r t o pr e va i l i n a qua t i c e nvi r onm e nt s s uc h a s l a ke w a t e r s e di m e nt s a nd g r oundw a t e r A pa r t f r om t h e s e c ont r a di c t or y r e s ul t s s om e ge ne r a i nc l udi ng M e t hy l obac t e r a nd M e t hy l oc y s t i s r e p r e s e nt a t i ve s of t ype I a nd I I m e t ha not r ophs r e s pe c t i ve l y, ha ve be e n de t e c t e d i n a w i de r a nge of ha bi t a t s I t ha s be e n s pe c ul a t e d t ha t t he i r a bi l i t y t o p r oduc e r e s i s t a nt c y s t s e na bl e s t he s e s t r a i ns t o pe r s i s t i n a w i de r a nge of ha bi t a t s ( K ni e f e t a l 2003; B ode l i e r e t a l 2005 )

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36 E n vi r on m e n t al f ac t or s af f e c t i n g m e t h an ot r op h s T he e f f e c t o f s e ve r a l e nvi r onm e nt a l va r i a bl e s on m e t ha not r oph c om pos i t i on, c om m uni t y s t r uc t ur e a nd a c t i vi t y ha s be e n s t udi e d i n a va r i e t y of ha bi t a t s w i t h s om e i nc ons i s t e nt r e s ul t s T he ou t c om e of t he s e s t udi e s s e e m s t o de pe nd on t he t ype o f ha bi t a t be i ng e va l ua t e d a nd on t he va r i e t y o f m e t hodol ogi e s us e d. S oi l t ype ha s be e n r e po r t e d a s t he pr i m a r y de t e r m i n a nt o f t he m e t ha not r oph c om m uni t y s t r uc t ur e i n a gr i c ul t u r a l s oi l s ( G i r va n e t a l 2003; S e g he r s e t a l 2005) H ow e v e r i n f or e s t s oi l s pH va l ue ha s be e n pos t ul a t e d a s t he pr i m a r y f a c t or a f f e c t i ng m e t ha not r oph di s t r i but i on ( K ni e f e t a l 2003) A t m os phe r i c C H 4 oxi da t i on a c t i vi t y ha s be e n r e po r t e d t o de pe nd on pl a nt c ove r a nd l a nd us e w he r e a c t i vi t y ha s be e n s h ow n t o de c r e a s e w i t h a n i nc r e a s e i n de gr e e o f di s t ur ba nc e ( w oodl a nd> gr a s s l a nds > f a r m l a nd) ( W i l l i s on e t a l 1995; K ni e f e t a l 2003) A l s o, m a na ge m e nt pr a c t i c e s i nc l udi ng f e r t i l i z e r t ype ( o r ga ni c v e r s us m i ne r a l ) a nd t ype of t r e e i n f or e s t s t a nds ha ve be e n r e por t e d t o i n f l ue nc e m e t ha not r oph a c t i vi t y a nd a bunda nc e ( R e a y e t a l 2001; G i r va n e t a l 2003 ) H ow e ve r s om e ge ne r a M e t hy l oc al dum M e t hy l os i nus a nd M e t hy l oc y s t i s a r e uni ve r s a l l y o bs e r ve d i n di f f e r e nt s oi l s i nde pe nde nt of l a nd us e or pl a nt c ove r ( K ni e f e t a l 2003) I n t he p r e s e nc e of pl a nt s m a i n l y i n s a t ur a t e d e nvi r onm e nt s t he s pa t i a l di s t r i but i on of m e t ha not r ophs i s de t e r m i ne d by t he s oi l c om pa r t m e nt ( r hi z os phe r e > bul k s oi l > ba r e s oi l ) or t he pos i t i on o f t he s oi l w a t e r i nt e r f a c e ( G i l be r t a nd F r e nz e l 1998; D ube y a nd S i ngh, 2001; M a c a l a dy e t a l 2002 ) I t ha s be e n p r opos e d t ha t be c a us e pl a nt s di f f e r i n t he i r a bi l i t y t o t r a ns por t oxyge n t o t he r hi z os phe r e di f f e r e nt f a c t or s c ont r ol t he i r a s s oc i a t e d m e t ha not r oph popul a t i ons ( K i ng, 199 4; M a c a l a dy e t a l 2002 ) S pa t i a l c ha nge s i n t he m e t ha not r oph c om m un i t y ha ve a l s o be e n obs e r ve d i n f or e s t s oi l s de pe ndi ng on s e a s on a nd s oi l de pt h ( H e nc ke l e t a l 2000; B ode l i e r e t a l 2005) I n

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37 w i nt e r a t m os phe r i c C H 4 oxi da t i on oc c ur s i n a w e l l de f i ne d s ubs ur f a c e l a ye r ( 6 14 c m de e p) a nd, dur i ng s um m e r t he c om pl e t e s oi l c or e ( 0 2 6 c m de e p) i s a c t i ve H ow e ve r no s e a s ona l s hi f t i n c om m uni t y c om pos i t i on w a s de t e c t e d, t he s a m e m e t ha not r oph popul a t i on w a s i de nt i f i e d i n s um m e r a nd w i nt e r ( H e nc ke l e t a l 2000) O t he r e nvi r onm e nt a l f a c t or s know n t o a f f e c t m e t h a not r ophs a r e t he pot e nt i a l i nhi bi t or y e f f e c t s of a m m on i um a nd/ or ni t r i t e t ha t a c t a s c om pe t i t i ve s ubs t r a t e s f or M M O ( D unf i e l d a nd K now l e s 1995 ; H a ns on a nd H a ns on 1996) H ow e ve r r e c e nt s t udi e s w i t h r i c e pl a nt s ha ve s how n t ha t ni t r oge n f e r t i l i z a t i on i n c r e a s e s C H 4 oxi da t i on i n de ns e l y r oot e d s oi l s be c a us e r hi z os phe r e m e t ha not r ophs f a c e i nt e ns e pl a nt a nd m i c r obi a l c om pe t i t i on f or ni t r oge n ( M a c a l a dy e t a l 2002 ; E l l e r e t a l 2005) M e t h an ot r op h s an d c h l or i n at e d c om p o u n d s M e t ha not r ophs oxi di z e t he l e s s c hl or i na t e d hydr oc a r bons a t ve r y di f f e r e nt r a t e s de pe ndi ng on t he f or m o f M M O e xpr e s s e d ( L e a db e t t e r a nd F os t e r 1959; L i t t l e e t a l 1988; F ox e t a l 1990; A l va r e z C ohe n a nd M c C a r t y, 1991a ; A l va r e z C ohe n a nd M c C a r t y, 1991b; H e nr y a nd G r bi G a l i 1991) T C E oxi da t i on by s M M O i s c om pa r a bl e t o t ha t o f C H 4 a nd up t o 700 f ol d h i gh e r t ha n t ha t r e por t e d f or ot he r M M O m i c r obi a l e nz ym e s ( t ol ue ne 4 m onoxyge na s e a m m oni a m onooxyge na s e a nd pr opa ne m onooxyge na s e ) ( F ox e t a l 1990) H ow e ve r T C E oxi da t i on c a t a l yz e d by pM M O oc c ur s a t m uc h l ow e r r a t e s t ha n s M M O ( D i S pi r i t o e t a l 1992) s M M O oxi di z e s T C E t o T C E e poxi de ( 95 % ) a nd c hl or a l hydr a t e ( 5% ) ( F i g. 1 3A ) ( O l de nhui s e t a l 1989; N e w m a n a nd W a c ke t t 19 91; F ox e t a l 19 90 ) T C E e poxi de r a pi dl y unde r goe s s pont a ne ous de c om pos i t i on; m e a nw hi l e c hl or a l hyd r a t e i s m o r e s t a bl e a nd unde r goe s bi ol ogi c a l t r a ns f or m a t i on w i t hi n 1 t o 24 h of i nc uba t i on t o t r i c hl or oe t ha nol a nd t r i c hl or oa c e t i c a c i d. A t hi gh t e m pe r a t ur e ( 60 C ) a nd pH o f 9 0, c hl or a l hyd r a t e i s

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38 e a s i l y de c om pos e d t o c hl or of or m a nd f o r m i c a c i d ( N e w m a n a nd W a c ke t t 1991) S i nc e T C E de gr a da t i on i s s t r i c t l y a c om e t a bol i c pr oc e s s no e ne r gy or c a r bon ga i n r e s ul t s f r o m i t s oxi da t i on; t he r e f o r e t he pr e s e nc e of a c os ubs t r a t e i s ne c e s s a r y t o m a i nt a i n c e l l bi om a s s a nd r e ge ne r a t e r e duc t a nt s uppl y. A l t houg h C H 4 oxi da t i on i s r e qui r e d f or gr ow t h a nd c a n pr ovi de e l e c t r ons i t a l s o f unc t i ons a s a c o m pe t i t i ve i nhi bi t or of T C E t r a ns f or m a t i on ( H e nr y a nd G r bi G a l i 1991) B ypr oduc t t oxi c i t y a l s o oc c ur s a s a r e s ul t of t hi s r e a c t i on w i t h a c onc om i t a nt de c r e a s e i n C H 4 oxi da t i on r a t e s r e s pi r a t or y a c t i vi t y a nd T C E de g r a da t i on r a t e s ( A l va r e z C ohe n a nd M c C a r t y, 1991b; H a ns on a nd H a ns on, 1996; C hu a nd A l va r e z C ohe n, 1999) A ddi t i ona l l y, T C E m e t a bol i t e s c a n bi nd nons pe c i f i c a l l y t o c e l l pr o t e i ns a nd i na c t i va t e M M O a c t i vi t y ( F ox e t a l 1990) T C E e poxi de ha s be e n pos t ul a t e d a s t he r e s pons i bl e c o m pound f or t he obs e r ve d t ox i c i t y due t o i t s r e a c t i vi t y or t ha t o f i t s de gr a da t i on pr oduc t s ( F o x e t a l 1990; C ha ng a nd A l va r e z C ohe n, 1996; V l i e g e t a l 1996 ; S ul l i va n e t a l 19 98) I nt e r m e di a t e t oxi c i t y c a n be r e duc e d by t he a ddi t i on of a n e xt e r na l s uppl y of r e duc i ng e qui va l e nt s uc h a s f or m a t e ( A l va r e z C ohe n a nd M c C a r t y, 1991b) H ow e ve r T C E oxi da t i on t oxi c i t y a ppe a r s t o ha ve a s e l e c t i ve e f f e c t ove r di f f e r e nt s pe c i e s of m e t ha no t r ophs ba s e d on obs e r va t i ons of di s t i nc t r a t e s of r e c ove r y ( H e nr y a nd G r b i G a l i 1991) U nde r a na e r obi c c ondi t i ons c hl or i na t e d c om pound s r e a di l y unde r go r e duc t i ve de c hl or i na t i on ( F i g 1 3B ) P C E a nd T C E a r e de gr a de d t o di c hl or oe t he ne i s om e r s ( c i s a nd t r ans 1, 2 D C E ) 1 1 D C E vi nyl c hl or i de e t he ne a nd e t ha ne D C E i s om e r s a nd vi nyl c hl or i de i n t he p r e s e nc e of T C E a nd no oxyg e n of t e n pe r s i s t i n t he e nvi r on m e nt be c a us e t he i r de c hl or ona t i on yi e l ds l e s s e ne r gy t ha n t ha t of t he i r pa r e nt c om pound ( F ox e t a l 1990; H a ns on a nd H a ns on, 1996) C ons i de r a bl e c onc e r n e xi s t s ove r t he bi ol o gi c a l

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39 pr oduc t i on of vi nyl c hl o r i de a know n hu m a n c a r c i noge n; how e ve r t h i s pr oduc t i s r e a di l y oxi di z e d by s M M O i n a e r obi c e nvi r onm e nt s ( F ox e t a l 1990) A ddi t i ona l l y w he n c ons or t i a of ba c t e r i a ( m e t ha not r ophs a nd he t e r ot r o phs ) a r e pr e s e nt f ur t he r oxi di z a t i on of c hl or a l hydr a t e c h l or i na t e d a c e t i c a c i ds a nd vi nyl c hl or i de ha s be e n obs e r ve d a l ong w i t h a pr ovi s i on of a ddi t i ona l r e duc i ng pow e r f or t he p r oc e s s ( A l va r e z C ohe n e t a l 1992; U c hi ya m a e t a l 1992; C ha ng a nd A l va r e z C ohe n, 1996) M e t h an ot r op h s an d p l an t s P l a nt m e t ha not r oph a s s oc i a t i ons s t udi e d t o da t e ha ve c ons i de r e d m a i nl y r i c e f i e l ds a nd w e t l a nds be c a us e of t he i r i m po r t a nc e a s m a j or a r e a s of C H 4 pr oduc t i on. D e B ont e t a l ( 1978 ) w a s t he f i r s t t o r e por t C H 4 ox i da t i on a s s oc i a t e d w i t h r i c e r oot s he not i c e d t ha t m os t of t he C H 4 di f f us e d t hr ough t he r hi z os phe r e w a s oxi di z e d. T hi s obs e r va t i on w a s of r e l e va nc e be c a us e a ny s m a l l c ha nge i n oxi da t i on p r oc e s s e s oc c ur r i ng a t t he r hi z os phe r e c oul d ha ve a gl oba l i m pa c t be c a us e r i c e f i e l ds c ont r i but e t o a ppr oxi m a t e l y 25 % of t he c ur r e nt C H 4 f l ux t o t he a t m os phe r e H ow e ve r s t u di e s t o da t e on t he s e i nt e r a c t i ons s how hi gh une xpl a i ne d va r i a bi l i t y w i t hi n pl a nt s pe c i e s a nd be t w e e n e nvi r onm e nt s I t ha s be e n obs e r ve d t ha t pl a nt s pe c i e s know n t o oxi di z e C H 4 i n t he i r r hi z os phe r e s w he n pl a nt e d i n a di f f e r e nt e nvi r onm e nt C H 4 c ons um pt i on r a nge d f r om de t e c t e d t o no ox i da t i on ( K i ng 1996) R oot s ur f a c e s a nd t he i r i nt e r i or z one s w he r e C H 4 i s t r a ns por t e d f r om t he m e t ha noge ni c s e di m e nt s t o t he a t m os phe r e a nd w h e r e a t m os phe r i c oxyge n i s t r a ns por t e d t o t he s e di m e nt s bot h s uppor t m e t ha not r oph popul a t i ons i n s a t ur a t e d e nvi r onm e nt s ( K i ng, 1996; G i l be r t e t a l 1998; E l l e r e t a l 2005) H ow e ve r m e t ha not r ophs a nd m e t hyl ot r ophs ha ve a l s o be e n de t e c t e d i n t he s e l oc a t i ons i n popl a r a nd pi ne t r e e s i n non s a t ur a t e d e nvi r onm e nt s ( B r i gm on e t a l 1999 ; P i l o n S m i t s 2005 ) M e t hyl ot r ophs

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40 pe r m a ne nt l y a s s oc i a t e d w i t h t he pl a nt a r e of t e n e nc ount e r e d i n t he phy l l os phe r e ( l e a f s ur f a c e ) a nd r hi z os phe r e P l a nt s c a n a l s o be ne f i t f r om t he s e a s s oc i a t i ons F or e xa m pl e m e t ha not r ophs c a n e xc r e t e or e xpe l by c e l l l ys i s phyt ohor m one s ( c yt oki ni ns a nd a uxi ns ) a nd ot he r bi oa c t i ve c om pounds A ddi t i ona l l y, t yp e I I a nd X m e t ha not r ophs c a n f i x ni t r oge n a nd, t he r e f or e c a n be c ons i de r e d phyt os ym bi ont s on t he s ur f a c e a nd i ns i de pl a nt t i s s ue s ( D or oni na e t a l 2004) T he pa t t e r n o f m e t ha not r oph r oot c ol oni z a t i on ha s be e n s t udi e d i n r i c e p l a nt s ( G i l be r t e t a l 1998; G i l be r t a nd F r e nz e l 1998) T he c ol oni z a t i on i s s pa t i a l l y ve r y he t e r oge ne ous ; s om e r oot s a r e not c ol on i z e d a t a l l w hi l e ot he r s pos s e s s m i c r oc ol oni e s a s c l um ps or t hi c k ba c t e r i a l l a ye r s A s know n f or ot h e r t ype s of ba c t e r i a m e t ha no t r oph r oo t c ol oni z a t i on f ol l ow e d t he pa t t e r n o f c e l l w a l l f o r m a t i on, pot e nt i a l l y due t o t he e xuda t i on of or ga ni c s ubs t r a t e s a nd oxyge n l e a ka ge a t t he s e s i t e s W hi l e m e t ha not r ophs c a nnot ut i l i z e c om pl e x or ga ni c s ubs t r a t e s f or gr ow t h t he y do ut i l i z e s om e a m i no a c i ds a s ni t r oge n s our c e s ( G i l be r t a nd F r e nz e l 1998 ) P h yl oge n e t i c s of m e t h a n ot r op h s M e t hyl ot r ophs a r e s c a t t e r e d a m ong t he P r ot e obac t e r i a w i t hi n t he a nd # s ubdi vi s i ons not f or m i ng a n e vol ut i ona r y c ohe r e nt gr oup. M ul t i ge ne ope r ons a ppe a r t o be r a r e a m ong i t s m e m be r s a nd, on t he c ont r a r y, p l a s m i ds a r e c om m on. H ow e ve r no f unc t i ons ha ve be e n a s c r i be d t o t he s e pl a s m i ds a n d t he y a r e e nt i r e l y c r yp t i c M e t ha not r ophs c l us t e r i nt o t he a nd # P r ot e obac t e r i a a nd a r e c ons i de r e d i de a l m i c r oor ga ni s m s f or m ol e c ul a r bi ol ogy s t udi e s M e t ha not r oph phyl oge ny a nd t he i r phe not ypi c a nd e c o phys i ol ogy c ha r a c t e r i z a t i on i n t o t ype s I I I a nd X va l i da t e e a c h ot he r ( L i ds t r om 2001) T he t ype c l a s s i f i c a t i on, i n i t i a l l y pr opos e d by W hi t t e nbu r y e t a l

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41 ( 1970) ha s be e n s uppor t e d by a na l ys i s of 5S a nd 1 6S r R N A ge ne s R e c e nt l y, w i t h t he i nc or por a t i on o f m ol e c ul a r m e t hods t o m e t ha not r o ph s t udi e s nove l s t r a i ns a r e be i ng r e c ogni z e d t ha t do not gr ow i n s t a nda r d l a bor a t or y c ondi t i ons ( e nr i c he d s ol i d a nd l i qui d m e di a w i t h hi gh c onc e nt r a t i ons of C H 4 ) F o r e xa m pl e t he up l a nd m e t ha not r oph s oi l c l us t e r s ( U S C a nd U S C # ) t ha t ox i di z e a t m os phe r i c C H 4 i n f or e s t s oi l s A not he r e xa m pl e i s t he ge nus M e t hy l oc e l l a s e ns i t i ve t o s a l t s i n r e gul a r c ul t i va t i on m e di a ( H ol m e s e t a l 1999; H e nc ke l e t a l 2000; J e ns e n e t a l 200 0; B our ne e t a l 2001; K ni e f e t a l 2003; T he i s e n e t a l 2005) M ol e c u l ar an al ys i s of m e t h an ot r op h s M e t ha not r oph phyl oge ne t i c s t udi e s ha ve be e n c onduc t e d w i t h bot h p hyl oge ne t i c a nd f unc t i ona l ge ne m a r ke r s F unc t i ona l ge ne m a r ke r s de t e c t t he a c t i ve s i t e s ubun i t o f bot h M M O f or m s pm o A f or pM M O a nd m m oX f o r s M M O a nd of m e t ha nol de hydr oge na s e by t he m x aF ge ne T he us e of t he s e m a r ke r s e na bl e s a s s e s s m e nt of t he pot e nt i a l f unc t i ona l di ve r s i t y of m e t hyl ot r ophs a nd m e t ha not r ophs w i t hi n a n e nvi r onm e nt T he uni ve r s a l phyl oge ne t i c 16S r D N A ( r R N A ) p r i m e r s e t a m pl i f i e s t he va r i a bl e V 3 r e gi on of t he ge ne e xt e ns i ve l y s t udi e d t o e na bl e i n f e r e nc e of phyl oge ne t i c r e l a t i ons hi ps a m ong m i c r oor ga ni s m s P hyl oge ne t i c a na l ys i s of m e t ha not r ophs us ua l l y c ons i de r s bot h pm oA a nd 16S r D N A a na l ys i s due t o t he f a c t t ha t m os t m e t ha not r ophs e xpr e s s t he pM M O ge ne a nd phyl oge ni e s be t w e e n t he s e t w o pr i m e r s e t s a r e c l os e l y r e l a t e d t o e a c h ot he r ( B ow m a n, 2000) I nt e r pr e t a t i on o f pm o A phyl oge ne t i c a na l ys i s m us t t a ke i nt o c ons i de r a t i on t ha t m ul t i pl e c opi e s of t he ge ne c a n e xi s t i n one o r ga ni s m ; t he r e f or e nove l c l us t e r s of pm oA s e que nc e s do not ne c e s s a r i l y i ndi c a t e t ha t nove l gr oups of unc ul t i va t e d m e t ha not r ophs e xi s t C opi e s of t he pm oA ge ne ( pm o A 2 ) c a n s how l e s s t ha n 80% i de nt i t y t o t he

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42 pr e vi ous l y know n pm oA ge ne ( pm o A 1 ) A l s o i n s om e c a s e s t he r e i s no c or r e l a t i on be t w e e n t he 16S r D N A a nd pm oA phyl oge ni e s a n d, f o r t he ge nus M e t hy l obac t e r s om e s t r a i ns poor l y a m pl i f y t he pm o A ge ne w i t h t he s t a n da r d pr i m e r s e t s unde r e s t i m a t i ng t he m e t ha not r oph di ve r s i t y. F i na l l y, one m us t ke e p i n m i nd t ha t t he ge nus M e t hy l oc e l l a t he onl y know n e xc e pt i on t o t he uni ve r s a l i t y a m ong m e t ha not r ophs of t he pm oA ge ne m us t be de t e c t e d us i ng a di f f e r e nt pr i m e r s e t O t he r pr i m e r s e t s t ha t c oul d be us e d a r e t he m m oX or m dh t ha t a m pl i f y t he s M M O a nd m e t ha nol de hydr oge na s e a c t i ve s i t e r e s pe c t i ve l y ( D e dys h e t a l 2000) I t i s of i m po r t a nc e t o r e c ogni z e t ha t t he s uc c e s s i n ge ne r e t r i e va l f r om e nvi r onm e nt a l s a m pl e s de pe nds on t he qua l i t y o f t he pr i m e r s e t s us e d. D i f f e r e nt l e ve l s of m e t ha n ot r oph di ve r s i t y ha ve be e n r e por t e d w i t h di f f e r e nt p r i m e r s s e t s ( B our ne e t a l 2001) F or e xa m pl e H ut c he ns e t a l ( 2004) r e por t e d t ha t by us i ng t he pm o A p r i m e r s e t A 189f / A 682r onl y 8 ope r a t i ona l t a xonom i c uni t s ( O T U s ) w e r e de t e c t e d, but w i t h t he A 189f / m b661r p r i m e r s e t 12 O T U s w e r e r e t r i e ve d ( H ut c he ns e t a l 2004 ) T he pm oA pr i m e r s e t A 189f / m b661r de t e c t s a l m os t a l l m e t ha not r ophi c ba c t e r i a e xc e pt s e que nc e s of M e t hy l om onas M e t hy l oc al dum a nd t he r e por t e d f or e s t c l one c l us t e r s bu t i t doe s e xc l ude a l l know n am oA s e que nc e s of a m m oni a oxi di z i ng ba c t e r i a e xc e pt f o r N i t r os oc oc c us ( K ol b e t a l 2003) W i t h t he i nc or po r a t i on of m ol e c ul a r t e c hni que s t o t he s t udy of m i c r obi a l e c ol ogy one of t he m os t i nt r i gui ng que s t i ons i s t he r e l a t i on s hi p be t w e e n w ha t ha s be e n r e por t e d pr e vi ous l y by c om m uni t y a s s e s s m e nt ba s e d on t r a di t i on a l c ul t ur i ng a nd w ha t ha s r e c e nt l y be e n de s c r i be d by c ul t ur e i nde pe nde nt t e c hni que s S e ve r a l m e t ha not r oph s t udi e s t ha t i m pl e m e nt e d de na t ur i ng g r a di e nt ge l e l e c t r opho r e s i s ( D G G E ) poi nt out m i s l e a di ng

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43 r e s ul t s of pr e vi ous t r a di t i ona l c ul t u r e de pe nde nt m e t hod s F o r e xa m pl e i n g r a s s l a nd s oi l s t he c ul t ur e de pe nde nt m os t pr oba bl e num be r ( M P N ) t e c hni que w a s c om pa r e d t o di r e c t s oi l s a m pl e D G G E a na l ys i s ( H or z e t a l 200 2) W hi l e M P N a na l ys i s de t e c t e d onl y one m e t ha not r oph s t r a i n D G G E r e ve a l e d a m o r e d i ve r s e a nd dyna m i c m e t ha not r oph c om m uni t y. I n a s i m i l a r m a t t e r e nr i c hm e nt s c ha r a c t e r i z e d by D G G E w e r e c om pa r e d t o r e s ul t s of m or phol og i c a l obs e r va t i ons a nd s t r a i n i s ol a t i on f r om a gr i c ul t u r a l s oi l ( J e ns e n e t a l 1998 ) T he D G G E pr of i l e of t he e nr i c hm e nt s s how e d hi ghe r di ve r s i t y ( 13 14 ba nds ) t ha n t he m or phol og i c a l obs e r va t i on a nd i s ol a t i on w he r e onl y 2 t o 4 dom i na nt m or phol ogi c a l t ype s w e r e de t e c t e d a nd onl y one c ol ony w a s i s ol a t e d ( J e ns e n e t a l 1998) A not he r m e t hodol ogy t ha t i s r e vol ut i oni z i ng m e t h a not r oph s t udi e s by l i nki ng m i c r obi a l i de nt i t y t o bi o l ogi c a l f unc t i on unde r c on di t i ons a ppr oa c hi ng t hos e i n s i t u i s s t a bl e i s ot ope pr obi ng ( S I P ) ( R a da j e w s ki e t a l 20 00) A l a be l e d s u bs t r a t e a l e s s na t ur a l l y f r e que nt i s ot ope i s i nc or por a t e d i n t o t he a c t i ve m i c r obi a l bi om a s s I n t he c a s e of m e t ha not r ophs 1 3 C H 4 ha s be e n us e d t o l a be l t he D N A of a c t i ve or ga ni s m s dur i ng D N A s ynt he s i s a nd r e pl i c a t i on. T he he a vi e r D N A ( 1 3 C D N A ) c a n t he n be s e pa r a t e d f r om t he na t ur a l l y oc c ur r i ng 1 2 C D N A T he m e t hodol o gy ha s be e n us e d t o s t udy m e t ha not r oph c om m uni t i e s of pe a t s oi l s ( M or r i s e t a l 2002) a c i di c f or e s t s oi l s ( R a da j e w s ki e t a l 2002 ) c a ve w a t e r ( H ut c he ns e t a l 2004 ) a nd s oda l a ke s e di m e nt s ( L i n e t a l 2004 ) R e s ul t s of t hi s S I P m e t hod c onf i r m e d t ha t m os t m e t ha not r oph c om m uni t i e s i n t he e nvi r onm e nt a r e a c t i ve a nd c ons t i t ut e a s m a l l f r a c t i o n of t he e nt i r e popu l a t i on r e s pons i bl e f or C H 4 oxi da t i on. S o i l c om m uni t y f r a c t i ons r e ve a l e d t ha t onl y a s m a l l pe r c e nt a ge or

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44 pos s i bl y no m e t ha not r ophs w e r e pr e s e nt i n t he 1 2 C D N A f r a c t i on w hi l e t he 1 3 C D N A f r a c t i on w a s c om pos e d of 32 % o r 96 % m e t ha not r o ph, i n pe a t a nd f or e s t s oi l s r e s pe c t i ve l y ( M or r i s e t a l 2002; R a da j e w s ki e t a l 2002) I nt e r e s t i ngl y, s e que nc e s ha ve be e n f ound t ha t m a y r e pr e s e nt nove l m e t ha not r oph s a nd m e t hyl ot r ophs s ugge s t i ng t ha t t he s e ba c t e r i a m os t pr oba bl y a s s i m i l a t e d m e t ha nol ( 1 3 C H 3 O H ) e xc r e t e d by m e t ha not r ophs dur i ng 1 3 C H 4 ox i da t i on. H ow e ve r i n s om e c a s e s t he a f f i l i a t i on t o m e t ha not r ophs of t he r e t r i e ve d s e que nc e s ( P r ot e oba c t e r i a ) c a n not be e xpl a i ne d, s ugge s t i ng t he pos s i bi l i t y t ha t ba c t e r i a not pr e vi ou s l y c ons i de r e d t o be i nvol ve d i n C H 4 oxi da t i on m a y de r i ve a s i gni f i c a nt p r opor t i on of t h e i r c a r bon f r om pr oduc t s of m e t ha not r oph m e t a bol i s m or pos s i bl y e ve n f r om C H 4 i t s e l f W he n t he f unc t i ona l pm o A ge ne ha s be e n e xa m i ne d be f or e a nd a f t e r S I P e xpe r i m e nt s t he di ve r s i t y w a s l ow e r i n t he 1 3 C D N A f r a c t i on i nd i c a t i ng t ha t no t a l l m e t ha not r ophs i n a n e nvi r onm e nt a r e a c t i ve ( M or r i s e t a l 2002; R a da j e w s ki e t a l 2002; L i n e t a l 2004) O ve r a l l t he s e s t udi e s us i ng S I P m e t hods ha ve r e ve a l e d t ha t t he a c t i ve m e t ha not r oph c om m uni t y i n pe a t a nd a c i di c f o r e s t s oi l s w a s dom i na t e d by t ype I I m e t ha not r ophs ( M or r i s e t a l 2002; R a da j e w s ki e t a l 2002 ) a nd i n s oda l a ke s e di m e nt s by t ype I m e t ha not r ophs ( L i n e t a l 2004 ) T he s e r e s ul t s gi ve i ns i ght i nt o t he e c ol ogi c a l ni c he s oc c upi e d by e a c h m e t ha not r oph t ype M e t h od s U s e d t o A s s e s s R h i z od e gr ad at i on P o t e n t i al i n P h yt or e m e d i a t i on T he m i c r obi a l c om pos i t i on of t he r hi z os phe r e i s kn ow n t o di f f e r bo t h qua l i t a t i ve l y a nd qua nt i t a t i ve l y f r om t ha t i n a non pl a nt e d s oi l H ow e ve r a pr e c i s e de t e r m i na t i on of t he m i c r obi a l di ve r s i t y i n s oi l or t he r h i z os phe r e c om pa r t m e nt r e m a i ns t o be e s t a bl i s he d, a s onl y up t o 10% o f s oi l m i c r obi a l s pe c i e s c a n c ur r e nt l y be c ul t u r e d i n t he l a bor a t o r y ( F r y, 2004) A l t hough t he a bi l i t y t o c ul t u r e t he ye t unc ul t ur e d ba c t e r i a i s of i m por t a nc e

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45 a num be r of i ndi r e c t m e t hods a r e c ur r e nt l y us e d t o e s t a bl i s h t he bi ode gr a da t i on pot e nt i a l of s oi l m i c r o or ga ni s m s B y t he us e of t he s e i ndi r e c t m e t hods not onl y i s t he m i c r obi a l c om pos i t i on a nd s t r uc t ur e o f a s pe c i f i c ha bi t a t be i n g de t e r m i ne d but a l s o i t i s pos s i bl e t o l i nk f unc t i on t o a c t i vi t y by t he us e of l a be l e d s ubs t r a t e s C u l t u r e d e p e n d e n t t e c h n i q u e s M i c r ob i al c ou n t s H i s t or i c a l l y c ol oni e s f o r m i ng uni t s ( C F U ) a nd m os t pr oba bl e num be r ( M P N ) t e c hni que ha ve be e n us e d t o e num e r a t e s e l e c t e d m i c r oor ga ni s m s a nd a s s e s s t he m i c r obi a l c om pos i t i on of a s i t e H ow e v e r i t i s r e c ogni z e d t ha t onl y a s m a l l por t i on of ba c t e r i a c a n f or m c ol oni e s w he n t r a di t i o na l pl a t i ng t e c hni que s a r e us e d. T he pr opor t i on a ppe a r s t o be de t e r m i ne d by t he ol i g ot r ophi c e xt e nt of t he e va l ua t e d e nvi r onm e nt w he r e t he m o r e ol i got r ophi c t he e nvi r onm e nt t he hi ghe r por t i on of ba c t e r i a t ha t do not gr ow unde r s t a nda r d c ul t i va t i on c ondi t i ons ( S m a l l a 2004) C ul t u r a bi l i t y, de f i ne d a s t he pe r c e nt a ge of c ul t ur a bl e ba c t e r i a t o t ot a l c e l l c ount s ( m i c r os c opi c a l l y a s s e s s ) ha s be e n de t e r m i ne d t o be a r ound 0 3% i n s oi l s ( A m a nn e t a l 1995 ) F u r t he r l i m i t a t i ons r e pr e s e nt or ga ni s m s t ha t unde r e nvi r on m e nt a l s t r e s s e nt e r t he v i a bl e but nonc ul t ur a bl e s t a t e a nd ba c t e r i a s t r ongl y a t t a c he d t o s oi l pa r t i c l e s t ha t c a nnot be di s l odge d ( S m a l l a 2004) W hi l e m i c r obi a l c ount s a r e w i de l y us e d a nd e a s y t o p r e pa r e t he y a r e t i m e c ons um i ng a nd r e qui r e m ul t i pl e r e pl i c a t e s a nd c ul t i va t i on pe r i ods of w e e ks or m ont hs A ddi t i ona l l y t he y do not di s c e r n r e l a t i ons hi ps a m ong ba c t e r i a a r e h i ghl y s e l e c t i ve a nd i na c c ur a t e unde r e s t i m a t i ng t he a bun da nc e of t he m i c r obi a l popul a t i ons ( L ync h, 2002) E n r i c h m e n t s A not h e r c om m onl y us e d a ppr oa c h f or m i c r obi a l c ha r a c t e r i z a t i on o f e nvi r onm e nt a l s a m pl e s i s t o obt a i n e nr i c hm e nt s of s e l e c t e d gr oups of ba c t e r i a U s ua l l y, t he hi ghe s t di l ut i on o f t he M P N t e c hni que i s us e d a s a n i noc ul um f o r f u r t he r c ul t i va t i on.

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46 T hi s pr oc e dur e a voi ds t he s e l e c t i on of onl y t he f a s t gr ow i ng a nd l e s s num e r ous ba c t e r i a w hi c h be ne f i t f r om t he f a c t t ha t s om e a bunda nt ba c t e r i a do not gr ow d i r e c t l y on c onve nt i ona l m e di a ( F r y, 2004) E nr i c hm e nt s o f f e r t he pos s i bi l i t y o f p r e s e r vi ng s ynt r ophi c r e l a t i ons hi ps a m ong ba c t e r i a a nd obt a i n i ng e nvi r onm e nt a l i s ol a t e s f r om w hi c h phys i ol ogi c a l a nd phyl oge ne t i c c ha r a c t e r i z a t i on c a n be pe r f o r m e d ( W i s e e t a l 1999) A ddi t i ona l l y, c ul t u r e s c a n be us e d t o a s s e s s pot e nt i a l m i c r obi a l a c t i vi t y op t i m um c ondi t i ons f or de g r a da t i on, a nd m i c r obi a l di ve r s i t y of a pa r t i c ul a r s a m pl e H ow e ve r c ondi t i ons f or e n r i c hm e nt do not r e s e m bl e t he f i e l d, a nd t he y a r e hi ghl y s e l e c t i ve w hi c h m a ke s e xt r a pol a t i on of r e s ul t s t o t he f i e l d di f f i c ul t F u r t he r m or e obt a i ni ng a s t a bl e c ul t ur e c a n t a ke m ont hs a nd, w he n s t udyi ng t he c ul t ur e s phyl oge ne t i c s t he r e i s no r e a l i ndi c a t i on of ge ne e xpr e s s i on i n s i t u w hi c h i s ul t i m a t e l y w ha t de t e r m i ne s e nvi r onm e nt a l i m pa c t a t t he f i e l d. B e c a us e t hi s t e c hni que r e l i e s o n t he c ul t ur a bi l i t y of t he m e m be r s of a pa r t i c ul a r s a m pl e i t i s a c om m on f i ndi ng t ha t i s ol a t e s r e pr e s e nt onl y a f e w of t he m os t a bunda nt ba c t e r i a H ow e ve r i n s om e e nvi r onm e nt s i s ol a t e s c a n r e pr e s e nt hi ghe r num be r s I n s e a w a t e r i s ol a t e s r e pr e s e nt e d 7 69 % of t he t ot a l ba c t e r i a l c l one s obt a i ne d f r om c ul t u r e i nde pe nde nt m e t hods ( F r y 2004) C u l t u r e i n d e p e n d e n t t e c h n i q u e s M ol e c u l ar m e t h od s S i nc e 1990, m i c r obi a l e c ol ogi s t s ha ve be e n s t udyi ng ba c t e r i a l d i ve r s i t y by i s ol a t i ng c om m uni t y D N A a m pl i f yi ng t he i r 16S r R N A ge ne s c l oni ng t he f r a gm e nt s a nd s e que nc i ng t he c l one s T he de ve l opm e nt of m ol e c ul a r m e t hods ove r t he pa s t t w o de c a de s ha s he l pe d r e s ol ve di f f i c ul t i e s i nhe r e nt i n s t udyi ng di ve r s i t y us i ng t r a di t i ona l a ppr oa c he s t ha t a r e ba s e d on obs e r va t i ons of phys i ol ogy a nd m or phol ogy. I t ha s l e d t o a n i nc r e a s e i n t he num b e r s of i de nt i f i e d ba c t e r i a di v i s i ons t o gr e a t e r t ha n 40 i n w hi c h onl y 23 di v i s i ons a r e r e pr e s e nt e d by i s ol a t e s ( S m a l l a 2004 )

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47 T he r e f or e m os t o f t he ba c t e r i a i n c ul t u r e c ol l e c t i o ns t ha t gr ow on c onve nt i ona l m e di a a r e not t he m os t a bunda nt i n na t ur a l ha bi t a t s F or t hi s r e a s on, t he r e i s a ne c e s s i t y t o i s ol a t e t he e c ol ogi c a l l y r e l e va nt ba c t e r i a t he a s ye t unc ul t ur e d ba c t e r i a a nd s t udy t h e i r phys i ol ogy. T he ba s i c pr obl e m i s t ha t m a ny num e r i c a l l y a bunda nt ba c t e r i a g r ow m o r e s l ow l y t ha n t he l e s s dom i na nt ba c t e r i a on m os t l a b or a t or y m e di a ( F r y 2004 ) I n s pi t e of t he a dva nc e s s om e c ha l l e nge s s t i l l r e m a i n f or t he m ol e c ul a r m i c r obi a l e c ol og i s t T he m os t pr e s s i ng c ha l l e nge s a r e obt a i n i ng nuc l e i c a c i ds s ui t a bl e f or m ol e c ul a r a na l ys i s a nd a c c e s s t o s uf f i c i e nt l y l a r ge hi gh qua l i t y da t a ba s e s ( S m a l l a 2004 ) E xt r a c t i on p r obl e m s w he n or ga ni c s a r e hi gh i n a n e nvi r onm e nt s uc h a s i n t he r hi z os phe r e s t i l l c ons t i t ut e a m a j or dr a w ba c k o f t hi s t e c hni que A l s o, t he e f f e c t i ve ne s s of ol i gonuc l e ot i de p r obe s t o de t e c t o r ga ni s m s m a y be unc e r t a i n be c a us e of t he pos s i bi l i t y of e nc ount e r i ng ne w ge ne s or ge ne s t ha t a r e not c o ns e r ve d i n a s i m i l a r m a t t e r w i t hi n r e l a t e d gr oups of ba c t e r i a a nd, a s a r e s ul t ge ne s obt a i ne d f r om c ul t ur e d o r ga ni s m s m a y not be s uf f i c i e nt l y s i m i l a r t o ge ne s i n t he e nvi r onm e nt ( H a ns on a nd H a ns on, 1996 ) A l s o, t he r e i s a l a c k of r D N A s e que nc e s of m a ny d e s c r i be d s pe c i e s A not he r l i m i t a t i on i s t ha t s om e m ol e c ul a r a ppl i c a t i ons do not a l l ow c onc l us i ons a bout t he m e t a bol i c a l l y a c t i ve popul a t i ons or on ge ne e xpr e s s i on be c a us e t he y do not di s t i ngui s h be t w e e n a c t i ve a nd non a c t i ve or ga ni s m s t hus l i m i t i ng t he us e of t he s e m e t hods N e ve r t he l e s s t hi s i nf or m a t i on m i ght be obt a i ne d f r om R N A a na l ys i s or by t he us e of l a be l e d s ubs t r a t e s D G G E an al ys i s T he t e c hni que i s ba s e d on t he s e pa r a t i on of P C R f r a gm e nt s of t he s a m e l e ngt h i n pol ya c r yl a m i de ge l s c ont a i ni ng a l i ne a r l y i nc r e a s i ng gr a di e nt o f c h e m i c a l de na t ur a nt s ( ur e a a nd f or m a m i de ) ( M uyz e r e t a l 1 993; M uyz e r e t a l 2004) S e pa r a t i on i s ba s e d on t he e l e c t r ophor e t i c m obi l i t y of t he pa r t i a l l y m e l t e d D N A m ol e c ul e w hi c h i s

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48 l ow e r c om pa r e d t o t ha t o f t he c om pl e t e l y he l i c a l f or m of t he m ol e c ul e T he di f f e r e nt f r a gm e nt s m e l t i n di s c r e t e m e l t i ng dom a i ns ( s t r e t c he s of ba s e pa i r s w i t h a n i de nt i c a l m e l t i ng t e m pe r a t ur e ) O nc e t he dom a i n w i t h t he l ow e s t m e l t i ng t e m pe r a t ur e r e a c he s i t s de na t ur i ng c onc e nt r a t i on a t a pa r t i c ul a r pos i t i on i n t he ge l a t r a n s i t i on f r om he l i c a l t o pa r t i a l l y m e l t e d m ol e c ul e oc c ur s a nd t he m ol e c ul e w i l l s t op m i gr a t i ng T he r e f o r e s e que nc e va r i a nt s ( di f f e r e nt i n ba s e pa i r s ) w i l l s t op m i gr a t i ng a t di f f e r e nt pos i t i ons f r om w hi c h D N A f r a gm e nt s a r e di f f e r e nt i a t e d a nd e xc i s e d f or s e que nc e a na l ys i s ( M uyz e r e t a l 1993 ) A G C r i c h s e que nc e ( G C c l a m p) i s i nc or por a t e d i nt o one of t he p r i m e r s t o m odi f y i t s m e l t i ng be ha vi or t o t he e xt e nt t o w hi c h c l os e t o 100% o f a l l pos s i bl e s e que nc e va r i a t i ons c a n be de t e c t e d. T he r e s ul t i ng ba ndi ng pa t t e r n r e pr e s e nt s a pr o f i l e of t he popul a t i ons i n t he s a m pl e a nd t he r e l a t i ve i nt e ns i t y of e a c h ba nd a nd pos i t i on r e pr e s e nt s t he r e l a t i ve a bunda nc e of a pa r t i c ul a r m e m be r of t he c om m uni t y ( M uyz e r e t a l 1993 ) T he m a i n a dva nt a ge o f D G G E i s t ha t i t pe r m i t s hi g h r e s ol ut i on phyl oge ne t i c a na l ys i s of a c om pl e t e c om m uni t y by i t s d i ve r s i t y pa t t e r n i n a qua l i t a t i ve a nd s e m i qua nt i t a t i ve m a t t e r L a r ge num be r s of s a m pl e s c a n be qui c kl y a na l yz e d a nd c om pa r e d, pe r m i t t i n g t e m po r a l a nd s pa t i a l a na l ys i s w i t hi n a nd be t w e e n c om m uni t i e s T he on l y c om pa r a bl e t e c hni que a t t he m o m e nt i s t e r m i na l r e s t r i c t i on f r a gm e nt l e ngt h pol ym or phi s m ( T R F L P ) ( B ode l i e r e t a l 2005) H ow e ve r i nt e r p r e t a t i on of T R F L P da t a r e qui r e s c ons t r uc t i ng a c l one l i br a r y t ha t c a n be t i m e c ons um i ng due t o t he c l oni ng s t e p, a nd l e s s a bunda nt s pe c i e s a r e not a l w a ys de t e c t e d. O n t he c ont r a r y, D G G E c a n de t e c t s pe c i e s r e pr e s e nt e d by a s l ow a s 1% of t he p opul a t i on, a nd ba nds a r e di r e c t l y e xc i s e d f r om t he ge l r e a m pl i f i e d a nd s e que nc e d w i t hout t he ne e d o f c l oni ng ( M uyz e r e t a l 1993 ) D G G E c a gn de t e c t up t o 95 % o f a l l pos s i bl e s i ngl e ba s e s ubs t i t ut i ons a m ong

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49 s e que nc e s of up t o 1, 000 bp i n l e ngt h, a nd i t c a n b e a dj us t e d t o na r r ow e d de na t ur a nt gr a di e nt s t o pr ovi de hi ghe r r e s ol ut i on. A l s o, D G G E pr o f i l e s c a n be t r a ns f e r r e d t o hybr i di z a t i on m e m br a ne s a nd p r obe d w i t h s pe c i f i c ol i gonuc l e ot i de s ( V a l l a e ys e t a l 1997) H ow e ve r t he m a i n c ons t r a i nt of D G G E i s t he a m o unt of phy l oge ne t i c i nf o r m a t i on i n t he l e ngt h of t he c om m onl y a m pl i f i e d f r a gm e nt s ( < 500 bp) T he s e pa r t i a l s e que nc e s m a y not be s uf f i c i e nt t o di s c r i m i na t e a m ong s t r a i n s ( B oon e t a l 2002 ; B ode l i e r e t a l 2005) A not he r l i m i t a t i on i s t he p r oduc t i on of m ul t i pl e ba nds by one o r ga ni s m be c a us e of m ul t i pl e he t e r oge ne ous ope r ons or c opi e s of t he t a r ge t ge ne o r due t o t he us e of de ge ne r a t e pr i m e r s A l s o, i f t he t a r ge t s e que nc e s i n a s a m pl e a r e p r e s e nt a t di s s i m i l a r c onc e nt r a t i ons t he l e s s a bunda nt s e que nc e s m a y not a m pl i f i e d s uf f i c i e nt l y t o be vi s ua l i z e d a s ba nds unde r e s t i m a t i ng t he di ve r s i t y of t he s a m pl e ( B oon e t a l 2002) A not he r pr obl e m of D G G E i s c o m i gr a t i on of ba n ds a nd ba nds a t i de nt i c a l pos i t i ons t ha t a r e not ne c e s s a r i l y de r i ve d f r om t he s a m e s pe c i e s ; how e ve r t he s e ba nds c a n be s c r e e ne d by r e duc i ng t he de na t ur a nt gr a di e nt a nd w he n ne c e s s a r y, ba nds i n s i m i l a r pos i t i ons m a y r e qui r e m ul t i p l e s e que nc i ng. F i na l l y, i n c om m un i t y a na l ys i s of hi ghl y r e l a t e d phyl oge ne t i c c l us t e r s ba nds c a n r e pr e s e nt he t e r od upl e xe s P C R a r t i f a c t s f r o m m i xe d D N A t e m pl a t e s t ha t r e s ul t f r om t w o s i m i l a r but no t c or r e s pondi n g s t r a nds a nne a l i ng t oge t he r T he s e a r t i f a c t s c a n be de t e c t e d be c a us e t he y pr oduc e ba nds a t l ow de na t ur a nt c onc e nt r a t i ons ( W i s e e t a l 1999) S t ab l e i s ot op e p r ob i n g m i c r oc os m s ( S I P ) S I P m i c r oc os m s pe r m i t t he i de nt i f i c a t i on of or ga ni s m s r e s pons i bl e f or c e r t a i n i n s i t u t r a ns f or m a t i on pr oc e s s e s by t he us e of a l a be l e d s ubs t r a t e ( a l e s s na t ur a l l y f r e que nt i s ot ope ) t ha t w i l l be i nc or po r a t e d i nt o

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50 t he a c t i ve m i c r obi a l bi om a s s ( R a da j e w s ki e t a l 20 00; M c D ona l d e t a l 2005 ) S ubs e que nt l y, t he l a be l e d D N A f r a c t i on i s s e pa r a t e d f r om t he na t u r a l l y oc c ur r i ng f r a c t i on by C s C l de ns i t y gr a di e nt c e nt r i f uga t i on T he l a be l e d f r a c t i on i s t he n a na l yz e d t o i de nt i f y t he a c t i ve m i c r obi a l c om m uni t y by c l oni ng, f ol l ow e d by r e s t r i c t i on f r a gm e nt l e ngt h pol ym or phi s m ( T R F L P ) o r by D G G E a na l ys i s T he m a j or l i m i t a t i on of t h e S I P m e t hodol ogy i s t he di l ut i on o f t he l a be l e d s ubs t r a t e be f or e i t s a s s i m i l a t i on a nd i nc o r por a t i on i nt o t he a c t i ve or ga ni s m s w hi c h c a n ha ppe n i f s i m ul t a ne ous gr ow t h on a n unl a be l e d s ubs t r a t e i s o c c ur r i ng i n t he m i c r oc os m s O t he r c ons t r a i nt s m a y be t he r e l a t i ve l ong i nc uba t i on pe r i ods ne e de d t o l a be l s uf f i c i e nt bi om a s s a nd, c ons e que nt l y, t he po t e nt i a l f o r t he us e of l a be l e d m e t a bol i t e s by non t a r ge t or ga ni s m s ( c r os s f e e di ng) A l s o, t he a r t i f i c i a l s pi k e a nd r e l a t i ve l y hi gh c onc e nt r a t i ons us e d of t he l a be l e d s ubs t r a t e m a y s t i m ul a t e m i c r o or ga ni s m s t ha t w e r e not a c t i ve i n s i t u a nd, a s a r e s ul t t he a na l ys i s m a y r e pr e s e nt t he pot e nt i a l a c t i ve popul a t i on, r a t he r t ha n t he a c t i ve m i c r obi a l c om m uni t y a t t he t i m e o f s a m pl i n g ( L i n e t a l 2004; M c D ona l d e t a l 2 005) T he r e f or e S I P s t udi e s s houl d pr ovi de a r a t i ona l ba s i s f or t he a ppl i c a t i on of m ol e c ul a r bi ol ogi c a l t e c hni que s t o s t udy t he r o l e o f s pe c i f i c or ga ni s m s t ha t a r e l i ke l y t o be i nvol ve d i n a de f i ne d p r oc e s s ( R a da j e w s ki e t a l 2003) F i na l l y t he t e c hni que i s e xpe ns i ve a nd r e qui r e s c e r t a i n e xpe r t i s e b ut i t i s o ne of t he m os t pow e r f ul m ol e c ul a r t e c hni que s a va i l a bl e pr ovi di ng i nf or m a t i on on t he a c t i ve m i c r obi a l popul a t i ons o f a n e nvi r onm e nt a l s a m pl e a nd l i nki ng f unc t i on t o i de n t i t y. S t u d y H yp ot h e s i s T he di ve r s i t y a nd a c t i vi t y o f m e t ha not r oph popul a t i ons a s s oc i a t e d w i t h t he r hi z os phe r e of pl a nt s us e d i n phyt or e m e di a t i on pr o c e s s e s a r e i m pa c t e d by pl a nt t ype s ys t e m de s i gn, a nd e nvi r onm e nt a l c ondi t i ons p r e s e nt a t t he s i t e

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51 S t u d y O b j e c t i ve s B r oad O b j e c t i ve s P r ovi de f ul l c ha r a c t e r i z a t i on o f a w e l l know n, pur e m e t ha not r oph, S t r a i n C S C 1, i s ol a t e d f r om a gr oundw a t e r a qui f e r know n t o ox i d i z e T C E a s a m e a ns of m e t hod de ve l opm e nt of phyt o r e m e di a t i on s t udi e s A s s e s s t he e f f e c t s of pl a nt e xuda t e s s pe c i f i c a l l y m onot e r pe ne s on T C E c om e t a bol i s m by m e t ha not r oph ba c t e r i a D e ve l op a pr ot oc ol us i ng s t a bl e i s ot ope pr obi ng ( S I P ) m e t hods s pe c i f i c t o r hi z os phe r e m i c r oor ga ni s m s A s s e s s di f f e r e nc e s i n m e t ha not r oph a bunda nc e a c t i vi t y, a nd di ve r s i t y obs e r ve d i n r hi z os phe r e s a m pl e s f r om s e ve r a l pl a nt s pe c i e s us e d i n phyt o r e m e di a t i on. D e t e r m i ne t he e f f e c t i ve ne s s of c ul t u r e de pe nde nt a nd c ul t ur e i nde pe nde nt m e t hods t o c ha r a c t e r i z e pot e nt i a l m i c r obi a l de gr a de r s U l t i m a t e l y pr ovi de gu i da nc e f or t he phyt o r e m e di a t i on pr a c t i t i one r t o m o r e a c c ur a t e l y pr e di c t t he e xt e nt of T C E r hi z ode gr a da t i on w he n us i ng m onot e r pe ne a nd non m onot e r pe ne r e l e a s i ng pl a nt s S p e c i f i c O b j e c t i ve s C ha r a c t e r i z e t he m e t ha not r oph S t r a i n C S C 1 us i ng phe not ypi c a nd phys i ol ogi c a l de s c r i pt i ons phyl oge ne t i c s of t he 16S r D N A a nd m ul t i pl e f unc t i ona l ge ne s ( m m oX pm oA m x a F ) a nd D N A D N A hyb r i di z a t i on. D e t e r m i ne t he e f f e c t of ( R ) pi ne ne on T C E c om e t a bol i s m by pur e c ul t u r e s of r e pr e s e nt a t i ve t ype I I I a nd X m e t ha not r ophs us i ng oxyge n upt a ke a na l ys i s C om bi ne a nd i m pl e m e nt S I P m e t hods w i t h m ol e c u l a r f i nge r p r i nt s t e c hni que s a s de na t ur i ng gr a di e nt ge l e l e c t r ophor e s i s ( D G G E ) u s i ng t he 16S r D N A a nd f unc t i ona l pm oA ge ne s t o de ve l op a pr e c i s e m e t ho dol ogy f or m e t ha not r oph r hi z os phe r e s t udi e s D e t e r m i ne by c ul t ur e de pe nde nt m i c r obi a l c ount s t he a bunda nc e of t he he t e r o t r oph a nd m e t ha not r oph c om m uni t i e s f r om r hi z os phe r e s oi l c om pa r t m e nt s of t w o phyt or e m e di a t i on s i t e s E nr i c h f or a nd c ha r a c t e r i z e m e t ha not r oph m i xe d c ul t ur e s f r om r hi z os phe r e s oi l c om pa r t m e nt s of t w o phyt or e m e di a t i o n s i t e s by t h e i r oxi da t i on po t e nt i a l us i ng oxyge n upt a ke a na l ys i s pr e s e nc e a nd a c t i vi t y of s ol ubl e m e t ha ne m onoxyge na s e a nd phyl oge ne t i c s of 16S r D N A D G G E a na l ys i s

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52 D e t e r m i ne t he e f f e c t of s e ve r a l e nvi r onm e nt a l va r i a bl e s ( l oc a t i on, t i m e t r e e t ype c ont a m i na nt t ype a nd c onc e nt r a t i on de pt h a nd s y s t e m de s i gn) on t he m e t ha not r oph popul a t i ons of di f f e r e nt r hi z os phe r e s oi l c om pa r t m e nt s a t t w o c ur e nt phyt or e m e di a t i on s i t e s

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53 T a bl e 1 1 P hys i c a l a nd c he m i c a l pr ope r t i e s of T C E a nd P C E 1 C ha r a c t e r i s t i c C om poun d T C E P C E M ol e c ul a r f or m ul a C 2 H C l 3 C 2 C l 4 M ol e c ul a r w e i ght ( g m o l 1 ) 131. 4 165. 8 S pe c i f i c gr a vi t y ( a t 20 C ) 1. 465 1. 623 V a por pr e s s ur e ( a t 25 C ) ( m m H g ) 74 18. 5 W a t e r s ol ubi l i t y ( a t 25 C ) ( m g l 1 ) 1 366 150 l og K o c 2. 03 2. 66 2. 20 2. 70 l og K o w 2. 42 3. 40 H e nr y s l a w c ons t a nt ( a t 25 C ) ( a t m m 3 m o l 1 ) 0. 011 0. 018 1 (A T S D R 1 9 9 9 ) F i gur e 1 1 S c he m a t i c of pr oc e s s e s i n a phyt or e m e di a t i on s ys t e m ( M c C ut c he on a nd S c hnoor 2003 )

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54 T a bl e 1 2 P hys i c a l a nd c he m i c a l pr ope r t i e s of pi ne ne 1 P r ope r t y V a l ue M ol e c ul a r f or m ul a C 1 0 H 1 6 M ol e c ul a r w e i ght ( g m o l 1 ) 136. 24 S pe c i f i c gr a vi t y ( a t 20 C ) 0. 8592 V a por pr e s s ur e ( a t 25 C ) ( m m H g ) 4. 75 W a t e r s ol ubi l i t y ( a t 25 C ) ( m g l 1 ) 2 2. 5 K o c 1 200 L og K o w 4. 83 H e nr y s l a w c ons t a nt ( a t 25 C ) ( a t m m 3 m o l 1 ) 0. 107 ( 1H S B D 1999; 2L i e t a l 1998) F i gu r e 1 2 C 1 m e t a bol i s m by m e t ha not r ophs a nd m e t hyl ot r ophs a s de s c r i be d by W a c ke t t ( 1995)

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55 T a bl e 1 3 C ha r a c t e r i s t i c s of di f f e r e nt m e t ha not r o ph t ype s 1 C ha r a c t e r i s t i c T ype I T ype I I T ype X R e c ogni z e d ge ne r a M e t hy l om onas M e t hy l obac t e r M e t hy l om i c r obi um M e t hy l oc al dum M e t hy l os phae r a M e t hy l os i nus M e t hy l oc y s t i s M e t hy l oc e l l a M e t hy l oc aps a M e t hy l oc oc c us C e l l ul a r s ha pe S hor t r ods s om e c oc c i or e l l i ps oi ds R ods c r e s c e nt or pe a r s ha pe d C oc c i R e s t i ng s t a ge s A z ot oba c t e r t ype c ys t s E xos por e s or l i pi d c ys t s A z ot oba c t e r t ype c ys t s I nt r a c yt opl a s m i c m e m br a n e s D i s c s ha pe d bundl e s of ve s i c l e s P a i r e d, pa r a l l e l t o t he c yt opl a s m a t i c m e m br a ne D i s c s ha pe d bundl e s of ve s i c l e s F or m a l de hyde pa t hw a y a s s i m i l a t i on ( C a s s i m i l a t i on) R uM P 2 S e r i ne R uM P ( m a j or ) / S e r i ne T C A c yc l e 2 I nc om pl e t e ( one e xc e pt i on) C om pl e t e I nc om pl e t e D N A G + C c ont e nt 50 54% 62. 5% 62. 5% P r e dom i na nt phos phol i pi d f a t t y a c i ds ( P L F A s ) 16 C a t om s 18 C a t om s 16 C a t om s K e y e nz ym e s : M e t ha ne m onooxyge na s e 3 he xul os e phos pha t e s ynt ha s e H ydr oxypyr uva t e r e duc t a s e N i t r oge na s e R i bul os e bi phos pha t e c a r boxyl a s e I s oc i t r a t e de hydr oge na s e 2 pM M O 2 + N A D + / N A D P + pM M O / s M M O 2 + + N A D P + pM M O / s M M O + + + + N A D + G r ow t h t e m pe r a t ur e 40 C > 40 C > > 45 C P hyl oge ny P r o t e oba c t e r i a # P r ot e oba c t e r i a P r o t e oba c t e r i a 1 (H an s o n an d H an s o n 1 9 9 6 ; Su l l i v an et al 1 9 9 8 ; G rah am et al 2 0 0 2 ) 2 Ru MP = ri b u l o s e mo n o p h o s p h at e cy cl e; T C A = ri c arb o x y l i c aci d cy cl e; p M M O = p art i cu l at e met h an e mo n o o x y g en as e; s M M O = s o l u b l e met h an e m o n o o x y g en as e; N A D + = n i co t i n a mi d e ad en i n e d i n u cl eo t i d e; N A D P + = n i co t i n ami d e ad en i n e d i n u cl eo t i d e p h o s p h at e.

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56 F i gur e 1 3 O xi da t i on o f T C E by a e r obi c m e t ha no t r oph de gr a da t i on ( A ) a nd a na e r obi c r e duc t i ve de ha l oge na t i on ( B ) ( 4 B a r r i o L a ge e t a l 1986; 3 F ox e t a l 1990; 1 V l i e g e t a l 1996; 2 L ont oh e t a l 2000)

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57 C H A P T E R 2 M E T H Y L O C Y ST I S A L D R I C H I I S P N O V A N O V E L M E T H A N O T R O P H I S O L A T E D F R O M A G R O U N D W A T E R A Q U I F E R N ot e : M anus c r i pt s ubm i t t e d t o t he I nt e r nat i onal J o ur nal of Sy s t e m at i c and E v ol u t i onar y M i c r obi ol ogy L i ndne r A S P a c he c o, A A l dr i c h H C C os t e l l o S t a ni e c A U z I a nd H ods on, D J 200 6. M e t hy l oc y s t i s al dr i c hi i s p. nov. a nove l m e t ha not r oph i s ol a t e d f r o m a gr oundw a t e r a qui f e r I nt e r nat i onal J our nal of Sy s t e m at i c and E v ol ut i onar y M i c r obi ol ogy X X : X X X X X X I n t r od u c t i on S pe c i e s of t he ge nus M e t hy l oc y s t i s a r e s t r i c t l y a e r o bi c gr a m ne ga t i ve ba c t e r i a t ha t a r e a bl e t o g r ow on one c a r bon c om pounds ( e g. m e t ha ne or m e t ha nol ) ( B ow m a n e t a l 1993a ) T he ge nus M e t hy l oc y s t i s be l ongs t o t he a l pha s ubc l a s s of t he P r ot e oba c t e r i a a nd c ur r e nt l y c ons i s t s of 2 s pe c i e s w i t h s t a ndi ng i n no m e nc l a t ur e M e t hy l oc y s t i s par v us a nd M e t hy l oc y s t i s e c hi noi de s ( W hi t t e nbur y e t a l 1970 ; G a l c he nko e t a l 1977; B ow m a n e t a l 1993a ) N um e r ous M e t hy l oc y s t i s s t r a i ns ha ve be e n i de nt i f i e d i n a va r i e t y of e nvi r onm e nt s i nc l u di ng l a ke oc e a n, m a r s h, a nd c r e e k s e di m e nt s a nd w a t e r c oa l m i ne dr a i na ge w a t e r t he r oot s o f pl a nt s e t c ( W hi t t e nbu r y e t a l 1970; G a l c he nko e t a l 1977; B ow m a n e t a l 1993a ; H a ns on a nd H a ns on, 1996; C a l houn a nd K i ng 1998; H e ye r e t a l 2002) S pe c i e s of t he ge nus M e t hy l oc y s t i s a r e T ype I I m e t ha not r ophs c l a s s i f i e d, i n pa r t by t he i r pos s e s s i on of pa i r e d m e m br a ne s a l i gne d w i t h t he c e l l pe r i phe r y, t he s e r i ne pa t hw a y, a nd pr e dom i na nt f a t t y a c i ds w i t h 18 c a r b ons ( H a ns on a nd H a ns on, 1996; G r a ha m e t a l 2002 ) A l l know n T ype I I m e t ha not r ophs i nc l udi ng t he M e t hy l oc y s t i s s pe c i e s e xpr e s s t he pa r t i c ul a t e f or m o f m e t ha ne m onooxyge na s e ( pM M O ) a nd, w i t h t he

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58 e xc e pt i on of M e t hy l oc y s t i s par v us a l l e xpr e s s t he s ol ubl e f or m o f m e t ha ne m onooxyge na s e ( s M M O ) unde r c ondi t i ons of l ow c oppe r c onc e nt r a t i ons ( S t a nl e y e t a l 1983; P r i o r a nd D a l t on 1985 ; C hoi e t a l 2003 ) M e t hy l oc y s t i s par v us doe s not pos s e s s ge ne s e nc odi ng f or s M M O ( T s i e n a nd H a ns on, 19 92; M c D ona l d e t a l 1997; L l oyd e t a l 1999) a nd i s t he r e f o r e i nc a pa bl e of oxi di z i ng a r o m a t i c c om po unds A l l M e t hy l oc y s t i s s pe c i e s pr oduc e oxi da s e a nd c a t a l a s e a r e nonm ot i l e a nd a r e c a pa bl e of f i xi ng a t m os phe r i c ni t r oge n ( H a ns on a nd H a ns on, 1996) T he f oc us of t hi s pa pe r i s S t r a i n C S C 1, a gr oup I I m e t ha not r oph pr e vi ous l y i s ol a t e d f r om a n unc ont a m i n a t e d gr oundw a t e r a qui f e r a t M of f e t N a va l A i r S t a t i on i n M ount a i n V i e w C A U S A ( H e nr y a nd G r bi G a l i 1990 ) T hi s m e t ha not r oph e xp r e s s e s s M M O unde r c oppe r l i m i t i ng c ondi t i ons a nd i s c a pa bl e of oxi di z i ng a l i pha t i c a nd a r om a t i c c om pounds ( H e nr y a nd G r b i G a l i 1991; A dr i a e ns a nd G r bi G a l i 1994; A d r i a e ns 1994; H r $ a k, 1996; H r $ a k a nd B e gonj a 1998) D e s pi t e i t s be i ng t he f oc us of t he s e num e r ous s t udi e s a i m e d pr i m a r i l y t ow a r ds c ont a m i na nt de gr a da t i on pot e nt i a l S t r a i n C S C 1 ha s not be e n c ha r a c t e r i z e d a nd di f f e r e nt i a t e d f r om ot he r know n T ype I I m e t ha not r ophs T hi s s t udy p r ovi de s phe not ypi c a n d ge not ypi c a na l ys i s of t hi s gr oundw a t e r i s ol a t e T he f or m a l t a xonom i c de s c r i pt i on of t hi s nove l M e t hy l oc y s t i s ba c t e r i um M e t hy l oc y s t i s al dr i c hi i s p. nov. s t r a i n C S C 1, i s r e po r t e d. D i f f e r e nc e s i n va r i ous c ha r a c t e r i s t i c s of S t r a i n C S C 1 c om pa r e d t o ot he r know n m e t ha not r ophs a r e de s c r i be d, a nd i t s uni que s ur f a c e f e a t ur e s br oa de n t he obs e r ve d phys i ol ogi c a l t r a i t s of m e t ha not r ophi c ba c t e r i a M at e r i al s an d M e t h od s S t r a i n C S C 1 w a s obt a i ne d f r om D r D ubr a vka H r $ a k a t t he R udj e r B os kovi c I ns t i t ut e i n Z a gr e b, C r oa t i a a nd M e t hy l os i nus t r i c hos por i um w a s obt a i ne d f r om D r

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59 J e r e m y S e m r a u i n t he D e pa r t m e nt of C i vi l a nd E n vi r onm e nt a l E ng i ne e r i n g a t t he U ni ve r s i t y of M i c hi ga n, A nn A r bo r U S A M e t hy l oc y s t i s par v u s a nd M e t hy l oc y s t i s e c hi noi de s w e r e obt a i ne d f r om N C I M B ( A be r de e n, E ng l a nd) T he ba s a l m e di um us e d f or g r ow t h w he n c ul t u r i ng f or s M M O e xpr e s s i on w a s ni t r a t e m i ne r a l s a l t s ( N M S ) m e di um w i t h no a dde d c oppe r a s de s c r i be d pr e vi o us l y ( W hi t t e nbur y e t a l 1970; L ont oh a nd S e m r a u, 1998) T e n m ol l 1 c oppe r ni t r a t e ( C u( N O 3 ) 2 ) w a s a dde d t o t he N M S m e di um t o p r ovi de c ondi t i ons f or pM M O e xpr e s s i on. L i qui d c ul t ur e s w e r e r out i ne l y gr ow n a t 250 r pm a nd 30 C i n e i t he r 50 o r 500 m l ba t c he s i n 250 m l E r l e nm e ye r o r 2800 m l F e r nba c h f l a s ks r e s pe c t i ve l y. T he f l a s ks w e r e f i t t e d w i t h r ubbe r s t oppe r s ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) e qui ppe d w i t h a r e s e a l a bl e gl a s s t ube f i l l e d w i t h gl a s s w ool t o a l l ow he a ds pa c e r e m ova l a nd f i l l i ng A po r t i on o f t he a i r he a ds pa c e w a s r e m ove d a nd r e f i l l e d w i t h m e t ha ne of 99. 99 % pur i t y ( S t r a t e W e l di ng, J a c ks onvi l l e F L U S A ) us i ng a va c uum pum p a s s e m bl y t o a c hi e ve a he a ds pa c e c onc e nt r a t i on of a i r w i t h 20% ( v/ v ) m e t ha ne F or s ol i d c ul t u r i ng, 1. 5 % ( w / v) o f B a c t o a ga r ( D i f c o L a bor a t or i e s D e t r oi t M I U S A ) w a s a dde d t o t he N M S m e di um A l l pl a t e s w e r e i nc uba t e d i n a s e a l e d de s i c c a t or c ont a i ni ng a nhydr ous C a S O 4 ( D r i e r i t e W A H a m m ond D r i e r i t e C om pa ny, X e ni a O H U S A ) unde r a n a t m os phe r e of 20% m e t ha ne a nd 8 0% a i r ( by vol um e ) a t 30 C t ha t w a s r e f r e s he d e ve r y f ou r t o f i ve da ys P ur i t y of t he c ul t ur e s w a s ve r i f i e d by r out i ne s t r e a ki ng on 2% ( w / v) nut r i e nt a ga r i n doubl y de i oni z e d w a t e r s M M O e xpr e s s i on w a s qua l i t a t i ve l y ve r i f i e d by a na pht ha l e ne a s s a y m odi f i e d f r om B r us s e a u e t a l ( 1990) F our ne ga t i ve c ont r ol s a u t oc l a ve d c e l l s c e l l s c ul t ur e d w i t h 10 m ol l 1 C u( N O 3 ) 2 ( f or e xpr e s s i on of pM M O ) c e l l f r e e a nd c e l l s t ha t ha ve be e n

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60 s ubj e c t e d t o a ddi t i on of one t o t w o m l of a c e t yl e ne ga s ( a know n i nhi bi t or of M M O P r i o r a nd D a l t on ( 1985) ) w e r e i nc l ude d w i t h t h r e e l i ve s a m pl e s of a c t i ve t e s t c ul t ur e di l ut e d t o a n a bs or ba nc e of 0 2 ( a t a w a ve l e ngt h of 600 n m ) a nd t r a ns f e r r e d t o a ut oc l a ve d 10 m l c a ppe d t e s t t ube s S e ve nt y m g o f c r us he d na pht ha l e ne ( S i gm a S t L o ui s M O U S A ) w e r e a dde d t o e a c h t ube A f t e r i nc uba t i on a t 30 C a nd 250 r pm f or a m i ni m um of one hour 0 1 m l of f r e s hl y pr e pa r e d 4 21 m m o l l 1 t e t r a z ot i z e d or t ho di a ni s i di ne ( S i gm a S t L oui s M O U S A ) s ol ut i on w a s a dd e d. A s ubs e que nt pi nk t o pu r pl e c ol or f o r m a t i on i n t he t ube s i ndi c a t e d pos i t i ve s M M O a c t i vi t y t ha t w a s ve r i f i e d us i ng s pe c t r ophot om e t r y ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) a t 550 nm G e nom i c D N A w a s i s ol a t e d f r om S t r a i n C S C 1, gr ow n t o e xpone nt i a l pha s e by a s t a nda r d m e t hod ( A us ube l e t a l 1989) 16S r R N A ge ne w a s a m pl i f i e d by P C R us i ng t he uni ve r s a l ba c t e r i a l p r i m e r s 27 f a nd 1492r ( L a ne 1 991) P C R pr i m e r s us e d f or s M M O w e r e m m oX A ( 5 A C C A A G G A R C A R T T C A A G 3 ) a nd m m oX B ( 5 T G G C A C T C R T A R C G C T C 3 ) ( A u m a n e t a l 20 00) ; f o r m e t ha nol de hydr oge na s e ( M D H ) m x a f 1003 ( 5 G C G G C A C C A A C T G G G G C T G G T 3 ) a nd m x a r 1561 ( 5 G G G C A G C A T G A A G G G C T C C C 3 ) ( M c D ona l d a nd M ur r e l l 1997) ; a nd f or pM M O A 18 9f ( 5 G G N G A C T G G G A C T T C T G G 3 ) a nd A 682r ( 5 G A A S G C N G A G A A G A A S G C 3 ) ( H ol m e s e t a l 1995) A l l P C R r e a c t i ons w e r e c a r r i e d out i n a P T C 200 T he r m o C yc l e r ( M J R e s e a r c h, M A U S A ) us i ng 25 l r e a c t i ons a nd P r e m i x T a q p ol ym e r a s e ( T a ka r a O t us u, S h i ga J a pa n) C ondi t i ons us e d f o r t he pr i m e r s e t s ha ve be e n de s c r i be d pr e vi ous l y ( H ol m e s e t a l 1995 ; C os t e l l o a nd L i ds t r om 1999; A um a n e t a l 2000 ) T he P C R a m pl i f i c a t i on pr oduc t s w e r e l i ga t e d t o ve c t or pC R 2. 1 ( I nv i t r oge n C a r l s ba d, C A U S A ) a nd

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61 t r a ns f or m e d t o c om pe t e nt E c ol i c e l l s ( T O P 10F ) a c c or di ng t o t he ve ndo r s i ns t r uc t i ons P l a s m i d D N A f r om t r a ns f or m a nt s w a s i s ol a t e d a nd t he i ns e r t s s e que nc e d by t he B i ot e c hnol ogy R e s our c e C e nt e r a t C or ne l l U ni ve r s i t y ( I t ha c a N Y U S A ) S e que nc e s w e r e c om pa r e d w i t h pr e vi ous l y i de nt i f i e d s e que nc e s i n t he N a t i ona l C e nt e r f or B i ot e c hnol ogy I nf o r m a t i on ( N C B I ) da t a ba s e us i ng B L A S T ( A l t s c hul e t a l 1990) T he 16S r R N A ge ne f r o m S t r a i n C S C 1 w a s a l s o a l i gne d w i t h s e que nc e s obt a i ne d f r om t he S e que nc e M a t c h pr ogr a m pr ovi de d by t he R i bos om a l D a t a ba s e P r oj e c t I I ( R D P I I ) ( C ol e e t a l 2005) P hyl oge ne t i c t r e e s w e r e ge ne r a t e d us i ng P H Y L I P ve r s i on 3 6 ( F e l s e ns t e i n, 2004) a nd vi e w e d us i ng T r e e vi e w ( P a ge 1996) T he G e nB a nk a c c e s s i on num be r s f or t h e 16S r R N A M D H s M M O a nd pM M O ge ne s e que nc e s obt a i ne d i n t hi s s t udy a r e D Q 364433, D Q 664499 D Q 664498 a nd D Q 364434, r e s pe c t i ve l y. D N A D N A hybr i di z a t i on w a s pe r f o r m e d on S t r a i n C S C 1 by D S M Z ( B r a uns c hw e i g, G e r m a ny) a ga i ns t M e t hy l oc y s t i s e c hi noi de s s t r a i n I M E T 10491 w a s pe r f or m e d us i ng 2 x S S C buf f e r ( 0. 3 M N a C l 0 03 M s odi um c i t r a t e pH 7 0) + 10% ( v/ v) f or m a m i de a t a n opt i m a l r e na t ur a t i on t e m pe r a t ur e of 68 C E a r l i e r s t udi e s r e por t e d S t r a i n C S C 1 a s gr a m ne ga t i ve non m ot i l e c oc c oba c i l l us pos s e s s i ng a n i nt e r na l m e m br a ne s t r uc t u r e c ha r a c t e r i s t i c of T ype I I m e t ha not r ophs ( pa i r e d m e m br a ne s i ns i de t he pe r i phe r y o f t he c e l l ) a nd f or m i ng l i pi d i nc l us i ons ( H e nr y a nd G r bi G a l i 1990; H e nr y a nd G r bi G a l i 19 91; H r $ a k a nd B e gonj a 1998 ) F a ng e t a l ( 2000 ) c onc l ude d t ha t t he i nt a c t phos phol i pi ds of S t r a i n C S C 1 c l us t e r e d w i t hi n t he T ype I I gr oupi ng c l e a r l y di s t i nc t f r om gr oupi ngs o f T ype I m e t ha not r ophs T hi s s t udy e xt e nde d t he pr e vi ous phe not ypi c c ha r a c t e r i z a t i on s t udi e s by a s s e s s i ng e xos por e a nd r os e t t e f or m a t i on gr ow t h a t 37 C t he p r e s e nc e of a s ur f a c e ( S ) l a ye r c a r bon a nd

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62 ni t r oge n s our c e ut i l i z a t i on a nd l ys i s by 2% ( w / v) s odi um dode c yl s ul f a t e ( S D S ) a l l i de nt i f i e d by B ow m a n e t a l ( 1993a ) or H a ns on a nd H a ns on ( 1996) a s di f f e r e nt i a t i ng c ha r a c t e r i s t i c s a m ong T ype I I m e t ha not r ophi c s pe c i e s E xos por e f or m a t i on w a s de t e r m i ne d w i t h one t o t w o w e e k ol d br ot h c ul t ur e s gr ow n a s pr e vi ous l y de s c r i be d f ol l ow i ng m e t hods of S m i be r t a nd K r i e g ( 1981 ) F i ve m l of c ul t ur e w e r e t r a ns f e r r e d i n dupl i c a t e t o f r e s h N M S m e di um f o r c ont r ol s A s e c ond s e t of dupl i c a t e s w a s he a t e d i n a w a t e r ba t h a t 80 C f o r 20 m i n f o r pa s t e ur i z a t i on. G r ow t h w a s m oni t or e d a f t e r s t r e a ki ng t he c ont r ol s a nd t r e a t e d c ul t ur e s ont o s ol i d N M S pl a t e s a nd i nc uba t i on ( a s pr e vi ous l y de s c r i be d) f or 21 da ys E xos por e s w e r e m o ni t or e d us i ng l i ght m i c r os c opy, a l s o us e d t o de t e r m i ne r os e t t e f o r m a t i on ( N or r i s a nd R i bbons 1971 ) G r ow t h i n l i qui d c ul t u r e w a s m oni t or e d us i ng 250 m l ne phl os f l a s ks w i t h t he s a m e s t oppe r a s s e m bl y de s c r i be d a bove a nd a s pe c t r ophot om e t e r ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) a t a w a ve l e ngt h of 600 nm N i t r oge n a nd c a r bon s our c e s w e r e t e s t e d us i ng N M S ba s a l m e di um T o t e s t f or a l t e r na t e ni t r oge n s our c e s K N O 3 w a s r e pl a c e d w i t h 0. 1% ( w / v ) of a nhydr ous L a s pa r a gi ne ( M P B i om e di c a l s I r vi ne C A U S A ) L a s pa r t a t e ( P f a l t z & B a ue r W a t e r bur y, C T U S A ) o r L gl ut a m i ne ( M P B i om e di c a l s I r vi n e C A U S A ) ( a l l s how n t o s uppor t gr ow t h of M e t hy l oc y s t i s e c hi noi de s a nd M e t hy l oc y s t i s par v us B ow m a n e t a l 1993a ) o r L l ys i ne m onohydr oc hl o r i de ( S i gm a A l dr i c h, S t L oui s M O U S A ) L o r ni t hi ne hydr o c hl or i de ( M P B i om e di c a l s I r v i ne C A U S A ) o r put r e s c i ne ( M P B i om e di c a l s I r vi ne C A U S A ) ( a l l s how n t o s uppor t gr ow t h of M e t hyl os i nus t r i c hos por i um B ow m a n e t a l 1993a ) N M S m e di um w i t h K N O 3 a nd w i t h out a ni t r oge n s our c e s e r ve d a s pos i t i ve a nd ne ga t i ve c ont r ol s r e s pe c t i ve l y, a nd t he l a t t e r c ont r ol a l s o s e r ve d a s a t e s t t o f i x

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63 a t m os phe r i c ni t r oge n. T o t e s t f o r a l t e r na t e c a r bon s our c e s 0. 2% ( w / v) of m e t hyl a m i ne hydr oc hl or i de ( A l f a A e s a r W a r d H i l l M A U S A ) di m e t hyl s ul f oxi de m e t ha nol o r gl uc os e ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) w a s a dde d. O ve r t he 30 da y t e s t pe r i od f l a s ks w e r e pr e pa r e d i n dup l i c a t e a nd t r a ns f e r s w e r e m a de t o f r e s h m e di um a nd ni t r oge n or c a r bon s our c e e ve r y 4 da ys G r ow t h m e a s ur e m e nt s w e r e pe r f or m e d a s de s c r i be d pr e vi ou s l y. L ys i s by 2% ( w / v) S D S ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) w a s de t e r m i ne d by di r e c t m i c r os c opi c obs e r va t i on us i ng c e l l s ha r v e s t e d a t % l og pha s e C e l l s w e r e c e nt r i f uge d a t 2460 x g f or 20 m i n, r e s us pe nde d i n t he 2% S D S s t oc k s ol ut i on f or a ppr oxi m a t e l y 2 hou r s a nd o bs e r ve d us i ng a n oi l i m m e r s i on pha s e c ont r a s t m i c r os c ope ( Z e i s s O be r koc he n, G e r m a ny) T r a ns m i s s i on e l e c t r on m i c r os c opy w a s us e d t o obs e r ve c e l l s of S t r a i n C S C 1 e xpr e s s i ng M M O l i pi d i nc l us i ons a nd ot he r f i ne s t r uc t ur a l f e a t u r e s i nc l u di ng S l a ye r s L i qui d c ul t u r e s w e r e i nc uba t e d f o r t w o t o t h r e e da ys a nd w e r e f i xe d f or 30 m i n a t r oom t e m pe r a t ur e w i t h c a c odyl a t e buf f e r e d gl ut a r a l de hyde bot h w i t h a nd w i t hout 0. 1% A l c i a n bl ue ( F a s s e l e t a l 1992 ) s t a i ne d f or 30 m i n a t r oo m t e m pe r a t ur e w i t h 1% c a c odyl a t e buf f e r e d os m i um t e t r oxi de a nd t he n s t a i ne d f or 50 m i n i n 1% a que ous ur a nyl a c e t a t e A f t e r de hydr a t i ng i n i nc r e a s i ng s t r e ngt hs of e t ha no l c e l l s w e r e e m be dde d i n bot h S pur r s a nd E pon r e s i ns ( D yks t r a 1993 ) T hi n s e c t i ons w e r e pr e pa r e d a nd s t a i ne d w i t h l e a d c i t r a t e a nd e xa m i ne d on a Z e i s s E M 10C A t r a ns m i s s i on e l e c t r on m i c r os c ope M e t hy l oc y s t i s e c hi noi de s w a s obs e r ve d by ne ga t i v e s t a i n us i ng 1% a que ous ur a nyl a c e t a t e a ppl i e d t o c e l l s us pe ns i ons on F or m va r c oa t e d gr i ds

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64 I n or de r t o pr ovi de e vi de nc e t ha t t he obs e r ve d S l a ye r i s gl yc opr ot e i n t w o a d di t i ona l c yt oc he m i c a l a ppr oa c he s w e r e ut i l i z e d. I m a ge s of A l c i a n bl ue s t a i ne d s pe c i m e ns w e r e c om pa r e d t o t hos e w i t h no A l c i a n bl ue i n t he g l ut a r a l de hyde f i xa t i ve s i nc e A l c i a n bl ue s t a i ns pol ys a c c ha r i de m oi e t i e s ( L e w i s a nd K ni ght 1977) S e c ondl y, t hi n E pon s e c t i ons o n F or m va r c oa t e d ni c ke l g r i ds w e r e f i r s t e xpos e d t o 3% hyd r oge n pe r oxi de ( H 2 O 2 ) f or 15 m i n a t r oo m t e m pe r a t u r e t o r e m ove os m i um a nd t he n e xpos e d t o 1% a que ous pr ona s e s ol ut i on ( S i gm a C he m i c a l C o m pa ny, S t L oui s M O U S A ) f or 60 90 m i n a t 35 C t o r e m ove pr o t e i n c om pone nt s f r o m t he s e c t i on ( M onne r on a nd B e r nha r d, 1966; L e w i s a nd K ni ght 1977 ) C ont r o l s i nc l ude d H 2 O 2 a l one a nd w a t e r s ubs t i t ut e d f or t he pr ona s e s t e p. R e s u l t s T he phyl oge ni e s of t he 16S r R N A s M M O M D H a nd pM M O ge ne s s how n i n F i g. 2 1 a nd F i g. 2 2 ( a c ) a r e c ons i s t e nt w i t h pl a c e m e nt of S t r a i n C S C 1 w i t h ot he r know n T ype I I m e t ha not r ophs T he 16S r R N A ph yl oge ny of S t r a i n C S C 1 c l e a r l y pl a c e s i t w i t hi n a br a nc h o f t he al pha P r ot e obac t e r i a do m i na t e d by M e t hy l oc y s t i s s pe c i e s T hi s m e t ha not r oph s ha r e s 98% 16S r R N A ge ne s e que n c e s i m i l a r i t y w i t h i t s ne a r e s t de f i ne d r e l a t i ve s a n unc ul t ur e d m e m be r o f t he M e t hy l oc y s t ac e ae ( A F 358021) a s w e l l a s t w o c ul t ur e d or ga ni s m s : M e t hy l oc y s t i s s p. L 32 ( A J 831 522) a n d M e t hy l oc y s t i s s p. S C 2 ( A J 431384) a l t hough 16S r R N A ge ne s i m i l a r i t y i s not s uf f i c i e nt t o p l a c e S t r a i n C S C 1 t o t he s pe c i e s l e ve l T he D N A D N A hybr i d i z a t i on r e s ul t s s how e d t ha t S t r a i n C S C 1 pos s e s s e s a 3. 8% D N A D N A s i m i l a r i t y w i t h M e t h y l oc y s t i s e c hi noi de s s t r a i n I M E T 10491. A s s how n i n T a bl e 2 1 r os e t t e f o r m a t i on by c e l l s of S t r a i n C S C 1 w a s not obs e r ve d. N o g r ow t h w a s e vi de nt a f t e r pa s t e ur i z a t i on, i ndi c a t i ng t ha t t hi s m e t ha not r oph

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65 i s not r e s i s t a nt t o he a t a nd g r ow t h w a s a l s o not ob s e r ve d a t 37 C O pt i m u m g r ow t h w a s obs e r ve d a t a ppr oxi m a t e l y 30 C S t r a i n C S C 1 w a s not l ys e d by a 2 % s ol ut i on ( w / v) of S D S but a 10 % s ol ut i on ( w / v) of S D S di d l ys e t he c e l l s I t w a s s how n t o be c a pa bl e of gr ow i ng on a l t e r na t e ni t r oge n s our c e s of L a s pa r a gi ne L a s pa r t a t e L gl ut a m i ne L or ni t hi ne a nd put r e s c i ne ; how e ve r no gr ow t h w a s vi s i bl e i n t he p r e s e nc e of L l ys i ne O f t he f our a l t e r na t e c a r bon s our c e s of m e t hyl a m i ne di m e t hyl s ul f i de m e t ha nol a nd g l uc os e t e s t e d, onl y m e t ha nol s uppor t e d gr ow t h of S t r a i n C S C 1. T he e xpr e s s i on of s M M O upon c ul t ur i ng S t r a i n C S C 1 i n N M S m e di um w i t h no c oppe r w a s c onf i r m e d by f or m a t i on of a pu r pl e c ol or a f t e r i nc uba t i on w i t h na pht ha l e ne a nd a ddi t i on of or t ho di ani s i di ne w he r e a s c ont r ol s w i t h a c e t yl e ne a nd w i t h c e l l s c ul t u r e d i n t he pr e s e nc e of c oppe r yi e l de d no c ol or T he s e r e s ul t s s t r ongl y s ugge s t t ha t s M M O w a s e xpr e s s e d i n S t r a i n C S C 1 w he n gr ow n w i t hou t c oppe r a nd w a s r e s pons i bl e f or na pht ha l e ne oxi da t i on. T r a ns m i s s i on e l e c t r on m i c r ogr a phs o f S t r a i n C S C 1 gr ow n i n t he p r e s e nc e of c oppe r ve r i f y t he T ype I I m e m br a ne s t r uc t ur e of pa i r e d m e m br a ne l a m e l l a e i n t he pe r i phe r a l c yt opl a s m ( F i g. 2 3a b ) I n t hi n s e c t i on, a va r i e t y of c e l l s ha pe s w e r e vi s i bl e a t l ow m a gni f i c a t i on ( F i g 2 3a ) but e l onga t e d or dum bb e l l s ha pe s of c e l l s pr e dom i na t e d. M a n y of t he ot he r pr o f i l e s c oul d r e p r e s e nt dum bbe l l s ha pe s s e c t i one d i n di f f e r e nt pl a ne s C e l l s gr ow n w i t hout c oppe r c ont a i ne d onl y a f e w i nt e r na l m e m br a nous l a m e l l a e ( da t a not s how n) P ol yphos pha t e bodi e s a nd l i pi d i nc l us i ons w e r e c om m on. A s s how n i n F i g s 2 3( a ) a nd 2 3 ( b) di s t i nc t i ve l y s t r i ki ng S l a ye r s l i ke l y c om pos e d of gl yc opr ot e i n, w e r e r e ve a l e d w i t h t r a ns m i s s i on e l e c t r on m i c r os c opy of ul t r a t hi n s e c t i ons of S t r a i n C S C 1 f i xe d w i t h A l c i a n bl ue T he s e s pi ke d S l a ye r s t r uc t ur e s

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66 50 75 nm i n he i ght c ove r e d t he e nt i r e s ur f a c e of t he c e l l w a l l W e ha ve s e e n t ha t t he c yt opl a s m of c e l l s of S t r a i n C S C 1 e m be dde d i n S p ur r r e s i n w i l l s om e t i m e s s hr i nk a w a y f r om t he w a l l l e ndi ng s uppor t t o t he i de a t ha t t he S l a ye r i s m o r e r i gi d t ha n t he r e s t o f t he w a l l ( da t a not s how n) T h i s s hr i nka ge doe s not oc c ur i n c e l l s e m be dde d i n E pon r e s i n ( e g. c e l l s i n F i g. 2 3a b) S l a ye r s ha ve be e n obs e r ve d i n bot h T ype I a nd I I m e t ha not r ophs i s ol a t e d f r om a w i de r a nge of e nvi r onm e nt s i nc l udi ng t he ge ne r a M e t hy l om i c r obi um M e t hy l om onas M e t hy l os i nus a nd M e t hy l oc y s t i s ( F a s s e l e t a l 199 2; S or oki n e t a l 2000; T r ot s e nko a nd K hm e l e ni na 2002) T ype I I M e t hy l os i nus t r i c hos por i um O B 3b w a s f ound t o ha ve be a d l i ke S l a ye r s t r uc t ur e s a nd oc c a s i ona l f i l a m e nt ous m a t e r i a l i n t he out e r e nve l ope ( F a s s e l e t a l 1990; F a s s e l e t a l 1992) S i m i l a r be a d l i ke S l a ye r s t r uc t ur e s w e r e obs e r ve d i n t he c e l l e nve l ope of M e t hy l oc y s t i s s p. s t r a i n L a ke W a s hi ngt on but not i n M e t hy l oc y s t i s par i s a nd t he a ut hor s c onc l ude d t ha t t he a bs e nc e of t he s e s t r uc t ur e s i n t he l a t t e r s pe c i e s c oul d be a s pe c i e s va r i a t i on. B ot h M e t hy l oc y s t i s s pe c i e s pos s e s s e d c ons i de r a bl e f i l a m e nt ous m a t e r i a l how e ve r ( F a s s e l e t a l 1992 ) M e t hy l oc y s t i s e c hi noi de s s t r a i n I M E T 10491 w a s r e por t e d t o ha ve r i gi d t ubul a r s t r uc t ur e s a r r a nge d r a di a l l y a t t he c e l l s ur f a c e ( G a l c he nko e t a l 1977) f e a t ur e s t ha t a r e a bs e nt f r om M e t hy l o c y s t i s par v u s ( H e ye r e t a l 2002 ) I n t hi s s t udy, ne ga t i ve s t a i n p r e pa r a t i ons of M e t hy l oc y s t i s e c hi noi de s s how e l l i ps oi d c e l l s w i t h s qua r e e nde d t ubul a r pr oj e c t i ons ( F i g. 2 3c ) t ha t a ppe a r s t r i a t e d a t hi gh m a gni f i c a t i on ( F i g 2 3d) T hi s s t r i a t i on w a s not r e por t e d i n p r e vi ous s t udi e s of t hi s s t r a i n ( G a l c he nko e t a l 1977; B ow m a n e t a l 1 99 3a ) a nd t he t ubul a r a ppe a r a nc e of t h i s S l a ye r i s m uc h di f f e r e nt f r om t he s ol i d s ha r p s pi n e s of S t r a i n C S C 1.

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67 T o f u r t he r e l uc i da t e t he na t ur e of t he s pi ke d S l a ye r i n S t r a i n C S C 1 c e l l s f i xe d w i t h gl ut a r a l de hyde a l one ( F i g 2 4a ) w e r e c om pa r e d w i t h t hos e f i xe d w i t h a n A l c i a n bl ue / gl ut a r a l de hyde m i xt ur e ( F i g 2 4b) A l c i a n bl ue i s a di f f e r e nt i a l s t a i n f or pol ys a c c ha r i de ( L e w i s a nd K ni ght 1977) a nd t he s pi ne s i n F i g. 2 4b w e r e c ons i de r a bl y da r ke r l onge r a nd m or e di s t i nc t t ha n t he s a m e s t r uc t ur e s i n F i g 2 4a e ve n t h o ugh F i g 2 4b i s a t a l ow e r m a gni f i c a t i on. T hi s s t r ongl y i nd i c a t e s pol ys a c c ha r i de c ont e nt A f t e r t r e a t m e nt w i t h H 2 O 2 t o r e m ove os m i um f r om t he E pon s e c t i ons s e c t i ons t r e a t e d w i t h pr ona s e a br oa d s pe c t r um pr ot e a s e l os t t he e nt i r e S l a ye r ( F i g. 2 4c d ) i ndi c a t i ng t ha t t he l a ye r c ont a i ns c ons i de r a bl e pr ot e i n D i s c u s s i on S e que nc e a na l ys i s of t he 16S r R N A ge ne ( F i g 2 1 ) t he s M M O ge ne ( F i g. 2 2a ) t he m e t ha nol de hydr oge na s e ge ne ( F i g 2 2b) a nd t he pM M O ge ne ( F i g. 2 2c ) s uppor t s pl a c e m e nt of S t r a i n C S C 1 w i t hi n t he c l os e l y r e l a t e d ge ne r a M e t hy l oc y s t i s a nd M e t hy l os i nus G i ve n t he s us pe c t e d pl a c e m e nt i n t he ge ne r a M e t h y l oc y s t i s D N A D N A hybr i di z a t i on w a s pe r f o r m e d w i t h M e t hy l oc y s t i s e c hi noi de s B a s e d on t he D N A D N A hybr i di z a t i on r e s ul t s s how i ng onl y 3. 8% s i m i l a r i t y S t r a i n C S C 1 doe s not be l ong t o t he s pe c i e s M e t hy l oc y s t i s e c hi noi de s f ol l ow i ng t he t hr e s hol d va l ue r e c om m e nda t i on of W a yne e t a l ( 1987) P he not ypi c r e s ul t s i n T a bl e 2 1 s how t ha t S t r a i n C S C 1 di f f e r e nt i a t e s f r o m M e t hy l os i nus t r i c hos por i um M e t hy l oc y s t i s e c hi noi de s a nd M e t hy l oc y s t i s par v us t hr e e c l os e l y m a t c hi ng c ul t u r e d s t r a i ns i n t he phyl oge ne t i c a na l ys i s i n va r i ous c ha r a c t e r i s t i c s A l l of t he s t r a i ns s how n i n T a bl e 2 1 ha ve be e n r e por t e d t o be oxi da s e a nd c a t a l a s e pos i t i ve pos s e s s c ol oni e s t ha t a r e of opa que t r a ns pa r e nc y, s m oot h e dge c onve x e l e va t i on, f or m pol y & hydr oxybut y r a t e g r ow on

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68 m e t ha nol a nd c a pa bl e of f i x i ng a t m os phe r i c ni t r og e n. U nl i ke t he know n s t r a i ns S t r a i n C S C 1 w a s not c a pa bl e of gr ow t h a t 37 C ; how e ve r a l l of t he m e t ha not r ophs g r ow opt i m a l l y ne a r 30 C I t i s i m por t a nt t o no t e t ha t G a l c he nko e t a l ( 1977) a nd B ow m a n e t a l ( 1993a ) r e por t c onf l i c t i n g i nf o r m a t i on c onc e r ni ng t he a bi l i t y of M e c hi noi de s t o a c c um ul a t e pol y & hydr oxybut yr a t e a nd gr ow a t 3 7 C t he f o r m e r r e por t i ng pos i t i ve r e s ul t s f or e a c h a nd t he l a t t e r r e por t i ng ne ga t i ve r e s ul t s O ur T E M a nd gr ow t h s t udi e s w i t h t hi s s t r a i n a gr e e d w i t h G a l c he nko e t a l ( 197 7) ( da t a no t s how n) T he e l onga t e d dum bbe l l s ha pe of S t r a i n C S C 1, l a c k of m o t i l i t y a nd a bi l i t y t o f or m pol yphos pha t e s e pa r a t e i t f r om t he M e t hy l os i nus t r i c hos por i um O t he r di s t i ngui s hi ng c ha r a c t e r i s t i c s be t w e e n S t r a i n C S C 1 a nd M t r i c ho s por i um i nc l ude s m a l l e r c e l l s i z e S l a ye r m or phol ogy a nd l a c k of he a t r e s i s t a nc e A l s o, unl i ke r e por t e d obs e r va t i ons of M t r i c hos por i um S t r a i n C S C 1 c a n us e L a s pa r a gi ne L a s pa r t a t e a nd L gl ut a m i ne a nd c a nnot us e L l ys i ne a s ni t r oge n s our c e s B ot h s t r a i ns s ha r e t he a bi l i t y t o us e L or ni t hi ne a nd put r e s c i ne M os t s i m i l a r i t i e s how e ve r a r e s ha r e d w i t h t he t w o M e t hy l oc y s t i s s t r a i ns i nc l udi ng l a c k of m o t i l i t y, he a t r e s i s t a nc e a nd r os e t t e f or m a t i on. S t r a i n C S C 1 s c e l l s ha pe a bi l i t y t o f or m pol yphos pha t e a nd c ol ony c ol or of ye l l ow w hi t e di f f e r f r om M e t hy l oc y s t i s e c hi noi de s a nd M e t hy l oc y s t i s par v us ( T a bl e 2 1) U nl i ke M e c hi noi de s S t r a i n C S C 1 i s c a pa bl e of us i ng L or ni t hi ne a nd put r e s c i ne a s ni t r oge n s our c e s w he r e a s unl i ke M par v us S t r a i n C S C 1 i s not c a pa bl e of us i ng L l ys i ne I n a ddi t i on a s r e por t e d f o r M t r i c hos por i um M e c hi noi de s a nd M par v us S t r a i n C S C 1 i s not l ys e d by a 2% s ol ut i on ( w / v) o f S D S

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69 W hi l e S t r a i n C S C 1 ha s p r e vi ous l y be e n s how n by T E M t o c ont a i n c ha r a c t e r i s t i c T ype I I m e m br a ne s ( H e nr y a nd G r bi G a l i 1990; H r $ a k a nd B e gonj a 1998 ) i t w a s r e ve a l e d he r e t o a c c um ul a t e bot h pol yphos pha t e b odi e s ( F i g. 2 2) a nd pol y & hydr oxybut yr a t e s t or a ge g r a nul e s c ons i s t e nt w i t h M e t hy l oc y s t i s par v us ( B ow m a n e t a l 1993) N o s t udy ha s r e por t e d t he s t r uc t ur e o f S t r a i n C S C 1 s c e l l e nve l ope i n c om pa r i s on t o t ha t of ot he r w e l l c ha r a c t e r i z e d m e t ha not r ophs O f s pe c i a l i nt e r e s t a r e t he s ur f a c e ( S ) l a ye r s r e gul a r c r ys t a l l i ne s ur f a c e l a ye r s i n A r c ha e ba c t e r i a a nd E uba c t e r i a c om pos e d of pr ot e i n or gl yc opr ot e i n s ubuni t s ( S l e yt r e t a l 1993 ; S i dhu a nd O l s e n, 1997) I t i s not know n w hy S l a ye r s de ve l op i n s om e s t r a i ns of c l os e l y r e l a t e d ba c t e r i a a nd not i n ot he r s H ow e ve r one hypot he s i s i s t ha t f o r m a t i on of t he s e s t r uc t ur e s r e f l e c t s a da pt a t i on t o a n e c ol ogi c a l ni c he ( E a s t e r br ook a nd A l e xa nde r 1983; E a s t e r br ook 1989 ) or a r e s pons e t o e xpos ur e t o ha r s h e nvi r onm e nt s ( M i ns ky e t a l 2002) O t he r s s ugge s t t ha t S l a ye r s m a y pr ovi de m i c r oor ga ni s m s w i t h a s e l e c t i ve a dva nt a ge by s e r vi ng a s a pr ot e c t i ve c oa t i ng or a s m ol e c ul a r po r i ns or s i e ve s a nd t r a ps f or s ubs t r a t e s i n m a i nt a i ni ng t he r i gi di t y of t he c e l l e nve l ope or pr ovi di ng a m e a ns of c e l l a dhe s i on a nd s ur f a c e r e c ogni t i on ( S r a a nd S l e yt r 1987; S l e yt r a nd M e s s ne r 1988; S r a e t a l 1992; S i dhu a nd O l s e n, 1997) E a s t e r br ook a nd S pe r ke r ( 1982 ) hypot he s i z e d t ha t s pi na e m a y s i m ul t a ne ous l y f ul f i l l m a ny f o r t ui t ous r ol e s a na l ogous t o a r m s w i t h m ul t i pot e nt i a l a c t i vi t i e s i nc l udi ng a t t a c hm e nt di s t a nc e ke e pi ng, a nd pr ot e c t i on. H ow e ve r w hy s om e s pe c i e s a r e pr one t o s pi ne f o r m a t i on a nd ot he r s not w hy S l a ye r s e xi s t i n a va r i e t y of s ha pe s a nd s ym m e t r i e s a nd w hy t he s e s t r uc t ur e s d e ve l op a m ong s pe c i e s of m e t ha not r ophs i s not c l e a r l y unde r s t ood.

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70 M e t hy l oc y s t i s e c hi noi de s s t r a i n I M E T 10491 w a s i s ol a t e d f r om l a ke m ud i n R us s i a ( G a l c he nko e t a l 1977) pos s i bl y a m or e nut r i e nt r i c h e nvi r on m e nt t ha n t he s e di m e nt s of t he unc ont a m i na t e d g r oundw a t e r a qui f e r i n C a l i f or ni a w he r e S t r a i n C S C 1 w a s i s ol a t e d. E c hi no i de s i s t he L a t i n i z e d a dj e c t i ve de r i ve d f r om t he G r e e k w o r d e c hi nos m e a ni ng he dge hog, na m e d f o r t he he dge hog l i k e a ppe a r a nc e of t hi s ba c t e r i um H ow e ve r a s r e por t e d by G a l c he nko e t a l ( 1977) a nd ve r i f i e d i n t hi s s t udy ( F i g 2 3c d) t he s pi ne s on t hi s m e t ha not r oph a ppe a r t o be t ubul a r a nd l e s s de ns e i n c om pa r i s on t o t he s pi ke s obs e r ve d on S t r a i n C S C 1, w hi c h w oul d be m or e a pt l y na m e d f or a he dge hog. D e s pi t e t he di f f e r e nt or i gi na t i ng e nvi r onm e nt s of t he s e t w o s t r a i ns pr oxi m i t y i n t he gr oupi ng of t he M e t hy l oc y s t i s ge nus a s s t r ongl y s ugge s t e d by t he r e s ul t s of t hi s s t udy, a dds c r e de nc e t o t he hypot he s i s t ha t phyl oge ny a n d e c ol ogy m a y bot h pl a y a r ol e i n S l a ye r f or m a t i on S i m i l a r c l us t e r i ng of S l a ye r p r od uc i ng s t r a i ns of B ac i l l us c e r e us ha s be e n obs e r ve d, a nd, s i m i l a r t o t he s e r e s ul t s s t r a i ns i n t hi s c l us t e r do not pos s e s s S l a ye r s w hi l e ot he r s do ( M i gnot e t a l 2001) T he s e a ut ho r s c onc l ude d t ha t e c ol ogi c a l pr e s s ur e i s a s s oc i a t e d w i t h t he a c qui s i t i on a nd m a i nt e na nc e of S l a ye r s i n hos t s t ha t f a l l i nt o a phyl oge ne t i c c l us t e r P hyl oge ne t i c a l l y, S t r a i n C S C 1 i s m os t c l os e l y r e l a t e d t o M e t hy l oc y s t i s s p. I t s c e l l s i z e r os e t t e f or m a t i on a nd pr e s e nc e of s ur f a c e l a y e r s a r e m os t s i m i l a r t o M e t hy l oc y s t i s e c hi noi de s H ow e ve r S t r a i n C S C 1 s how e d onl y 3 8% s i m i l a r i t y w i t h M e t hy l oc y s t i s e c hi noi de s by D N A D N A hybr i di z a t i on, a nd t he s e t w o s t r a i ns s how e d di f f e r e nc e s i n s ur f a c e l a ye r m or phol ogy c e l l s ha pe c ol ony c ol or f o r m a t i on o f pol yphos pha t e a nd a bi l i t y t o us e L or n i t hi ne or put r e s c i ne a s a ni t r oge n s our c e C ha r a c t e r i s t i c s of c e l l s ha pe a nd t he pr e s e nc e of s ur f a c e l a ye r s ge ne s e nc odi ng f or s M M O e xpr e s s i on, a nd a bi l i t y t o

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71 us e L l ys i ne a s a ni t r oge n s our c e a r e di ve r ge nt f r o m t hos e of M e t hy l oc y s t i s par v us T he l a c k of pol a r f l a ge l l a s m a l l e r c e l l s i z e di f f e r e nt c e l l s ha pe l a c k of he a t r e s i s t a nc e pr e s e nc e of pol yph os pha t e a bi l i t y t o us e L a s pa r a gi ne L a s pa r t a t e or L gl ut a m i ne a nd i na bi l i t y t o us e L l ys i ne a s a n i t r oge n s our c e di f f e r e nt i a t e S t r a i n C S C 1 f r om M e t hy l os i nus t r i c hos por i um A c c e pt i ng t he s e di f f e r e nc e s S t r a i n C S C 1 c oul d be de s c r i be d a s a ne w s pe c i e s i n t he M e t hy l oc y s t i s ge nus W e pr opos e d t hi s s pe c i e s be na m e M e t hy l oc y s t i s al dr i c hi i s p. nov. D e s c r i p t i on of M e t h y l oc y s t i s al dr i c h i i s p n ov. M e t hy l oc y s t i s al dr i c hi i s p. nov. ( a l d r i c h i i M L g e n. N al dr i c hi i of A l dr i c h; na m e d a f t e r H C A l dr i c h a n A m e r i c a n m i c r obi ol ogi s t de c e a s e d A ugus t 9, 2005) C e l l s a r e a e r obi c gr a m ne ga t i ve 0 3 0 6 x 0. 7 1 m i n s i z e t ha t oc c ur s i ngl y o r i n c l us t e r s R e pr oduc e s by nor m a l c e l l di vi s i on. B ud di ng di vi s i on doe s not oc c ur C e l l s a r e not m ot i l e but pos s e s s a s pi n e y s ur f a c e l a ye r c o m pos e d of pol ys a c c ha r i de P r oduc e s oxi da s e a nd c a t a l a s e F or m s l i pi d c ys t s P ol y & h ydr oxybut yr a t e a c c um ul a t e s C ont a i ns T ype I I i nt r a c yt opl a s m i c m e m br a ne s w hi c h a r e a l i gne d pa r a l l e l t o t he c e l l w a l l T ype I I m e t ha not r oph. M e t ha ne a nd m e t ha nol a r e t he s ol e s our c e s of c a r bon a nd e ne r gy. C a pa bl e of us i ng K N O 3 L a s pa r a gi ne L a s pa r t a t e L gl ut a m i ne L or ni t hi ne a nd put r e s c i ne a s ni t r oge n s our c e s C a pa bl e of f i xi ng a t m os phe r i c ni t r oge n. E xpr e s s e s s M M O unde r l ow c oppe r c onc e nt r a t i ons C a pa bl e of c om e t a bol i c a l l y oxi di z i ng a va r i e t y of a l i pha t i c a nd a r om a t i c c om pounds N ot r e s i s t a nt t o pa s t e ur i z a t i on. I s not l ys e d by 2% ( w / v) S D S I s l ys e d by 10% ( w / v) S D S C ol oni e s a r e w hi t e / ye l l ow s l ow g r ow i ng a nd 0. 8 1. 5 m m i n di a m e t e r a f t e r 17 18 d a ys a t 30 C o n N M S a ga r pl a t e s i nc uba t e d i n t he pr e s e nc e of 20% by vol um e m e t ha ne i n t he he a ds pa c e of a s e a l e d de s i c c a t or N o gr ow t h

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72 on c om pl e x or ga ni c m e di a O pt i m a l pH f o r gr ow t h i s 7. 0; doe s not g r ow a t pH 4. 0 or 9. 0. I s no t c a pa bl e of gr ow t h a t 37 C O pt i m a l t e m pe r a t ur e f o r gr ow t h i s a ppr oxi m a t e l y 30 C T he t ype s t r a i n S t r a i n C S C 1 ( A T C C B A A 1344) w a s i s ol a t e d f r om a n unc ont a m i na t e d gr oundw a t e r a qui f e r i n t he m i d 19 80s f r om M of f e t t N a va l A i r S t a t i on i n M ount a i n V i e w C A U S A

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73 F i gur e 2 1 16 S r R N A phyl oge ny of S t r a i n C S C 1 a nd r e l a t e d M e t hy l os i nus a nd M e t hy l oc y s t i s s pe c i e s N um be r s a t br a nc h po i nt s r e pr e s e nt boot s t r a p va l ue s ba s e d on 100 r e pl i c a t e s

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74 F i gur e 2 2 F unc t i ona l ge ne s phyl oge ni e s of S t r a i n C S C 1. ( a ) P hyl o ge ne t i c t r e e of S t r a i n C S C 1 s ol ubl e m e t ha ne m onooxyge na s e ( s M M O ) ge ne s e que nc e ( b) P hyl oge ne t i c t r e e of S t r a i n C S C 1 m e t ha nol de hyd r oge na s e ( M D H ) ge ne s e que nc e ( c ) P hyl oge ne t i c t r e e of S t r a i n C S C 1 pa r t i c ul a t e m e t ha ne m onooxyge na s e ( pM M O ) ge ne s e que nc e N u m be r s a t br a nc h poi nt s r e pr e s e nt boot s t r a p va l ue s ba s e d on 100 r e pl i c a t e s

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75 T a bl e 2 1 P he not ypi c c ha r a c t e r i s t i c s di f f e r e nt i a t i n g S t r a i n C S C 1 f r om M e t hy l os i nus t r i c hos por i um M e t hy l oc y s t i s e c hi noi de s a nd M e t hy l oc y s t i s par v u s 1 C ha r a c t e r i s t i c S t r a i n C S C 1 M t r i c hos por i um M e c hi noi de s M par v us C ol ony m or phol ogy: C ol or Y e l l ow / w hi t e W hi t e / buf f W hi t e / pa l e pi nk W hi t e / pa l e pi nk/ t a n C e l l m or phol ogy: W i dt h ( m ) 0. 3 0. 6 0. 5 1. 5 0. 6 0. 8 0. 3 0. 5 L e ngt h ( m ) 0. 7 1 2 3 0. 8 1. 2 0. 5 1. 5 S ha pe D um bbe l l R ods P yr i f or m P e a r s ha pe O voi d P e a r s ha pe O voi d S l a ye r s S ha r p, s ol i d s pi ne s B e a d l i ke / f i l a m e nt ous T ubul a r s pi ne s P ol yphos pha t e + + P ol y & hydr oxybut yr a t e + + + + M ot i l i t y P ol a r f l a ge l l a R os e t t e s + H e a t r e s i s t a nc e + 2% ( w / v) S D S l ys e d G r ow t h a t 37 o C + + + N 2 s our c e a nd us e : L a s pa r a gi ne + + + # L a s pa r t a t e + + + # L gl ut a m i ne + + + # L l ys i ne + + # L or ni t h i ne + + + # P ut r e s c i ne + + + # N M S no N 2 s our c e + + + + C s our c e a nd us e : M e t hyl a m i ne D i m e t hyl s ul f i de M e t ha nol + + + + G l uc os e 1 Refer en ces : W h i t t en b u ry 1 9 7 0 ; G al ch en k o et al 1 9 7 7 ; Fas s el et al 1 9 9 0 Fas s el et al 1 9 9 2 ; H en ry an d G rb i G al i 1 9 9 1 ; Bo w m an et al 1 9 9 3 a; H an s o n an d H an s o n 1 9 9 6 ; H r $ ak an d Beg o n j a, 1 9 9 8 T h i s s t u d y G al ch en k o et al (1 9 7 7 ) rep o rt ed t h at M e ch i n o i d es fo rms l i p i d cy s t s an d d o es g ro w t o a l i mi t ed ex t en t at 3 7 o C. Bo w m an et al (1 9 9 3 a ), h o w ev er rep o rt ed t h at t h i s s t rai n d o es n o t accu mu l at e p o l y & h y d ro x y b u t y rat e an d o n l y 0 1 0 % o f t h e s t rai n s t es t ed g re w at 3 7 o C. # Rep o rt ed b y Bo w man et al (1 9 9 3 a ) as 7 5 8 7 % o f t h e s t rai n s w e re p o s i t i v e.

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76 F i gur e 2 3 T r a ns m i s s i on e l e c t r on m i c r os c opy pho t ogr a phs of S t r a i n C S C 1 a nd M e t hy l oc y s t i s e c hi noi de s P a ne l s ( a ) a nd ( b) s how t he m or phol ogy of c e l l s o f S t r a i n C S C 1 gr ow n w i t h 10 M C u. I n pa ne l ( b) num e r ous l a m e l l a e ( L a ) a r e pr e s e nt L i pi d i nc l us i ons ( L i ) a nd pol yphos p ha t e ( P ) s t or a ge i nc l us i ons a r e a l s o pr e s e nt S i ndi c a t e s a s ur f a c e vi e w o f t he s pi ny s ur f a c e of a c e l l C e l l s of M e t hy l oc y s t i s e c hi noi de s vi e w e d w i t h ne ga t i ve s t a i n ( pa ne l s ( c ) a nd ( d ) ) a r e e l l i pt i c a l i n p r of i l e a nd ha ve num e r ous t ubul a r pr o j e c t i ons f r o m t he s ur f a c e S om e m a y be s e e n i n c i r c ul a r e nd on p r of i l e a t t he uppe r r i ght of pa ne l ( c ) T he hi gh m a gni f i c a t i on vi e w i n pa ne l ( d) s how s t ha t t he t ube s a r e s t r i a t e d M a r ke r s i n pa ne l s ( a ) a nd ( b) i ndi c a t e 1 m ; i n pa n e l ( c ) 0. 5 m ; i n pa ne l ( d) 0. 1 m

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77 F i gur e 2 4 E l e c t r on m i c r os c ope c yt oc he m i s t r y o f t he S l a ye r of S t r a i n C S C 1 P a ne l ( a ) s how s t he s ur f a c e of a c e l l f i xe d i n i t i a l l y w i t h gl ut a r a l de hyde a l one S pi ny l a ye r i s i ndi s t i nc t a nd l i ght l y s t a i ne d. P a ne l ( b) s h ow s t he s ur f a c e of a c e l l f i xe d i ni t i a l l y w i t h a gl ut a r a l de hyde / A l c i a n bl ue m i xt ur e t o s e l e c t i ve l y s t a i n pol ys a c c ha r i de C om pa r e d t o pa ne l ( a ) t he s pi ny l a ye r s t a i ns da r ke r a nd i s t hi c ke r a nd i s m o r e di s t i nc t P a ne l s ( c ) a nd ( d) s how c e l l s a f t e r p r ona s e di ge s t i on. I n pa ne l ( c ) a c r os s s e c t i on, a l i ght l a ye r a r ound t he c e l l ha s be e n l e f t w he r e t he pr ona s e r e m ove d t he pr ot e i n i n t he S l a ye r I n pa ne l ( d ) a gr a z i ng s e c t i on of t he s pi ne s a t t he c e l l s ur f a c e nu m e r ous l i ght s pot s i n t he pl a s t i c s how w he r e t he pr ona s e r e m ove d t he s p i ke s M a r ke r s r e pr e s e nt 0. 2 m

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78 C H A P T E R 3 E F F E C T S O F A L P H A P I N E N E A N D T R I C H L O R O E T H Y L E N E O N O X I D A T I O N P O T E N T I A L S O F M E T H A N O T R O P H I C B A C T E R I A N ot e : P ubl i s he d m anus c r i pt ( P ac he c o and L i ndne r 2005) P a c he c o, A a nd L i ndne r A S ( 2005) E f f e c t s of a l pha pi ne ne a nd t r i c hl or oe t hyl e ne on oxi da t i on pot e nt i a l s of m e t ha not r ophi c ba c t e r i a B ul l e t i n of E nv i r onm e nt al C ont am i nat i on and T ox i c ol ogy 74 : 133 140. I n t r od u c t i on T r i c hl or oe t hyl e ne ( T C E ) a w i de l y us e d s ol ve nt no t a bl e f or i t s de gr e a s i ng pr ope r t i e s i s a c om m on e nvi r on m e nt a l c ont a m i na nt t ha t pos e s s i gni f i c a nt r i s k t o publ i c he a l t h ( ( A T S D R ) 1999) T C E ha s be e n s how n t o be e f f e c t i ve l y r e m ove d f r om s oi l a nd w a t e r by phyt or e m e di a t i on o f t e n f a vo r e d ove r ot h e r m e t hods be c a us e of i t s e f f e c t i ve ne s s l ow c os t a nd a e s t he t i c be ne f i t s M o r e r a pi d T C E r e m ova l ha s be e n obs e r ve d i n t he r oo t z one o f pl a nt s ( r hi z os phe r e ) u s e d i n phyt or e m e di a t i on ( W a l t on a nd A nde r s on, 1990; A nde r s on a nd W a l t on, 1995; B r i gm on e t a l 1999 ) a nd m e t ha not r o phs m e t ha ne oxi di z i ng ba c t e r i a t ha t t hr i ve on oxyge n a nd m e t ha ne a nd a r e c a pa bl e o f c o oxi di z i ng T C E ( W i l s on a nd W i l s on, 1985; L i t t l e e t a l 1988) ha ve be e n i m pl i c a t e d i n t hi s i nc r e a s e d a c t i vi t y ( B r i gm on e t a l 1999) L obl ol l y pi ne s ( P i nus t ae da ) s how n t o s uppor t l a r ge r hi z os phe r e popul a t i ons of m e t ha not r ophs ( B r i gm on e t a l 1999 ) ha ve be e n c ons i de r e d f or T C E r e m e di a t i on. T he s e t r e e s pr oduc e a nd r e l e a s e s i gni f i c a nt qua nt i t i e s o f m onot e r pe ne s t he m os t p r e dom i na nt be i ng ( R ) pi ne ne c om pos i ng ove r 65% of t he t o t a l ol e or e s i n c om pos i t i on i n di f f e r e nt pl a nt t i s s ue s ( P hi l l i ps e t a l 1999) S i nc e c onc e nt r a t i ons of ( R ) pi ne ne ha ve be e n obs e r ve d t o be a s hi gh a s 1 4 m g g 1 i n f r e s h l i t t e r l a ye r s of pi ne f or e s t s oi l s ( W hi t e

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79 1994) t he pr oba bi l i t y t ha t s oi l m i c r oor ga ni s m s e nc ount e r t he s e c om pounds i n na t ur e i s hi gh. P r e vi ous s t udi e s ha ve s how n t ha t ( R ) pi ne ne ha s a c onc e nt r a t i on de pe nde nt i nhi bi t or y e f f e c t on m e t ha ne oxi da t i on by m e t ha no t r ophs ( A m a r a l a nd K now l e s 1997; A m a r a l e t a l 1998 ; A m a r a l a nd K now l e s 1998 ) a nd, t hus m a y i m pa c t no t on l y t he gr ow t h of t he s e ba c t e r i a i n t he r hi z os phe r e but a l s o t he i r a bi l i t y t o c o oxi di z e T C E W hi l e m e t ha not r ophs w e r e s how n t o r e ga i n m e t ha ne oxi da t i on a c t i vi t y one t o t hr e e da ys a f t e r e xpos ur e t o ( R ) p i ne ne ( A m a r a l e t a l 1998 ) t he i m pl i c a t i ons of t he l ong t e r m pr e s e nc e of t hi s m onot e r pe ne on m e t ha not r ophi c a c t i vi t y i n t he r hi z os phe r e i n pa r t i c ul a r c onc e nt r a t i on e f f e c t s of t hi s c he m i c a l a nd i t s i nf l ue nc e on T C E r e m ova l po t e nt i a l s a r e not c l e a r T o t hi s e nd t hi s s t udy s ought t o f i r s t a s s e s s t he a bi l i t y of r e pr e s e nt a t i ve T ype I I I a nd X m e t ha not r ophs gr oupe d by t he i r di f f e r e nc e s i n c a r bon a s s i m i l a t i on pa t hw a ys i nt r a c yt opl a s m i c m e m br a ne s t r uc t ur e s f a t t y a c i d c a r bon l e ngt hs a nd phyl oge ny ( B ow m a n e t a l 1993a ) t o oxi di z e ( R ) pi ne ne o ve r a r a nge o f c onc e nt r a t i ons us i ng oxyge n upt a ke a na l ys i s S e c ondl y, t hi s s t udy s oug ht t o ga i n a be t t e r pr e l i m i na r y unde r s t a ndi ng of t he va r i a t i on i n oxyge n upt a ke r e s pons e s t o m i xt ur e s of ( R ) pi ne ne a nd T C E by r e pr e s e nt a t i ve m e t ha not r ophs t hus ul t i m a t e l y pr ovi di ng i ns i ght i nt o t he e f f e c t of ( R ) pi ne ne on T C E oxi da t i on pot e nt i a l s of t he s e ba c t e r i a a nd gui da nc e f o r t he phyt or e m e di a t i on pr a c t i t i one r t o m or e a c c ur a t e l y p r e di c t t he e xt e nt o f T C E r hi z ode gr a da t i on w he n us i ng m onot e r pe ne r e l e a s i ng pl a nt s W e r e por t he r e i n obs e r va t i ons of t he pot e nt i a l of m e t ha not r ophs t o o xi di z e ( R ) pi ne ne ove r a br oa d r a nge of c onc e nt r a t i ons a nd ( R ) pi ne ne / T C E m i xt ur e e f f e c t s on m e t ha not r ophi c oxyge n upt a ke a c t i vi t y.

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80 M at e r i al s an d M e t h od s M e t ha not r oph s t r a i ns us e d i n t hi s s t udy i nc l ude d T ype I M e t hy l om i c r obi um al bum B G 8 ( A T C C 33003) a nd T ype I I M e t hy l os i nus t r i c hos por i um O B 3b ( A T C C 35070) obt a i ne d f r om D r J e r e m y S e m r a u ( U ni ve r s i t y of M i c hi ga n, A nn A r bor M I U S A ) a nd T ype X M e t hy l oc oc c us c aps ul at us ( B a t h) ( A T C C 33009) pu r c ha s e d f r om t he A m e r i c a n T ype C ul t ur e C ol l e c t i on ( M a na s s a s V A U S A ) C ul t ur e s w e r e gr ow n i n ni t r a t e m i ne r a l s a l t s ( N M S ) m e di um ( W hi t t e nbur y e t a l 1970 ) w i t h or w i t hout 10 M C u( N O 3 ) 2 t o pr ovi de c ondi t i ons f o r e xpr e s s i on of pM M O or s M M O r e s pe c t i ve l y. W i t h t he e xc e pt i on of M c aps ul at us ( B a t h) i nc u ba t e d a t 45 o C w i t h 5 0% m e t ha ne ( 99 99% pur e S t r a t e W e l di ng, J a c ks onvi l l e F L U S A ) i n t he he a ds pa c e a l l or ga ni s m s w e r e r out i ne l y s ubc ul t ur e d i n s e a l e d e r l e nm e ye r f l a s ks c ont a i ni ng 20% m e t ha ne i n t he he a ds pa c e a nd i nc uba t e d a t 30 o C i n a r ot a r y s ha ke r a t 250 r p m a s pr e vi ous l y de s c r i be d ( L i ndne r e t a l 2000) P ur i t y of t he c ul t u r e s w a s ve r i f i e d by r out i ne s t r e a ki ng on 2% ( w / v ) nut r i e nt a ga r pl a t e s ( D i f c o, S pa r ks M D U S A ) E xpr e s s i on of s M M O w a s qua l i t a t i ve l y ve r i f i e d by a na pht ha l e ne a s s a y m odi f i e d f r om B r us s e a u e t a l ( 1990) a nd de s c r i be d by L i ndne r e t a l ( 2000) O xyge n upt a ke a na l ys i s w a s pe r f or m e d i n t hi s s t ud y, a s i t ha s be e n s how n t o be a r a pi d, e f f e c t i ve m e a ns of a s s e s s i ng oxi da t i ve pot e nt i a l of w hol e c e l l s ( L i ndne r e t a l 2000; L i ndne r e t a l 2003) ( R ) # p i ne ne w a s c hos e n t o r e pr e s e nt m onot e r pe ne s be c a us e i t i s a m a j o r c om pone nt o f l obl o l l y pi ne ol e or e s i n ( P hi l l i ps e t a l 1999) ( R ) # pi ne ne a nd T C E w e r e obt a i ne d i n t he hi ghe s t pu r i t y a va i l a bl e f r om A l dr i c h C he m i c a l C o. ( M i l w a uke e W I U S A ) S t a nda r d s ol ut i ons o f 10 m ol m l 1 w e r e p r e pa r e d i n 1, 4 di oxa ne ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) us e d a s t he c a r r i e r s ol ve nt be c a us e i t e a s i l y s ol ubi l i z e d t he s ubs t r a t e s w a s not oxi di z e d by a ny of t he c ul t ur e s s t udi e d, a nd

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81 c a us e d no pr obe e f f e c t s dur i ng oxyge n upt a ke a na l ys i s ( L i ndne r e t a l 2000 ) R e s t i ng c e l l s us pe ns i on s w e r e pr e pa r e d f r om 500 m l c ul t ur e s ha r ve s t e d a t % l og pha s e by c e nt r i f uga t i on i n a J 2 H S B e c km a n f l oor m ode l c e nt r i f uge ( B e c km a n C oul t e r F u l l e r t on, C A U S A ) a t 2460 x g 4 o C f or 20 m i n. T o e ns ur e r e m ova l o f a l l m e t ha ne t he c e l l s w e r e w a s he d w i t h N M S m e di um r e c e nt r i f uge d a nd r e s us pe nde d i n t he N M S m e di um t o a w e t c e l l c onc e nt r a t i on of 0. 2 g m l 1 T he oxyge n upt a k e s ys t e m w a s c om pos e d of a 1. 9 m l w e l l s t i r r e d, e nc l os e d r e a c t or he l d a t r oom t e m pe r a t ur e a s de s c r i be d by L i ndne r e t a l ( 2000) A f t e r a s s e s s i ng t he a bi l i t y of m e t ha not r op hs t o oxi di z e T C E a nd ( R ) # pi ne ne a l one t he s t udy p r oc e e de d t o i nve s t i ga t e t he e f f e c t o f ( R ) # pi ne ne on T C E oxi da t i on by a ddi ng bot h s ubs t r a t e s s i m ul t a ne ous l y i nt o t he oxy ge n upt a ke s ys t e m be f or e a ddi t i on of t he r e s t i ng c e l l s D e s pi t e s t or a ge of t he r e s us pe nde d c e l l s on i c e t hr oughout t he oxyge n up t a ke e xpe r i m e nt s l os s of c e l l a c t i vi t y ove r t i m e w a s obs e r ve d. T o e ns ur e c om pa r a bi l i t y o f m e a s ur e m e nt s t hr oughout t he 2 3 da y t e s t i ng pe r i o d, a l l r a t e s of oxyge n upt a ke w e r e nor m a l i z e d t o t he r a t e s obs e r ve d w i t h 4 m l of m e t h a ne ga s m e a s ur e d j us t pr i o r t o a c ha nge t o a ne w s ubs t r a t e c onc e nt r a t i on D e t a i l s o f t hi s nor m a l i z a t i on pr oc e dur e a r e pr e s e nt e d i n L i ndne r e t a l ( 2000) T he e l e c t r ode w a s c a l i br a t e d a t l e a s t da i l y w i t h a s a t ur a t e d s odi um s ul f i t e s ol ut i on, a nd l i ve r uns w e r e pe r f or m e d a t l e a s t i n t r i pl i c a t e f or e a c h c onc e nt r a t i on t e s t e d. A l l r uns w e r e c or r e c t e d f or e ndoge nous m e t a bol i s m C ont r ol s w i t hout c e l l s a nd w i t h 4 m l of a c e t yl e ne g a s a know n i nhi bi t or o f M M O ( P r i or a nd D a l t on, 1985 ) w e r e r out i ne l y r un t o ve r i f y t ha t de pl e t i on of oxyge n, he nc e oxi da t i on a c t i vi t y, w a s a r e s ul t of M M O a c t i vi t y. I ni t i a l r a t e s of oxyge n upt a ke w e r e c a l c ul a t e d by

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82 l i ne a r or pol ynom i a l f i t s t o t he da t a poi nt s us i ng M i c r os of t E xc e l s of t w a r e ( M i c r os of t C or p. R e d m ond, W A U S A ) R e s u l t s an d D i s c u s s i on M t r i c hos por i um O B 3b a nd M c aps ul at us ( B a t h) w he n c ul t ur e d w i t h no c oppe r e xpr e s s e d pos i t i ve s M M O a c t i vi t y, a s e vi de nc e d by a br i ght pi nk t o pur pl i s h c ol o r i n t he a s s a y. A l l of t he s t r a i ns t e s t e d ne ga t i ve f o r s M M O a c t i vi t y ( no c ol or c ha nge obs e r ve d) w he n c ul t ur e d w i t h 10 M C u( N O 3 ) 2 s M M O a nd pM M O e xpr e s s i on unde r c ul t ur i ng c ondi t i ons w i t hout a nd w i t h c oppe r r e s pe c t i ve l y, w a s t hus a s s um e d, a r e a s ona bl e c onc l us i on gi ve n t ha t e nz ym e e xpr e s s i on i n t he s e m e t ha not r op hs unde r t he s e c ondi t i ons i s w e l l c ha r a c t e r i z e d. A c t i ve r e s t i ng c e l l s o f a l l t hr e e r e pr e s e nt a t i ve m e t ha not r ophs c ons um e d oxyge n ove r a r a nge of T C E a nd ( R ) # pi ne ne c onc e nt r a t i ons r e ga r dl e s s of t he t ype of M M O e xpr e s s e d ( F i g. 3 1 A E ) N o oxyg e n upt a ke w a s obs e r ve d a f t e r a ddi t i on of a c e t yl e ne or w i t hout c e l l s pr e s e nt ve r i f yi ng M M O a c t i vi t y i n a l l c a s e s A s s how n i n F i gur e 3 1 r e ga r dl e s s of t he m e t ha n ot r oph o r s ubs t r a t e t e s t e d, a m a xi m um r a t e of oxyge n upt a ke w a s obs e r ve d, f ol l ow e d by a r a pi d de c r e a s e i n r a t e s s ugge s t i ng t oxi c e f f e c t s o f e i t he r t he s ubs t r a t e i t s e l f o r o f oxi da t i on pr oduc t s f or m e d. T hi s oxyge n upt a ke be ha vi or ha s be e n r e por t e d pr e vi ou s l y f or m e t ha not r ophs w i t h a r om a t i c s ubs t r a t e s ( L i ndne r e t a l 2000) a nd w hi l e bot h s ub s t r a t e s ha ve be e n s how n t o ha ve t oxi c e f f e c t s on m e t ha not r ophi c a c t i vi t y ( F ox e t a l 1990; A l va r e z C ohe n a nd M c C a r t y, 1991a ; H e nr y a nd G r bi G a l i 1991; O l de nhui s e t a l 1991; W h i t e 1994; A m a r a l a nd K now l e s 1997; A m a r a l e t a l 1998 ; A m a r a l a nd K now l e s 1998 ) t he r e ha ve be e n no p r e vi ous r e por t s on t he e f f e c t s of a r a ng e o f s ubs t r a t e c onc e nt r a t i ons o n r e l a t i ve a c t i vi t i e s A s s how n i n F i gur e 3 1, m e t ha not r ophs e xpr e s s i ng s M M O ( pl ot s A C ) oxi di z e d T C E a t hi ghe r m a xi m u m r a t e s t ha n t hos e e xpr e s s i ng pM M O ( pl ot s B D E ) a s

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83 pr e vi ous l y r e por t e d ( L i t t l e e t a l 1988; D i S pi r i t o e t a l 1992; L ont oh a nd S e m r a u 1998 ) T he m a xi m um nor m a l i z e d r a t e s of oxi da t i on by M t r i c hos por i um O B 3b a nd M c aps ul at us ( B a t h) e xpr e s s i ng s M M O or pM M O w e r e 0. 11 0. 01 a nd 0. 03 0. 00 a nd 0. 05 0 01 a nd 0 03 0. 01 r e s pe c t i ve l y ( F i g. 3 1 A D ) w h i l e t he m a xi m um r a t e e xpr e s s e d by M al bum B G 8, c a pa bl e o f pM M O e xpr e s s i on onl y, w a s 0. 05 0. 01 ( F i g. 3 1, E ) T he T C E c onc e nt r a t i ons w he r e t he obs e r ve d nor m a l i z e d oxyge n up t a ke r a t e w a s t he hi ghe s t r a nge d f r om 20 t o 35 ppm f or t he t e s t e d s t r a i ns M t r i c hos por i um O B 3b a nd M c aps ul at us ( B a t h) e xpr e s s i ng s M M O e xhi bi t e d oxyge n upt a ke m a xi m a a t h i ghe r T C E c onc e nt r a t i ons ( 35 ppm ) t ha n w he n e xp r e s s i ng pM M O ( 20 25 ppm ) a nd M al bum B G 8 e xpr e s s i ng pM M O s how e d a m a xi m um obs e r ve d r a t e a t 35 ppm T C E T he s e r e s u l t s do s ugge s t di f f e r i ng s e ns i t i vi t y l e ve l s t o T C E de pe nd i ng on t he m e t ha not r oph a nd t ype of M M O e xpr e s s i on. A s obs e r ve d w i t h T C E bot h s M M O e xpr e s s i ng m e t ha not r ophs w e r e a l s o c a pa bl e of oxi di z i ng ( R ) pi ne ne a t hi ghe r r a t e s t ha n t he i r pM M O e xpr e s s i ng c o unt e r pa r t s ( F i g. 3 1, A D ) T he m a xi m um nor m a l i z e d r a t e of oxyg e n upt a ke by M t r i c hos por i um O B 3b e xpr e s s i ng s M M O w a s a l m os t 10 t i m e s t he r a t e ob s e r ve d w i t h pM M O e xpr e s s i ng c e l l s ( 0. 28 0 04 a nd 0. 02 0. 01 r e s pe c t i ve l y) ; how e v e r bot h r a t e m a xi m a oc c ur r e d a t 20 ppm ( R ) pi ne ne ( F i g 3 1 A B ) T he m a xi m um nor m a l i z e d oxyge n up t a ke r a t e w i t h M c aps ul at us ( B a t h) e xpr e s s i ng s M M O w a s 0. 10 + 0. 02 a t 20 ppm ( R ) pi ne ne c om pa r e d t o 0 08 + 0 01 a t 50 ppm ( R ) p i ne ne u nde r pM M O e xpr e s s i on ( F i g. 3 1, C D ) T he obs e r ve d m a xi m um no r m a l i z e d r a t e of ox yge n upt a ke by M al bum B G 8 w a s 0. 04 0 00, be t w e e n t he va l ue s obs e r ve d f or t he o t he r t w o s t r a i ns unde r pM M O e xpr e s s i on ( F i g. 3 1E ) P r e vi ous s t udi e s ha ve r e po r t e d hi ghe r T C E oxi da t i on r a t e s by

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84 pur e m e t ha not r ophs unde r s M M O e x pr e s s i on ( W i l s on a nd W i l s on, 1985; L i t t l e e t a l 1988; D i S pi r i t o e t a l 1992; L ont oh a nd S e m r a u 1 998) ; how e ve r t hi s i s t he f i r s t r e por t of s uc h a t r e nd w i t h ( R ) pi ne ne T he s e r e s ul t s br i ng di r e c t r e l e va nc e t o t he e nvi r onm e nt a s s M M O e xpr e s s i on i n m e t ha not r op hs oc c ur s onl y a t ve r y l ow c oppe r c onc e nt r a t i ons ( L ont oh a nd S e m r a u 1998) M e a s ur e m e nt o f bi oa va i l a bl e c oppe r i s e s s e nt i a l t he r e f or e f or e f f e c t i ve p r e di c t i on of m e t ha not r ophi c a c t i vi t y pot e nt i a l T he r e s pons e of e a c h m e t ha not r oph i n t he pr e s e nc e of 20 ppm T C E ove r a r a nge of ( R ) pi ne ne c onc e nt r a t i ons i s s how n i n F i gur e 3 2, A E T hi s pl ot pr e s e nt s t he c ha nge i n nor m a l i z e d oxyge n upt a ke r a t e w i t h 20 ppm T C E a l one c a us e d by t he pr e s e nc e of di f f e r e nt c onc e nt r a t i ons o f ( R ) pi ne ne a nd t hus r e pr e s e nt s t he i nf l ue nc e of ( R ) # pi ne ne on T C E oxi da t i on a nd pr ovi de s i ns i ght i nt o m i xt ur e e f f e c t s on m e t ha not r oph a c t i vi t y. T he c onc e nt r a t i on of 20 ppm T C E w a s c hos e n be c a us e i t w a s not obs e r ve d t o be t oxi c t o a ny o f t he m e t ha not r ophs t e s t e d pr e vi ous l y ( F i g 3 1) T he r e s pons e s t o ( R ) pi ne ne w e r e hi ghl y de pe nd e nt on t he t ype of m e t ha not r oph a nd M M O e xpr e s s i on, w i t h M t r i c hos por i um O B 3b s how i ng de c r e a s e d r a t e s r e l a t i ve t o 20 ppm T C E a l one r e ga r dl e s s of ( R ) pi ne ne c on c e nt r a t i on ( F i g 3 2, A B ) a nd M c aps ul at us ( B a t h) a nd M al bum B G 8 s how i ng m o s t l y i nc r e a s e d r a t e s ( F i g. 3 2 C D E ) W i t h t he e xc e pt i on of M c aps ul at us ( B a t h) unde r pM M O e xpr e s s i on, t he hi ghe s t obs e r ve d r a t e s i n t he p r e s e nc e of t he m i xt u r e w e r e l ow e r t ha n t hos e obs e r ve d w i t h ( R ) pi ne ne a l one M t r i c hos por i um O B 3b e xpr e s s i ng pM M O s how e d c ons i s t e nt l y s m a l l de c r e a s e s i n oxyge n upt a ke a c t i vi t y i n t he pr e s e nc e of t he m i xt ur e c om pa r e d t o 20 ppm T C E a l one ; how e ve r t he a c t i vi t y o f t hi s s t r a i n w he n e xpr e s s i ng s M M O a ppe a r e d t o be i nhi bi t e d t o a gr e a t e r e xt e nt i n t he pr e s e nc e of a l l t e s t e d c onc e nt r a t i ons ( 2 t o 20 ppm ) of

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85 ( R ) # pi ne ne i n t he m i x t ur e s ( F i g 3 2, A B ) R e g a r dl e s s of M M O e xpr e s s i on, M c aps ul at us ( B a t h) yi e l de d i nc r e a s e d nor m a l i z e d ox yge n upt a ke r a t e s i n t he pr e s e nc e of t he m i xt ur e a bove a ppr oxi m a t e l y 20 ppm ( R ) # pi ne ne r e l a t i ve t o i t s obs e r ve d r a t e a t 20 ppm T C E a l one T he gr e a t e s t r a t e i nc r e a s e s how n by M c aps ul at us ( B a t h) e xpr e s s i ng s M M O i n t he pr e s e nc e of t he m i xt u r e w a s obs e r ve d a t 40 pp m ( R ) # pi ne ne T hi s m a xi m um r a t e obs e r ve d w i t h t he m i xt ur e w a s 1 8 t i m e s t he r a t e w i t h 20 ppm T C E a l one s ugge s t i ng a l e s s oni ng of t oxi c i t y e f f e c t s on t he c e l l s T he m a xi m um i nc r e a s e w i t h t hi s s t r a i n unde r pM M O e xpr e s s i on w a s obs e r ve d a t 30 ppm ( R ) # pi ne ne a nd w a s a ppr oxi m a t e l y 3. 5 t i m e s hi ghe r t ha n w i t h 20 ppm T C E a l one a nd 1. 5 t i m e s hi ghe r t ha n obs e r ve d a t 50 ppm ( R ) p i ne ne a l one I nc r e a s e i n oxi da t i on pot e nt i a l of M al bum B G 8 w a s a l s o obs e r ve d w he n ( R ) # pi ne ne w a s i n t he p r e s e nc e of 20 ppm T C E ( F i g 3 2, E ) A t t he hi ghe s t c onc e nt r a t i on o f ( R ) # pi ne ne t e s t e d ( 30 ppm ) w i t h t hi s s t r a i n t he i nc r e a s e i n nor m a l i z e d oxyge n upt a ke r a t e w a s 1 8 t i m e s t he r a t e obs e r ve d w i t h T C E a l one I n c onc l us i on, a l l of t he t e s t e d m e t ha not r ophs e xp r e s s i ng e i t he r s M M O or pM M O w e r e c a pa bl e of oxi di z i ng ( R ) # pi ne ne ove r a r a n ge of e nvi r on m e nt a l l y r e l e va nt c onc e nt r a t i ons H ow e ve r t ox i c i t y e f f e c t s of t hi s m onot e r pe ne s i m i l a r t o t hos e s how n w i t h T C E w e r e obs e r ve d. W he n bot h ( R ) pi ne ne a nd T C E w e r e i nt r oduc e d t o t he r e pr e s e nt a t i ve m e t ha not r ophs va r yi ng r e s pons e s i n t he r a t e s de c r e a s e s w i t h t he T ype I I m e t ha not r oph a nd i nc r e a s e s w i t h t he T ype s I a nd X m e t ha not r ophs w e r e obs e r ve d i n c om pa r i s on t o t hos e obs e r ve d i n t he T C E onl y e xp e r i m e nt s W he t he r T C E a nd/ or ( R ) pi ne ne w e r e oxi di z e d i n t he m i xt u r e i s not know n, gi ve n t he i ndi r e c t m e a s ur e m e nt m e t hod of oxyge n upt a ke a na l ys i s ; how e ve r i t i s s ugge s t e d he r e t ha t t he t ot a l oxi da t i on pot e nt i a l of m e t ha not r ophs i s a f f e c t e d, e i t he r a nt a goni s t i c a l l y or s yne r gi s t i c a l l y, i n t he

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86 pr e s e nc e of T C E a nd ( R ) p i ne ne m i xt ur e s T he s e r e s ul t s e m pha s i z e t he i m por t a nc e of not onl y a s s e s s i ng t he c onc e nt r a t i on l e ve l s of bot h c ont a m i na nt s a nd m onot e r pe ne s a nd but a l s o of m e a s ur i ng t he oxi da t i on pot e nt i a l s a nd di ve r s i t y of r hi z os phe r e m e t ha not r ophs a t phyt or e m e di a t i on s i t e s w he r e pl a nt s t ha t r e l e a s e l a r ge a m ount s of m onot e r pe ne s a r e be i ng c ont e m pl a t e d f or u s e F i gur e 3 1 N o r m a l i z e d r a t e o f oxyge n upt a ke by t he r e pr e s e nt a t i ve m e t ha not r ophs i n t he pr e s e nc e of va r yi ng c onc e nt r a t i ons o f T C E ( ) a nd ( R ) pi ne ne ( ) ( A ) ( B ) : M t r i c hos por i um O B 3b c ul t ur e d w i t hou t a nd w i t h c oppe r r e s pe c t i ve l y. ( C ) ( D ) : M c aps ul at us ( B a t h) c ul t u r e d w i t hout a n d w i t h C u, r e s pe c t i ve l y. ( E ) : M al bum B G 8 c ul t ur e d w i t h C u. E r r o r ba r s r e pr e s e nt t he s t a nda r d de vi a t i on f or t r i pl i c a t e s a m pl e s

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87 F i gur e 3 2 C ha nge i n t he no r m a l i z e d oxyge n upt a ke r a t e by r e pr e s e nt a t i ve m e t ha not r ophs obs e r ve d i n t he p r e s e nc e of 20 pp m T C E a t va r yi ng c onc e nt r a t i ons of ( R ) pi ne ne ( A ) ( B ) : M t r i c hos por i um O B 3b c ul t ur e d w i t hout a nd w i t h C u r e s pe c t i ve l y. ( C ) ( D ) : M c a ps ul at us ( B a t h) c ul t ur e d w i t hout a nd w i t h C u, r e s pe c t i ve l y. ( E ) : M al bum B G 8 c ul t ur e d w i t h C u E r r o r ba r s r e pr e s e nt e d t he s t a nda r d de vi a t i on f or t r i pl i c a t e s a m pl e s

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88 C H A P T E R 4 S T A B L E I S O T O P E P R O B I N G F O R C H A R A C T E R I Z A T I O N O F M E T H A N O T R O P H I C B A C T E R I A I N T H E R H I Z O S P H E R E O F P H Y T O R E M E D I A T I N G P L A N T S N ot e : M anus c r i pt t o be s ubm i t t e d t o B i ol ogy L e t t e r s I n t r od u c t i on P hyt or e m e di a t i on, t he us e of pl a nt s t o r e m ove a va r i e t y of c ont a m i na nt s f r om s oi l a nd a que ous e nvi r onm e nt s ha s be e n s how n t o be m or e e c onom i c a l a nd a e s t he t i c a l l y pl e a s i ng t ha n t r a di t i ona l r e m e di a t i on m e t hods s uc h a s pum p a nd t r e a t a ppr oa c he s ( M c C ut c he on a nd S c hnoor 2003 ) D e s pi t e i t s obs e r ve d e f f e c t i ve ne s s i n r e m ova l o f c ont a m i na nt s i nc l udi ng t r i c hl or oe t hyl e ne ( T C E ) a nd t e t r a c hl or oe t hyl e ne ( P C E ) t w o w i de l y di s t r i but e d c hl or i na t e d s ol ve nt s t ha t c a us e c onc e r n be c a us e of t he i r pot e nt i a l he a l t h e f f e c t s ( A T S D R 2006) phy t or e m e di a t i on i s s t i l l l i m i t e d by a l a c k o f unde r s t a ndi ng of t he pr i m a r y r e m ova l p r oc e s s e s i nvol ve d. I n pa r t i c ul a r t he pot e nt i a l r ol e s t ha t r oot z one ( r hi z os phe r e ) ba c t e r i a c a n a s s um e i n t he ove r a l l r e m ova l of c ont a m i na nt s i s not f ul l y a ppr e c i a t e d ( W a l t on a nd A nde r s on, 1990; A nde r s on a nd W a l t on, 1995; B r i gm on e t a l 1998; B r i gm on e t a l 1999) O ne r e a s on f or t he l a c k of s pe c i f i c i nf o r m a t i on on t he de gr a da t i on pot e nt i a l s of r oot z one ba c t e r i a i s t ha t t r a di t i ona l c ul t ur e de pe nde nt m e t hods a r e not c a pa bl e of di r e c t l y a s s e s s i ng t he a c t i vi t y a nd di ve r s i t y of m i c r oor ga ni s m s i n s i t u F ur t he r m or e t he s e m e t hods pr ovi de l i m i t e d i nf o r m a t i on be c a us e of t h e i r a s s oc i a t e d i nhe r e nt c ul t i va t i on bi a s ( F r y, 2004; S m a l l a 2004) T he de ve l opm e nt of c u l t ur e i nde pe nde nt m ol e c ul a r m e t hods s uc h a s s t a bl e i s ot ope pr obi ng ( S I P ) ha s e na bl e d s c i e nt i s t s t o s t udy i n s i t u c ondi t i ons

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89 m or e e f f e c t i ve l y a nd, m o r e i m po r t a nt l y, t o c ha r a c t e r i z e t he a c t i ve m i c r obi a l popul a t i ons T he pr om i s i ng S I P t e c hni que r e l i e s on t he i nc or po r a t i on of a l a be l e d s ubs t r a t e w i t h a l e s s na t ur a l l y f r e que nt i s ot ope i n t o t he a c t i ve m i c r obi a l c om m uni t y o f a s a m pl e t ha t l a t e r c a n be s e pa r a t e d f r om t he unl a be l e d bi om a s s ( R a da j e w s ki e t a l 2000) R e c e nt s t udi e s of m e t ha not r ophi c ba c t e r i a ha ve s h ow n s uc c e s s f ul r e s ul t s us i ng t he S I P a ppr oa c h i n e nvi r on m e nt s s uc h a s pe a t s oi l s a c i di c f or e s t s oi l s c a ve w a t e r a nd s oda l a ke s e di m e n t s ( M or r i s e t a l 2002 ; R a da j e w s ki e t a l 2002 ; H ut c he ns e t a l 2004; L i n e t a l 2004 ) M e t ha not r ophs a r e a m ong t he a e r ob i c b a c t e r i a t ha t a r e know n t o r e s i de i n t he r hi z os phe r e of pl a nt s a nd t ha t a r e c a pa bl e of oxi di z i ng c hl or i na t e d c ont a m i na nt s s uc h a s T C E ( W i l s on a nd W i l s on, 1985 ; H a ns on a nd H a ns on, 1996; B r i gm on e t a l 1999; D or oni na e t a l 2004; P i l on S m i t s 2005) B e c a us e m e t ha ne s e r ve s a s t he s ol e s our c e o f c a r bon a nd e ne r gy f or m e t ha not r ophs i t i s o f t e n us e d a s t he m e a s ur e of m e t ha not r oph a c t i vi t y i n t he e nvi r onm e nt a nd i s a na t ur a l s ubs t r a t e f or S I P t e s t i ng. L a be l e d m e t ha ne ( 1 3 C C H 4 ) ha s be e n s uc c e s s f ul l y i nc or por a t e d i nt o t he D N A of g r ow i ng c e l l s o f m e t ha not r ophs ( 1 3 C D N A ) a nd s e pa r a t e d f r om t he na t ur a l l y oc c ur r i ng 1 2 C D N A by de ns i t y gr a di e nt c e nt r i f uga t i on ( R a da j e w s ki e t a l 2000; M or r i s e t a l 2002; R a da j e w s ki e t a l 2002; M c D ona l d e t a l 2005) M ol e c ul a r f i nge r pr i nt i ng t e c hni que s s uc h a s de na t ur i ng gr a di e nt ge l e l e c t r ophor e s i s ( D G G E ) w i t h D N A f r a gm e nt s of s pe c i f i c m e t ha not r oph e nz ym e s s uc h a s pa r t i c ul a t e m e t ha n e m onoxyge na s e ( pM M O ) c a n be s ubs e que nt l y us e d t o i de nt i f y a nd a s s e s s t he r e l a t i ve a bunda nc e of t he a c t i ve popul a t i ons ( M uyz e r e t a l 1993 ) W i t h t he a dve nt o f t hi s s ophi s t i c a t e d m ol e c ul a r b i o l ogy m e t hod t ha t l i nk s i de nt i t y w i t h f unc t i on de ve l opm e nt o f a p r ot oc ol us i ng S I P m e t hods t ha t i s s pe c i f i c t o t he

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90 r hi z os phe r e hol ds pr om i s e i n be t t e r unde r s t a ndi ng t he r hi z ode gr a da t i on p r oc e s s of phyt or e m e di a t i on s ys t e m s T hi s s t udy pr ovi de s a f i r s t ba s i s i n m e t hod de ve l opm e nt a nd a na l ys i s of t he S I P t e c hni que f o r t he m e a s ur e m e nt of pot e nt i a l i n s i t u a c t i vi t y a nd di ve r s i t y of m e t ha not r ophi c ba c t e r i a i n t he r oot z o ne of t r e e s us e d f or r e m e di a t i on of T C E f r om c ont a m i na t e d gr oundw a t e r a nd s oi l M at e r i al s an d M e t h od s S i t e D e s c r i p t i on T hi s s t udy w a s l oc a t e d a t a S upe r f und s i t e t he f or m e r L a S a l l e E l e c t r i c a l U t i l i t i e s i n L a S a l l e I L ( U S A ) T he c om pa ny m a nuf a c t ur e d c a pa c i t or s f r om 1943 t o 1 982, r e s ul t i ng i n s oi l a nd g r oundw a t e r c ont a m i na t i on of m os t l y p ol yc hl or i na t e d bi phe nyl s a nd t he c hl or i na t e d s ol ve nt s T C E a nd P C E C ur r e nt l y i n t he f i na l s t a ge s of t he c l e a nup pr oc e s s a t t he s i t e t w o phyt or e m e di a t i on pl ot s ha ve be e n i m pl e m e nt e d t o e nha nc e c hl or i na t e d s ol ve nt r e m ova l ( L a nge 2004 ) T he f i r s t pl ot ( 0. 2 5 ha ) c ont a m i na t e d w i t h T C E ( 0 254 ppb) w a s i ns t a l l e d i n S e pt e m be r 2002 ( l a be l e d a s T C E S i t e ) P opl a r ( 18 c l one s ) a nd w i l l ow ( 24 c l one s ) ge not ype s w e r e pl a nt e d by l ow e r i ng 1. 8 m r oot e d c ut t i ngs t o t he bot t om of bor e hol e s ( 0 6 m di a m e t e r ) l i ne d w i t h hi gh de ns i t y pol ye t hyl e ne pi pe a nd f i l l e d w i t h a n e qua l m i x of s a nd, s oi l ba r k a nd pe a t ( pH 7. 8) T he s e c ond phyt or e m e di a t i on pl ot ( 0 21 ha ) c ont a m i na t e d w i t h P C E ( 0 838 ppb ) w a s e s t a bl i s he d i n M a r c h 2002 ( l a be l e d a s P C E S i t e ) A t t hi s pl ot popl a r t r e e s w e r e pl a nt e d di r e c t l y i nt o t he i m pr ove d s oi l ( pH 7 3) w i t h m ul c h c om pos e d of t r e e c hi ps o n t he t op 0. 5 m o f t he s oi l s ur f a c e S am p l i n g A l l r hi z os phe r e s oi l s a m pl e s w e r e c ol l e c t e d us i ng a s m a l l di a m e t e r ( 1 9 c m ) ha nd s oi l a uge r t o m i ni m i z e di s t ur ba nc e i n t he pot s S a m pl e s w e r e t a ke n a t t h r e e t i m e pe r i ods J ul y 2003, J ul y 2004 a nd N ove m be r 2004 i n or de r t o c om pa r e s um m e r a nd f a l l

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91 c ondi t i ons a nd t i m e e f f e c t s on t he a c t i vi t y a nd pop ul a t i on di ve r s i t y o f m e t ha not r ophs E a c h s a m pl i ng pe r i od m i d s um m e r ( J ul y) a nd e a r l y f a l l ( N ove m be r ) f e l l i n t he w e t s e a s on of A pr i l t o D e c e m be r w he n 642 m m of pr e c i pi t a t i on w e r e c ol l e c t e d a t t he s i t e dur i ng 2004. T he a ve r a ge da i l y a i r a nd s oi l t e m pe r a t ur e s w e r e 21C a nd 11C r e s pe c t i ve l y, i n t he 2004 s um m e r s a m pl i ng a nd 20 C a nd 9C r e s pe c t i ve l y, i n t he 2004 f a l l s a m pl i ng. G r oundw a t e r l e ve l s be l ow t he p l a nt e d pl ot s f l uc t ua t e d i n 2004 f r om 2. 1 t o 3. 1 m a nd 1. 8 t o 3. 3 m f r om t he s oi l s ur f a c e a t t he T C E a nd P C E S i t e r e s pe c t i ve l y. R oot gr ow t h of t he t r e e s a t t he T C E a nd P C E S i t e w a s obs e r ve d t o e xt e nd f r om t he s ur f a c e t o 90 120 c m be l ow s ur f a c e A c om pos i t e s a m pl e i n r e gi ons o f hi gh c ont a m i na nt c onc e nt r a t i on, w a s t a ke n f r om t w o oppos i t e l oc a t i o ns a r ound t he t r e e ba s e a t a de pt h of 30 60 c m T hi s s oi l l a ye r w a s c hos e n a s a pot e nt i a l z one of i nt e r m e di a c y r h i z os phe r e a c t i vi t y be t w e e n s ur f a c e a nd de e pe r s oi l l a ye r s A t t he T C E S i t e s a m pl e s w e r e a l s o r e m ove d f r om non pl a nt e d pot s i n t he c ont a m i na t e d a r e a t o s e r ve a s a c ont r ol w he n c om pa r e t o t he p l a nt e d pot s A l s o di f f e r e nt t r e e c l one s s how i ng t he gr e a t e s t vi gor w e r e s a m pl e d a t t he T C E S i t e T he y w e r e one popl a r c l o ne I 45/ 51 ( P opul us de l t oi de s x P ni gr a ; or i gi n, N or t h A m e r i c a x E ur ope ) a nd t hr e e w i l l ow c l one s S X 61 ( Sal i x s ac hal i ne ns i s ; or i gi n, J a pa n; e xot i c ) S 365 ( S. di s c ol or 18; or i gi n U ni ve r s i t y of T or on t o) a nd 94014 ( S pur pur e a ; or i gi n S t a t e U ni ve r s i t y o f N e w Y or k; e xot i c ) W hi l e i t i s w e l l know n t ha t m e t ha not r ophs a r e no t c a pa bl e of oxi di z i ng P C E s a m pl e s w e r e r e m ove d f r om t he P C E S i t e t o s e r ve a s a m e a n of c om pa r i ng m e t ha not r oph a c t i vi t y a nd di ve r s i t y w i t h t he T C E S i t e s a m pl e s O ne popl a r c l one I 45/ 51 w a s s a m pl e d a t t he P C E S i t e a l ong w i t h a non pl a nt e d s a m pl e r e m ove d f r om out s i de t he pl ot i n a n

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92 unc ont a m i na t e d r e gi on t ha t w a s e ve nt ua l l y c onve r t e d t o a n i r r i ga t e d, f e r t i l i z e d s oc c e r f i e l d a f t e r t he f i r s t s a m pl i ng i n J ul y 2003 T o pr e ve nt c r os s c ont a m i na t i on, t he s a m pl i ng a ug e r w a s w a s he d w i t h s t e r i l e w a t e r r i ns e d w i t h 95% e t ha nol a nd w a s he d a ga i n be f or e e a c h s a m pl e w a s t a ke n. S a m pl e s w e r e i m m e di a t e l y pl a c e d i n s t e r i l e ba gs ( N a s c o W hi r l P a k, F or t A t ki ns on, W I U S A ) pl a c e d on i c e a nd t r a ns por t e d t o t he U F l a bor a t or y w he r e t he y w e r e s ubs e que nt l y s t or e d a t 4 C unt i l t e s t i ng. I n t he l a bo r a t or y, s a m pl e s w e r e ge nt l y hom oge ni z e d us i ng a s t e r i l e s pa t ul a a nd f i ne r oo t s of l e s s t ha n 2 m m di a m e t e r w e r e s e pa r a t e d f r om t he s oi l f or s e pa r a t e t e s t i ng. S t ab l e I s ot op e P r ob i n g ( S I P ) S oi l M i c r oc os m s E xp e r i m e n t al c on d i t i on s I n or de r t o a s s e s s a c t i vi t y a nd di ve r s i t y of t he a c t i ve m e t ha not r oph popul a t i ons i n popl a r a nd w i l l ow t r e e r hi z os phe r e s dur i ng T C E r e m e di a t i on, s oi l m i c r oc os m s w e r e pr e pa r e d f r om s a m pl e s c ol l e c t e d ove r t i m e M i c r oc os m s c ons i s t e d of 10 g w e t s oi l ( pl a nt m a t e r i a l r e m ove d) nor m a l i z e d t o 16% w a t e r c ont e nt w i t h s t e r i l e w a t e r T hi s w a t e r c ont e nt r e pr e s e nt e d a ppr oxi m a t e l y 40% o f t he f i e l d c a pa c i t y of t he T C E a nd P C E S i t e s oi l s w he r e t he gr e a t e s t e xt e nt of C H 4 oxi da t i on w a s obs e r ve d i n pr e l i m i na r y e xpe r i m e n t s a s pr e vi ous l y de s c r i be d by R e a y e t a l ( 2001 ) T he w e t t e d s oi l w a s pl a c e d i n s t e r i l e 160 m l s e r um v i a l s w hi c h w e r e s ubs e que nt l y s e a l e d w i t h gr a y but y l r ubbe r s t oppe r s a nd c r i m p t ops T e n m l ( 0 4 m m ol ; $ 7% v/ v ) o f f i l t e r s t e r i l i z e d 1 3 C H 4 ( 99. 9 % ; I s ot e c M i a m i s bur g, O H U S A ) or 1 2 C H 4 ( 99 9% ; A i r c o B O C M ur r a y H i l l N J U S A ) us e d i n pr e l i m i na r y e xpe r i m e nt s t o opt i m i z e c ondi t i ons a nd a s s e s s a ny e f f e c t s of t he l a be l e d s ubs t r a t e by c om p a r i ng t o t he 1 3 C H 4 m i c r oc os m s r a t e s w a s t he n a dde d a s pr e vi ous l y de s c r i be d ( M or r i s e t a l 2002 ; R a da j e w s ki e t a l 2002 ) a nd e a c h vi a l w a s w r a ppe d w i t h a l um i nu m f oi l f o r s ub s e que nt i nc uba t i on i n t he da r k a t r oom

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93 t e m pe r a t ur e ( $ 25C ) H e a ds pa c e C H 4 de pl e t i on w a s m oni t or e d e ve r y 2 t o 5 da ys by r e m ova l of 25 ul of C H 4 w i t h a ga s t i ght s yr i nge a n d a na l ys i s us i ng a ga s c hr om a t ogr a ph ( M ode l H P 5809A G C / T C D ; H e w l e t t P a c ka r d, P a l oA l t o, C A U S A ) e qui ppe d w i t h a G S C a r bon pl ot c ol um n ( A gi l e nt T e c hnol ogy, P a l oA l t o, C A U S A ) T he ga s c hr om a t og r a ph w a s m a i nt a i ne d a t a he a d pr e s s ur e of 5 ps i a nd pr o gr a m m e d i n a 4 m i n r un w i t h t e m pe r a t ur e s of 25 120 a nd 200 C i n t he ove n i nj e c t or a nd de t e c t or r e s pe c t i ve l y. W he n m or e t ha n 90 % o f t he C H 4 w a s c ons um e d, v i a l s w e r e ope ne d, ge nt l y f l us he d w i t h f i l t e r s t e r i l i z e d a i r f o r 5 s t o r e m ov e a ny a c c u m ul a t e d 1 3 C O 2 a nd t o m a i nt a i n a e r obi c c ondi t i ons r e s e a l e d, a nd t he s a m e i ni t i a l a m ount o f 1 3 C H 4 a dde d. T he pr oc e dur e w a s r e pe a t e d f i ve t i m e s unt i l a t o t a l of 2. 0 m m ol of C H 4 w a s c ons um e d ( R a da j e w s ki e t a l 2002) A pos i t i ve c ont r ol w i t h pur e m e t ha not r oph M e t hy l oc y s t i s t r i c hos por i um O B 3b, a nd t hr e e ne ga t i ve c ont r ol s w i t h no C H 4 a dde d, w i t h t w i c e a ut oc l a ve d ( ki l l e d) s oi l a nd w i t h 20% ( v/ v) e a c h of C H 4 a nd a c e t yl e ne ( a know n i nhi bi t or of m e t ha ne m onooxyge na s e P r i or a nd D a l t on ( 1985) ) i n t he h e a ds pa c e w e r e a l s o i nc l ude d. A ddi t i ona l l y, s om e o f t he m i c r oc os m s w e r e s e t i n r e pl i c a t e s t o a s s ur e r e pr oduc i bl e r e s ul t s I ni t i a l C H 4 de pl e t i on r a t e s w e r e c a l c ul a t e d f r om da t a t a ke n du r i ng i nc uba t i on a f t e r t he f i r s t C H 4 a ddi t i on by l i ne a r r e gr e s s i on a na l ys i s of t he c ons um pt i on c ur ve D N A e xt r ac t i on an d u l t r ac e n t r i f u ga t i on T he c ont e nt of t he m i c r oc os m s ( 10 g s oi l ) w a s pr oc e s s e d us i ng a P ow e r M a x S oi l D N A E xt r a c t i on K i t ( M o B i o, C a r l s ba d, C A U S A ) D N A e xt r a c t s w e r e r e s ol ve d by C s C l de ns i t y gr a di e nt c e nt r i f uga t i on. B r i e f l y, 1 g m l 1 of C s C l ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) w a s di s s ol ve d i n t he D N A s ol ut i on a nd 100 l of e t h i di um b r om i de ( 10 m g m l 1 ; B i o R a d, H e r c ul e s C A U S A ) w a s a dde d be f or e l oa di ng t he s ol ut i on i nt o 5. 1 m l qui c k s e a l p ol ya l l om e r ul t r a c e nt r i f uge t ube s

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94 ( B e c km a n C oul t e r F ul l e r t on, C A U S A ) U l t r a c e nt r i f uga t i on w a s pe r f or m e d us i ng a V T i 65 ve r t i c a l r ot or i n a M ode l L 8 80 ul t r a c e nt r i f uge ( B e c km a n I ns t r um e nt s F ul l e r t on C A U S A ) a t 265, 000 x g f o r 16 h a t 20 C A f t e r ul t r a c e nt r i f uga t i on, f r a c t i ons w e r e vi s ua l i z e d w i t h U V l i ght a t 365 nm ( S a m br ook e t a l 1989; R a da j e w s ki e t a l 2002 ) T hr e e D N A ba nds w e r e ge ne r a l l y obs e r ve d a nd c o l l e c t e d: ( 1 ) a l i ght D N A uppe r ba nd ( 1 2 C D N A ) ; ( 2) a m i dd l e ba nd, s m e a r of 1 2 C a nd 1 3 C D N A ; a nd ( 3 ) a he a vy D N A l ow e r ba nd ( 1 3 C D N A ) D N A f r a c t i ons w e r e c ol l e c t e d a nd pur i f i e d a s de s c r i be d by S a m b r ook e t a l ( 1989) E t hi di um br om i de w a s e xt r a c t e d f r o m t he D N A w i t h 1 but a nol ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) s a t ur a t e d w i t h w a t e r F ol l ow i ng f i ve e xt r a c t i ons t he D N A s ol ut i on w a s di l ut e d i n w a t e r a nd p r e c i pi t a t e d w i t h e t ha nol ove r ni ght a t 20 C a nd di s s ol ve d i n 100 ul T E buf f e r ( S a m br ook e t a l 19 89) A s e c ond ul t r a c e nt r i f uga t i on s t e p w a s not ne c e s s a r y a f t e r c onf i r m i ng t ha t t he p r ot oc ol of D N A ba nd e xt r a c t i on w a s e xa c t R e r uns ve r i f i e d t he pr e s e nc e of onl y o ne di s t i nc t ba nd i n t he ne w c ol um ns P ol ym e r as e c h ai n r e ac t i on ( P C R ) a m p l i f i c at i on T he pur i f i e d D N A f r a c t i ons ( 1 2 C a nd 1 3 C D N A ) w e r e us e d a s a t e m pl a t e f o r P C R a na l ys i s T he phyl oge ne t i c a na l ys i s w a s pe r f or m e d w i t h t he f unc t i ona l pm o A ge ne t a r ge t e d us i ng t he pr i m e r s e t A 189f ( 5 G G N G A C T G G G A C T T C T G G 3 ) a nd m b661 ( 5 C C G G M G C A A C G T C Y T T A C C 3 ) ( I nt e gr a t e d D N A T e c hnol ogi e s C or a l vi l l e I A U S A ) s pe c i f i c t o t he pM M O a c t i ve s i t e ( C os t e l l o a nd L i ds t r om 1999) A G C c l a m p ( 5 c c c c c c c c c c c c c g c c c c c c gc c c c c c gc c c c c gc c gc c c 3 ) w a s a t t a c he d t o t he A 189f pr i m e r a s de s c r i be d by H e nc ke l e t a l ( 1999) P C R a m pl i f i c a t i on w a s pe r f o r m e d a c c or di ng t o t he pr oc e dur e de s c r i be d by K ni e f e t a l ( 2003 ) P C R r e a c t i ons c ons i s t e d of t he M a s t e r A m p 2X P C R pr e m i xt u r e F ( E pi c e nt r e

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95 T e c hnol ogi e s M a di s on, W I U S A ) c ont a i ni ng 100 m M T r i s H C l ( pH 8. 3 ) 100 m M K C l 400 M e a c h of dN T P 3 7 m M M gC l 2 a nd t he e nha nc e r be t a i ne ( 0 8 X ) c om bi ne d w i t h 0. 5 M e a c h p r i m e r 1U T a q pol ym e r a s e a nd s t e r i l e w a t e r t o a t ot a l r e a c t i on vol um e of 50 l ( K ni e f e t a l 2003 ) A l l r e a c t i ons w e r e a s s e m bl e d on i c e a nd t he c ool e d t ube s w e r e pl a c e d i n a p r e he a t e d ( 94C ) t he r m a l bl oc k f or P C R i ni t i a t i on ( H e nc ke l e t a l 1999) T he P C R pr ot oc ol c ons i s t e d of a t ouc hdow n pr ogr a m us i ng a t he r m oc yc l e r ( M a s t e r c yc l e r P e r s ona l 5332; E ppe ndo r f W e s t bur y, N Y U S A ) w i t h t he f ol l ow i ng pa r a m e t e r s : i ni t i a l de na t ur a t i on o f 5 m i n a t 94C f ol l ow e d by 35 c yc l e s of 1 m i n a t 94C f or de na t ur a t i on 1 5 m i n a t 6 2 t o 55C i n 0. 5C i nc r e m e nt s f or a nne a l i ng, 1 m i n a t 72C f or e l onga t i on w i t h a f i na l e xt e ns i on s t e p of 7 m i n a t 72C ( K ni e f e t a l 2003) P C R pr oduc t s i z e ( 540 bp ) w a s e xa m i ne d by hor i z ont a l a ga r os e e l e c t r ophor e s i s P C R pos i t i ve c ont r ol s i nc l ude d r e pr e s e nt a t i ve s of a l l m e t ha not r o phs t ype s ( t ype X M e t hy l oc oc c u s c aps ul at us ( B a t h) ( A T C C 33009) t ype I I s t r a i ns M e t hy l os i nus t r i c hos por i um O B 3b ( A T C C 35070) S t r a i n C S C 1, M e t hy l oc y s t i s e c hi noi de s ( I M E T 10491) M e t hy l oc y s t i s par v us O B B P ( N C I M B 11129) a nd t ype I M e t hy l om i c r obi um al bum B G 8 ( A T C C 33003) ) D e n at u r i n g G r ad i e n t G e l E l e c t r op h or e s i s A n al ys i s ( D G G E ) S e q u e n c i n g, an d P h yl oge n e t i c A n a l ys i s D G G E P C R pr oduc t s w e r e s e pa r a t e d by D G G E i n t he D C ode S ys t e m ( B i o R a d, H e r c ul e s C A U S A ) a s de s c r i be d by H e nc ke l e t a l ( 1999) B r i e f l y 1 m m t h i c k 6. 5 % ( w / v) pol ya c r i l a m i de ge l s ( 37 5: 1 a c r yl a m i de bi s a c r yl a m i de ) ( F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) w e r e pr e pa r e d a nd e l e c t r oph or e s e d i n 1X T A E bu f f e r a t 61C a nd 180 V f o r 5 h i n a 35 65 % l i ne a r de na t ur a nt g r a di e nt ( 65% i s 4. 5 M ur e a a nd 26% ( v/ v )

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96 de i oni z e d f or m a m i de ) G e l s w e r e l oa de d w i t h 25 45 l o f P C R p r oduc t a c c or di ng t o ba nd i nt e ns i t y i n a ga r os e ge l s a nd ( vol um e o f l o a di ng buf f e r G e l s w e r e s t a i ne d w i t h e t hi di um br om i de a c c or di ng t o t he m a nuf a c t ur e s i ns t r uc t i ons ( B i o R a d, H e r c ul e s C A U S A ) vi s ua l i z e d a t 312 nm on a U V t r a ns i l l um i na t or ( M ode l 88A F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) a nd phot o doc um e nt e d w i t h t he s ys t e m D i gi D oc I T ( D a i gge r V e r non H i l l I L U S A ) us i ng t he D oc I t s of t w a r e v 2 4 ( U V P U pl a nd C A U S A ) D G G E ba nds w e r e e xc i s e d f r om t he m i dd l e pa r t of t he ba nd w i t h a s t e r i l e s c a l pe l a nd t he D N A e l ut e d a c c or di ng t o t he pr ot oc ol de s c r i be d by C hor y a nd P ol l a r d ( 1999) T he e l ut e d D N A w a s r e a m pl i f i e d a nd r e a na l yz e d on D G G E t o ve r i f y s a m pl e pur i t y B a nd r e a m pl i f i c a t i on w a s pe r f o r m e d by m odi f yi ng t he P C R pr ot oc ol t o 30 c yc l e s of 30 s a t 94C f or de na t ur a t i on 45 s a t 66C f or a nne a l i ng ( t o a voi d s e que nc e a m bi gui t y a s r e por t e d by D un f i e l d e t a l ( 20 02) ) a nd 30 s a t 72 C f or e l onga t i on w i t h t he s a m e i ni t i a l a nd f i na l s t e ps S e ve r a l ba nds w i t h t he s a m e m obi l i t y w e r e e xc i s e d f r o m di f f e r e nt l a ne s t o c he c k f or s e que nc e i de nt i t y. S e q u e n c i n g. R e a m pl i f i e d P C R pr oduc t s of e xc i s e d D G G E ba nds w e r e pu r i f i e d w i t h a P C R pur i f i c a t i on ki t ( M o B i o, C a r l s ba d, C A U S A ) be f or e s e que nc i ng. P C R pr oduc t c onc e nt r a t i on a nd pur i t y w a s de t e r m i ne d b y U V a bs or pt i on s pe c t r ophot om e t r y ( 1: 20 di l ut i on ) P C R p r oduc t s w e r e s e que nc e d by t he I nt e r di s c i pl i na r y C e nt e r f or B i ot e c hnol og y R e s e a r c h ( I C B R ) U ni ve r s i t y of F l o r i da ( G a i ne s vi l l e F l or i da U S A ) P h yl oge n e t i c a n al ys i s S e que nc e s w e r e c om pa r e d i n t he N a t i ona l C e nt e r f o r B i ot e c hnol ogy I nf or m a t i on ( N C B I ) da t a ba s e us i ng B L A S T ( A l t s c hul e t a l 1990) R e l a t e d s e que nc e s i de nt i f i e d i n B L A S T a s w e l l a s s e que nc e s of e xt a nt m e t ha not r ophs w e r e a l i gne d a nd a dj us t e d m a nua l l y w i t h C L U S T A L X v. 1. 8 ( T h om ps on e t a l 1997)

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97 P hyl oge ne t i c t r e e s w e r e ge ne r a t e d by t he ne i ghbor j oi ni ng ( N J ) m e t hod w i t h C L U S T A L X a nd di s pl a ye d i n T r e e V i e w v. 1. 6 6 ( P a ge 1996) N uc l e ot i de a c c e s s i on num be r s of a l l obt a i ne d s e que nc e s w e r e pl a c e d i n t he G e nB a nk f or f ut ur e a c c e s s ( A Y X X X A Y X X X ) S t at i s t i c s C H 4 de pl e t i on r a t e s obs e r ve d i n t he S I P m i c r oc os m s w e r e a na l yz e d by c om pa r i ng t he i ni t i a l s l ope s of t he l i n e a r r e gr e s s i on c ur ve s f i t t e d t o t he c ons um pt i on c ur ve dur i ng i nc uba t i on a f t e r t he f i r s t C H 4 a ddi t i on W he n a s e t of s a m pl e s s how e d no s i gni f i c a nt di f f e r e nc e s i n r a t e s a n a ve r a ge de pl e t i on r a t e ( c om m on r e gr e s s i on c oe f f i c i e nt ) w a s c a l c ul a t e d a s a n e s t i m a t e of t he C H 4 de pl e t i on r a t e unde r l yi ng a l l r a t e s of a pa r t i c ul a r s e t of s a m pl e s ( f or e xa m pl e r a t e s a m ong t he s a m e pl a nt t ype i n e a c h s a m pl i ng pe r i od) A ddi t i ona l l y, di f f e r e nc e s a m ong s a m pl e m e a ns a n d be t w e e n s a m pl e s a nd t he c ont r ol w e r e a na l yz e d by T uke y s a nd D unne t t s t e s t r e s pe c t i ve l y ( Z a r 1984 ) S A S s of t w a r e v 7 ( S A S I ns t i t ut e C a r y, N C U S A ) w a s us e d f or a l l t he a na l ys i s R e s u l t s S I P P r ot oc ol I m p l e m e n t at i on T he S I P t e c hni que w a s s uc c e s s f ul l y a ppl i e d t o t he r hi z os phe r e s oi l s T he r a t e s obs e r ve d i n t he 1 3 C H 4 a nd 1 2 C H 4 m i c r oc os m s w e r e c om pa r a bl e a nd va r i a bi l i t y a m ong t he r e pl i c a t e s w a s l ow T he M t r i c hos por i um O B 3b c ont r ol w a s e f f e c t i ve l y l a be l e d by t he 1 3 C H 4 a nd no 1 3 C H 4 c ons um pt i on w a s obs e r ve d i n t he ne ga t i ve c ont r ol s D N A e xt r a c t s f r om t he m i c r oc os m s w e r e e f f e c t i ve l y s e pa r a t e d b y C s C l de ns i t y gr a di e nt s ( F i g. 4 1A ) W he n a s m e a r w a s pr e s e nt be t w e e n t he unl a be l e d ( 1 2 C D N A ) a nd l a be l e d ( 1 3 C D N A ) f r a c t i ons i t w a s c ol l e c t e d a s a 1 2 1 3 C D N A c om bi ne d f r a c t i on a nd w a s not i nc l ude d i n t he s t udy. C or r e c t r e c ove r y o f t he s e f r a c t i ons w a s ve r i f i e d by a s e c ond

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98 ul t r a c e nt r i f uga t i on unde r t he s a m e pr ot oc ol e ve n t hough ba nd pos i t i on va r i e d be c a us e of c ha nge s i n t he de ns i t y of t he ne w s ol ut i on ( F i g 4 1, B C ) T he e xt r a c t e d 1 3 C D N A f r a c t i ons pr oduc e d pm oA ge ne f r a gm e nt s of t he e x pe c t e d s i z e ( 54 0 bp) T he m e t ha not r oph pos i t i ve c ont r ol c ul t ur e s r e ve a l e d m ul t i pl e D G G E ba nds ( F i g. A 1 A ppe ndi x) i n ke e pi ng w i t h e a r l i e r r e por t s o f t he h i gh pr oba bi l i t y of e nc ount e r i n g m ul t i pl e c opi e s of t he pm oA ge ne i n m e t ha not r oph s ( S e m r a u e t a l 1995; D unf i e l d e t a l 2002) B e c a us e D G G E ba nd pur i f i c a t i on w a s di f f i c ul t i n m os t s a m pl e s yi e l di ng a m bi guous pos i t i ons a f t e r s e que nc i ng, t he a nne a l i n g t e m pe r a t ur e w a s i nc r e a s e d f r om 62 t o 66C a nd onl y t he r e v e r s e pr i m e r w a s us e d f or s e que nc i ng ( D unf i e l d e t a l 2002) F ur t he r m or e 16S r D N A D G G E pr o f i l e s r e ve a l e d c om pl e x ba nd pa t t e r ns a nd s m e a r s t ha t w e r e di f f i c ul t t o e xa m i ne ( da t a not s how n) I n ge ne r a l pm oA D G G E pr of i l e s of t he 1 3 C D N A f r a c t i ons of t he di f f e r e nt r hi z os phe r e s oi l m i c r oc os m s di d not di f f e r g r e a t l y a m ong s i t e s pl a nt t ype or s a m pl i ng pe r i od. C ons e que nt l y, i t w a s us e f ul t o s e t a r e f e r e nc e pr of i l e f or t he a na l ys i s ( F i g 4 3A l a ne 1) R e f e r e nc e ba nds 1, 3, 9, 10 a nd 11 w e r e n ot pos s i bl e t o s e q ue nc e M e t h an ot r op h A c t i vi t y an d C o m p os i t i on i n t h e T C E S i t e 1 3 C H 4 a dde d i n f i ve a ddi t i ons t o a t ot a l o f a ppr oxi m a t e l y 2. 0 m m ol w a s c ons um e d w i t hi n 31 37 da ys i n a l l T C E S i t e s oi l m i c r oc os m s I ni t i a l C H 4 de pl e t i on r a t e s ( F i g. 4 2A ) c a l c ul a t e d a s a m e a s ur e o f t he oxi da t i ve pot e n t i a l a t t he t i m e of s a m pl i ng, s how e d no s i gni f i c a nt di f f e r e nc e s w i t hi n pl a nt t ype ove r t h e 16 m ont h s a m pl i ng pe r i od. A c om pa r i s on of a ve r a ge r a t e s pe r pl a nt t ype w hi c h r e pr e s e nt s pl a nt t ype s ove r a l l a c t i vi t y t hr oughout t he s t ud y, s how s a l s o no s i gni f i c a nt di f f e r e nc e i n C H 4 de pl e t i on r a t e s a m ong pl a nt t ype s a nd w i t h t he non pl a nt e d s a m pl e ( P < 0 05) T he ove r a l l a ve r a ge C H 4 de pl e t i on r a t e a t t he T C E S i t e w a s 0. 11 m ol h 1 g 1 dr y w e i ght s oi l ( 0. 01 S E )

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99 A s s how n i n F i g. 4 3 T C E S i t e ( pa ne l A t o C ) a nd P C E S i t e ( pa ne l D ) pm o A D G G E pr of i l e s w e r e m os t l y de s c r i be d by t he s a m e gr oup o f ba nds num be r e d f r om 1 t o 11 i n t he r e f e r e nc e p r of i l e ( F i g 4 3A l a ne 1) C o nc e nt r i c c i r c l e s w i t h l e t t e r s de not e d ba nds di f f e r e nt f r om t he r e f e r e nc e pr of i l e P r o f i l e s w e r e l a be l e d s t a r t i ng w i t h t he pl ot l oc a t i on ( T C E or P C E ) f ol l ow e d by t he t r e e t ype ( popl a r or w i l l ow ) s oi l c om pa r t m e nt a na l yz e d ( r hi z os phe r e r h i z opl a ne or non pl a nt e d s oi l ) a nd s a m pl i ng pe r i od. W i l l ow c l one s S X 61 a nd 94019 w e r e not s a m pl e d i n t he N ove m be r 2004 s a m pl i ng pe r i od, c ons e que nt l y, t he y onl y s how e d t w o pr of i l e s e a c h ( F i g. 4 3C l a ne s 1 4) T he pm o A a m pl i f i e d s e que nc e s f r om t he 1 3 C D N A f r a c t i ons o f t he T C E S i t e m i c r oc os m s r e ve a l e d D G G E pr of i l e s c om pos e d by 2 t o 11 ba nds ( F i g. 4 3, B C ) T he s e pr o f i l e s w e r e m a i nl y de s c r i be d by t hr e e gr oups o f hi gh l y s i m i l a r s e que nc e s a c c or di ng t o ba nd pos i t i on a nd B L A S T a l i gnm e nt s i ndi c a t i ng t ha t s om e ba nds m a y r e pr e s e nt c opi e s of m ul t i pl e pm o A ge ne s H ow e ve r s om e p r of i l e s t ha t e xhi bi t e d t he s e gr oupi ngs di d no t r e ve a l a l l of t he ba nds T he T C E S i t e D G G E p r of i l e s t hr ough out t he s t udy di d not va r y t o a g r e a t e xt e nt a m ong pl a nt t ype ( F i g 4 3 B C ) bu t t he y w e r e di s t i nc t f r om t he non pl a nt e d s oi l ( F i g. 4 3B l a ne 4 t o 6) R hi z os phe r e D G G E p r of i l e s f r om popl a r ( F i g. 4 3B l a ne 1 2) a nd a l l w i l l ow c l one s ( F i g 4 3 pa ne l B l a ne s 7 8 a nd, pa ne l C l a ne s 1 t o 4) c ol l e c t e d i n J ul y 2003 a nd 2004 r e ve a l e d t he s a m e c om m uni t y of o r ga ni s m s T he s e pr of i l e s w e r e c om pos e d of t w o g r oupi ngs of ba nds r e p r e s e nt e d b y r e f e r e nc e ba nds 4 a nd 6 t o 8, a nd ba nds 2 a nd 5 ( F i g. 4 3A l a ne 1 ) W i t hi n e a c h gr o upi ng, ba nds s ha r e d 100% s e que nc e s i m i l a r i t y a nd a l i gne d i n B L A S T ( > 99% s i m i l a r i t y ) w i t h t w o c l one s of unc ul t ur e d M e t hy l oc al dum s p. i s ol a t e d f r om a l a ndf i l l c o ve r s oi l T he phyl oge ne t i c t r e e c l us t e r e d

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100 t he s e s e que nc e s a s t he de s c r i be d gr oupi ngs ( F i g 4 4 G r oup 1 a nd 2) a nd w i t hi n t he M e t hy l oc al dum br a nc h ( t ype X m e t ha not r oph) T h e s e gr oupi ngs a r e c l os e l y r e l a t e d t o t he c ul t ur e d or ga ni s m M gr ac i l e A l s o, a t hi r d g r oup of ba nds de s i gna t e d by r e f e r e nc e ba nds 9 t o 11 ( F i g 4 3A l a ne 1) w a s de t e c t e d i n a l l pr of i l e s H ow e ve r be c a us e r e a m pl i f i c a t i on of t hi s g r oup w a s not pos s i bl e a nd s i nc e t he y w e r e t he onl y ba nds pr e s e nt i n t he J ul y 2003 T C E S i t e non pl a nt e d p r of i l e ( F i g 4 3B l a ne 4 ) i t i s pos s i bl e t he y r e pr e s e nt a not he r s e t of s i m i l a r pm o A ge ne s F r om t he pl a nt e d m i c r oc os m s a t t he T C E S i t e onl y t he pol a r a nd w i l l ow c l one 94014 e xhi bi t e d c ha nge s i n t he i r m e t ha not r oph c o m m uni t y t hr oughou t t he s t udy T he popl a r t r e e i n t he N ove m be r 2004 s a m pl i ng ( F i g. 4 3B l a ne 3 ) s how e d l e s s t ha n ha l f of t he ba nds i n t he J ul y s a m pl i ngs ( F i g. 4 3B l a ne 1 2) I t onl y r e ve a l e d t hr e e ba nds t w o a t ve r y l ow i nt e ns i t y a nd t he s a m e m a j or ba nd o f pr e vi ous s a m pl i ngs ( r e f e r e nc e ba nd 5 ) W i l l ow c l one 94014, i n t he J ul y 2004 s a m pl i ng ( F i g. 4 3C l a ne 4 ) di d not e xhi bi t t hi s m a j or ba nd a nd s how e d a n e xt r a ba nd of a n unc ul t ur e d m e t ha not r oph t ha t c l os e l y r e l a t e d ( 88% s i m i l a r i t y) t o a not he r t ype X m e t ha not r oph, M e t hy l oc oc c us c aps u l at us ( F i g. 4 4 ) T he T C E S i t e non pl a nt e d m i c r oc os m s ove r t he t h r e e s a m pl i ng t i m e s e xhi bi t e d va r i a bl e pr of i l e s a t ve r y l ow i nt e ns i t y ( F i g. 4 3B l a ne 4 t o 6 ) I n t he J ul y 2003 s a m pl i ng, onl y t he 9 t o 11 gr oupi ng w a s r e t r i e ve d. H ow e ve r i n t he J ul y a nd N ove m be r 2004 s a m pl i ngs t he 9 t o 11 g r oupi ng w a s obs e r ve d a l on g w i t h s om e of t he ba nds t ha t de s c r i be d t he M e t hy l oc al dum c l one s f ound i n t he p l a nt e d s a m pl e s a nd unc ul t ur e d ba c t e r i a f r om r i c e f i e l ds a nd upl a nd s oi l s ( > 87% s i m i l a r i t y ) T he unc ul t u r e d ba c t e r i a ( F i g. 4 3B l a ne 5 6) w e r e pl a c e d w i t hi n t he M e t hy l oc al dum a nd M e t hy l oc oc c us br a nc h ( F i g 4 4)

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101 c l os e l y r e l a t e d t o t he t ype s pe c i e s M e t hy l oc al dum s z e ge di e n s e a nd M e t hy l oc oc c us c aps ul at us r e s pe c t i ve l y. A ddi t i ona l l y, one of t he 1 2 C D N A f r a c t i ons f r om t h e w i l l ow c l one S 365 m i c r oc os m s w a s a m pl i f i e d t o c om pa r e t o t he 1 3 C D N A f r a c t i on ( F i g. 4 3C l a ne 6 7) O nl y one ba nd w a s di f f e r e nt be t w e e n t he s e pr o f i l e s t he f i r s t ba nd i n t he 1 2 C pr o f i l e ( F i g. 4 3C l a ne 7 ) w hi c h a l i gne d i n B L A S T t o a n unc u l t ur e d ba c t e r i um f r om a n upl a nd s oi l ( 97% s i m i l a r i t y) T hi s ba nd w a s pl a c e d i n t he phy l oge ne t i c t r e e w i t hi n t he t ype X m e t ha not r ophs c l os e l y r e l a t e d t o M e t hy l oc al dum s z e ge di e ns e ( F i g. 4 4) M e t h an ot r op h A c t i vi t y an d C om p os i t i on i n t h e P C E S i t e A t t he P C E S i t e s oi l m i c r oc os m s a c t i vi t y di f f e r e d gr e a t l y be t w e e n pl a nt e d a nd non pl a nt e d s a m pl e s ( F i g. 4 2B ) H ow e ve r i t w a s t he a c t i vi t y of t he non pl a nt e d s oi l t ha t s i gni f i c a nt l y c ha nge d a m ong s a m pl i ng pe r i ods ( P < 0 0005) N on pl a nt e d m i c r oc os m s r a t e s r a nge d f r om 0. 01 t o 0 29 m ol h 1 g 1 d r y w e i ght s oi l ( 0 00 S E ) f r om t he f i r s t t o t he l a s t s a m pl i ng pe r i od. A s m e nt i one d pr e vi ous l y, t he P C E c ont r ol w a s l oc a t e d i n a gr a s s y a r e a t ha t w a s t r a ns f or m e d i n t o a s oc c e r f i e l d, t he s hi f t m a y ha ve i m pr ove d c ondi t i ons f o r m i c r ob i a l popul a t i ons t o t hr i ve dur i ng t he l a s t s a m pl i ng pe r i od. A s i n t he T C E S i t e pl a nt e d m i c r oc os m s r a t e s w e r e c om pa r a bl e dur i ng t he s t udy ( P < 0 05) a nd a ve r a ge d 0. 03 m ol h 1 g 1 dr y w e i ght s oi l ( 0 01 S E ) H ow e ve r t he s e r a t e s w e r e s i gni f i c a nt l y l ow e r t h a n t he r a t e s o f t he p l a nt e d m i c r oc os m s a t t he T C E S i t e ( T uke y s t e s t P < 0. 0005 ) a nd r e s ul t e d i n l onge r pe r i ods of e xpos ur e t o t he 1 3 C H 4 ( 86 20 da ys ) T he a c t i ve m e t ha not r oph c om pos i t i on of t he popl a r t r e e s a t t he P C E S i t e r e ve a l e d i n e a c h s a m pl i ng pe r i od a di s t i nc t D G G E pr o f i l e c om pos e d of 3 t o 7 ba nds ( F i g. 4 3D l a ne 1 2) I n t he J ul y 2003 s a m pl i ng ( F i g 4 3D l a ne 1) t he pr o f i l e o f t he P C E S i t e

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102 popl a r w a s c om pos e d of t he s a m e gr oup o f ba nds t ha t de s c r i be d t he unc ul t ur e d M e t hy l oc al dum s p. c l one f ound a t t he T C E S i t e t r e e s ( F i g. 4 3, B C ) T he pl a c e m e nt o f t he s e s e que nc e s i n t he phyl oge ne t i c t r e e ( F i g 4 4, G r oup 2) c onf i r m e d t ha t t he r e f e r e nc e ba nds f ound a t t he P C E S i t e c or r e s ponde d t o t he b a nds a t T C E S i t e c l os e l y r e l a t e d t o M gr ac i l e H ow e ve r i n t he J ul y 2004 s a m pl i ng t he c om pos i t i on of t he popl a r t r e e c ha nge d, t he obs e r ve d ba nds ( 97% s i m i l a r i t y) a l i gne d i n B L A S T t o unc ul t ur e d ba c t e r i a ( > 87% s i m i l a r i t y) f r om upl a nd s oi l s i n T ha i l a nd ( F i g. 4 3 D l a ne 2) T he s e ba nds w e r e pl a c e d ne xt t o t he M e t h y l oc oc c us br a nc h i n t he phyl oge ne t i c t r e e c l os e s t t o M c aps ul at us ( F i g 4 4) T he onl y P C E non pl a nt e d s oi l m i c r oc os m s a na l yz e d by D G G E w a s c ol l e c t e d dur i ng N ove m be r 2004 ( F i g. 4 3D l a ne 3 ) a nd e x hi bi t e d ba nds of t he s a m e unc ul t ur e d M e t hy l oc al dum s p. c l one a s t he pl a nt e d s a m pl e dur i ng J ul y 2003 ( F i g. 4 3D l a ne 1 ) I n a ddi t i on, t w o ba nds t ha t s ha r e d 99% s i m i l a r i t y a nd a l i gne d i n B L A S T w i t h di f f e r e nt M e t hy l oc y s t i s s p. c l one s ( 99% s i m i l a r i t y) w e r e r e t r i e ve d. T he phyl oge ne t i c a na l ys i s c onf i r m e d t he i r pl a c e m e nt i n t he M e t hy l oc y s t i s br a nc h ( t ype I I m e t ha not r ophs ) c l os e l y r e l a t e d t o M e t hy l oc y s t i s s p. s t r a i n S C 2. H ow e ve r di f f e r e nc e s be t w e e n t r e e s a nd t he non pl a nt e d s oi l w e r e di f f i c ul t t o a s s e s s a t t hi s s i t e be c a us e va r i ous s a m pl e s w e r e l os t dur i ng a na l ys i s D i s c u s s i on I n t hi s s t udy t he S I P t e c hni que w a s s uc c e s s f ul l y i m pl e m e nt e d t o c ha r a c t e r i z e t he a c t i ve m e t ha not r oph popul a t i ons p r e s e nt i n t he r hi z os phe r e of di f f e r e nt t r e e s t ype s us e d i n t w o phyt o r e m e di a t i on pl ot s w i t h di s t i nc t e nvi r o nm e nt a l c ondi t i ons A t t he T C E S i t e be c a us e t he t r e e s w e r e gr ow n i n pot s c ont a i ni ng pl a nt i ng m a t e r i a l a c om pos t e f f e c t gove r ne d t he a c t i vi t y a nd c om pos i t i on of t he m e t ha not r oph popul a t i ons w hi c h m a y

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103 e xpl a i n w hy no s i gni f i c a nt t r e nd w a s obs e r ve d a m ong c l one s t i m e s a m pl e s o r t he non pl a nt e d s oi l A ddi t i ona l l y, t he ye a r s o f e s t a bl i s hm e nt of t he pl ot s f i r s t a nd s e c ond gr ow i ng s e a s ons m a y ha ve a l s o c ont r i but e d t o t he hom oge ne i t y of t he T C E S i t e s a m pl e s I t ha s be e n r e por t e d t ha t d i f f e r e nc e s be t w e e n popl a r c l one s c a n t a ke t i m e t o r e ve a l i n phyt or e m e di a t i on a ppl i c a t i ons ( E be r t s e t a l 2003) A l s o, s t udi e s on t he a bove gr ound pe r f or m a nc e of t he s e t r e e s a t t he s i t e ha ve not be e n a bl e t o de t e c t a ny di f f e r e nc e s i n ge not ype s a f t e r t w o ye a r of e s t a bl i s hm e nt ( R oc kw ood e t a l 2005 ) C ons e que nt l y, di f f e r e nc e s i n c om m uni t y s t r uc t ur e a nd a c t i vi t y o f t he r hi z os phe r e m e t ha not r ophi c ba c t e r i a m a y de ve l op w i t h t i m e a s t he di f f e r e nt pl a nt t ype s e xe r t e d a g r e a t e r i n f l ue nc e ove r t he s ys t e m s uc h a s r oot e xpa ns i on a nd pl a nt b i om a s s a c c um ul a t i on. T he phyl oge ne t i c a na l ys i s a t t he T C E S i t e r e ve a l e d t ha t t ype X m e t ha not r ophs ( t he r m ot ol e r a nt ba c t e r i a ) of t he ge nus M e t hy l oc al dum a nd M e t hy l oc oc c us dom i na t e d t he a c t i ve m e t ha not r oph popul a t i ons T hi s r e s ul t s upp or t s t he c om pos t e f f e c t m e nt i one d a bove I t i s know n t ha t M e t hy l oc al dum s t r a i ns a r e c om m onl y f ound a m ong t he m e t ha not r oph popul a t i ons i n c om pos t pi l e s ( 10 9 c e l l s g dw 1 ) ( E s hi ni m a e v e t a l 2004 ; J a c ke l e t a l 2005 ) A l s o, i nc r e a s e d m e t ha not r oph a c t i vi t y ha s be e n r e po r t e d w he n c om pos t i s i nc or por a t e d t o s oi l s ( S e ghe r s e t a l 20 05) C ons e que nt l y, by u t i l i z i ng a pl a nt i ng m a t e r i a l h i gh a c t i vi t y of t ype X m e t ha not r ophs c a n be e xpe c t e d a s i t w a s obs e r ve d i n t hi s s t udy w he n bot h phyt or e m e di a t i o n s i t e s w e r e c om pa r e d. H ow e ve r M e t hy l oc al dum s t r a i ns do not pr oduc e t he s ol ubl e f or m o f M M O ( B odr os s y e t a l 1997 ) pr e f e r r e d be c a us e of i t s hi ghe r oxi da t i ve pot e nt i a l ( O l de nhui s e t a l 1989; H a ns on a nd H a ns on, 1996) N e v e r t he l e s s M e t hy l oc oc c us s t r a i ns c a n e xpr e s s bot h f or m s o f M M O

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104 a nd de gr a de c hl or i na t e d c om pounds a t hi ghe r r a t e s t ha n ot he r t ype s of m e t ha not r ophs ( D i S pi r i t o e t a l 1992; H a ns on a nd H a ns on, 1996) D G G E f i nge r p r i nt s w hi c h s how e d gr e a t e r s e ns i t i vi t y i n de t e c t i ng di f f e r e nc e s a m ong s oi l m i c r oc os m s r e ve a l e d t ha t a t t he T C E S i t e no n pl a nt e d p r of i l e s w e r e s i gni f i c a nt l y di f f e r e nt f r om t he pl a nt e d. T C E S i t e non pl a nt e d s a m pl e s va r i e d gr e a t l y w i t h t i m e a nd s how e d l ow e r r e l a t i ve a bunda nc e of i t s c om m uni t y m e m be r s t ha n t he pl a nt e d pot s s ugge s t i ng a pl a nt e f f e c t o f t he t r e e s ove r t he m e t ha not r oph i c popul a t i ons a t t he s i t e A ddi t i ona l l y, D G G E p r of i l e s r e ve a l e d t ha t t he T C E S i t e popl a r i n t he N ove m be r 2004 s a m pl i ng e xhi bi t e d s i gni f i c a nt l y f e w e r ba nds t ha n pr e vi ous s a m pl i ngs ( J ul y 2003 a nd 2004) w hi c h m a y i m pl y t ha t popl a r r hi z os phe r e m e t h a not r oph popul a t i on e xhi bi t e d a s e a s ona l e f f e c t ( s um m e r v e r s us f a l l ) D ur i ng 2 004 t he m ont hs of J ul y a nd N ove m be r e xpe r i e nc e d s oi l t e m pe r a t ur e s of 22 a nd 9C r e s pe c t i ve l y. T he r e f o r e t he N ove m be r s a m pl i ng c ondi t i ons m a y not be t he opt i m um f or g r ow t h of t he r m ot ol e r a nt m e t ha not r ophs ( B odr os s y e t a l 1997 ) A l s o, dur i ng t he s e c ond gr ow i ng s e a s on w i l l ow c l one 94014 di d no t s how t he m a j o r ba nd a s i n t he pr e vi ous s a m pl i ng, w hi c h c or r e s ponde d t o t he M e t hy l oc al dum c l one c l os e l y r e l a t e d t o M gr ac i l e H ow e ve r i n t he popl a r t r e e i t w a s t hi s c l one t he onl y one t ha t pe r s i s t e d i n t he l a s t s a m pl i ng pe r i od. T hi s r e s ul t m a y s ugge s t c e r t a i n s e l e c t i on of M e t hy l oc al dum c l one s by t he t ype of p l a nt a s f or e a c h t r e e t ype a di f f e r e nt c l one of M e t hy l oc al dum pe r s i s t e d i n t he i r D G G E pr o f i l e s A l s o, i t w a s i nt e r e s t i ng t ha t w i l l ow c l one s S 365 a n d S X 61 s how e d e xa c t l y t he s a m e pr of i l e t h r oughout t he s t udy. W i l l ow t r e e s pr oduc e d hi ghe r f i ne r oot bi om a s s t ha n popl a r t r e e s ( da t a not s how n) t hus pos s i bl y a s s i s t i ng i n t h e m a i nt e na nc e of hi ghe r t e m pe r a t ur e s

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105 i n t he pot t e d s oi l c om pa r e d t o t he popl a r or t he no n pl a nt e d s oi l s w hi c h m a y ha ve be e n m or e s us c e pt i bl e t o c ha nge s i n t he e nvi r onm e nt M e t hy l oc al dum s t r a i ns c a n gr ow i n a w i de r a nge o f t e m pe r a t ur e s M gr ac i l e gr ow s i n a l ow e r t e m pe r a t ur e r a nge ( 20 47C ) t ha n M s z e ge di e ns e ( 37 62C ) ( B odr os s y e t a l 1997) w hi c h m a y e xpl a i n w hy M s z e ge di e ns e w a s onl y f ound a t l ow a bunda nc e a nd i n t he i na c t i ve f r a c t i on ( 1 2 C ba nd) o f w i l l ow c l one S 3 65. H ow e ve r s om e t he r m ot ol e r a nt m e t ha not r ophs a s t he ge nus M e t hy l oc oc c us pr e s e nt a t bot h phyt or e m e di a t i on s i t e s a r e know n t o c ont a i n a ddi t i ona l gl yc opr ot e i n s t r uc t ur e s on t he out e r s ur f a c e of t he i r c e l l w a l l s w hi c h a ppa r e nt l y p r ovi de s hi ghe r r e s i s t a nc e t o s t r e s s f a c t or s s uc h a s t e m pe r a t ur e f l uc t ua t i ons a nd c onc e nt r a t i on o f s ol ut e s ( E s hi ni m a e v e t a l 2004) H ow e ve r e ve n i f t ype X m e t ha not r ophs pos s e s s hi gh a da pt a bi l i t y t o c ha nge s i n t he e nvi r onm e nt c ondi t i ons a t t he s i t e a r e not t he opt i m um f or t he s e s pe c i e s P a r t of t he ye a r t he s oi l f r e e z e s a nd t he hi g he s t s oi l a nd a i r t e m pe r a t u r e r e c or de d dur i ng t he s t udy w e r e 25 a nd 32C r e s pe c t i ve l y. T he r e f o r e t he que s t i on r e m a i ns a s t o w he t he r t he s e a c t i ve m e t ha not r oph popul a t i ons de t e c t e d a f t e r onl y one a nd t w o ye a r s of e s t a bl i s hm e nt w i l l pe r s i s t a t t he s e phyt or e m e di a t i on pl ot s C ont r a r y t o r e s ul t s a t t he T C E S i t e D G G E p r of i l e s i n t he P C E S i t e popl a r t r e e s w e r e di s t i nc t f or e a c h s a m pl i ng pe r i od G i ve n t ha t m e t ha not r oph popul a t i o ns a r e not c a pa bl e of oxi di z i ng P C E i t i s pr oba bl e t ha t t he y s how e d hi ghe r s us c e pt i bi l i t y t o t he t oxi c e f f e c t s of t he c ont a m i na nt a nd c ons e que nt l y, a r e m or e vul ne r a bl e t o e nvi r onm e nt a l c ha nge s a t t he s i t e ( O l de nhui s e t a l 1991; H a ns on a nd H a ns on, 1996) A ddi t i ona l l y no di f f e r e nc e s be t w e e n pl a nt e d a nd non p l a nt e d m i c r oc os m s w e r e de t e c t e d a t t he P C E S i t e

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106 H ow e ve r i t w a s not pos s i bl e t o de t e r m i ne a t i m e e f f e c t of t he non pl a nt e d s oi l be c a us e of t h e l os s of s e ve r a l s a m pl e s P hyl oge ne t i c a na l ys i s of t he P C E S i t e popl a r s a m pl e s r e ve a l e d uni que pr of i l e s c om pos e d of onl y, c l one s of t he M e t hy l oc al dum ge nus or unc ul t ur e d ba c t e r i um c l os e l y r e l a t e d t o M e t hy l oc oc c us O n t he c ont r a r y t he P C E S i t e c ont r ol w a s m or e di ve r s e c om pos e d of one m e m be r of t he ge ne r a M e t hy l oc al dum a nd M e t hy l oc y s t i s C ons e que nt l y a c t i ve m e t ha not r oph popul a t i ons o f t he popl a r t r e e r hi z os phe r e w e r e l e s s di ve r s e a nd m or e va r i a bl e a t t he P C E S i t e t ha n a t t he T C E S i t e A l s o, t he P C E S i t e m e t ha not r oph c om m uni t y w a s not dom i na t e d by M e t hy l oc al dum s t r a i ns H ow e ve r M e t hy l oc al dum i s f r e que nt l y f ound i n na t ur a l s oi l s a l ong w i t h M e t hy l os i nus a nd M e t hy l oc y s t i s ( B odr os s y e t a l 1997 ; K ni e f e t a l 2003) I n c onc l us i on, t he S I P t e c hni que w a s e f f e c t i ve l y i m pl e m e nt e d i n r hi z os phe r e s oi l s of phyt or e m e di a t i on s i t e s a nd t he pr o t oc ol de s c r i b e d i n de t a i l B y c om bi ni ng t he S I P t e c hni que w i t h t he pm o A D G G E a na l ys i s t he e xt e nt a nd r e s ol ut i on o f t he t e c hni que w a s br oa de n t o pos s i bl y de t e c t di f f e r e nc e s ove r t i m e a n d a m ong t he r e l a t i ve a bunda nc e of t he a c t i ve m e m be r s of t he m e t ha not r oph c om m uni t y. A l t hough, i t i s e xpe ns i ve a nd r e qui r e s c e r t a i n e xpe r t i s e t he m e t hod i s a pow e r f u l t e c hni qu e t ha t a l l ow s t he a s s e s s m e nt of t he pot e nt i a l de gr a de r s by a s s e s s i ng t he a c t i ve m i c r obi a l popul a t i on a t a ny phyt or e m e di a t i on s i t e C ons e que n t l y, by us i ng t hi s pr o t oc ol t he phy t or e m e di a t i on pr a c t i t i one r c a n a de qua t e l y m oni t or a nd i m pl e m e nt t he i r r e m e di a t i on t e c hnol ogy t o t he ne e ds of t he a s s e s s e d a c t i ve r hi z os phe r e popul a t i ons

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107 F i gur e 4 1 E qui l i br i um c e nt r i f uga t i on of i s ot opi c a l l y l a be l e d D N A i n C s C l de ns i t y gr a di e nt c ol um ns ( A ) 1 2 C a nd 1 3 C D N A f i r s t s e p a r a t i on c ol um n f r om T C E S i t e popl a r S I P m i c r oc os m s i n t he J ul y 2003 s a m pl i ng. ( B ) 1 2 C D N A s e c ond s e pa r a t i on c ol um n. ( C ) 1 3 C D N A s e c ond s e pa r a t i o n c ol um n.

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108 F i gur e 4 2 I ni t i a l 1 3 C H 4 de pl e t i on r a t e s ( ba r s ) obs e r ve d i n S I P m i c r oc os m s a f t e r t he t hr e e s a m pl i ng pe r i ods a t t he T C E S i t e ( A ) a nd P C E S i t e ( B ) T r i a ngl e s r e pr e s e nt a ve r a ge of i ni t i a l r a t e s ove r t he t h r e e t i m e pe r i ods E r r or ba r s r e pr e s e nt t he s t a nda r d e r r o r o f t he l i ne a r r e gr e s s i on a na l ys i s

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109 F i gur e 4 3 D G G E ge l s of pm o A P C R p r oduc t s de r i ve d f r o m t he 1 3 C D N A f r a c t i on o f S I P m i c r oc os m s a t t he T C E S i t e ( A C ) a nd P C E S i t e ( D ) R e f e r e nc e ba nds ( w hi t e c i r c l e s ) s e que nc e d ba nds ( e m pt y c i r c l e s ) a n d s e que nc e d ba nds di f f e r e nt f r om t he r e f e r e nc e pr of i l e ( c onc e nt r i c c i r c l e s ) a r e de not e d. P r of i l e na m e s = c ont a m i na nt t ype ( T = T C E P = P C E ) t r e e t ype ( P = popl a r W = w i l l ow ) w i l l ow c l one ( S 365, S X 61 94014) s oi l c om pa r t m e nt ( R H = r hi z os phe r e N P = non pl a nt e d) s a m pl i ng pe r i od ( 1 = J ul 2003, 2= J ul 2004, 3= N ov 2004) a nd r e pl i c a t e s ( R ) B a nds f r om r e f e r e n c e pr of i l e a r e de not e d by num be r s ( 1 11) a nd ba nds di f f e r e nt f r om t he r e f e r e nc e pr of i l e by l e t t e r s ( a h) a nd c onc e nt r i c c i r c l e s

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110 F i gur e 4 4 N e i ghbor j oi ni ng phyl oge ne t i c t r e e o f pm oA s e que nc e s de r i ve d f r om t he 1 3 C D N A f r a c t i on o f 1 3 C H 4 S I P m i c r oc os m s T r e e c on s t r uc t e d i n r e l a t i on t o e xt a nt m e t ha not r ophs a nd hi ghe s t s c or e B L A S T a l i gne d s e que nc e s L e ngt h of br a nc he s i s pr opor t i ona l t o % di s s i m i l a r i t y ( 0. 1 s ubs t i t ut i on pe r nuc l e ot i de s i t e ) B r a nc h c i r c l e s r e p r e s e nt % 90% ( ) o r % 68% ( ) boot s t r a p va l ue s f r om 1000 r e pl i c a t e s S e que nc e na m e = c ont a m i na nt t yp e ( T = T C E P = P C E ) t r e e t ype ( P = popl a r W = w i l l ow ) w i l l ow c l one ( S 365, S X 61 94014) s oi l c om pa r t m e nt ( R H = r hi z os phe r e N P = non pl a nt e d) s a m pl i ng pe r i od ( 1= J ul 2003, 2= J ul 2 004, 3= N ov 2004 ) a nd ba nd num be r ( 1 11= ba nds f r o m r e f e r e nc e pr of i l e a h= ba nds di f f e r e nt f r om t he r e f e r e nc e pr of i l e )

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111 C H A P T E R 5 C H A R A C T E R I Z A T I O N O F R H I Z O S P H E R E M E T H A N O T R O P H I C B A C T E R I A I N T C E P H Y T O R E M E D I A T I O N : I M P A C T O F T H E D E S I G N N ot e : M anus c r i pt t o be s ubm i t t e d t o J our nal of A p pl i e d M i c r obi ol ogy I n t r od u c t i on A s t he us e of pl a nt s t o r e m e di a t e c ont a m i na t e d s i t e s ( phyt or e m e di a t i on) ha s e m e r ge d a s a n a t t r a c t i ve t e c hnol ogy, di f f e r e nt de s i gns a r e be i ng t e s t e d t o i m pr ove t he e f f e c t i ve ne s s of t he c l e a nup pr a c t i c e G i ve n t ha t p hyt or e m e di a t i on i s t he r e s ul t of m ul t i pl e pl a nt r e l a t e d c ont a m i na nt r e m ova l p r oc e s s e s i t i s of i m por t a nc e t o a s s e s s how di f f e r e nt de s i gns m a y a f f e c t t he e xt e nt of c ont r i but i on of e a c h m e c ha ni s m R hi z ode gr a da t i on, m i c r obi a l br e a kdow n of pol l ut a nt s a t t he r oot z one of t he pl a nt ( r hi z os phe r e ) ha s be e n pr opos e d a s t he m a i n r e m o va l m e c ha ni s m c ont r i but i ng t o t he ove r a l l r e m e di a t i on o f g r oundw a t e r a nd s oi l s c ont a m i na t e d w i t h c hl or i na t e d s ol ve nt s ( A nde r s on a nd W a l t on, 1995; B r i g m on e t a l 1999 ) A l t hough phyt or e m e di a t i on i s be i ng t e s t e d i n t he f i e l d f oc us e d s t udi e s on r hi z ode gr a da t i on a nd, e ve n t o a l e s s e r e xt e nt t he e f f e c t of t he de s i gn on r hi z os phe r e m i c r obi a l pr oc e s s e s a r e s c a r e P l a nt r oot z one pr oc e s s e s ha ve l ong be e n r e c ogni z e d a nd e va l ua t e d i n a gr i c ul t ur a l f i e l ds be c a us e of t he i r be ne f i c i a l e f f e c t s on pl a nt s F ur t he r m o r e a t a l a r ge r s c a l e t hi s z one of hi ghe r m i c r obi a l a c t i vi t y ha s a m a j or i m pa c t on t he gl oba l c yc l i ng o f nut r i e nt s ( C ur l a nd T r ue l ove 1986; W a l t on e t a l 1994) A c t i vi t y, c om pos i t i on a nd a bunda nc e o f r hi z os phe r e m i c r oor ga ni s m s a r e d r i ve n by c om p l e x i nt e r a c t i ons be t w e e n t he e nvi r on m e nt ( s oi l t ype nut r i e nt s t a t us pH m oi s t ur e ) a s w e l l a s pl a nt va r i a bl e s ( s pe c i e s a ge )

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112 A ddi t i ona l l y, s i nc e s oi l s a r e c ha r a c t e r i z e d f or be i n g ol i got r ophi c e nvi r onm e nt s pl a nt r e l a t e d or ga ni c c om pounds ( r oot e xuda t e s c e l l l ys a t e s ) c a n gr e a t l y i nf l ue nc e s oi l m i c r obi a l popul a t i ons a s t he y c ont r i but e t o t he c a r bon pool of t he s oi l T h e va r i e t y of t he s e c om pounds de t e r m i ne d by t he t ype o f pl a nt ha s be e n pos t ul a t e d a s a ke y f a c t or i nf l ue nc i ng s oi l m i c r ob i a l c om m uni t i e s P l a nt s pe c i e s e f f e c t s m a y f ur t he r va r y w i t h r oot z one c om pa r t m e nt pl a nt a ge a nd m yc or r hi z a l f un gi i nt e r a c t i ons H o w e ve r c ont r a s t i ng r e s ul t s ha ve be e n r e por t e d on t he r o l e t ha t e nvi r on m e nt a l a nd pl a nt va r i a bl e s pl a y a s de t e r m i na nt s of s oi l m i c r obi a l c om m uni t y c om pos i t i on ( M a r s c hne r e t a l 2001) M i c r oo r ga ni s m s ha ve t he a bi l i t y t o pe r s i s t i n t he e nvi r onm e nt de s pi t e unf a vor a bl e gr ow t h c ondi t i ons a nd, a s a r e s ul t a n a l m os t uni v e r s a l m i c r obi a l c om pos i t i on a nd s t r uc t ur e a r e f ound i n s oi l ( M a ha f f e e a nd K l oe ppe r 1997) i nc l ud i ng t hos e i n m i c r oe nvi r om e nt s f or m e d by t he i nf l ue nc e o f t he r oot s ys t e m of a p l a nt T he r e i s gr e a t ne e d, t he r e f or e f or e xpe r i m e nt a l a ppr oa c he s t ha t r e ve a l not onl y t he m i c r obi a l c om pos i t i on of a s i t e but a l s o i t s r e l a t i ve a bunda nc e a nd i n s i t u a c t i vi t y of i t s c om pone nt s t o e f f e c t i ve l y p r e di c t m i c r ob i a l r hi z os phe r e p r oc e s s e s a nd a s s e s s di f f e r e nc e s a m ong pl a nt e d s ys t e m s ( M a ha f f e e a nd K l oe ppe r 1997 ) H ow e ve r a c um ul a t i ve e f f e c t o f l a nd us e on t he m i c r obi a l c om m uni t y s t r uc t ur e of a s i t e ha s a l s o be e n r e por t e d ( F e l s ke a nd A kke r m a ns 1997; B uc kl e y a nd S c hm i dt 2001) F or e xa m pl e s i t e s w i t h a l ong t e r m hi s t or y of a g r i c ul t ur a l m a na ge m e nt s ha r e s i m i l a r i t i e s de s pi t e di f f e r e nc e s i n pl a nt c om pos i t i on, s oi l t ype a nd m a na ge m e nt p r a c t i c e s I n t he phyt o r e m e di a t i on s c e na r i o, m i c r oor ga ni s m s r e s pons i bl e f or t he b r e a kdow n of c ont a m i na nt s a r e o f s pe c i a l i nt e r e s t M e t ha not r ophs ( a e r obi c m e t ha ne oxi di z i ng ba c t e r i a ) pr e s e nt i n t he r hi z os phe r e o f pl a nt s a r e k now n f or t he i r c a pa bi l i t i e s t o de gr a de a

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113 va r i e t y of xe nobi ot i c c om pounds a nd t hus pl a y a n i m por t a nt r o l e i n c hl or i na t e d c om pound de gr a da t i on ( H a ns on a nd H a ns on, 1996 ; B r i gm on e t a l 1999 ) T he m e t ha not r oph c om m uni t y c om pos i t i on a nd s t r uc t u r e of a s oi l ha ve be e n s how n t o be a f f e c t e d by l a nd us e s oi l t ype pl a nt t ype w a t e r c o nt e nt a nd m a na ge m e nt p r a c t i c e s ( D ube y a nd S i ngh 2001; R e a y e t a l 2001 ; K ni e f e t a l 2005; S e ghe r s e t a l 2005) H ow e ve r i n s a t ur a t e d ha bi t a t s ot he r f a c t or s s uc h a s pl a nt t ype a nd t he i r a bi l i t y t o t r a ns por t oxyge n t o t he r hi z os phe r e a l ong w i t h t he l oc a t i on of t he s oi l w a t e r i n t e r f a c e ga i n m or e i m po r t a nc e i n de t e r m i ni ng t he m e t ha not r oph c o m m uni t y ( K i ng 1994; M a c a l a dy e t a l 2002 ) A ddi t i ona l l y t he m e t ha not r oph a bunda nc e va r i e s a c c or di ng t o t he s oi l c om pa r t m e nt H i ghe r num be r s ha ve be e n obs e r ve d i n t he r h i z os phe r e c om pa r e d t o t he bul k s oi l ( B r i g m on e t a l 1999) I m pr ove d p r a c t i c e s i n phyt or e m e di a t i on s ys t e m s ha ve i nc l ude d pl a nt a nd c l one t ype s e l e c t i on, m a ni pul a t i on o f t he pl a nt i ng m e t ho d, a nd a ddi t i on of s oi l a m e ndm e nt s ( M c C ut c he on a nd S c hnoor 2003; N e gr i e t a l 200 3; R oc kw ood e t a l 2005 ) T o t r e a t s i t e s c ont a m i na t e d w i t h c hl or i na t e d s ol ve nt s phyt or e m e di a t i on s ys t e m de s i gns ha ve r a nge d f r om na t ur a l s e t t i ngs ( na t ur a l a t t e nua t i on ) t o c om pl e t e l y e ngi ne e r e d s ys t e m s w i t h c oni f e r s popl a r s a nd w i l l ow s t r e e s ( M c C ut c he on a nd S c hnoor 2003; N e gr i e t a l 2003) I n t he e ngi ne e r e d s ys t e m s t r e e c ut t i ngs a r e c a s t i n t he s oi l t o r e s t r i c t w a t e r us a ge t o t he c ont a m i na t e d gr oundw a t e r a nd p r om ot e de e p r oot pr ol i f e r a t i on A l s o, t r e e s c a n be pl a nt e d i n t r e nc he s w i t h t he i nc or po r a t i on of s oi l a m e ndm e nt s r e s ul t i ng i n a hom oge ni z e d s oi l r i c h i n nut r i e nt s T he i m pa c t of t he s e phyt or e m e di a t i on pr a c t i c e s ha ve not ye t be e n f ul l y c ha r a c t e r i z e d, e s pe c i a l l y i n t e r m s of t he i r e f f e c t on r hi z os phe r e m i c r obi a l popul a t i ons a nd t he i r bi ode g r a da t i on pot e nt i a l

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114 T hi s s t udy de s c r i be s t he r hi z os phe r e m e t ha not r oph c om m uni t y o f t h r e e di f f e r e nt phyt or e m e di a t i on s e t t i ngs a s a m e a ns of c om pa r i n g t he i r r hi z ode gr a da t i on pot e nt i a l T he phyt or e m e di a t i on s e t t i ngs r a nge d f r om a na t ur a l r e ve ge t a t e d a r e a t o a n e ngi ne e r e d s ys t e m l oc a t e d i n t w o d i f f e r e nt s i t e s t ha t va r y s i gni f i c a n t l y by s oi l t ype c l i m a t e z one a nd pl a nt t ype M or e s pe c i f i c a l l y, t hi s s t udy de s c r i be s t he a bunda nc e a c t i vi t y, a nd phyl oge ne t i c c om pos i t i on of t he m e t ha not r oph c o m m uni t y by c ul t ur e ba s e d m e t hods m i c r obi a l c ount s a nd e nr i c hm e nt s c om bi ne d w i t h c ul t ur e i nde pe nde nt m ol e c ul a r a na l ys i s of phyl oge ne t i c a nd f unc t i ona l ge ne s by de na t u r i n g gr a di e nt ge l e l e c t r ophe r e s i s ( D G G E ) a nd s t a bl e i s ot ope pr obi ng m i c r oc os m s ( S I P ) T he ul t i m a t e goa l of t hi s w or k i s t o e s t a bl i s h f or e a c h phyt or e m e di a t i on s e t t i ng t he m a i n f a c t or s i nf l ue nc i ng m e t ha not r ophi c r hi z ode gr a da t i on pot e nt i a l t o f u r t he r i m pr ove phyt or e m e di a t i on e f f i c i e nc y. M at e r i al s an d M e t h od s S i t e D e s c r i p t i on T w o S upe r f und s i t e s c ont a m i na t e d w i t h c hl or i na t e d s ol ve nt s w e r e s t udi e d. A na t ur a l a t t e nua t i on s e t t i ng a t C A r e a of t he S a va nna h R i ve r S i t e ( S R S ) A i ke n S C a nd t w o di f f e r e nt e ngi ne e r e d s ys t e m s a t t he f o r m e r L a S a l l e E l e c t r i c U t i l i t i e s L a S a l l e I L ( F i g. 5 1) T he pr i m a r y c l i m a t e a nd s oi l c ha r a c t e r i s t i c s of e a c h phyt or e m e di a t i on s i t e a r e pr e s e nt e d i n T a bl e 5 1 a nd T a bl e A 1 ( A ppe ndi x) r e s pe c t i ve l y. A de t a i l e d de s c r i pt i on of e a c h s i t e a nd i t s phyt or e m e di a t i on de s i gn f ol l ow s S R S S C T he S R S ( 80 289 ha ) w a s c ons t r uc t e d i n 1950s by t he U S D O E f o r t he pr oduc t i on of ba s i c m a t e r i a l s us e d i n nuc l e a r de f e n s e pr ogr a m s P r oduc t i on c e a s e d i n 1988 w i t h s uf f i c i e nt c he m i c a l a nd r a di oa c t i ve w a s t e t ha t r e s ul t e d i n s oi l a nd g r oundw a t e r c ont a m i na t i on. T he s t udy w a s l oc a t e d i n a na t u r a l r e ve ge t a t e d a r e a dom i na t e d by 20 ye a r ol d l obl ol l y pi ne t r e e s ( P i nus t ae da ) w he r e a T C E pl um e ( 6 20 ppb) r e s ur f a c e s f r om

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115 t he c ont a m i na t e d gr oundw a t e r i nt o t he F our M i l e B r a nc h C r e e k. V i nyl c hl or i de a nd c i s 1, 2 di c hl or oe t hyl e ne ( c i s D C E ) bot h pos s i bl e T C E a na e r obi c m e t a bol i t e s ha ve a l s o be e n de t e c t e d i n t he a r e a ( P uns hon e t a l 2002) L aS al l e I L T he f or m e r L a S a l l e E l e c t r i c a l U t i l i t i e s ( 4 ha ) m a nu f a c t ur e d c a pa c i t or s f r om 1943 unt i l 1982, w he n i t f i l e d ba nk r upt c y. T he a c t i vi t y c a us e d m a j or c ont a m i na t i on of s ur f a c e s oi l s a nd gr ou ndw a t e r w i t h pol yc hl or i na t e d bi phe nyl s a nd c hl or i na t e d s ol ve nt s T w o phyt or e m e di a t i on pl ot s w e r e i m pl e m e nt e d t o e nha nc e c hl or i na t e d s ol ve nt r e m ova l a f i na l s t a ge of t he c l e a nup pr oc e s s T he f i r s t pl o t ( 0 25 ha ) c ont a m i na t e d w i t h T C E w a s i ns t a l l e d i n S e pt e m be r 2002 ( l a be l e d a s T C E S i t e ) P opl a r a nd w i l l ow t r e e s w e r e pl a nt e d by l ow e r i ng 1. 8 m r oot e d w hi ps t o t he bot t om of 0 6 m di a m e t e r bor e hol e s l i ne d w i t h hi gh de ns i t y pol ye t hyl e ne p i pe a nd f i l l e d w i t h a n e qua l m i x of s a nd, s oi l ba r k, a nd pe a t ( pH 7. 8) ( T a bl e 5 1) L ow a nd hi gh c onc e nt r a t i on r e gi ons e x i s t a t t he s i t e e xhi bi t i ng a br oa d r a nge o f T C E c onc e nt r a t i ons ( 0 254 ppb) ( F i g 5 1 ) I n e a c h r e gi on 18 popl a r a nd 24 w i l l ow c l one s w e r e pl a nt e d. T he s e c ond phyt or e m e di a t i on pl ot ( 0 21 ha ) c ont a m i na t e d w i t h P C E w a s e s t a bl i s he d i n M a r c h 2002 ( P C E S i t e ) T he s oi l ( pH 7. 3) w a s i m p r ove d by i nc or por a t i ng a m ul c h c om p os e d of t r e e c hi ps on t he t op 0. 5 m o f t he s oi l s ur f a c e a nd popl a r p i ne a nd w i l l ow t r e e s w e r e pl a nt e d di r e c t l y i nt o t he s oi l T hi s s i t e a l s o pr e s e nt s a c onc e nt r a t i on gr a di e nt w h e r e hi gh a nd l ow P C E r e gi ons ha ve be e n de l i ne a t e d ( 0 838 ppb ) ( F i g. 5 1 ) A t bot h ph yt or e m e di a t i on pl ot s t he r e i s e vi de nc e t ha t s uppor t s t r e e upt a ke o f t he c ont a m i na t e d gr ou ndw a t e r a pa r t f r om c om pl e t e br e a kdow n of t he c hl or i na t e d c om pounds ( R L a ng e a nd J I s e br a n ds pe r s ona l c om m uni c a t i on) S i nc e 2002 c ont a m i na nt c onc e nt r a t i ons ha ve de c r e a s e d s ubs t a nt i a l l y. I n 2005, T C E c onc e nt r a t i ons w e r e r e duc e d f r om 2 54 t o 11 ppb a nd P C E f r om 838 t o 300

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116 ppb. O nl y one a na e r obi c m e t a bol i t e of de gr a da t i o n ha s be e n de t e c t e d a t t he s i t e ( c i s D C E ) a nd i t s c onc e nt r a t i on ha s a l s o de c r e a s e d w i t h t i m e ( 353 t o 3. 6 ppb) N e ve r t he l e s s i ns uf f i c i e nt da t a e xi s t t o a c c ur a t e l y a s s e s s t he r ol e of phyt or e m e di a t i on be c a us e a dua l s ol i d pha s e w a t e r e xt r a c t i on s ys t e m i s a l s o ope r a t e d a t t he s i t e ( L a nge 2004) S am p l i n g S oi l s a m pl i ng oc c ur r e d i n O c t obe r 2003 a t S R S a n d i n J ul y 2003 J ul y 2004 a nd N ove m be r 2004 a t L a S a l l e A t t he S R S t h r e e pi ne t r e e s w e r e s a m pl e d w i t h a l a r ge di a m e t e r ( 7. 6 c m ) ha nd s oi l a uge r a l l i n t he s a m e di r e c t i on ( w e s t ) a nd a t a ppr oxi m a t e l y 1. 5 m f r om t he t r e e ba s e T he s a m pl e d a r e a pr e s e nt s a de c l i ni ng s l ope t ow a r ds t he s e e pl i ne out f l ow ; c ons e que nt l y, t r e e s w e r e de s i gna t e d a s pi ne 1 t o 3 ( pi ne 1 a t t he t op of t he gr a di e nt a nd p i ne 3 a t t he bot t o m ) ( F i g 5 1 ) P i ne 2 a nd pi ne 3 w e r e unde r s a t ur a t e d c ondi t i ons dur i ng s a m pl i ng ( > 30 c m de pt h ) T o t e s t t he e f f e c t o f s a m pl i ng de pt h t he c om pl e t e s oi l pr of i l e i nf l ue nc e d by t he r oot s ys t e m w a s s a m pl e d a t de pt hs of 0 15, 15 30 30 60, 60 90 a nd 90 120 c m A c ont r ol ne xt t o t he r e ve ge t a t e d a r e a w a s a l s o s a m pl e d. A t t he L a S a l l e s i t e t r e e s w e r e s a m pl e d a t a ppr oxi m a t e l y 0. 3 m f r om t he t r e e ba s e w i t h a s m a l l di a m e t e r ( 1 9 c m ) ha nd s oi l a uge r t o m i ni m i z e s oi l di s t ur ba nc e T he s a m e s oi l de pt hs a s a t t he S R S w e r e s a m pl e d; how e ve r t he f i r s t s a m pl i ng ( J ul y 2003) onl y e xt e nde d t o t he 60 90 c m de pt h due t o l i m i t e d r oot e xpa ns i on. B e c a us e of l i m i t e d s a m pl e d a r e a a t t he P V C pot s a nd t o c ol l e c t e nough r hi z os phe r e m a t e r i a l t o a na l yz e d, t he f i r s t t w o s oi l l a ye r s t ha t w e r e 15 c m i n de pt h ( 0 15 a nd 15 30 c m ) c ons i s t e d of a c om pos i t e s a m pl e f r om a l l f our c a r di na l po i nt s a r o und t he t r e e ba s e T he r e s t o f s oi l l a ye r s s a m pl e d ( 30 c m i n de pt h) w e r e c ol l e c t e d f r o m t w o oppos i t e l oc a t i ons a r ound t he t r e e ba s e t o obt a i n t he s a m e s a m pl e d vol um e pe r s oi l l a ye r

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117 T o de t e r m i ne i f di f f e r e nc e s e xi s t a m ong t he s upe r i or t r e e ge not ype s ( c l one s w i t h vi gor ous a bove gr ound gr ow t h ) a t t he T C E S i t e on e popl a r c l one ( c l one I 45/ 51 ; P op ul us de l t oi de s x P ni gr a ; o r i gi n, N or t h A m e r i c a x E ur o pe ) a nd t hr e e w i l l ow c l one s w e r e s t udi e d ( c l one S X 61, Sal i x s ac hal i ne ns i s J a pa n, e xot i c ; c l one S 365 S di s c ol or 18, U ni ve r s i t y of T or ont o; c l one 94014 S pu r pur e a S t a t e U ni ve r s i t y of N e w Y or k e xot i c ) A ddi t i ona l l y, a non pl a nt e d s a m pl e w a s t a ke n f r o m a n e s t a bl i s he d bor e hol e t h a t w a s not pl a nt e d. A t t he P C E S i t e t he s a m e popl a r c l one I 4 5/ 95 w a s s a m pl e d a l ong w i t h a non pl a nt e d s a m pl e r e m ove d f r om out s i de t he c ont a m i na t e d pl ot i n a gr a s s y a r e a ( s e r vi ng a s a c ont r ol ) T hi s c ont r ol a r e a w a s t r a ns f or m e d t o a w e l l m a i nt a i ne d s oc c e r f i e l d be t w e e n J ul y 2003 a nd 2004 I n o r de r t o obs e r ve e f f e c t s of t he de gr e e of T C E a nd P C E c onc e nt r a t i on, s a m pl i ng w a s c onduc t e d i n bot h t he l ow a nd hi gh c onc e nt r a t i on r e gi ons a t e a c h s i t e ( F i g. 5 1) T o pr e ve nt c r os s c ont a m i na t i on, t he a uge r w a s w a s he d w i t h s t e r i l e w a t e r r i ns e d w i t h 95% e t ha nol a nd w a s he d a ga i n s e ve r a l t i m e s S a m pl e s w e r e c ol l e c t e d i n s t e r i l e 0. 025 m m ba gs ( N a s c o W hi r l P a k F o r t A t k i ns on, W I U S A ) pl a c e d on i c e t r a ns por t e d t o t he U ni ve r s i t y o f F l or i da w i t hi n 1 t o 2 da ys a nd s t or e d a t 4 C I n or de r t o c om pa r e a c t i vi t y a nd di ve r s i t y i n t he s oi l ( i n t he r hi z os phe r e ; de not e d a s R H ) i n t he s oi l a dhe r e d t o t he r oo t s ( i n t he r hi z opl a ne ; de not e d a s R P ) a nd i n s oi l no t i nf l ue nc e d by t he p l a nt s ( i n t he non pl a nt e d s oi l ; de not e d a s N P ) s a m pl e s w e r e hom oge ni z e d us i ng a s t e r i l e s pa t ul a a nd f i ne r oo t s ( < 2 m m i n di a m e t e r ) w e r e s e pa r a t e d f r om t he s oi l f or s e pa r a t e t e s t i ng. S oi l C h ar ac t e r i z at i on S oi l a na l ys i s w a s c onduc t e d a t t he A na l yt i c a l R e s e a r c h L a bor a t or y i n t he I ns t i t ut e of F ood a nd A g r i c ul t u r a l S c i e nc e ( I F A S ) a t t he U n i ve r s i t y of F l or i da W a t e r e xt r a c t a bl e P C a M g, a nd C u, a nd K C l e xt r a c t e d a m m oni a w e r e a na l yz e d by i nduc t i ve l y c oupl e d

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118 pl a s m a ( I C P ) i n c om bi na t i on w i t h c ol or i m e t r i c a na l ys i s f or P de t e r m i na t i on O r ga ni c m a t t e r w a s a na l yz e d by l os s on i gni t i on a nd w a t e r c ont e nt by t he gr a vi m e t r i c m e t hod. S oi l pH w a s r e s ol ve d i n de i oni z e d w a t e r a nd t he s oi l f i e l d c a pa c i t y w a s qua l i t a t i ve l y de t e r m i ne d ( M yl a va r a pu a nd K e nne l l e y, 2002) T he a na l ys i s of t he s a m pl e s w a s c onduc t e d i n t w o s e pa r a t e a ppr oa c he s by t r a di t i ona l c ul t ur e de pe nde nt m e t hods m i c r obi a l c ount s a nd e nr i c hm e nt s a nd, by a c ul t ur e i nde pe nde nt m e t hod, s t a bl e i s ot ope pr obi n g ( S I P ) us i ng s oi l m i c r oc os m s A ddi t i ona l l y, phyl oge ne t i c a na l ys i s of t he e nr i c hm e nt s a nd S I P m i c r oc os m s w e r e pe r f or m e d t o c ha r a c t e r i z e t he s t udi e d m i c r obi a l po pul a t i ons w i t hi n e a c h a ppr oa c h. M i c r ob i al C ou n t s H e t e r ot r ophs a nd m e t ha not r ophs p r e s e nt i n s oi l a n d r oot s a m pl e s w e r e e num e r a t e d by c ol ony f o r m i ng uni t s ( C F U ) a nd m os t p r oba bl e num be r t e c hni q ue s ( M P N ) r e s pe c t i ve l y. C ount s w e r e pe r f or m e d a s de s c r i be d by B a ke r e t a l ( 2001 ) a nd W oom e r ( 1994) B r i e f l y, 1 g w e t w e i ght s oi l or r oot s a m pl e w e r e pl a c e d i n 9 m l of phos pha t e buf f e r ( 10 m M ; pH 7. 0 ) a nd s ha ke n w i t h 0. 8 g gl a s s be a ds ( 0. 5 m m di a m e t e r ) a t 250 r pm a nd 30 C f or 20 m i n t o di s a ggr e ga t e pa r t i c l e s f or s ubs e que nt di l ut i ons S e r i a l di l u t i ons w e r e pl a t e d i n t r i pl i c a t e on t r ypt i c s oy a ga r pl a t e s ( 1/ 10 s t r e ngt h) a nd i nc uba t e d a t 30 o C f or 1 w e e k be f or e c ount i ng c ol oni e s M P N vi a l s ( 10 6 di l ut i ons ) i n t r i pl i c a t e w e r e i noc ul a t e d w i t h di l ut i on a l i quot s i nt o ni t r a t e m i ne r a l s a l t s m e di um ( N M S ) w i t h 10 M C u( N O 3 ) 2 a nd 20 % ( v/ v) C H 4 ( W hi t t e nbur y e t a l 1970) V i a l s w e r e i nc uba t e d f or a m ont h a t r oom t e m pe r a t u r e a nd gr ow t h w a s r e c or de d a s pos i t i ve w he r e t ur bi di t y w a s obs e r ve d t o i nc r e a s e M i c r obi a l c ount s w e r e a dj us t e d t o a d r y w e i ght s oi l ba s i s a nd w he n l e s s t ha n 1 g r oot m a t e r i a l w a s a va i l a bl e

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119 C h ar ac t e r i z at i on of E n r i c h m e n t s T he l ow e s t pos i t i ve di l ut i on f r om t he M P N t e c hni q ue w a s s ubs e que nt l y c ul t ur e d i n N M S w i t h a nd w i t hout C u, i nc uba t e d on a r e c i pr oc a l s ha ke r ( 250 r pm ) a t 30C w i t h 20% ( v/ v) C H 4 f or m or e t ha n t w o m ont hs M os t of t he c ha r a c t e r i z a t i on of t he e nr i c hm e nt s w a s r e s t r i c t e d t o e nr i c hm e nt s obt a i ne d f r om t he 30 60 c m s oi l l a ye r t o l i m i t s a m pl e s t e s t e d t o a m a na ge a bl e num be r E nr i c hm e n t s w e r e c ha r a c t e r i z e d by gr ow t h c ur ve s s M M O a c t i vi t y, a nd oxyge n upt a ke i n t he pr e s e nc e of C H 4 a s f o l l ow s G r ow t h c u r ve s T he c ul t u r e m e di um w a s i noc ul a t e d w i t h t he c o r r e s pondi ng e nr i c hm e nt t o a n i ni t i a l op t i c a l de ns i t y of 0. 035 a t 600 nm a nd i nc uba t e d a t 250 r pm a nd 30 C w i t h 20% ( v/ v) C H 4 C ul t u r e s i n t r i pl i c a t e s a nd a ne ga t i ve c ont r ol ( no C H 4 a dde d) w e r e us e d t o c ons t r uc t c ur ve s a nd de t e r m i ne e xpon e nt i a l gr ow t h r a t e s by m oni t o r i ng t ur bi di t y a t 600 nm i n a s pe c t r ophot om e t e r ( S pe c t r oni c 21, M i l t on R oy C om pa ny, P A U S A ) s M M O as s ay. P r e s e nc e a nd a c t i vi t y of s ol ubl e m e t ha ne m onooxyge na s e ( s M M O ) w a s qua l i t a t i ve l y ve r i f i e d by a na pht ha l e ne a s s a y m odi f i e d f r om B r us s e a u e t a l ( 1990 ) T r i pl i c a t e s of t he a c t i ve t e s t c ul t u r e a nd c ont r ol s w e r e i nc l ude d a s de s c r i be d by L i ndne r e t a l ( 2002 ) C ont r ol s i nc l ude d one he a t ki l l e d, one c e l l f r e e a nd one c ul t u r e d w i t h l i ve c e l l s i n t he pr e s e nc e of 1 M C uS O 4 # 6H 2 O know n t o r e p r e s s s M M O s ynt he s i s ( P r i or a nd D a l t on, 1985) T he i nt e ns i t y of t he c ol or i ndi c a t e d t he de gr e e o f s M M O a c t i vi t y. O xyge n u p t a k e e xp e r i m e n t s T he o xi da t i on pot e nt i a l of t he e nr i c hm e nt s w a s de t e r m i ne d by m e a s ur i ng oxyge n upt a ke i n t he pr e s e nc e of C H 4 e m pl oyi ng a C l a r ke e l e c t r ode a nd a n a ut om a t e d da t a a c qui s i t i on s ys t e m a s de s c r i be d by L i ndne r e t a l ( 2000 ) T he s ubs t r a t e w a s i nt r oduc e d t o t he t e s t c ul t u r e by bubbl i ng C H 4 ( 99 9% ; A i r c o B O C

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120 M ur r a y H i l l N J U S A ) f o r 5 m i n i nt o s t e r i l e w a t e r a nd i m m e di a t e l y i nj e c t i ng 40 0 l i nt o t he r e a c t or I n t hi s s t udy, t he r e a c t or w a s s e t a t r oo m t e m pe r a t ur e ( $ 25C ) T r i pl i c a t e c ur ve s w e r e c onduc t e d pe r s a m pl e pl us a ne ga t i ve c ont r ol c ur ve p r e pa r e d by a ddi ng 4 m l of a c e t yl e ne ( 99. 6 % P r a xa i r D a m bu r y, C T U S A ) a n M M O i nhi bi t o r ( P r i o r a nd D a l t on 1985) i n t he p r e s e nc e of C H 4 P ot e nt i a l r a t e s of o xi da t i on w e r e c a l c ul a t e d by r e gr e s s i on a na l ys i s of t he i ni t i a l l i ne a r po r t i on o f t he c ur ve a f t e r a c c ount i ng f o r e ndoge nous m e t a bol i s m D N A e xt r ac t i on G e n om i c D N A w a s e xt r a c t e d f r om 10 m l of e nr i c hm e nt c ul t u r e w i t h a c om m e r c i a l m i c r obi a l D N A i s ol a t i on ki t ( M o B i o L a b. C a r l s ba d, C A U S A ) T he e nr i c hm e nt s f r om a l l t i m e s a m pl e s r e m ove d f r o m t he L a S a l l e T C E S i t e popl a r t r e e a nd t he non pl a nt e d s a m pl e w e r e a na l yz e d, w he r e a s on l y t he N ove m be r 2 004 s a m pl e s r e m ove d f r om t he w i l l ow c l one S 365 a nd P C E S i t e popl a r t r e e w e r e a na l yz e d. D N A t e m pl a t e s w e r e s t or e d a t 40C unt i l t he a na l ys i s Q ua nt i f i c a t i on a nd pur i t y of D N A t e m pl a t e s w a s de t e r m i ne d by U V s pe c t r ophot om e t r y a s de s c r i be d by A us ube l e t a l ( 1992) S t ab l e I s ot op e P r ob i n g ( S I P ) S oi l M i c r oc os m s T he S I P pr ot oc ol w a s pr e vi ous l y de s c r i be d i n C ha pt e r 4. B r i e f l y, 10 g w e t w e i ght of s oi l ( 16 % w a t e r c ont e nt ) f r om t he 30 60 c m s oi l l a ye r a nd h i gh c ont a m i na nt r e gi on w e r e pl a c e d i n 160 m l s e r um vi a l s a nd 10 m l of 1 3 C H 4 ( 99. 9 % I s ot e c M i a m i s bur g, O H U S A ) w e r e a s e pt i c a l l y a dde d ( M or r i s e t a l 2002; R a da j e w s ki e t a l 2002) V i a l s w e r e i nc uba t e d i n t he da r k a t r oom t e m pe r a t u r e H e a ds pa c e C H 4 de pl e t i on w a s m oni t or e d e ve r y 2 5 da ys by ga s c hr om a t ogr a phy. A f t e r m or e t ha n 90% of t he C H 4 w a s c ons um e d, vi a l s w e r e ope ne d, ge nt l y f l us he d w i t h a i r f or 5 s r e s e a l e d, a nd r e pl e ni s he d w i t h 1 3 C H 4

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121 T he pr oc e dur e w a s r e pe a t e d f i ve t i m e s ( R a da j e w s k i e t a l 2002 ) I ni t i a l C H 4 de pl e t i on r a t e s w e r e c a l c ul a t e d f r om da t a t a ke n du r i ng i nc ub a t i on a f t e r t he f i r s t C H 4 a ddi t i on by l i ne a r r e gr e s s i on a na l ys i s of t he c ons um pt i on c ur v e D N A e xt r a c t i on f r om m i c r oc os m s oi l s w a s pe r f or m e d us i ng t he P ow e r M a x S oi l D N A E xt r a c t i on K i t ( M o B i o L a b C a r l s ba d, C A U S A ) T he D N A e xt r a c t s w e r e r e s o l ve d by C s C l de ns i t y gr a di e nt c e nt r i f uga t i on ( B e c km a n V T i 65 r o t or 6 h 20C 2 65 000 x g) ( S a m br ook e t a l 1989; R a da j e w s ki e t a l 2002) T h r e e f r a c t i ons w e r e ge n e r a l l y c ol l e c t e d a s t he f o l l ow i ng: ( 1) a l i ght D N A uppe r ba nd ( 1 2 C D N A ) ; ( 2) a m i ddl e ba nd, c o m bi ne 1 2 C a nd 1 3 C D N A ; a nd ( 3) a he a vy D N A l ow e r ba nd ( 1 3 C D N A ) D N A f r a c t i ons w e r e pur i f i e d a s de s c r i be d by S a m br ook e t a l ( 1989) P h yl oge n e t i c A n a l ys i s of E n r i c h m e n t s an d S I P M i c r oc os m s P r i m e r s e t s an d p ol ym e r as e c h ai n r e ac t i o n ( P C R ) am p l i f i c at i on D N A e xt r a c t e d f r om e nr i c hm e nt s a nd S I P m i c r oc os m s w a s us e d a s t e m pl a t e s f or P C R a m pl i f i c a t i on a s pr e vi ous l y de s c r i be d ( C ha pt e r 4) T he phyl oge ne t i c a na l ys i s w a s pe r f or m e d w i t h t he 16 S r D N A pr i m e r s e t 533f / 907 r t a r ge t i ng a l l l i f e ( H e nc ke l e t a l 1999) T he f unc t i ona l pm o A p r i m e r s e t A 189f / m b 661 w a s us e d t o s pe c i f i c a l l y t a r ge t t he pM M O a c t i ve s i t e ( C os t e l l o a nd L i ds t r om 1999) G C c l a m ps w e r e a t t a c he d t o t he 907 r 16S r D N A p r i m e r a nd A 189f pm oA pr i m e r a s de s c r i be d by H e nc ke l e t a l ( 1999) P C R r e a c t i ons a nd a m pl i f i c a t i on p r ot oc ol f or t he 16S r D N A a nd pm oA ge ne w e r e f ol l ow e d a c c or di ng t o H e nc ke l e t a l ( 1999) a nd K ni e f e t a l ( 2003) r e s pe c t i ve l y. P C R p r oduc t s w e r e ve r i f i e d by 1. 5 % a ga r os e hor i z ont a l e l e c t r oph or e s i s P C R pr oduc t s w e r e s e pa r a t e d by D G G E i n t he D C ode S ys t e m ( B i o R a d L a b. H e r c ul e s C A U S A ) a s de s c r i be d by H e nc ke l e t a l ( 1999) B r i e f l y 1 m m t h i c k 6. 5 % ( w / v) pol ya c r i l a m i de ge l s ( 37 5: 1 a c r yl a m i de bi s a c r yl a m i de ) ( F i s he r S c i e nt i f i c

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122 P i t t s bur gh, P A U S A ) w e r e pr e pa r e d a nd e l e c t r oph or e s e i n 1X T A E bu f f e r a t 60C a nd 150 V f o r 5 h i n a 35 80 % l i ne a r de na t ur a nt g r a di e nt ( 80% de na t ur a nt r e pr e s e nt s 5. 6 M ur e a a nd 32% ( v/ v) de i oni z e d f o r m a m i de ) D i f f e r e nt c ondi t i ons f o r r unni ng t he ge l s ( 61C a nd 180V f or 5 h i n a 35 65% l i ne a r de na t ur a nt gr a di e nt ) w e r e us e d w he n w or ki ng w i t h t he pm oA pr i m e r s G e l s w e r e l oa de d w i t h 25 45 l o f t he P C R pr oduc t a c c or di ng t o a ga r os e ge l ba nd i nt e ns i t y a nd 1/ 4 vol um e o f l oa di ng buf f e r A f t e r ge l s w e r e s t a i ne d w i t h e t hi di um b r om i de a c c or di ng t o t he m a nuf a c t u r e s pr ot oc ol vi s ua l i z e d on a U V t r a ns i l l um i na t or a t 312 nm ( M ode l 88A ; F i s he r S c i e nt i f i c P i t t s bur gh, P A U S A ) a nd phot ogr a phe d w i t h t he di gi t a l pho t odoc um e nt a t i on s ys t e m D i gi D oc I T ( D a i gge r V e r non H i l l I L U S A ) D G G E ba nds w e r e e xc i s e d f r om t he m i dd l e pa r t of t he ba nd w i t h a s t e r i l e s c a l pe l a nd D N A w a s e l ut e d a c c or di ng t o t he pr ot oc ol de s c r i be d by C hor y a nd P ol l a r d ( 1999 ) T he e l ut e d D N A w a s r e a m pl i f i e d a nd r e a na l yz e d o n D G G E t o ve r i f y s a m pl e pur i t y. R e a m pl i f i c a t i on o f 16S r D N A ba n ds w a s pe r f or m e d by m odi f yi ng t he P C R pr ot oc ol t o a n a nne a l i ng t e m pe r a t ur e of 60C w i t h no t ouc hdow n p r ogr a m a nd 25 c yc l e s F or pm oA ba nd r e a m pl i f i c a t i on, t he P C R p r ot oc ol w a s c ha nge d t o a n i ni t i a l de na t ur i ng s t e p of 5 m i n a t 94C f ol l ow e d by 30 c yc l e s of 30 s a t 94C f or de na t ur a t i on 45 s a t 66C f o r a nne a l i ng ( t o a voi d s e que nc e a m bi gui t y a s r e por t e d by D unf i e l d e t a l ( 2002) ) 30 s a t 72C f or e l onga t i on a nd a f i na l e l onga t i on s t e p of 7 m i n a t 72C S e ve r a l ba nds w i t h t he s a m e m obi l i t y w e r e e xc i s e d f r om di f f e r e nt l a ne s t o c he c k f or s e que nc e i de nt i t y. S e q u e n c i n g. R e a m pl i f i e d P C R pr oduc t s w e r e pur i f i e d w i t h a c om m e r c i a l P C R pur i f i c a t i on ki t ( M o B i o L a bor a t or i e s C a r l s ba d, C A U S A ) be f or e s e que nc i ng. Q ua nt i f i c a t i on a nd pur i t y of t he P C R pr oduc t ( 1: 20 di l ut i on) w a s de t e r m i ne d a s

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123 m e nt i one d a bove ( A u s ube l e t a l 1992) P C R pr o duc t s w e r e s e que nc e d by t he I nt e r di s c i pl i na r y C e nt e r f or B i ot e c hnol ogy R e s e a r c h ( I C B R ) a t t he U ni ve r s i t y of F l o r i da i n G a i ne s vi l l e F L P h yl oge n e t i c a n al ys i s S e que nc e s w e r e c om pa r e d w i t h da t a ba s e s e que nc e s i n t he N a t i ona l C e nt e r f or B i ot e c hnol ogy I n f or m a t i on ( N C B I ) us i ng B L A S T ( A l t s c hul e t a l 1990) R e l a t e d s e que nc e s i de nt i f i e d i n B L A S T a n d s e que nc e s of e xt a nt m e t ha not r ophs w e r e m a nua l l y a l i gne d w i t h C L U S T A L X v 1 8 ( T hom ps on e t a l 1997) P hyl oge ne t i c t r e e s w e r e c ons t r uc t e d by t he ne i ghbor j o i ni ng ( N J ) m e t hod i n C L U S T A L X a nd di s pl a ye d i n T r e e V i e w v 1 6. 6 ( P a ge 1996) A l l nuc l e ot i de a c c e s s i on num be r s of t he obt a i ne d s e que nc e s w e r e pl a c e d i n t he G e nB a nk f o r f ut u r e a c c e s s ( A Y X X X A Y X X X ) S t at i s t i c s M i c r obi a l c oun t s ( l og 1 0 t r a ns f or m e d) w e r e a na l yz e d f or s i gni f i c a nt di f f e r e nc e s ( P < 0. 05) a m ong s a m pl i ng pe r i ods a nd s oi l de pt hs by r e pe a t e d m e a s ur e m e nt s a nd a s pl i t pl ot A N O V A de s i gn, r e s pe c t i ve l y. A ddi t i ona l l y d i f f e r e nc e s a m ong s a m pl e m e a ns a nd be t w e e n c ont r ol s w e r e a na l yz e d by T uke y s a nd D unne t t s t e s t r e s pe c t i ve l y. A l s o, t he S t ude nt s t t e s t w a s us e d t o c om pa r e di f f e r e nc e s be t w e e n t he r hi z os phe r e a nd r hi z opl a ne s a m pl e s M e t ha ne de pl e t i on r a t e s of S I P s oi l m i c r oc os m s w e r e a na l yz e d by c om pa r i ng t he i ni t i a l s l ope s of t he l i ne a r r e gr e s s i on c ur ve s of e a c h pl a nt t ype a nd c ont r ol pe r s a m pl i ng pe r i od. W he n a s e t o f s a m pl e s s how e d no s i gni f i c a nt di f f e r e nc e s i n r a t e s a n a ve r a ge de pl e t i on r a t e w a s c a l c ul a t e d a s a n e s t i m a t e of t he i r C H 4 de pl e t i on r a t e A ve r a ge r e gr e s s i on r a t e s w e r e c om pa r e d by a m odi f i e d T uk e y s t e s t a nd di f f e r e nc e s be t w e e n t he c ont r ol s by a m odi f i e d D une t t s t e s t ( Z a r 1984) S A S s of t w a r e v. 7 ( 1998) ( S A S I ns t i t ut e I nc C a r y N C U S A ) w a s us e d f or a l l a na l ys e s

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124 A t e a c h phyt or e m e di a t i on s e t t i ng pr i nc i p a l c om po ne nt a na l ys i s ( P C A ) , w a s pe r f or m e d f or c ul t ur e de pe nde nt a nd i nde pe nde nt da t a P C A a l l ow s m ul t i va r i a t e da t a t o be c ha r a c t e r i z e d by a s m a l l e r num be r o f va r i a bl e s w i t h no bi a s f r om a pr i or i a s s um pt i ons a bout t r e a t m e nt e f f e c t s ( B a l s e r e t a l 2002 ) T o vi s ua l i z e e a c h phyt or e m e di a t i on s e t t i ng, pr i nc i pa l c om pone nt s P C 1 a nd P C 2 w e r e pl ot t e d i n a t w o d i m e ns i ona l pl o t S P S S v 8. 0. 0 ( 1997) w a s us e d a s t he s t a t i s t i c a l s of t w a r e f o r t hi s a na l ys i s ( S P S S I nc C hi c a go, I L U S A ) R e s u l t s D e s c r i p t i on of S i t e s T he t w o phyt o r e m e di a t i on s i t e s s t udi e d va r y s i gni f i c a nt l y i n c l i m a t e z one s oi l t ype a nd pl a nt e d s ys t e m T he S R S i s l oc a t e d i n a s e m i t r opi c a l c l i m a t e w i t h r a i nf a l l di s t r i but e d t hr oughout t he ye a r w i t h r e l a t i ve l y hi g h t e m pe r a t ur e s hum i di t y, a nd pr e c i pi t a t i on ( T a bl e 5 1 ) S R S s oi l s a r e a c i di c f r e que nt l y f l oode d, w i t h s ur f a c e s a nd l a ye r s a nd l oa m y s ub s oi l of c l a ys ( < 52% s a nd, 28 50% s i l t a nd 7 27 % c l a y) ( U S D A 1990) ( T a bl e 5 1 ) C l i m a t e a t t he L a S a l l e s i t e i s t e m pe r a t e w i t h l ow e r t e m pe r a t ur e s a nd a ppr oxi m a t e l y ha l f t he pr e c i pi t a t i on r e po r t e d a t S R S S oi l s i n t he L a S a l l e r e gi on pos s e s s ne ut r a l pH a nd a r e pr i m a r i l y s i l t y f or m e d i n l oe s s a nd c a l c a r e ous l a c us t r i ne s e di m e nt s ( > 80% s i l t a nd < 12% c l a y) ( U S D A 1996 ) H ow e ve r onl y t he P C E S i t e phyt or e m e di a t i on pl ot a t L a S a l l e c ont a i ne d na t i ve s oi l ( T a bl e 5 1) w he r e a s t he T C E S i t e c ont a i ne d pl a nt i ng m a t e r i a l S oi l phys i c oc he m i c a l c ha r a c t e r i s t i c s a t t he S R S w e r e i nf l ue nc e d by t he de pt h o f t he s oi l pr of i l e ( T a bl e A 1, A ppe ndi x) N ut r i e nt s a nd or ga ni c m a t t e r c ont e nt de c r e a s e d dr a s t i c a l l y be t w e e n t he 0 15 a nd 15 30 c m s oi l l a ye r a nd t he non pl a nt e d s oi l e xhi bi t e d t he l ow e s t va l ue s c om pa r e d t o t ha t of t he pl a nt e d s oi l O n t he c ont r a r y s oi l c ondi t i ons a t

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125 L a S a l l e w e r e r e l a t i ve l y hom oge ne ous t hr oughout t he s oi l pr of i l e w i t h s i m i l a r phys i c oc he m i c a l va l ue s be t w e e n pl a nt e d a nd non pl a nt e d a r e a s ( T a bl e A 1) M a c r o a nd m i c r o nut r i e nt s ( P C a M g, a nd N H 3 N ) a t t he S R S w e r e l ow e r t ha n a t L a S a l l e e xc e pt f or C u c onc e nt r a t i ons ( T a bl e A 1 ) P hos phor us w a s t he onl y s oi l nut r i e nt de t e c t e d be l ow t he l ow r a nge i n s oi l f e r t i l i t y t a bl e s ( M yl a va r a pu a nd K e nne l l e y, 2002) B ot h t he S R S a nd L a S a l l e P C E S i t e s oi l s c ont a i ne d l ow P l e ve l s a t 0. 4 1 4 a nd 0. 7 1 3, r e s pe c t i ve l y. M i c r ob i al C ou n t s S av an n ah R i v e r S i t e ( S R S ) M i c r obi a l a bunda nc e i n pi ne t r e e s va r i e d d e pe ndi ng on t he m i c r obi a l gr oup s t udi e d t r e e l oc a t i on, a nd s oi l c om pa r t m e nt ( F i g 5 2A ) H ow e ve r c ount s w e r e not s i gni f i c a nt l y a f f e c t e d by s oi l de pt h. R hi z os phe r e he t e r ot r oph c ount s ( 10 6 10 9 C F U g 1 dw s oi l ) w e r e s i gni f i c a nt l y di f f e r e nt a m ong t he t hr e e pi ne t r e e s s a m pl e d ( P < 0. 0001 n= 20) H ow e ve r t he he t e r ot r oph c ount s i n t he non pl a nt e d s oi l s a m pl e w a s not di f f e r e nt f r om P i ne 3. C ount s a pp e a r e d t o de c r e a s e a c c or di ng t o t he l oc a t i on of t he t r e e s on t he s l ope t ow a r ds t he s e e pl i ne out f l ow P i ne 1, a t t he t op o f t he s l ope e xhi bi t e d t he hi ghe s t nu m be r s w he r e a s P i ne 3, a t t he bot t om e xhi bi t e d t he l ow e s t C onve r s e l y, he t e r ot r oph c ount s i n t he r hi z opl a ne w e r e s i m i l a r a m ong t r e e s ( 10 6 10 7 C F U g 1 f r e s h r oot m a t e r i a l ) M e t ha not r oph c ount s i n t he r hi z os phe r e ( 10 1 10 4 c e l l s g 1 dw s oi l ) di d no t va r y a m ong t r e e s a nd onl y P i ne 3 c ount s di f f e r e d f r om t hos e of t he ot he r t r e e s a nd t he non pl a nt e d bul k s oi l ( P = 0. 003 n= 20 ) H ow e ve r a t t h e r hi z opl a ne m e t ha not r oph c ount s ( 10 3 10 5 c e l l s g 1 f r e s h r oo t m a t e r i a l ) w e r e s i gni f i c a nt l y di f f e r e nt ( P = 0. 0155 n= 8) I nt e r e s t i ngl y, P i ne 3 a t t he bot t om of t he s l ope ( un de r s a t ur a t e d c ondi t i ons ) s how e d t he l ow e s t num be r s of m e t ha not r ophs i n t he r hi z os phe r e ( 5 0 4 X 10 1 c e l l s g 1 dw s oi l ) but t he hi ghe s t a t t he r hi z opl a ne ( 2 5 5 X 10 4 c e l l s g 1 f r e s h r oot m a t e r i a l ) ( F i g 5 2A )

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126 L aS al l e S i t e A l t hough m i c r ob i a l a bunda nc e w a s de t e r m i ne d a t L a S a l l e i n di f f e r e nt s a m pl i ng pe r i ods ( J ul y 2003 J ul y 2004, a nd N ove m be r 2004) onl y t he N ove m be r 2004 da t a a r e pr e s e nt e d i n F i gur e 5 2 t o de s c r i be c om pa r a bl e t i m e s i n t he ye a r ( O c t obe r a nd N ove m be r ) be t w e e n phyt or e m e di a t i o n s i t e s H ow e ve r c ount s a t L a S a l l e di d not s i gni f i c a nt l y va r y w i t h t i m e de pt h o r a m o ng t he t r e e t ype s or t he non pl a nt e d s oi l D e pt h s how e d a n e f f e c t on m e t ha not r op h a bu nda nc e onl y i n t he T C E S i t e r hi z os phe r e s oi l s a m pl e r e m ove d i n t he J ul y m on t h s ( T a bl e A 2) M e t ha not r oph c ount s i nc r e a s e d f r om t he 0 15 t o t he 30 60 c m s oi l l a ye r f r om a n a ve r a ge o f 4 9 t o 5. 7 c e l l s g 1 dw s oi l A l s o, a t t he T C E S i t e a ne ga t i ve e f f e c t o n m e t ha not r oph c ount s i n t he r hi z opl a ne w a s obs e r ve d i n t he hi gh c ont a m i na nt e xpos ur e z one ( T a bl e A 2 ) A l t hough, not s i gni f i c a nt l y di f f e r e nt t hr oughout t he s t udy, w i l l ow c l one S X 61 r hi z os phe r e s a m pl e s e xhi bi t e d bot h he t e r ot r oph a nd m e t ha not r oph c oun t s t ha t w e r e pos i t i ve l y a f f e c t e d by t he hi gh c ont a m i na nt e xpos ur e a nd va l ue s t ha t w e r e hi ghe r t ha n t he non pl a nt e d s a m pl e A s s how n i n F i gur e 5 2, t he hi ghe s t nu m be r s of bo t h m i c r obi a l gr oups s t udi e d w e r e obs e r ve d a t t he L a S a l l e T C E S i t e w i t h no s i gni f i c a nt di f f e r e nc e s be t w e e n s oi l c om pa r t m e nt s ( r hi z os phe r e v e r s us r hi z opl a ne ) or a m ong t r e e t ype s o r t he non pl a nt e d s oi l a s pr e vi ous l y m e nt i one d ( F i g 5 2B ) H e t e r ot r ophs r a nge d f r om 10 8 10 9 C F U g 1 dw s oi l or f r e s h r oot m a t e r i a l a nd m e t ha not r ophs r a ng e d f r om 10 5 10 6 c e l l s g 1 dw s oi l o r f r e s h r oot m a t e r i a l T he L a S a l l e P C E S i t e ( F i g 5 2C ) s how e d a s i m i l a r pa t t e r n a s t he T C E S i t e but w i t h l ow e r num be r s of m e t ha not r oph s ( 10 2 10 5 c e l l s g 1 dw s oi l o r f r e s h r oot m a t e r i a l ) R oot B i om as s F i ne r oot ( < 2 m m di a m e t e r ) bi om a s s pr oduc t i on a nd di s t r i but i on t hr oughout t he s oi l pr of i l e d i f f e r e d f or e a c h t r e e t ype e va l ua t e d ( d a t a not s how n) A t t he S R S pi ne t r e e s

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127 unde r s a t ur a t e d c ondi t i ons s how e d m a xi m um r oot bi om a s s i n t he 15 30 c m s oi l l a ye r M e a nw hi l e t r e e s not di r e c t l y i nf l ue nc e d by t he s e e pl i ne out f l ow e xhi bi t e d a m o r e hom oge ne ous r oot di s t r i but i on i n t he s oi l pr o f i l e w i t h a m a xi m um a t t he 30 60 c m s oi l l a ye r T r e e c ut t i ngs a t L a S a l l e a f t e r 2 ye a r s of e s t a bl i s hm e nt ( J ul y 2004 s a m pl i ng pe r i od) e xhi bi t e d m a xi m um r oot bi om a s s i n t he s ur f a c e s oi l l a ye r ( 0 15 c m ) O ve r a l l f i ne r oot bi om a s s w a s hi ghe r i n t he w i l l ow t r e e s c o m pa r e d t o popl a r s A ddi t i ona l l y, t he T C E S i t e popl a r t r e e s e xhi bi t e d hi ghe r r oot bi om a s s t ha n t he P C E S i t e pop l a r t r e e s E n r i c h m e n t s A c t i vi t y E nr i c hm e nt s w e r e s uc c e s s f ul i n N M S m e di um w i t h C u a dde d f r o m a l l s oi l c om pa r t m e nt s e va l ua t e d a t bot h phyt o r e m e di a t i on s i t e s H ow e ve r a t t he L a S a l l e va r i ous e nr i c hm e nt s i n N M S m e di um w i t hout C u a dde d w e r e not obt a i ne d f r om t he r hi z opl a ne s a m pl e s r e m ove d f r o m t he hi gh T C E a nd P C E c onc e nt r a t i on r e gi ons t hr oughou t t he s t udy ( da t a not s how n) F i gur e 5 3 s how s t he a c t i vi t y of onl y t he e nr i c hm e nt s obt a i ne d f r om s a m pl e s r e m ove d dur i ng t he l a s t s a m pl i ng a t L a S a l l e O nl y t he s e L a S a l l e e nr i c hm e nt a c t i vi t i e s a r e s how n i n or de r t o c om pa r e t he r e s pe c t i ve S R S a c t i vi t i e s of e nr i c hm e nt s obt a i ne d f r om s a m pl e s r e m ove d dur i ng t he s a m e f a l l s a m pl i ng pe r i od ( N ove m be r a nd O c t obe r a t L a S a l l e a nd S R S r e s pe c t i ve l y) O ve r a l l a c t i vi t y be t w e e n r hi z os phe r e a nd r hi z opl a ne e nr i c hm e nt s w a s c om pa r a bl e M a xi m um gr ow t h r a t e s f or a l l e nr i c hm e nt s i n e a c h c ul t ur e m e di um w e r e c om pa r a bl e G r ow t h r a t e s r a nge d f r o m 0 014 t o 0. 027 h 1 a nd 0. 003 t o 0 008 h 1 i n N M S w i t h C u a nd w i t hout C u, r e s pe c t i ve l y. G r e a t e r di f f e r e nc e s w e r e obs e r ve d i n oxy ge n upt a ke r a t e s i n t he p r e s e nc e of C H 4 ( 0. 009 0. 065 a nd 0. 003 0 023 m ol O 2 s 1 f or t he N M S w i t h C u a nd N M S w i t hout C u e nr i c hm e nt s r e s pe c t i ve l y) w hi c h w a s c onf i r m e d by t he r a nge i n s M M O a c t i vi t y a s obs e r ve d by c ol or i nt e ns i t y, a s pr e vi ous l y de s c r i be d ( d a t a

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128 not s how n) E n r i c hm e nt s i n N M S w i t h C u s how e d t he hi ghe s t gr ow t h a nd oxyge n upt a ke r a t e s w he n c om pa r e d t o t he N M S w i t hout C u e nr i c hm e nt s ( F i g. 5 3 pa ne l s A C v e r s us pa ne l s D F ) H ow e ve r C H 4 oxi da t i on r a t e s w e r e a l w a ys l ow e r t ha n t he non pl a nt e d e nr i c hm e nt s ( F i g. 5 3 ) w i t h t he e xc e pt i on of P i ne 3 N M S w i t h C u r hi z os phe r e e nr i c hm e nt ( F i g 5 3A ) a nd P C E S i t e popl a r r h i z opl a ne N M S w i t hout C u e nr i c hm e nt ( F i g. 5 3F ) e a c h of w hi c h s how e d s i m i l a r r a t e s t o t ha t of t he non pl a nt e d s oi l S av an n ah R i v e r S i t e ( S R S ) A na l ys i s of e a c h pi n e t r e e r e ve a l e d t ha t e nr i c hm e nt s a c t i vi t y i n N M S w i t h C u i nc r e a s e d a c c or di ng t o t h e i r pos i t i on i n t he s l ope o f t he s a m pl e d a r e a f r o m P i ne 1 ( a t t he t op) t o P i ne 3 ( a t t he bot t o m ) ( F i g. 5 3A ) O n t he c ont r a r y N M S w i t hout C u e nr i c hm e nt s s how e d de c r e a s i ng oxi da t i on r a t e s a nd s M M O a c t i vi t y w i t h t he de c r e a s i ng s a m pl i ng a r e a s l ope ( F i g. 5 3D ) E n r i c hm e nt s f r om t he S R S s i t e ge ne r a l l y s how e d hi ghe r r a t e s c om pa r e d t o t he L a S a l l e e nr i c hm e nt s L aS al l e S i t e E n r i c hm e nt a c t i vi t y f r o m t he di f f e r e nt t r e e t ype s a t t he L a S a l l e va r i e d t hr oughout t he s t udy w i t h no de t e c t a bl e t r e n ds O nl y e nr i c hm e nt s f r o m t he T C E S i t e popl a r t r e e s c ons i s t e nt l y s how e d hi ghe r r a t e s i n t he r hi z os phe r e t ha n r hi z opl a ne ( F i g. 5 3, B a nd E ) A t t e m pt s t o e nr i c hm e nt s o m e r hi z o pl a ne s a m pl e s f r om t he h i gh c onc e nt r a t i on T C E z one i n N M S w i t hout C u m e di um w e r e not pos s i bl e pos s i bl y ne ga t i ve l y a f f e c t e d by t he hi gh c ont a m i na nt e xpos ur e ( da t a not s how n) I n bot h e nr i c hm e nt m e di a po pl a r r hi z os phe r e a nd w i l l ow c l one S 365 r hi z o pl a ne s how e d t he hi ghe s t r a t e s i n e a c h s oi l c om pa r t m e nt a na l yz e d. P opl a r s a t t he L a S a l l e P C E S i t e s how e d hi ghe r r hi z opl a ne r a t e s t ha n popl a r t r e e s a t t he T C E S i t e a nd a l l N M S w i t hout C u r hi z opl a ne e nr i c hm e nt s w e r e s uc c e s s f ul us i ng t he s e s a m pl e s

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129 A c t i vi t y of t he e nr i c hm e nt s f r om s a m pl e s t a ke n t h r oughout t he s oi l p r of i l e w a s de t e r m i ne d f or t he L a S a l l e T C E S i t e popl a r t r e e s i n N M S w i t h C u c ondi t i ons dur i ng J ul y 2003 a nd N ove m be r 2004 s a m pl i ngs ( F i g A 2 ) A s hi f t i n a c t i vi t y w a s obs e r ve d be t w e e n s a m pl i ngs I n J ul y 2003, t he hi ghe s t a c t i vi t y w a s o bs e r ve d a t t he s ur f a c e s oi l l a ye r ( 0 15 c m ) H ow e ve r i n N ove m be r 2004 r a t e s i nc r e a s e d w i t h de pt h, a nd t he a c t i vi t y of t he non pl a nt e d s oi l e xhi bi t e d a m a xi m um r a t e i n t he m i ddl e s oi l l a ye r ( 30 60 c m ) P h yl oge n e t i c s of E n r i c h m e n t s T he uni ve r s a l pr i m e r s e t w a s s uc c e s s f ul l y a m pl i f i e d f r om a l l e n r i c hm e nt s T he va r i a bi l i t y of t he P C R a nd D G G E a na l ys i s w a s l o w w i t h a l m os t i de nt i c a l pr o f i l e s obt a i ne d i n r e pl i c a t e r uns ( F i g A 3 ) D G G E pr o f i l e s f r om e a c h s i t e a nd t r e e t ype e va l ua t e d w e r e uni que a nd c ons i s t e d of m ul t i pl e b a nds ( 1 7 ba nds ) ( F i g. A 3) T he l e a s t num be r of ba nds w a s f ound i n t he r h i z opl a ne e nr i c hm e nt s a nd t he gr e a t e s t i n t he non pl a nt e d s oi l e nr i c hm e nt s S av an n ah R i v e r S i t e ( S R S ) T he S R S e nr i c hm e nt s s ho w e d D G G E pr of i l e s w i t h a gr e a t e r num be r of ba nds ( 5 7 ba nds ) c om pa r e d t o t he L a S a l l e s i t e s e nr i c hm e nt s ( F i g. A 3) B L A S T a l i gnm e nt s c onf i r m e d t ha t t he m os t a bunda nt m e m be r s of t he e nr i c hm e nt s be l onge d t o t ype I I m e t ha not r ophs R hi z os phe r e a nd non pl a nt e d e nr i c hm e nt ba nds a l i gne d w i t h M e t hy l oc y s t i s s p. s t r a i n 18 2 ( 100% s i m i l a r i t y) f r om a c ons or t i um t ha t r a pi dl y de gr a de s T C E M e a nw hi l e t he dom i na nt m e m be r i n t he r hi z opl a ne e nr i c hm e nt r e ve a l e d a 100% s i m i l a r i t y t o a n unc ul t u r e d M e t hy l os i nus s p. f r om a m i ne e nvi r onm e nt T he r e s t of t he e n r i c hm e nt ba nds a l i gne d t o s pe c i e s of Sphi ngobac t e r i a B ac t e r oi de t e s P r ot e obac t e r i a a nd unc ul t ur e d ba c t e r i a A ddi t i on a l l y, one m e t hy l ot r oph ( H y phom i c r obi um s p. ) w a s r e t r i e ve d f r om t he r hi z opl a ne e nr i c hm e nt

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130 L aS al l e S i t e T he phyl oge ne t i c a na l ys i s of T C E S i t e popl a r t r e e e nr i c hm e nt s w a s c onduc t e d t hr oughout t he s t udy a l ong w i t h t he non pl a nt e d s a m pl e s ( F i g. A 3) H ow e ve r w i l l ow c l one S 365 a nd P C E S i t e pop l a r t r e e e nr i c h m e nt s w e r e onl y a na l yz e d dur i ng t he l a s t s a m pl i ng pe r i od ( N ove m be r 2004) T he m i c r obi a l c om pos i t i on of t he T C E S i t e popl a r t r e e e nr i c hm e nt s w a s di s t i nc t i n s oi l c om pa r t m e nt a na l yz e d a nd s a m pl i ng pe r i od. A r e duc t i on i n t he c om pone nt s of t he r hi z os phe r e e nr i c hm e nt w a s obs e r ve d i n t he N ove m be r 2004 e nr i c hm e nt s ( F i g A 3) P r of i l e s f r om t he J ul y 2003 a nd 2004 e nr i c hm e nt s s how e d 5 6 ba nds w he r e a s t he N ove m be r 2004 pr o f i l e e xhi bi t e d onl y t w o ba nds T he non pl a nt e d e nr i c hm e nt pr o f i l e s s how e d no not i c e a bl e t r e nd i n s e a s ona l e f f e c t s E nr i c hm e nt s f r om bot h phyt or e m e di a t i on pl ot s a t L a S a l l e e xhi bi t e d num e r ous B L A S T a l i gnm e nt s w i t h unc ul t ur e d ba c t e r i a t ha t w e r e of pa r t i c ul a r i nt e r e s t a s m os t o f t he s e s e que nc e s be l onge d t o t he m a j or ba nds of t h e pr of i l e s O nl y t hr e e out of t he 14 e nr i c hm e nt s a na l yz e d e xhi bi t e d a s do m i na nt m e m be r s of t he i r c om m uni t y e xt a nt m e t ha not r ophs O ve r a l l m e t ha not r ophs r e t r i e ve d f r om t he L a S a l l e e nr i c hm e nt s a l i gne d ( > 98% s i m i l a r i t y ) w i t h s e ve r a l s t r a i ns of M e t hy l os i nus a nd M e t hy l oc y s t i s f r om a va r i e t y of e nvi r onm e nt s ( r i ve r f l oodpl a i n s oi l s f o r e s t s oi l s gr oundw a t e r a nd a c ons or t i um t ha t r a pi dl y de gr a de s T C E ) A ddi t i ona l l y f r om t he P C E S i t e popl a r r hi z os phe r e e nr i c hm e nt one m e t hyl ot r oph w a s r e t r i e ve d t ha t r e ve a l e d a 10 0% s i m i l a r i t y t o unc ul t u r e d M e t hy l obac t e r i um s p. S t i l l a s i n t he S R S onl y t y pe I I m e t ha not r ophs f r om t he ge nus M e t hy l oc y s t i s a nd M e t hy l os i nus w e r e de t e c t e d a t L a S a l l e I n ge ne r a l a l l e nr i c hm e nt s f r om t he di f f e r e nt phyt or e m e di a t i on s e t t i ngs e xhi bi t e d a s m a j or phyl a P r ot e obac t e r i a ( 44 58 % ) B ac t e r oi de t e s ( 13 33% ) a nd un c ul t ur e d

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131 ba c t e r i a ( 8 38% ) ( F i g. 5 4 ) I n t e r e s t i ngl y, t he m a j or c l a s s of B ac t e r oi de t e s a t L a S a l l e w a s F l av obac t e r i a a nd, a t S R S Sphi ngobac t e r i a T he phyl oge ne t i c t r e e c onf i r m e d t hi s gr oupi ng; how e ve r i t di d not s how a ny ot he r s pe c i f i c c l us t e r i ng a c c or di ng t o s oi l c om pa r t m e nt t r e e t ype s i t e or phyt or e m e di a t i on s e t t i ng ( F i g A 4 ) I n e a c h m a j or phyl oge ne t i c gr oup, a l l phyt o r e m e di a t i on s e t t i ngs w e r e r e pr e s e nt e d by s e que nc e s of t he i r e nr i c hm e nt s T he s e que nc e s t ha t a l i gne d w i t h unc ul t ur e d ba c t e r i a m os t l y f r om t he L a S a l l e e nr i c hm e nt s w e r e pl a c e d i n i t s m a j or i t y w i t hi n B ac t e r oi de t e s S I P S oi l M i c r oc os m s A s pr e vi ous l y r e por t e d ( C ha pt e r 4) S I P m i c r oc os m s w e r e e f f e c t i ve l y i m pl e m e nt e d t o r hi z os phe r e s t udi e s of phy t or e m e di a t i on s e t t i ngs I n t h i s s t udy, t he t e c hni que i s f u r t he r a ppl i e d t o a di f f e r e nt s ys t e m t he S a va nna h R i ve r S i t e w hi c h pr e s e nt s t he s a m e c ont a m i na nt s c e na r i o i n a m o r e na t ur a l s e t t i ng c om pa r e d t o t he e ngi ne e r e d L a S a l l e pl ot s S I P ac t i v i t y I n i t i a l C H 4 de pl e t i on r a t e s f r om m i c r oc os m s of e a c h phyt or e m e di a t i on s e t t i ng di d not s i gni f i c a nt l y va r y a m ong s a m pl i ng pe r i ods t r e e t ype s or w i t h t he non pl a nt e d s oi l T he onl y e xc e pt i on w a s t he P C E S i t e non pl a nt e d m i c r oc os m s t ha t s how e d i nc r e a s e d r a t e s w i t h t i m e w hi c h m a y c or r e s pond t o t he c ha nge of t hi s a r e a t o a w e l l m a i nt a i ne d s oc c e r f i e l d C ons e que nt l y, w i t hout c ons i de r i ng t he P C E S i t e non pl a nt e d s a m pl e s a n a ve r a ge C H 4 de pl e t i on r a t e w a s c a l c ul a t e d f o r e a c h phyt or e m e di a t i on s e t t i ng. A ve r a ge r a t e s s i gni f i c a nt l y di f f e r e d a m ong phy t or e m e di a t i on s e t t i ngs a nd f ol l ow e d t he o r de r L a S a l l e T C E S i t e ( 0. 52 0 06 m o l g dw 1 h 1 n= 13 ) > S R S ( 0. 28 0. 05 m ol g dw 1 h 1 n= 4) > L a S a l l e P C E S i t e ( 0. 12 0. 03 m ol g dw 1 h 1 n= 3)

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132 S I P ph y l oge n e t i c an a l y s i s S av an n ah R i v e r S i t e ( S R S ) L a be l e d D N A ( 1 3 C D N A ) s uc c e s s f ul l y s e pa r a t e d f r om t he non a c t i ve po r t i on, w a s f ur t he r pur i f i e d i n t he S R S m i c r oc os m s a s onl y w e a k ba nds w e r e i ni t i a l l y obt a i ne d f r om t he pm o A D G G E pr of i l e s S i nc e p ur i f i c a t i on a t t e m pt s f a i l e d a nd be c a us e of t he l i m i t e d a m ount of D N A t e m pl a t e n o f ur t he r pu r i f i c a t i on w a s pur s ue d. C ons e que nt l y, gi ve n t ha t a n une xpl a i ne d s m e a r o f t he P C R pr oduc t s w a s vi s ua l i z e d i n a ga r os e ge l s P C R f r a gm e nt s w e r e e xt r a c t e d f r o m t hi s ge l a nd r e a m pl i f i e d. B y us i ng t hi s pr oc e dur e de t e c t i o n of t w o ba nds i n t he pm oA D G G E pr of i l e s w a s pos s i bl e a nd t he s e pr of i l e s w e r e t he s a m e f or a l l pi ne t r e e s a nd non pl a nt e d s a m pl e s T he s e ba nds a l i gne d i n B L A S T ( 98% s i m i l a r i t y) w i t h a n unc ul t ur e d M e t hy l oc al dum s p. c l one f r om a l a ndf i l l c ove r s oi l ( T a bl e 5 2) T o c onf i r m t hi s f i n di ng, t he 16S r D N A pr i m e r s e t w a s a l s o us e d t o r e a m pl i f y t he s e D N A t e m pl a t e s T he a c t i ve ( 1 3 C D N A ) a nd non a c t i ve ( 1 2 C D N A ) f r a c t i ons of t w o S R S m i c r oc os m s w e r e e f f e c t i ve l y a m pl i f i e d w i t h t he 16 S r D N A p r i m e r s e t T hi s r e s ul t s ugge s t e d t ha t t he D N A t e m pl a t e di d not c ont a i n a ny P C R i nhi bi t i ng s ubs t a nc e s ( a s hum i c a c i ds ) D G G E p r of i l e s of t he 16 S r D N A f r a gm e nt s r e ve a l e d l ow D N A di ve r s i t y of t he r hi z os phe r e s oi l a nd di s t i nc t pr of i l e s a m ong t he pi ne t r e e s oi l s ( F i g 5 5A ) H ow e ve r t he pi ne t r e e m i c r oc om s s h a r e d t he m a j o r ba nds i n t he 1 3 C a nd 1 2 C D N A pr of i l e s T he m a j or ba nd s e que nc e d f r om t he 1 3 C D N A pr o f i l e a l i gne d i n B L A S T w i t h unc ul t ur e d P r ot e obac t e r i a f r om a pa s t ur e s oi l ( 99 % s i m i l a r i t y ) a nd, s e c ondl y, w i t h M e t hy l oc e l l a s p. B L 2 f r om a f or e s t s oi l ( 98% s i m i l a r i t y) T he phy l oge ne t i c t r e e ( F i g 5 6B ) c onf i r m e d pl a c e m e nt of t hi s s e que nc e a l ong w i t h a not he r m a j or ba nd o f t he s a m e p r of i l e w i t hi n t ype I I m e t ha not r ophs of t he ge ne r a M e t hy l oc e l l a a nd M e t hy l oc aps a A ddi t i ona l l y a m e t hyl ot r oph ( 94 % s i m i l a r i t y) c l os e l y r e l a t e d t o t he ge nus M e t hy l ophi l us w a s r e t r i e ve d

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133 f r om t he s a m e pr of i l e T hi s ba nd w a s obs e r ve d a s t he m a j or c om pone nt of t he n on a c t i ve pr of i l e s ( F i g 5 5A l a ne 1 a nd 3) w hi c h m a y s ugge s t c r os s f e e di ng of t he l a be l e d bypr oduc t s of m e t ha ne oxi da t i on ( m e t ha nol ) O ve r a l l t he dom i na nt c om pone nt of t he a c t i ve m e t ha not r oph popul a t i on a t t he S R S w a s c l o s e l y r e l a t e d t o t he ge nus M e t h y l oc e l l a ( T a bl e 5 2 ) pos s i bl y e xpl a i ni ng t he di f f i c ul t y i n o bt a i ni ng a pm oA P C R pr oduc t s i nc e t hi s ge nus doe s not e xpr e s s pM M O ( D e dys h e t a l 2000) S I P ph y l oge n e t i c an a l y s i s L aS al l e C ont r a r y t o t h e l ow D N A di ve r s i t y obs e r ve d i n t he S R S m i c r oc os m s a n a t t e m pt t o s t udy t he L a S a l l e 16S r D N A D G G E p r of i l e s r e ve a l e d D N A s m e a r s w he r e ba nds c oul d not be di f f e r e nt i a t e d, pr oba bl y due t o t he hi gh c ont e nt a nd di ve r s i t y o f t he D N A i n t he s e s oi l s ( da t a not s how n) A s a l r e a dy de s c r i be d i n C ha pt e r 4, pm o A D G G E a na l ys i s of t he a c t i ve L a S a l l e popul a t i ons r e ve a l e d t hr e e g r oups c om pos e d of hi ghl y s i m i l a r s e que nc e s a c c or di ngl y t o ba nd pos i t i on a nd B L A S T a l i gnm e nt s T w o of t he g r oups de s c r i be d un c ul t ur e d M e t hy l oc al dum s p. c l one s f r om l a ndf i l l c ove r s oi l a nd t he ot he r g r oup w a s not pos s i bl e t o a m pl i f i e d ( T a bl e 5 3 ) A ddi t i ona l l y, s e ve r a l di s t i nc t ba nds w e r e a l s o obs e r ve d t ha t a l i gne d i n B L A S T w i t h unc ul t ur e d ba c t e r i a of di ve r s e e nvi r onm e nt s ( r i c e f i e l d, upl a nd s oi l s a nd r i c e s t r a w ) T he s e ba nds c l us t e r e d i n t he phyl oge ne t i c t r e e w i t hi n M e t hy l oc oc c us a nd M e t hy l oc al dum ( T a bl e 5 3 ) A l t hough p r of i l e s a m ong t r e e t ype s w e r e c om pa r a bl e di f f e r e nc e s be t w e e n pl a nt e d a nd non pl a nt e d s a m pl e s w e r e e vi de nt N on pl a nt e d s oi l m i c r oc os m s e xhi bi t e d D G G E pr of i l e s t ha t va r i e d gr e a t l y w i t h t i m e a nd s how e d l ow r e l a t i ve a bunda nc e of i t s c om pone nt s A t t he L a S a l l e P C E S i t e popl a r t r e e p r of i l e s s hi f t e d gr e a t l y w i t h t i m e I n t he f i r s t s a m pl i ng ( J ul y 2003) onl y t h e M e t hy l oc al dum c l o ne s f ound a t t he T C E S i t e w e r e

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134 r e t r i e ve d a nd, i n t he s ubs e que nt s a m pl i ng ( J ul y 20 04) onl y unc ul t ur e d ba c t e r i a c l os e l y r e l a t e d t o t he ge nus M e t hy l oc oc c us w e r e obs e r ve d ( T a bl e 5 3 ) T he non p l a nt e d s oi l e xhi bi t e d t he s a m e M e t hy l oc al dum s p. c l one a s t he popl a r t r e e a nd a gr oup o f ba nds t ha t de s c r i be d M e t hy l oc y s t i s s p. ( 99% s i m i l a r i t y) D i f f e r e nc e s a m ong t r e e s a nd t he non pl a nt e d s oi l c oul d not be de t e r m i ne d be c a us e s om e s a m pl e s w e r e l os t dur i ng a na l ys e s O ve r a l l a do m i na nc e of t he r m ot ol e r a nt m e t ha not r ophs ( M e t hy l oc al dum a nd M e t hy l oc oc c us ) ; w a s obs e r ve d i n t he L a S a l l e T C E S i t e s a m pl e s a nd, e ve n t hough t he s a m e ge ne r a w e r e f ound a t t he L a S a l l e P C E S i t e t h e y w e r e not c ons i s t e nt l y r e t r i e ve d i n a l l s a m pl i ng pe r i ods a s obs e r ve d i n t he T C E S i t e s a m pl e s ( T a bl e 5 3 ) P r i n c i p al C o m p on e n t A n al ys i s ( P C A ) P C A t he m e a s ur e m e nt s obt a i ne d f r om t he c ul t u r e de pe nde nt m e t hods m i c r obi a l c ount s a nd e nr i c hm e nt a c t i vi t y of t he di f f e r e nt s oi l c om pa r t m e nt s e va l ua t e d s ugge s t e d t ha t t he va r i a bi l i t y a m ong d i f f e r e nt da t a s e t s w a s e xpl a i ne d by t he phyt o r e m e di a t i on s e t t i ng ( F i g. 5 6 ) T he di f f e r e nt phyt o r e m e di a t i on s e t t i ngs w e r e s e pa r a t e d by t he s e c ond a xi s ( P C 2) w hi c h e xpl a i ne d 25% of t he va r i a bi l i t y ( F i g 5 6A ) H ow e ve r t he s e gr oups w e r e not c om pl e t e l y s e pa r a t e d on t he f i r s t a xi s ( P C 1) w hi c h e xpl a i ne d a hi ghe r va r i a bi l i t y ( 38 % ) T he L a S a l l e T C E S i t e s e pa r a t e d c om pl e t e l y f r om t he S R S a nd t he L a S a l l e P C E S i t e s how e d a n i nt e r m e di a t e pos i t i on be t w e e n a c om pl e t e l y e ngi ne e r e d s ys t e m ( L a S a l l e T C E S i t e ) a nd a na t ur a l r e ve ge t a t e d s e t t i ng ( S R S ) A l s o, i t w a s obs e r ve d t ha t m os t of t he r h i z opl a ne s a m pl e s f r om t he T C E S i t e gr oupe d i n t he ne ga t i ve qua dr a nt of t he f i r s t a xi s w hi c h m a y i ndi c a t e f ur t he r di f f e r e nc e s a c c or di ngl y t o s oi l c om pa r t m e nt a na l yz e d. T he s a m e P C A r e s ul t s w e r e obt a i ne d by i nc or po r a t i ng i nt o t he a na l ys i s t he c ul t ur e i nde pe nde nt C H 4 de pl e t i on r a t e s o f t he S I P s oi l m i c r oc os m s ( F i g. 5 6B ) T hi s t i m e t he

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135 f i r s t a xi s w hi c h e xpl a i ne d 36% of t he va r i a bi l i t y, s e pa r a t e d t he L a S a l l e T C E S i t e f r om t he gr oup be t w e e n t he L a S a l l e P C E S i t e a nd S R S T he s e c ond a xi s w hi c h e xpl a i ne d 27% of t he va r i a bi l i t y, s e pa r a t e d c om pl e t e l y t he T C E S i t e f r om t he S R S but a ga i n t he P C E S i t e di d not s e pa r a t e f r om e i t he r t he T C E S i t e or t he S R S T he P C E S i t e gr oupe d i n one a xi s w i t h t he S R S da t a a nd i n t he ot he r w i t h t h e T C E S i t e D i s c u s s i on T he t hr e e s t udi e d phy t or e m e di a t i on s e t t i ngs r e p r e s e nt di f f e r e nt r e m e di a t i on s t r a t e gi e s i n di s t i nc t e nvi r onm e nt s H ow e ve r t he a bi l i t y t o di s c e r n m e a ni ngf ul t r e nds i n popul a t i ons a t t he s e s i t e s va r i e d w i t h t he m e t hod u s e d. M i c r ob i al A b u n d an c e T he m i c r obi a l c ount m e t hodol ogy r e l yi ng on t he c ul t ur a bi l i t y o f t he m i c r oor ga ni s m s w a s not s e ns i t i ve e nough t o de t e c t di f f e r e nc e s a m ong t r e e t ype s t i m e or non pl a nt e d s a m pl e s A t t he S R S w he r e s oi l ph ys i c oc he m i c a l c ha r a c t e r i s t i c s dr a s t i c a l l y c ha nge d w i t h t he s oi l pr of i l e a nd w he r e nut r i e nt s i n t he ve ge t a t e d e c os ys t e m w e r e hi ghe r i n c onc e nt r a t i on t ha n i n t he non pl a nt e d a r e a s no di f f e r e nc e s w e r e a s s e s s e d i n m i c r obi a l c ount s w i t h r e s pe c t t o s oi l pr o f i l e o r p r e s e nc e of pl a nt i ngs H ow e ve r be c a us e t he s oi l s a r e a c i di c a nd f r e que nt l y e xpe r i e nc e s a t ur a t e d c ondi t i ons r a pi d l e a c hi ng of nut r i e nt s m a y oc c ur t hus e xpl a i ni ng t he va r i a bi l i t y obs e r ve d a m ong t he pi ne t r e e s pos i t i one d dow n t he s l ope t ow a r ds t he c ont a m i na t e d s e e pl i ne out f l ow A s w a t e r s a t ur a t i on i nc r e a s e d, r hi z os phe r e s oi l he t e r ot r oph a nd m e t ha not r oph c ount s de c r e a s e d w hi l e c ount s of m e t ha not r ophs i n t he r oot s i nc r e a s e d. M e t ha not r ophs i n t hi s e nvi r onm e nt m a y be ne f i t f r om t he i nput of oxyge n by t he pl a n t s ( W hi pps a nd L ync h, 1983 ; W a l t on e t a l 1994 ) a nd t he i r pr oxi m i t y t o t he a noxi c z one s w he r e C H 4 i s be i ng p r oduc e d by m e t ha noge ns ( H a ns on a nd H a ns on, 1993; G i l be r t a nd F r e nz e l 1998) T he s e r e s ul t s m a y

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136 i m pl y a hi ghe r bi ode gr a da t i on pot e nt i a l o f t he s a t u r a t e d e nvi r onm e nt s i nc e m e t ha not r ophs pr ol i f e r a t e i n t he r oot s o f t he pl a nt s i n di r e c t c ont a c t w i t h t he c ont a m i na t e d gr oundw a t e r A s e xpe c t e d, t he de s i gn a t t he L a S a l l e T C E S i t e ( P V C pot s f i l l e d w i t h pl a nt i ng m a t e r i a l ) c ons t i t ut e d a hom oge ne ous e nvi r onm e n t c ha r a c t e r i z e d by i t s ne ut r a l pH a nd a de qua t e nut r i e nt c ont e nt T he s e c ondi t i ons r e f l e c t e d m i c r obi a l c ount s t ha t s l i ght l y va r i e d t hr oughout t he s t udy, by s e a s ons by t r e e t y pe or be t w e e n pl a nt e d a nd non pl a nt e d pot s s ugge s t i ng t ha t t he pl a nt i ng m a t e r i a l us e d i n t hi s r e m e di a t i on s ys t e m m a y ha ve a c t e d a s t he s our c e of m i c r obi a l i noc ul um H ow e ve r t hi s t r e nd m a y de pe nd on pl a nt gr ow t h a nd ye a r s o f e s t a bl i s hm e nt of t he s i t e ( 1 2 ye a r s a t t he t i m e of t he s t udy) T he w i l l ow t r e e s s how e d hi ghe r r oot bi o m a s s t ha n popl a r s ; de s pi t e s a m pl e s f r om t he s e t w o t r e e t ype s not s how i ng hi ghe r m i c r ob i a l c ount s a c t i vi t y, o r s oi l d i ve r s i t y f o r t he w i l l ow t r e e s O ve r a l l m e t ha not r oph a bunda nc e w a s hi gh e r a t t he T C E S i t e a r e s ul t a nt i c i pa t e d s i nc e gr ow t h of m e t ha not r ophs w a s not e xpe c t e d a t t he P C E S i t e gi ve n t he i r i na bi l i t y t o oxi di z e P C E ( U c hi ya m a e t a l 1989 ; B ow m a n e t a l 1993b) A c t i vi t y an d P h yl oge n e t i c s of E n r i c h m e n t s A l l phyt or e m e di a t i on s e t t i ngs c ont a i ne d m e t ha not r ophs c a pa bl e of e xpr e s s i ng bot h f or m s of M M O H ow e ve r e nr i c hm e nt s i n N M S w i t hout C u f r om r hi z opl a ne s a m pl e s a t t he hi gh c ont a m i na nt r e gi ons of t he L a S a l l e T C E S i t e w e r e not s uc c e s s f ul pr oba bl y a s t he r e s ul t of a ne ga t i ve e f f e c t of t he hi gh c ont a m i n a nt e xpos ur e i n t he r oot s a s t he y a r e i n di r e c t c ont a c t w i t h t he c ont a m i na nt du r i ng pl a nt w a t e r upt a ke N M S w i t h C u e nr i c hm e nt s s how e d hi ghe r oxi da t i on r a t e s t ha n N M S a s e xpe c t e d f or pM M O e xpr e s s i ng m e t ha not r ophs w hi c h pos s e s s a l ow e r e ne r gy de m a nd a nd hi ghe r a f f i ni t y f or C H 4 ( H a ns on a nd H a ns on, 1996) E nr i c hm e nt s i n N M S w i t h C u f r om t he S R S s how e d t he

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137 hi ghe s t oxyge n upt a ke r a t e s i n t he pr e s e nc e of C H 4 c or r e l a t i ng w i t h t he hi ghe r bi oa va i l a bi l i t y of C u f ound a t t hi s s i t e a s i t i s kno w n t ha t pM M O oxi di z e s C H 4 f a s t e r t ha n s M M O ( S e m r a u e t a l 1995 ) A l s o t he non p l a nt e d s oi l i n m os t of t he e nr i c hm e nt s s how e d hi ghe r oxi da t i ve a c t i vi t y t ha n t he p l a nt r e l a t e d c ul t ur e s e ve n t hough m i c r obi a l c ount s w e r e c om pa r a bl e T he h i ghe r a c t i vi t y obs e r ve d i n t he non pl a nt e d s a m pl e s m a y be r e l a t e d t o a ne ga t i ve e f f e c t o f h i gh c ont a m i na nt e xpos ur e a s pr e vi ous l y m e nt i one d. T he e f f e c t be i ng m or e pr onounc e d a t t he T C E S i t e w he r e c onc e nt r a t i ons a r e a ppr oxi m a t e l y 100 f o l d gr e a t e r t ha n t hos e a t t he S R S H ow e ve r r h i z os phe r e s a m pl e s f r om de e pe r s oi l l a ye r s ( > 30 60 c m ) du r i ng t he N ove m be r s a m pl i ng s how e d hi ghe r a c t i vi t y t ha n t he non pl a nt e d s oi l i m pl yi ng t ha t m e t ha not r oph a c t i vi t y i s be ne f i t e d by t he pr e s e nc e of t he pl a nt w hi c h e xt e nt s de e pe r i n t he s oi l pr of i l e P hyt o r e m e di a t i on pl ot di f f e r e nc e s m a y r e l a t e t o t he t ype of m e t ha not r oph pr e s e nt a t t he s i t e a nd t he i r a bi l i t y t o e xpr e s s e i t he r oxi da t i ve e nz ym e a s T C E a nd i t s m e t a bol i t e s ha ve be e n s how n t o ha ve a s e l e c t i ve e f f e c t on pa r t i c ul a r gr oups o f ba c t e r i a a n d on di f f e r e nt t ype s o f m e t ha not r ophs ( K a na z a w a a nd F i l i p, 1987; A l va r e z C ohe n a nd M c C a r t y, 1991a ; H e n r y a nd G r bi G a l i 1991) T r e e t ype di f f e r e nc e s w e r e di f f i c ul t t o a s s e s s e d by t hi s c ul t ur e de pe nde nt m e t hod; how e ve r popl a r t r e e s a t t he T C E S i t e c ons i s t e nt l y e xhi bi t e d hi ghe r oxyge n upt a ke r a t e s i n t he r h i z os phe r e e nr i c hm e nt s c om pa r e t o t he r hi z opl a ne w hi c h m a y be r e l a t e d t o a hi ghe r e xpos ur e t o t he c ont a m i na nt a nd i t s by p r od uc t s know n t o be t oxi c t o m e t ha not r ophs ( F ox e t a l 1990; A l va r e z C ohe n a nd M c C a r t y, 1991a ) H ow e ve r t he s a m e be ha vi or w a s not obs e r ve d on w i l l ow s w hi c h a r e r e po r t e d t o pos s e s s c om pa r a bl e t r a ns pi r a t i on a nd c ont a m i na nt upt a ke r a t e s a s popl a r s ( S nyde r a nd W i l l i a m s 20 00)

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138 T hough, w i l l ow t r e e s s how e d hi ghe r f i ne r oot bi o m a s s t ha t m a y ha ve c ont r i but e d t o a hi ghe r t ol e r a nc e t o t he c ont a m i na nt i n t he r hi z opl a ne C om pa r a bl e r a t e s b e t w e e n s oi l c om pa r t m e nt s m a y s ugge s t hi ghe r r e s i s t a nt t o t he c ont a m i na nt by t he t r e e t ype a s w i t h pi ne a nd w i l l ow t r e e s T he phyl oge ne t i c a na l ys i s of a l l N M S w i t h C u e nr i c hm e nt s r e ve a l e d di s t i nc t c om m uni t y pr of i l e s c om pos e d of a s s oc i a t i ons of m e t ha not r oph s w i t h ot he r ba c t e r i a l gr oups m a i nl y f r om t he phyl u m B a c t e r oi de t e s a nd unc ul t ur e d ba c t e r i um T he num be r o f m e t ha not r ophs r e t r i e ve d f r om e a c h c ul t ur e di d not c or r e l a t e w i t h t he m i c r obi a l a bunda nc e a s s e s s e d f or t he s i t e ne i t he r w i t h a c t i vi t y H ow e ve r a l l de t e c t e d m e t ha not r ophs w e r e t ype I I m a i nl y f r o m t he ge nus M e t hy l oc y s t i s t ha t a r e know n t o dom i na t e i n s oi l e nvi r onm e nt s w i t h a b r oa d r a nge o f pH ( D e dys h e t a l 2001; K ni e f e t a l 2003) H ow e ve r s i nc e t he r e i s a n i nhe r e nt bi a s w i t h c ul t u r e de pe nde nt m e t hods t he hi gh c onc e nt r a t i on of C H 4 us e d ( 20% v/ v ) m a y h a ve pr om ot e d t he dom i na nc e of t ype I I m e t ha not r ophs know n f or t he i r l ow a f f i ni t y t o C H 4 ( H a ns on a nd H a ns on, 1996) C ul t i va t i on bi a s t ow a r ds t ype I I m e t ha not r ophs ha s be e n r e por t e d i n s e ve r a l s t udi e s ( J e ns e n e t a l 1998; B a ke r e t a l 2001; H or z e t a l 20 02) A t t he S R S a nd L a S a l l e P C E S i t e a M e t hy l oc y s t i s s t r a i n de s c r i be d a s one t ha t r a pi dl y de gr a de s T C E w a s r e t r i e ve d, w hi c h m a y c onf e r a hi gh bi ode gr a da t i on pot e nt i a l t o t he s e pl ot s H ow e ve r t h i s r e s ul t doe s not i m pl y t ha t t ype I I m e t ha not r ophs a r e t he a c t i ve popul a t i ons a t t he s e s i t e s T he f a c t t ha t not a l l of t he e nr i c hm e nt s s how e d s i m i l a r oxi da t i ve po t e nt i a l or c om m uni t y pr of i l e s e ve n t hough a l l o f t he m pos s e s s e d t he s a m e t ype of m e t ha not r oph popul a t i ons i s i nt r i gui ng I t doe s s ugge s t t ha t a t t h e S R S c ha r a c t e r i z e d a s a n ol i got r ophi c e nvi r onm e nt w i t h l ow m i c r obi a l bi om a s s ( N e vi us e t a l 2004) m e t ha not r ophs m a y be a

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139 dom i na nt ba c t e r i a l gr oup a s t he m a j o r m e m be r s o f t he i r c om m uni t y pr of i l e s w e r e m e t ha not r ophs H ow e ve r t hi s w a s not t he c a s e i n t he L a S a l l e T C E S i t e e ve n t hough i t s ho w e d t he hi ghe s t a bunda nc e of m e t ha not r ophs A c t i vi t y an d P h yl oge n e t i c s of S I P S oi l M i c r oc os m s S I P s oi l m i c r oc os m s of e a c h phyt o r e m e di a t i on s e t t i ng s how e d, e ve n a t no r m a l i z e d l a bor a t or y c ondi t i ons a nd w a t e r c ont e nt s i gni f i c a n t di f f e r e nc e s i n C H 4 de pl e t i on r a t e s a m ong pl ot s H ow e ve r no di f f e r e nc e s i n a c t i vi t y w e r e obs e r ve d be t w e e n pl a nt e d a nd non pl a nt e d s a m pl e s w i t hi n t he pl o t s A l t hough, c or r e l a t i ng w i t h t he hi ghe r m e t ha not r oph a bunda nc e a s s e s s e d by c ul t ur e de pe nde nt m e t hods but not w i t h t he a c t i vi t y of t he e nr i c hm e nt s t he L a S a l l e T C E S i t e s oi l m i c r oc os m s s how e d t he hi ghe s t C H 4 de pl e t i on r a t e s f ol l ow e d by t he S R S a nd t he P C E S i t e O nl y by ut i l i z i ng t he S I P t e c hni que c om bi ne d w i t h c ul t u r e i nde pe nde nt m ol e c ul a r m e t hods i t w a s pos s i bl e t o a c c ur a t e l y di s c e r ne d on t he t ype s of a c t i ve m e t ha n ot r oph popul a t i ons p r e s e nt a t e a c h phyt or e m e di a t i on pl ot A l s o, t hi s p r ot oc ol w a s t he onl y t o a s s e s s e d di f f e r e nc e s be t w e e n pl a nt e d a nd non pl a nt e d s a m pl e s E a c h phyt or e m e di a t i on s e t t i ng w a s c om pos e d of di f f e r e nt a c t i ve m e t ha not r oph c om m uni t i e s A t t he S R S t he a c t i ve m e t ha not r op h c om m uni t y w a s dom i na t e d by t he M e t hy l oc e l l a M e t hy l oc aps a gr oup, m ode r a t e l y a c i dophi l i c m e t ha not r ophs t ha t gr ow be t w e e n pH va l ue s of 4. 5 t o 7 ( D e dys h e t a l 2000 ) T hi s f i ndi ng c or r e l a t e s w i t h t he e nvi r onm e nt a l c ondi t i ons pr e s e nt a t t he s i t e a nd w i t h pr e vi ous r e por t s of a c t i ve m e t ha not r ophs pr e s e nt i n m ode r a t e l y a c i di c e nvi r o nm e nt s ( R a da j e w s ki e t a l 2002) T he M e t hy l oc e l l a gr oup a ppe a r s t o pos s e s s hi gh a da pt a bi l i t y t o e nvi r onm e nt a l c ondi t i ons a s i t ha s be e n r e c e nt l y r e por t e d t ha t one o f i t s s t r a i ns i s t he onl y know n e xc e pt i on t o obl i ga t e m e t hyl ot r ophy, pr e s e nt i ng f a c ul t a t i ve g r ow t h on m ul t i c a r bon s our c e s ( D e dys h e t a l

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140 2005) A l s o, h i gh bi o r e m e di a t i on pot e nt i a l m a y be e xpe c t e d by t hi s m e t ha not r oph ge nus a s i t onl y e xpr e s s e s t he hi gh oxi da t i ve f or m of M M O ( s M M O ) w hi c h i s not r e gul a t e d by t he pr e s e nc e of C u ( D un f i e l d e t a l 2003; T he i s e n e t a l 2005) T he r e f o r e M e t hy l oc e l l a m a y be pr e s e nt i n e nvi r onm e nt s w i t h hi ghe r C u c o nc e nt r a t i ons t ha n ot he r s M M O e xpr e s s i ng m e t ha n ot r ophs H ow e ve r no T C E de g r a da t i on s t udi e s ha ve be e n pe r f or m e d w i t h t he s e s t r a i ns due t o t he di f f i c ul t y o f c ul t ur i ng t hi s m i c r oor ga ni s m i n ba t c h ( s a l t s e ns i t i ve ) A t L a S a l l e T C E S i t e t he t he r m o t ol e r a nt t ype X m e t ha not r ophs ( M e t hy l oc al dum M e t hy l oc oc c us ) dom i na t e d t he a c t i ve popul a t i ons I t i s s pe c ul a t e d t ha t t he pl a nt i ng m a t e r i a l be c a us e of i t s or i gi n i n m ul c h pi l e s a t hi g h t e m pe r a t ur e s pos s e s s e d a dom i na nc e of t he r m ot ol e r a nt m e t ha not r ophs w hi c h ha ve be e n c om m onl y i s ol a t e d f r om t hi s e nvi r onm e nt ( E s hi ni m a e v e t a l 2004; J a c ke l e t a l 2005) T he r e f or e i t i s pr opos e d t ha t t he pl a nt i ng m a t e r i a l f unc t i one d a s t he m e t ha not r o ph i noc ul um i n t hi s pl ot ( c om pos t e f f e c t ) H ow e ve r t he c om m uni t y pr o f i l e of t he n on pl a nt e d pot w a s not i de nt i c a l t o t ha t of t he pl a nt e d pot s L ow r e l a t i ve a bunda nc e a nd h i ghe r va r i a bi l i t y of i t s c om pone nt s w a s obs e r ve d i n t he non p l a nt e d s a m pl e s C ons e que nt l y, t he p l a nt m us t e xe r t s om e e f f e c t t ha t a l l ow e d M e t hy l oc al dum s t r a i ns pe r s i s t i n t he pl a nt e d pot s e s pe c i a l l y i n t he w i l l ow c l one s w hi c h m a i nt a i ne d t he s a m e p r o f i l e t hr ough out t he s t udy. A s m e nt i one d pr e vi ous l y, w i l l ow c l one s pr oduc e d hi ghe r s ur f a c e r oot bi om a s s t ha n t he pop l a r t r e e s t ha t m a y ha ve a s s i s t e d i n ke e pi ng hi ghe r t e m pe r a t u r e s i n t he pot s c om pa r e t o t he popl a r s or t he non pl a nt e d s oi l A s a r e s ul t t h e popl a r t r e e s a nd t he non pl a nt e d pot s m a y ha ve be e n m or e s us c e pt i bl e t o e nvi r onm e nt a l c ha nge s a t t he s i t e

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141 T he c ha nge s obs e r ve d w i t h t i m e i n t he T C E S i t e p opl a r t r e e c om m un i t y p r of i l e s dur i ng t he f a l l s a m pl i ng m a y e xe m pl i f i e d t he e f f e c t of t e m pe r a t ur e ove r t he a c t i ve m e t ha not r oph popul a t i ons i n t hi s pl ot A l t hough M e t hy l oc al dum s t r a i ns c a n gr ow i n a w i de r a nge of t e m pe r a t ur e s ( m ode r a t e t he r m ophi l e s ) s om e s pe c i e s c a n s us t a i n l ow e r t e m pe r a t ur e s t ha n ot he r s ( B odr os s y e t a l 1997) F or e xa m pl e M gr ac i l e c a n gr ow be t w e e n 20 47C but M s z e ge di e ns e onl y gr ow s f r om 37 62C M e m be r s of t he a c t i ve m e t ha not r oph popul a t i ons of t he t r e e s a t t he T C E S i t e c l os e l y r e l a t e d t o M gr ac i l e m or e t ha n M s z e ge di e ns e w hi c h m a y s ugge s t t ha t c ondi t i ons a t t he s i t e a r e not t he opt i m um f or s om e of t he s e t he r m ot ol e r a nt s pe c i e s A ddi t i o na l l y, t he M e t hy l oc oc c us ge nus a l s o r e t r i e ve d a s a n a c t i ve m e m be r of t he T C E S i t e pos s e s s e xt r a gl yc opr ot e i n s t r uc t u r e s on t he out e r s ur f a c e o f t he c e l l w a l l w hi c h a ppa r e nt l y pr ovi de hi ghe r r e s i s t a nc e t o s t r e s s f a c t or s i nc l udi ng t e m pe r a t ur e f l uc t ua t i ons a nd c on c e nt r a t i on of s ol ut e s ( E s hi ni m a e v e t a l 2004 ) I n ge ne r a l t ype X m e t ha not r ophs e xhi b i t s t r uc t ur a l a nd f unc t i ona l a da pt a bi l i t y t o c ha nge s i n e nvi r onm e nt a l c ondi t i ons t ha t m a y e xpl a i n t he i r p r e s e nc e a f t e r t w o ye a r s of e s t a bl i s hm e nt of t he s i t e s H ow e ve r e ve n i f t ype X m e t ha not r ophs pos s e s s hi gh a da pt a bi l i t y, c ondi t i ons a t t he s i t e a r e not opt i m um f or t he s e s pe c i e s ( pa r t of t he ye a r t he s oi l f r e e z e s ) T he r e f o r e t he que s t i on r e m a i ns t o w he t h e r t he s e a c t i ve m e t ha not r oph popul a t i ons de t e c t e d onl y 1 2 ye a r s a f t e r e s t a bl i s hm e nt w i l l pe r s i s t i n t hi s pl ot A ddi t i ona l l y, t he de gr a da t i ve pot e nt i a l of t he s e s pe c i e s m a y be hi gh a s M e t hy l oc oc c us s pe c i e s a r e know n t o e xpr e s s bot h f o r m s of t he M M O e nz ym e a nd s how e d hi gh T C E bi ode gr a da t i on pot e nt i a l ( H a ns on a nd H a ns on, 199 6) H ow e ve r M e t hy l oc al dum s pe c i e s do not pr oduc e t he s ol ubl e f o r m o f t he M M O t ha t s how s hi ghe r oxi da t i on c a pa c i t y ( B odr os s y e t a l 1997)

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142 A t t he P C E S i t e t he ge nus M e t hy l oc al dum w a s a l s o f ound but not a s dom i na nt a s i n t he T C E S i t e a nd M e t hy l oc y s t i s a l s o de s c r i be t he a c t i ve m e t ha not r oph popul a t i ons a t t hi s pl ot T he s e ge ne r a a l ong w i t h M e t hy l os i nus r e pr e s e nt c om m onl y f ound m e t ha not r ophs i n s oi l s ( K ni e f e t a l 2003) P opl a r t r e e s p l a nt e d i n t he T C E a nd P C E pl ot s s how e d di s t i nc t m e t ha not r oph c om m uni t i e s t he r e f or e m e t ha not r ophs a ppe a r e d t o be i nf l ue nc e d a t t hi s t i m e of t he s t udy m or e by t he phyt or e m e di a t i on de s i gn t ha n by t he pl a nt t ype P r i n c i p al C o m p on e n t A n al ys i s ( P C A ) P C A of c ul t ur e de pe nde nt a nd c ul t ur e i nde pe nde nt m e a s ur e m e nt s s ugge s t e d t ha t t he va r i a bi l i t y a m ong m e a s ur e m e nt s m i ght be e xpl a i ne d by s e pa r a t i ng t he L a S a l l e T C E S i t e f r o m bot h t he L a S a l l e P C E S i t e a nd S R S T he L a S a l l e P C E S i t e s e e m e d t o ha ve a n i nt e r m e di a t e pos i t i on be t w e e n t he T C E S i t e a nd t h e S R S da t a s e t s C ons e que nt l y, t he m e t ha not r oph c om m uni t y of t he s e phyt or e m e di a t i on pl ot s m a y be e xpl a i ne d by t he de gr e e of de pa r t u r e of t he de s i gn f r om a na t ur a l s e t t i ng a nd not be c a us e of l oc a t i on o r t r e e t ype H ow e ve r t h e us e of pl a nt i ng m a t e r i a l a t L a S a l l e T C E S i t e s e e m e d t o di c t a t e t he a c t i ve m e t ha not r oph popu l a t i ons a t t he t i m e of t he s t udy, w hi c h e xe m pl i f i e d a n e a r l y s t a ge of pl a nt de ve l opm e nt M e t ha not r oph c om m u ni t y s hi f t s a s pl a nt g r ow a nd e xe r t s hi ghe r pr e s s ur e ov e r t he r hi z os phe r e e nvi r on m e nt m a y be e xpe c t e d. R e s ul t s of m a na ge m e nt pr a c t i c e s m a y t a ke t i m e t o a f f e c t s oi l m i c r obi a l c om m uni t i e s a s s oi l s s how a n i m m e ns e c a pa c i t y f or d i ve r s i t y. A l s o, di f f e r e nc e s a m ong t r e e c l one s ha ve be e n s how n t o t a ke t i m e t o de ve l o p ( G i r va n e t a l 2003 ) I n c onc l us i on, t he r e s ul t s of t hi s s t udy a s s e s s e d a s i m por t a nt va r i a bl e s f o r t he e s t a bl i s hm e nt of di f f e r e nt t ype s of m e t ha not r oph, t he de gr e e of s oi l s a t ur a t i on, c ont a m i na nt c onc e nt r a t i on, s oi l c om pa r t m e nt a nd t he us e of pl a nt i ng m a t e r i a l H ow e ve r

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143 r e s ul t s w e r e not c ons i s t e nt w i t hi n t he t r a di t i ona l m e t hods us e d or be t w e e n c ul t ur e de pe nde nt a nd i nde pe nde nt t e c hni que s L i m i t a t i o ns i nhe r e nt i n c ul t i va t i on w e r e e vi de nt M e t hods t ha t c ons i de r t he a c t i ve m i c r obi a l popul a t i ons w e r e m or e e f f e c t i ve i n a s s e s s i ng di f f e r e nc e s a m ong pl a nt e d a nd non pl a nt e d s a m pl e s w i t h r e s pe c t t o m i c r obi a l c om m uni t y c om pos i t i on, s t r uc t ur e a nd a c t i vi t y a t e a c h pl ot a n d a r e r e c om m e nde d f or f ut ur e e va l ua t i ons H ow e ve r a t t he t i m e of t he s t udy t he r e w a s a s t r ong e f f e c t of t he phyt or e m e di a t i on de s i gn i n s om e o f t he pl ot s e va l u a t e d. T he e a r l y s t a ge of pl a nt de ve l opm e nt i n t he s e pl ot s m a y s ugge s t t ha t t he ob s e r ve d pa t t e r ns m a y pot e nt i a l l y c ha nge w i t h t i m e a nd c ont i nue d m oni t or i ng i s r e c om m e n de d.

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144 F i gur e 5 1 L oc a t i on of phyt or e m e di a t i on s i t e s a n d di a gr a m o f s a m pl i ng a r e a s a t t he ( A ) S a va nna h R i ve r S i t e ( S R S ) S C a nd ( B ) L a S a l l e I L

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145 T a bl e 5 1 G e ne r a l c ha r a c t e r i s t i c s of t he di f f e r e nt phyt or e m e di a t i on pl ot s a t t he S R S a nd L a S a l l e s i t e s Si te S R S 1 La Sa l l e C ha ra ct eri s ti c T C E Si t e P C E Si te Lo ca ti o n 3 3 1 5 N ; 8 1 4 2 W 4 1 2 0 N ; 8 8 7 0 W El ev a ti o n ( m ) 5 3 2 0 0 3 0 0 A nn ua l cl i m a te da ta 3 T e mp er at u re ( C) 1 8 1 1 Preci p i t at i o n ( mm y ear 1 ) 1 5 5 4 6 4 2 P hy to re m edi a ti o n s etti ng Pl an t t y p e L o b l o l l y p i n e (mat u re t re es i n rev eg et at ed ar ea) Po p l ar, w i l l o w (t ree cu t t i n g s i n P V C p o t s ) Po p l ar (t ree cu t t i n g s i n n at i v e s o i l ) So i l mat e ri al N at i v e Pl an t i n g mat e ri al N at i v e mu l ch So i l ty pe Fl u v aq u en t s V au cl u s e A i l ey A s s o c s an d : s o i l : b ark : p eat Pat t o n H arco A s s o c. 2 an d t re e b ark mu l ch So i l pH 4 5 4 7 6 7 3 C o n ta m i na n t co nce nt ra ti o n (p pb) 5 6 2 0 4 2 5 4 8 3 8 1 So i l an d cl i mat e d at a fro m U S D A (1 9 9 0 ) an d H u n t er ( 2 0 0 4 ) res p ect i v el y ; 2 A p p ro x i m at i o n fro m t h e are a ( U S D A 1 9 9 6 ); 3 Y e ar 2 0 0 4 ; 4 A t t h e 1 5 3 0 c m s o i l l ay er; 5 Y ear 2 0 0 2

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146 F i gur e 5 2 M i c r obi a l c ount s pe r t r e e t ype f r om t h e di f f e r e nt phyt or e m e di a t i on pl ot s a t S R S a nd L a S a l l e M e t ha not r oph ( c i r c l e s ) a nd he t e r ot r oph ( t r i a ngl e s ) c ount s i n t he r h i z os phe r e ( R H bl a c k s ym bol s ) r h i z opl a ne ( R P w hi t e s ym bol s ) a nd non pl a nt e d s oi l ( N P g r a y s ym bol s ) D a t a c ol l e c t e d i n O c t obe r 2003 a nd N ove m be r 2004 a t t he S R S ( A ) a nd L a S a l l e ( B C ) hi gh c ont a m i na nt r e gi ons r e s pe c t i ve l y. V a l ue s r e pr e s e nt a ve r a ge S D ove r t he s oi l pr of i l e ( S R S : R H n= 15, R P n= 12, N P n= 3; L a S a l l e : R H n= 15, R P n = 12, N P n= 3) 1 P i ne 2 R P c ount s not de t e r m i ne d. M e a ns w i t h t he s a m e l e t t e r a r e not s i gni f i c a nt l y di f f e r e nt ( P < 0. 05 ) R H a nd R P r e s ul t s de not e d by l ow e r c a s e a nd c a pi t a l l e t t e r s r e s pe c t i ve l y. S i gni f i c a nt d i f f e r e nc e s be t w e e n pl a nt e d a nd non pl a nt e d s a m pl e s ( D unne t t s t e s t P < 0. 05 )

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147 F i gur e 5 3 O xyge n upt a ke r a t e s of e nr i c hm e nt s f r om di f f e r e nt t r e e t ype s i n t he pr e s e nc e of C H 4 E n r i c hm e nt s f r om t he 30 60 c m s oi l l a ye r i n N M S w i t h C u ( A B C ) a nd w i t hout C u ( D E F ) a t t he S R S a nd L a S a l l e hi g h c ont a m i na nt r e gi ons dur i ng O c t obe r 2003 a nd N ove m be r 2004, r e s pe c t i ve l y. N D = N ot de t e r m i ne d. N E = N ot e nr i c he d V a l ue s r e pr e s e nt a ve r a ge s t a nda r d de vi a t i on of t r i pl i c a t e r uns

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148 F i gur e 5 4 F r e que nc y o f phyl um a f f i l i a t i ons pe r t r e e t ype of t he N M S w i t h C u e nr i c hm e nt c om pone nt s P hyl oge ne t i c a na l ys i s of t he 16S r D N A ge ne of pl a nt e d a nd non pl a nt e d s a m pl e e nr i c hm e nt s a c c or di ng t o B L A S T a l i gn m e nt s S a m pl e s w e r e obt a i ne d f r om t he 30 60 c m s oi l l a y e r dur i ng O c t obe r 200 3 a nd N ove m be r 2004 a t t he S R S a nd L a S a l l e hi gh c ont a m i na nt r e gi ons r e s pe c t i ve l y.

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149 F i gur e 5 5 P hyl oge ne t i c a na l ys i s of t he S R S S I P s oi l m i c r oc os m s ( A ) 16S r D N A D G G E ge l of t he 1 2 C a nd 1 3 C D N A f r a c t i ons o f pi ne 1 a nd 2 a t t he 30 60 c m s oi l l a ye r W hi t e a nd e m pt y c i r c l e s r e pr e s e nt m os t a bunda nt a nd s e que nc e d ba nds r e s pe c t i ve l y. ( B ) R oot e d phyl oge ne t i c t r e e ba s e d on t he nuc l e ot i de s e que nc e of 16S r D N A pa r t i a l f r a gm e nt s f r o m t he 1 3 C D N A f r a c t i on of pi ne 2 i n r e l a t i on t o t ype I a nd I I m e t ha not r ophs a n d t he h i ghe s t s c or i ng B L A S T a l i gne d s e q ue nc e s L e ngt h of br a nc he s i s pr opo r t i ona l t o % di s s i m i l a r i t y ( 0. 1 s ubs t i t ut i on pe r nuc l e ot i de s i t e ) B l a c k ( ) 90% ) a nd w hi t e c i r c l e s ( ) 50% ) on br a nc he s r e pr e s e nt % of boot s t r a p va l ue s f r om 100 0 r e pl i c a t i ons S e que nc e na m e = D N A f r a c t i on ( 1 2 C 1 3 C ) t r e e t ype ( P i = pi ne ) s oi l c om pa r t m e nt ( R H = r hi z os phe r e ) pi ne t r e e nu m be r ( 1, 2) ba nd n um be r ( 1 2, 3 )

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150 T a bl e 5 2 S um m a r y of phyl oge ne t i c a s s i gnm e nt s ( B L A S T s e a r c h) of t he pm oA ge ne s e que nc e s of t he a c t i ve m e t ha not r oph popul a t i ons ( 1 3 C D N A f r a c t i on) f r om t he S I P s oi l m i c r oc os m s i n e a c h phy t or e m e di a t i on pl ot *A t t he 30 60 c m s oi l l a ye r a nd h i gh c ont a m i na nt r e gi ons 1 + = P o t e nt i a l s t r a i n o r c l one ; 2 L ow r e l a t i ve a bunda nc e ; 3 N D = N ot de t e r m i ne d; 4 D e t e r m i ne d by 16S r D N A D G G E a na l ys i s ; 5 B a nds not s e que nc e d.

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151 F i gur e 5 6 P r i nc i pa l c om pone nt a na l ys i s ( P C A ) o f c ul t ur e de pe nde nt a nd c ul t ur e i nde pe nde nt m e a s ur e m e nt s ( A ) D a t a f r om c ul t u r e de pe nde nt m e t hods of r hi z os phe r e ( f i l l e d s ym bol s ) a nd r h i z opl a ne ( e m pt y s ym bol s ) s a m pl e s ( n= 34) ( B ) D a t a f r om c ul t u r e de pe nde nt a nd c ul t u r e i nde pe nde nt m e t hods of onl y r hi z os phe r e s a m pl e s ( n= 23) T he s e m e t hods w e r e us e d t o de s c r i be m i c r obi a l a bunda nc e a nd a c t i vi t y of e n r i c hm e nt s a nd S I P s oi l m i c r oc os m s a t e a c h phyt or e m e di a t i on pl ot V a l ue s r e pr e s e n t m e a n of r e gr e s s i on c oe f f i c i e nt s s t a nda r d e r r or

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152 C H A P T E R 6 C O N C L U S I O N S L a bor a t or y a nd f i e l d s t udi e s w e r e c onduc t e d t o c ha r a c t e r i z e c om m uni t i e s of m e t ha not r ophs i n t he r hi z os phe r e of phyt or e m e di a t i ng pl a nt s us e d t o r e m e di a t e s oi l a nd gr oundw a t e r c ont a m i na t i on o f c hl or i na t e d s ol ve nt s B y us i ng c ul t ur e de pe nde nt m e t hods a nd de ve l opi ng c ul t ur e i nde pe nde nt p r ot oc ol s s pe c i f i c t o t he r hi z os phe r e e nvi r onm e nt m e t ha not r oph a bunda nc e a c t i vi t y a nd phyl oge ne t i c s w e r e de t e r m i ne d. A l s o, va r i a bl e s t ha t pot e nt i a l l y a f f e c t m e t ha not r oph pr oc e s s e s i n t he f i e l d w e r e a s s e s s e d. U l t i m a t e l y, t he s e r e s ul t s c a n a s s i s t i n t he de ve l opm e nt o f m o r e e f f i c i e nt r e m e di a t i on t e c hnol ogi e s L a bor a t or y s t udi e s w i t h pur e c ul t u r e s s e r ve d t o de s c r i be a nove l s t r a i n of m e t ha not r ophs w i t h uni que m or phol ogi c a l c ha r a c t e r i s t i c s a nd know n t o de gr a de a w i de r a nge of c ont a m i na nt s S e c ondl y t he a bi l i t y o f m e t ha not r ophs t o de gr a de c hl o r i na t e d a nd pl a nt r e l a t e d c om pounds t he l a t e r not p r e vi ou s l y r e por t e d i n t he l i t e r a t ur e w a s a s s e s s e d f or di f f e r e nt t ype s of m e t ha not r ophs F i e l d e xpe r i m e nt s pe r m i t t e d t he de ve l opm e nt of a p r ot oc ol us i ng c ul t ur e i nde pe nde nt s t a bl e i s ot ope pr obi ng ( S I P ) m e t hods t ha t hol ds pr om i s e i n be t t e r c ha r a c t e r i z i ng t he pot e nt i a l l y a c t i ve r hi z os phe r e m e t ha not r oph c om m uni t y M o r e ove r due t o t he c om bi ne us e of c ul t u r e de pe nde nt a nd c ul t ur e i nde pe nde nt t e c hni que s i t w a s pos s i bl e t o c onf i r m t he a c c ur a c y a nd r e l e va nc e of t he S I P t e c hni que A ddi t i ona l l y t h r e e ongoi ng ph yt or e m e di a t i on pl ot s t ha t va r i e d i n t he i r de s i gn w e r e c ha r a c t e r i z e d a c c or di ngl y t o a bunda nc e a c t i vi t y, a nd phyl oge ne t i c s of t he i r m e t ha not r oph c om m uni t i e s F i e l d c ha r a c t e r i z a t i on s a nd l a bor a t or y s t udi e s r e ve a l e d t he c a pa bi l i t y of m e t ha not r ophi c ba c t e r i a t o a da pt t o a va r i e t y of e nvi r onm e nt s a nd a s s e s s e d

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153 t he pot e nt i a l f a c t or s d r i vi ng t he s e popul a t i ons T h e s e f i ndi ngs pr ovi de a ba s i s f o r i m pr ovi ng t he e f f i c a c y of t hi s bi ol og i c a l r e m e di a t i on m e t hod T he f ol l ow i ng pa r a gr a phs de s c r i be i n de t a i l t he f i ndi ngs of e a c h c ha pt e r i n t h i s s t udy. I n C ha pt e r 2 a nove l m e t ha ne o xi di z i ng ba c t e r i um i s ol a t e d f r om a n unc ont a m i na t e d a qui f e r w a s de s c r i be d, M e t hy l oc y s t i s al dr i c hi i s p. nov S t r a i n C S C 1. T hi s m i c r oor ga ni s m e xe m pl i f i e d t he ubi qui t y o f m e t ha not r ophs i n na t ur e a nd t he i r c a pa bi l i t y t o de gr a de xe nobi ot i c s f r om a r om a t i c t o a l i pha t i c c om pounds A ddi t i ona l l y no s t udy ha s r e por t e d t he s t r uc t ur e of S t r a i n C S C 1 s c e l l e nve l ope t ha t pos s e s s e s a uni que s pi ny S l a ye r T he r o l e of t hi s uni que s ur f a c e f e a t ur e ha s not be e n e l uc i da t e d t o da t e ; how e ve r t he s e r e s ul t s s uppor t e d t he hypo t he s i s t ha t phyl oge ny a nd e c ol ogy m a y bot h pl a y a r ol e i n S l a ye r f or m a t i on O ve r a l l t hi s s t ud y br oa de ns t he obs e r ve d phys i ol ogi c a l t r a i t s of m e t ha not r ophs a nd br i ngs up t opi c s t ha t c oul d be a ddr e s s e d i n f ut u r e w or k r e l a t e d t o t he r ol e o f t hi s S l a ye r i n na t ur e a nd i t s e f f e c t on c ont a m i na nt de gr a da t i on pot e nt i a l L a bor a t or y s t udi e s c ont i nue d i n C ha pt e r 3 w i t h d i f f e r e nt t ype s of pur e m e t ha not r oph c ul t ur e s t ha t w e r e a s s e s s e d f or t he i r a bi l i t y t o oxi di z e m onot e r pe ne s ( pi ne ne ) a nd de t e r m i ne i t s e f f e c t s on T C E oxi da t i on M e t ha not r ophs e xpr e s s i ng e i t he r s M M O or pM M O w e r e c a pa bl e of oxi di z i ng # pi n e ne a bunda nt i n pl a nt s s uc h a s pi ne t r e e s ove r a r a nge of e nvi r onm e nt a l l y r e l e va nt c on c e nt r a t i ons O xi da t i on pot e nt i a l o f m e t ha not r ophs w e r e a f f e c t e d, e i t he r a nt a g oni s t i c a l l y or s yne r gi s t i c a l l y, i n t he pr e s e nc e of T C E a nd pi ne ne m i xt ur e s A c t i vi t y de c r e a s e d w i t h t he t ype I I m e t ha not r ophs a nd i nc r e a s e d w i t h t he t ype s I a nd X m e t ha not r ophs T he s e r e s ul t s e m pha s i z e t he i m por t a nc e of not on l y a s s e s s i ng c onc e nt r a t i on l e ve l s of c ont a m i na nt s a nd m onot e r pe ne s but a l s o o f

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154 m e a s ur i ng t he oxi da t i on pot e nt i a l s a nd di ve r s i t y o f r hi z os phe r e m e t ha not r ophs a t phyt or e m e di a t i on s i t e s w he r e m onot e r pe ne r e l e a s i ng pl a nt s a r e c ons i de r e d f o r us e F ur t he r r e s e a r c h m us t c onc e nt r a t e on t h e m e c ha ni s m s of de gr a da t i on w he n T C E a nd a pi ne ne a r e bot h p r e s e nt i n t he s ys t e m A l s o, i f m o not e r pe ne s r e l e a s e d by pl a nt s do s t i m ul a t e m e t ha not r ophi c T C E de gr a da t i on t he n t he s e c om pounds c oul d be i nc or por a t e d i n t he f i e l d t o pot e nt i a l l y i nc r e a s e T C E r e m ova l I n C ha pt e r 4 a p r ot oc ol us i ng t he S I P m e t hod s pe c i f i c t o r hi z os phe r e s t udi e s of phyt or e m e di a t i ng pl a nt s w a s de ve l ope d. B y c om b i ni ng t hi s t e c hni que w i t h pm oA D G G E a na l ys i s t he e xt e nt a nd r e s ol ut i on of t he S I P m e t ho d w a s br oa de ne d t o de t e c t di f f e r e nc e s ove r t i m e a nd a m ong t he r e l a t i ve a bunda nc e of t he a c t i ve m e m be r s of t he c om m uni t y A l t hough i t i s e xpe ns i ve a nd r e qui r e s c e r t a i n e xpe r t i s e t he pow e r of t hi s m e t hod i s a l m os t i nc om pa r a bl e a t t he pr e s e nt t i m e C ont r a r y t o t r a di t i ona l m e t hods w hi c h a r e l a bor i nt e ns i ve a nd de pe nde nt on t he c ul t ur a bi l i t y of t he m i c r oor ga ni s m s t he S I P m e t hod a l l ow s t he a s s e s s m e nt of t he pot e nt i a l l y r e l e va nt bi ode gr a da t i ng or ga ni s m s C ons e que nt l y, by us i ng t hi s pr ot oc ol t he phyt or e m e di a t i on pr a c t i t i one r c oul d be t t e r m oni t or a nd i m pl e m e nt t he r e m e di a t i on t r e a t m e nt t o t he ne e ds of t he a s s e s s e d a c t i ve r hi z os phe r e popul a t i ons F i na l l y, i n C ha pt e r 5 t he r hi z os phe r e m e t ha not r op h c om m uni t y o f t h r e e di f f e r e nt phyt or e m e di a t i on s e t t i ngs w a s c ha r a c t e r i z e d. C ul t ur e de pe nde nt a nd c u l t ur e i nde pe nde nt t e c hni que s w e r e us e f ul i n e l uc i da t i ng f a c t or s s uc h a s s oi l w a t e r c ont e nt c ont a m i na nt c onc e nt r a t i on, a nd s ys t e m de s i gn w hi c h gr e a t l y a f f e c t e d m e t ha not r ophs a t t he f i e l d. H ow e ve r r e s ul t s be t w e e n m e t hodol ogi e s w e r e not c ons i s t e nt a nd l i m i t a t i ons i nhe r e nt t o c ul t i va t i on de pe nde nt t e c hni que s w e r e e vi de nt B e c a us e t he ul t i m a t e goa l of t he s t udy

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155 w a s t o a s s e s s m e t ha not r oph r hi z ode gr a da t i on pot e nt i a l a t e a c h phyt or e m e di a t i on s e t t i ng, t he S I P t e c hni que c om bi ne d w i t h m ol e c ul a r c om m uni t y a na l ys i s i s r e c om m e nde d a s t he onl y m e t hodol ogy t ha t e f f e c t i ve l y de s c r i be d t he a c t i ve m e t ha not r oph c om m un i t y of t he s i t e s a nd de t e c t e d di f f e r e nc e s be t w e e n pl a nt e d a nd non pl a nt e d s a m pl e s A ddi t i ona l l y, e a c h o f t he r hi z os phe r e m e t ha not r op h popul a t i ons i n t he di f f e r e nt phyt or e m e di a t i on s e t t i ngs w a s di s t i nc t f r om t he o t he r s T he L a S a l l e S i t e pr e s e nt e d hi ghe r m i c r obi a l a bunda nc e a c t i vi t y a nd di ve r s i t y of t he a c t i ve m e t ha not r oph popul a t i ons H ow e ve r t he r e w a s a s t r ong e f f e c t of t he pl a nt i ng m a t e r i a l a t t he t i m e of t he s t udy. T he S R S m e t ha not r oph c om m un i t y s how e d c om pa r a bl e r e s ul t s t o t he hi ghl y c ont r ol l e d a nd r i c h e nvi r onm e nt of t he L a S a l l e pl o t s e ve n w he n m i c r ob i a l di ve r s i t y a nd a bunda nc e w e r e l ow Y e a r s of e s t a bl i s hm e nt a t t h e S R S pl ot m a y ha ve s e l e c t e d f or m e t ha not r ophs w e l l a da pt e d t o t he c ont a m i na t e d e nvi r onm e nt P hyt o r e m e di a t i on pl ot s w e r e s e pa r a t e d be t w e e n na t ur a l a nd c om pl e t e l y e ngi ne e r e d s ys t e m s by P C A a na l ys i s a nd, t he r e f or e t he s ys t e m de s i gn s e e m e d t o be m o r e i m por t a nt t ha n pl a nt s pe c i e s or l oc a t i on i n de s c r i bi ng t he m e t ha not r oph r h i z os phe r e c om m uni t y of t he di f f e r e nt s e t t i ngs H ow e ve r be c a us e of t he e a r l y s t a ge of pl a nt de ve l opm e nt a t t he L a S a l l e pl ot s t he obs e r ve d pa t t e r ns m a y pot e nt i a l l y c ha nge w i t h t i m e F ut ur e w or k i s ne e de d t o e f f e c t i ve l y a s s e s s c ont a m i na nt de gr a da t i on r a t e s o f t he a l r e a dy c ha r a c t e r i z e d r hi z os phe r e m e t ha not r oph c om m uni t i e s a t e a c h phyt or e m e di a t i on pl ot B y a s s e s s i ng t he r hi z ode gr a da t i on pot e nt i a l a t e a c h pl ot t he r ol e t ha t t hi s m e c ha ni s m pl a ys i n t he ove r a l l c hl or i na t e d s ol v e nt phyt or e m e di a t i on s ys t e m c a n be de t e r m i ne d T hi s s t udy c ont r i but e s t o a be t t e r kno w l e dge of m e t ha not r oph i nt e r a c t i ons i n t he r hi z os phe r e of pl a nt s a nd pr ovi de s a ba s i s f or i m pr ovi ng t he e f f i c a c y of t hi s bi ol ogi c a l

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156 r e m e di a t i on t e c hni que S i nc e t h i s w or k s how s t ha t pl a nt r e l a t e d c om pounds a f f e c t t he oxi da t i on a c t i vi t y o f m e t ha not r ophs i n t he pr e s e nc e of T C E f ur t he r r e s e a r c h i s ne e de d t o i nve s t i ga t e t he m e c ha ni s m s t ha t s e l e c t a nd r e gul a t e m i c r obi a l a c t i vi t y i n t he r h i z os phe r e A ddi t i ona l l y, c ha nge s m a y oc c ur a t t he r e c e nt l y e s t a bl i s he d L a S a l l e phyt or e m e di a t i on pl ot s due t o l ong t e r m c ont a m i na nt e xpos ur e a nd p l a nt gr ow t h a nd t he r e f or e c ont i nui ng m oni t or i ng i s r e qui r e d t o a s s e s s s uc h c ha nge s i n m e t ha not r oph popul a t i ons

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157 A P P E N D I X A D D I T I O N A L T A B L E S A N D F I G U R E S T a bl e A 1. S oi l c ha r a c t e r i s t i c s of t he S R S a nd L a S a l l e phyt or e m e di a t i on pl ot s f r om hi gh c ont a m i na nt r e gi ons * S a m pl e s c ol l e c t e d i n O c t obe r 2003 a nd J ul y 2003 a t t he S R S a nd L a S a l l e r e s pe c t i ve l y. b O M = or ga ni c m a t t e r ; W C = w a t e r c ont e nt ; F C = f i e l d c a pa c i t y.

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158 T a bl e A 2. A na l ys i s of va r i a nc e r e s ul t s ( P va l ue s ) f or t he e f f e c t o f t i m e a nd de pt h on r hi z os phe r e ( R H ) a nd r hi z opl a ne ( R P ) m i c r obi a l a bunda nc e of t he L a S a l l e phyt or e m e di a t i on pl ot s N S= n o t s i g n i fi can t ( P v al u e> 0 0 5 ); N A = n o t an al y zed 1 A t t h e 3 0 6 0 c m s o i l l ay er r ep eat ed m eas u r emen t d e s i g n 2 [T C E / PC E ] = Co n t a mi n an t co n c en t rat i o n 3 A t e ach s a mp l i n g t i me (J u l 2 0 0 3 / J u l 2 0 0 4 / N o v 2 0 0 4 ) s p l i t p l o t d es i g n

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159 F i gur e A 1 D G G E ge l o f P C R a m pl i f i e d pa r t i a l p m oA f r a gm e nt s of di f f e r e nt m e t ha not r op h t ype s

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160 F i gur e A 2. E f f e c t of de pt h on oxyge n upt a ke r a t e s of N M S w i t h C u e nr i c hm e nt s M e t ha not r oph a c t i vi t y i n t he pr e s e nc e of C H 4 f or t he T C E S i t e popl a r r hi z os phe r e ( R H s ol i d ba r s ) a nd non pl a nt e d s oi l ( N P c r os s e d ba r s ) a t t he hi gh c ont a m i na nt r e gi on a nd dur i ng J ul y 2003 ( e m pt y ba r s ) a nd N ove m be r 2004 ( f i l l e d ba r s ) V a l ue s r e p r e s e nt a ve r a ge s t a nda r d de vi a t i on of t r i pl i c a t e r uns

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161 F i gur e A 3. D G G E ge l s of 16S r D N A pa r t i a l s e que nc e s f r om N M S w i t h C u e nr i c hm e nt s S a m pl e s c ol l e c t e d f r om t he hi gh c ont a m i na nt r e gi on a nd 30 60 c m s oi l l a ye r P r of i l e na m e = phy t or e m e di a t i on pl o t ( S = S R S T = L a S a l l e T C E S i t e # P = L a S a l l e P C E S i t e ) t r e e t ype ( P i = pi ne P = po pl a r W = w i l l ow ) w i l l ow c l one ( S 365) s oi l c om pa r t m e nt ( R H = r hi z os phe r e R P = r hi z opl a ne N P = non pl a nt e d s oi l ) s a m pl i ng pe r i od ( 1= J ul y 2003 2= J ul y 2004, 3= O c t obe r 2003 a nd N ove m be r 2004, a t t he S R S a nd L a S a l l e s i t e s r e s pe c t i ve l y) ( R ) = r e pl i c a t e pr of i l e W hi t e f i l l e d c i r c l e = m os t a bunda nt ba nd. E m pt y c i r c l e = s e que nc e d ba nd. M a r ke r ( a = M e t hy l oc oc c us c aps ul at us [ B a t h] b= M e t hy l om i c r obi um al bum B G 8, c = M e t hy l oc y s t i s t r i c hos por i um O B 3b)

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162 F i gur e A 4. P hyl oge ne t i c t r e e o f 16S r D N A pa r t i a l s e que nc e s f r om N M S w i t h C u e nr i c hm e nt s A na l ys i s ba s e d i n r e l a t i on t o e xt a nt m e t ha not r ophs a nd hi g he s t s c or i ng B L A S T a l i gne d s e que nc e s S a m pl e s c ol l e c t e d f r om t he 30 60 c m s oi l l a ye r a t t he h i gh c ont a m i na nt r e gi ons L e ngt h of br a nc he s i s pr opor t i ona l t o % di s s i m i l a r i t y ( 0. 01 s ubs t i t ut i on pe r nuc l e ot i de s i t e ) N um be r s on b r a nc he s r e pr e s e nt % of boot s t r a p va l ue s f r om 1000 r e pl i c a t i ons S e que nc e na m e = phyt or e m e di a t i on pl ot ( S = S R S + T = L a S a l l e T C E S i t e , P = L a S a l l e P C E S i t e ) t r e e t ype ( P i = pi ne P = popl a r W = w i l l ow ) w i l l ow c l one ( S 365 ) s oi l c om pa r t m e nt ( R H = r hi z os phe r e R P = r hi z opl a ne N P = non pl a nt e d s oi l ) s a m pl i ng pe r i od ( 3= O c t obe r 2003 a nd N ove m be r 2004, a t t he S R S a nd L a S a l l e s i t e s r e s pe c t i ve l y) ba nd num be r

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163 L I S T O F R E F E R E N C E S A dr i a e ns A a nd G r bi G a l i D ( 1994 ) C om e t a bol i c t r a ns f or m a t i on of m ono a nd di c hl or obi phe nyl s a nd c hl or ohydr oxyb i phe nyl s by m e t ha not r ophi c gr oundw a t e r i s ol a t e s E nv i r onm e nt al Sc i e nc e and T e c hnol ogy 2 8 : 1325 1330 A dr i a e ns P ( 1994) E v i de nc e f or c hl or i ne m i gr a t i o n dur i ng ox i da t i on of 2 c hl o r obi phe nyl by a t ype I I m e t ha not r oph. A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 60 : 1658 1662. A ge nc y f or T oxi c S ubs t a nc e s D i s e a s e R e gi s t r y ( A T S D R ) ( 1999 ) T oxi c ol ogy p r of i l e f or t r i c hl oe t hyl e ne U S D e pa r t m e nt o f H e a l t h & H u m a n S e r vi c e s P ubl i c H e a l t h S e r vi c e A t l a nt a G A A ge nc y f or T oxi c S ubs t a nc e s D i s e a s e R e gi s t r y ( A T S D R ) ( 2006 ) 2005 C E R C L A P r i or i t y L i s t of H a z a r dous S ubs t a nc e s U S D e pa r t m e nt o f H e a l t h & H um a n S e r vi c e s P ubl i c H e a l t h S e r vi c e A t l a nt a G A A l t s c hul S F G i s h, W M i l l e r E W a nd L i pm a n D J ( 1990 ) B a s i c l oc a l a l i gnm e nt s e a r c h t ool J our nal of M ol e c ul ar B i ol ogy 215 : 40 3 410. A l va r e z C ohe n, L a nd M c C a r t y, P L ( 1991a ) E f f e c t s of t oxi c i t y, a e r a t i on, a nd r e duc t a nt s uppl y on t r i c hl o r oe t hyl e ne t r a ns f or m a t i on by a m i xe d m e t ha not r ophi c c ul t ur e A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 57 : 228 235. A l va r e z C ohe n, L a nd M c C a r t y, P L ( 1991b) P r o duc t t oxi c i t y a nd c om e t a bol i c c om pe t i t i ve i nhi bi t i on m ode l i ng o f c hl or o f or m a nd t r i c hl or oe t hyl e ne t r a ns f or m a t i on by m e t ha not r ophi c r e s t i ng c e l l s A p pl i e d and E nv i r onm e nt al M i c r obi ol ogy 57 : 1031 1 037 A l va r e z C ohe n, L M c C a r t y, P L B oul ygi na E H a ns on, R S B r us s e a u, G A a nd T s i e n, H C ( 1992 ) C ha r a c t e r i z a t i on o f a m e t ha ne ut i l i z i ng ba c t e r i um f r om a ba c t e r i a l c ons or t i um t ha t r a pi dl y de g r a de s t r i c hl or oe t hyl e ne a nd c hor of or m A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 58 : 188 6 1893. A m a nn, R I L udw i g W a nd S c hl e i f e r K H ( 19 95) P hyl oge ne t i c i de nt i f i c a t i on a nd i n s i t u de t e c t i on of i n di vi dua l m i c r obi a l c e l l s w i t hout c ul t i va t i on. M i c r obi ol ogi c al R e v i e w s 59 : 143 169. A m a r a l J A a nd K now l e s R ( 1997 ) I nhi bi t i on o f m e t ha ne c ons um pt i on i n f or e s t s oi l s a nd pur e c ul t ur e s o f m e t ha not r ophs by a que ous f or e s t s oi l e xt r a c t s Soi l B i ol ogy and B i oc he m i s t r y 29 : 1713 1720

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164 A m a r a l J A a nd K now l e s R ( 1998 ) I nhi bi t i on o f m e t ha ne c ons um pt i on i n f or e s t s o i l s by m onot e r pe ne s J our nal of C he m i c al E c ol ogy 24 : 723 733. A m a r a l J A E ki ns A R i c ha r ds S R a nd K now l e s R ( 1998 ) E f f e c t of s e l e c t e d m onot e r pe ne s on m e t ha ne oxi da t i on, de ni t r i f i c a t i o n, a nd a e r obi c m e t a bol i s m by ba c t e r i a i n pur e c ul t ur e A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 64 : 520 525. A nde r s on, T A G ut hr i e E A a nd W a l t on, B T ( 1993) B i or e m e di a t i on i n t he r hi z os phe r e E nv i r onm e nt al Sc i e nc e and T e c hnol o gy 27 : 2630 2636 A nde r s on, T A a nd W a l t on B T ( 1995 ) C om pa r a t i ve f a t e of [ 14C ] t r i c hl or oe t hyl e ne i n t he r oot z one of pl a nt s f r om a f o r m e r s ol ve nt di s pos a l s i t e E nv i r onm e nt al T ox i c ol ogy and C he m i s t r y 14 : 2041 2047. A r c i e r o, D V a nne l l i T L oga n, M a nd H oope r A B ( 1989 ) D e gr a da t i on of t r i c hl or oe t hyl e ne by t he a m m oni a oxi d i z i ng ba c t e r i um N i t r os om as e ur opae a B i oc he m i c al and B i ophy s i c al R e s e ar c h C om m uni c at i ons : 640 643. A um a n, A J S t ol ya r S C os t e l l o, A M a nd L i ds t r om M E ( 2000 ) M ol e c ul a r c ha r a c t e r i z a t i on of m e t ha not r o phi c i s ol a t e s f r om f r e s hw a t e r l a ke s e di m e nt A ppl i e d and E n v i r onm e nt al M i c r obi ol ogy 66 : 525 9 5266. A us ube l F M B r e nt R K i ngs t on, R E M oor e D D S m i t h J A S e i dm e n, J G a nd S t r uhl K ( e ds ) ( 1989) C ur r e nt pr ot oc ol s i n m ol e c ul ar bi ol ogy N e w Y o r k, N Y : J ohn W i l e y & S ons I nc A us ube l F M B r e nt R K i ngs t on, R E M oor e D D S e i dm a n, J G S m i t h, J A a nd S t r uhl K ( e ds ) ( 1992) Shor t pr ot oc ol s i n m ol e c ul a r bi ol ogy N e w Y or k : J ohn W i l e y & S ons B a ke r P F ut a m a t a H H a r a ya m a S a nd W a t a n a be K ( 2001) B a c t e r i a l popul a t i ons oc ur r i ng i n a t r i c hl or oe t hyl e ne c ont a m i na t e d a qui f e r dur i ng m e t ha ne i nj e c t i on. E nv i r onm e nt al M i c r obi ol ogy 3 : 187 193. B a l s e r T C K i r c hne r J W a nd F i r e s t one M K ( 2002) M e t hodol ogi c a l va r i a bi l i t y i n m i c r obi a l c om m uni t y l e ve l phys i ol ogi c a l p r of i l e s Soi l Sc i Soc A m J 66 : 519 523. B a r oc s i A C s i nt a l a n, Z K oc s a nyi L D us he nkov, S K upe r be r g, J M K uc ha r s ki R a nd R i c ht e r P I ( 2003) O pt i m i z i ng phyt o r e m e di a t i on of he a vy m e t a l c ont a m i na t e d s oi l by e xpl oi t i ng pl a nt s s t r e s s a da pt a t i on. I n t e r nat i onal J our nal of P hy t or e m e di at i on 5 : 13 23 B a r r i o L a ge G P a r s ons F Z N a s s a r R S a nd L or e nz o, P A ( 1986) S e que nt i a l de ha l oge na t i on of c hl or i na t e d e t he ne s E nv i r onm e nt al Sc i e nc e and T e c hnol ogy 20 : 96 99.

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165 B e nj a m i n, M T S udol M B l oc h, L a nd W i ne r A M ( 1996 ) L ow e m i t t i ng ur ba n f or e s t s : A t a xonom i c m e t hodol ogy f o r a s s i gni ng i s opr e ne a nd m onot e r pe ne e m i s s i on r a t e s A t m os phe r i c E nv i r onm e nt 30 : 1437 1452. B l a yl oc k, M J S a l t D E D us he nkov, S Z a kha r o va O G us s m a n, C K a pul ni k Y e t a l ( 1997 ) E nha nc e d a c c um ul a t i on of P b i n I ndi a n m us t a r d by s oi l a ppl i e d c he l a t i ng a ge nt s E nv i r onm e nt al Sc i e nc e & T e c hnol ogy 31 : 860 865 B ode l i e r P L E M e i m a F r a nke M Z w a r t G a nd L a a nbr oe k, H J ( 2005) N e w D G G E s t r a t e gi e s f or t he a na l ys e s of m e t ha not r ophi c m i c r obi a l c om m un i t i e s us i ng di f f e r e nt c om bi na t i ons o f e xi s t i ng 16S r R N A ba s e d pr i m e r s F E M S M i c r obi ol ogy E c ol ogy 52 : 163 174. B odr os s y, L H ol m e s E M H ol m e s A J K ova c s K L a nd M ur r e l l J C ( 1997) A na l ys i s of 16S r R N A a nd m e t ha ne m onooxyge na s e ge ne s e que nc e s r e v e a l s a nove l gr oup o f t he r m ot ol e r a nt a nd t he r m ophi l i c m e t ha not r ophs M e t hy l oc al dum ge n. nov. A r c hi v e s of M i c r obi ol ogy 168 : 493 503 B oon, N D e W i ndt W V e r s t r a e t e W a nd T op E M ( 2002) E va l ua t i on of ne s t e d P C R D G G E ( de na t ur i ng gr a di e nt ge l e l e c t r op hor e s i s ) w i t h gr oup s pe c i f i c 16 S r R N A pr i m e r s f o r t he a na l ys i s of ba c t e r i a l c om m u ni t i e s f r om d i f f e r e nt w a s t e w a t e r t r e a t m e nt pl a nt s F e m s M i c r obi ol ogy E c ol ogy 39 : 101 112. B our ne D G M c D ona l d, I R a nd M ur r e l l J C ( 2001) C om pa r i s on of pm oA P C R pr i m e r s e t s a s t ool s f o r i nve s t i ga t i ng m e t ha not r oph di ve r s i t y i n t hr e e D a ni s h s oi l s A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 67 : 380 2 3809. B ow m a n, J ( 2000 ) T he M e t ha not r ophs : T he F a m i l i e s M e t hy l oc oc c ac e ae a nd M e t hy l oc y s t ac e ae I n T he P r ok ar y ot e s : A n E v ol v i n g E l e c t r oni c R e s our c e f or t he M i c r obi ol ogi c al C om m uni t y D w or ki n M ( e d) N e w Y or k: S p r i nge r V e r l a g B ow m a n, J P S l y, L I N i c hol s P D a nd H a yw a r d, A C ( 1993a ) R e vi s e d t a xonom y o f t he m e t ha not r ophs : de s c r i pt i on o f M e t hy l obac t e r g e n. nov. e m e nda t i on of M e t hy l oc oc c us va l i da t i on of M e t hy l os i nus a nd M e t hy l oc y s t i s s pe c i e s a nd a pr opos a l t ha t t he f a m i l y M e t hy l oc oc c ac e ae i nc l ude s onl y t he gr oup I m e t ha not r ophs I nt e r nat i onal J our nal of Sy s t e m at i c B ac t e r i ol ogy 43 : 735 753. B ow m a n, J P J i m e ne z L R os a r i o, I H a z e n, T C a nd S a yl e r G S ( 1993b) C ha r a c t e r i z a t i on of t he m e t ha not r ophi c ba c t e r i a l c om m uni t y pr e s e nt i n a t r i c hl or oe t hyl e ne c ont a m i na t e d s ubs ur f a c e gr ound w a t e r s i t e A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 59 : 2380 2387. B r i gm on, R L B e l l N C F r e e dm a n, D L a nd B e r r y, C J ( 1998) N a t u r a l a t t e nua t i on o f t r i c hl or oe t hyl e ne i n r hi z os phe r e s oi l s a t t he S a va nna h R i ve r S i t e J our nal of Soi l C ont am i nat i on 7 : 433 453.

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166 B r i gm on, R L A nde r s on, T A a nd F l i e r m a ns C B ( 1999) M e t ha not r ophi c ba c t e r i a i n t he r hi z os phe r e of t r i c hl or oe t hyl e ne de g r a di ng pl a nt s I nt e r nat i onal J our nal of P hy t or e m e di at i on 1 : 241 253 B r i gm on, R L A l t m a n, D W i l de E B e r r y, C J F r a nc k, M W a s hbur n, F e t a l ( 2001) P hyt or e m e di a t i on of t r i c hl o r oe t hyl e ne a nd pe r c hl o r oe t hyl e n e a t t he S a va nna h R i ve r S i t e I n W M 01 C onf e r e nc e T uc s on, A Z p. 22. B r i gm on, R L S a unde r s F M A l t m a n, D W i l de E B e r r y C J F r a nc k, M e t a l ( 2003) F Y 02 F i na l r e por t on phyt o r e m e di a t i on of c hl or i na t e d e t he ne s i n s out he r n s e c t or s e e pl i ne s e di m e nt s of t he S a va nna h R i ve r S i t e W S R C T R 2002 00557 M a r c h, W e s t i nghous e S a va nna h R i ve r C om pa ny. A i ke n, S C B r us s e a u, G A T s i e n, H C H a ns on, R S a nd W a c ke t t L P ( 1990 ) O pt i m i z a t i on of t r i c hl or oe t hyl e ne oxi da t i on by m e t ha not r ophs a nd t he us e of a c ol o r i m e t r i c a s s a y t o de t e c t s ol ubl e m e t ha ne m onooxyge na s e a c t i vi t y. B i ode gr adat i on 1 : 19 29 B r us s e a u, G A B ul ygi na E S a nd H a ns on, R S ( 1994) P hy l oge ne t i c a na l ys i s a nd de ve l opm e nt of pr obe s f or di f f e r e nt i a t i ng m e t hyl ot r ophi c ba c t e r i a A ppl i e d and E n v i r onm e nt al M i c r obi ol ogy 60 : 626 636. B uc kl e y, D H a nd S c hm i dt T M ( 2001) T he s t r u c t ur e of m i c r obi a l c om m un i t i e s i n s oi l a nd t he l a s t i ng i m pa c t o f c ul t i va t i on M i c r obi al E c ol ogy 42 : 11 21 B ur ke n, J G a nd S c hnoor J L ( 1998) P r e di c t i ve r e l a t i ons hi ps f or upt a ke of or ga ni c c ont a m i na nt s by hybr i d pop l a r t r e e s E nv i r onm e nt al Sc i e nc e and T e c hnol ogy 32 : 3379 3385. C a l houn, A a nd K i ng G M ( 1998) C ha r a c t e r i z a t i on of r oot a s s oc i a t e d m e t ha not r ophs f r om t h r e e f r e s hw a t e r m a c r ophyt e s : P ont e de r i a c o r dat a, Spar gani um e ur y c ar pu m a nd Sagi t t ar i a l at i f ol i a A pp l i e d and E nv i r onm e nt al M i c r obi ol ogy 64 : 1099 1105 C ha ng, H a nd A l va r e z C ohe n, L ( 1996) B i ode gr a da t i on of i ndi v i dua l a nd m u l t i pl e c hl or i na t e d a l i pha t i c hydr oc a r bons by m e t ha ne oxi di z i ng c ul t ur e s A ppl i e d a nd E nv i r onm e nt al M i c r obi ol ogy 62 : 3371 3377. C ha udhr y, Q B l om Z a nds t r a M G upt a S a nd J one r E J ( 2005) U t i l i s i ng t he s yne r gy be t w e e n pl a nt s a nd r hi z os phe r e m i c r oor ga ni s m s t o e nha nc e br e a kdow n of o r ga ni c pol l ut a nt s i n t he e nvi r onm e nt E nv i r onm e nt al Sc i e nc e and P ol l ut i on R e s e ar c h 12 : 34 48. C he r e m i s i nof f N P ( 2001 ) S pot l i ght on c hl or i na t e d hydr oc a r bons : t hr e e t ype s of c hl or i na t e d hydr oc a r bons c a n pos e s e r i ous he a l t h r i s ks by c ont a m i na t i ng dr i nki ng w a t e r P o l l ut i on E ngi ne e r i ng N ov : 22 26. C hoi D W K unz R C B oyd, E S S e m r a u J D A nt hol i ne W E H a n, J I e t a l ( 2003) T he m e m br a ne a s s oc i a t e d m e t ha ne m onoo xyge na s e ( pM M O ) a nd pM M O

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1 69 E nvi r onm e nt a l P r ot e c t i on A ge nc y ( E P A ) ( 2000a ) D r i nki ng w a t e r s t a nda r ds a nd he a l t h a dvi s or i e s E P A 822 8 00 001 S um m e r U S E nv i r onm e nt a l P r o t e c t i on A ge nc y. W a s hi ngt on, D C E nvi r o nm e nt a l P r ot e c t i on A ge nc y ( E P A ) ( 2000b) I nt r oduc t i on t o phyt or e m e di a t i on E P A / 600/ R 99/ 107, F e br ua r y U S E nvi r onm e nt a l P r ot e c t i on A ge nc y. C i nc i nna t i O H E nvi r onm e nt a l P r ot e c t i on A ge nc y ( E P A ) ( 2001) G r ound w a t e r i s s ue : phyt or e m e di a t i on of c ont a m i na t e d s o i l a nd gr ound w a t e r a t ha z a r dou s w a s t e s i t e s E P A / 540/ S 01/ 500, F e br ua r y, U S E nvi r onm e nt a l P r ot e c t i on A ge nc y. A da O K E nvi r onm e nt a l P r ot e c t i on A ge nc y ( E P A ) ( 2002) N P L F a c t s he e t s f or I l l i noi s : L a S a l l e E l e c t r i c U t i l i t i e s E P A I D # I L D 980794333 F e br ua r y, U S E nv i r onm e nt a l P r ot e c t i on A ge nc y. L a S a l l e I L E s hi ni m a e v, B T M e dve dkova K A K hm e l e ni n a V N S uz i na N E O s i pov, G A L ys e nko, A M a nd T r o t s e nko, Y A ( 2004) N e w t he r m ophi l i c m e t ha not r ophs o f t he ge nus M e t hy l oc al dum M i c r obi ol ogy 73 : 448 4 56 E w e r s J F r e i e r s c hr ode r D a nd K na c km us s H J ( 1990) S e l e c t i on o f t r i c hl or oe t he ne ( T C E ) de gr a di ng ba c t e r i a t ha t r e s i s t i na c t i va t i on b y T C E A r c hi v e s of M i c r obi ol ogy 154 : 410 413 F a ng, J B a r c e l ona M J a nd S e m r a u J D ( 2000 ) C ha r a c t e r i z a t i on of m e t ha not r ophi c ba c t e r i a on t he ba s i s of i nt a c t phos phol i pi d p r of i l e s F E M S M i c r obi ol ogy E c ol ogy 189 : 67 72. F a s s e l T A S c ha l l e r M J L i ds t r om M E a nd R e m s e n, C C ( 1990 ) E f f e c t of f i xa t i on r e s i n c om bi na t i ons a nd r ut he ni um r e d on e l uc i da t i ng out e r e nve l ope s t r uc t u r e a nd s ur f a c e m or phol ogy of 2 m e t ha not r ophi c ba c t e r i a J our nal of E l e c t r on M i c r os c opy T e c hni que 14 : 52 62. F a s s e l T A S c ha l l e r M J a nd R e m s e n, C C ( 19 92) C om pa r i s on of A l c i a n bl ue a nd r ut he ni um r e d e f f e c t s on p r e s e r va t i on of out e r e nv e l ope ul t r a s t r uc t ur e i n m e t ha not r ophi c ba c t e r i a M i c r os c opy R e s e ar c h T e c hni que s 20 : 87 94. F e l s e ns t e i n, J ( 2004) P H Y L I P ( P hyl oge ny I nf e r e n c e P a c ka ge ) ve r s i on 3 6b. U ni ve r s i t y of W a s hi ngt on, S e a t t l e F e l s ke A a nd A kke r m a ns A D L ( 1997 ) S pa t i a l hom o ge ne i t y of a bunda nt ba c t e r i a l 16S r R N A m ol e c ul e s i n g r a s s l a nd s oi l s M i c r obi al E c ol ogy 36 : 31 36. F l e t c he r J S a nd H e gde R S ( 1995) R e l e a s e of p he nol s by pe r e nni a l pl a nt r oot s a nd t he i r pot e nt i a l i m por t a nc e i n bi or e m e di a t i on C he m os phe r e 31 : 3009 3016

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170 F ol s om B R C ha pm a n, P J a nd P r i t c ha r d, P H ( 1990) P he nol a nd t r i c hl o r oe t hyl e ne de gr a da t i on by P s e udom onas c e pac i a G 4 ki ne t i c s a nd i nt e r a c t i ons be t w e e n s ubs t r a t e s A ppl i e d and E nv i r onm e nt al M i c r obi ol o gy 56 : 1279 1285 F ox, B G B or ne m a n, J G W a c ke t t L P a nd L i p s c om b, J D ( 1990) H a l oa l ke ne oxi da t i on by t he s ol ubl e m e t ha ne m onooxyge na s e f r om M e t hyl os i nus t r i c hos por i um O B 3b: m e c ha ni s t i c a nd e nvi r onm e nt a l i m pl i c a t i ons B i oc he m i s t r y 29 : 6419 6427 F r y, J C ( 2004) C ul t u r e de pe nde nt m i c r obi o l ogy. I n M i c r obi al d i v e r s i t y and bi opr os pe c t i ng B ul l A T ( e d) W a s hi ngt on, D C : A S M P r e s s pp. 80 87. G a l c he nko, V F S hi s hki na V N S uz i na N E a nd T r ot s e nko, Y A ( 1977 ) I s ol a t i on a nd pr ope r t i e s of ne w s t r a i ns of obl i ga t e m e t ha not r ophs M i c r obi ol ogy 46 : 898 903. G e r on, C H a r l e y P a nd G ue nt he r A ( 2001) I s opr e ne e m i s s i on c a pa c i t y f or U S t r e e s pe c i e s A t m os phe r i c E nv i r onm e nt 35 : 3341 3352. G i l be r t B A B m us B H a r t m a nn A a nd F r e nz e l P ( 1998) I n s i t u l oc a l i z a t i on of t w o m e t ha not r ophi c s t r a i ns i n t he r hi z os phe r e of r i c e pl a nt s F E M S M i c r obi ol ogy E c ol ogy 25 : 117 128. G i l be r t B a nd F r e nz e l P ( 1998) R i c e r oot s a nd C H 4 oxi da t i on: t he a c t i vi t y of ba c t e r i a t he i r di s t r i but i on a nd t he m i c r oe nvi r onm e nt Soi l B i ol ogy and B i oc he m i s t r y 30 : 1903 1916. G i r va n, M S B ul l i m or e J P r e t t y, J N O s bor n A M a nd B a l l A S ( 2003 ) S o i l t ype i s t he pr i m a r y de t e r m i na nt o f t he c om pos i t i on of t he t ot a l a nd a c t i ve ba c t e r i a l c om m uni t i e s i n a r a bl e s oi l s A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 69 : 1800 1809. G r a ha m D W K i m H J a nd L i ndne r A S ( 200 2) M e t ha not r ophi c ba c t e r i a I n E nc y c l ope di a of E nv i r onm e nt al M i c r obi ol ogy B i t t on, G ( e d) N e w Y or k, N Y : J ohn W i l e y & S ons I nc pp 1923 1936. H a by, P A a nd C r ow l e y, D E ( 1996 ) B i ode gr a da t i on of 3 c hl or obe n z oa t e a s a f f e c t e d by r hi z ode pos i t i on a nd s e l e c t e d c a r bon s ubs t r a t e s J our nal of E nv i r onm e nt al Q ual i t y 25 : 304 310. H a ns on, R S a nd H a ns on, T E ( 1996) M e t ha not r ophi c ba c t e r i a M i c r obi ol ogi c al R e v i e w s 60 : 439 471. H a r de r J a nd P r obi a n C ( 1995) M i c r ob i a l de g r a da t i on of m o not e r pe ne s i n t he a bs e nc e of m ol e c ul a r oxyge n A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 61 : 3804 3808

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174 L a ne D J ( 1991 ) 16S / 23S r R N A s e que nc i ng. I n N uc l e i c ac i d t e c hni que s i n bac t e r i al s y s t e m at i c s S t a c ke br a ndt E a nd G oodf e l l ow M ( e ds ) N e w Y or k, N Y : J ohn W i l e y & S ons I nc pp 115 175. L a nge R ( 2004) S e c ond f i ve ye a r r e vi e w r e por t f or L a S a l l e E l e c t r i c U t i l i t i e s S i t e S e pt e m be r U S E P A L a S a l l e I l l i noi s L a s h, L H F i s he r J W L i ps c om b, J C a nd P a r k e r J C ( 2000 ) M e t a bol i s m of t r i c hl or oe t hyl e ne E nv i r onm e nt al H e al t h P e r s pe c t i v e s 108 : 177 200. L e a dbe t t e r E R a nd F os t e r J W ( 1959) O xi da t i o n pr oduc t s f o r m e d f r om g a s e ous a l ka ne s by t he ba c t e r i um P s e udom ona s m e t ha ni c a A r c hi v e s of B i oc he m i s t r y and B i ophy s i c s 82 : 491 492. L e i gh, M B F l e t c he r J S F u, X O a nd S c hm i t z F J ( 2002) R oot t u r nove r : A n i m por t a nt s our c e of m i c r obi a l s ubs t r a t e s i n r hi z os p he r e r e m e di a t i o n o f r e c a l c i t r a nt c ont a m i na nt s E nv i r onm e nt al Sc i e nc e and T e c hnol ogy 36 : 1579 1583 L e w i s P R a nd K ni ght D P ( 1977) St ai ni ng m e t hods f or s e c t i one d m at e r i al A m s t e r da m T he N e t he r l a nds : N or t h H ol l a nd L i J P e r due E M P a vl os t a t hi s S G a nd A r a uj o R ( 1998) P hys i c oc he m i c a l pr ope r t i e s of s e l e c t e d m onot e r pe ne s E nv i r onm e nt I nt e r nat i onal 24 : 353 358. L i ds t r om M E ( 2001) A e r obi c m e t hyl ot r ophi c pr oka r yot e s r e l e a s e 3. 7, N ove m be r 2, 2001, ht t p: / / l i nk. s pr i nge r ny. c om / l i nk/ s e r vi c e / books / 10125/ I n T he P r ok ar y ot e s : A n E v ol v i ng E l e c t r oni c R e s our c e f or t he M i c r obi ol ogi c al C om m uni t y D w or ki n M B a l ow s A T r upe r H G H a r de r W a nd S c h l e i f e r K H ( e ds ) N e w Y o r k, N Y : S pr i nge r V e r l a g. L i e be r m a n, R L a nd R os e nz w e i g, A C ( 2004) B i ol ogi c a l m e t ha ne oxi da t i on: R e gul a t i on, bi oc he m i s t r y, a nd a c t i ve s i t e s t r uc t ur e of pa r t i c ul a t e m e t ha ne m onooxyge na s e C r i t i c al R e v i e w s i n B i oc he m i s t r y and M ol e c ul ar B i ol ogy 39 : 147 164. L i n, J L R a da j e w s ki S E s hi ni m a e v, B T T r ot s e nko, Y A M c D ona l d, I R a nd M ur r e l l J C ( 2004 ) M ol e c ul a r di ve r s i t y of m e t ha n ot r ophs i n T r a ns ba i ka l s oda l a ke s e di m e nt s a nd i de nt i f i c a t i on of pot e nt i a l l y a c t i ve popul a t i ons by s t a bl e i s ot ope pr obi ng. E nv i r onm e nt al M i c r ob i ol ogy 16 : 1049 1060. L i ndne r A S A dr i a e ns P a nd S e m r a u, J D ( 200 0) T r a ns f or m a t i on o f o r t ho s ubs t i t ut e d bi phe nyl s by M e t hy l os i nus t r i c hos por i um O B 3b: s ubs t i t ue nt e f f e c t s on oxi da t i on ki ne t i c s a nd pr oduc t f o r m a t i on. A r c hi v e s of M i c r obi ol ogy 174 : 35 4 1. L i ndne r A S W hi t f i e l d C C he n, N S e m r a u, J D a nd A dr i a e ns P ( 2003) Q ua nt i t a t i ve s t r uc t ur e bi ode gr a da t i on r e l a t i ons hi ps f or or t ho s ubs t i t ut e d bi phe nyl c om pounds oxi di z e d by M e t hy l os i nus t r i c hos por i um O B 3b. E nv i r onm e nt al T ox i c ol ogy and C he m i s t r y 22 : 2251 2257.

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180 S hi m H C ha uha n, S R yoo, D B ow e r s K T ho m a s S M C a na da K A e t a l ( 2000 ) R hi z os phe r e c om pe t i t i ve ne s s of t r i c hl or oe t hyl e ne de gr a di ng, popl a r c ol oni z i ng r e c om bi na nt ba c t e r i a A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 66 : 4673 4678 S i dhu, M S a nd O l s e n, I ( 1997 ) S l a ye r s of B ac i l l us s pe c i e s M i c r obi ol ogy U k 143 : 1039 1052. S l e yt r U B a nd M e s s ne r P ( 1988) C r ys t a l l i ne s ur f a c e l a ye r s i n p r oka r yot e s J our nal of B ac t e r i ol ogy 17 0 : 2891 2897 S l e yt r U B M e s s ne r P P um D a nd S a r a M ( 1 993) C r ys t a l l i ne ba c t e r i a l c e l l s ur f a c e l a ye r s ge ne r a l pr i nc i pl e s a nd a ppl i c a t i on pot e nt i a l J our nal of A ppl i e d B ac t e r i ol ogy 74 : S 21 S 32. S m a l l a K W i e l a nd, G B uc hne r A Z oc k, A P a r z y J K a i s e r S e t a l ( 2001 ) B ul k a nd r hi z os phe r e s oi l ba c t e r i a l c om m un i t i e s s t udi e d by de na t ur i ng gr a di e nt ge l e l e c t r ophor e s i s : pl a nt de pe nde nt e nr i c hm e nt a nd s e a s ona l s hi f t s r e ve a l e d. A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 67 : 4742 4751 S m a l l a K ( 20 04 ) C ul t u r e i nde pe nde nt m i c r obi ol o gy. I n M i c r obi al D i v e r s i t y and B i opr os pe c t i ng B ul l A T ( e d) W a s hi ngt on, D C : A S M P r e s s pp 88 99. S m i be r t R M a nd K r i e g, N R ( 1981) G e ne r a l c h a r a c t e r i z a t i on. I n M anual of m e t hods f or ge ne r al bac t e r i ol ogy P hi l l i p s G B ( e d) W a s hi ngt on, D C : A m e r i c a n S oc i e t y f or M i c r obi ol ogy pp 409 443. S m i t h, K S C os t e l l o, A M a nd L i ds t r om M E ( 1997) M e t ha ne a nd t r i c hl o r oe t hyl e ne oxi da t i on by a n e s t ua r i ne m e t ha not r oph, M e t hy l ob ac t e r s p. s t r a i n B B 5. 1. A ppl i e d and E nv i r onm e nt al M i c r obi ol ogy 63 : 4617 4620 S nyde r K A a nd W i l l i a m s D G ( 2000) W a t e r s o ur c e s us e d by r i pa r i a n t r e e s va r i e s a m ong s t r e a m t ype s on t he S a n P e dr o R i ve r A r i z o na A gr i c ul t ur al and F or e s t M e t e or ol ogy 105 : 227 240 S or oki n, D Y J one s B E a nd K ue ne n, J G ( 200 0) A n obl i ga t e m e t hyl ot r ophi c m e t ha ne oxi di z i ng M e t hy l om i c r obi um s pe c i e s f r o m a hi ghl y a l ka l i ne e nvi r onm e nt E x t r e m ophi l e s 4 : 145 155 S t a c pool e P W H e nde r s on, G N Y a n, Z M a nd J a m e s M O ( 1998) C l i ni c a l pha r m a c ol ogy a nd t oxi c ol ogy of di c hl o r oa c e t a t e E nv i r onm e nt al H e al t h P e r s pe c t i v e s 106 : 989 994. S t a nl e y, S H P r i or S D L e a k, D J a nd D a l t on H ( 1983 ) C oppe r s t r e s s unde r l i e s t he f unda m e nt a l c ha nge i n i nt r a c e l l ul a r l oc a t i on of m e t ha ne m ono oxyge na s e i n m e t ha ne oxi di z i ng or ga ni s m s : s t udi e s i n ba t c h a nd c ont i nuous c ul t ur e s B i ot e c hnol ogy L e t t e r s 5 : 487 492

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184 Z i m m e r m a n, P R C ha t f i e l d, R B F i s hm a n, J a n d H a ns t P L ( 1978 ) E s t i m a t e s on t he pr oduc t i on of C O a nd H 2 f r om t he oxi da t i on o f hy dr oc a r bon e m i s s i ons f r om ve ge t a t i on. G e ophy s i c al R e s e ar c h L e t t e r s 5 : 679 6 82.

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185 B I O G R A P H I C A L S K E T C H I ha ve a l w a ys be e n i nt e r e s t e d i n t he r e l a t i ons hi p be t w e e n hum a ns a nd t he i r e nvi r onm e nt a nd how w e c a n o f f e r s oc i e t y s us t a i na bl e a l t e r na t i v e s t o ut i l i z e t he na t ur a l s ur r oundi ngs I be ga n m y a ppr oa c h t o m y pr of e s s i ona l goa l by s t udyi ng f or e s t r y i n m y hom e c ount r y, C os t a R i c a i n 1994 A s I pr ogr e s s e d t hr ough m y B S de g r e e I r e a l i z e d how i m por t a nt i t w a s t o f ul l y unde r s t a nd how na t u r a l s ys t e m s f unc t i on. T ha t i s w hy I de c i de d t o c ont i nue m y s t udi e s a nd f ur t he r e xpl or e m y unde r s t a ndi ng o f t he na t ur a l s ys t e m s by pur s ui ng a M S de gr e e i n bi ol ogy i n 1 998 a t t he U ni ve r s i t y of C os t a R i c a W he n I c om pl e t e d m y M S de gr e e i n 2001 I kne w I w a nt e d t o a ppl y t he know l e dge I ha d a c qui r e d i n f i ndi ng w a ys t o r e m e di a t e t he hum a n i m pa c t e d e nvi r onm e nt by ut i l i z i ng na t ur a l s ys t e m s T he r e f or e I pur s ue d m y P h. D de gr e e a t t he U ni ve r s i t y o f F l o r i da i n e nvi r onm e nt a l e ngi ne e r i ng s c i e nc e s i n 2001 i n t he a r e a o f bi o r e m e di a t i on a nd phyt or e m e di a t i on, t he us e of m i c r oor ga ni s m s a nd pl a nt s t o r e m e di a t e c ont a m i na t e d s i t e s T he s ki l l s a nd e xpe r i e nc e I ha ve obt a i ne d a l ong t h e w a y of pu r s ui ng m y P h. D a r e unpa r a l l e l e d. N ow I l ook f or w a r d t o a c a r e e r i n r e s e a r c h of r e m e di a t i on t e c hnol ogi e s t o s oc i e t y c ont a m i na t i on pr obl e m s f r om t he m ol e c ul a r t o t he e c os ys t e m l e ve l a ppr oa c h, i n pa r t i c ul a r t he ut i l i z a t i on of na t ur a l p r oc e s s e s a nd s ys t e m s a s s us t a i n a bl e s ol ut i ons


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Title: Contribution of Methanotrophic Groundwater and Rhizosphere Bacteria to Phytoremediation
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
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CONTRIBUTION OF METHANOTROPHIC GROUNDWATER AND
RHIZOSPHERE BACTERIA TO PHYTOREMEDIATION












By

ADRIANA PACHECO


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2006









































Copyright 2006

by

Adriana Pacheco


































This dissertation is dedicated to my parents.
















ACKNOWLEDGMENTS

I thank Dr. Gabriel Bitton, Dr. Robin L. Brigmon, Dr. Donald L. Rockwood, Dr.

Willian R. Wise, and Dr. Angela S. Lindner for their time and assistance offered as my

advisory committee. I especially thank Dr. Angela S. Lindner, my advisor professor, for

her mentoring, her patience, and, especially, for sharing her enthusiasm towards teaching

and scientific research that will always be a source of inspiration. Also, I would like to

thank Dr. Jud Isebrands and Dr. Madeline Rasche for their absolute support. I also thank

our research group for all their support; Jessica Strate, Chance Lauderdale, and Callie

Whitfield. Finally, I thank my family and friends for their unconditional support, without

which none of this would have been possible.




















TABLE OF CONTENTS


I W Le

ACKNOWLEDGMENT S ................. ................. 4..............

LIST OF TABLES ................. ..............8........... .....


LI ST OF FIGURE S ................. ................. 9......... ...


AB STRACT .........._.._.._ ................ 1...._._. 1...


CHAPTER

1 INTRODUCTION ................ ................. 13..............


Significance of the Study ................ ................. 16.............
Literature Review............... ................ 17
Chlorinated Compounds ................. .......... ......... ..... .....1
Phytoremediation. ........._.. ..... ._ ._ .............._ 20...
The Rhizosphere ........._.. ..... ._ ._ .............._ 24...
M onoterpenes ................ ................. 29..............
Methanotrophic Bacteria............... .. .. ..............3
Ecology and habitats of methanotrophs. ........._..._.. ...._.._ ........._.... 34
Environmental factors affecting methanotrophs. ........._..._.. ......_..._.......36
Methanotrophs. and chlorinated compounds ........._..._.. ......_._. ..........37
Methanotrophs. and plants ........._..._.._ ...._._. ...._._ ............3
Phylogenetics of methanotrophs. .....__. ............... ........._.._...... 40
Molecular analysis of methanotrophs. ........._.._.... ........._._. ........._.........41
Methods Used to Assess Rhizodegradation Potential in Phytoremediation .......44
Culture-dependent techniques .....___.....__.___ .......____ .............4
Culture-independent techniques .....__.___ ........_._ ........__.........46
Study Hypothesis.............. ............... 50
Study Objectives ........._.. ..... ._ ._ .............._ 51...
Broad Obj ectives ........._.. ..... ._ ._ .............._ 5 1..
Specific Obj ectives ........._.. ..... ._ ._ .............._ 51...

2 M~ETHYLOCYSTIS ALDRICHII SP. NOV., A NOVEL METHANOTROPH
ISOLATED FROM A GROUNDWATER AQUIFER.............. ............ .... 57

Introduction............... ............. 57
M materials and M ethods ................. ................. 58......... ...












Results .............. .. ..............64.......... ......
Di scus si on ................ ..............67. ..............


3 EFFECTS OF ALPHA-PINENE AND TRICHLOROETHYLENE ON
OXIDATION POTENTIALS OF METHANOTROPHIC BACTERIA ............... 78


Introduction............... ............. 78
Materials and Methods ............. ...... .............. 80...
Results and Discussion ............. ...... ..............82...


4 STABLE ISOTOPE PROBING FOR CHARACTERIZATION OF
METHANOTROPHIC BACTERIA INT THE RHIZOSPHERE OF
PHYTOREMEDIATING PLANT S ......... ............... ......... ............8


Introduction............... ............. 88
M material s and M ethod s ................. ................. 90......... ...
Site Description ................ ................. 90..............
Sam pling .................. ........ ......... .......... ............ 9
Stable Isotope Probing (SIP) Soil Microcosms.................... ........... 92
Denaturing Gradient Gel Electrophoresis Analysis (DGGE), Sequencing, and
Phylogenetic Analysis ................ ................. 95..............
Statistics ................ ................. 97..............
Results .............. ... ........... ................. 97....
SIP Protocol Implementation ................ ... ........... ..... .............. 97....
Methanotroph Activity and Composition in the TCE Site ........._.._... ..............98
Methanotroph Activity and Composition in the PCE Site. ........._.._... ............ 101
Discussion ........._.._.. ...._..._ ............... 102..


5 CHARACTERIZATION OF RHIZOSPHERE METHANOTROPHIC
BACTERIA INT TCE PHYTOREMEDIATION: IMPACT OF THE DESIGN...... 111


Introducti on ............. ...... ._ ............._ 111...
Materials and Methods ............. ...... __ ............._ 114..
Site Description ............. ...... __ ............._ 114...
Sampling ............. ...... __ ............._ 116...
Soil Characterization ............. ....._ __ .....__ ... ......17
M icrobial Counts .............. ......_ ............._ 118...
Characterization of Enrichments ...._._._._ ..... ..__.. ....._............. 1
Stable Isotope Probing (SIP) Soil Microcosms.............. .............. 120
Phylogenetic Analysis of Enrichments and SIP Microcosms............._.._.. ....... 121
Statistics ........._.._.. ...._..._ ............... 123..
Results ........._.... ......_.._ ............... 124...
Description of Sites ........._.._.. ...._..._ ............... 124..
Microbial Counts ........._.._.. ...._..._ ............... 125..
Root Biomass ........._.._.. ...._..._ ............... 126..
Enrichments Activity ........._.._.._ ...._.._......_._ ..........12
Phylogenetics of Enrichments ................ ................ 129........ ....












SIP Soil M icrocosms .........._.._.. .........._ __ ............._. 131...
Principal Component Analysis (PCA).............. ................. 134
Discussion .........._.. .. ...._.. ............._. 135...
Microbial Abundance ............... ..... ..._ _....._ .... .. .....13
Activity and Phylogenetics of Enrichments. ...._.._.._ ..... ...... ............ 136
Activity and Phylogenetics of SIP Soil Microcosms ................. ................ 139
Principal Component Analysis (PCA).............. ................. 142


6 CONCLUSIONS ................ ................ 152........ .....


APPENDIX ADDITIONAL TABLES AND FIGURES ................ .................... 157


LIST OF REFERENCES.............. .............. 163


BIOGRAPHICAL SKETCH ................. ................ 185........ ....

















LIST OF TABLES


Table pg

1-1. Physical and chemical properties of TCE and PCE .............. ......_ ............53

1-2. Physical and chemical properties of a-pinene. ......___. .... ..._. .............. 54

1-3. Characteristics of different methanotroph types. ........._.._.. ...._.._ ........._.... 55

2-1. Phenotypic characteristics differentiating Strain CSC1 from M~ethylosinus
trichosporium, M~ethylocystis echinoides, and M\~ethylocystis parvus ................... .. 75

5-1. General characteristics of the different phytoremediation plots at the SRS and
LaSalle sites. ........._._.. ...._._._ ............... 145...

5-2. Summary of phylogenetic assignments (BLAST search) of the pmoA gene
sequences of the active methanotroph populations (13C-DNA fraction) from the
SIP soil microcosms in each phytoremediation plot.............. ................. 150

Al. Soil characteristics of the SRS and LaSalle phytoremediation plots from high-
contaminant regions.* ........... 157.......................

A2. Analysis of variance results (P-values) for the effect of time and depth on
rhizosphere (RH) and rhizoplane (RP) microbial abundance of the LaSalle
phytoremediation plots. ........._.._ ...._._ .....___ ............ 5

















LIST OF FIGURES


Fign age

1-1. Schematic of processes in a phytoremediation system. .........._.__ ........._._ .... 53

1-2. Cl metabolism by methanotrophs and methylotrophs as described by Wackett
(1995). .............. .. ................. 54..............

1-3. Oxidation of TCE by aerobic methanotroph degradation (A) and anaerobic
reductive dehalogenation (B).............. .................. 56

2-1. 16S rRNA phylogeny of Strain CSC1 and related M~ethylosinus and M~ethylocystis
species.. ........._ ......._. ..............73....

2-2. Functional genes phylogenies of Strain CSC1 ................ ................ ......... 74

2-3. Transmission electron microscopy photographs of Strain CSC1 and
M~ethylocystis echinoides.. ................ ......... ......... .... ......7

2-4. Electron microscope cytochemistry of the S-layer of Strain CSC1 ................... ...... 77

3-1. Normalized rate of oxygen uptake by the representative methanotrophs in the
presence of varying concentrations of TCE (*) and (R)-a-pinene. ........._............. 86

3-2. Change in the normalized oxygen uptake rate by representative methanotrophs
observed in the presence of 20 ppm TCE at varying concentrations of (R)-a-
pinene.. ........._.._._ ............... 87..._._. ....

4-1. Equilibrium centrifugation of isotopically labeled DNA in CsCl density gradient
colum ns............... ............... 107

4-2. Initial 13CH4 depletion rates (bars) observed in SIP microcosms after the three
sampling periods at the TCE Site (A) and PCE Site (B).............. .................. 108

4-3. DGGE gels of pmoA PCR products derived from the 13C-DNA fraction of SIP
microcosms at the TCE Site (A-C) and PCE Site (D). ................ ................ ..109

4-4. Neighbor joining phylogenetic tree of pmoA sequences derived from the 13C
DNA fraction of 13CH4 SIP microcosms. ........._.._.. ..........__ ....._.. ... 110











5-1. Location of phytoremediation sites and diagram of sampling areas at the (A)
Savannah River Site (SRS), S.C., and (B) LaSalle, IL. ................ ................. 144

5-2. Microbial counts per tree type from the different phytoremediation plots at SRS
and LaSalle. .......___..........___ .......___..........14

5-3. Oxygen uptake rates of enrichments from different tree types in the presence of
CH 4. ........._.. ......___ ............._ 147...

5-4. Frequency of phylum affiliations per tree type of the NMS with Cu enrichment
components. ................ ................ 148........ .....

5-5. Phylogenetic analysis of the SRS SIP soil microcosms .................... ............. 149

5-6. Principal component analysis (PCA) of culture-dependent and culture-
independent measurements. ................ ................ 151........ .....

Al. DGGE gel of PCR-amplified partial pmoA fragments of different methanotroph
types. ............. .................... 159

A2. Effect of depth on oxygen uptake rates of NMS with Cu enrichments. ................. 160

A3. DGGE gels of 16S rDNA partial sequences from NMS with Cu enrichments....... 161

A4. Phylogenetic tree of 16S rDNA partial sequences from NMS with Cu
enrichments. ................ ................ 162........ .....
















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CONTRIBUTION OF METHANOTROPHIC GROUNDWATER AND
RHIZOSPHERE BACTERIA TO PHYTOREMEDIATION

By

Adriana Pacheco

August 2006

Chair: Angela S. Lindner
Major Department: Environmental Engineering Sciences

Trichloroethylene (TCE), a widely used solvent and ubiquitous contaminant, is

effectively removed from soil and groundwater by the use of plants (phytoremediation).

Rapid removal has been reported at the root-zone (rhizosphere), where methanotrophs

(methane-oxidizing bacteria) capable of co-oxidizing TCE are present. The objective of

the study was to determine, by the development of an adequate protocol, how plant type,

system design, and environmental conditions present at two phytoremediation sites

impacts methanotroph's biodegradation potential. To develop characterization methods,

phenotypic and genotypic analyses of an uncharacterized methanotroph, Strain CSC1,

isolated from an uncontaminated groundwater aquifer, were performed. Field sites

represented an engineered system, with poplar and willow trees, and a natural loblolly

pine re-growth area. Laboratory studies were conducted to assess the ability of

methanotrophs to oxidize pine exudates (monoterpenes) and its effects on TCE oxidation.

Field samples were analyzed by culture-dependent microbial counts and enrichments, and









culture-independent stable isotope probing (SIP) microcosms and molecular methods.

Strain CSC1 possessed a unique spiny S-layer and was shown to be a novel strain of the

genus M~ethylocystis and was named M~ethylocystis aldrichii sp. nov. Characterization

methods developed with Strain CSC1 were successfully applied to phytoremediation field

samples and isolates. Different types of methanotrophs were capable of oxidizing

monoterpenes (a-pinene) and, in the presence of TCE; antagonistic and synergistic

responses were observed depending on methanotroph type. Rhizosphere samples

analyzed by culture-dependent methods confirmed the presence of methanotrophs at both

sites; however, enrichments were biased towards type II methanotrophs and did not

correspond with the active populations. Active populations were more diverse and

abundant in the planted samples and strongly influenced by the design, especially the use

of planting material that resulted in a dominance of thermotolerant methanotrophs.

Variable results between the engineered and natural settings highlight the importance of

measuring oxidation potentials and diversity of rhizosphere methanotrophs at any

phytoremediation site, especially if monoterpene-releasing plants are contemplated for

use. Also, study of active populations was shown to be the most accurate

characterization method. Phylogenetic analysis combined with SIP microcosms offers

powerful analytical tools that can ultimately aid practitioners in optimizing

phytoremediation for more effective treatment.















CHAPTER 1
INTTRODUCTION

Chlorinated solvents, such as trichloroethylene (TCE), are a major source of

groundwater and soil pollution throughout the United States. It is well known that soil

microorganisms in the presence and absence of oxygen are capable of degrading these

compounds (Barrio-Lage et al., 1986; Fox et al., 1990; Hanson and Hanson, 1996). TCE

can be metabolized to vinyl chloride, a potent carcinogen, if oxygen is not present

(Barrio-Lage et al., 1986; Ensley, 1991). Therefore, this pathway of degradation is

undesirable given conditions where vinyl chloride can accumulate with no further

breakdown. In contrast, methane-oxidizing bacteria (methanotrophs), aerobic

microorganisms known for their bioremediation potential and prevalence in the

environment, can co-metabolize TCE to CO2 at higher rates than other microorganisms

(Wilson and Wilson, 1985; Little et al., 1988; Fox et al., 1990). However, the pathway of

TCE degradation under aerobic conditions is not without risk of forming toxic

intermediates, including chloral hydrate, dichloroacetate, and ethylene glycol (Oldenhuis

et al., 1989; Alvarez-Cohen and McCarty, 1991a; Stacpoole et al., 1998; Lash et al.,

2000).

Recently, the possibility of using vegetation to enhance degradation of organic

contaminants in soil systems (phytoremediation) has received attention as an attractive

low-cost alternative to the traditional engineering approaches of soil excavation and

incineration, air stripping, and pump-and-treat (EPA, 2000b; EPA, 2001; McCutcheon

and Schnoor, 2003). When plants are used for this purpose, a series of mechanisms are









involved, including phyto-volatilization, -accumulation, -degradation, -stabilization, and

rhizodegradation. However, the role and contribution of each of these processes to the

overall remediation system has not been accurately characterized (Orchard et al., 2000b;

Shang et al., 2003).

It is well known that the microenvironment surrounding the root-zone of plants

(rhizosphere) is characterized by higher numbers of bacteria and increased microbial

activity (Curl and Truelove, 1986). Therefore, rhizosphere metabolism can significantly

contribute or govern the remedial potential of vegetation. Pilot studies of

phytoremediation systems are being tested in the field in areas contaminated with

chlorinated compounds. The two sites studied in this project are the Savannah River Site

(SRS) in Aiken, South Carolina, and the former LaSalle Electrical Utilities in LaSalle,

Illinois, both involving active phytoremediation of TCE and PCE (Brigmon et al., 2001;

EPA, 2002; Lange, 2004). Research conducted at the SRS has demonstrated that TCE

degradation occurs faster in the rhizosphere of trees (Walton and Anderson, 1990;

Anderson and Walton, 1995; Brigmon et al., 2001). Thus, vegetation may be used to

actively promote microbial restoration of contaminated soils and enhanced hazardous

contaminant biodegradation.

In these sites several tree species are being studied for their phytoremediation

potential. Loblolly pine (Pinus taeda) is being tested as a promising species that is

capable of up to 90% TCE removal from soils and groundwater at the SRS (Brigmon et

al., 2001). This species is characterized by the production of significant quantities of oil

extracts, composed mainly of monoterpenes (Amaral et al., 1998; Savithiry et al., 1998;

Phillips et al., 1999). Therefore, these compounds may influence the microbial processes









occurring in the rhizosphere either as root exudates or leachate from the decaying foliage

in the surface soil layers. The importance and occurrence of these interactions on

microbial metabolism have been addressed previously in several studies (White, 1986;

Misra et al., 1996; Amaral and Knowles, 1997; Ward et al., 1997). Conflicting findings

on the role of plant exudates on microbial natural processes in the upper soil layers, such

as nitrification and methane consumption, have claimed inhibition as well as stimulation

by these compounds (White, 1986; Misra et al., 1996; Amaral and Knowles, 1997; Ward

et al., 1997). In planted soils at phytoremediation sites, there is no evidence that connects

the increased microbial mineralization of contaminants with the presence of plant

compounds.

The lack of understanding of the potential roles that rhizosphere bacteria can

assume in the overall removal of contaminants is hindered by the inability to directly

assess the activity and diversity of microorganisms in situ using traditional culture-

dependent methods (Fry, 2004; Smalla, 2004). The recent development of culture-

independent methods that involve soil microcosms and labeled substrates combined with

molecular techniques have enabled scientists to more effectively test in situ conditions

and, more importantly, accurately identify and characterize active microbial populations

(Radajewski et al., 2000).

The main purpose of this study is to determine, by the development of an adequate

protocol, how plant type, system design, and environmental conditions present at two

phytoremediation sites impact the potential ability of methanotrophic bacteria to achieve

biodegradation of chlorinated solvents in contaminated rhizosphere soils and

groundwater. The characterization of this mechanism and the provision of an adequate









technique specific to the rhizosphere can ultimately lead to more efficient

phytoremediation for more effective environmental restoration.

Significance of the Study

Any substance that poses a significant threat to human health and the environment

deserves prioritized attention not only to the study of its toxicology profile but also to its

environmental fate and development of remediation technologies. The U.S. EPA has

classified chlorinated solvents, TCE and PCE, as priority pollutants on the basis of its

widespread contamination in groundwater, its possible carcinogenic nature, and its

potential to be biologically converted to the more potent carcinogen vinyl chloride under

anaerobic conditions. Therefore, the majority of the National Priority List (NPL) sites

are dealing with this type of contamination.

Groundwater and soil contamination by chlorinated solvents presents unique

challenges to remediation technologies. Due to the chemical and physical properties of

these compounds, small amounts of these solvents can contaminate a large volume of

groundwater. The remediation of contaminated soil usually involves excavation and

disposal of the impacted media. However, if the contaminant has reached the

groundwater, the risk to the public, the remedial cost, and the amount of time required to

remove the contaminants can increase substantially (Cheremisinoff, 2001).

Alternatively, in situ remediation technologies, such as bioremediation and

phytoremediation are being tested at several NPL field sites. One example is the SRS

where the potential for phytoremediation of chlorinated solvents has been demonstrated

with loblolly pines capable of up to 90% TCE removal in soil and groundwater (Walton

and Anderson, 1990; Anderson and Walton, 1995). These results are encouraging for the

application of more sustainable remediation technologies that do not require large









amounts of inputs and promote the application of biological systems already in nature to

environmental problems created by humans.

In order to better understand and monitor these biological processes occurring

during phytoremediation, this study combined a laboratory and field approach. The focus

of the study is on the potential contribution of methanotrophic bacteria, capable of co-

oxidizing chlorinated compounds, to the rhizodegradation mechanism in

phytoremediation systems. Additionally, the proj ect concentrates efforts on method

development specific to the rhizosphere environment and compares traditionally used

methodologies with more recent culture-independent methods. Laboratory-based studies

involved characterization of isolated pure and mixed cultures, including a groundwater

isolate, Strain CSC1, which served as a means to develop phenotypic and genotypic

protocols used in the phytoremediation portion of this study. In addition, the role of plant

exudates and their impact on TCE biodegradation was determined with representative

methanotrophs. in the laboratory. Field-based studies involved characterization of the

methanotrophic community in rhizosphere samples from two current TCE- and PCE-

contaminated sites undergoing phytoremediation with different tree types.

Literature Review

Chlorinated Compounds

The widespread use of chlorinated hydrocarbons as solvents and degreasers in the

metal and dry cleaning industries and their indiscriminate disposal have resulted in a

significant adverse effect on the environment. Trichloroethylene (TCE) and

tetrachloroethylene (PCE), primary chlorinated solvents found at hazardous waste sites,

occupied the 16th and 31s~t position, respectively, in the Priority List of the United States

Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA),









known as Superfund (ATSDR, 2006). The pollutants rank according to their presence at

National Priority List facilities, possible carcinogenic nature, and potential to be

converted to more toxic byproducts, as vinyl chloride that occupies the 4th pOSition in the

CERCLA Priority List (Vogel and McCarty, 1985). A recent report from the National

Academies of Science's Committee on Human Health Risks of Trichloroethylene

concluded that the evidence of TCE' s carcinogenic risk has increased since 2001 (NAS,

2006). Consequently, these compounds are heavily regulated by federal and state

standards. The Safe Drinking Water Act regulates the national maximum contaminant

level (MCL) of TCE and PCE in drinking water at 5 ppb, with a zero maximum

contaminant level goal (MCLG) (EPA, 2000a).

The fate of TCE and PCE released into the environment through a variety of waste

streams will be dictated by their physical and chemical properties (Table 1-1). Because

their densities are greater than 1 g ml l, PCE and TCE are considered dense nonaqueous

phase liquids (DNAPLs). These compounds, considered volatile organic compounds

(VOCs), because of their high values of vapor pressure (74-18.5 mm Hg) and Henry's

law constants (0.011-0.018 atm m3 mOl-1), when released into the atmosphere or surface

water and soil, will volatilize into the atmosphere. In the atmosphere, both compounds

are subj ected to photooxidation with a half-life of a couple of months to days. Also, they

both would be predicted to reach the groundwater given their low partitioning coeffieient

values (log Koc and log Kow of 2-3), high specific gravity (>1), and resulting low

tendencies to adsorb to sediments or soils and to bioconcentrate in animals and plants.

Nevertheless, specific site conditions such as organic soil content can readily contribute

to transient sorption of TCE (Brigmon et al., 1998; Sheremata et al., 2000). PCE may









move slower than TCE in soil infiltration processes, because of its lower water solubility

(150 mg 1- ) compared to TCE (1,366 mg 1- ) (ATSDR, 1999).

As a result of their chemical and physical characteristics, groundwater

contamination by chlorinated compounds presents several challenges for remediation.

When TCE and PCE reach the groundwater, they are anticipated to sink deeper into the

subsurface until they reach a less permeable stratum (confining layer). In this layer they

will spread out or escape through fractures of the rock or clay (Kueper and McWhorter,

1991). Therefore, remediation is more difficult than spills of light NAPLs (LNAPLs),

such as gasoline fuels, that float near the surface of the water table as a compact mass and

do not act as a slow-releasing, continuous source of pollution (Cheremisinoff, 2001).

Chlorinated compounds in the environment are prone to microbial degradation;

however, the rate and extent of oxidation in the presence of oxygen is inversely related to

the chlorine-to-carbon ratio (Hanson and Hanson, 1996). Highly chlorinated

hydrocarbons, such as PCE, are not degraded aerobically. PCE is reductively

dehalogenated under anaerobic conditions (Uchiyama et al., 1989; Bowman et al.,

1993b).

In aerobic environments, TCE and the metabolites of the reductive dehalogenation

of PCE and TCE, such as dichloroethylene (DCE) and vinyl chloride (VC), are

cometabolically oxidized to CO2 by bacteria that possess oxygenase enzymes. Some of

these enzymes are the methane monooxygenase (MMO) of methanotrophs, toluene

dioxygenase (TDO) of Pseudomona~sputida F l, toluene 2-monooxygenases (TMO) of

Burkholderia cepacia G4, propane monoxygenase of Mycobacterium vaccae JOB5,

phenol hydroxylase (PH) of Alcaligenes eutrophus JMPl34 and Burkholderia cepacia










G4, alkene monooxygenase (AMO) ofAlcaligenes denitrificans spp., ammonia

monooxygenase of Nitrosomona~s europaea, and isopropylbenzene dioxygenase (IPB) of

Pseudomona~s sp. JR1 (Arciero et al., 1989; Wackett et al., 1989; Ewers et al., 1990;

Folsom et al., 1990; Fox et al., 1990; Dabrock et al., 1992; Kim et al., 1996; Smith et al.,

1997). From this cometabolic process, microorganisms do not gain energy or carbon of

the oxidized pollutant. Therefore, an external source of carbon (electron donor) must be

present apart from oxygen that serves as the electron acceptor in the reaction.

In anaerobic conditions, pathways of degradation occur via the process of

dehalorespiration catalyzed by the reductive dehalogenase enzyme. The chlorinated

compound functions as the electron acceptor and, commonly, hydrogen as the electron

donor. The only known microorganism that performs reductive dechlorination of TCE

and PCE to completion is Dehalococcoides ethenogenes)11) strain 195 (Maymo-Gatell et al.,

1999). Other anaerobes and facultative anaerobes, such as sulfate reducers and

methanogens, degrade TCE and PCE incompletely to cis-DCE and VC (Holliger et al.,

1998). Desulfuromona~s chloroethenica, a sulfur-reducing bacterium utilizes pyruvate or

acetate as the electron donor and degrades PCE or TCE to cis-DCE (Krumholz, 1997).

Phytoremediation

Phytoremediation, the use of plants to remediate contaminated sites, takes

advantage of the ability of plants to extract, sequester or degrade pollutants by the

mechanisms of phyto-extraction, -volatilization, -degradation, -stabilization, and

rhizodegradation (Fig. 1-1). The last mechanism is of special interest because it involves

plant-microbe interactions occurring in the root system (rhizosphere). The role of

rhizosphere microorganisms in the overall breakdown and removal of pollutants is

influenced by the type of contaminant and plant species utilized. Rhizodegradation has

20









been reported as the main process in organic pollutant remediation of toluene, phenol,

and TCE (Narayanan et al., 1999).

The use of plants represents an alternative technology to traditional waste

management practices, such as incineration, excavation and landfilling, and pump-and-

treat-systems. The effectiveness of phytoremediation has been demonstrated in a wide

range of applications, such as herbicides, petroleum hydrocarbons, metals, radionuclides,

leachates from landfills and sewage, nutrients, pentachlorophenol, polycyclic aromatic

hydrocarbons, and chlorinated solvents. Phytoremediation offers multiple advantages,

including being a low cost in situ technology that is environment-friendly and publicly

accepted. Most importantly there is no need to disturb the site and, after the treatment,

the soil is left fertile for further use. However, some limitations and concerns dictate the

potential applications of this technology, including the time necessary for acceptable

effects to take place, the limited depth of the root system, the sensitivity of plants and

microbes towards the contaminant, the seasonal variability in the rate of treatment, and

the potential of contaminant bioaccumulation or transport into the food chain.

Nevertheless, some of these limitations can be overcome by selecting the appropriate

plant species or by combining other technologies, such as pumping and irrigating the

trees with the deeper contaminated groundwater (EPA, 2000b; McCutcheon and Schnoor,

2003).

Phytoremediation efficiency is still limited by a lack of knowledge of many basic

plant processes and interactions with other organisms such as bacteria and fungi.

Pollutant degradation by bacteria and fungi have been studied extensively and, even

though plants can also express similar metabolic pathways, it is only recently that efforts









have been concentrated towards understanding the plant system. Most enzymes involved

in organic xenobiotic degradation, such as cytochrome P450 oxidases, peroxidases, and

glutathione-S-transferase, are known to be present in both microorganisms and plants

(Sandermann, 1994; Shang et al., 2003; Chaudhry et al., 2005).

Phytoremediation of chlorinated solvents from groundwater and soil have reported

up to 90% contaminant removal by the use of different plant species (Walton and

Anderson, 1990; Newman et al., 1999; Brigmon et al., 2001; Nevius et al., 2004).

However, when assessing the responsible mechanisms of contaminant removal, studies

are not consistent. The main contradiction in phytoremediation of chlorinated solvents

regards the magnitude of plant uptake, phytovolatilization, and rhizodegradation

(Orchard et al., 2000a). Several studies have reported that TCE disappearance in planted

systems is mainly due to plant uptake, followed by phytovolatilization and diffusion

through the stem and/or metabolism by the plant (Schroll et al., 1994; Anderson and

Walton, 1995; Newman et al., 1997; Burken and Schnoor, 1998). On the contrary, other

studies have observed TCE degradation occurring mainly as a result of rhizosphere

microbial metabolism (Walton and Anderson, 1990; Anderson et al., 1993; Schnabel et

al., 1997; Orchard et al., 2000a). Contradictory results may be the outcome of

experimental artifacts caused by high exposure to TCE concentrations, use of co-

solvents, the short duration of many studies, and plant stress originated by the use of

static chambers to assess a mass balance of the system. Additionally, problems exist in

the separation of the above- and below-ground compartments, selection of adequate

controls, and lack of methods to correlate bench-scale studies to the field (Orchard et al.,

2000b; Orchard et al., 2000a).









Poplar and willow trees are the preferred plant species in temperate climates for

TCE and PCE phytoremediation. They have also been used for the remediation of heavy

metals, salts, pesticides, explosives, radionuclides, hydrocarbons, and landfill leachates

(Isebrands and Karnosky, 2001). Valuable poplar and willow characteristics that make

them ideal for this application are that they are fast-growing, easily propagated, tolerant

to high levels of contaminants (<550 ppm TCE), resistant to saturated conditions, and

they are phreatophytes (deep-rooted plant where water uptake is mainly from the

groundwater) (Isebrands and Karnosky, 2001; Pilon-Smits, 2005). In particular, willows

have been found to consistently utilize groundwater sources even during periods of

rainfall (Snyder and Williams, 2000). Additionally, poplar and willow trees possess

specialized root vessels (aerenchyma) that may comprise up to 60% of the intracellular

volume and mediate oxygen diffusion deeper into the soil profile (Chaudhry et al., 2005).

It has been hypothesized that willow trees may contain a higher concentration of

oxidative enzymes. When poplar and willow trees were dosed with PCE, only by-

products of degradation were found in willow and no TCE was detected, as it was

commonly found in poplar tissue and its rhizosphere (Nzengung and Jeffers, 2001).

The large surface area and porous wood of poplar trees allows water transport

through the entire cross-section of the stem, which can result in 3 m year- growth under

optimal conditions (Landmeyer, 2001). Transpiration rates can increase from 19 to 200-

1000 L of water day-l in young to mature trees (Newman et al., 1997; Pilon-Smits, 2005).

These high transpiration rates can extract enough water to depress the water table locally,

inducing flow toward the trees and, consequently, containing the contaminant plume

(hydraulic control). Additionally, poplar trees possess endophytic bacteria, including









methanotrophs, that live symbiotically within the plant. Some of these bacteria isolated

from plants are known for their bioremediation potential, including members of

Pseudomona~s sp., Enterobacter-Clostridium species, and methylotroph species such as

M~ethylobacterium populi sp. nov. (Brigmon et al., 1999; Van Aken et al., 2004a; Van

Aken et al., 2004b).

The effectiveness of chlorinated solvent phytoremediation by poplar and willow

trees is strongly influenced by the choice of genotypes (clones). Consideration of the

adequate clone is an essential selection criterion, as the choice must be compatible with

the intended use, the site characteristics (soil type, microclimate, pests and diseases) and

with the local opinion concerning use of native versus exotic trees (Isebrands and

Karnosky, 2001).

Other potential tree species that have been studied for chlorinated solvent

phytoremediation are conifers, in particular the loblolly pine (Anderson and Walton,

1995; Punshon et al., 2002; Brigmon et al., 2003). In a study where pine, willow, and

poplar trees were compared for their TCE phytoremediation potential, undegraded TCE

was found primarily in the vascular system and leaves of pine, whereas plant metabolites

of TCE were found within the leaf tissue of poplar and willow trees, suggesting plant

degradation potential of these type of trees (Punshon et al., 2002). Meanwhile, for pines,

it has been postulated that rhizodegradation is the main phytoremediation mechanism

(Anderson and Walton, 1995).

The Rhizosphere

The rhizosphere is the root-zone under the influence of the plant (Curl and

Truelove, 1986). This zone is constantly enriched with a variety of plant-derived

compounds, and, as a result, higher microbial densities (5-20 times) and rates of activity
24










(2-3 orders) occurred in this area compared to non-vegetated soil (Walton et al., 1994).

In rhizosphere studies, the "rhizoplane" is defined as the soil adhered to the roots, the

"rhizosphere" as the soil under the influence of the plant, and the "rhizosphere effect"

(R/S ratio) as the ratio between the abundance of microbial populations in the rhizosphere

to that in bulk soil. However, the effect of the plant is not only translated in higher

abundance but also in higher activity. Therefore, when plants are present, selective

enrichment of populations may or may not translate to higher R/S ratios, although higher

degradation activity is observed (Haby and Crowley, 1996).

Up to 10-40% of the assimilated carbon may be exuded by plants into the

rhizosphere (rhizodeposition) in the form of compounds that are readily utilized by

microorganisms (Whipps and Lynch, 1983). Plant exudates include sugars, amino acids,

organic acids, nucleotides, flavonones, phenolic compounds, terpenes, and certain

enzymes. The rate of exudation depends on the age of the plant, soil nutrient availability,

presence of contaminants, and seasonality. These compounds have been shown to be

released into the rhizosphere in greater amounts at the end of the growing season during

leaf senescence (Hegde and Fletcher, 1996). During this period, about 58% of the

produced fine root biomass dies (root turnover), and, as a result, an increase of up to 2-

fold in phenolic compounds has been observed at the rhizosphere (Leigh et al., 2002).

These compounds are known to stimulate polychlorinated biphenyl (PCB) biodegradation

(Donnelly et al., 1994). Apart from the variety of carbon sources, the rhizosphere

provides steady redox conditions and ideal attachment sites for bacterial proliferation

(Curl and Truelove, 1986; Shim et al., 2000).









Plants benefit from the presence of rhizosphere microorganisms because they can

increase nutrient availability through biosurfactant production (solubilizes soil-bound

nutrients) and N2 Eixation, produce hormones that promote plant growth, suppress

deleterious microorganisms by the production of antibiotics, and degrade phytotoxic soil

contaminants (Smalla et al., 2001). Thus, there is also considerable interest in

characterizing the structure and function of rhizosphere microbial communities for the

advantageous effects to plants.

Phytoremediation may exploit the beneficial effect of moderate plant stress

(Barocsi et al., 2003; Chaudhry et al., 2005). Certain levels of nutrient and water

deficiencies and chemical toxicity may induce stress adaptation, root proliferation and

exudation, and enhance root hair density. For example, P or K deficiency is known to

stimulate exudation of organic acids and certain enzymes. Meanwhile, Fe or Zn

deficiency induces the production of metal chelators (phytosiderophores) (Chaudhry et

al., 2005). Plant tolerance to heavy metals was enhanced when a synthetic chelate

(ethylenediamintetraacetic acid, EDTA), which rapidly increases metal bioavailability,

was applied in several low doses avoiding plant detrimental effects and securing time for

plant adaptation (Blaylock et al., 1997; Barocsi et al., 2003).

Within the diversity of rhizosphere microorganisms, there are strains capable of

degrading xenobiotic compounds (Curl and Truelove, 1986; Walton and Anderson, 1990;

Walton et al., 1994; Anderson and Walton, 1995; Brigmon et al., 1999). The diversity of

heterotroph microorganisms may enhance stepwise transformation of contaminants by

microbial consortium and/or provide an environment that is favorable for genetic

exchange and gene rearrangements of the degradative traits. The presence of structural









analogs to contaminants in root exudates, cell wall components, and lysates, as well as

secondary products of degradation of these materials, might fortuitously select for

microbes that metabolize (accompanied by energy gain) or cometabolize (involving no

energy gain) xenobiotics. Terpenes (secondary plant metabolites) and PCBs plant

analogs (phenolic compounds) have been reported to play an important role in activating

or transforming specific bacterial habitats by inducing biphenyl dioxygenase in PCB-

degrading bacteria and increase populations of this degraders by up to 100-fold (Donnelly

et al., 1994; Fletcher and Hegde, 1995; Haby and Crowley, 1996).

The rhizosphere provides stable sources of oxygen and methane that can support

the activity of methane-oxidizing bacteria (methanotrophs), known to cometabolically

oxidize TCE at higher rates than other bacteria (Little et al., 1988; Fox et al., 1990;

Brigmon et al., 1999). It has been demonstrated that TCE degradation occurs faster in the

rhizosphere of plants (Walton and Anderson, 1990; Anderson and Walton, 1995), where

the presence and density of methanotrophs has been shown to play an important role in

TCE degradation (Brigmon et al., 1999).

Microbial degradation of contaminants is usually not driven by energy needs, but

by a necessity to reduce toxicity for which microbes may experience an energy deficit.

Therefore, the process may be assisted and driven by the abundant energy available in the

rhizosphere environment as root exudates and accumulated plant biomass. The type of

compounds, the species of plant, and the degree of contamination may have the potential

to exert pressure and thus select for specialized degrading bacterial populations.

Consequently, rhizosphere microbial populations may change considerably with time in

response to the type and degree of contamination (Fletcher and Hegde, 1995; Hernandez









et al., 1997; Brigmon et al., 1999; Kozdroj and van Elsas, 2000). However, there is a

lack of information on specific plant characteristics that promote microbial degradation

of organic pollutants (Chaudhry et al., 2005).

Another unexplored area of rhizosphere microenvironments is the interaction of

plant and microbial populations with mycorrhizae. Mycorrhizae, symbiotic root-fungi,

play an important role in plant establishment and survival. Some of these symbiotic

associations are specific to plant species, such as with loblolly pine trees used in

phytoremediation. In these pines higher densities of methanotrophic bacteria were

observed to be associated with the fungi (Brigmon et al., 1999). Therefore, mycorrhizae

may contribute significantly to the remediation potential of several plant species. The

fungi provides the plant and rhizosphere bacteria protection against drought and toxic

pollutants because of the physical barrier created by their extensive hyphae network.

Also, this network can increase the surface area over which the plants and associated

microorganisms explore for water, nutrients, and pollutant uptake. Additionally,

mycorrhizae is known for the extraction of heavy metals and degradation of organic

pollutants from soil, including 2-4-D, atrazine, and PCBs (Donnelly and Fletcher, 1995;

Meharg and Cairney, 2000; Chaudhry et al., 2005).

Rhizosphere-enhanced microbial degradation processes are poorly understood and

certainly vary according to soil conditions, plant species, and type of contaminant (Haby

and Crowley, 1996). It is relevant to study these processes, as they have the potential to

completely mineralize contaminants. As a result, contaminants are not transported into

the plant, reducing the possibility of passing the toxic compound into other organisms in

the food chain and the release of potentially harmful pollutants into the atmosphere. This









scenario may represent the ideal in situ remediation system, where the role of the plant is

to support and stimulate microorganisms capable of contaminant degradation.

Monoterpenes

More than 70 hydrocarbons, including isoprene, mono- and sesqui-terpenes and a

substantial number of oxygenated organic, are the predominant chemical species emitted

by vegetation (Benjamin et al., 1996). Monoterpenes and isoprene are the major natural

volatile organic compounds (VOCs) and a- and (S-pinene are the representative

monoterpenes (Kim, 2001). Monoterpenes are the simplest constituents of the plant

essential oils and the maj or non-methane hydrocarbon emitted to the atmosphere (4.8 X

1014 -1yar), which contributes to the formation of tropospheric ozone (Zimmerman et

al., 1978).

Emissions from conifer forests are predominantly monoterpenes (Amaral and

Knowles, 1998; Savithiry et al., 1998). For Pinus taeda (loblolly pine), monoterpene

emissions are 5.1 Epg g leaf dw-l h-l with greater than 60% represented by a-pinene

(Benjamin et al., 1996; Kim, 2001). On the contrary, broad leaf species, such as Populus

deltoides (poplar) and Salix nigra (willow), are among the high isoprene-emitting species

(Geron et al., 2001) with 37.0 and 25.2 Epg leaf dw-l h- respectively, with no detected

monoterpene emissions (Lamb et al., 1985).

Apart from tree emissions, monoterpenes can be released into the environment

from discharge effluents of the pulp-manufacturing industry, as monoterpenes are the

predominant component of turpentine (Kleinheinz et al., 1999). Monoterpenes are also

being used in the food, perfume, pharmaceutical industries, and, recently, at a larger scale

in an effort to substitute for chlorofluorocarbons and halogenated solvents (Amaral et al.,










1998). Therefore, their environmental fate and interactions with other substances are of

importance.

These compounds possess a broad range of functions in nature, from ecological

interactions that extend from allelopathy agents antimicrobialss and fungicides) to

pollinator attractants (Tooker et al., 2002). Potential sources of monoterpenes in soils

include leachate from leaf litter and canopy leaves, root exudation, and deposition from

the atmosphere. The role of monoterpenes and their effect on soil microbial communities

is complex and has not been fully elucidated. It is known that certain microbial enzymes

are stimulated by the presence of monoterpenes and that several microorganisms,

including Pseudomona~s sp., Alcaligenes xylosoxidans,~~~~ddd~~~~ddd and Bacillus sp., can use these

compounds as carbon and energy sources (Vokou et al., 1984; Misra et al., 1996; Vokou

and Liotiri, 1999; Yoo et al., 2001). Also, it has been reported that monoterpenes, first

introduced as decaying plant material or exudates of monoterpene-releasing plants, may

enhance biotransformation of PCBs (Hernandez et al., 1997). However, there are also

reports of inhibition of different microbial processes by these compounds (Vokou et al.,

1984; White, 1986; Ward et al., 1997).

Nitrogen mineralization and nitriaication is inhibited in the presence of

monoterpenes, but the precise mode of action has not yet been elucidated (White, 1986;

White, 1988; White, 1994). White (1988) proposed that monoterpenes hinder

nitrifieation by inhibiting the enzymatic activity of ammonium monooxygenase (AMO),

the first enzyme in the ammonia oxidation pathway, and that the degree of inhibition was

determined by the structure of the compound. These results have provoked other studies

on the effect of monoterpenes on methane oxidation because of the similarity between the









monooxygenase enzymes of these two groups of bacteria (Amaral and Knowles, 1997;

Amaral et al., 1998). The speculation is that monoterpenes inhibit IVMO similarly to

AMO, resulting in the inhibition of CH4 uptake. The authors also support this hypothesis

by mentioning that methanotrophs are not that commonly found in the surface layers of

forest soils, where monoterpene concentrations are the highest (Amaral and Knowles,

1997; Amaral et al., 1998). However, methanotrophs in soil surfaces oxidized

atmospheric CH4 (at concentrations of 1.7 ppm), and their isolation has proven to be

difficult because of the competitive advantage of low affinity methanotrophs in generally

used laboratory conditions at high CH4 COncentrations (usually 20% (v/v) CH4). To date,

high affinity methanotrophs have been phylogenetically identified but not isolated

(Holmes et al., 1999; Jensen et al., 2000). Consequently, the low abundance of

methanotrophs in soil surfaces may be the result of inadequate cultivation techniques.

Alpha-pinene, one of the most abundant monoterpenes, exists predominantly in

North America as the right enantiomer, (+)-a-pinene (Savithiry et al., 1998). This

compound is a bicyclic alkene composed of two isoprene units-CsHs. When released into

the environment, the fate of this monoterpene is dictated by its physical and chemical

properties (Table 1-2). Alpha-pinene released into the atmosphere exists solely as a

vapor that can be degraded by the reaction with photochemically produced hydroxyl

radicals (half-life= 4 h), ozone (half-life= 40 min), and nitrate radicals and in a nighttime

reaction (half-life= 6 min). In soils, a-pinene shows low mobility because it adsorbs to

soil particles (Koc of 1 200 and log Kow of 4.83). However, in moist soil surfaces,

volatilization is expected to be an important process based on its Henry's law constant

(0.107 atm m3 mOl-1). In water, a-pinene will adsorb to suspended solids and sediments









and exhibit a high potential for bioconcentration in aquatic organisms (bioconcentration

factor of 2, 800) (HSDB, 1999). Biodegradation of a-pinene occurs in soils, whereas a

variety of bacteria (Pseudomonas sp., Alcaligenes xylosoxid ans,~~~~ddd~~~~ddd Bacillus sp.) and fungi

(Cladosporium sp.) partially degrade this compound in both aerobic and anaerobic

conditions (Harder and Probian, 1995; Misra et al., 1996; Misra and Pavlostathis, 1997;

Kleinheinz et al., 1999; Pavlostathis and Misra, 1999; Yoo et al., 2001).

Methanotrophic Bacteria

Methanotrophs belong to the physiological group of methylotrophs. Methylotrophs

are aerobic microorganisms that utilize as their sole source of carbon and energy reduced

carbon substrates with no C-C bonds (C1 compounds) and assimilate carbon via

formaldehyde (Fig. 1-2) (Hanson and Hanson, 1996). Methanotrophs. are considered

obligate methylotrophs because they only grow on C1 compounds, including methane and

methanol (Lidstrom, 2001). However, recently, a new species was described with the

capability of facultative growth on multi-carbon compounds, M~ethylocella silvestris BL2

(Theisen et al., 2005).

The ability to grow on CH4 is almost exclusive to methanotrophs, except for a

gram-positive methylotroph of the genus M~ycobacterium (Reed and Dugan, 1987).

Methanotrophs. possess complex intracytoplasmic membrane systems, which appear to be

involved in CH4 uptake. The configuration of these membranes apart from other

characteristics separates methanotrophs. into two groups. Those that possess membranes

as bundles of disks stacked throughout the center of the cell (type I) and those with

membranes arranged as rings at the periphery of the cell (type II) (Table 1-3). Other

characteristics that correlate to the type classification include DNA GC content, pathways

of C-assimilation, rosette formation, types of cysts, and ability to fix N2. A small number









of methanotrophs from the genus M~ethylococcus possess characteristics of both groups;

therefore, they have been classified into the type X category (Hanson and Hanson, 1996;

Graham et al., 2002). The existence of this type X grouping is, however, a point of

debate within the Cl research community.

The mechanism by which methanotrophs oxidize CH4 to methanol and

cometabolize (the microorganism gains no carbon or energy from the substrate it

oxidizes, as previously defined) many other compounds including chlorinated solvents is

facilitated by the enzyme methane monooxygenase (1VMVO), unique to methanotrophs.

1V1VO exists in the soluble (slVMVO) or particulate (plV1VO) form depending on the

bioavailability of copper in the environment. plV1VO is a Cu- and Fe-containing enzyme

bound to the intracytoplasmic membrane (Nguyen et al., 1994; Lieberman and

Rosenzweig, 2004), whereas slVMVO, with a unique di-iron site at its catalytic center, is

located in the cytoplasm (Lipscomb, 1994; Kopp and Lippard, 2002). Although both

forms of 1V1VO exhibit a lack of substrate specifieity, the soluble form has been shown to

display a broader range, including alkanes, alkenes, and aromatic compounds. slV1VO is

among the most nonspecific enzymes known to date and exhibit high substrate turnover

rates. Therefore, slVMVO is more suitable for the degradation of a wider variety of

contaminants. However, slVMVO is only synthesized by type II and X methanotrophs in

environments with Cu concentrations less than 50 nM (< 0.89 -1 Epmol Cu gl dw cells)

(Oldenhuis et al., 1989; Hanson and Hanson, 1996).

The oxygen and methane levels also influence the expression of either form of

1V1VO. In environments with abundant oxygen and limiting concentrations of CH4,

methanotrophs express plV1VO, regardless of whether Cu is limiting. On the contrary, in










oxygen-limited environments with high CH4 COncentrations, the expression of either

enzyme is dictated solely by the Cu availability. At low Cu concentrations and high cell

densities, sMMO is expressed. Cells that express pMMO have higher growth yields and

greater affinity for CH4 because pMMO employs an abundant high-energy electron donor

for CH4 Oxidation. Meanwhile, sMMO possesses a high-energy demand, because of the

involvement of NADH+H' as an electron donor that catalyzes this reaction (Hanson and

Hanson, 1996; Sullivan et al., 1998).

Ecology and habitats of methanotrophs

Methanotrophs. have been widely studied for their role in the carbon cycle. They

intercept and oxidize CH4 that escapes from anaerobic environments, thus preventing

large quantities from escaping into the atmosphere (Hanson and Hanson, 1996). Thus,

methanotrophs. are considered the principal biological sink of atmospheric CH4 by

regulating the amount of CH4 preSent in the atmosphere and, consequently, decreasing

the impact that CH4, 23 times more potent than CO2, has on global warming (Houghton

et al., 2001).

Methanotrophs. are widespread in nature, found in any environment where CH4 and

oxygen are present. Under flooded conditions, such as rice paddies, wetland soils,

swamps, and bogs, they are restricted to the soil surface layers and to the rhizosphere of

plants where they intercept the CH4 being produced nearby under anaerobic conditions.

Meanwhile, in upland soils, non-flooded habitats, such as forest, grasslands, and arable

land, methanotrophs. are found in the top soil layers where they oxidize atmospheric CH4

(Hanson and Hanson, 1996; Horz et al., 2002). Also, in these habitats, they are found

deeper in the soil profile, stratified in a narrow band at the oxic-anoxic interface where

concentrations of CH4 and oxygen are the highest. Methanotrophs. have been isolated









from marine, freshwater, and terrestrial habitats, under conditions of high and low pH,

and temperatures up to 550C. They exist as symbionts with invertebrates and plants.

However, little attention has been paid to symbiotic relationships between plants and

methanotrophs, even though the first methanotroph isolated was from the leaves of a

macrophyte in 1906 by Soihngen (Hanson and Hanson, 1996).

It is well known that different types of methanotrophs. adapt better to different

environmental conditions. Methane, oxygen, and nitrogen concentrations are the primary

determinants of the type of methanotroph present in an environment. Type I

methanotrophs. outcompete type II species at low CH4 COncentrations (<2 ppmy in soils),

whereas growth of type II methanotrophs. is favored under low oxygen (<0.2 ppm in deep

waters) and high CH4 COnditions (>1 000 ppmy in sediments) (Hanson and Hanson,

1996). However, because of habitat heterogeneity or differences in experimental

techniques used, a consistent pattern concerning the competitive dominance of certain

types of methanotrophs. has been difficult to discern. Type II methanotrophs. have been

reported to be dominant in soils; however, an abundance of type I or of both types has

also been reported in the soil environment (Vecherskaya et al., 1993; Brusseau et al.,

1994; Sundh et al., 1995; Hanson and Hanson, 1996; Seghers et al., 2005). On the

contrary, type I methanotrophs. appear to prevail in aquatic environments such as lake

water, sediments, and groundwater. Apart from these contradictory results, some genera,

including M~ethylobacter and M~ethylocystis, representatives of type I and II

methanotrophs, respectively, have been detected in a wide range of habitats. It has been

speculated that their ability to produce resistant cysts enables these strains to persist in a

wide range of habitats (Knief et al., 2003; Bodelier et al., 2005).









Environmental factors affecting methanotrophs

The effect of several environmental variables on methanotroph composition,

community structure, and activity has been studied in a variety of habitats with some

inconsistent results. The outcome of these studies seems to depend on the type of habitat

being evaluated and on the variety of methodologies used. Soil type has been reported as

the primary determinant of the methanotroph community structure in agricultural soils

(Girvan et al., 2003; Seghers et al., 2005). However, in forest soils pH value has been

postulated as the primary factor affecting methanotroph distribution (Knief et al., 2003).

Atmospheric CH4 Oxidation activity has been reported to depend on plant cover and land

use, where activity has been shown to decrease with an increase in degree of disturbance

(woodland>grasslands>farmland) (Willison et al., 1995; Knief et al., 2003). Also,

management practices, including fertilizer type (organic versus mineral) and type of tree

in forest stands, have been reported to influence methanotroph activity and abundance

(Reay et al., 2001; Girvan et al., 2003). However, some genera, M~ethylocaldum,

M~ethylosinus, and M~ethylocystis, are universally observed in different soils, independent

of land use or plant cover (Knief et al., 2003).

In the presence of plants, mainly in saturated environments, the spatial distribution

of methanotrophs is determined by the soil compartment (rhizosphere> bulk soil> bare

soil) or the position of the soil-water interface (Gilbert and Frenzel, 1998; Dubey and

Singh, 2001; Macalady et al., 2002). It has been proposed that, because plants differ in

their ability to transport oxygen to the rhizosphere, different factors control their

associated methanotroph populations (King, 1994; Macalady et al., 2002). Spatial

changes in the methanotroph community have also been observed in forest soils

depending on season and soil depth (Henckel et al., 2000; Bodelier et al., 2005). In









winter, atmospheric CH4 Oxidation occurs in a well-defined subsurface layer (6-14 cm

deep), and, during summer, the complete soil core (0-26 cm deep) is active. However, no

seasonal shift in community composition was detected, the same methanotroph

population was identified in summer and winter (Henckel et al., 2000).

Other environmental factors known to affect methanotrophs are the potential

inhibitory effects of ammonium and/or nitrite that act as competitive substrates for MMO

(Dunfield and Knowles, 1995; Hanson and Hanson, 1996). However, recent studies with

rice plants have shown that nitrogen fertilization increases CH4 Oxidation in densely

rooted soils because rhizosphere methanotrophs face intense plant and microbial

competition for nitrogen (Macalady et al., 2002; Eller et al., 2005).

Methanotrophs and chlorinated compounds

Methanotrophs oxidize the less-chlorinated hydrocarbons at very different rates

depending on the form of MMO expressed (Leadbetter and Foster, 1959; Little et al.,

1988; Fox et al., 1990; Alvarez-Cohen and McCarty, 1991a; Alvarez-Cohen and

McCarty, 1991b; Henry and Grbic-Galic, 1991). TCE oxidation by sMMO is

comparable to that of CH4 and up to 700-fold higher than that reported for other MMO

microbial enzymes (toluene 4-monoxygenase, ammonia monooxygenase, and propane

monooxygenase) (Fox et al., 1990). However, TCE oxidation catalyzed by pMMO

occurs at much lower rates than sMMO (DiSpirito et al., 1992).

sMMO oxidizes TCE to TCE epoxide (95%) and chloral hydrate (5%) (Fig. 1-3A)

(Oldenhuis et al., 1989; Newman and Wackett, 1991; Fox et al., 1990). TCE epoxide

rapidly undergoes spontaneous decomposition; meanwhile, chloral hydrate is more stable

and undergoes biological transformation within 1 to 24 h of incubation to trichloroethanol

and trichloroacetic acid. At high temperature (600C) and pH of 9.0, chloral hydrate is









easily decomposed to chloroform and formic acid (Newman and Wackett, 1991). Since

TCE degradation is strictly a cometabolic process, no energy or carbon gain results from

its oxidation; therefore, the presence of a cosubstrate is necessary to maintain cell

biomass and regenerate reductant supply. Although CH4 Oxidation is required for growth

and can provide electrons, it also functions as a competitive inhibitor of TCE

transformation (Henry and Grbic-Galic, 1991). Byproduct toxicity also occurs as a result

of this reaction, with a concomitant decrease in CH4 Oxidation rates, respiratory activity,

and TCE degradation rates (Alvarez-Cohen and McCarty, 1991b; Hanson and Hanson,

1996; Chu and Alvarez-Cohen, 1999). Additionally, TCE metabolites can bind

nonspecifically to cell proteins and inactivate MMO activity (Fox et al., 1990). TCE

epoxide has been postulated as the responsible compound for the observed toxicity due to

its reactivity or that of its degradation products (Fox et al., 1990; Chang and Alvarez-

Cohen, 1996; Vlieg et al., 1996; Sullivan et al., 1998). Intermediate toxicity can be

reduced by the addition of an external supply of reducing equivalent such as format

(Alvarez-Cohen and McCarty, 1991b). However, TCE oxidation toxicity appears to have

a selective effect over different species of methanotrophs based on observations of

distinct rates of recovery (Henry and Grbic-Galic, 1991).

Under anaerobic conditions chlorinated compounds readily undergo reductive

dechlorination (Fig. 1-3B). PCE and TCE are degraded to dichloroethene isomers (cis-

and trans-1,2-DCE),tr~r~r~r~r~r~ 1,1-DCE, vinyl chloride, ethene, and ethane. DCE isomers and

vinyl chloride in the presence of TCE and no oxygen often persist in the environment

because their dechloronation yields less energy than that of their parent compound (Fox

et al., 1990; Hanson and Hanson, 1996). Considerable concern exists over the biological










production of vinyl chloride, a known human carcinogen; however, this product is readily

oxidized by sMMO in aerobic environments (Fox et al., 1990). Additionally, when

consortia of bacteria (methanotrophs and heterotrophs) are present, further oxidization of

chloral hydrate, chlorinated acetic acids, and vinyl chloride has been observed along with

a provision of additional reducing power for the process (Alvarez-Cohen et al., 1992;

Uchiyama et al., 1992; Chang and Alvarez-Cohen, 1996).

Methanotrophs and plants

Plant-methanotroph associations studied to date have considered mainly rice Hields

and wetlands because of their importance as maj or areas of CH4 prOduction. DeBont et

al. (1978) was the first to report CH4 Oxidation associated with rice roots, he noticed that

most of the CH4 diffused through the rhizosphere was oxidized. This observation was of

relevance because any small change in oxidation processes occurring at the rhizosphere

could have a global impact because rice Hields contribute to approximately 25% of the

current CH4 flux to the atmosphere. However, studies to date on these interactions show

high unexplained variability within plant species and between environments. It has been

observed that plant species, known to oxidize CH4 in their rhizospheres, when planted in

a different environment, CH4 COnSumption ranged from detected to no oxidation (King,

1996).

Root surfaces and their interior, zones where CH4 is transported from the

methanogenic sediments to the atmosphere and where atmospheric oxygen is transported

to the sediments, both support methanotroph populations in saturated environments

(King, 1996; Gilbert et al., 1998; Eller et al., 2005). However, methanotrophs and

methylotrophs have also been detected in these locations in poplar and pine trees in non-

saturated environments (Brigmon et al., 1999; Pilon-Smits, 2005). Methylotrophs










permanently associated with the plant are often encountered in the phyllosphere (leaf

surface) and rhizosphere. Plants can also benefit from these associations. For example,

methanotrophs can excrete or expel by cell lysis phytohormones (cytokinins and auxins)

and other bioactive compounds. Additionally, type II and X methanotrophs can Eix

nitrogen and, therefore, can be considered phytosymbionts on the surface and inside plant

tissues (Doronina et al., 2004).

The pattern of methanotroph root colonization has been studied in rice plants

(Gilbert et al., 1998; Gilbert and Frenzel, 1998). The colonization is spatially very

heterogeneous; some roots are not colonized at all, while others possess microcolonies as

clumps or thick bacterial layers. As known for other types of bacteria, methanotroph root

colonization followed the pattern of cell wall formation, potentially due to the exudation

of organic substrates and oxygen leakage at these sites. While, methanotrophs cannot

utilize complex organic substrates for growth, they do utilize some amino acids as

nitrogen sources (Gilbert and Frenzel, 1998).

Phylogenetics of methanotrophs

Methylotrophs are scattered among the Proteobacteria within the a-, (3-, and y-

subdivisions, not forming an evolutionary coherent group. Multi-gene operons appear to

be rare among its members, and, on the contrary, plasmids are common. However, no

functions have been ascribed to these plasmids, and they are entirely cryptic.

Methanotrophs cluster into the a- and y-Proteobacteria and are considered ideal

microorganisms for molecular biology studies. Methanotroph phylogeny and their

phenotypic and eco-physiology characterization into types I, II, and X validate each other

(Lidstrom, 2001). The type classification, initially proposed by Whittenbury et al.









(1970), has been supported by analysis of 5S and 16S rRNA genes. Recently, with the

incorporation of molecular methods to methanotroph studies, novel strains are being

recognized that do not grow in standard laboratory conditions (enriched solid and liquid

media with high concentrations of CH4). For example, the upland methanotroph soil

clusters (USC-a and USC-y) that oxidize atmospheric CH4 in forest soils. Another

example is the genus M~ethylocella, sensitive to salts in regular cultivation media (Holmes

et al., 1999; Henckel et al., 2000; Jensen et al., 2000; Bourne et al., 2001; Knief et al.,

2003; Theisen et al., 2005).

Molecular analysis of methanotrophs

Methanotroph phylogenetic studies have been conducted with both phylogenetic

and functional gene markers. Functional gene markers detect the active-site subunit of

both MMO forms, pnzoA for pMMO and namoX for sMMO, and of methanol

dehydrogenase by the nzxa~F gene. The use of these markers enables assessment of the

potential functional diversity of methylotrophs and methanotrophs within an

environment. The universal phylogenetic 16S rDNA (rRNA) primer set amplifies the

variable V3 region of the gene, extensively studied to enable inference of phylogenetic

relationships among microorganisms. Phylogenetic analysis of methanotrophs usually

considers both pnzoA and 16S rDNA analysis due to the fact that most methanotrophs

express the pMMO gene and phylogenies between these two primer sets are closely

related to each other (Bowman, 2000).

Interpretation of pnoA phylogenetic analysis must take into consideration that

multiple copies of the gene can exist in one organism; therefore, novel clusters of pnoA

sequences do not necessarily indicate that novel groups of uncultivated methanotrophs

exist. Copies of the pnoA gene (pnzoA2) can show less than 80% identity to the










previously known pnoA gene (pnoA1). Also, in some cases, there is no correlation

between the 16S rDNA and pnoA phylogenies, and, for the genus M~ethylobacter, some

strains poorly amplify the pnzoA gene with the standard primer sets, underestimating the

methanotroph diversity. Finally, one must keep in mind that the genus M~ethylocella, the

only known exception to the universality among methanotrophs of the pnoA gene, must

be detected using a different primer set. Other primer sets that could be used are the

namoX or nadh that amplify the sMMO and methanol dehydrogenase active site,

respectively (Dedysh et al., 2000).

It is of importance to recognize that the success in gene retrieval from

environmental samples depends on the quality of the primer sets used. Different levels of

methanotroph diversity have been reported with different primers sets (Bourne et al.,

2001). For example, Hutchens et al. (2004) reported that, by using the pnoA primer set

Al89f/A682r, only 8 operational taxonomic units (OTUs) were detected, but, with the

Al89f/mb661r primer set, 12 OTUs were retrieved (Hutchens et al., 2004). The pnoA

primer set Al89f/mb661r detects almost all methanotrophic bacteria, except sequences of

M~ethylonona~s, Methylocaldunt, and the reported forest clone clusters, but it does exclude

all known a~noA sequences of ammonia-oxidizing bacteria, except for Nitrosococcus

(Kolb et al., 2003).

With the incorporation of molecular techniques to the study of microbial ecology,

one of the most intriguing questions is the relationship between what has been reported

previously by community assessment based on traditional culturing and what has recently

been described by culture-independent techniques. Several methanotroph studies that

implemented denaturing gradient gel electrophoresis (DGGE) point out misleading









results of previous traditional culture-dependent methods. For example, in grassland

soils the culture-dependent most probable number (MPN) technique was compared to

direct soil sample DGGE analysis (Horz et al., 2002). While MPN analysis detected only

one methanotroph strain, DGGE revealed a more diverse and dynamic methanotroph

community. In a similar matter, enrichments characterized by DGGE were compared to

results of morphological observations and strain isolation from agricultural soil (Jensen et

al., 1998). The DGGE profile of the enrichments showed higher diversity (13-14 bands)

than the morphological observation and isolation, where only 2 to 4 dominant

morphological types were detected and only one colony was isolated (Jensen et al.,

1998).

Another methodology that is revolutionizing methanotroph studies by linking

microbial identity to biological function under conditions approaching those in situ is

stable isotope probing (SIP) (Radajewski et al., 2000). A labeled substrate, a less

naturally frequent isotope, is incorporated into the active microbial biomass. In the case

of methanotrophs, 13CH4 has been used to label the DNA of active organisms during

DNA synthesis and replication. The heavier DNA (13C-DNA) can then be separated from

the naturally occurring 12C-DNA. The methodology has been used to study

methanotroph communities of peat soils (Morris et al., 2002), acidic forest soils

(Radajewski et al., 2002), cave water (Hutchens et al., 2004), and soda lake sediments

(Lin et al., 2004).

Results of this SIP method confirmed that most methanotroph communities in the

environment are active and constitute a small fraction of the entire population responsible

for CH4 Oxidation. Soil community fractions revealed that only a small percentage or









possibly no methanotrophs were present in the 12C-DNA fraction, while the 13C-DNA

fraction was composed of 32% or 96% methanotroph, in peat and forest soils,

respectively (Morris et al, 2002; Radajewski et al., 2002). Interestingly, sequences have

been found that may represent novel methanotrophs and methylotrophs, suggesting that

these bacteria most probably assimilated methanol (13CH30H) excreted by

methanotrophs during 13CH4 Oxidation. However, in some cases, the affiliation to

methanotrophs of the retrieved sequences ((p-Proteobacteria) can not be explained,

suggesting the possibility that bacteria not previously considered to be involved in CH4

oxidation may derive a significant proportion of their carbon from products of

methanotroph metabolism or possibly even from CH4 itself.

When the functional pmoA gene has been examined before and after SIP

experiments, the diversity was lower in the 13C-DNA fraction indicating that not all

methanotrophs in an environment are active (Morris et al., 2002; Radajewski et al., 2002;

Lin et al., 2004). Overall these studies using SIP methods have revealed that the active

methanotroph community in peat and acidic forest soils was dominated by type II

methanotrophs (Morris et al., 2002; Radajewski et al., 2002) and in soda lake sediments

by type I methanotrophs (Lin et al., 2004). These results give insight into the ecological

niches occupied by each methanotroph type.

Methods Used to Assess Rhizodegradation Potential in Phytoremediation

The microbial composition of the rhizosphere is known to differ both qualitatively

and quantitatively from that in a non-planted soil. However, a precise determination of

the microbial diversity in soil or the rhizosphere compartment remains to be established,

as only up to 10% of soil microbial species can currently be cultured in the laboratory

(Fry, 2004). Although the ability to culture the yet-uncultured bacteria is of importance,
44









a number of indirect methods are currently used to establish the biodegradation potential

of soil microorganisms. By the use of these indirect methods, not only is the microbial

composition and structure of a specific habitat being determined but also it is possible to

link function to activity by the use of labeled substrates.

Culture-dependent techniques

Microbial counts. Historically, colonies forming units (CFU) and most probable

number (MPN) technique have been used to enumerate selected microorganisms and

assess the microbial composition of a site. However, it is recognized that only a small

portion of bacteria can form colonies when traditional plating techniques are used. The

proportion appears to be determined by the oligotrophic extent of the evaluated

environment, where the more oligotrophic the environment, the higher portion of bacteria

that do not grow under standard cultivation conditions (Smalla, 2004). Culturability,

defined as the percentage of culturable bacteria to total cell counts (microscopically

assess), has been determined to be around 0.3% in soils (Amann et al., 1995). Further

limitations represent organisms that, under environmental stress, enter the viable but

nonculturable state and bacteria strongly attached to soil particles that cannot be

dislodged (Smalla, 2004). While microbial counts are widely used and easy to prepare,

they are time-consuming and require multiple replicates and cultivation periods of weeks

or months. Additionally, they do not discern relationships among bacteria, are highly

selective, and inaccurate, underestimating the abundance of the microbial populations

(Lynch, 2002).

Enrichments. Another commonly used approach for microbial characterization of

environmental samples is to obtain enrichments of selected groups of bacteria. Usually,

the highest dilution of the MPN technique is used as an inoculum for further cultivation.









This procedure avoids the selection of only the fast-growing and less-numerous bacteria,

which benefit from the fact that some abundant bacteria do not grow directly on

conventional media (Fry, 2004). Enrichments offer the possibility of preserving

syntrophic relationships among bacteria and obtaining environmental isolates from which

physiological and phylogenetic characterization can be performed (Wise et al., 1999).

Additionally, cultures can be used to assess potential microbial activity, optimum

conditions for degradation, and microbial diversity of a particular sample. However,

conditions for enrichment do not resemble the Hield, and they are highly selective, which

makes extrapolation of results to the Hield difficult. Furthermore, obtaining a stable

culture can take months, and, when studying the culture's phylogenetics, there is no real

indication of gene expression in situ, which is ultimately what determines environmental

impact at the Hield. Because this technique relies on the culturability of the members of a

particular sample, it is a common finding that isolates represent only a few of the most

abundant bacteria. However, in some environments, isolates can represent higher

numbers. In seawater, isolates represented 7-69% of the total bacterial clones obtained

from culture-independent methods (Fry, 2004).

Culture-independent techniques

Molecular methods. Since 1990, microbial ecologists have been studying

bacterial diversity by isolating community DNA, amplifying their 16S rRNA genes,

cloning the fragments, and sequencing the clones. The development of molecular

methods over the past two decades has helped resolve difficulties inherent in studying

diversity using traditional approaches that are based on observations of physiology and

morphology. It has led to an increase in the numbers of identified bacteria divisions to

greater than 40, in which only 23 divisions are represented by isolates (Smalla, 2004).
46









Therefore, most of the bacteria in culture collections that grow on conventional media are

not the most abundant in natural habitats. For this reason, there is a necessity to isolate

the ecologically relevant bacteria, the as-yet-uncultured bacteria, and study their

physiology. The basic problem is that many numerically abundant bacteria grow more

slowly than the less-dominant bacteria on most laboratory media (Fry, 2004).

In spite of the advances, some challenges still remain for the molecular microbial

ecologist. The most pressing challenges are obtaining nucleic acids suitable for

molecular analysis and access to sufficiently large, high quality databases (Smalla, 2004).

Extraction problems when organic are high in an environment, such as in the

rhizosphere, still constitute a maj or draw back of this technique. Also, the effectiveness

of oligonucleotide probes to detect organisms may be uncertain because of the possibility

of encountering new genes or genes that are not conserved in a similar matter within

related groups of bacteria, and, as a result, genes obtained from cultured organisms may

not be sufficiently similar to genes in the environment (Hanson and Hanson, 1996).

Also, there is a lack of rDNA sequences of many described species. Another limitation is

that some molecular applications do not allow conclusions about the metabolically active

populations or on gene expression because they do not distinguish between active and

non-active organisms, thus limiting the use of these methods. Nevertheless, this

information might be obtained from RNA analysis or by the use of labeled substrates.

DGGE analysis. The technique is based on the separation of PCR fragments of the

same length in polyacrylamide gels containing a linearly increasing gradient of chemical

denaturants (urea and formamide) (Muyzer et al., 1993; Muyzer et al., 2004). Separation

is based on the electrophoretic mobility of the partially melted DNA molecule, which is









lower compared to that of the completely helical form of the molecule. The different

fragments melt in discrete melting domains (stretches of base pairs with an identical

melting temperature). Once the domain with the lowest melting temperature reaches its

denaturing concentration at a particular position in the gel, a transition from helical to

partially melted molecule occurs, and the molecule will stop migrating. Therefore,

sequence variants (different in base pairs) will stop migrating at different positions from

which DNA fragments are differentiated and excised for sequence analysis (Muyzer et

al., 1993). A GC-rich sequence (GC clamp) is incorporated into one of the primers to

modify its melting behavior to the extent to which close to 100% of all possible sequence

variations can be detected. The resulting banding pattern represents a profile of the

populations in the sample, and the relative intensity of each band and position represents

the relative abundance of a particular member of the community (Muyzer et al., 1993).

The main advantage of DGGE is that it permits high-resolution phylogenetic

analysis of a complete community by its diversity pattern in a qualitative and semi-

quantitative matter. Large numbers of samples can be quickly analyzed and compared,

permitting temporal and spatial analysis within and between communities. The only

comparable technique at the moment is terminal restriction fragment length

polymorphism (T-RFLP) (Bodelier et al., 2005). However, interpretation of T-RFLP

data requires constructing a clone library that can be time-consuming due to the cloning

step, and less abundant species are not always detected. On the contrary, DGGE can

detect species represented by as low as 1% of the population, and bands are directly

excised from the gel, reamplified, and sequenced without the need of cloning (Muyzer et

al., 1993). DGGE cagn detect up to 95% of all possible single base substitutions among










sequences of up to 1, 000 bp in length, and it can be adjusted to narrowed denaturant

gradients to provide higher resolution. Also, DGGE profies can be transferred to

hybridization membranes and probed with specific oligonucleotides (Vallaeys et al.,

1997).

However, the main constraint of DGGE is the amount of phylogenetic information

in the length of the commonly amplified fragments (< 500 bp). These partial sequences

may not be sufficient to discriminate among strains (Boon et al., 2002; Bodelier et al.,

2005). Another limitation is the production of multiple bands by one organism because

of multiple heterogeneous operons or copies of the target gene, or due to the use of

degenerate primers. Also, if the target sequences in a sample are present at dissimilar

concentrations, the less abundant sequences may not amplified sufficiently to be

visualized as bands, underestimating the diversity of the sample (Boon et al., 2002).

Another problem of DGGE is co-migration of bands and bands at identical positions that

are not necessarily derived from the same species; however, these bands can be screened

by reducing the denaturant gradient, and, when necessary, bands in similar positions may

require multiple sequencing. Finally, in community analysis of highly related

phylogenetic clusters, bands can represent heteroduplexes, PCR artifacts from mixed

DNA templates that result from two similar, but not corresponding strands, annealing

together. These artifacts can be detected because they produce bands at low denaturant

concentrations (Wise et al., 1999).

Stable isotope probing microcosms (SIP). SIP microcosms permit the

identification of organisms responsible for certain in situ transformation processes by the

use of a labeled substrate (a less naturally frequent isotope) that will be incorporated into









the active microbial biomass (Radajewski et al., 2000; McDonald et al., 2005).

Subsequently, the labeled DNA fraction is separated from the naturally occurring fraction

by CsCl density gradient centrifugation. The labeled fraction is then analyzed to identify

the active microbial community by cloning, followed by restriction fragment length

polymorphism (T-RFLP) or by DGGE analysis.

The maj or limitation of the SIP methodology is the dilution of the labeled substrate

before its assimilation and incorporation into the active organisms, which can happen if

simultaneous growth on an unlabeled substrate is occurring in the microcosms. Other

constraints may be the relative long incubation periods needed to label sufficient biomass

and, consequently, the potential for the use of labeled metabolites by non-target

organisms (cross-feeding). Also, the artificial spike and relatively high concentrations

used of the labeled substrate, may stimulate microorganisms that were not active in situ,

and, as a result, the analysis may represent the potential active population, rather than the

active microbial community at the time of sampling (Lin et al., 2004; McDonald et al.,

2005). Therefore, SIP studies should provide a rational basis for the application of

molecular biological techniques to study the role of specific organisms that are likely to

be involved in a defined process (Radajewski et al., 2003). Finally, the technique is

expensive and requires certain expertise, but it is one of the most powerful molecular

techniques available, providing information on the active microbial populations of an

environmental sample and linking function to identity.

Study Hypothesis

The diversity and activity of methanotroph populations associated with the

rhizosphere of plants used in phytoremediation processes are impacted by plant type,

system design, and environmental conditions present at the site.









Study Objectives


Broad Objectives

*Provide full characterization of a well-known, pure methanotroph, Strain CSC1,
isolated from a groundwater aquifer known to oxidize TCE as a means of method
development of phytoremediation studies.

*Assess the effects of plant exudates, specifically monoterpenes, on TCE
cometabolism by methanotroph bacteria.

*Develop a protocol using stable isotope probing (SIP) methods specific to
rhizosphere microorganisms.

*Assess differences in methanotroph abundance, activity, and diversity observed in
rhizosphere samples from several plant species used in phytoremediation.

*Determine the effectiveness of culture-dependent and culture-independent methods
to characterize potential microbial degraders.

*Ultimately provide guidance for the phytoremediation practitioner to more
accurately predict the extent of TCE rhizodegradation when using monoterpene-
and non-monoterpene releasing plants.

Specific Objectives

*Characterize the methanotroph Strain CSC1 using phenotypic and physiological
descriptions, phylogenetics of the 16S rDNA and multiple functional genes (mmoX,
pmoA, mxaF), and DNA-DNA hybridization.

*Determine the effect of (R)-a-pinene on TCE cometabolism by pure cultures of
representative type I, II, and X methanotrophs using oxygen uptake analysis.

*Combine and implement SIP methods with molecular fingerprints techniques, as
denaturing gradient gel electrophoresis (DGGE), using the 16S rDNA and
functional pmoA genes, to develop a precise methodology for methanotroph
rhizosphere studies.

*Determine by culture-dependent microbial counts the abundance of the heterotroph
and methanotroph communities from rhizosphere soil compartments of two
phytoremediation sites.

*Enrich for and characterize methanotroph mixed cultures from rhizosphere soil
compartments of two phytoremediation sites by their oxidation potential using
oxygen uptake analysis, presence and activity of soluble methane monoxygenase,
and phylogenetics of 16S rDNA-DGGE analysis.










*Determine the effect of several environmental variables (location, time, tree type,
contaminant type and concentration, depth, and system design) on the
methanotroph populations of different rhizosphere soil compartments at two current
phytoremediation sites.









Table 1-1. Physical and chemical properties of TCE and PCE.1
Characteristic Compound
TCE PCE
Molecular formula C2HCl3 2 C4
Molecular weight (g mol l) 131.4 165.8
Specific gravity (at 20oC) 1.465 1.623
Vapor pressure (at 25oC) (mm Hg) 74 18.5
Water solubility (at 25oC) (mg I 1) 1 366 150
log Koc 2.03-2.66 2.20-2.70
log Kow 2.42 3.40
Henry's law constant (at 25oC) (atm m3 mOl-1) 0.011 0.018
'(ATSDR, 1999)


SPhytovolatilization
(removal of contaminants
and release to the
atmosphere)
Phytodegradation
(plant metabolism of
contaminants)



Phytoextraction
(extraction of contaminants,
can result in plant
accumulation)


SRhizodegradation
(microbial metabolism
Phytostabilization of contaminants in the
(soil/vegetation binding or rhizosphere)
containment of contaminant plume
by plant water uptake)


Figure 1-1. Schematic of processes in a phytoremediation system (McCutcheon and
Schnoor, 2003).









Table 1-2. Physical and chemical properties of a-pinene.l
Property Value
Molecular formula CloH16
Molecular weight (g mol l) 136.24
Specific gravity (at 20oC) 0.8592
Vapor pressure (at 25oC) (mm Hg) 4.75
Water solubility (at 25oC) (mg 1- ) 2 2.5
Koc 1 200
Log Kow 4.83
Henry's law constant (at 25oC) (atm m3 mOl-1) 0.107
(1HSBD, 1999; 2Li et al., 1998)



IMethanotrophs
Methylotrophs
Methane
Monooxygenas e Formaldehyde
(MMO) Dehydrogenase Formate
H20 Methanol Dehydrogenase
02 Dehydrogenase H20 rHOHAH++0
CH4 /C3H HH CO O
H +NADH NAD ""5 HH
2H+ 2H+ A
Formaldehyde
C-Assimilation

Figure 1-2. Cl metabolism by methanotrophs and methylotrophs as described by
Wackett (1995).










Table 1-3. Characteristics of different methanotroph types .
Characteri sti c Type I Type II Type X
M~ethylomona~s
M~ethylosinus
M~ethylobacter
M~ethylocystis
Recognized genera M~ethylomicrobium M~ethylococcus
M~ethylocella
M~ethylocaldum
M~ethylosphaera ~tyo~s
Short rods, some Rods, crescent- or
Cellular shape Cocci
cocci or ellipsoids pear-shaped

Azotobacter-type Exospores or lipid Azotobacter-type
Resting stages
cysts cysts cysts
Paired, parallel to Disc-shaped
Intracytoplasmic Disc-shaped
membranes bundles of vesicles .h yolsatc bnlso
membrane vesicles
Formaldehyde
pathway assimilation RuMP2 Serine u (ao)
Serine
(C assimilation)

TCA cycle Competencomlet
(one exception)Cmlt Icmlt
DNA G+C content 50-54% 62.5% 62.5%
Predominant
phospholipid fatty 16 C-atoms 18 C-atoms 16 C-atoms
acids (PLFAs)
Key enzymes:
-Methane pMMO2 pMMO/sMMO2 pMMO/sMMO
monooxygenase
-3-hexulose phosphate + +
synthase
-Hydroxypyruvate + +
reductase
-Nitrogenase + +
-Ribulose-biphosphate +
carboxylase
-Isocitrate NAD /NADP' NADP' NAD
dehydrogenase2
Growth temperature 40oC> 40oC> >45oC

Phylogeny 6 Proteobacteria at Proteobacteria 6 Proteobacteria
'(Hanson and Hanson, 1996: Sullivan et al., 1998: Graham et al., 2002).
2RuMP= ribulose monophosphate cycle: TCA= ricarboxylic acid cycle: pMMO= particulate
methane monooxygenase: sMMO= soluble methane monooxygenase: NAD = nicotinamide
adenine dinucleotide: NADP = nicotinamide adeni le dinucleotide phosphate.












A. C~HPc H


Figure 1-3. Oxidation of TCE by aerobic methanotroph degradation (A) and anaerobic
reductive dehalogenation (B) (4Barrio-Lage et al., 1986; 3Fox et al., 1990;
1Vlieg et al., 1996; 2Lontoh et al., 2000).


H~c
Cl C


sMMO1,2
pMMO3


CIC OOHH
chloral hydrate



CI CI O

tr chloroacetate tric loroethanH






O O-
oxalate


H H
cis-1 ,2-DCE


CI H
trans-1 ,2-DCE


TCE epoxide

SsMMO


CIH O
dichloroacetate


CH--COH
glyoxylate


HO CH
format


H


H H
Vinyl chloride



H H-

H H
Ethylene


CH- OH
glyoxylate





HOCH + CO2
format














CHAPTER 2
M~ETHYLOCYSTIS ALDRICHII SP. NOV., A NOVEL IVETHANOTROPH ISOLATED
FROM A GROUNDWATER AQUIFER

Note: Manuscript submitted to the International Journal of Systematic and Evolutionary
Microbiology

Lindner, A.S., Pacheco, A., Aldrich, H.C., Costello Staniec, A., Uz, I. and Hodson, D.J.
2006. M~ethylocystis aldrichii sp. nov., a novel methanotroph isolated from a
groundwater aquifer. International Journal of Systematic and Evolutionary
Microbiology XX: XXX-XXX.

Introduction

Species of the genus M~ethylocystis are strictly aerobic, gram-negative bacteria that

are able to grow on one-carbon compounds (e.g., methane or methanol) (Bowman et al.,

1993a). The genus M~ethylocystis belongs to the alpha-subclass of the Proteobacteria and

currently consists of 2 species with standing in nomenclature, M\~ethylocystis parvus and

M~ethylocystis echinoides (Whittenbury et al., 1970; Gal'chenko et al., 1977; Bowman et

al., 1993a). Numerous M~ethylocystis strains have been identified in a variety of

environments, including lake, ocean, marsh, and creek sediments and water, coal mine

drainage water, the roots of plants, etc. (Whittenbury et al., 1970; Gal'chenko et al.,

1977; Bowman et al., 1993a; Hanson and Hanson, 1996; Calhoun and King, 1998; Heyer

et al., 2002).

Species of the genus M~ethylocystis are Type II methanotrophs, classified, in part,

by their possession of paired membranes aligned with the cell periphery, the serine

pathway, and predominant fatty acids with 18 carbons (Hanson and Hanson, 1996;

Graham et al., 2002). All known Type II methanotrophs, including the M~ethylocystis

species, express the particulate form of methane monooxygenase (plV1VO), and, with the










exception of 2ethylocystis parvus, all express the soluble form of methane

monooxygenase (sMMO) under conditions of low copper concentrations (Stanley et al.,

1983; Prior and Dalton, 1985; Choi et al., 2003). M~ethylocystis parvus does not possess

genes encoding for sMMO (Tsien and Hanson, 1992; McDonald et al., 1997; Lloyd et al.,

1999) and is, therefore, incapable of oxidizing aromatic compounds. All M~ethylocystis

species produce oxidase and catalase, are nonmotile and are capable of fixing

atmospheric nitrogen (Hanson and Hanson, 1996).

The focus of this paper is Strain CSC1, a group II methanotroph previously isolated

from an uncontaminated groundwater aquifer at Moffet Naval Air Station in Mountain

View, CA, USA (Henry and Grbid-Galid, 1990). This methanotroph expresses sMMO

under copper-limiting conditions and is capable of oxidizing aliphatic and aromatic

compounds (Henry and Grbid-Galid, 1991; Adriaens and Grbid-Galid, 1994; Adriaens,

1994; Hriak, 1996; Hriak and Begonja, 1998). Despite its being the focus of these

numerous studies aimed primarily towards contaminant degradation potential, Strain

CSC1 has not been characterized and differentiated from other known Type II

methanotrophs. This study provides phenotypic and genotypic analysis of this

groundwater isolate. The formal taxonomic description of this novel M~ethylocystis

bacterium, M~ethylocystis aldrichii sp. nov. strain CSC1, is reported. Differences in

various characteristics of Strain CSC1 compared to other known methanotrophs are

described, and its unique surface features broaden the observed physiological traits of

methanotrophic bacteria.

Materials and Methods

Strain CSC1 was obtained from Dr. Dubravka Hriak at the Rudj er Boskovic

Institute in Zagreb, Croatia, and M~ethylosinus trichosporium was obtained from Dr.









Jeremy Semrau in the Department of Civil and Environmental Engineering at the

University of Michigan, Ann Arbor, USA. M\~ethylocystis parvus and M~ethylocystis

echinoides were obtained from NCIMB (Aberdeen, England). The basal medium used

for growth when culturing for sMMO expression was nitrate mineral salts (NMS)

medium with no added copper, as described previously (Whittenbury et al., 1970; Lontoh

and Semrau, 1998). Ten Cpmol 1-1 copper nitrate (Cu(NO3)2) WAS added to the NMS

medium to provide conditions for pMMO expression. Liquid cultures were routinely

grown at 250 rpm and 300C in either 50- or 500-ml batches in 250-ml Erlenmeyer or

2800-ml Fernbach flasks, respectively. The flasks were fitted with rubber stoppers

(Fisher Scientifie, Pittsburgh, PA, USA) equipped with a resealable glass tube filled with

glass wool to allow headspace removal and filling. A portion of the air headspace was

removed and refilled with methane of 99.99% purity (Strate Welding, Jacksonville, FL,

USA) using a vacuum pump assembly to achieve a headspace concentration of air with

20% (v/v) methane.

For solid culturing, 1.5% (w/v) of Bacto agar (Difco Laboratories, Detroit, MI,

USA) was added to the NMS medium. All plates were incubated in a sealed desiccator,

containing anhydrous CaSO4 (Drierite, W.A. Hammond Drierite Company, Xenia, OH,

USA) under an atmosphere of 20% methane and 80% air (by volume) at 300C that was

refreshed every four to Hyve days. Purity of the cultures was verified by routine streaking

on 2% (w/v) nutrient agar in doubly deionized water.

sMMO expression was qualitatively verified by a naphthalene assay modified from

Brusseau et al. (1990). Four negative controls--autoclaved cells, cells cultured with 10

Cpmol 1-1 Cu(NO3)2 (fOr expression of pMMO), cell-free, and cells that have been









subj ected to addition of one to two ml of acetylene gas (a known inhibitor of MMO, Prior

and Dalton (1985))--were included with three live samples of active test culture diluted

to an absorbance of 0.2 (at a wavelength of 600 nm) and transferred to autoclaved 10-ml

capped test tubes. Seventy mg of crushed naphthalene (Sigma, St. Louis, MO, USA)

were added to each tube. After incubation at 300C and 250 rpm for a minimum of one

hour, 0. 1 ml of freshly prepared 4.21 mmol 11 tetrazotized ortho-dianisidine (Sigma, St.

Louis, MO, USA) solution was added. A subsequent pink-to-purple color formation in

the tubes indicated positive sMMO activity that was verified using spectrophotometry

(Fisher Scientifie, Pittsburgh, PA, USA) at 550 nm.

Genomic DNA was isolated from Strain CSC1, grown to exponential phase, by a

standard method (Ausubel et al., 1989). 16S rRNA gene was amplified by PCR using the

universal bacterial primers 27f and 1492r (Lane, 1991). PCR primers used for sMMO

were mmoXA (5 '-ACCAAGGARCARTTCAAG-3 ') and mmoXB (5'-

TGGCACTCRTARCGCTC-3 ') (Auman et al., 2000); for methanol dehydrogenase

(MDH), mxa fl003 (5' -GCGGCACCAACTGGGGCTGGT-3 ') and mxa rl561 (5'-

GGGCAGCATGAAGGGCTCCC-3 ') (McDonald and Murrell, 1997); and for pMMO,

Al89f (5' -GGNGACTGGGACTTCTGG-3 ') and A682r (5'-

GAASGCNGAGAAGAASGC-3 ') (Holmes et al., 1995).

All PCR reactions were carried out in a PTC-200 Thermo Cycler (MJ Research,

MA, USA) using 25 Cl1 reactions and Premix Taq polymerase (Takara, Otusu, Shiga,

Japan). Conditions used for the primer sets have been described previously (Holmes et

al., 1995; Costello and Lidstrom, 1999; Auman et al., 2000). The PCR amplification

products were ligated to vector pCR2. 1 (Invitrogen, Carlsbad, CA, USA) and









transformed to competent E. coli cells (TOP 10F') according to the vendor' s instructions.

Plasmid DNA from transformants was isolated and the inserts sequenced by the

Biotechnology Resource Center at Cornell University (Ithaca, NY, USA).

Sequences were compared with previously identified sequences in the National

Center for Biotechnology Information (NCBI) database using BLAST (Altschul et al.,

1990). The 16S rRNA gene from Strain CSC1 was also aligned with sequences obtained

from the Sequence Match program provided by the Ribosomal Database Proj ect II (RDP-

II) (Cole et al., 2005). Phylogenetic trees were generated using PHYLIP version 3.6

(Felsenstein, 2004) and viewed using Treeview (Page, 1996). The GenBank accession

numbers for the 16S rRNA, MDH, sMMO and pMMO gene sequences obtained in this

study are DQ364433, DQ664499, DQ664498, and DQ364434, respectively.

DNA-DNA hybridization was performed on Strain CSC1 by DSMZ

(Braunschweig, Germany) against M~ethylocystis echinoides strain IMET 10491 was

performed using 2 x SSC buffer (0.3 M NaC1, 0.03 M sodium citrate, pH 7.0) + 10%

(v/v) formamide at an optimal renaturation temperature of 680C.

Earlier studies reported Strain CSC1 as gram-negative, non-motile, coccobacillus,

possessing an internal membrane structure characteristic of Type II methanotrophs

(paired membranes inside the periphery of the cell), and forming lipid inclusions (Henry

and Grbic-Galic, 1990; Henry and Grbic-Galic, 1991; Hrliak and Begonja, 1998). Fang et

al. (2000) concluded that the intact phospholipids of Strain CSC1 clustered within the

Type II grouping, clearly distinct from groupings of Type I methanotrophs. This study

extended the previous phenotypic characterization studies by assessing exospore and

rosette formation, growth at 370C, the presence of a surface (S-) layer, carbon and









nitrogen source utilization, and lysis by 2% (w/v) sodium dodecyl sulfate (SDS), all

identified by Bowman et al. (1993a) or Hanson and Hanson (1996) as differentiating

characteristics among Type II methanotrophic species.

Exospore formation was determined with one- to two-week-old broth cultures

grown as previously described following methods of Smibert and Krieg (1981). Five ml

of culture were transferred in duplicate to fresh NMS medium for controls. A second set

of duplicates was heated in a water bath at 800C for 20 min for pasteurization. Growth

was monitored after streaking the controls and treated cultures onto solid NMS plates and

incubation (as previously described) for 21 days. Exospores were monitored using light

microscopy, also used to determine rosette formation (Norris and Ribbons, 1971).

Growth in liquid culture was monitored using 250-ml nephlos flasks with the same

stopper assembly described above and a spectrophotometer (Fisher Scientific, Pittsburgh,

PA, USA) at a wavelength of 600 nm.

Nitrogen and carbon sources were tested using NMS basal medium. To test for

alternate nitrogen sources, KNO3 WAS replaced with 0. 1% (w/v) of anhydrous L-

asparagine (MP Biomedicals, Irvine, CA, USA), L-aspartate (Pfaltz & Bauer, Waterbury,

CT, USA), or L-glutamine (MP Biomedicals, Irvine, CA, USA) (all shown to support

growth of Methylocystis echinoides and M\~ethylocystis parvus, Bowman et al., 1993a) or

L-lysine monohydrochloride (Sigma-Aldrich, St. Louis, MO, USA), L-ornithine

hydrochloride (MP Biomedicals, Irvine, CA, USA), or putrescine (MP Biomedicals,

Irvine, CA, USA) (all shown to support growth of Methylosinus trichosporium, Bowman

et al., 1993a). NMS medium with KNO3 and without a nitrogen source served as positive

and negative controls, respectively, and the latter control also served as a test to fix









atmospheric nitrogen. To test for alternate carbon sources, 0.2% (w/v) of methylamine

hydrochloride (Alfa Aesar, Ward Hill, MA, USA), dimethyl sulfoxide, methanol, or

glucose (Fisher Scientifie, Pittsburgh, PA, USA) was added. Over the 30-day test period,

flasks were prepared in duplicate, and transfers were made to fresh medium and nitrogen

or carbon source every 4 days. Growth measurements were performed as described

previously.

Lysis by 2% (w/v) SDS (Fisher Scientifie, Pittsburgh, PA, USA) was determined

by direct microscopic observation using cells harvested at %/-log phase. Cells were

centrifuged at 2460 x g for 20 min, resuspended in the 2% SDS stock solution for

approximately 2 hours, and observed using an oil immersion phase contrast microscope

(Zeiss, Oberkochen, Germany).

Transmission electron microscopy was used to observe cells of Strain CSC1

expressing MMO, lipid inclusions, and other Eine structural features, including S-layers.

Liquid cultures were incubated for two to three days and were Eixed for 30 min at room

temperature with cacodylate-buffered glutaraldehyde both with and without 0. 1% Alcian

blue (Fassel et al., 1992), stained for 30 min at room temperature with 1% cacodylate-

buffered osmium tetroxide, and then stained for 50 min in 1% aqueous uranyl acetate.

After dehydrating in increasing strengths of ethanol, cells were embedded in both Spurr' s

and Epon resins (Dykstra, 1993). Thin sections were prepared and stained with lead

citrate and examined on a Zeiss EM-10CA transmission electron microscope.

M~ethylocystis echinoides was observed by negative stain using 1% aqueous uranyl

acetate applied to cell suspensions on Formvar-coated grids.









In order to provide evidence that the observed S-layer is glycoprotein, two

additional cytochemical approaches were utilized. Images of Alcian blue-stained

specimens were compared to those with no Alcian blue in the glutaraldehyde Eixative,

since Alcian blue stains polysaccharide moieties (Lewis and Knight, 1977). Secondly,

thin Epon sections on Formvar-coated nickel grids were first exposed to 3% hydrogen

peroxide (H202) for 15 min at room temperature to remove osmium and then exposed to

1% aqueous pronase solution (Sigma Chemical Company, St. Louis, MO, USA) for 60-

90 min at 350C to remove protein components from the section (Monneron and Bernhard,

1966; Lewis and Knight, 1977). Controls included H202 alOne and water substituted for

the pronase step.

Results

The phylogenies of the 16S rRNA, sMMO, MDH, and pMMO genes, shown in

Fig. 2-1 and Fig. 2-2 (a-c), are consistent with placement of Strain CSC1 with other

known Type II methanotrophs. The 16S rRNA phylogeny of Strain CSC1 clearly places

it within a branch of the alpha-Proteobacteria dominated by M~ethylocystis species. This

methanotroph shares 98% 16S rRNA gene sequence similarity with its nearest defined

relatives, an uncultured member of the M~ethylocystaceae (AF358021), as well as two

cultured organisms: M~ethylocystis sp. L32 (AJ83 1522) and M~ethylocystis sp. SC2

(AJ4313 84), although 16S rRNA gene similarity is not sufficient to place Strain CSC1 to

the species level. The DNA-DNA hybridization results showed that Strain CSC1

possesses a 3.8% DNA-DNA similarity with 2ethylocystis echinoides strain IMET

10491.

As shown in Table 2-1, rosette formation by cells of Strain CSC1 was not

observed. No growth was evident after pasteurization, indicating that this methanotroph









is not resistant to heat, and growth was also not observed at 370C. Optimum growth was

observed at approximately 300C. Strain CSC1 was not lysed by a 2% solution (w/v) of

SDS, but a 10% solution (w/v) of SDS did lyse the cells. It was shown to be capable of

growing on alternate nitrogen sources of L-asparagine, L-aspartate, L-glutamine, L-

ornithine and putrescine; however, no growth was visible in the presence of L-lysine. Of

the four alternate carbon sources of methylamine, dimethylsulfide, methanol, and glucose

tested, only methanol supported growth of Strain CSC1.

The expression of sMMO upon culturing Strain CSC1 in NMS medium with no

copper was confirmed by formation of a purple color after incubation with naphthalene

and addition of ortho-dianisidine, whereas controls with acetylene and with cells cultured

in the presence of copper yielded no color. These results strongly suggest that sMMO

was expressed in Strain CSC1 when grown without copper and was responsible for

naphthalene oxidation.

Transmission electron micrographs of Strain CSC1 grown in the presence of copper

verify the Type II membrane structure of paired membrane lamellae in the peripheral

cytoplasm (Fig. 2-3a,b). In thin section, a variety of cell shapes were visible at low

magnifieation (Fig. 2-3a), but elongated or dumbbell shapes of cells predominated.

Many of the other profies could represent dumbbell shapes sectioned in different planes.

Cells grown without copper contained only a few internal membranous lamellae (data not

shown). Polyphosphate bodies and lipid inclusions were common.

As shown in Figs. 2-3(a) and 2-3(b), distinctively striking S-layers, likely

composed of glycoprotein, were revealed with transmission electron microscopy of

ultrathin sections of Strain CSC1 Eixed with Alcian blue. These spiked S-layer structures,









50-75 nm in height, covered the entire surface of the cell wall. We have seen that the

cytoplasm of cells of Strain CSC1 embedded in Spurr resin will sometimes shrink away

from the wall, lending support to the idea that the S-layer is more rigid than the rest of the

wall (data not shown). This shrinkage does not occur in cells embedded in Epon resin

(e.g., cells in Fig. 2-3a,b).

S-layers have been observed in both Type I and II methanotrophs isolated from a

wide range of environments, including the genera M~ethylomicrobium, Methylomona~s,

M~ethylosinus, and M~ethylocystis (Fassel et al., 1992; Sorokin et al., 2000; Trotsenko and

Khmelenina, 2002). Type II 2ethylosinus trichosporium OB3b was found to have bead-

like S-layer structures and occasional filamentous material in the outer envelope (Fassel

et al., 1990; Fassel et al., 1992). Similar bead-like S-layer structures were observed in the

cell envelope of2~ethylocystis sp. strain Lake Washington but not in M\~ethylocystis paris,

and the authors concluded that the absence of these structures in the latter species could

be a species variation. Both M~ethylocystis species possessed considerable filamentous

material, however (Fassel et al., 1992). M~ethylocystis echinoides strain IlVET 10491 was

reported to have rigid tubular structures arranged radially at the cell surface (Gal'chenko

et al., 1977), features that are absent from M\~ethylocystis parvus (Heyer et al., 2002). In

this study, negative stain preparations of2~ethylocystis echinoides show ellipsoid cells

with square-ended tubular proj sections (Fig. 2-3c) that appear striated at high

magnification (Fig. 2-3d). This striation was not reported in previous studies of this

strain (Gal'chenko et al., 1977; Bowman et al. 1993a), and the tubular appearance of this

S-layer is much different from the solid-sharp spines of Strain CSC1.









To further elucidate the nature of the spiked S-layer in Strain CSC1 cells Eixed with

glutaraldehyde alone (Fig. 2-4a) were compared with those Eixed with an Alcian

blue/glutaraldehyde mixture (Fig. 2-4b). Alcian blue is a differential stain for

polysaccharide (Lewis and Knight, 1977), and the spines in Fig. 2-4b were considerably

darker, longer, and more distinct than the same structures in Fig. 2-4a, even though Fig.

2-4b is at a lower magnification. This strongly indicates polysaccharide content. After

treatment with H202 to remove osmium from the Epon sections, sections treated with

pronase, a broad-spectrum protease, lost the entire S-layer (Fig. 2-4c,d), indicating that

the layer contains considerable protein.

Discussion

Sequence analysis of the 16S rRNA gene (Fig. 2-1), the sMMO gene (Fig. 2-2a),

the methanol dehydrogenase gene (Fig. 2-2b), and the pMMO gene (Fig. 2-2c) supports

placement of Strain CSC1 within the closely related genera 2ethylocystis and

M~ethylosinus.

Given the suspected placement in the genera M~ethylocystis, DNA-DNA

hybridization was performed with M~ethylocystis echinoides. Based on the DNA-DNA

hybridization results showing only 3.8% similarity, Strain CSC1 does not belong to the

species M~ethylocystis echinoides, following the threshold value recommendation of

Wayne et al. (1987). Phenotypic results in Table 2-1 show that Strain CSC1

differentiates from M~ethylosinus trichosporium, M~ethylocystis echinoides, and

M\~ethylocystis parvus, three closely matching cultured strains in the phylogenetic

analysis, in various characteristics. All of the strains shown in Table 2-1 have been

reported to be oxidase- and catalase-positive, possess colonies that are of opaque

transparency, smooth edge, convex elevation, form poly-P-hydroxybutyrate, grow on









methanol, and capable of fixing atmospheric nitrogen. Unlike the known strains, Strain

CSC1 was not capable of growth at 370C; however, all of the methanotrophs grow

optimally near 300C. It is important to note that Gal'chenko et al. (1977) and Bowman et

al. (1993a) report conflicting information concerning the ability ofM~ echinoides to

accumulate poly-P-hydroxybutyrate and grow at 370C, the former reporting positive

results for each and the latter reporting negative results. Our TEM and growth studies

with this strain agreed with Gal'chenko et al. (1977) (data not shown).

The elongated dumbbell shape of Strain CSC1, lack of motility, and ability to form

polyphosphate separate it from the M~ethylosinus trichosporium. Other distinguishing

characteristics between Strain CSC1 and M~ trichosporium include smaller cell size, S-

layer morphology, and lack of heat resistance. Also, unlike reported observations ofM~

trichosporium, Strain CSC1 can use L-asparagine, L-aspartate and L-glutamine and

cannot use L-lysine as nitrogen sources. Both strains share the ability to use L-ornithine

and putrescine.

Most similarities, however, are shared with the two M~ethylocystis strains, including

lack of motility, heat resistance, and rosette formation. Strain CSC1's cell shape, ability

to form polyphosphate, and colony color of yellow-white differ from M~ethylocystis

echinoides and M\~ethylocystis parvus (Table 2-1). Unlike M~ echinoides, Strain CSC1 is

capable of using L-ornithine and putrescine as nitrogen sources, whereas, unlike M~

parvus, Strain CSC1 is not capable of using L-lysine. In addition, as reported for M

trichosporium, M~ echinoides, and M~ parvus, Strain CSC1 is not lysed by a 2% solution

(w/v) of SDS.









While Strain CSC1 has previously been shown by TEM to contain characteristic

Type II membranes (Henry and Grbic-Galic, 1990; Hrliak and Begonja, 1998), it was

revealed here to accumulate both polyphosphate bodies (Fig. 2-2) and poly-P-

hydroxybutyrate storage granules, consistent with M\~ethylocystisparvus (Bowman et al.,

1993). No study has reported the structure of Strain CSC1's cell envelope in comparison

to that of other well-characterized methanotrophs. Of special interest are the surface- (S-)

layers, regular crystalline surface layers in Archaebacteria and Eubacteria, composed of

protein or glycoprotein subunits (Sleytr et al., 1993; Sidhu and Olsen, 1997).

It is not known why S-layers develop in some strains of closely related bacteria and

not in others. However, one hypothesis is that formation of these structures reflects

adaptation to an ecological niche (Easterbrook and Alexander, 1983; Easterbrook, 1989)

or a response to exposure to harsh environments (Minsky et al., 2002). Others suggest

that S-layers may provide microorganisms with a selective advantage by serving as a

protective coating or as molecular porins or sieves and traps for substrates, in maintaining

the rigidity of the cell envelope, or providing a means of cell adhesion and surface

recognition (Sara and Sleytr, 1987; Sleytr and Messner, 1988; Sara et al., 1992; Sidhu

and Olsen, 1997). Easterbrook and Sperker (1982) hypothesized that spinae may

simultaneously fulfill many fortuitous roles, analogous to "arms" with multipotential

activities, including attachment, distance-keeping, and protection. However, why some

species are prone to spine formation and others not, why S-layers exist in a variety of

shapes and symmetries, and why these structures develop among species of

methanotrophs is not clearly understood.










M~ethylocystis echinoides strain IlVET 10491 was isolated from lake mud in Russia

(Gal'chenko et al., 1977), possibly a more nutrient-rich environment than the sediments

of the uncontaminated groundwater aquifer in California where Strain CSC1 was

isolated. Echinoides is the Latinized adjective derived from the Greek word echinos,

meaning "hedgehog," named for the hedgehog-like appearance of this bacterium.

However, as reported by Gal'chenko et al. (1977), and verified in this study (Fig. 2-3c,d),

the spines on this methanotroph appear to be tubular and less dense in comparison to the

spikes observed on Strain CSC1, which would be more aptly named for a hedgehog.

Despite the different originating environments of these two strains, proximity in the

grouping of the M~ethylocystis genus, as strongly suggested by the results of this study,

adds credence to the hypothesis that phylogeny and ecology may both play a role in S-

layer formation. Similar clustering of S-layer-producing strains of Bacillus cereus has

been observed, and, similar to these results, strains in this cluster do not possess S-layers,

while others do (Mignot et al., 2001). These authors concluded that ecological pressure

is associated with the acquisition and maintenance of S-layers in hosts that fall into a

phylogenetic cluster.

Phylogenetically, Strain CSC1 is most closely related to 2ethylocystis sp. Its cell

size, rosette formation, and presence of surface layers are most similar to M~ethylocystis

echinoides. However, Strain CSC1 showed only 3.8% similarity with2~ethylocystis

echinoides by DNA-DNA hybridization, and these two strains showed differences in

surface-layer morphology, cell shape, colony color, formation of polyphosphate, and

ability to use L-ornithine or putrescine as a nitrogen source. Characteristics of cell shape

and the presence of surface layers, genes encoding for slV1VO expression, and ability to










use L-lysine as a nitrogen source are divergent from those of 2ethylocystis parvus. The

lack of polar flagella, smaller cell size, different cell shape, lack of heat resistance,

presence of polyphosphate, ability to use L-asparagine, L-aspartate, or L-glutamine and

inability to use L-lysine as a nitrogen source differentiate Strain CSC1 from 2ethylosinus

trichosporium. Accepting these differences, Strain CSC1 could be described as a new

species in the M~ethylocystis genus. We proposed this species be name M~ethylocystis

aldrichii sp. nov.

Description of Methylocystis aldrichii sp. nov.

M~ethylocystis aldrichii sp. nov. (al.drich'i.i ML gen. N. aldrichii of Aldrich;

named after H. C. Aldrich, an American microbiologist, deceased August 9, 2005).

Cells are aerobic, gram-negative, 0.3-0.6 x 0.7-1 Clm in size that occur singly or in

clusters. Reproduces by normal cell division. Budding division does not occur. Cells

are not motile but possess a spiney surface layer composed of polysaccharide. Produces

oxidase and catalase. Forms lipid cysts. Poly-P-hydroxybutyrate accumulates. Contains

Type II intracytoplasmic membranes which are aligned parallel to the cell wall. Type II

methanotroph. Methane and methanol are the sole sources of carbon and energy.

Capable of using KNO3, L-asparagine, L-aspartate, L-glutamine, L-ornithine, and

putrescine as nitrogen sources. Capable of fixing atmospheric nitrogen. Expresses

sMMO under low copper concentrations. Capable of cometabolically oxidizing a variety

of aliphatic and aromatic compounds. Not resistant to pasteurization. Is not lysed by 2%

(w/v) SDS. Is lysed by 10% (w/v) SDS. Colonies are white/yellow, slow-growing and

0.8-1.5 mm in diameter after 17-18 days at 300C on NMS agar plates, incubated in the

presence of 20% by volume methane in the headspace of a sealed desiccator. No growth









on complex organic media. Optimal pH for growth is 7.0; does not grow at pH 4.0 or

9.0. Is not capable of growth at 370C. Optimal temperature for growth is approximately

300C. The type strain, Strain CSC1 (ATCC BAA-1344), was isolated from an

uncontaminated groundwater aquifer in the mid-1980s from Moffett Naval Air Station in

Mountain View, CA, USA.












Rhodouvacus upacus
(AFO95715)

Methylocystis sp. KS33
(AJ458506)

Methylocystis sp. 39
98 (AJ458501)

Methylocystis sp. SC2
(AJ431384)

Methylocystis sp. L32
80 (AJ831522)
Strain CSC1


Uncultured bacterium L013.7
(AF358021)

Methylocystis sp. 5FB2
(AJ868420)

Methylocystis sp. 50/54
54 (AJ458510)
Methylocystis sp. SV97
(AJ414656)

Methylocystis sp. DWT
(AJ868423)

Methylocystis echinoides
(AJ458502)

58 Methylosinus parvus (Y18945)

55 Methylocystis echinoides str.
IMET 10491 (AJ458473)

Methylosinus trichosporium
67(Y18947)

Methylosinus sporium SK13
(AJ458488)






Figure 2-1. 16S rRNA phylogeny of Strain CSC1 and related M~ethylosinus and
M~ethylocystis species. Numbers at branch points represent bootstrap values
based on 100 replicates.





Methlom~ob~m~ump
BG8 (U70513)
Methylorystis sp M
(U70517)


Methylococcus capsulatus
(L40804)
Methylomicrobiumalbum
BG8 (U31654)
Unutrdclone MLM23
59 (AY571994)
Uncultured clone LOPB13
691 (AF358051)
51 1 Strain CSC1

Mehlcsisp. 50/42a
63 (AJ459016)
100 Methylocystis sp. 50/54
(AJ459017)
Mehlcsisp. B16
(AJ489799)
100 1 Methylocystissp. B2/7
(AJ459025)
Methylocystissp. SC2
(AJ584611)
100 Mthyloystiechinoides
71 (J500
Methylocystis parvus OBBP
(AF533665)

99 Mehlsnssporium str. KS16
(AJ459030)
Mehlsnssp. LW4
(AY007282)


b.
'""'


(AY007291)

Bath (M90050)
Methylosinus sporium

Methylocystis sp. M
78 (U81594)
Methylosinus sp. LW4
59(AY007288)
10 Methylosinus sp. LW3
(AY007287)
M~et 1702ins ssp.LW8
100 Methylocystis sp. 45/7a

S rain CS 1

Uncultured bacterium clone W9
73 (DQ~008430)
56rMethylocystis sp. 51
(AJ458517)
-o Methbr~ylocstssp.5FB2


Methylocystis sp. 41
(AJ458532)
53U~n Itr~ed bacterium clone 9
Methylocystis sp. IM ET 10486
(AJ458516)


(AJ459074)
Me~~l~~sisp. SC2

641 Methylocystissp.
~IMET 10486 (AJ459077)

(Met86y cystis sp. 5FB2

(AJ8h6y ytisp L

~Methylocystis sp. DWT
(AJ868411)
Methylocystis sp. SK28
(AJ459841)

StanCSC1

Mehlcsisp.
100 IMET 10489 (AF488302)

IM~E1004 1AJ59078)


96


Figure 2-2. Functional genes phylogenies of Strain CSC1. (a) Phylogenetic tree of Strain
CSC1 soluble methane monooxygenase (sMMO) gene sequence. (b)

Phylogenetic tree of Strain CSC1 methanol dehydrogenase (MDH) gene

sequence. (c) Phylogenetic tree of Strain CSC1 particulate methane

monooxygenase (pMMO) gene sequence. Numbers at branch points represent

bootstrap values based on 100 replicates.









Table 2-1. Phenotypic characteristics differentiating Strain CSC1 from M~ethylosinus
trichosporiurm, Me~cthyloc:ystis echinoidesy, and Miethylocy:~ stis palrvus.'
Characteristic Strain CSC1
trichosporium echmnoldes parvus
Colony morphology:
Yellow/ White/pale White/pale
Color White/buff .
white pink pink/tan
Cell morphology:
Width (Cim) 0.3-0.6 0.5-1.5 0.6-0.8 0.3-0.5
Length (Cim) 0.7-1 2-3 0.8-1.2 0.5-1.5
Sha e Dumbbell Rods, Pear-shape, Pear-shape,
Pyriform Ovoid Ovoid
Sharp, solid Bead-like/ Tubular
S-layersspinestf filamentous spines
Polyphosphate +f +
Poy--+ + +TI +
hydroxybutyrate
Motility -Polar flagella
Rosettes -Jf +
Heat resistance -Jf +

2%(w/v) SDS lysed -Jf
Growth at 37oC -Jf + +TI +
N,-source and use:
L-asparagine +t + +#
L-aspartate +f + +#
L-glutamine +t + +#
L-lysine -"f + -+#
L-ornithine +"r + -+#
Putrescine +"r + -+#
NMS no N2-SOUTCe "
C-source and use:
Methylamine -Jf -f
Dimethylsulfide -Jf
Methanol +"r + +"r +
Glucose -Jf -Jf
'References: Whittenbury, 1970; Gal'chenko et al., 1977; Fassel et al., 1990, Fassel et al. 1992;
Henry and Grbic-Galic, 1991; Bowman et al., 1993a; Hanson and Hanson, 1996; Hrliak and
Begonja, 1998. This study. Gal'chenko et al. (1977) reported that M echinoides forms lipid
cysts and does grow to a limited extent at 37oC. Bowman et al. (1993a), however, reported that
this strain does not accumulate poly- B-hydroxybutyrate, and only 0-10% of the strains tested
grew at 37oC. #Reported by Bowman et al. (1993a) as 75-87% of the strains were positive.























r ~F~


C


S
'
:,~.,3~II I
,. %~


Figure 2-3. Transmission electron microscopy photographs of Strain CSC1 and
M~ethylocystis echinoides. Panels (a) and (b) show the morphology of cells of
Strain CSC1 grown with 10 ELM Cu. In panel (b), numerous lamellae (La) are
present. Lipid inclusions (Li) and polyphosphate (P) storage inclusions are
also present. S indicates a surface view of the spiny surface of a cell. Cells of
M~ethylocystis echinoides viewed with negative stain (panels (c) and (d)) are
elliptical in profile and have numerous tubular projections from the surface.
Some may be seen in circular end-on profile at the upper right of panel (c).
The high magnification view in panel (d) shows that the tubes are striated.
Markers in panels (a) and (b) indicate 1 Epm; in panel (c), 0.5 Epm; in panel (d),
0.1 Epm.
















































Figure 2-4. Electron microscope cytochemistry of the S-layer of Strain CSC1. Panel (a)
shows the surface of a cell fixed initially with glutaraldehyde alone. Spiny
layer is indistinct and lightly stained. Panel (b) shows the surface of a cell
fixed initially with a glutaraldehyde/Alcian blue mixture to selectively stain
polysaccharide. Compared to panel (a), the spiny layer stains darker and is
thicker and is more distinct. Panels (c) and (d) show cells after pronase
digestion. In panel (c), a cross section, a light layer around the cell has been
left where the pronase removed the protein in the S-layer. In panel (d), a
grazing section of the spines at the cell surface, numerous light spots in the
plastic show where the pronase removed the spikes. Markers represent 0.2



77














CHAPTER 3
EFFECTS OF ALPHA-PINENE AND TRICHLOROETHYLENE ON OXIDATION
POTENTIALS OF IVETHANOTROPHIC BACTERIA

Note: Published manuscript (Pacheco and Lindner, 2005)

Pacheco, A., and Lindner, A.S. (2005) Effects of alpha-pinene and trichloroethylene on
oxidation potentials of methanotrophic bacteria. Bulletin ofEnvironmental
Conttttttttttttttttttamnt io and Toxicology 74:133-140.

Introduction

Trichloroethylene (TCE), a widely used solvent notable for its degreasing

properties, is a common environmental contaminant that poses significant risk to public

health ((ATSDR), 1999). TCE has been shown to be effectively removed from soil and

water by phytoremediation, often favored over other methods because of its

effectiveness, low cost, and aesthetic benefits. More rapid TCE removal has been

observed in the root zone of plants (rhizosphere) used in phytoremediation (Walton and

Anderson, 1990; Anderson and Walton, 1995; Brigmon et al., 1999), and methanotrophs,

methane-oxidizing bacteria that thrive on oxygen and methane and are capable of co-

oxidizing TCE (Wilson and Wilson, 1985; Little et al., 1988), have been implicated in

this increased activity (Brigmon et al., 1999).

Loblolly pines (Pinus taeda), shown to support large rhizosphere populations of

methanotrophs. (Brigmon et al., 1999), have been considered for TCE remediation. These

trees produce and release significant quantities of monoterpenes, the most predominant

being (R)-a-pinene, composing over 65% of the total oleoresin composition in different

plant tissues (Phillips et al., 1999). Since concentrations of (R)-a-pinene have been

observed to be as high as 1.4 mg gl in fresh litter layers of pine forest soils (White,










1994), the probability that soil microorganisms encounter these compounds in nature is

high. Previous studies have shown that (R)-a-pinene has a concentrate on-dependent

inhibitory effect on methane oxidation by methanotrophs (Amaral and Knowles, 1997;

Amaral et al., 1998; Amaral and Knowles, 1998), and, thus, may impact not only the

growth of these bacteria in the rhizosphere but also their ability to co-oxidize TCE.

While methanotrophs. were shown to regain methane oxidation activity one to three days

after exposure to (R)-a-pinene (Amaral et al., 1998), the implications of the long-term

presence of this monoterpene on methanotrophic activity in the rhizosphere, in particular

concentration effects of this chemical and its influence on TCE removal potentials, are

not clear.

To this end, this study sought to first assess the ability of representative Type I, II,

and X methanotrophs, grouped by their differences in carbon assimilation pathways,

intracytoplasmic membrane structures, fatty acid carbon lengths, and phylogeny

(Bowman et al., 1993a), to oxidize (R)-a-pinene over a range of concentrations using

oxygen uptake analysis. Secondly, this study sought to gain a better preliminary

understanding of the variation in oxygen uptake responses to mixtures of (R)-a-pinene

and TCE by representative methanotrophs, thus ultimately providing insight into the

effect of (R)-a-pinene on TCE oxidation potentials of these bacteria and guidance for the

phytoremediation practitioner to more accurately predict the extent of TCE

rhizodegradation when using monoterpene-releasing plants. We report herein

observations of the potential of methanotrophs. to oxidize (R)-a-pinene over a broad

range of concentrations and (R)-a-pinene/TCE mixture effects on methanotrophic

oxygen uptake activity.









Materials and Methods

Methanotroph strains used in this study included Type I2~ethylomicrobium album

BG8 (ATCC 33003) and Type II 2ethylosinus trichosporiunt OB3b (ATCC 35070),

obtained from Dr. Jeremy Semrau (University of Michigan, Ann Arbor, MI, USA), and

Type X M~ethylococcus capsulatus (Bath) (ATCC 33009), purchased from the American

Type Culture Collection (Manassas, VA, USA). Cultures were grown in nitrate mineral

salts (NMS) medium (Whittenbury et al., 1970), with or without 10 CtM Cu(NO3)2 to

provide conditions for expression of pMMO or sMMO, respectively. With the exception

of M capsulatus (Bath), incubated at 45 oC with 50% methane (99.99% pure, Strate

Welding, Jacksonville, FL, USA) in the headspace, all organisms were routinely

subcultured in sealed erlenmeyer flasks containing 20% methane in the headspace and

incubated at 30oC in a rotary shaker at 250 rpm, as previously described (Lindner et al.,

2000). Purity of the cultures was verified by routine streaking on 2% (w/v) nutrient agar

plates (Difco, Sparks, MD, USA). Expression of sMMO was qualitatively verified by a

naphthalene assay modified from Brusseau et al. (1990) and described by Lindner et al.

(2000).

Oxygen uptake analysis was performed in this study, as it has been shown to be a

rapid, effective means of assessing oxidative potential of whole cells (Lindner et al.,

2000; Lindner et al., 2003). (R)-a-pinene was chosen to represent monoterpenes because

it is a major component of loblolly pine oleoresin (Phillips et al., 1999). (R)-a-pinene

and TCE were obtained in the highest purity available from Aldrich Chemical Co.

(Milwaukee, WI, USA). Standard solutions of 10 Eomol mlF were prepared in 1,4-

dioxane (Fisher Scientifie, Pittsburgh, PA, USA), used as the carrier solvent because it

easily solubilized the substrates, was not oxidized by any of the cultures studied, and









caused no probe effects during oxygen uptake analysis (Lindner et al., 2000). Resting-

cell suspensions were prepared from 500 ml cultures harvested at %/-log phase by

centrifugation in a J2-HS Beckman floor model centrifuge (Beckman-Coulter, Fullerton,

CA, USA) at 2460 x g, 4oC, for 20 min. To ensure removal of all methane, the cells were

washed with NMS medium, recentrifuged, and resuspended in the NMS medium to a wet

cell concentration of 0.2 g ml l. The oxygen uptake system was composed of a 1.9 ml,

well-stirred, enclosed reactor held at room temperature, as described by Lindner et al.

(2000). After assessing the ability of methanotrophs to oxidize TCE and (R)-a-pinene

alone, the study proceeded to investigate the effect of (R)-a-pinene on TCE oxidation by

adding both substrates simultaneously into the oxygen uptake system before addition of

the resting cells.

Despite storage of the resuspended cells on ice throughout the oxygen uptake

experiments, loss of cell activity over time was observed. To ensure comparability of

measurements throughout the 2-3-day testing period, all rates of oxygen uptake were

normalized to the rates observed with 4 ml of methane gas, measured just prior to a

change to a new substrate concentration. Details of this normalization procedure are

presented in Lindner et al. (2000). The electrode was calibrated at least daily with a

saturated sodium sulfite solution, and "live" runs were performed at least in triplicate for

each concentration tested. All runs were corrected for endogenous metabolism.

Controls without cells and with 4 ml of acetylene gas, a known inhibitor of MMO (Prior

and Dalton, 1985), were routinely run to verify that depletion of oxygen, hence, oxidation

activity, was a result of MMO activity. Initial rates of oxygen uptake were calculated by










linear or polynomial fits to the data points using Microsoft Excel software (Microsoft

Corp., Redmond, WA, USA).

Results and Discussion

M. trichosporium OB3b and M~ capsulatus (Bath), when cultured with no copper,

expressed positive sMMO activity, as evidenced by a bright pink-to-purplish color in the

assay. All of the strains tested negative for sMMO activity (no color change observed)

when cultured with 10 CIM Cu(NO3)2. SMMO and pMMO expression under culturing

conditions without and with copper, respectively, was thus assumed, a reasonable

conclusion given that enzyme expression in these methanotrophs under these conditions

is well characterized. Active resting cells of all three representative methanotrophs

consumed oxygen over a range of TCE and (R)-a-pinene concentrations, regardless of the

type of MMO expressed (Fig. 3-1, A-E). No oxygen uptake was observed after addition

of acetylene or without cells present, verifying MMO activity in all cases. As shown in

Figure 3-1, regardless of the methanotroph or substrate tested, a maximum rate of oxygen

uptake was observed, followed by a rapid decrease in rates, suggesting toxic effects of

either the substrate itself or of oxidation products formed. This oxygen uptake behavior

has been reported previously for methanotrophs with aromatic substrates (Lindner et al.,

2000), and, while both substrates have been shown to have toxic effects on

methanotrophic activity (Fox et al., 1990; Alvarez-Cohen and McCarty, 1991a; Henry

and Grbic-Galic, 1991; Oldenhuis et al., 1991; White, 1994; Amaral and Knowles, 1997;

Amaral et al., 1998; Amaral and Knowles, 1998), there have been no previous reports on

the effects of a range of substrate concentrations on relative activities.

As shown in Figure 3-1, methanotrophs expressing sMMO (plots A, C) oxidized

TCE at higher maximum rates than those expressing pMMO (plots B, D, E), as










previously reported (Little et al., 1988; DiSpirito et al., 1992; Lontoh and Semrau, 1998).

The maximum normalized rates of oxidation by M~ trichosporium OB3b and M~

capsulatus (Bath) expressing sMMO or pMMO were 0. 11 + 0.01 and 0.03 + 0.00 and

0.05 + 0.01 and 0.03 + 0.01, respectively (Fig. 3-1, A-D), while the maximum rate

expressed by M~ album BG8, capable of pMMO expression only, was 0.05 + 0.01 (Fig. 3-

1, E). The TCE concentrations where the observed normalized oxygen uptake rate was

the highest ranged from 20 to 35 ppm for the tested strains. M~ trichosporium OB3b and

M~ capsulatus (Bath) expressing sMMO exhibited oxygen uptake maxima at higher TCE

concentrations (35 ppm) than when expressing pMMO (20-25 ppm), and M~ album BG8

expressing pMMO showed a maximum observed rate at 35 ppm TCE. These results do

suggest differing sensitivity levels to TCE, depending on the methanotroph and type of

MMO expression.

As observed with TCE, both sMMO-expressing methanotrophs were also capable

of oxidizing (R)-a-pinene at higher rates than their pMMO-expressing counterparts (Fig.

3-1, A-D). The maximum normalized rate of oxygen uptake by M~ trichosporium OB3b

expressing sMMO was almost 10 times the rate observed with pMMO-expressing cells

(0.28 + 0.04 and 0.02 + 0.01, respectively); however, both rate maxima occurred at 20

ppm (R)-a-pinene (Fig. 3-1, A, B). The maximum normalized oxygen uptake rate with

M~ capsulatus (Bath), expressing sMMO, was 0. 10 + 0.02 at 20 ppm (R)-a-pinene,

compared to 0.08 + 0.01 at 50 ppm (R)-a-pinene under pMMO expression (Fig. 3-1, C,

D). The observed maximum normalized rate of oxygen uptake by M. album BG8 was

0.04 + 0.00, between the values observed for the other two strains under pMMO

expression (Fig. 3-1E). Previous studies have reported higher TCE oxidation rates by










pure methanotrophs under sMMO expression (Wilson and Wilson, 1985; Little et al.,

1988; DiSpirito et al., 1992; Lontoh and Semrau, 1998); however, this is the first report

of such a trend with (R)-a-pinene. These results bring direct relevance to the

environment, as sMMO expression in methanotrophs occurs only at very low copper

concentrations (Lontoh and Semrau, 1998). Measurement of bioavailable copper is

essential, therefore, for effective prediction of methanotrophic activity potential.

The response of each methanotroph in the presence of 20 ppm TCE over a range of

(R)-a-pinene concentrations is shown in Figure 3-2, A-E. This plot presents the change

in normalized oxygen uptake rate with 20 ppm TCE alone caused by the presence of

different concentrations of (R)-a-pinene and thus represents the influence of (R)-a-

pinene on TCE oxidation and provides insight into mixture effects on methanotroph

activity. The concentration of 20 ppm TCE was chosen because it was not observed to be

toxic to any of the methanotrophs tested previously (Fig. 3-1).

The responses to (R)-a-pinene were highly dependent on the type of methanotroph

and MMO expression, with M~ trichosporium OB3b showing decreased rates relative to

20 ppm TCE alone regardless of (R)-a-pinene concentration (Fig. 3-2, A, B) and M~

capsulatus (Bath) and M~ album BG8 showing mostly increased rates (Fig. 3-2, C, D, E).

With the exception ofM~ capsulatus (Bath) under pMMO expression, the highest

observed rates in the presence of the mixture were lower than those observed with (R)-a-

pinene alone. M~ trichosporium OB3b expressing pMMO showed consistently small

decreases in oxygen uptake activity in the presence of the mixture compared to 20 ppm

TCE alone; however, the activity of this strain when expressing sMMO appeared to be

inhibited to a greater extent in the presence of all tested concentrations (2 to 20 ppm) of










(R)-a-pinene in the mixtures (Fig. 3-2, A, B). Regardless of 1VMO expression, M~

capsulatus (Bath) yielded increased normalized oxygen uptake rates in the presence of

the mixture above approximately 20 ppm (R)-a-pinene relative to its observed rate at 20

ppm TCE alone. The greatest rate increase shown by M~ capsulatus (Bath) expressing

slV1VO in the presence of the mixture was observed at 40 ppm (R)-a-pinene. This

maximum rate observed with the mixture was 1.8 times the rate with 20 ppm TCE alone,

suggesting a lessoning of toxicity effects on the cells. The maximum increase with this

strain under plV1VO expression was observed at 30 ppm (R)-a-pinene and was

approximately 3.5 times higher than with 20 ppm TCE alone and 1.5 times higher than

observed at 50 ppm (R)-a-pinene alone. Increase in oxidation potential ofM~ album BG8

was also observed when (R)-a-pinene was in the presence of 20 ppm TCE (Fig. 3-2, E).

At the highest concentration of (R)-a-pinene tested (30 ppm) with this strain, the increase

in normalized oxygen uptake rate was 1.8 times the rate observed with TCE alone.

In conclusion, all of the tested methanotrophs expressing either slVMVO or plV1VO

were capable of oxidizing (R)-a-pinene over a range of environmentally relevant

concentrations. However, toxicity effects of this monoterpene, similar to those shown

with TCE, were observed. When both (R)-a-pinene and TCE were introduced to the

representative methanotrophs, varying responses in the rates--decreases with the Type II

methanotroph and increases with the Types I and X methanotrophs-were observed in

comparison to those observed in the TCE-only experiments. Whether TCE and/or (R)-a-

pinene were oxidized in the mixture is not known, given the indirect measurement

method of oxygen uptake analysis; however, it is suggested here that the total oxidation

potential of methanotrophs is affected, either antagonistically or synergistically, in the










presence of TCE and (R)-a-pinene mixtures. These results emphasize the importance of

not only assessing the concentration levels of both contaminants and monoterpenes and

but also of measuring the oxidation potentials and diversity of rhizosphere methanotrophs

at phytoremediation sites where plants that release large amounts of monoterpenes are

being contemplated for use.


0.35

0.30

0.25

0.20

0.15

0.10

0.05

0.00


0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60

TCE and (R)-co-pinene concentration (ppm)


Figure 3-1. Normalized rate of oxygen uptake by the representative methanotrophs in the
presence of varying concentrations of TCE (*) and (R)-a-pinene (0). (A),
(B): M~ trichosporium OB3b cultured without and with copper, respectively.
(C), (D): M~ capsulatus (Bath) cultured without and with Cu, respectively.
(E): M~ album BG8 cultured with Cu. Error bars represent the standard
deviation for triplicate samples.













-A -- B -- C- -- D -
















0 20 4060 0 20 4060 0 20 400 0~ 20 4060 0 20 4080


xo





- E

Eo


0.09




0.03


0.00

-0.03


-0.06


(R)-a.-pinene concentration (ppm)


Figure 3-2. Change in the normalized oxygen uptake rate by representative
methanotrophs observed in the presence of 20 ppm TCE at varying
concentrations of (R)-a-pinene. (A), (B): M. trichosporium OB3b cultured
without and with Cu, respectively. (C), (D): M~ capsulatus (Bath) cultured
without and with Cu, respectively. (E): M. album BG8 cultured with Cu.
Error bars represented the standard deviation for triplicate samples.















CHAPTER 4
STABLE ISOTOPE PROBING FOR CHARACTERIZATION OF
IVETHANOTROPHIC BACTERIA INT THE RHIZOSPHERE OF
PHYTORE1VEDIATING PLANT S

Note: Manuscript to be submitted to Biology Letters

Introduction

Phytoremediation, the use of plants to remove a variety of contaminants from soil

and aqueous environments, has been shown to be more economical and aesthetically

pleasing than traditional remediation methods, such as pump-and-treat approaches

(McCutcheon and Schnoor, 2003). Despite its observed effectiveness in removal of

contaminants, including trichloroethylene (TCE) and tetrachloroethylene (PCE), two

widely distributed chlorinated solvents that cause concern because of their potential

health effects (ATSDR, 2006), phytoremediation is still limited by a lack of

understanding of the primary removal processes involved. In particular, the potential

roles that root-zone (rhizosphere) bacteria can assume in the overall removal of

contaminants is not fully appreciated (Walton and Anderson, 1990; Anderson and

Walton, 1995; Brigmon et al., 1998; Brigmon et al., 1999).

One reason for the lack of specific information on the degradation potentials of

root-zone bacteria is that traditional culture-dependent methods are not capable of

directly assessing the activity and diversity of microorganisms in situ. Furthermore, these

methods provide limited information because of their associated inherent cultivation bias

(Fry, 2004; Smalla, 2004). The development of culture-independent molecular methods

such as stable isotope probing (SIP), has enabled scientists to study in situ conditions









more effectively and, more importantly, to characterize the active microbial populations.

The promising SIP technique relies on the incorporation of a labeled substrate with a less

naturally frequent isotope, into the active microbial community of a sample that later can

be separated from the unlabeled biomass (Radaj ewski et al., 2000).

Recent studies of methanotrophic bacteria have shown successful results using the

SIP approach in environments, such as peat soils, acidic forest soils, cave water, and soda

lake sediments (Morris et al., 2002; Radajewski et al., 2002; Hutchens et al., 2004; Lin et

al., 2004). Methanotrophs. are among the aerobic bacteria that are known to reside in the

rhizosphere of plants and that are capable of oxidizing chlorinated contaminants such as

TCE (Wilson and Wilson, 1985; Hanson and Hanson, 1996; Brigmon et al., 1999;

Doronina et al., 2004; Pilon-Smits, 2005). Because methane serves as the sole source of

carbon and energy for methanotrophs, it is often used as the measure of methanotroph

activity in the environment, and is a natural substrate for SIP testing. Labeled methane

(13C-CH4) has been successfully incorporated into the DNA of growing cells of

methanotrophs. (13C-DNA) and separated from the naturally occurring 12C-DNA by

density gradient centrifugation (Radajewski et al., 2000; Morris et al., 2002; Radajewski

et al., 2002; McDonald et al., 2005). Molecular fingerprinting techniques, such as

denaturing gradient gel electrophoresis (DGGE) with DNA fragments of specific

methanotroph enzymes, such as particulate methane monoxygenase (pMMO), can be

subsequently used to identify and assess the relative abundance of the active populations

(Muyzer et al., 1993).

With the advent of this sophisticated molecular biology method that links identity

with function, development of a protocol using SIP methods that is specific to the









rhizosphere holds promise in better understanding the rhizodegradation process of

phytoremediation systems. This study provides a first-basis in method development and

analysis of the SIP technique for the measurement of potential in situ activity and

diversity of methanotrophic bacteria in the root-zone of trees used for remediation of

TCE from contaminated groundwater and soil.

Materials and Methods

Site Description

This study was located at a Superfund site, the former LaSalle Electrical Utilities in

LaSalle, IL (USA). The company manufactured capacitors from 1943 to 1982, resulting

in soil and groundwater contamination of mostly polychlorinated biphenyls and the

chlorinated solvents, TCE and PCE. Currently, in the final stages of the cleanup process

at the site, two phytoremediation plots have been implemented to enhance chlorinated

solvent removal (Lange, 2004). The first plot (0.25 ha), contaminated with TCE (0-254

ppb), was installed in September 2002 (labeled as "TCE Site"). Poplar (18 clones) and

willow (24 clones) genotypes were planted by lowering 1.8 m rooted cuttings to the

bottom of boreholes (0.6 m diameter) lined with high-density polyethylene pipe and filled

with an equal mix of sand, soil, bark, and peat (pH 7.8). The second phytoremediation

plot (0.21 ha), contaminated with PCE (0-838 ppb), was established in March 2002

(labeled as "PCE Site"). At this plot, poplar trees were planted directly into the improved

soil (pH 7.3) with mulch composed of tree chips on the top 0.5 m of the soil surface.

Sampling

All rhizosphere soil samples were collected using a small diameter (1.9 cm) hand

soil auger to minimize disturbance in the pots. Samples were taken at three time periods,

July 2003, July 2004, and November 2004, in order to compare summer and fall









conditions and time effects on the activity and population diversity of methanotrophs.

Each sampling period, mid-summer (July) and early fall (November), fell in the "wet

season" of April to December when 642 mm of precipitation were collected at the site

during 2004. The average daily air and soil temperatures were 210C and 110C,

respectively, in the 2004 summer sampling, and 200C and 90C, respectively, in the 2004

fall sampling. Groundwater levels below the planted plots fluctuated in 2004 from 2.1 to

3.1 m and 1.8 to 3.3 m from the soil surface at the TCE and PCE Site, respectively.

Root growth of the trees at the TCE and PCE Site was observed to extend from the

surface to 90-120 cm below surface. A composite sample, in regions of high contaminant

concentration, was taken from two opposite locations around the tree base at a depth of

30-60 cm. This soil layer was chosen as a potential zone of intermediacy rhizosphere

activity between surface and deeper soil layers. At the TCE Site, samples were also

removed from non-planted pots in the contaminated area to serve as a control when

compare to the planted pots. Also different tree clones, showing the greatest vigor, were

sampled at the TCE Site. They were one poplar clone 145/51 (Populus deltoides x P.

nigra; origin, North America x Europe) and three willow clones, SX61 (Salix

sachalinensis; origin, Japan; exotic), S365 (S. discolor 18; origin, University of Toronto),

and 94014 (S. purpurea; origin, State University of New York; exotic).

While it is well known that methanotrophs. are not capable of oxidizing PCE,

samples were removed from the PCE Site to serve as a mean of comparing methanotroph

activity and diversity with the TCE Site samples. One poplar clone, 145/51, was sampled

at the PCE Site, along with a non-planted sample removed from outside the plot in an









uncontaminated region that was eventually converted to an irrigated, fertilized soccer

Hield after the first sampling in July 2003.

To prevent cross-contamination, the sampling auger was washed with sterile water,

rinsed with 95% ethanol, and washed again before each sample was taken. Samples were

immediately placed in sterile bags (Nasco Whirl-Pak, Fort Atkinson, WI, USA), placed

on ice, and transported to the UF laboratory where they were subsequently stored at 4oC

until testing. In the laboratory, samples were gently homogenized using a sterile spatula,

and Eine roots of less than 2 mm diameter were separated from the soil for separate

testing.

Stable Isotope Probing (SIP) Soil Microcosms

Experimental conditions. In order to assess activity and diversity of the active

methanotroph populations in poplar and willow tree rhizospheres, during TCE

remediation, soil microcosms were prepared from samples collected over time.

Microcosms consisted of 10 g wet soil (plant material removed) normalized to 16% water

content with sterile water. This water content represented approximately 40% of the Hield

capacity of the TCE and PCE Site soils, where the greatest extent of CH4 Oxidation was

observed in preliminary experiments, as previously described by Reay et al. (2001). The

"wetted" soil was placed in sterile 160 ml serum vials, which were subsequently sealed

with gray butyl rubber stoppers and crimp tops. Ten ml (0.4 mmol; ~7%, v/v) of filter-

sterilized 13CH4 (99.9%; Isotec, Miamisburg, OH, USA) or 12CH4 (99.9%; Airco-BOC,

Murray Hill, NJ, USA), used in preliminary experiments to optimize conditions and

assess any effects of the labeled substrate by comparing to the 13CH4 miCTOCOSms rates,

was then added as previously described (Morris et al., 2002; Radajewski et al., 2002), and

each vial was wrapped with aluminum foil for subsequent incubation in the dark at room









temperature (~250C). Headspace CH4 depletion was monitored every 2 to 5 days by

removal of 25 ul of CH4 with a gastight syringe and analysis using a gas chromatograph

(Model HP5809A GC/TCD; Hewlett Packard, PaloAlto, CA, USA) equipped with a GS-

Carbon plot column (Agilent Technology, PaloAlto, CA, USA). The gas chromatograph

was maintained at a head pressure of 5 psi and programmed in a 4 min run with

temperatures of 25, 120 and 2000C in the oven, injector and detector, respectively.

When more than 90% of the CH4 WAS COnSumed, vials were opened, gently flushed

with fi1ter-sterilized air for 5 s to remove any accumulated 13CO2 and to maintain aerobic

conditions, resealed, and the same initial amount of 13CH4 added. The procedure was

repeated Hyve times until a total of 2.0 mmol of CH4 WAS COnSumed (Radaj ewski et al.,

2002). A positive control with pure methanotroph, M~ethylocystis trichosporium OB3b,

and three negative controls with no CH4 added, with twice-autoclaved (killed) soil, and

with 20% (v/v) each of CH4 and acetylene (a known inhibitor of methane

monooxygenase, Prior and Dalton (1985)) in the headspace, were also included.

Additionally, some of the microcosms were set in replicates to assure reproducible

results. Initial CH4 depletion rates were calculated from data taken during incubation

after the first CH4 addition by linear regression analysis of the consumption curve.

DNA extraction and ultracentrifugation. The content of the microcosms (10 g

soil) was processed using a PowerMax Soil DNA Extraction Kit (Mo-Bio, Carlsbad, CA,

USA). DNA extracts were resolved by CsCl density gradient centrifugation. Briefly, 1 g

mll of CsCl (Fisher Scientifie, Pittsburgh, PA, USA) was dissolved in the DNA solution,

and 100 El1 of ethidium bromide (10 mg ml l; Bio-Rad, Hercules, CA, USA) was added

before loading the solution into 5.1 ml quick-seal polyallomer ultracentrifuge tubes









(Beckman Coulter, Fullerton, CA, USA). Ultracentrifugation was performed using a

VTi65 vertical rotor in a Model L8-80 ultracentrifuge (Beckman Instruments, Fullerton,

CA, USA) at 265,000 x g for 16 h at 200C. After ultracentrifugation, fractions were

visualized with UV light at 365 nm (Sambrook et al., 1989; Radajewski et al., 2002).

Three DNA bands were generally observed and collected: (1) a light-DNA upper band

(12C-DNA); (2) a middle band, smear of 12C- and 13C-DNA; and (3) a heavy-DNA lower

band (13C-DNA). DNA fractions were collected and purified as described by Sambrook

et al. (1989). Ethidium bromide was extracted from the DNA with 1-butanol (Fisher

Scientific, Pittsburgh, PA, USA) saturated with water. Following fiye extractions the

DNA solution was diluted in water and precipitated with ethanol overnight at -200C and

dissolved in 100 ul TE buffer (Sambrook et al., 1989). A second ultracentrifugation step

was not necessary after confirming that the protocol of DNA band extraction was exact.

Re-runs verified the presence of only one distinct band in the new columns.

Polymerase chain reaction (PCR) amplification. The purified DNA fractions

(12C- and 13C-DNA) were used as a template for PCR analysis. The phylogenetic

analysis was performed with the functional pmoA gene, targeted using the primer set

Al89f (5' -GGNGACTGGGACTTCTGG-3 ') and mb661 (5'-

CCGGMGCAACGTCYTTACC-3 ') (Integrated DNA Technologies, Coralville, IA,

USA), specific to the pMMO active site (Costello and Lidstrom, 1999). A GC-clamp (5'-

cccccccccccccgccccccgccccccgcccccgccgccc-3') was attached to the Al 89f primer as

described by Henckel et al. (1999).

PCR amplification was performed according to the procedure described by Knief et

al. (2003). PCR reactions consisted of the MasterAmp 2X PCR premixture F (Epicentre









Technologies, Madison, WI, USA) containing 100 mM Tris-HCI (pH 8.3), 100 mM KC1,

400 ELM each of dNTP, 3-7 mM MgCl2, and the enhancer betaine (0-8 X), combined with

0.5 ELM each primer, lU Taq polymerase, and sterile water to a total reaction volume of

50 El1 (Knief et al., 2003). All reactions were assembled on ice, and the cooled tubes

were placed in a preheated (940C) thermal block for PCR initiation (Henckel et al.,

1999).

The PCR protocol consisted of a touchdown program using a thermocycler

(Mastercycler Personal 5332; Eppendorf, Westbury, NY, USA) with the following

parameters: initial denaturation of 5 min at 940C, followed by 35 cycles of 1 min at 940C

for denaturation, 1.5 min at 62 to 550C in -0.50C increments for annealing, 1 min at 720C

for elongation, with a final extension step of 7 min at 720C (Knief et al., 2003). PCR

product size (540 bp) was examined by horizontal agarose electrophoresis. PCR positive

controls included representatives of all methanotrophs types (type X M~ethylococcus

capsulatus (Bath) (ATCC 33009), type II strains, M~ethylosinus trichosporium OB3b

(ATCC 35070), Strain CSC1, M~ethylocystis echinoides (IMET 10491), M~ethylocystis

parvus OBBP (NCIMB 11129), and type I2~ethylomicrobium album BG8 (ATCC

33003)).

Denaturing Gradient Gel Electrophoresis Analysis (DGGE), Sequencing, and
Phylogenetic Analysis

DGGE. PCR products were separated by DGGE in the DCode System (Bio-Rad,

Hercules, CA, USA) as described by Henckel et al. (1999). Briefly, 1 mm thick 6.5%

(w/v) polyacrilamide gels (37.5:1 acrylamide-bisacrylamide) (Fisher Scientific,

Pittsburgh, PA, USA) were prepared and electrophoresed in lX TAE buffer at 610C and

180 V for 5 h in a 35-65% linear denaturant gradient (65% is 4.5 M urea and 26% (v/v)









deionized formamide). Gels were loaded with 25-45 El1 of PCR product, according to

band intensity in agarose gels, and '/ volume of loading buffer. Gels were stained with

ethidium bromide according to the manufacture' s instructions (Bio-Rad, Hercules, CA,

USA), visualized at 312 nm on a UV transilluminator (Model 88A, Fisher Scientifie,

Pittsburgh, PA, USA), and photo-documented with the system DigiDoc-IT TM (Daigger,

Vernon Hill, IL, USA) using the Doc-It software v. 2.4 (UVP, Upland, CA, USA).

DGGE bands were excised from the middle part of the band with a sterile scalpel and the

DNA eluted according to the protocol described by Chory and Pollard (1999). The eluted

DNA was reamplified and reanalyzed on DGGE to verify sample purity. Band

reamplifieation was performed by modifying the PCR protocol to 30 cycles of 30 s at

940C for denaturation, 45 s at 660C for annealing (to avoid sequence ambiguity as

reported by Dunfield et al. (2002)) and 30 s at 720C for elongation, with the same initial

and Einal steps. Several bands with the same mobility were excised from different lanes

to check for sequence identity.

Sequencing. Reamplified PCR products of excised DGGE bands were purified

with a PCR purification kit (Mo-Bio, Carlsbad, CA, USA) before sequencing. PCR

product concentration and purity was determined by UV absorption spectrophotometry

(1:20 dilution). PCR products were sequenced by the Interdisciplinary Center for

Biotechnology Research (ICBR), University of Florida (Gainesville, Florida, USA).

Phylogenetic analysis. Sequences were compared in the National Center for

Biotechnology Information (NCBI) database using BLAST (Altschul et al., 1990).

Related sequences identified in BLAST, as well as sequences of extant methanotrophs,

were aligned and adjusted manually with CLUSTALX v. 1.8 (Thompson et al., 1997).









Phylogenetic trees were generated by the neighbor j oining (NJ) method with

CLUSTALX and displayed in TreeView v. 1.6.6 (Page, 1996). Nucleotide accession

numbers of all obtained sequences were placed in the GenBank for future access

(AYXXX-AYXXX) .

Statistics

CH4 depletion rates observed in the SIP microcosms were analyzed by comparing

the initial slopes of the linear regression curves fitted to the consumption curve during

incubation after the first CH4 addition. When a set of samples showed no significant

differences in rates, an average depletion rate (common regression coefficient) was

calculated as an estimate of the CH4 depletion rate underlying all rates of a particular set

of samples (for example, rates among the same plant type in each sampling period).

Additionally, differences among sample means and between samples and the control

were analyzed by Tukey's and Dunnett' s test, respectively (Zar, 1984). SAS software v.

7 (SAS Institute, Cary, NC, USA) was used for all the analysis.

Results

SIP Protocol Implementation

The SIP technique was successfully applied to the rhizosphere soils. The rates

observed in the 13CH4 and 12CH4 miCTOCOSms were comparable, and variability among

the replicates was low. The M. trichosporium OB3b control was effectively labeled by

the 13CH4 and no 13CH4 COnSumption was observed in the negative controls.

DNA extracts from the microcosms were effectively separated by CsCl density

gradients (Fig. 4-1A). When a smear was present between the unlabeled (12C-DNA) and

labeled (13C-DNA) fractions, it was collected as a 12-13C-DNA combined fraction and was

not included in the study. Correct recovery of these fractions was verified by a second









ultracentrifugation under the same protocol, even though band position varied because of

changes in the density of the new solution (Fig. 4-1, B-C). The extracted 13C-DNA

fractions produced pmoA gene fragments of the expected size (540 bp). The

methanotroph positive control cultures revealed multiple DGGE bands (Fig. Al,

Appendix), in keeping with earlier reports of the high probability of encountering

multiple copies of the pmoA gene in methanotrophs (Semrau et al., 1995; Dunfield et al.,

2002). Because DGGE band purification was difficult in most samples yielding

ambiguous positions after sequencing, the annealing temperature was increased from 62

to 660C and only the reverse primer was used for sequencing (Dunfield et al., 2002).

Furthermore, 16S rDNA-DGGE profies revealed complex band patterns and smears that

were difficult to examine (data not shown).

In general, pmoA-DGGE profies of the 13C-DNA fractions of the different

rhizosphere soil microcosms did not differ greatly among sites, plant type or sampling

period. Consequently, it was useful to set a reference profie for the analysis (Fig. 4-3A,

lane 1). Reference bands 1, 3, 9, 10 and 11 were not possible to sequence.

Methanotroph Activity and Composition in the TCE Site

13CH4 added in Hyve additions to a total of approximately 2.0 mmol, was consumed

within 31-37 days in all TCE Site soil microcosms. Initial CH4 depletion rates (Fig. 4-

2A), calculated as a measure of the oxidative potential at the time of sampling, showed

no significant differences within plant type over the 16-month sampling period. A

comparison of average rates per plant type, which represents plant type's overall activity

throughout the study, shows also no significant difference in CH4 depletion rates among

plant types and with the non-planted sample (P<0.05). The overall average CH4

depletion rate at the TCE Site was 0. 11 Epmol h-l g-l dry weight soil (+ 0.01, SE).









As shown in Fig. 4-3, TCE Site (panel A to C) and PCE Site (panel D) pnzoA-

DGGE profies were mostly described by the same group of bands numbered from 1 to

11 in the reference profie (Fig. 4-3A, lane 1). Concentric circles with letters denoted

bands different from the reference profie. Profies were labeled starting with the plot

location (TCE or PCE) followed by the tree type (poplar or willow), soil compartment

analyzed (rhizosphere, rhizoplane, or non-planted soil), and sampling period. Willow

clones SX61 and 94019 were not sampled in the November 2004 sampling period,

consequently, they only showed two profies each (Fig. 4-3C, lanes 1-4). The pnoA-

amplified sequences from the 13C-DNA fractions of the TCE Site microcosms revealed

DGGE profies composed by 2 to 11 bands (Fig. 4-3, B-C). These profies were mainly

described by three groups of highly similar sequences according to band position and

BLAST alignments, indicating that some bands may represent copies of multiple pnzoA

genes. However, some profies that exhibited these groupings did not reveal all of the

bands.

The TCE Site DGGE profies throughout the study did not vary to a great extent

among plant type (Fig. 4-3, B-C), but they were distinct from the non-planted soil (Fig. 4-

3B, lane 4 to 6). Rhizosphere DGGE profies from poplar (Fig. 4-3B, lane 1-2) and all

willow clones (Fig. 4-3, panel B, lanes 7-8, and, panel C, lanes 1 to 4) collected in July

2003 and 2004 revealed the same community of organisms. These profiles were

composed of two groupings of bands represented by reference bands 4 and 6 to 8, and

bands 2 and 5 (Fig. 4-3A, lane 1). Within each grouping, bands shared 100% sequence

similarity and aligned in BLAST (>99% similarity) with two clones of uncultured

M~ethylocaldunt sp. isolated from a landfill cover soil. The phylogenetic tree clustered









these sequences as the described groupings (Fig. 4-4, Group 1 and 2) and within the

M~ethylocaldunt branch (type X methanotroph). These groupings are closely related to

the cultured organism, M~ gracile. Also, a third group of bands, designated by reference

bands 9 to 11 (Fig. 4-3A, lane 1), was detected in all profiles. However, because

reamplifieation of this group was not possible, and since they were the only bands present

in the July 2003 TCE Site non-planted profie (Fig. 4-3B, lane 4), it is possible they

represent another set of similar pnoA genes.

From the planted microcosms at the TCE Site, only the polar and willow clone

94014 exhibited changes in their methanotroph community throughout the study. The

poplar tree, in the November 2004 sampling (Fig. 4-3B, lane 3), showed less than half of

the bands in the July samplings (Fig. 4-3B, lane 1-2). It only revealed three bands, two at

very low intensity and the same maj or band of previous samplings (reference band 5).

Willow clone 94014, in the July 2004 sampling (Fig. 4-3C, lane 4), did not exhibit this

maj or band and showed an extra band of an uncultured methanotroph that closely related

(88% similarity) to another type X methanotroph, M~ethylococcus capsulatus (Fig. 4-4).

The TCE Site non-planted microcosms, over the three sampling times, exhibited

variable profies at very low intensity (Fig. 4-3B, lane 4 to 6). In the July 2003 sampling,

only the 9 to 11 grouping was retrieved. However, in the July and November 2004

samplings, the 9 to 11 grouping was observed along with some of the bands that

described the M~ethylocaldunt clones found in the planted samples and uncultured bacteria

from rice Hields and upland soils (>87% similarity). The uncultured bacteria (Fig. 4-3B,

lane 5-6) were placed within the M~ethylocaldunt and M~ethylococcus branch (Fig. 4-4),


100