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Methanotrophic Cultures from Landfill Environments: Promise for Bioremediation of Hazardous Chemicals

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Methanotrophic Cultures from Landfill Environments: Promise for Bioremediation of Hazardous Chemicals
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Strate, Jessica
Lindner, Angela ( Mentor )
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Gainesville, Fla.
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University of Florida
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English

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Methanotrophic Cultures from Landfill Environments: Promise
for Bioremediation of Hazardous Chemicals

Jessica Strate


ABSTRACT


The recent explosive population growth in the state of Florida has resulted in a massive increase in the amount

of municipal solid waste (MSW) and associated hazardous chemicals disposed in landfills. As a result of this

rapid loading of landfills, an urgent need exists for controlling landfill environments so that natural

attenuation processes can more efficiently degrade this waste. In a direct response to this need, we are

investigating bioremediation as a possible method for MSW hazardous chemical degradation. We have isolated

and characterized a methanotrophic-heterotrophic mixed culture (GW 60,13') from the Alachua County Landfill

and are comparing these results to a well-defined, previously isolated groundwater mixed culture, MM1. Initial

results from this work show that these Type II methanotrophic cultures exhibit different characteristics but

are capable of degrading representative volatile organic compounds that are prevalent in landfill environments.



INTRODUCTION


In the last decade, the state of Florida has experienced a growth rate of approximately 40%, and projections

of Florida population growth rates into the year 2010 show values that are on average 92% higher than values

for the rest of the country (1). As a result, the municipal solid waste (MSW) and associated hazardous

chemicals generated in Florida each year have increased to over 24 million tons (2). In order to eliminate this

waste, environmental engineers have resorted to landfilling up to 40%, despite efforts to promote recycling

and composting (2). As a result of the rapid loading of landfills, there is an urgent need to control

landfill environments for rapid degradation of this waste before leaching into potential drinking water sources occurs.



The predominant degradation processes in landfill environments are biological and differ depending on the levels

of oxygen present (3). Current landfill design promotes the formation of anaerobic processes (with no

oxygen present), and the primary microorganisms involved in these processes are known as "methanogens."

These microbes are responsible for breaking MSW down to methane, a key component of greenhouse gasses.

The actual amount of methane released into the atmosphere from landfills is approximately 10-70 Tg per year

(4), with a significant portion intercepted by methanotrophs, aerobic microorganisms that reside near
the methanogenic zones and require stable sources of both methane and oxygen (4). Certain

methanotrophs (expressing soluble methane monooxygenase, sMMO) have been suggested to play a role





in transforming a variety of other compounds of environmental interest, including polychlorinated biphenyls

(PCBs) (5,6,7, 8) and trichloroethylene (TCE) (9,10). However, such studies have not been directly targeted to

solving contamination problems specific to landfill environments. Thus, further research is needed to address

our limited understanding of the diversity, distribution, and potential for landfill microorganisms to transform

leachate chemicals into harmless forms in an optimized fashion.



The aims of this study were (i) to isolate a novel methanotrophic-heterotrophic mixed culture from samples

taken from the Alachua County Landfill, (ii) to characterize this mixed culture based on cellular and

colony characteristics and activity, (iii) to compare these characterization results to a well-defined

mixed methanotrophic-heterotrophic culture (MM1) previously isolated from an uncontaminated

groundwater environment. Based on the results reported herein, these two mixed cultures exhibit

divergent characteristics, yet both have been shown to transform representative organic contaminants prevalent

in landfill environments.



MATERIALS AND METHODS


Growth Media


Both solid and liquid media were used for culturing methanotrophic populations expressing sMMO (expressed

under low or no copper concentrations). The liquid media used was based on Whittenbury's nitrate mineral

salts (NMS) media with no copper added (Table 1) (11). Methanotrophic bacteria were isolated on solid plates

using NMS medium and Bacto� agar (Difco, Detroit, MI), and heterotrophic bacteria were isolated on nutrient

agar (Difco, Detroit, MI).



Table 1

Chemicals Needed for NMS Preparation Chemical Components in NMS Medium

Chemical Amount

MgSO4 7H20 (magnesium sulfate) � 1.0 g/1.0 Liter

KNO3 (potassium nitrate)� 1.0 g/1.0 Liter

CaCI2 (calcium chloride)� 0.2 g/1.0 Liter

FeEDTA" 0.1 mL/1.0 Liter

NaMolybdate- 4 H20 0.5 mL/1.0 Liter

Whittenbury Trace Elements (already prepared) 1.0 mL/1.0 Liter

Phosphate Stock Solution (already prepared)* 10.0 mL/1.0 Liter

Vitamin Stock Solution (already prepared)* " 10.0 mL/1.0 Liter






* indicates that these chemicals are added to the media after autoclaving (phase 2)

� indicates that the chemical is in solid form

" indicates that the chemical is in liquid form


Isolation and Maintenance of Liquid Landfill Mixed Cultures


To isolate this culture, one gram of a soil sample, taken 13 feet below the landfill surface, was added to 25 mL

of NMS liquid medium in a 250 mL Erlenmeyer flask, equipped with a rubber stopper and a glass wool-packed

filling tube. Headspace was removed from the flasks through the filling tubes using a vacuum pump apparatus,

and an equivalent amount of 99.9% methane (Strate Welding, Jacksonville, FL) was added to achieve a methane:

air ratio of approximately 30:70. The cultures were incubated and shaken at 30 oC and 270 rpm. After

detecting sufficient visible turbidity (~7 days), transfers were prepared using a 10-20% inoculum and repeated

until a stable culture was detected (determined visually on solid medium). Once verified as stable, it was given

the name "GW 60, 13" in reference to the well number and depth of sampling.



Maintenance of Solid cultures


Both MM1 and GW 60,13' cultures were serially diluted and spread plated onto nutrient and NMS agar plates.

NMS plates were incubated under methane and air (30:70) using airtight dessicators (at 300 C), and nutrient

plates were stored in tupperware containers at 300 C, with routine swabbing of the containers with ethanol to

prevent fungal contamination.



Colony and Cellular Morphology Characterization


Characterization of colonies involved visual observations of colony size, overall shape, margin, elevation,

color, transmittance of light and any other noticeable details (i.e., changes in growth over time).

Cellular characterization methods involved Gram and methyl violet staining and scanning electron

microscopy techniques. Samples of both mixed cultures were prepared for scanning electron microscopy by

fixing with Trumps fixative (buffered 1% glutaraldyhyde, 4% formalin), postfixing with 4% oxmium tetroxide,

rinsing with ethanol solutions of increasing concentration, and mounting the dried samples by sputter coating with

a gold/palladium mixture. Prepared samples were then viewed on a Hitachi S-4000 scanning electron microscope.



Measurement of Growth and sMMO Activity


Growth curves, measured using side-armed flasks equipped with the rubber stopper design described

previously, were prepared using a UV/VIS spectrophotometer (Fischer Technical Cpy., Schaumburg, IL) at

a wavelength of 600 nm. sMMO assay involved incubation of suspended cultures with naphthalene

crystals. Suspensions expressing sMMO tested positive by the formation of a purple color upon addition of

tetra-azotized ortho-dianisidine.






Oxygen Uptake Measurements


The oxidation potential of MM1 and GW 60,13' against representative environmental contaminants was

measured using oxygen uptake methods. Rates were measured with an oxygen electrode (YSI Co., Yellow

Springs, OH, USA), mounted onto a 2.0-mL jacketed reaction vessel held at a constant temperature (30C) and

linked to an oxygen analyzer (YSI Co.. Yellow Springs, OH). Data were collected at 10 Hz and converted to

digital input by an A/D converter board (CIA-DAS08-PGL, Computer Boards, Inc, Mansfield, MA, USA), mounted in

an Hewlett Packard 486 computer equipped with Labtech Notebook software (Wilmington, MA, USA). Rates

were normalized to oxygen uptake rates in the presence of methane.




RESULTS



Increasing turbidity in the initial landfill sediment liquid cultures was visible after approximately 7 days of

incubation. Subsequent routine transfers of the stable consortium showed significant growth in approximately 3

days after inoculation. Growth on nutrient agar plates was rapid, with visible colony formation after 1 day.

Isolation of the GW 60,13' methanotroph(s) is still in progress; however, consistent growth appears on NMS plates

3-4 days after streaking.



Table 2 summarizes the characteristics of the GW 60,13' heterotrophic community. Upon streaking onto

nutrient agar, 12 different colonies were initially isolated; however, after repeated transfers onto fresh nutrient

agar plates, only 6 colonies appeared to be distinct from the rest. Whether this was due to the inability to

visually distinguish the colonies together on nutrient plates or to the loss of colonies upon repeated streaking is

not known. Table 3 describes the cellular and colony morphologies and the growth characteristics of these 6

stable colonies. The liquid mixed culture MM1 showed 4 heterotrophs on nutrient agar. Characteristics of

these colonies are summarized in Table 4.



Table 2

Colony and Cellular Characteristics of GW 60, 13 Heterotrophs

SHAPE COLONY AMOUNT
GRAM CELL COLONY SHAPE COLONY COLONY COLONY L AMOUNT CATALASE
COLONY OF TRANSMITTANCE OF
REACTION SHAPE PIGMENTATION MARGIN ELEVATION CONSISTENCY REACTION
COLONY TO LIGHT GROWTH

1 Cream, Smooth
B White/ cream Circular Convex Mucoid glossy Translucent 1+ +
Glossy Entire

B and Smooth
Tan - Tan/ dark orange Circular Convex Mucoid glossy Translucent 1+ +
S Entire

Cream Light orange/ Smooth
+ CB Circular Convex Mucoid glossy Translucent 2+ +
clump cream Entire






White 1 Smooth
B White/ cream Circular Convex Mucoid glossy Translucent 3+ +
clump Entire

Yellow -
Smooth
White 1 - B Light Yellow Circular? Convex Mucoid glossy Translucent 3+ +
Entire
clump

Rhizoidal
Orange -
+ B Electric Pink Circular or Umbonate Dry Opaque 3+ +
Pink*
irregular


Yellow
+ CB Pastel Yellow Circular Irregular Umbonate Dry Opaque 3+ +
Glob

C - It. Light orange/ Smooth
B Circular Convex Mucoid glossy Translucent 2+ +
Orange cream Entire

Smooth
A - beige - B White cream Circular Convex Mucoid glossy Translucent 2+ +
Entire

Smooth
Cloudy - CB White Cream Circular Raised Mucoid glossy Translucent 2+ +
Entire

1-tan/ Smooth
B Tan/ Dark Orange Circular Convex Mucoid glossy Translucent 2+ +
orange Entire

Smooth
2 - white - B White Cream Circular Convex Mucoid glossy Translucent 2+ +
Entire


1+ = growth on 1/3 of plate; 2+ =growth on 2/3 of plate; 3+ = growth on entire plateC= cocci CB= coccobacilli B= bacilli S= spirilla V= vibri



Table 3

GW 60,13' Heterotrophic Colonies

Number Colony Name Colony Color

1 Yellow-white 1 clump Light yellow

2 Yellow glob Pastel yellow

3 Orange/pink Electric pink

Tan
4 Tan/ dark orange
1-tan/orange

C - It. Orange
5 Light orange/ cream
Cream clump






Cloudy

White 1 clump

1 cream glossy

2 white

A-beige


Table 4

Colony and Cellular Characteristics of MM1 Heterotrophs


CULTURE GRAM CELL COLONY SHAPE COLONY COLONY COLONY COLONY
OF TRANSMITTANCE
NAME REACTION SHAPE PIGMENTATION OL MARGIN ELEVATION CONSISTENCY TOANSMIT
COLONY TO LIGHT


AMOUNT CATALASE
OF
O REACTION
GROWTH


Smooth
B White cream Circular
Entire

Smooth
B Cream Circular
Entire

Smooth
B Yellow Circular
Entire

Smooth
B Beige Circular
Entire


Convex Mucoid glossy Translucent


Convex

umbonate

Gumdrop

Irregular


Mucoid glossy Translucent


Translucent


Convex Mucoid glossy Translucent


2+= growth on 2/3 of plate; 3+= growth on entire plate



Scanning electron microscopy (SEM) photos (Figures 1A, B, C, and D) reveal the diversity of the mixed GW

60,13' culture as is expected of isolates from environments rich in substrates. The photos show a variety of

cell shapes, sizes, and appendages, including filamentous extensions and flagella. Figures 2A, B, C show

SEM photos of MM1. As is evident, this culture is less diversified than GW 60,13'. Cells in this culture were

typically bacilli and cocci in shape, most with smooth surfaces and convex in nature.


Figure id. SEM Photograph of GW60, 13' (8,000x).


White/ cream


4 - White

cream



3 - Cream




2 - yellow



1 - cream

beige


2+ +




3+ +




2+ +




2+ +


































Figure id. SEM Pnotograpn ot uwoo, 13 (11,uuox).


Figure id. SEM Photograph of GW60, 13' (18,000x).





Figure id. SEM Photograph of GW60, 13' (13,000x).


Figure 2a. SEM Photograph of Strain MM1 (2,000x).


Figure 2b. SEM Photograph of Strain MM1 (5,000x).
































Figure 2c. SEM Photograph of Strain MM1 (18,000x).


Figure 3 shows the growth curve obtained for the GW 60, 13' liquid culture grown under a 30:70 methane:air

ratio. The corresponding growth rate for GW 60, 13' under these conditions was calculated to be 0.342 d -1 with
a doubling time of 2 days. Both mixed cultures showed positive expression of the sMMO and were
tentatively characterized as Type II methanotrophs, capable of expression of pMMO or sMMO, depending on
the concentration of copper present in growth medium.


0.3
0.25
0.2
0.15
0.1
0.05
0


- = [1l/(t2-tl)][I nx2/xl]
~ -342 day



I I I^


0 10 20 30


40 50 60
Time (hr)


90 100


Figure 3. GW60, 13' Growth Curve.



Oxygen uptake rates were measured at varying concentrations of selected contaminants. Figures 4 and 5
show oxygen uptake curves for MM1 in the presence of biphenyl and GW60, 13' in the presence of
toluene, respectively. As is shown, oxidation activity was shown by each culture with possible inhibition occurring,





as evidenced by the maximum rate followed by a sharp rate decrease.


07



06



05


0.
I






0 3
C 04
�










o 01
z


0 75 150 225 300 375 40 525
Toluene Concentration, PM
Figure 4. Oxygen Uptake Curve for GW60, 13' with Varying Concentrations of Toluene.


07


06

DS /


04


03


02


01
u2 * I "----------------------------




tt8------------------------------------------


Concentrations of Biphenyl.


0 50 100 1S0 2 3IV 3MO
Biphonyl Concentratiou. PM
Figure 5. Oxygen Uptake Plot of Strain MM1 with Varying








DISCUSSION AND CONCLUSIONS


In this paper, we describe a new mixed methanotrophic-heterotrophic culture, tentatively referred to as GW60,

13'. Isolated thirteen feet below the surface of a landfill in the cover soil zone, this culture shows 6

stable heterotrophic populations and one or more methanotrophs. The difficulty in separating the methanotroph

from the heterotrophs is possibly due to the positive interactions that exist between the two types of bacteria

as hypothesized previously by researchers (12). Work is ongoing to elucidate the roles that the

individual methanotrophs play in these mixed cultures.



Upon comparison of the heterotrophic populations isolated from the landfill culture with those isolated from

an uncontaminated groundwater environment, a divergence in the complexity of the mixtures is evident. Whereas

the heterotrophs isolated from GW60, 13' showed a wide variety of cellular and colony characteristics (as shown

in Table 3), those isolated from the groundwater environment were fewer in number (only four) and more similar

in cellular and colony size and shape. This decrease in complexity of populations is expected for cultures derived

from relatively pristine environments in comparison to those derived from environments rich in organic carbon.

Both mixed cultures tested positive for the expression of sMMO, and, therefore, their potential for oxidizing a

broad range of substrates, including aliphatic and aromatic compounds, is high. Initial screening of

the biodegradative activity of these mixed cultures by oxygen uptake experiments demonstrated that the cultures

are capable of transforming aromatic compounds, such as toluene and biphenyl, with possible inhibition

indicating that either the substrate or intermediates formed may be inhibitory to successful bioremediation.



Future work will address more thorough characterization methods for the isolated landfill mixed and pure

cultures, including genetic analysis. Also the optimum conditions (pH, temperature, concentrations of

nutrients, oxygen, and methane) for growth and contaminant degradation will be assessed. Ultimately, we expect

our research to provide a better understanding of the community structure and interactions of

microorganisms present in landfill environments. This knowledge will then be applied to MSW sites as a

possible method for controlling the landfill environment to effect maximum natural bioattenuation of

hazardous chemicals.






ACKNOWLEDGEMENTS


We thank Scott Whitaker for his time and patience in preparing samples for SEM viewing in the Electron

Microscopy Core Laboratory (UF campus). We also thank Strate Welding for supplying methane and acetylene tanks.


REFERENCES








1. "Strategic Assessment of Florida's Environment: Florida Population Growth". Internet. December 5,

1999. www.fsu.edu/~cpm/safe/safepopu.html.

2. U.S. EPA. 1994. Characterization of Municipal Solid Waste in the United States. EPA 530-94-042.

3. Barlaz, M.A. 1997.Microbial Studies of Landfills and Anaerobic Refuse Decomposition. Chapter 60: 541-557.

From Manual of Environmental Microbiology. ASM press, Washington D.C. (eds.) Christon J. Hurst, Guy

Knudsen, Michael McInerney, Linda Stetzenbach, and Michael Walters.

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9. Alvarez-Cohen L., P.L. McCarty, E. Boulygina, R.S. Hanson, G.A. Brusseau, and H.C. Tsien.

1992. Characterization of a Methane-Utilizing Bacterium from a Bacterial Consortium That Rapidly

Degrades Trichloroethylene and Chloroform. Appl. Environ.Microbiol. 58: 1886-1893.

10. Uchiyama H., T. Nakajima, 0. Yagi and T. Tabuchi. 1989. Aerobic Degradation of Trichloroethylene by a

New Type II Methane- Utilizing Bacterium, Strain M. Agric. Biol. Chem. 53: 2903-2907.

11. Whittenbury R., K.C. Phillips and J.F. Wilkinson. 1970. Enrichment, Isolation and Some Properties of

Methane-Utilizing Bacteria. J. of Gen. Microbiol. 61: 205-218.

12. Hrsak, D. and A. Begonja. (1998) Growth Characteristics and Metabolic Activities of a

Methanotrophic-Heterotrophic Groundwater Community. J. of Appl. Microbiol. 85:448-456.





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