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Isolation of butyrate-utilizing bacteria from thermophilic and mesophilic methane-producing ecosystems

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Isolation of butyrate-utilizing bacteria from thermophilic and mesophilic methane-producing ecosystems
Added title page title:
Butyrate-utilizing bacteria
Added title page title:
Thermophilic and mesophilic methane-producing ecosystems
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Mesophilic methane-producing ecosystems
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Henson, John Michael, 1952-
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vi, 63 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Acetates ( jstor )
Bacteria ( jstor )
Butyrates ( jstor )
Coculture techniques ( jstor )
Ecosystems ( jstor )
Hydrogen ( jstor )
Methane ( jstor )
Methane production ( jstor )
Methanosarcina ( jstor )
Vapor phases ( jstor )
Dissertations, Academic -- Microbiology and Cell Science -- UF
Gas producers ( lcsh )
Methane ( lcsh )
Microbiology and Cell Science thesis Ph. D
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1983.
Bibliography:
Bibliography: leaves 60-62.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by John Michael Henson.

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ISOLATION OF BUTYRATE-UTILIZING BACTERIA FROM THERMOPHILIC AND
MESOPHILIC METHANE-PRODUCING ECOSYSTEMS











BY

JOHN MICHAEL HENSON
















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

1983






























To Ellen















ACKNOWLEDGMENTS


I would like to express sincere gratitude to Dr. Paul Smith,

chairman of my supervisory committee, for the time, space, and, most

of all, his patience which enabled me to learn that "The experiment

is everything." I also thank Dr. Arnold Bleiweis and Dr. Roger

Nordstedt for their contributions as members of my supervisory

committee.

In addition, I thank Dr. Dave Boone for helpful discussions about

the isolation of cocultures, Dr. K. Shanmugam for helpful discussions

about hydrogenase, and Butch Bordeaux for technical assistance and

advice.
























iii















TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . iii

ABSTRACT . . . . . . . . . . . . . . . . . . . . .... .... v

INTRODUCTION . . . . . . . . . . . . . . . . . . . . .... . ... 1

MATERIALS AND METHODS . . . . . . . . . . . . . . . .. . . . .14

Organisms, Media, and Growth Conditions . . . . . . . . . .. 14
Preparation of Butyrate Medium . . . . . . . . . . . . . . .. 16
Anaerobic Techniques . . . . . . . . . . . . . . . . . . . . . 16
Descriptions of Ecosystems Studied . . . . . . . . . . . . .. 17
Descriptions of Butyrate Enrichments . . . . . . . . . . . .. 18
Thermophilic Coculture Isolation . . . . . . . . . . . . . . . 18
Mesophilic Coculture Isolation . . . . . . . . . . . . . . .. 19
Gas Chromatography Methods . . . . . . . . . . . . . . .... .19
Preparation and Use of E. coli Membrane Fragments To Attempt
Isolation of Pure Cultures of Hydrogenogenic Bacteria . . . . 20
Microscopy and Photomicroscopy . . . . . .. . . ...... .21

RESULTS . . . . . . . . . . . . . . . . ... . . . . . . . . . . . 22

Production of Methane When Various Ecosystems Were Enriched
with Butyrate . . . . . . . . . . . . . . . . . . . . . . . . 22
Description of Thermophilic Butyrate Enrichments . . . . . .. 22
Isolation of Thermophilic Butyrate-Utilizing Cocultures . . . 26
Studies on the Thermophilic Coculture . ........... .32
Effects of Pumping Butyrate into a Thermophilic Digester . . . 41
Enrichments from Mesophilic Ecosystems . . . . . . . . .... .48
Isolation of Mesophilic Butyrate-Utilizing Cocultures . . .. 49
Attempts To Isolate Butyrate-Utilizing Hydrogenogens in Pure
Culture with E. Coli Membrane Fragments . . . . . . . . . .. 52

DISCUSSION . . . . . . . . . . . . . . . . ... . ... .. . . .53

REFERENCES . . . . . . . . . . . . . . . . ... . ..... . . .60

BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . .... ..... ..63




iv














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


ISOLATION OF BUTYRATE-UTILIZING BACTERIA FROM THERMOPHILIC AND
MESOPHILIC METHANE-PRODUCING ECOSYSTEMS


By

John Michael Henson

December 1983

Chairman: Dr. Paul H. Smith
Major Department: Microbiology and Cell Science

The ability of various ecosystems to convert butyrate to methane

was studied in order to isolate the bacteria responsible for the conver-

sion. When thermophilic digester sludge was enriched with butyrate,

methane was produced without a lag period. Marine sediments enriched

with butyrate required a 2-week incubation period before methanogenesis

began. When hypersaline sediments were enriched with butyrate, methane

was not produced after 3 months. A thermophilic digester was studied

in more detail and found by most-probable-number enumeration to have

ca. 5 x 106 butyrate-utilizing bacteria/ml of sludge. A thermophilic

butyrate-utilizing bacterium was isolated in coculture with

Methanobacterium thermoautotrophicum and a Methanosarcina sp. This

bacterium was a gram-negative, slightly curved rod that occurred singly,

was nonmotile, and did not appear to produce spores. When this

coculture was incubated with Methanospirillum hungatei at 370C, the

quantity of methane produced was less than 5% of that produced when the


v










coculture was incubated at 550C, the routine incubation temperature.

The coculture required clarified digester fluid (CDF), which could not

be replaced by rumen fluid (RF). The addition of yeast extract to a

medium containing 5% CDF stimulated methane production when the

Methanosarcina sp. was present. Hydrogen in the gas phase prevented

butyrate utilization. However, when the hydrogen was removed, butyrate

utilization began. Penicillin G and D-cycloserine caused the complete

inhibition of butyrate utilization by the coculture.

The thermophilic digester was infused with butyrate at the rate

of 10 pmoles/ml of sludge per day. Biogas production increased by 150%,

with the percentage of methane increasing from 58% to 68%. Acetate,

propionate, and butyrate did not accumulate. When the infusion rate was

increased to 20 pmoles/ml of sludge per day, an unstable digestion

resulted.

Butyrate-utilizing enrichments from mesophilic ecosystems were

used in obtaining cocultures of butyrate-utilizing bacteria. These

cocultures served as inocula for attempts to isolate pure cultures of

butyrate-utilizing bacteria by use of hydrogenase-containing membrane

fragments of Escherchia coli. After a 3-week incubation period,

colonies appeared onlyin inoculated tubes that contained membrane frag-

ments and butyrate.









vi














INTRODUCTION


In anaerobic ecosystems where light, sulfate, and nitrate are

absent, such as digesters and freshwater sediments, organic matter is

degraded exclusively to methane and carbon dioxide. This degradation

is mediated by several groups of bacteria and is regulated by the hydro-

gen concentration (21,29). Hydrogen does not accumulate and is diffi-

cult to detect in methane-producing ecosystems because of rapid inter-

species hydrogen transfer. Interspecies hydrogen transfer is the

utilization of hydrogen by one bacterial species (hydrogenotroph) that

is produced by another bacterial species (hydrogenogen) (33,34). This

phrase was introduced by lannotti et al. (15) when they compared the

fermentation products formed by a pure culture of Ruminococcus albus

with the fermentation products formed when this organism was grown with

a hydrogenotroph. R. albus is a carbohydrate-fermenting organism that

produces acetate, ethanol, and hydrogen when it is grown in continuous

culture with glucose as the sole carbon and energy source. However,

when R. albus is grown in a mixed continuous culture with Vibrio

succinogenes, a hydrogenotroph, the only products detected are acetate

and succinate. Succinate is formed when V. succinogenes uses the

hydrogen produced by R. albus to reduce fumarate which R. albus alone

cannot use as an electron acceptor. Thus, because of the interspecies

transfer of hydrogen from R. albus to V. succinogenes, there is a shift




1






2



in fermentation products from the less reduced ethanol to the more

reduced acetate and hydrogen.

Selenomonas ruminantium is a carbohydrate-fermenting organism that

when grown in pure culture produces trace amounts of hydrogen (27).

Hydrogen production, however, as indicated by the amount of methane

formed, is stimulated almost 100-fold when cocultured with methanogenic

bacteria (27). Subsequent analysis (9) shows that the fermentation

products formed from glucose are altered by the presence of hydrogen-

utilizing methanogens. The electron sink fermentation products, lactate

and propionate, are produced in decreased amounts, whereas acetate and

hydrogen formation increases. It has been suggested (9) that the pro-

duction of hydrogen results from reduced nicotinamide adenine dinucleo-

tide (NADH); thus hydrogen becomes a major electron sink product and

alters the fermentation products formed.

Similar results are observed when cellulose is fermented by

Ruminococcus flavefacians (16). In the presence of Methanobacterium

ruminantium, a hydrogenotropic methanogen, major fermentation products

shift from succinate and acetate to acetate and hydrogen, which is

evidenced by the large amounts of methane formed.

These studies confirm Hungate's hypothesis (13) that methanogenesis

in the rumen provides for the removal of electrons from pyruvate via

the formation of hydrogen. This hypothesis arose from observations

that, when many rumen bacteria are grown in pure culture, a variety of

products are found that are not found in the rumen. These products are

electron sink products that result from the oxidation of NADH. The

removal of electrons released by catabolism via hydrogen formation






3



results in the net removal of electrons from the rumen because the

methanogenic bacteria maintain a low partial pressure of hydrogen by

using it to reduce carbon dioxide to methane. The methane then leaves

the rumen when the ruminent eructates.

All of the fermentative bacteria described above, as well as

other fermentative bacteria, are able to produce alternative electron

sink products in place of hydrogen when the partial pressure (pH2) of

hydrogen is not maintained sufficiently low for oxidation of NADH.

Table 1 illustrates the required lowered pH2 to oxidize NADH. Forma-

tion of H2 from NADH, when products and reactants are equimolar, will

not occur as indicated by the positive AGo' for the reaction. As the

concentration of H2 approaches zero, the AGO' for the reaction will be-

come negative. If all the electrons carried by NADH are ultimately

used to form CH4 via H2, as in the summation reactions in Table 1, the

overall reaction has a AGO' of -63.6 kJ. Thus, alternate electron

sink products are not formed.

There are, however, other hydrogen-producing bacteria whose growth

is dependent upon the constant removal of the hydrogen produced by the

oxidation of NADH. Being unable to produce alternate electron sink

products, these bacteria are obligate hydrogenogens. They are involved

in the utilization of ethanol (8), propionate (5), butyrate (22,23),

other fatty acids (23), benzoate (11,26), and possibly other substrates.

Methanobacillus omelianskii has been described by Barker (4) as

being able to convert ethanol and carbon dioxide to acetate and

methane. In 1967, Bryant et al. (8) were able to separate cultures of

M. omelianskii into two bacterial species. Their studies reveal






4



Table 1. Oxidation of reduced nicotinamide adenine dinucleotide (NADH)
by removal of H2 via methane formation



AGo,
Reaction (kJ/reaction)a



1. NADH + H+ - H2 + NAD+ +18.0

2. 4H2 + HCO3 + H+ - CH4 + 3H20 -135.6

Summation:
4NADH + 4H+ - 4H2 + 4NAD+ +72.0

4H2 + HCO3 + H + - CH4 + 3H20 -135.6

4NADH + HCO3 + 5H+ 4NAD+ + CH4 + 3H20 -63.6


avalues taken from or calculated from data in Thauer et al. (31).




5



that the presence of the two separate bacteria are necessary for the

conversion to occur. One of the two bacteria, the S organism, oxidizes

ethanol with the production of acetate and H2, and will not grow alone.

The second species is a methanogenic bacterium which utilizes H2 but

not ethanol for growth and methane production. Bryant et al.(8) also

proposed that fatty acids other than formate and acetate are anaerobi-

cally degraded by nonmethanogenic bacteria similar to the S organism

with the production of H2.

As is the case for the oxidation of NADH described above, the

pH2 must be maintained at a low level for the oxidation of ethanol to

occur in the coculture just described. Shown in Table 2 are the equa-

tions and free-energy changes for the oxidations of ethanol as well as

propionate and butyrate. At equimolar concentrations of products and

reactants, propionate has the highest AG0': +76.1 kJ/reaction.

Butyrate is intermediate with a +48.1 kJ/reaction, whereas ethanol is

the lowest with a +19.2 kJ/reaction. When the concentrations of pro-

ducts are lowered, as by the removal of H2, the reactions have a nega-

tive AG0' and, hence, become thermodynamically possible. The most diffi-

cult reaction to make possible via H2 removal is the oxidation of pro-

pionate; the reaction is less difficult with butyrate and least diffi-

cult with ethanol. The pH2 must be 10-, 10-, and 10- atm., respec-

tively (21). The separation of Methanobacillus omelianskii cultures

into two components (8) was an important contribution to the under-

standing of microbial interactions in the fermentation of organic matter

to methane because it gave initial insight into the physiological

dependence of one group of bacteria on another group of bacteria.






6



Table 2. Equations and free-energy changes for oxidation of ethanol,
propionate, and butyrate



RAGo
Reaction (kJ/reaction)a



1. CH3CH20H + 2H20 - 2CH3COO + 4H2 + 2H+ +19.2


2. CH3CH2COO + 3H20 CH3COO" + HCO3 + H + 3H2 +76.1

3. CH3CH2CH2COO + 2H20 + 2CH3COO + 2H2 + H +48.1


avalues taken from or calculated from data in Thauer et al. (31).





7



Smith's report (29) summarized the results obtained when domestic

sludge was enriched with propionate or butyrate. When these enrichments

were rapidly sparged, H2 was detected, indicating the presence of a

physiological group of bacteria that produces H2 from propionate or

butyrate. In nonsparged enrichments, H2 was not detected because of the

interspecies transfer of H2 to the methanogens and the subsequent produc-

tion of CH4. Both enrichments utilized H2-CO2 without a lag, indicating

the existence of a population of hydrogenotrophs. Propionate or buty-

rate utilization was inhibited by the presence of H2, indicating that

removal of H2 must take place in order for utilization to occur.

The first report of a fatty acid-utilizing bacterium is that of

an anaerobic bacterium that utilizes butyrate as well as other fatty

acids (23). This organism, which has subsequently been named Syn-

trophomonas wolfei (22), B-oxidizes saturated fatty acids (butyrate

through octanoate) to acetate and H2 or to acetate, propionate, and H2.

S. wolfei is a gram-negative helical rod with laterally inserted flagella

and a sluggish twitching motility (23). When S. wolfei is cocultured

with a Desulfovibrio sp., a generation time of 54 hours is obtained,

which is less than the generation time of 87 hours when it is cocultured

with M. hungatei. Compounds tested and found not to act as electron

acceptors are dimethyl sulfoxide, fumarate, malate, nitrate, oxygen,

sulfate, sulfite, sulfur, and thiosulfate (23). In addition, manganese

oxide, methyl viologen, palladium chloride, phenosafranin, tetrazolium

chloride, and trimethylamine-N-oxide are not utilized as electron accep-

tors when butyrate is the electron donor (22). The presence of 80% H2

in the gas phase inhibits growth and butyrate utilization when S. wolfeiis






8



cocultured with Mi. hungatei (22). S. wolfei did not grow alone when the

gas phase was continually recycled through hot copper oxide filings to

remove H2 (22). Cells contained peptidoglycan and poly-g-hydroxybutyrate

(22). A morphologically similar bacterium was isolated from rumen

fluid (24).

Syntrophobacter wolinii is a nonmotile, gram-negative rod that

degrades propionate,but not other fatty acids, to acetate and, presumably,

H2 and CO2 (or formate) only in the presence of a hydrogenotrophic bac-

terium (5). When S. wolinii is cocultured with a Desulfovibrio sp., the

hydrogenotroph, the doubling time is about 87 hours. The doubling time

increases to 161 hours when the Desulfovibrio sp. is present as a minor

component of the coculture and Methanospirillum hungatei is the major

hydrogenotrophic organism. S. wolinii is a strict anaerobe.

Another example of an obligate hydrogenogenic bacterium is one

that does not utilize fatty acids. Benzoate is degraded to acetate and,

presumably, CO2 and H2 (or formate) by a gram-negative,motile, rod-shaped

bacterium in coculture with the hydrogenotrophic Desulfovibrio sp.(26).

The benzoate utilizer does not use other common aromatic compounds,

C3-C7 monocarboxylic acids, or C4-C6 dicarboxylic acids for growth. It

is unable to use nitrate, sulfate, or fumarate as alternate electron

acceptors. In coculture with the Desulfovibrio sp., the generation time

is 132 hours, whereas in coculture with M. hungatei, the generation time

is 166 hours. The presence of 80% H2 in the gas phase inhibits utiliza-

tion of benzoate.

Descriptions of the two species of fatty acid-utilizing, hydro-

genogenic bacteria are the only information available concerning the






9



bacteria involved in the degradation of fatty acids in the fermentation

of organic matter to methane. There are at least three major groups of

bacteria involved in the fermentation of the polymeric substrates,

cellulose, starch, protein, lipids, etc., to methane (6,18,21,29). The

methane fermentation is divided into three distinct stages (6,21,29),

as shown in Figure 1. Fermentative bacteria are the first of the three

major groups to react. This group is a complex mixture of many obligate

and facultative anaerobic bacterial species (12,21) which act on the

polymeric substrates. Polysaccharides are initially hydrolyzed to their

component sugars, which are then transported into the fermentative bac-

teria. The sugars are fermented primarily by the Embden-Meyerhof-Parnas

pathway with the production of a variety of electron sink products such

as acetate, propionate, and butyrate (6), as is shown in Figure 1. Pro-

teins are hydrolyzed to amino acids which are then fermented to the

products shown in Figure 1, in addition to other products such as

isobutyrate, phenylacetate, and phenylpropionate (6). Lipids are

hydrolyzed to glycerol, a fermentable substrate, and long-chain fatty

acids which are not fermented by the fermentative bacteria (6). The

fermentative bacteria have been studied in detail, but additional re-

search is needed.

The terminal group of bacteria, the methanogens, is generally

understood although not as well as the fermentative bacteria. These

bacteria are essential to the complete anaerobic degradation of organic

matter. Otherwise, organic acids that contain about as much energy as

the polymeric substrates would accumulate and anaerobic degradation

would cease (6,21). As shown in Figure 1, these unique bacteria utilize
























Figure 1. Schematic representation of the three stages of anaerobic degradation of organic matter to
methane.







Polymeric Organic Substrates





Propionate ydrogen
Acetate- and and
Butyrate Carbon Dioxide





METHANE






12



a limited number of substrates, mainly acetate and H2-CO2, to produce

methane, which contains 90% of the energy of the polymeric substrates

(6,21).

The third group of bacteria is the least understood of the three

groups. These bacteria, the hydrogenogens, utilize the fermentation

products of the first group and produce substrates that are utilizable

by the methanogenic group. In Figure 1, this reaction is shown by the

degradation of propionate and butyrate to acetate and H2-CO2. The primary

reason for the lack of information concerning these bacteria is the

difficulty in isolating and manipulating them. Existing studies are of

cocultures because the bacteria have not been isolated in an axenic

culture.

The hydrogenogens are obligate proton-reducing bacteria and pro-

duce H2 with the concomitant oxidation of NADH (21). Therefore, the

enzyme hydrogenase may be useful in the isolation of axenic cutlures of

hydrogenogens. Hydrogenase is the collective term for enzymes which

catalyze the reversible reaction (1,28):


H2 -- 2H+ + 2e


Thus, a cell-free extract containing hydrogenase, and other components

necessary to transfer electrons from H2 to an electron acceptor, may

make possible the isolation of axenic cultures of hydrogenogenic bac-

teria.

The utilization of fatty acids may be the rate-restricting step

in the fermentation of organic matter to methane (19,21). The bacteria

responsible for these reactions have not been well studied. Therefore,






13



to better exploit the production of methane from biomass, these bacteria

must be better understood. It is the objective of this study to increase

the understanding of the fatty acid-utilizing bacteria by attempting to

isolate cocultures of new species and to attempt the isolation of pure

cultures of these bacteria. The knowledge gained, it is hoped, may be

used to bring about the more efficient degradation of biomass to methane.














MATERIALS AND METHODS

Organisms, Media, and Growth Conditions

Methanobacterium thermoautotrophicum strain AH, Methanospirillum

hungatei strain JF-1, and Escherichia coli ATCC 11303 were obtained from

our culture collection. Desulfovibrio sp. strain G-11 was a gift grate-

fully received from Dr. Dave Boone.

Mb. thermoautotrophicum was incubated at 550C in medium number 2

of Balch et al. (3). Ms. hungatei was incubated at 370C in medium

number 1 of Balch et al. (3). To prepare these media, stock solutions

of trace minerals, trace vitamins, and various salt solutions were pre-

pared and frozen until used. The stock solution of trace minerals con-

tained in grams per 100 ml of distilled water, the following: nitrilo-

triacetic acid, 1.5 (to pH 7.0 with 4M KOH); MgSO4*7H20, 3.0; MnSO4'2H20,

0.5; NaCl, 1.0; FeSO4-7H20, 0.1; CaCl2, 0.12; CaC12.2H20, 0.1; ZnSO4*

7H2), 0.18; CuSO4.5H20, 0.01; A1K(SO4).12H20, 0.02; H3803, 0.01; NaMoO4
2H20, 0.11; and NiC12*6H20, 0.24. The stock solutions of trace vitamins

contained, in milligrams per liter of distilled water, the following:

biotin, 2; folic acid, 2; pyridoxine hydrochloride, 10; thiamine

hydrochloride, 5; riboflavin, 5; nicotinic acid, 5; DL-calcium pentothe-

nate, 5; vitamin B12, 0.1; p-aminobenzoic acid, 5; lipoic acid, 5; and

2-mercaptoethanesulfonic acid, 100. The salts stock solution contained,

in grams per 100 ml distilled water, the following: (NH4)2S04, 3.0;

NaC1, 6.0; MgSO4*7H20, 1.3; CaC12, 0.06; and KH2PO4, 3.0. The phosphate


14






15



stock solution contained 3.0 g K2HPO4 per 10 ml distilled water. The

iron stock solution contained 0.02 g Fe(SO4)2.7H20 per 10 ml distilled

water. The resazurin stock solution contained 0.5 mg of resazurin per

ml of distilled water.

The medium for Mb. thermoautotrophicum was composed of the follow-

ing: trace minerals solution, 1 ml; salts solution, 10 ml; phosphate

solution, 1 ml; (NH4)2SO4, 2.7 g; resazurin solution, 1 ml; trace

vitamins solution, 10 ml; iron solution, 1 ml; NaHCO3, 5.0 g; cysteine

hydrochloride-H20, 0.5 g; and distilled water to 1000 ml. The final gas

phase was 80% H2-20% Co2. The pH was 7.2.

The medium for Ms. hungatei was composed of the following: trace

minerals stock solution, 1 ml; salts solution, 10 ml; phosphate solution,

1 ml; sodium acetate, 2.5 g; sodium formate, 2.5 g; yeast extract (Difco),

2.0 g; Trypticase (BBL), 2.0 g; resazurin solution, 1 ml; trace vitamins

solution, 10 ml; iron solution, 1 ml; NaHCO3, 5.0 g; cysteine hydro-

chloride-H20, 0.5 g; and distilled water to 1000 ml. The final gas

phase was 80% H2-20% CO2. The pH was 7.0.

The medium for Desulfovibrio sp. strain G-11 was composed of the

following: trace minerals solution, 1 ml; salts solution, 10 ml;

phosphate solution, 1 ml; sodium acetate-3H20, 0.68 g; sodium sulfate,

2.84 g; sodium formate, 2.0 g; Trypicase (BBL), 2.0 g; resazurin solu-

tion, 1 ml; trace vitamins solution, 10 ml; iron solution, 1 ml;

NaHCO3, 5.0 g; cysteine hydrochloride*H20, 0.5g; and distilled water

to 1000 ml. The final gas phase was 80% H2-20% CO2. The pH was 7.0.

Escherichia coli was incubated statically at 370C in 200 ml of

the medium in a stoppered 500-ml serum bottle. The medium for E. coli






16



was Luria broth, which was composed of yeast extract, 5 g; Bacto-

tryptone (Difco), 10 g; NaC1, 5 g; glucose, 1 g; and distilled water

to 1000 ml. The final gas phase was N2. The pH was 6.8.

Agar Noble (Difco) was added, final concentration 1.5%, when a

solid medium was required. All of the media were sterilized by auto-

claving for 20 minutes at 1210C.


Preparation of Butyrate Medium

The control medium for butyrate enrichments and butyrate studies

was composed of the following: trace minerals solution, 1 ml; salts

solution, 10 ml; phosphate solution, 1 ml; resazurin solution, 1 ml;

clarified digester fluid, 50 ml; NaHCO3, 5.0 g; and cysteine hydro-

chloride-H20, 0.5 g. The gas phase was 80% N2-20%C02. The pH was 7.2

to 7.4. After the control medium was dispensed to several serum tubes,

sodium n-butyrate (Pfaltz and Bauer, Inc., Stamford, Conn.) was added

to the remaining control medium to yield a final concentration of 0.3%.

This medium was then dispensed, stoppered, and autoclaved for 20 minutes

at 121C .


Anaerobic Techniques

Principles of anaerobic techniques, as described by Hungate (14),

were utilized to prepare the media-except Luria broth-and during ex-

perimental procedures. To prepare the media, all components, except

trace vitamins, NaHCO3, cysteine hydrochloride-H20, clarified digester

fluid, and butyrate, were added to distilled water in a round-bottom

flask, boiled for several minutes, and cooled to room temperature in an

ice bath while being sparged with 80% N2-20% CO2. When the components






17



were cool, the remainder of the components were added in accordance to

the medium being prepared. After the pH was adjusted, if necessary,

the media were dispensed into serum tubes (Bellco Glass Co., Vineland,

N.J.) or serum bottles (Wheaton Scientific, Millville, N.J.) in which

the gas phase had been replaced with 80% H2-20% CO2 or 80% N2-20% CO2.

The serum tubes or serum bottles were closed with butyl rubber stoppers

(Bellco No. 2048-11800) which were held in place by crimped aluminum

seals (Wheaton No. 224193).

Gas mixtures were purchased from Matheson (Morrow, Ga.) and trace

oxygen was removed by passing these gases over heated (350�C) copper

turnings.

Transfers of cultures were made with sterile needle and syringe

units that had been made anoxic by aspirating sterile reduced medium or

sterile anoxic gas.

Prior to the use of all media, except Luria broth, a volume of

1.25% Na2S-9H20 was injected into the media so that the sodium sulfide

was diluted 1:50.

Luria broth was prepared and sparged with N2 gas before being

stoppered and sealed into 500 ml serum bottles (Wheaton).


Descriptions of Ecosystems Studied

Thermophilic and mesophilic digesters served as sources of inoculum

for various studies. The digesters were similar in design and opera-

tion with the exception of source of initial inoculum and incubation

temperature. The thermophilic digester was maintained at 550C whereas

the mesophilic digester was maintained at 40�C. The digesters were con-

structed from aspirator bottles and were stirred semicontinuously. Each











day they received 16 g of feed consisting of 75% bermuda grass and 25%

Universal cattle feed (Seminole Brands). The hydraulic detention times

were 20 days.

Freshwater sediment samples were taken at Bivens Arm, a eutrophic

lake located near the University of Florida. Marine sediment samples

were taken from Halodule sp.and Thalassia sp. seagrass beds at Seahorse

Key, located near Cedar Key, Florida. Hypersaline sediment samples

were taken from Great Salt Lake, Utah, and salterns, from San Francisco

Bay, California.

Descriptions of Butyrate Enrichments

Enrichments from thermophilic and mesophilic digesters were begun

by placing sludge from those digesters into butyrate medium. Enrich-

ments from Bivens Arm were initiated by placing sediments into butyrate

medium. Enrichments with marine sediments were begun by using sulfate-

free artificial seawater (SF-ASW) instead of distilled water as given in

the description for butyrate medium. The SF-ASW was composed, in grams

per liter of distilled water, of the following: NaC1, 21.15; MgC12-

6H20, 9.65; CaCl2, 1.0; NH4C1, 0.25; KC1, 0.5; KBr, 0.086; SrC12*6H20,

0.022; and H3BO3, 0.023. Enrichments with hypersaline sediments were

begun by adding sodium n-butyrate, final concentration 0.3%, to the

hypersaline sediments.

Thermophilic Coculture Isolation

A stable thermophilic enrichment was used as a source of inoculum

for coculture isolation attempts. The enrichment was serially diluted

in the control medium, and roll tubes were prepared from these dilutions.






19



Each dilution was a source of inoculum for two butyrate control

(BC) roll tubes, as well as for three butyrate experimental (BE) roll

tubes which contained 0.3% butyrate. Before the tubes were rolled out,

they received 0.5 ml of turbid, active Mb. thermoautotrophicum, which

served as the hydrogenotroph. The roll tubes were incubated at 550C.


Mesophilic Coculture Isolation

A stable mesophilic enrichment was used as a source of inoculum

for coculture isolation attempts. The enrichment was serially diluted

as described for thermophilic coculture isolation attempts. Also, dupli-

cate BC and triplicate BE roll tubes were prepared at the appropriate

dilutions. Desulfovibrio sp. strain G-11, 0.5 ml of active culture per

roll tube, was used as the hydrogenotroph. These roll tubes were in-

cubated at 370C.


Gas Chromatography Methods

Methane concentrations were measured by use of a Hewlett-Packard

model 5880A gas chromatograph. Gases were separated in a 1.8-m by 1.0-

mm stainless steel column packed with Carbosphere mesh 80/100 (Alltech

Associates, Inc., Deerfield, Ill.), and were measured with a thermal

conductivity detector. Helium was the carrier gas. Column and detec-

tor temperatures were maintained at 130 and 145�C, respectively. Methane

concentrations were determined by comparison to standards (Ultra High

Purity Methane, Matheson). Gas pressures in stoppered vessels were de-

termined by use of a pressure transducer (Setra System, Inc., Acton, Mass.).

Volatile fatty acids (VFAs) were measured by use of a Hewlett-

Packard 5880A gas chromatograph. They were separated in a 1.8-m by






20



1.0-mm glass column packed with 8% SP1000, 2% SP1200, and 1.5% H3PO4 on

80/100 mesh Chromosorb W AW 8100 (Supelco, Bellefonte, Pa.), and were

measured with a flame ionization detector. Helium was the carrier gas.

Injector, oven, and detector temperatures were 145, 130, and 1750C,

respectively. Samples were mixed with an equal volume of 4% o-phosphoric

acid, centrifuged at 12,800 x g for 2 minute (22�C), and the super-

natant was frozen until VFA determinations were made. Each VFA concen-

tration was determined by comparison to standards.


Preparation and Use of E. coli Membrane Fragments To Attempt
Isolation of Pure Cultures of Hydrogenogenic Bacteria

Statically grown E. coli were centrifuged at 5,000 x g for 10

minutes (5�C) and washed twice with buffer. The buffer was composed of

the following: NaCl, 0.4M; MgSO4.7H20, 0.02M; and KH2PO4, 0.1 M. The

pH was 7.0. Cells and buffer were prechilled to 50C. Membrane frag-

ments were prepared by passing the cell suspension twice through a

French pressure cell at 20,000 psi. The lysate was centrifuged at 5,000

x g for 10 minutes (5�C), and the supernatant was frozen at -200C until

used.

The lysate was sterilized by being passed through a sterile 0.2-um

membrane filter. Fumarate, final concentration 20 mM, was filter

sterilized (Falcon 7103) and aseptically added to the BC or BE medium.

The source of inoculum was the stable mesophilic butyrate enrichment.

Anaerobic roll tubes were prepared so that they contained lysate and

fumarate, lysate or fumarate, or no addition. The roll tubes were in-

cubated at 370C.






21



Microscopy and Photomicroscopy

A Carl Zeiss Standard WL microscope equipped for epifluorescence

was used for observation of wet mounts and photomicroscopy. Light of

the 420-nm wavelength was provided by a mercury light source (HBO 50

DC 3) and a filter set comprised of an exciter filter (BP 390-440), a

chromatic beam splitter (FT 460), and a barrier filter (LP 475). A

Leica camera back was attached to the microscope for photomicroscopy.

Kodak Technical Pan film 2415 was exposed for times in accordance to

previously exposed test rolls. The film was developed according to

Kodak instructions and printed on Kodak F5 RC or Polycontrast RC paper.















RESULTS


Production of Methane When Various Ecosystems
Were Enriched with Butyrate

When various ecosystems were enriched with butyrate, not all pro-

duced methane (Table 3). Anaerobic digesters and freshwater sediments

produced methane with little or no lag, whereas marine sediments in

sulfate-free artificial seawater required about 2 weeks for methane

production to begin. Methane was not produced after several months of

incubation when hypersaline sediments were enriched with butyrate.


Description of Thermophilic Butyrate Enrichments

The population of butyrate-utilizing bacteria in a 550C digester

was enumerated by the 5-tube most-probable-number (MPN) method. After

a 4-week incubation period, 4.5 x 106 butyrate-utilizing bacteria per

ml sludge were found. The lower dilution MPN tubes produced signifi-

cantly more methane than did the higher dilution MPN tubes. Examina-

tion revealed that the lower dilution MPN tubes contained a Methanosar-

cina sp. Butyrate enrichments were established by use of each of these

distinct dilution types as an inoculum. The greater methane production

by the enrichment containing the Nethanosarcina sp. is shown in Figure 2.

Acetate accumulated in the enrichment without the Methanosarcina sp.

but disappeared in the enrichment with the Methanosarcina sp. Butyrate

was utilized by both enrichments. The Methanosarcina sp. was isolated

and would not grow alone when H2-CO2 was the only methanogenic substrate


22






23



Table 3. Examination of various ecosystems for methane production from
butyrate enrichments


Lag period before
Source of enrichment Methane produceda onset of methane
production


Thermophilic digester Yes None

Mesophilic digester Yes None

Bivens Arm Yes 3 days

Halodule, sp. seagrass bed Yes 14 days

Thalassia, sp. seagrass bed Yes 14 days

Great Salt Lake No

San Franscisco Bay saltern No


alndicates production of methane in medium with butyrate minus methane
production in medium without butyrate.

























Figure 2. Methane production by thermophilic butyrate-utilizing enrichments with and without Methanosarcina
sp. present.







3200 ,

2800 -

2 2400 -
*s ME M A? sp


E
S1600 -

z1200
I-,
LJ 800 - T i/./T
,IET/Ml#OSARCIV,/#A
400


0 5 10 15 20 25 30 35
DAYS
IN)
(yn






26



present, growing only in the presence of acetate. This organism was

tentatively identified as Methanosarcina strain TM-1 (35). Both enrich-

ments contained rod-shaped bacteria that autofluoresced under examina-

tion by 420 nm epifluorescence microscopy, indicating the presence of

Factor F420 found in methanogens (10,25). This methanogenic rod-shaped

bacterium, tentatively identified as a strain of Methanobacterium

thermoautotrophicum, utilized H2-CO2 for growth. There were several

other rod-shaped nonfluorescing bacteria present. Therefore, it was

difficult to know which bacterium utilized butyrate. The enrichments

were transferred every 7 to 10 days after being analyzed and were found

positive for methane production. After 4 months, a stable enrichment

was obtained. A stable enrichment was defined as having a few morpho-

types present consistently. M. thermoautotrophicum was always present

as the largest population of bacteria,generally comprising 90% of the

bacteria in each microscopic field.


Isolation of Thermophilic Butyrate-Utilizing Cocultures

The stable thermophilic enrichment was used as a source of inoculum

for attempts to isolate butyrate-utilizing cocultures. M. thermoauto-

trophicum was used as a hydrogenotrophic partner. Anaerobic roll tubes

were incubated for about 4 weeks until methane was detected in the gas

phase and colonies appeared. Basically, two colony types were present.

One colony type was brownish, granular, and irregular in shape; averaged

2 mm in diameter (Figure 3(A)); and autofluoresced when exposed to 420

nm light. This colony resembled Methanosarcina TM-1, but, when a wet

mount of the colony was examined, it was found to be composed of the






























Figure 3. Photomicrographs of thermophilic coculture colony types.
(A) Colony type that contains Methanosarcina sp., Methano-
bacterium thermoautotrophicum, and a curved rod. (B) Colony
type that contains Mb. thermoautotrophicum and a curved rod.






28






29



Methanosarcina sp., Mb. thermoautotrophicum, and a nonfluorescing, rod-

shaped bacterium. The second colony type (Figure 3(B)) was white and

circular with an entire margin, average 1 mm in diameter, and auto-

fluoresced when exposed to 420 nm light. Upon microscopic examination,

the colony was found to be composed of Mb. thermoautotrophicum and a

nonfluorescing rod-shaped bacterium. The two colony types were found to

be predominately composed of Mb. thermoautotrophicum. The colonies

appeared only in the BE medium and not in the BC medium.

A photomicrograph of the coculture is shown in Figure 4. The

Methanosarcina sp. is not shown in this photomicrograph. Mb. thermoauto-

trophicum, as shown in the photomicrograph, was rod shaped, autofluoresced

at 420 nm, and had greater contrast than the butyrate-utilizing bacterium.

Several examples are indicated by the single arrows. The butyrate-

utilizing bacterium was a slightly curved, gram-negative rod that aver-

aged 2 to 3 pm in length, occurred singly, was nonmotile, and did not

contain spores. Several examples are indicated by the double arrows in

Figure 4.

The colonies were picked and placed into fresh medium and were

rolled out. Once colonies appeared again, they were picked and placed

onto slants prepared in serum tubes. After 6 weeks methane was not

present in the gas phase and growth had not occurred. The enrichment

was rolled out again, and once colonies appeared they were picked and

placed onto slants. Again, methane was not produced and growth did not

occur. The entire process was repeated with the same results. The

colonies were viable because they could be placed into liquid medium and

would grow. Because this culture only produced two colony types that






















Figure 4. Photomicrograph showing Methanobacterium thermoautotrophicum, indicated by a single arrow,
and the butyrate-utilizing bacterium, indicated by the double arrows.





31








IF



























A






32



could not be routinely grown on slants, it should probably be referred

to as a highly purified enrichment. For the sake of brevity, however,

it is referred to as a coculture. The coculture was maintained on slants

by the injection of active enrichments onto the slants.


Studies on the Thermophilic Coculture

The coculture was incubated at various temperatures to determine

whether the butyrate-utilizing coculture was capable of growth at other

temperatures. Table 4 shows the differing temperatures tested and the

results. When Methanospirillum hungatei, a mesophilic, hydrogen-

utilizing methanogen, was added to the coculture, only trace amounts of

methane were formed at 370C. The amount of methane formed was less than

5% of that formed when the coculture was incubated at 550C. Methane was

not produced when the coculture was incubated at 45 or 700C.

The coculture was examined to see whether clarified digester fluid

(CDF) could be replaced by rumen fluid (RF) or deleted from the medium.

Table 5 shows that when neither CDF nor RF was a component of the medium,

methane production was greatly diminished. The addition of rumen fluid

did not stimulate methane production and was inhibitory at concentra-

tions of 20% and above on day 17. Clarified digester fluid addition

resulted in consistent methane production at all concentrations tested.

When the basal medium without butyrate (BE) contained RF, methane was

produced in greater quantities as compared to when the BC medium con-

tained CDF. This result indicated that greater quantities of methano-

genic substrates were present in the RF. The quantity of methanogenic

substrates in the media containing 20 and 30% RF inhibited the produc-

tion of methane from butyrate (Table 5).









Table 4. Effects of various temperatures on the methane production by a thermophilic butyrate-utilizing
coculture


Methane production (pmoles)a

Day 2 Day 5 Day 8 Day 13 Day 26


370Cb A 0.87 � 0.6 2.4 � 3.3 4.83 � 3.02 7.9 � 5.4 14.4 � 4.9
B MNDc MND MND MND MND
C 1.01 0.99 1.83 1.02 0.96

450C MND MND MND MND MND

550C 1.07 � 0.4 2.59 � 1.6 51.58 � 10.5 304.7 � 19.4 298.8 � 15.0

70�C MND MND MND MND MND


avalues represent a mean of triplicate determination plus or minus standard deviation, except for
M. hungatei alone, where they are single determinations.
(A) coculture with M. hungetei; (B) coculture without M. hungatei; (C) M. hungatei alone.

cMND equals methane not detected.






34



Table 5. Effects of various concentrations of rumen fluid (RF) or
clarified digester fluid (CDF) on the percentage of methane
production by thermophilic coculture


Methane production (%)

Day 3 Day 9 Day 17

No addition 0 1.10 � 0.02a 6.25 � 0.42


Rumen fluid (RF)
5% LTCb 3.63 � 2.57 38.11 � 10.57
10% LTC LTC 41.30 � 0.46
20% LTC LTC 24.00 � 4.62
30% LTC LTC LTC


Clarified digester
fluid (CDF)
5% LTC 7.24 � 1.1 42.87 � 2.25
10% LTC 8.16 � 1.2 41.34 � 2.00
20% LTC 9.38 � 0.7 40.04 � 3.27
30% LTC 8.03 � 3.1 36.61 � 3.36


Basal medium
without butyrate
RF NDc 9.80 � 2.2 22.62 � 11.45
CDF ND 3.60 � 1.1 5.10 � 1.69


aMean of three tubes plus or minus standard deviation.

Less than control.

CNot determined.






35



The effect of the addition of 0.1% yeast extract to the thermo-

philic coculture is shown in Table 6. The addition of yeast extract re-

sulted in a 142% increase in methane produced after 18 days' incubation

by the coculture containing the acetate-utilizing Methanosarcina sp.

At day 22, the increase in methane production by the coculture with the

Methanosarcina sp. was 28%. Yeast extract did not stimulate methane

production and showed a slight inhibition of methane production in the

coculture when the Methanosarcina sp. was absent.

Antibiotics known to affect cell wall synthesis of eubacteria but

not archaebacteria were added to the coculture. Figure 5 shows that

penicillin G (3000U/ml) and D-cycloserine (0.1 mg/ml) caused the complete

inhibition of methane production by the coculture.

The presence of hydrogen in the gas phase (80% H2-20% CO2) inhib-

ited the utilization of butyrate by the coculture (Figure 6). The gas

phase was replaced every 2 days until day 8. At that time, indicated

by the arrow, hydrogen was not detected in the gas phase because of its

removal by Mb. thermoautotrophicum. The culture was allowed to continue

incubating in the absence of hydrogen to determine if the butyrate

utilizers had been killed or merely inhibited by the hydrogen. After a

lag period, butyrate utilization began with the butyrate being rapidly

utilized (Figure 6).

In general, the enrichments utilized butyrate faster and produced

methane quicker when they were incubated without shaking. When the

enrichments were shaken, methane production showed a longer lag period,

but if the shaking was stopped, methane production increased.






36



Table 6. Effects of the addition of 0.1% yeast extract to thermophilic
butyrate-utilizing enrichments


Methane production (pmoles)a

Without Methanosarcina With Methanosarcina

-YE +YE -YE +YE


Day 2 0 0 0 0

Day 11 0.9 1.8 1.0 0.1

Day 18 36.9 54.6 99.4 240.8

Day 22 62.1 53.5 244.4 313.3


avalues represent means of duplicate tubes.






















Figure 5. The effect of eubacterial antibiotics on methane production by a thermophilic butyrate-
utilizing coculture. The antibiotics were penicillin G (3000 U/ml) and D-cycloserine
(0.1 mg/ml).






650
600
550 -
500 - AV AfT/!forfCS
u450 -
0400
E
3350
u300
<250
H-
wL200 -
150
100
50 A- ATiB/OTICS
0 - '
0 4 8 12 16 20

DAYS






















Figure 6. The effect of hydrogen in the gas phase on butyrate utilization by a thermophilic butyrate-
utilizing coculture. The hydrogen was replaced every 2 days until day 8.







20

18 -

. 16 -
E
14 -
a)
0 12
E
3 10 mo HW~
LLJ
8-
>- 6 -

m 4-

2- +

0 4 8 12 16 20 24

DAYS






41



Effects of Pumping Butyrate into a Thermophilic Digester

Volatile fatty acids were found in low concentrations in thethermo-

philic digester. Acetate concentration was 3 to 4 imoles/ml of sludge,

whereas n-butyrate concentration was less than 0.5 pmoles/ml of sludge.

A stock solution of sodium n-butyrate was prepared so that it could be

pumped into the digester at the rate of 10 pmoles/ml of sludge per day.

Figure 7 shows the theoretical accumulation of butyrate if it were not

utilized by the digester and the actual concentrations measured. Buty-

rate did not accumulate in the digester when pumped at this concentra-

tion. The concentrations of acetate and propionate are shown in Figure

8. Acetate concentration increased from about 3 pmoles/ml of sludge to

about 35 pmoles/ml of sludge by day 23. Propionate concentration in-

creased to about 3.3 pmoles/ml of sludge by day 23. The ratio of gas

produced by the butyrate-amended digester versus the control digester

was initially 1.5 and by day 23 had stabilized at about 1.4 (Figure 9).

The percentage of methane in the gas phase increased from 58% to 68%.

The pH increased from 7.3 to 7.8, where it remained stable.

The digester maintained consistent levels of acetate (33 to 35

pmoles/ml of sludge), butyrate (0.7 to 0.75 pmoles/ml of sludge),and

ratio of gas production (1.36 to 1.37) from day 18 to day 23. These levels

indicated that a stable digestion had been attained and that the rate

of addition of butyrate was not exceeding the capability of the digester

for utilization of butyrate. To determine the concentration of butyrate

that would have to be infused into the digester in order to exceed the

ability of the digester to maintain a stable digestion, the rate of

addition would need to be increased. Therefore, on day 24, as indicated









250
225
200 -
-175 - /EO PECA!
0150
E
3125 -
100 -
>- 75 -
m 50 -
25 - C/ML
0 "
0 4 8 12 16 20 24 28 32 36 40 44 48
DAYS
c-4:























Figure 8. Concentration of acetate and propionate in a thermophilic digester supplemented with butyrate.
See legend for Figure 7 for conditions.







180



-140 - A L-rE
()
cu
0120 -
E
:D
v100 -
CD 80 -
S80 -

S60/

0 40

S20

00 4 8 12 16 20 24 28 32 36 40 44 48
DAYS
CX2.























Figure 9. Biogas production by a thermophilic digester supplemented with butyrate. See legend for
Figure 7 for conditions.






10 I I 1
9- Crw
8





CD

m3 3
LD
2
1 -

0 4 8 12 16 20 24 28 32 36 40 44 48
DAYS






48



by the arrows in Figures 5, 7, and 8, the concentration of sodium

n-butyrate infused into the digester was doubled so that the rate of

addition would be 20 pmoles butyrate/ml of sludge per day. The levels

of all VFAs began to increase. By day 36 the concentrations, in Pmoles/

ml, were acetate, 167; propionate 16.9; and n-butyrate, 8.9. During

this increase of VFAs, the biogas production ratio remained between 1.4

and 1.5 and on day 36 was 1.35. Because of the rapid increase in VFAs,

the addition of butyrate was stopped.


Enrichments from Mesophilic Ecosystems

Butyrate enrichments were begun with a mesophilic, 400C digester

as the source of inoculum. These enrichments were analyzed each week

for methane production, and the enrichments that produced the greatest

quantities of methane were transferred to fresh medium. After about 8

transfers the enrichment was analyzed for VFA concentrations. The

acetate concentration was 17.2 pmoles/ml (acetate not detected in un-

inoculated medium), whereas the butyrate concentration was 0.16 Pmoles/

ml (about 22 pmoles/ml in uninoculated medium). Microscopic examina-

tion showed that the predominant bacterium was an irregular coccus that

autofluoresced when exposed to 420 nm of light. A bacterium resembling

Methanosarcina barkeri was also observed. In addition, nonfluorescing,

short, nonmotile, curved-rod shaped and long, nonmotile, rod-shaped

bacteria were observed.

Sediments from a eutrophic freshwater lake, Bivens Arm, were used

to begin butyrate enrichments. After weekly transfers for several

months, the enrichments were examined by phase-contrast microscopy. The

bacteria in the most predominant numbers were Methanospirillum hungatei






49



and a Methanococcus sp. There were also small numbers of Methanosarcina

barkeri present. In addition, there were nonfluorescing long rods and

short curved rods present.

Eubacterial antibiotics were added to mesophilic sludge enriched

with butyrate. In the enrichment without antibiotics, about 300 umoles

of methane were found in the gas phase, whereas when antibiotics were

present, the gas phase contained only about 40 inoles of methane (Figure

10). The effect of the antibiotics was not on the H2-CO2-utilizing

methanogens because methane was formed in the presence of the anti-

biotics when 80% H2-20% CO2 was the gas phase. All of the butyrate

in the enrichment without antibiotics was utilized, whereas 87% of the

butyrate remained in the enrichment with antibiotics.


Isolation of Mesophilic Butyrate-Utilizing Cocultures

A stable mesophilic enrichment was the source of inoculum for

attempts to isolate butyrate-utilizing cocultures. Desulfovibrio G-11

was used as the hydrogenotrophic partner. After 28 days of incubation

at 370C, colonies with blackened centers appeared in roll tubes with

butyrate and sulfate, but not in roll tubes with butyrate alone or sul-

fate alone. Also, colonies which fluoresced when exposed to 420 nm of

light were observed. Both types of colonies were picked from the roll

tubes and placed onto slants of butyrate-containing medium. The fluo-

rescent colonies grew within 10 days on the slants and produced methane.

However, there were four morphotypes of bacteria present. Therefore,

this culture was rolled out again in an attempt to isolate colonies

composed of two members. The black-centered colonies produced visible

growth on slants and were transferred to liquid broth for further study.






















Figure 10. Effect of eubacterial antibiotics on methane production by butyrate enrichments from a
mesophilic digester. The antibiotics were penicillin G (3000 U/ml) and D-cycloserine
(0.1 mg/ml).






300
275 -
250 - Y/Tmio
,225 Af//
c200 -
E175
150
=125
t100
LU
7 75
50- 17T drNT/r/fCS
25
n;o== |----------- i ----- - ------
0 4 8 12 16 20
DAYS
U"






52



Attempts To Isolate Butyrate-Utilizing Hydrogenogens in
Pure Culture with E. coli Membrane Fragments

E. Coli were statically grown for 6 hours at which time hydrogen

was present in the gas phase. The lysate from French Pressure-cell

passage was filter sterilized and injected into molten agar tubes con-

taining stable mesophilic enrichment. The amount injected was 0.1 ml

(57.0 � 1.6 mg dry weight per ml). The roll tubes were incubated at

37�C. After about 4 weeks, colonies were present in the tubes contain-

ing butyrate and E. coli membrane fragments and fumarate. These colonies

were not present in tubes with butyrate alone or E. coli membrane frag-

ments alone. The colonies were white, circular with entire margins, and

1 to 2 mm in diameter.















DISCUSSION


Organic matter is degraded exclusively to methane and carbon

dioxide in nongastrointestinal ecosystems where light, nitrate, oxygen,

and sulfate are absent. This degradation requires at least three groups

of bacteria (21,29): fermentative, hydrogenogenic, and methanogenic.

The hydrogenogenic bacteria are the least understood of the three groups,

with only two species being known (5,22,23).

When several ecosystems were enriched with butyrate, only digesters

and freshwater sediments produced methane in a short or no lag period.

These ecosystems continually receive organic matter which undergoes

degradation to methane. Thus, these ecosystems have a population of

butyrate-utilizing bacteria and can readily degrade butyrate to methane.

When seagrass beds were enriched with butyrate in sulfate-free artifi-

cial seawater, methane was not produced until after a 2-week lag period.

The lag in methane production may have resulted from butyrate being

utilized by fatty acid-utilizing,sulfate-reducing bacteria (SRB) which

were reducing the sulfate that remained in the sediments. These enrich-

ments smelled strongly of H2S before and after methane production began.

Desulfovibrio spp. will degrade lactate (7,20) or ethanol (7) in the

absence of sulfate when methanogens are present to remove hydrogen, the

electron sink product. In these marine sediments, once sulfate is de-

pleted the methanogens may participate in the degradation of butyrate

by removing H2 produced by SRB. The inability of the microflora


53






54



of hypersaline sediments to produce methane when enriched with butyrate

indicates that butyrate may not be a typical substrate in the ecosystems

examined.

The thermophilic digesters were chosen for more detailed study.

Thermophilic digestion may have several advantages over mesophilic di-

gestion. Varel et al. (32) reported the advantage of being able to

reduce retention times to less than 6 days.at thermophilic (>450C)

temperatures. Also, different microflora of various plant biomasses

added to a thermophilic digester would not compete with the digester

microflora. If competition were allowed, then it might be possible for

destabilization of the digester to occur.

The thermophilic digester had ca. 5 x 106 butyrate-utilizing

bacteria/ml of sludge, a finding similar to that in another study (17).

When a thermophilic Methanosarcina sp. that utilized acetate, but not

H2-CO2, was present in butyrate enrichments, greater quantities of

methane were produced and the enrichments seemed more stable. The en-

hanced methane production resulted from the decarboxylation of acetate

with the concomitant production of methane by the Methanosarcina sp.

Table 2 shows the equation for the oxidation of butyrate which resulted

in the production of acetate and hydrogen. The removal of hydrogen made

it thermodynamically possible for the reaction to occur and allowed the

hydrogenogen to continue metabolic processes because of the recycling

of NADH. The additional removal of acetate made the thermodynamics of

the equation even more negative, thus enhancing the stability of the

enrichments. Thermophilic methanogenic butyrate enrichments were com-

posed primarily of the H2-utilizing Methanobacterium thermoautotrophicum,

with other morphotypes present in smaller numbers. Since M.






55



thermoautotrophicum was present in large numbers in the butyrate enrich-

ments, it was the bacterium of choice as a hydrogenotrophic partner for

coculture isolation attempts. When butyrate enrichments were rolled

out with M. thermoautotrophicum, two colony types were observed. When

transferred onto slants, the colonies did not grow, possibly because of

the change in the oxidation-reduction potential occurring when the serum

tubes were opened for inoculation. This lack of growth might be over-

come if a reducing agent were used which would maintain a lowered oxida-

tion-reduction potential for a greater length of time.

The two previously described hydrogenogenic bacteria are mesophilic

(5,22,23). In order to determine whether the butyrate-utilizing bac-

terium isolated from the thermophilic digester was in fact a thermophile,

the coculture was incubated at several temperatures. The data for this

experiment are found in Table 4. When the coculture was incubated at

370C, methane was not detected. The lack of methane production could

possibly have resulted from the inability of M. thermoautotrophicum

to grow. Therefore, a mesophilic H2-CO2-utilizing methanogen, Methano-

spirillum hungatei, used in the study of the two reported hydrogenogens

(5,22,23), was added to the coculture. If the butyrate-utilizing bac-

terium could produce H2 at 370C. then M. hungatei would oxidize it and

methane should be detected. Table 4 shows that a small amount, ca. 14

pmoles, of methane was produced after 26 days of incubation. When the

coculture was incubated at 55�C, the temperature of isolation, ca. 300

pmoles of methane were produced by day 13, half the time required for

production of 14 pmoles of methane by the coculture with 1. hungatei at

37�C. This indicated that the butyrate utilizer was a thermophilic






56



bacterium and, hence, was different from S. wolfei, a mesophile. In

addition, the thermophilic butyrate utilizer was a nonmotile, slightly

curved rod, 2 to 3 vm in length, whereas S. wolfei exhibited sluggest

motility and was 7 pm in length (22). Therefore it appears that this

bacterium is a new species of anaerobic hydrogenogens. It required a

growth factor (or factors) that was present in clarified digester fluid

(CDF) but not in rumen fluid (RF) (Table 5). The addition of CDF in

increasing concentrations resulted in a stimulation of methane production

which may have aided in the isolation and maintenance of these bacteria.

The addition of 0.1% yeast extract to the cocultures containing Methano-

sarcina sp. resulted in a 142% increase in methane production after 18

days of incubation. Yeast extract can replace CDF for growth of methano-

sarcina TM-1 (P. A. Murray and S. H. Zinder, Abstr. Annu. Meet. Am. Soc.

Microbiol., 1983, 18, p. 14). It appeared that the addition of yeast

extract to medium with CDF resulted in the enhancement of methane pro-

duction from acetate by the Methanosarcina sp. It may also be of benefit

in the isolation and maintenance of the butyrate-utilizing bacteria.

The addition of penicillin and D-cycloserine completely inhibited

methane production by the thermophilic coculture (Figure 5). S. wolfei

was inhibited by the addition of penicillin and has been shown to possess

a peptidoglycan cell wall (22). Since the thermophilic coculture was

inhibited by antibiotics that inhibit eubacteria, it appeared that the

butyrate-utilizing bacteria were eubacterial and possessed a peptidoglycan

call wall like that of S. wolfei.

The presence of 80% hydrogen in the gas phase inhibited butyrate

utilization by S. wolfei (22). When the thermophilic coculture was

placed under an 80% hydrogen gas phase, butyrate was not utilized






57



(Figure 6). However, when hydrogen was removed from the thermophilic

coculture, butyrate was utilized (Figure 6). Thus it is indicated that

hydrogen inhibited but did not kill the thermophilic butyrate-utilizing

bacteria.

The addition of butyrate to the thermophilic digester at the rate

of 10 pmoles/ml of sludge per day did not result in the accumulation

of butyrate. Thus, it is possible that if this digester could be loaded

at a higher rate, the result would be the production of a higher concen-

tration of butyrate. The addition of butyrate at the concentration of

20 pmoles/ml of sludge per day resulted in an unstable digestion. There-

fore, the maximum concentration of butyrate that could be infused into

this digester and still result in a stable digestion was between 10 and

20 pmoles/ml of sludge per day.

Hydrogenogenic bacteria have been isolated only in cocultures with

hydrogenotrophic bacteria. In order to better study these bacteria, the

isolation of pure cultures is necessary. Because the hydrogenogenic

bacteria are inhibited by hydrogen, the concentration of hydrogen must

be maintained close to zero. The use of hydrogenase-containing, cell-

free systems may make it possible to maintain the lowered concentration

of hydrogen. As a test of this possibility, butyrate-utilizing enrich-

ments from mesophilic ecosystems were initiated to provide inoculum for

the isolation of cocultures from which a pure culture might be isolated

directly or from the enrichments. An active enrichment was diluted and

rolled out in media with various additions. These additions were varied

to allow for a number of controls. After 21 days of incubation at 370C,

white colonies were found in anaerobic roll tubes that contained E. coli






58



membrane fragments, fumarate, and butyrate. If any one of these com-

ponents was not present in the anaerobic roll tubes, the white colonies

did not appear. Thus, it appears that the membrane fragments were able

to remove H2 and may be useful in isolating pure cultures of hydro-

genogens.

In 1951, Stadtman and Barker (30) reported a highly purified cul-

ture that degraded butyrate to acetate and methane. The methane-produc-

ing bacterium was named Methanobacterium suboxydans. This culture has

since been lost, and attempts to reisolate it have been unsuccessful.

In the present study, an experiment was attempted to isolate a butyrate-

utilizing methanogen. Because methanogens are capable of growing in

the presence of eubacterial antibiotics that act on the cell wall,

sludge from a mesophilic digester was enriched with butyrate in the

presence and absence of antibiotics. Figure 10 indicates that a small

amount of methane was produced in the presence of antibiotics. The

methane may have resulted from the inactivation of antibiotics by the

high concentration of organic matter in the digester sludge. Such a

result does not exclude the possibility that a butyrate-utilizing

methanogen existed. However, additional research is needed to deter-

mine with certainty whether one did.

Populations of bacteria in microbial communities exist in a variety

of symbiotic relationships. The relationship between hydrogenogenic and

hydrogenotrophic bacteria is described as syntrophic by several authors

(5,18,21,22,23,24). Alexander (2) defines syntrophism as a relationship

which entails a bilateral exchange of growth factors. As presently

understood, interspecies hydrogen transfer involves the unidirectional






59



transfer of a substrate. When one organism uses the excretion of a

second as a substrate for growth, the relationship is commensalistic

(2). Therefore, what is presently referred to as a syntrophic rela-

tionship may in fact be a commensalistic one. As more information be-

comes available, it may be necessary to redefine the relationship that

exists between hydrogenogens and hydrogenotrophs.

In summary, several ecosystems were studied to determine their

methane-producing capability when enriched with butyrate. A new species

of a thermophilic butyrate-utilizing bacterium was established in co-

culture. This bacterium was a gram-negative, slightly curved rod that

measured 2 to 3 pm in length. Most cells occurred singly. Spores were

not observed and cells did not exhibit motility. Growth was inhibited

by penicillin. Hydrogen inhibited growth but this inhibition was re-

versed upon the removal of the hydrogen. Growth did not occur at 37�C

in the presence of a mesophilic hydrogen-utilizing methanogen.

Mesophilic methane-producing enrichments were initiated to isolate co-

cultures of butyrate-utilizing bacteria and to attempt the isolation

of pure cultures of these bacteria. The isolation of pure cultures was

not completely successful and is still being attempted.















REFERENCES


1. Adams, M. W., L. E. Mortenson, and J.-S. Chen. 1981. Hydrogenase.
Biochem. Biophys. Acta 594:105-176.

2. Alexander, M. 1971. Microbiol ecology. John Wiley & Sons, Inc.,
New York.

3. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe.
1979. Methanogens: reevaluation of a unique biological group.
Microbiol. Rev. 43:260-296.

4. Barker, H. A. 1940. Studies upon the methane fermentation. IV.
The isolation and culture of Methanobacterium omelianskii.
Antonie van Leeuwenhoek 6:201-220.

5. Boone, D. R., and M. P. Bryant. 1980. Propionate-degrading
bacterium Syntrophobacter wolinii sp. nov. gen. nov., from
methanogenic ecosystems. Appl. Environ. Micobiol. 40:626-632.

6. Bryant, M. P. 1979. Microbial methane production-theoretical
aspects. J. Anim. Sci. 48:193-201.

7. Bryant, M. P. L. L. Campbell, C. A. Reddy, and M. R. Crabill. 1977.
Growth of desulfovibrio in lactate or ethanol media low in
sulfate in association with H2-utilizing methanogenic bacteria.
Appl. Environ. Microbiol. 33:1162-1169.

8. Bryant, M. P., E. A. Wolin, M. J. Wolin, and R. S. Wolfe. 1967.
Methanobacillus omelianskii, a symbiotic association of two species
of bacteria. Arch. Mikrobiol. 59:20-31.

9. Chen, M., and M. J. Wolin. 1977. Influence of CH4 production by
Methanobacterium ruminantium on the fermentation of glucose and
lactose by Selenomonas ruminantium. Appl. Environ. Microbiol.
34:756-759.

10. Doddema, H. J., and G. D. Vogels. 1978. Improved identification
of methanogenic bacteria by fluorescence microscopy. Appl.
Environ. Microbiol. 36:752-745.

11. Ferry, J. G., and R. S. Wolfe. 1976. Anaerobic degradation of
benzoate to methane by a microbial consortium. Arch. Microbiol.
197:33-40.



60






61



12. Hobson, P. N., S. Bousfield, and R. Summers. 1974. Anaerobic
digestion of organic matter, p. 131. In CRC critical reviews
in environmental control. Chemical Rubber Co., Cleveland, Ohio.

13. Hungate, R. E. 1966. The rumen and its microbes. Acad. Press,
Inc., New York.

14. Hungate, R. E. 1969. A roll tube method for cultivation of
strict anaerobes. Methods Microbiol. 3B:117-132.

15. lannotti, E. L., D. Kafkewitz, M. J. Wolin, and M. P. Bryant.
1973. Glucose fermentation products of Ruminococcus albus grown
in continuous culture with Vibrio succinogenes: changes caused
by interspecies transfer of H2. J. Bacteriol. 114:1231-1240.

16. Lathem, M. J., and M. J. Wolin. 1977. Fermentation of cellulose
by Ruminococcus flavefaciens in the presence and absence of
Methanobacterium ruminantium. Appl. Environ. Microbiol. 34:297-
301.

17. Mackie, R. S., and M. P. Bryant. 1981. Metabolic activity of
fatty acid-oxidizing bacteria and the contribution of acetate,
propionate, butyrate, and CO2 to methanogenesis in cattle waste
at 40 and 60�C. Appl. Environ. Microbiol. 41:1363-1373.

18. Mah, R. A. 1982. Methanogenesis and methanogenic partnerships.
Phil. Trans. R. Soc. Lond. B. 297:599-616.

19. McCarty, P. L. 1971. Energetics and kinetics of anaerobic treat-
ment, p. 91. In R. F. Gould (ed.), Anaerobic biological treat-
ment processes. Advances in Chemistry Series 105. Amer. Chem.
Soc., Washington, D.C.

20. McInerney, M. J., and M. P. Bryant. 1981. Anaerobic degradation
of lactate by syntrophic associations of Methanosarcina barkeri
and Desulfovibrio species and effect of H2 on acetate degradation.
Appl. Environ. Microbiol. 41:346-354.

21. Mclnerney, M. J., and M. P. Bryant. 1981. Basic principles of
bioconversions in anaerobic digestion and methanogenesis, p. 277-
296. In S. S. Sofar and 0. Zaborsky (ed.), Biomass conversion
processes for energy and fuels. Plenum Publishing Corp.,
New York.

22. Mclnerney, M. J., M. P. Bryant, R. B. Hespell, and J. W. Costerton.
1981. Syntrophomonas wolfei gen. nov. sp. nov., an anaerobic,
syntrophic, fatty acid-oxidizing bacterium. Appl. Environ.
Microbiol. 41:1029-1039.

23. Mclnerney, M. J., M. P. Bryant, and N. Pfenning. 1979. Anaerobic
bacterium that degrades fatty acids in syntrophic association
with methanogens. Arch. Microbiol. 122:129-135.






62



24. Mclnerney, M. J., R. I. Mackie, and M. P. Bryant. 1981. Syn-
trophic association of a butyrate-degrading bacterium and
Methanosarcina enriched from bovine rumen fluid. Appl. Environ.
Microbiol. 41:826-828.

25. Mink, R. W., and P. R. Dugan. 1977. Tentative identification of
methanogenic bacteria by fluorescence microscopy. Appl. Environ.
Microbiol. 33:713-717.

26. Mountfort, D. 0., and M. P. Bryant. 1982. Isolation and charac-
terization of an anaerobic syntrophic benzoate-degrading bac-
terium from sewage sludge. Arch. Microbiol. 133:249-256.

27. Scheifinger, C. C., B. Lineham, and M. J. Wolin. 1975. H2 produc-
tion by Selenomonas ruminantium in the absence and presence of
methanogenic bacteria. Appl. Environ. Microbiol. 29:480-483.

28. Schlegel, H. G., and K. Schneider. 1979. Introductory report:
Distribution and physiological role of hydrogenases in micro-
organisms, p. 15-44. In H. G. Schlegel and K. Schneider (ed.),
Hydrogenases: Their catalytic activity, structure and function.
E. Goltze KG, Gottingen.

29. Smith, P. H. 1980. Studies of methanogenic bacteria in sludge.
EPA-600/2-80-093. Research report, U.S. Government Printing
Office, Washington, D.C.

30. Stadtman, T. C., and H. A. Barker. 1951. Studies on the methane
fermentation. VIII. Tracer experiments on the fatty acid oxida-
tion by methane bacteria. J. Bacteriol. 61:67-80.

31. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy con-
servation in chemotrophic anaerobic bacteria. Bacteriol. Rev.
41:100.

32. Varel, V. H., A. G. Hashimoto, and Y. R. Chen. 1980. Effect of
temperature and retention time on methane production from beef
cattle waste. Appl. Environ. Microbiol. 40:217-222.

33. Wolin, M. J. 1982. Hydrogen transfer in microbial communities,
p. 323-356. In A. T. Bull and J. H. Slater (ed.), Microbial
interactions and communities, vol. 1. Academic Press, London.

34. Wolin, M. J., and T. L. Miller. 1982. Interspecies hydrogen
transfer: 15 years later. ASM News 48:561-565.

35. Zinder, S. H., and R. A. Mah. 1979. Isolation and characteriza-
tion of a thermophilic strain of Methanosarcina unable to use
H2-CO2 for methanogenesis. Appl. Environ. Microbiol. 38:996-
1008.














BIOGRAPHICAL SKETCH


John Michael Henson, son of John T. and Martha J. Henson, was

born on August 11, 1952, in Greer, South Carolina. After graduating

from Greer Senior High School in June 1970, he attended Furman Univer-

sity, Greenville, South Carolina. In 1971 he transferred to the

University of South Carolina, Columbia, where in August 1975 he

received the degree of Bachelor of Science with a major in biology.

In 1976 he entered the graduate program at Clemson University, Clemson,

South Carolina, and in 1978 received the degree of Master of Science

with a major in microbiology. That fall he joined the faculty at

Presbyterian College, Clinton, South Carolina, and taught general

biology, microbiology, and cell biology. In 1980 he moved to

Gainesville, Florida, and entered the graduate program in the Depart-

ment of Microbiology and Cell Science at the University of Florida.

On January 4, 1975, he married Ellen Adams Dobson. They have

two children, Martha Anne ("Marne"), 6, and Jonathan Adams, 16 months.

Henson served in the U.S. Naval Reserve from 1972 to 1978 as a field

medical service technician attached to the U.S. Marine Corps Reserve.











63











I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


Paul H. Smith, Chairman
Professor of Microbiology and
Cell Science


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


Arnold S. Bleiweis
Professor of Microbiology and
Cell Science


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.


RoggeA. Nordstedt
Associate Professor of Agricultural
Engineering

This dissertation was submitted to the Graduate Faculty of the College
of Agriculture and to the Graduate School, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

December 1983


Dean ollege of Agricul 'e



Dean for Graduate Studies and
Research




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ISOLATION OF BUTYRATE-UTILIZING BACTERIA FROM THERMOPHILIC AND MESOPHILIC METHANE-PRODUCING ECOSYSTEMS BY JOHN MICHAEL HENSON 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 1983

PAGE 2

To Ellen

PAGE 3

ACKNOWLEDGMENTS I would like to express sincere gratitude to Dr. Paul Smith, chairman of my supervisory committee, for the time, space, and, most of all, his patience which enabled me to learn that "The experiment is everything." I also thank Dr. Arnold Bleiweis and Dr. Roger Nordstedt for their contributions as members of my supervisory committee. In addition, I thank Dr. Dave Boone for helpful discussions about the isolation of cocultures, Dr. K. Shanmugam for helpful discussions about hydrogenase, and Butch Bordeaux for technical assistance and advice. i i i

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS iii ABSTRACT v INTRODUCTION 1 MATERIALS AND METHODS 14 Organisms, Media, and Growth Conditions 14 Preparation of Butyrate Medium 16 Anaerobic Techniques 16 Descriptions of Ecosystems Studied 17 Descriptions of Butyrate Enrichments 18 Thermophilic Coculture Isolation 18 Mesophilic Coculture Isolation 19 Gas Chromatography Methods 19 Preparation and Use of E_. col i Membrane Fragments To Attempt Isolation of Pure Cultures of Hydrogenogenic Bacteria .... 20 Microscopy and Photomicroscopy 21 RESULTS 22 Production of Methane When Various Ecosystems Were Enriched with Butyrate 22 Description of Thermophilic Butyrate Enrichments 22 Isolation of Thermophilic Butyrate-Utilizing Cocultures ... 26 Studies on the Thermophilic Coculture 32 Effects of Pumping Butyrate into a Thermophilic Digester ... 41 Enrichments from Mesophilic Ecosystems 48 Isolation of Mesophilic Butyrate-Utilizing Cocultures .... 49 Attempts To Isolate Butyrate-Utilizing Hydrogenogens in Pure Culture with E. Col i Membrane Fragments 52 DISCUSSION 53 REFERENCES 60 BIOGRAPHICAL SKETCH 53 i v

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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 ISOLATION OF BUTYRATE-UTILIZING BACTERIA FROM THERMOPHILIC AND MESOPHILIC METHANE-PRODUCING ECOSYSTEMS By John Michael Henson December 1983 Chairman: Dr. Paul H. Smith Major Department: Microbiology and Cell Science The ability of various ecosystems to convert butyrate to methane was studied in order to isolate the bacteria responsible for the conver sion. When thermophilic digester sludge was enriched with butyrate, methane was produced without a lag period. Marine sediments enriched with butyrate required a 2-week incubation period before methanogenesis began. When hypersaline sediments were enriched with butyrate, methane was not produced after 3 months. A thermophilic digester was studied in more detail and found by most-probable-number enumeration to have ca. 5 x 10° butyrate-util izing bacteria/ml of sludge. A thermophilic butyrate-util izing bacterium was isolated in coculture with Methanobacterium thermoautotrophicum and a Methanosarcina sp. This bacterium was a gram-negative, slightly curved rod that occurred singly was nonmotile, and did not appear to produce spores. When this coculture was incubated with Methanospirillum hungatei at 37°C, the quantity of methane produced was less than 5% of that produced when the v

PAGE 6

coculture was incubated at 55°C, the routine incubation temperature. The coculture required clarified digester fluid (CDF), which could not be replaced by rumen fluid (RF). The addition of yeast extract to a medium containing 5% CDF stimulated methane production when the Methanosarcina sp. was present. Hydrogen in the gas phase prevented butyrate utilization. However, when the hydrogen was removed, butyrate utilization began. Penicillin G and D-cycloserine caused the complete inhibition of butyrate utilization by the coculture. The thermophilic digester was infused with butyrate at the rate of 10 umoles/ml of sludge per day. Biogas production increased by 150%, with the percentage of methane increasing from 58% to 68%. Acetate, propionate, and butyrate did not accumulate. When the infusion rate was increased to 20 umoles/ml of sludge per day, an unstable digestion resul ted. Butyrate-utilizing enrichments from mesophilic ecosystems were used in obtaining cocultures of butyrate-utilizing bacteria. These cocultures served as inocula for attempts to isolate pure cultures of butyrate-utilizing bacteria by use of hydrogenase-containing membrane fragments of Escherchia col i . After a 3-week incubation period, colonies appeared only in inoculated tubes that contained membrane fragments and butyrate.

PAGE 7

INTRODUCTION In anaerobic ecosystems where light, sulfate, and nitrate are absent, such as digesters and freshwater sediments, organic matter is degraded exclusively to methane and carbon dioxide. This degradation is mediated by several groups of bacteria and is regulated by the hydrogen concentration (21,29). Hydrogen does not accumulate and is difficult to detect in methane-producing ecosystems because of rapid interspecies hydrogen transfer. Interspecies hydrogen transfer is the utilization of hydrogen by one bacterial species (hydrogenotroph) that is produced by another bacterial species (hydrogenogen) (33,34). This phrase was introduced by Iannotti et al. (15) when they compared the fermentation products formed by a pure culture of Ruminococcus a! bus with the fermentation products formed when this organism was grown with a hydrogenotroph. R_. albus is a carbohydrate-fermenting organism that produces acetate, ethanol , and hydrogen when it is grown in continuous culture with glucose as the sole carbon and energy source. However, when R^. al bus is grown in a mixed continuous culture with Vibrio succinogenes , a hydrogenotroph, the only products detected are acetate and succinate. Succinate is formed when V^. succinogenes uses the hydrogen produced by R. al bus to reduce fumarate which J}, albus alone cannot use as an electron acceptor. Thus, because of the interspecies transfer of hydrogen from R. al bus to V. succinogenes , there is a shift 1

PAGE 8

2 in fermentation products from the less reduced ethanol to the more reduced acetate and hydrogen. Selenomonas ruminantium is a carbohydrate-fermenting organism that when grown in pure culture produces trace amounts of hydrogen (27). Hydrogen production, however, as indicated by the amount of methane formed, is stimulated almost 100-fold when cocultured with methanogenic bacteria (27). Subsequent analysis (9) shows that the fermentation products formed from glucose are altered by the presence of hydrogenutilizing methanogens. The electron sink fermentation products, lactate and propionate, are produced in decreased amounts, whereas acetate and hydrogen formation increases. It has been suggested (9) that the production of hydrogen results from reduced nicotinamide adenine dinucleotide (NADH); thus hydrogen becomes a major electron sink product and alters the fermentation products formed. Similar results are observed when cellulose is fermented by Ruminococcus flavefacians (16). In the presence of Methanobacterium ruminantium , a hydrogenotropic methanogen, major fermentation products shift from succinate and acetate to acetate and hydrogen, which is evidenced by the large amounts of methane formed. These studies confirm Hungate's hypothesis (13) that methanogenesi s in the rumen provides for the removal of electrons from pyruvate via the formation of hydrogen. This hypothesis arose from observations that, when many rumen bacteria are grown in pure culture, a variety of products are found that are not found in the rumen. These products are electron sink products that result from the oxidation of NADH. The removal of electrons released by catabolism via hydrogen formation

PAGE 9

3 results in the net removal of electrons from the rumen because the methanogenic bacteria maintain a low partial pressure of hydrogen by using it to reduce carbon dioxide to methane. The methane then leaves the rumen when the ruminent eructates. All of the fermentative bacteria described above, as well as other fermentative bacteria, are able to produce alternative electron sink products in place of hydrogen when the partial pressure (pl^) of hydrogen is not maintained sufficiently low for oxidation of NADH. Table 1 illustrates the required lowered pH^ to oxidize NADH. Formation of H£ from NADH, when products and reactants are equimolar, will not occur as indicated by the positive AG°' for the reaction. As the concentration of H£ approaches zero, the AG 01 for the reaction will become negative. If all the electrons carried by NADH are ultimately used to form CH^ via W^, as in the summation reactions in Table 1, the overall reaction has a AG°' of -63.6 kJ. Thus, alternate electron sink products are not formed. There are, however, other hydrogen-producing bacteria whose growth is dependent upon the constant removal of the hydrogen produced by the oxidation of NADH. Being unable to produce alternate electron sink products, these bacteria are obligate hydrogenogens. They are involved in the utilization of ethanol (8), propionate (5), butyrate (22,23), other fatty acids (23), benzoate (11,26), and possibly other substrates. Methanobacillus omelianskii has been described by Barker (4) as being able to convert ethanol and carbon dioxide to acetate and methane. In 1967, Bryant et al. (3) were able to separate cultures of M. omel ianski i into two bacterial species. Their studies reveal

PAGE 10

4 Table 1. Oxidation of reduced nicotinamide adenine dinucleotide (NADH) by removal of Hp via methane formation AG° 1 Action (kJ/reaction) a 1. NADH + H + * H 2 + NAD + +18.0 2. 4H 2 + HC0~ + H + + CH 4 + 3H 2 0 -135.6 Summation: 4NADH + 4H + * 4H 2 + 4NAD + +72.0 4H 2 + HCO3 + H + + CH 4 + 3H 2 0 -135.6 4NADH + HCO3 + 5H + + 4NAD + + CH 4 + 3H 2 0 . -63.6 a Values taken from or calculated from data in Thauer et al . (31).

PAGE 11

5 that the presence of the two separate bacteria are necessary for the conversion to occur. One of the two bacteria, the S organism, oxidizes ethanol with the production of acetate and W^, and will not grow alone. The second species is a methanogenic bacterium which utilizes but not ethanol for growth and methane production. Bryant et al.(8) also proposed that fatty acids other than formate and acetate are anaerobically degraded by nonmethanogenic bacteria similar to the S organism with the production of f^. As is the case for the oxidation of NADH described above, the pH£ must be maintained at a low level for the oxidation of ethanol to occur in the coculture just described. Shown in Table 2 are the equations and free-energy changes for the oxidations of ethanol as well as propionate and butyrate. At equimolar concentrations of products and reactants, propionate has the highest AG 01 : +76.1 kJ/reaction. Butyrate is intermediate with a +48.1 kJ/reaction, whereas ethanol is the lowest with a +19.2 kJ/reaction. When the concentrations of products are lowered, as by the removal of H^, the reactions have a negative AG°' and, hence, become thermodynamically possible. The most difficult reaction to make possible via H 2 removal is the oxidation of propionate; the reaction is less difficult with butyrate and least diffi-6 -5 -3 cult with ethanol. The pH^ must be 10 , 10 , and 10 atm. , respectively (21). The separation of Methanobacillus omelianskii cultures into two components (8) was an important contribution to the understanding of microbial interactions in the fermentation of organic matter to methane because it gave initial insight into the physiological dependence of one group of bacteria on another group of bacteria.

PAGE 12

Table 2. Equations and free-energy changes for oxidation of ethanol , propionate, and butyrate AG 01 Reactlon (kJ/reaction) a 1. CH 3 CH 2 0H + 2H 2 0 2CH 3 C00" + 4H 2 + 2H + +19.2 2. CH 3 CH 2 C00" + 3H 2 0 + CH 3 C00" + HC0~ + H + + 3H 2 +76.1 3. CH 3 CH 2 CH 2 C00" + 2H 2 0 + 2CH 3 C00' + 2H 2 + H + +48.1 a Values taken from or calculated from data in Thauer et al . (31).

PAGE 13

7 Smith's report (29) summarized the results obtained when domestic sludge was enriched with propionate or butyrate. When these enrichments were rapidly sparged, H 2 was detected, indicating the presence of a physiological group of bacteria that produces H 2 from propionate or butyrate. In nonsparged enrichments, was not detected because of the interspecies transfer of H 2 to the methanogens and the subsequent production of CH 4 Both enrichments utilized H 2 -C0 2 without a lag, indicating the existence of a population of hydrogenotrophs. Propionate or butyrate utilization was inhibited by the presence of H 2 , indicating that removal of H 2 must take place in order for utilization to occur. The first report of a fatty acid-utilizing bacterium is that of an anaerobic bacterium that utilizes butyrate as well as other fatty acids (23). This organism, which has subsequently been named Syntrophomonas wolfei (22), B-oxidizes saturated fatty acids (butyrate through octanoate) to acetate and H 2 or to acetate, propionate, and H,,. S_. wolfei is a gram-negative helical rod with laterally inserted flagella and a sluggish twitching motility (23). When S_. wolfei is cocultured with a Desulfovibrio sp., a generation time of 54 hours is obtained, which is less than the generation time of 87 hours when it is cocultured with M. hungatei . Compounds tested and found not to act as electron acceptors are dimethyl sulfoxide, fumarate, malate, nitrate, oxygen, sulfate, sulfite, sulfur, and thiosulfate (23). In addition, manganese oxide, methyl viologen, palladium chloride, phenosafranin, tetrazolium chloride, and trimethylamine-N-oxide are not utilized as electron acceptors when butyrate is the electron donor (22). The presence of 80% H 2 in the gas phase inhibits growth and butyrate utilization when S_. wolfei is

PAGE 14

8 cocultured with M. hungatei (22). S^. wolfei did not grow alone when the gas phase was continually recycled through hot copper oxide filings to remove (22). Cells contained peptidoglycan and poly-B-hydroxybutyrate (22). A morphologically similar bacterium was isolated from rumen fluid (24). Syntrophobacter wolinii is a nonmotile, gram-negative rod that degrades propionate, but not other fatty acids, to acetate and, presumably, H 2 and C0 2 (or formate) only in the presence of a hydrogenotrophic bacterium (5). When S^. wol inii is cocultured with a Desulfovibrio sp., the hydrogenotroph, the doubling time is about 87 hours. The doubling time increases to 161 hours when the Desulfovibrio sp. is present as a minor component of the coculture and Methanospi rill urn hungatei is the major hydrogenotrophic organism. S^. wol inii is a strict anaerobe. Another example of an obligate hydrogenogenic bacterium is one that does not utilize fatty acids. Benzoate is degraded to acetate and, presumably, CC^ and (or formate) by a gram-negative, motile, rod-shaped bacterium in coculture with the hydrogenotrophic Desulfovibrio sp. (26). The benzoate utilizer does not use other common aromatic compounds, C-j-Cy monocarboxyl ic acids, or C^-Cg dicarboxylic acids for growth. It is unable to use nitrate, sulfate, or fumarate as alternate electron acceptors. In coculture with the Desulfovibrio sp., the generation time is 132 hours, whereas in coculture with M. hungatei , the generation time is 166 hours. The presence of 80% H 2 in the gas phase inhibits utilization of benzoate. Descriptions of the two species of fatty acid-utilizing, hydrogenogenic bacteria are the only information available concerning the

PAGE 15

9 bacteria involved in the degradation of fatty acids in the fermentation of organic matter to methane. There are at least three major groups of bacteria involved in the fermentation of the polymeric substrates, cellulose, starch, protein, lipids, etc., to methane (6,18,21,29). The methane fermentation is divided into three distinct stages (6,21,29), as shown in Figure 1. Fermentative bacteria are the first of the three major groups to react. This group is a complex mixture of many obligate and facultative anaerobic bacterial species (12,21) which act on the polymeric substrates. Polysaccharides are initially hydrolyzed to their component sugars, which are then transported into the fermentative bacteria. The sugars are fermented primarily by the Embden-Meyerhof-Parnas pathway with the production of a variety of electron sink products such as acetate, propionate, and butyrate (6), as is shown in Figure 1. Proteins are hydrolyzed to amino acids which are then fermented to the products shown in Figure 1, in addition to other products such as isobutyrate, phenyl acetate, and phenyl propionate (6). Lipids are hydrolyzed to glycerol, a fermentable substrate, and long-chain fatty acids which are not fermented by the fermentative bacteria (6). The fermentative bacteria have been studied in detail, but additional research is needed. The terminal group of bacteria, the methanogens, is generally understood although not as well as the fermentative bacteria. These bacteria are essential to the complete anaerobic degradation of organic matter. Otherwise, organic acids that contain about as much energy as the polymeric substrates would accumulate and anaerobic degradation would cease (6,21). As shown in Figure 1, these unique bacteria utilize

PAGE 16

sCD +-> to cn SO c o r— +-> IB -a «3 Scn Oi T3 -Q O i. 0) ia c 10 on cn (T3 0J QJ i~ +-> CD +J l*o c o 5 c QJ 00 CD 5CD -i-> a» E
PAGE 18

12 a limited number of substrates, mainly acetate and F^-CC^, to produce methane, which contains 90% of the energy of the polymeric substrates (6,21). The third group of bacteria is the least understood of the three groups. These bacteria, the hydrogenogens, utilize the fermentation products of the first group and produce substrates that are utilizable by the methanogenic group. In Figure 1, this reaction is shown by the degradation of propionate and butyrate to acetate and h^-COg. The primary reason for the lack of information concerning these bacteria is the difficulty in isolating and manipulating them. Existing studies are of cocultures because the bacteria have not been isolated in an axenic cul ture. The hydrogenogens are obligate proton-reducing bacteria and produce H 2 with the concomitant oxidation of NADH (21). Therefore, the enzyme hydrogenase may be useful in the isolation of axenic cutlures of hydrogenogens. Hydrogenase is the collective term for enzymes which catalyze the reversible reaction (1,28): H 2 7± 2H+ + 2e ~ Thus, a cell-free extract containing hydrogenase, and other components necessary to transfer electrons from to an electron acceptor, may make possible the isolation of axenic cultures of hydrogenogenic bacteria. The utilization of fatty acids may be the rate-restricting step in the fermentation of organic matter to methane (19,21). The bacteria responsible for these reactions have not been well studied. Therefore,

PAGE 19

13 to better exploit the production of methane from biomass, these bacteria must be better understood. It is the objective of this study to increase the understanding of the fatty acid-utilizing bacteria by attempting to isolate cocultures of new species and to attempt the isolation of pure cultures of these bacteria. The knowledge gained, it is hoped, may be used to bring about the more efficient degradation of biomass to methane.

PAGE 20

MATERIALS AND METHODS Organisms, Media, and Growth Conditions Methanobacterium thermoautotrophicum strain AH, Methanospiril Turn hungatei strain JF-1 , and Escherichia coli ATCC 11303 were obtained from our culture collection. Desulfovibrio sp. strain G-ll was a gift gratefully received from Dr. Dave Boone. Mb . thermoautotrophicum was incubated at 55°C in medium number 2 of Balch et al . (3). Ms_. hungatei was incubated at 37°C in medium number 1 of Balch et al . (3). To prepare these media, stock solutions of trace minerals, trace vitamins, and various salt solutions were prepared and frozen until used. The stock solution of trace minerals contained in grams per 100 ml of distilled water, the following: nitrilotriacetic acid, 1.5 (to pH 7.0 with 4M KOH); MgSO^HpO, 3.0; MnS0 4 «2H 2 0, 0.5; NaCl, 1.0; FeS0 4 «7H 2 0, 0.1; CaCl 2 , 0.12; CaCl^H^, 0.1; ZnS0 4 « 7H 2 ), 0.18; CuS0 4 -5H 2 0, 0.01; A1K(S0 4 )-12H 2 0, 0.02; H 3 B0 3 , 0.01; NaMo0 4 « 2H 2 0, 0.11; and NiCl 2 «6H 2 0, 0.24. The stock solutions of trace vitamins contained, in milligrams per liter of distilled water, the following: biotin, 2; folic acid, 2; pyridoxine hydrochloride, 10; thiamine hydrochloride, 5; riboflavin, 5; nicotinic acid, 5; DL-calcium pentothenate, 5; vitamin B-j 2 , 0.1; p-aminobenzoic acid, 5; lipoic acid, 5; and 2-mercaptoethanesulfonic acid, 100. The salts stock solution contained, in grams per 100 ml distilled water, the following: (NH 4 ) 2 S0 4 , 3.0; NaCl, 6.0; MgS0 4 «7H 2 0, 1.3; CaCl 2 , 0.06; and KH 2 P0 4 , 3.0. The phosphate 14

PAGE 21

15 stock solution contained 3.0 g K 2 HP0 4 per 10 ml distilled water. The iron stock solution contained 0.02 g Fe(S0 4 ) 2 «7H 2 0 per 10 ml distilled water. The resazurin stock solution contained 0.5 mg of resazurin per ml of distilled water. The medium for Mb. thermoautotrophicum was composed of the following: trace minerals solution, 1 ml; salts solution, 10 ml; phosphate solution, 1 ml; (NH 4 ) 2 S0 4 , 2.7 g; resazurin solution, 1 ml; trace vitamins solution, 10 ml; iron solution, 1 ml; NaHC0 3> 5.0 g; cysteine hydrochloride-!^, 0.5 g; and distilled water to 1000 ml. The final gas phase was 80% H 2 ~20% Co 2 The pH was 7.2. The medium for Ms_. hungatei was composed of the following: trace minerals stock solution, 1 ml; salts solution, 10 ml; phosphate solution, 1 ml; sodium acetate, 2.5 g; sodium formate, 2.5 g; yeast extract (Difco), 2.0 g; Trypticase (BBL), 2.0 g; resazurin solution, 1 ml; trace vitamins solution, 10 ml; iron solution, 1 ml; NaHC0 3 , 5.0 g; cysteine hydrochloride*^, 0.5 g; and distilled water to 1000 ml. The final gas phase was 80% H 2 ~20% C0 2 The pH was 7.0. The medium for Desulfovibrio sp. strain G-ll was composed of the following: trace minerals solution, 1 ml; salts solution, 10 ml; phosphate solution, 1 ml; sodium acetate-3H 2 0, 0.68 g; sodium sulfate, 2.84 g; sodium formate, 2.0 g; Trypicase (BBL), 2.0 g; resazurin solution, 1 ml; trace vitamins solution, 10 ml; iron solution, 1 ml; NaHC0 3 , 5.0 g; cysteine hydrochloride*!-!,^, 0.5g; and distilled water to 1000 ml. The final gas phase was 80% H 2 -20% C0 2> The pH was 7.0. Escherichia coli was incubated statically at 37°C in 200 ml of the medium in a stoppered 500-ml serum bottle. The medium for E_. col i

PAGE 22

16 was Luria broth, which was composed of yeast extract, 5 g; Bactotryptone (Difco), 10 g; NaCl , 5 g; glucose, 1 g; and distilled water to 1000 ml. The final gas phase was N 2 . The pH was 6.8. Agar Noble (Difco) was added, final concentration 1.5%, when a solid medium was required. All of the media were sterilized by autoclaving for 20 minutes at 121°C. Preparation of Butyrate Medium The control medium for butyrate enrichments and butyrate studies was composed of the following: trace minerals solution, 1 ml; salts solution, 10 ml; phosphate solution, 1 ml; resazurin solution, 1 ml; clarified digester fluid, 50 ml; NaHC0 3 , 5.0 g; and cysteine hydrochloride-^, 0.5 g. The gas phase was 80% N 2 -20%C0 2 The pH was 7.2 to 7.4. After the control medium was dispensed to several serum tubes, sodium n-butyrate (Pfaltz and Bauer, Inc., Stamford, Conn.) was added to the remaining control medium to yield a final concentration of 0.3%. This medium was then dispensed, stoppered, and autoclaved for 20 minutes at 121°C . Anaerobic Techniques Principles of anaerobic techniques, as described by Hungate (14), were utilized to prepare the media— except Luria broth— and during experimental procedures. To prepare the media, all components, except trace vitamins, NaHCO-j, cysteine hydrochloride-^, clarified digester fluid, and butyrate, were added to distilled water in a round-bottom flask, boiled for several minutes, and cooled to room temperature in an ice bath while being sparged with 80% N ? -20% C0 ? . When the components

PAGE 23

17 were cool, the remainder of the components were added in accordance to the medium being prepared. After the pH was adjusted, if necessary, the media were dispensed into serum tubes (Bellco Glass Co., Vineland, N.J.) or serum bottles (Wheaton Scientific, Millville, N.J.) in which the gas phase had been replaced with 80% H 2 -20% C0 2 or 80% N 2 ~20% C0 2< The serum tubes or serum bottles were closed with butyl rubber stoppers (Bellco No. 2048-11800) which were held in place by crimped aluminum seals (Wheaton No. 224193). Gas mixtures were purchased from Matheson (Morrow, Ga.) and trace oxygen was removed by passing these gases over heated (350°C) copper turnings. Transfers of cultures were made with sterile needle and syringe units that had been made anoxic by aspirating sterile reduced medium or sterile anoxic gas. Prior to the use of all media, except Luria broth, a volume of 1.25% Na 2 S-9H 2 0 was injected into the media so that the sodium sulfide was di luted 1 :50. Luria broth was prepared and sparged with N« gas before being stoppered and sealed into 500 ml serum bottles (Wheaton). Descriptions of Ecosystems Studied Thermophilic and mesophilic digesters served as sources of inoculum for various studies. The digesters were similar in design and operation with the exception of source of initial inoculum and incubation temperature. The thermophilic digester was maintained at 55°C whereas the mesophilic digester was maintained at 40°C. The digesters were constructed from aspirator bottles and were stirred semicontinuously . Each

PAGE 24

18 day they received 16 g of feed consisting of 75% bermuda grass and 25% Universal cattle feed (Seminole Brands). The hydraulic detention times were 20 days. Freshwater sediment samples were taken at Bivens Arm, a eutrophic lake located near the University of Florida. Marine sediment samples were taken from Halodule sp. and Thalassia sp. seagrass beds at Seahorse Key, located near Cedar Key, Florida. Hypersaline sediment samples were taken from Great Salt Lake, Utah, and salterns, from San Francisco Bay, California. Descriptions of Butyrate Enrichments Enrichments from thermophilic and mesophilic digesters were begun by placing sludge from those digesters into butyrate' medium. Enrichments from Bivens Arm were initiated by placing sediments into butyrate medium. Enrichments with marine sediments were begun by using sulfatefree artificial seawater (SF-ASW) instead of distilled water as given in the description for butyrate medium. The SF-ASW was composed, in grams per liter of distilled water, of the following: NaCl , 21.15; MgCl 2 « 6H 2 0, 9.65; CaCl 2 , 1.0; NH 4 C1 , 0.25; KC1 , 0.5; KBr, 0.086; SrCl 2 «6H 2 0, 0.022; and H 3 B0 3 , 0.023. Enrichments with hypersaline sediments were begun by adding sodium n-butyrate, final concentration 0.3%, to the hypersaline sediments. Thermophilic Coculture Isolation A stable thermophilic enrichment was used as a source of inoculum for coculture isolation attempts. The enrichment was serially diluted in the control medium, and roll tubes were prepared from these dilutions.

PAGE 25

19 Each dilution was a source of inoculum for two butyrate control (BC) roll tubes, as well as for three butyrate experimental (BE) roll tubes which contained 0.3% butyrate. Before the tubes were rolled out, they received 0.5 ml of turbid, active Mb. thermoautotrophicum , which served as the hydrogenotroph. The roll tubes were incubated at 55°C. Mesophilic Coculture Isolation A stable mesophilic enrichment was used as a source of inoculum for coculture isolation attempts. The enrichment was serially diluted as described for thermophilic coculture isolation attempts. Also, duplicate BC and triplicate BE roll tubes were prepared at the appropriate dilutions. Desulfovibrio sp. strain G-ll , 0.5 ml of active culture per roll tube, was used as the hydrogenotroph. These roll tubes were incubated at 37°C. Gas Chromatography Methods Methane concentrations were measured by use of a Hewlett-Packard model 5880A gas chroma tograph. Gases were separated in a 1.8-m by 1.0mm stainless steel column packed with Carbosphere mesh 80/100 (All tech Associates, Inc., Deerfield, 111.), and were measured with a thermal conductivity detector. Helium was the carrier gas. Column and detector temperatures were maintained at 130 and 145°C, respectively. Methane concentrations were determined by comparison to standards (Ultra High Purity Methane, Matheson). Gas pressures in stoppered vessels were determined by use of a pressure transducer (Setra System, Inc., Acton, Mass.). Volatile fatty acids (VFAs) were measured by use of a HewlettPackard 5880A gas chromatograph. They were separated in a 1.8-m by

PAGE 26

20 1.0-itm glass column packed with 8% SP1000, 2% SP1200, and 1.5% H 3 P0 4 on 80/100 mesh Chromosorb W AW 8100 (Supelco, Bellefonte, Pa.), and were measured with a flame ionization detector. Helium was the carrier gas. Injector, oven, and detector temperatures were 145, 130, and 175°C, respectively. Samples were mixed with an equal volume of 4% o-phosphoric acid, centrifuged at 12,800 x g for 2 minute (22°C), and the supernatant was frozen until VFA determinations were made. Each VFA concentration was determined by comparison to standards. Preparation and Use of E. coli Membrane Fragments To Attempt Isolation of Pure Cultures of H.ydrogenoqenic Bacteria Statically grown E_. coli were centrifuged at 5,000 x g for 10 minutes (5°C) and washed twice with buffer. The buffer was composed of the following: NaCl , 0.4M; MgS0 4 «7H 2 0, 0.02M; and KH 2 P0 4 , 0.1 M. The pH was 7.0. Cells and buffer were prechilled to 5°C. Membrane fragments were prepared by passing the cell suspension twice through a French pressure cell at 20,000 psi. The lysate was centrifuged at 5,000 x g for 10 minutes (5°C), and the supernatant was frozen at -20°C until used. The lysate was sterilized by being passed through a sterile 0.2-um membrane filter. Fumarate, final concentration 20 mM, was filter sterilized (Falcon 7103) and aseptically added to the BC or BE medium. The source of inoculum was the stable mesophilic butyrate enrichment. Anaerobic roll tubes were prepared so that they contained lysate and fumarate, lysate or fumarate, or no addition. The roll tubes were incubated at 37°C.

PAGE 27

21 Microscopy and Photomicroscopy A Carl Zeiss Standard WL microscope equipped for epi fluorescence was used for observation of wet mounts and photomicroscopy. Light of the 420-nm wavelength was provided by a mercury light source (HBO 50 DC 3) and a filter set comprised of an exciter filter (BP 390-440), a chromatic beam splitter (FT 460), and a barrier filter (LP 475). A Leica camera back was attached to the microscope for photomicroscopy. Kodak Technical Pan film 2415 was exposed for times in accordance to previously exposed test rolls. The film was developed according to Kodak instructions and printed on Kodak F5 RC or Polycontrast RC paper.

PAGE 28

RESULTS Production of Methane When Various Ecosystems Were Enriched with Butyrate When various ecosystems were enriched with butyrate, not all produced methane (Table 3). Anaerobic digesters and freshwater sediments produced methane with little or no lag, whereas marine sediments in sulfate-free artificial seawater required about 2 weeks for methane production to begin. Methane was not produced after several months of incubation when hypersaline sediments were enriched with butyrate. Description of Thermophilic Butyrate Enrichments The population of butyrate-util izing bacteria in a 55°C digester was enumerated by the 5-tube most-probable-number (MPN) method. After a 4-week incubation period, 4.5 x 10 butyrate-util izing bacteria per ml sludge were found. The lower dilution MPN tubes produced significantly more methane than did the higher dilution MPN tubes. Examination revealed that the lower dilution MPN tubes contained a Methanosarcina sp. Butyrate enrichments were established by use of each of these distinct dilution types as an inoculum. The greater methane production by the enrichment containing the Hethanosarcina sp. is shown in Figure 2 Acetate accumulated in the enrichment without the Methanosarcina sp. but disappeared in the enrichment with the Methanosarcina sp. Butyrate was utilized by both enrichments. The Methanosarcina sp. was isolated and would not grow alone when H 2 -C0 2 was the only methanogenic substrate 22

PAGE 29

23 Table 3. Examination of various ecosystems for methane production from butyrate enrichments Source of enrichment a Methane produced Lag period before Onset OT mctnailc production Thermophilic digester Yes None Mesophilic digester Yes None Bivens Arm Yes 3 days Halodule, sp. seagrass bed Yes 14 days Thalassia, sp. seagrass bed Yes 14 days Great Salt Lake No San Franscisco Bay saltern No indicates production of methane in medium with butyrate minus methane production in medium without butyrate.

PAGE 30

C\J sCD

PAGE 31

25

PAGE 32

26 present, growing only in the presence of acetate. This organism was tentatively identified as Methanosarcina strain TM-1 (35). Both enrichments contained rod-shaped bacteria that autof 1 uoresced under examination by 420 nm epi fluorescence microscopy, indicating the presence of Factor F420 found in methanogens (10,25). This methanogenic rod-shaped bacterium, tentatively identified as a strain of Methanobacterium thermoautotrophicum , utilized H 2 -C0 2 for growth. There were several other rod-shaped nonfluorescing bacteria present. Therefore, it was difficult to know which bacterium utilized butyrate. The enrichments were transferred every 7 to 10 days after being analyzed and were found positive for methane production. After 4 months, a stable enrichment was obtained. A stable enrichment was defined as having a few morphotypes present consistently. M. thermoautotrophicum was always present as the largest population of bacteria, generally comprising 90% of the bacteria in each microscopic field. Isolation of Thermophilic Butyrate-Utilizing Cocultures The stable thermophilic enrichment was used as a source of inoculum for attempts to isolate butyrate-uti 1 izing cocultures. M. thermoautotrophicum was used as a hydrogenotrophic partner. Anaerobic roll tubes were incubated for about 4 weeks until methane was detected in the gas phase and colonies appeared. Basically, two colony types were present. One colony type was brownish, granular, and irregular in shape; averaged 2 mm in diameter (Figure 3(A)); and autof 1 uoresced when exposed to 420 nm light. This colony resembled Methanosarcina TM-1, but, when a wet mount of the colony was examined, it was found to be composed of the

PAGE 33

Figure 3. Photomicrographs of thermophilic coculture colony types. (A) Colony type that contains Methanosarcina sp., Methanobacterium thermoautotrophicum , and a curved rod. (B) Colony type that contains Mb. thermoautotrophicum and a curved rod.

PAGE 34

28

PAGE 35

29 Methanosarcina sp. , Mb . thermoautotrophi cum , and a nonfluorescing, rodshaped bacterium. The second colony type (Figure 3(B)) was white and circular with an entire margin, average 1 mm in diameter, and autofluoresced when exposed to 420 nm light. Upon microscopic examination, the colony was found to be composed of Mb. thermoautotrophi cum and a nonfluorescing rod-shaped bacterium. The two colony types were found to be predominately composed of Mb. thermoautotrophi cum . The colonies appeared only in the BE medium and not in the BC medium. A photomicrograph of the coculture is shown in Figure 4. The Methanosarcina sp. is not shown in this photomicrograph. Mb. thermoautotrophicum , as shown in the photomicrograph, was rod shaped, autof luoresced at 420 nm, and had greater contrast than the butyrate-util izing bacterium. Several examples are indicated by the single arrows. The butyrateuti 1 izing bacterium was a slightly curved, gram-negative rod that averaged 2 to 3 urn in length, occurred singly, was nonmotile, and did not contain spores. Several examples are indicated by the double arrows in Figure 4. The colonies were picked and placed into fresh medium and were rolled out. Once colonies appeared again, they were picked and placed onto slants prepared in serum tubes. After 6 weeks methane was not present in the gas phase and growth had not occurred. The enrichment was rolled out again, and once colonies appeared they were picked and placed onto slants. Again, methane was not produced and growth did not occur. The entire process was repeated with the same results. The colonies were viable because they could be placed into liquid medium and would grow. Because this culture only produced two colony types that

PAGE 36

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PAGE 37

31

PAGE 38

32 could not be routinely grown on slants, it should probably be referred to as a highly purified enrichment. For the sake of brevity, however, it is referred to as a coculture. The coculture was maintained on slants by the injection of active enrichments onto the slants. Studies on the Thermophilic Coculture The coculture was incubated at various temperatures to determine whether the butyrate-utilizing coculture was capable of growth at other temperatures. Table 4 shows the differing temperatures tested and the results. When Methanospi ri 1 1 urn hungatei , a mesophilic, hydrogenutilizing methanogen, was added to the coculture, only trace amounts of methane were formed at 37°C. The amount of methane formed was less than 5% of that formed when the coculture was incubated at 55°C. Methane was not produced when the coculture was incubated at 45 or 70°C. The coculture was examined to see whether clarified digester fluid (CDF) could be replaced by rumen fluid (RF) or deleted from the medium. Table 5 shows that when neither CDF nor RF was a component of the medium, methane production was greatly diminished. The addition of rumen fluid did not stimulate methane production and was inhibitory at concentrations of 20% and above on day 17. Clarified digester fluid addition resulted in consistent methane production at all concentrations tested. When the basal medium without butyrate (BE) contained RF, methane was produced in greater quantities as compared to when the BC medium contained CDF. This result indicated that greater quantities of methanogenic substrates were present in the RF. The quantity of methanogenic substrates in the media containing 20 and 30% RF inhibited the production of methane from butyrate (Table 5).

PAGE 39

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PAGE 40

34 Table 5. Effects of various concentrations of rumen fluid (RF) or clarified digester fluid (CDF) on the percentage of methane production by thermophilic coculture Methane production (%) Day 3 Day 9 Day 17 No addition 1 1
PAGE 41

35 The effect of the addition of 0.1% yeast extract to the thermophilic coculture is shown in Table 6. The addition of yeast extract resulted in a 142% increase in methane produced after 18 days' incubation by the coculture containing the acetate-utilizing Methanosarcina sp. At day 22, the increase in methane production by the coculture with the Methanosarcina sp. was 28%. Yeast extract did not stimulate methane production and showed a slight inhibition of methane production in the coculture when the Methanosarcina sp. was absent. Antibiotics known to affect cell wall synthesis of eubacteria but not archaebacteria were added to the coculture. Figure 5 shows that penicillin G(3000U/ml) and D-cycloserine (0.1 mg/ml ) caused the complete inhibition of methane production by the coculture. The presence of hydrogen in the gas phase (80% ^-20% C0 2 ) inhibited the utilization of butyrate by the coculture (Figure 6). The gas phase was replaced every 2 days until day 8. At that time, indicated by the arrow, hydrogen was not detected in the gas phase because of its removal by Mb. thermoautotrophicum . The culture was allowed to continue incubating in the absence of hydrogen to determine if the butyrate utilizers had been killed or merely inhibited by the hydrogen. After a lag period, butyrate utilization began with the butyrate being rapidly utilized (Figure 6). In general, the enrichments utilized butyrate faster and produced methane quicker when they were incubated without shaking. When the enrichments were shaken, methane production showed a longer lag period, but if the shaking was stopped, methane production increased.

PAGE 42

36 Table 6. Effects of the addition of 0.1% yeast extract to thermophilic butyrate-utilizing enrichments Methane production (umoles) a Without Methanosarcina With Methanosarcina -YE +YE -YE +YE Day 2 0 0 0 0 Day 11 0.9 1.8 1.0 0.1 Day 18 36.9 54.6 99.4 240.8 Day 22 62.1 53.5 244.4 313.3 a Values represent means of duplicate tubes.

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41 Effects of Pumping Butyrate into a Thermophilic Digester Volatile fatty acids were found in low concentrations in the thermophilic digester. Acetate concentration was 3 to 4 ymoles/ml of sludge, whereas n-butyrate concentration was less than 0.5 ymoles/ml of sludge. A stock solution of sodium n-butyrate was prepared so that it could be pumped into the digester at the rate of 10 ymoles/ml of sludge per day. Figure 7 shows the theoretical accumulation of butyrate if it were not utilized by the digester and the actual concentrations measured. Butyrate did not accumulate in the digester when pumped at this concentration. The concentrations of acetate and propionate are shown in Figure 8. Acetate concentration increased from about 3 ymoles/ml of sludge to about 35 ymoles/ml of sludge by day 23. Propionate concentration increased to about 3.3 ymoles/ml of sludge by day 23. The ratio of gas produced by the butyrate-amended digester versus the control digester was initially 1.5 and by day 23 had stabilized at about 1.4 (Figure 9). The percentage of methane in the gas phase increased from 58% to 68%. The pH increased from 7.3 to 7.8, where it remained stable. The digester maintained consistent levels of acetate (33 to 35 ymoles/ml of sludge), butyrate (0.7 to 0.75 ymoles/ml of sludge), and ratio of gas production (1.36 to 1.37) from day 18 to day 23. These levels indicated that a stable digestion had been attained and that the rate of addition of butyrate was not exceeding the capability of the digester for utilization of butyrate. To determine the concentration of butyrate that would have to be infused into the digester in order to exceed the ability of the digester to maintain a stable digestion, the rate of addition would need to be increased. Therefore, on day 24, as indicated

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48 by the arrows in Figures 5, 7, and 8, the concentration of sodium n-butyrate infused into the digester was doubled so that the rate of addition would be 20 umoles butyrate/ml of sludge per day. The levels of all VFAs began to increase. By day 36 the concentrations, in umoles/ ml, were acetate, 167; propionate 16.9; and n-butyrate, 8.9. During this increase of VFAs, the biogas production ratio remained between 1.4 and 1.5 and on day 36 was 1.35. Because of the rapid increase in VFAs, the addition of butyrate was stopped. Enrichments from Mesophilic Ecosystems Butyrate enrichments were begun with a mesophilic, 40°C digester as the source of inoculum. These enrichments were analyzed each week for methane production, and the enrichments that produced the greatest quantities of methane were transferred to fresh medium. After about 8 transfers the enrichment was analyzed for VFA concentrations. The acetate concentration was 17.2 umoles/ml (acetate not detected in uninoculated medium), whereas the butyrate concentration was 0.16 umoles/ ml (about 22 umoles/ml in uninoculated medium). Microscopic examination showed that the predominant bacterium was an irregular coccus that autofluoresced when exposed to 420 nm of light. A bacterium resembling Methanosarcina barkeri was also observed. In addition, nonf luorescing , short, nonmotile, curved-rod shaped and long, nonmotile, rod-shaped bacteria were observed. Sediments from a eutrophic freshwater lake, Bivens Arm, were used to begin butyrate enrichments. After weekly transfers for several months, the enrichments were examined by phase-contrast microscopy. The bacteria in the most predominant numbers were Methanospi rill urn hungatei

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49 and a Methanococcus sp. There were also small numbers of Methanosarcina barkeri present. In addition, there were nonfluorescing long rods and short curved rods present. Eubacterial antibiotics were added to mesophilic sludge enriched with butyrate. In the enrichment without antibiotics, about 300 umoles of methane were found in the gas phase, whereas when antibiotics were present, the gas phase contained only about 40 umoles of methane (Figure 10). The effect of the antibiotics was not on the hL-^-utilizing methanogens because methane was formed in the presence of the antibiotics when 80% H 2 ~20% C0 2 was the gas phase. All of the butyrate in the enrichment without antibiotics was utilized, whereas 87% of the butyrate remained in the enrichment with antibiotics. Isolation of Mesophilic Butyrate-Util izing Cocultures A stable mesophilic enrichment was the source of inoculum for attempts to isolate butyrate-util izing cocultures. Desulfovi brio G-11 was used as the hydrogenotrophic partner. After 28 days of incubation at 37°C, colonies with blackened centers appeared in roll tubes with butyrate and sulfate, but not in roll tubes with butyrate alone or sulfate alone. Also, colonies which fluoresced when exposed to 420 nm of light were observed. Both types of colonies were picked from the roll tubes and placed onto slants of butyrate-containing medium. The fluorescent colonies grew within 10 days on the slants and produced methane. However, there were four morphotypes of bacteria present. Therefore, this culture was rolled out again in an attempt to isolate colonies composed of two members. The black-centered colonies produced visible growth on slants and were transferred to liquid broth for further study.

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52 Attempts To Isolate Butyrate-Utilizing Hydrogenogens in Pure Culture with E. coli Membrane Fragments E. Coli were statically grown for 6 hours at which time hydrogen was present in the gas phase. The lysate from French Pressure-cell passage was filter sterilized and injected into molten agar tubes containing stable mesophilic enrichment. The amount injected was 0.1 ml (57.0 ± 1.6 mg dry weight per ml). The roll tubes were incubated at 37°C. After about 4 weeks, colonies were present in the tubes containing butyrate and E_. coli membrane fragments and fumarate. These colonies were not present in tubes with butyrate alone or E_. coli membrane fragments alone. The colonies were white, circular with entire margins, and 1 to 2 mm in diameter.

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DISCUSSION Organic matter is degraded exclusively to methane and carbon dioxide in nongastrointestinal ecosystems where light, nitrate, oxygen, and sulfate are absent. This degradation requires at least three groups of bacteria (21,29): fermentative, hydrogenogenic, and methanogenic. The hydrogenogenic bacteria are the least understood of the three groups, with only two species being known (5,22,23). When several ecosystems were enriched with butyrate, only digesters and freshwater sediments produced methane in a short or no lag period. These ecosystems continually receive organic matter which undergoes degradation to methane. Thus, these ecosystems have a population of butyrate-utilizing bacteria and can readily degrade butyrate to methane. When seagrass beds were enriched with butyrate in sul fate-free artificial seawater, methane was not produced until after a 2-week lag period. The lag in methane production may have resulted from butyrate being utilized by fatty acid-utilizing, sulfate-reducing bacteria (SRB) which were reducing the sulfate that remained in the sediments. These enrichments smelled strongly of H2S before and after methane production began. Desulfovibrio spp. will degrade lactate (7,20) or ethanol (7) in the absence of sulfate when methanogens are present to remove hydrogen, the electron sink product. In these marine sediments, once sulfate is depleted the methanogens may participate in the degradation of butyrate by removing H ? produced by SRB. The inability of the microflora 53

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54 of hypersaline sediments to produce methane when enriched with butyrate indicates that butyrate may not be a typical substrate in the ecosystems examined. The thermophilic digesters were chosen for more detailed study. Thermophilic digestion may have several advantages over mesophilic digestion. Varel et al . (32) reported the advantage of being able to reduce retention times to less than 6 days, at thermophilic (>45°C) temperatures. Also, different microflora of various plant biomasses added to a thermophilic digester would not compete with the digester microflora. If competition were allowed, then it might be possible for destabilization of the digester to occur. The thermophilic digester had ca. 5 x 10 butyrate-util izing bacteria/ml of sludge, a finding similar to that in another study (17). When a thermophilic Methanosarcina sp. that utilized acetate, but not H 2 -C0 2 , was present in butyrate enrichments, greater quantities of methane were produced and the enrichments seemed more stable. The enhanced methane production resulted from the decarboxylation of acetate with the concomitant production of methane by the Methanosarcina sp. Table 2 shows the equation for the oxidation of butyrate which resulted in the production of acetate and hydrogen. The removal of hydrogen made it thermodynamically possible for the reaction to occur and allowed the hydrogenogen to continue metabolic processes because of the recycling of NADH. The additional removal of acetate made the thermodynamics of the equation even more negative, thus enhancing the stability of the enrichments. Thermophilic methanogenic butyrate enrichments were composed primarily of the Hp-utilizing Methanobacterium thermoautotrophicum , with other morphotypes present in smaller numbers. Since M.

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55 thermoautotrophicum was present in large numbers in the butyrate enrichments, it was the bacterium of choice as a hydrogenotrophic partner for coculture isolation attempts. When butyrate enrichments were rolled out with M. thermoautotrophicum , two colony types were observed. When transferred onto slants, the colonies did not grow, possibly because of the change in the oxidation-reduction potential occurring when the serum tubes were opened for inoculation. This lack of growth might be overcome if a reducing agent were used which would maintain a lowered oxidation-reduction potential for a greater length of time. The two previously described hydrogenogenic bacteria are mesophilic (5,22,23). In order to determine whether the butyrate-utilizing bacterium isolated from the thermophilic digester was in fact a thermophile, the coculture was incubated at several temperatures. The data for this experiment are found in Table 4. When the coculture was incubated at 37°C, methane was not detected. The lack of methane production could possibly have resulted from the inability of M. thermoautotrophicum to grow. Therefore, a mesophilic l^-CCL-utilizing methanogen, Methanospi rill urn hungatei , used in the study of the two reported hydrogenogens (5,22,23), was added to the coculture. If the butyrate-utilizing bacterium could produce H 2 at 37°C. then M. hungatei would oxidize it and methane should be detected. Table 4 shows that a small amount, ca. 14 umoles, of methane was produced after 26 days of incubation. When the coculture was incubated at 55°C, the temperature of isolation, ca. 300 umoles of methane were produced by day 13, half the time required for production of 14 ymoles of methane by the coculture with M. hungatei at 37°C. This indicated that the butyrate utilizer was a thermophilic

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56 bacterium and, hence, was different from S^. wolfei , a mesophile. In addition, the thermophilic butyrate utilizer was a nonmotile, slightly curved rod, 2 to 3 urn in length, whereas S_. wolfei exhibited sluggest motility and was 7 urn in length (22). Therefore it appears that this bacterium is a new species of anaerobic hydrogenogens. It required a growth factor (or factors) that was present in clarified digester fluid (CDF) but not in rumen fluid (RF) (Table 5). The addition of CDF in increasing concentrations resulted in a stimulation of methane production which may have aided in the isolation and maintenance of these bacteria. The addition of 0.1% yeast extract to the cocultures containing Methanosarcina sp. resulted in a 142% increase in methane production after 18 days of incubation. Yeast extract can replace CDF for growth of methano sarcina TM-1 (P. A. Murray and S. H. Zinder, Abstr. Annu. Meet. Am. Soc. Microbiol., 1983, 18, p. 14). It appeared that the addition of yeast extract to medium with CDF resulted in the enhancement of methane production from acetate by the Methanosarcina sp. It may also be of benefit in the isolation and maintenance of the butyrate-utilizing bacteria. The addition of penicillin and D-cycloserine completely inhibited methane production by the thermophilic coculture (Figure 5). wolfei was inhibited by the addition of penicillin and has been shown to possess a peptidoglycan cell wall (22). Since the thermophilic coculture was inhibited by antibiotics that inhibit eubacteria, it appeared that the butyrate-utilizing bacteria were eubacterial and possessed a peptidoglycan call wall like that of S. wol fei . The presence of 80% hydrogen in the gas phase inhibited butyrate utilization by S. wol fei (22). When the thermophilic coculture was placed under an 80% hydrogen gas phase, butyrate was not utilized

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57 (Figure 6). However, when hydrogen was removed from the thermophilic coculture, butyrate was utilized (Figure 6). Thus it is indicated that hydrogen inhibited but did not kill the thermophilic butyrate-util izing bacteria. The addition of butyrate to the thermophilic digester at the rate of 10 umoles/ml of sludge per day did not result in the accumulation of butyrate. Thus, it is possible that if this digester could be loaded at a higher rate, the result would be the production of a higher concentration of butyrate. The addition of butyrate at the concentration of 20 umoles/ml of sludge per day resulted in an unstable digestion. Therefore, the maximum concentration of butyrate that could be infused into this digester and still result in a stable digestion was between 10 and 20 umoles/ml of sludge per day. Hydrogenogenic bacteria have been isolated only in cocultures with hydrogenotrophic bacteria. In order to better study these bacteria, the isolation of pure cultures is necessary. Because the hydrogenogenic bacteria are inhibited by hydrogen, the concentration of hydrogen must be maintained close to zero. The use of hydrogenase-containing, cellfree systems may make it possible to maintain the lowered concentration of hydrogen. As a test of this possibility, butyrate-util izing enrichments from mesophilic ecosystems were initiated to provide inoculum for the isolation of cocultures from which a pure culture might be isolated directly or from the enrichments. An active enrichment was diluted and rolled out in media with various additions. These additions were varied to allow for a number of controls. After 21 days of incubation at 37°C, white colonies were found in anaerobic roll tubes that contained E. coli

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58 membrane fragments, fumarate, and butyrate. If any one of these components was not present in the anaerobic roll tubes, the white colonies did not appear. Thus, it appears that the membrane fragments were able to remove and may be useful in isolating pure cultures of hydrogenogens. In 1951, Stadtman and Barker (30) reported a highly purified culture that degraded butyrate to acetate and methane. The methane-producing bacterium was named Methanobacterium suboxydans . This culture has since been lost, and attempts to reisolate it have been unsuccessful. In the present study, an experiment was attempted to isolate a butyrateutilizing methanogen. Because methanogens are capable of growing in the presence of eubacterial antibiotics that act on the cell wall, sludge from a mesophilic digester was enriched with butyrate in the presence and absence of antibiotics. Figure 10 indicates that a small amount of methane was produced in the presence of antibiotics. The methane may have resulted from the inactivation of antibiotics by the high concentration of organic matter in the digester sludge. Such a result does not exclude the possibility that a butyrate-utilizing methanogen existed. However, additional research is needed to determine with certainty whether one did. Populations of bacteria in microbial communities exist in a variety of symbiotic relationships. The relationship between hydrogenogenic and hydrogenotrophic bacteria is described as syntrophic by several authors (5,18,21,22,23,24). Alexander (2) defines syntrophism as a relationship which entails a bilateral exchange of growth factors. As presently understood, interspecies hydrogen transfer involves the unidirectional

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59 transfer of a substrate. When one organism uses the excretion of a second as a substrate for growth, the relationship is commensal istic (2). Therefore, what is presently referred to as a syntrophic relationship may in fact be a commensal istic one. As more information becomes available, it may be necessary to redefine the relationship that exists between hydrogenogens and hydrogenotrophs . In summary, several ecosystems were studied to determine their methane-producing capability when enriched with butyrate. A new species of a thermophilic butyrate-utilizing bacterium was established in coculture. This bacterium was a gram-negative, slightly curved rod that measured 2 to 3 urn in length. Most cells occurred singly. Spores were not observed and cells did not exhibit motility. Growth was inhibited by penicillin. Hydrogen inhibited growth but this inhibition was reversed upon the removal of the hydrogen. Growth did not occur at 37°C in the presence of a mesophilic hydrogen-utilizing methanogen. Mesophilic methane-producing enrichments were initiated to isolate cocultures of butyrate-utilizing bacteria and to attempt the isolation of pure cultures of these bacteria. The isolation of pure cultures was not completely successful and is still being attempted.

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REFERENCES 1. Adams, M. W. , L. E. Mortenson, and J.-S. Chen. 1981. Hydrogenase. Biochem. Biophys. Acta 594:105-176. 2. Alexander, M. 1971. Microbiol ecology. John Wiley & Sons, Inc., New York. 3. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: revaluation of a unique biological group. Microbiol. Rev. 43:260-296. 4. Barker, H. A. 1940. Studies upon the methane fermentation. IV. The isolation and culture of Methanobacterium omelianskii . Antonie van Leeuwenhoek 6:201-220. 5. Boone, D. R. , and M. P. Bryant. 1980. Propionate-degrading bacterium Syntrophobacter wolinii sp. nov. gen. nov. , from methanogenic ecosystems. Appl . Environ. Micobiol. 40:626-632. 6. Bryant, M. P. 1979. Microbial methane production— theoretical aspects. J. Anim. Sci. 48:193-201. 7. Bryant, M. P. L. L. Campbell, C. A. Reddy, and M. R. Crabill. 1977. Growth of desulfovibrio in lactate or ethanol media low in sulfate in association with ^-utilizing methanogenic bacteria. Appl. Environ. Microbiol. 33:1162-1169. 8. Bryant, M. P., E. A. Wolin, M. J. Wolin, and R. S. Wolfe. 1967. Methanobacillus omelianskii , a symbiotic association of two species of bacteria. Arch. Mikrobiol. 59:20-31. 9. Chen, M. , and M. J. Wolin. 1977. Influence of CH4 production by Methanobacterium ruminantium on the fermentation of glucose and lactose by Selenomonas ruminantium . Appl. Environ. Microbiol. 34:756-759. 10. Doddema, H. J., and G. D. Vogels. 1978. Improved identification of methanogenic bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 36:752-745. 11. Ferry, J. G. , and R. S. Wolfe. 1976. Anaerobic degradation of benzoate to methane by a microbial consortium. Arch. Microbiol. 197:33-40. 60

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61 12. Hobson, P. N. , S. Bousfield, and R. Summers. 1974. Anaerobic digestion of organic matter, p. 131. In CRC critical reviews in environmental control. Chemical Rubber Co., Cleveland, Ohio. 13. Hungate, R. E. 1966. The rumen and its microbes. Acad. Press, Inc. , New York. 14. Hungate, R. E. 1969. A roll tube method for cultivation of strict anaerobes. Methods Microbiol. 3B : 1 1 7-1 32 . 15. Iannotti, E. L., D. Kafkewitz, M. J. Wolin, and M. P. Bryant. 1973. Glucose fermentation products of Ruminococcus albus grown in continuous culture with Vibrio succinogenes : changes caused by interspecies transfer of H2J. Bacterid. 114:1231-1240. 16. Lathem, M. J., and M. J. Wolin. 1977. Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacteri urn rumi nanti urn . Appl . Environ. Microbiol. 34:297301. 17. Mackie, R. S. , and M. P. Bryant. 1981. Metabolic activity of fatty acid-oxidizing bacteria and the contribution of acetate, propionate, butyrate, and CO2 to methanogenesis in cattle waste at 40 and 60°C. Appl. Environ. Microbiol. 41:1363-1373. 18. Mah, R. A. 1982. Methanogenesis and methanogenic partnerships. Phil. Trans. R. Soc. Lond. B. 297:599-616. 19. McCarty, P. L. 1971. Energetics and kinetics of anaerobic treatment, p. 91. lr\_ R. F. Gould (ed.), Anaerobic biological treatment processes. Advances in Chemistry Series 105. Amer. Chem. Soc, Washington, D.C. 20. Mclnerney, M. J., and M. P. Bryant. 1981. Anaerobic degradation of lactate by syntrophic associations of Methanosarcina barken' and Desulfovibrio species and effect of H2 on acetate degradation. Appl. Environ. Microbiol. 41:346-354. 21. Mclnerney, M. J., and M. P. Bryant. 1981. Basic principles of bioconversions in anaerobic digestion and methanogenesis, p. 277296. J_n S. S. Sofar and 0. Zaborsky (ed.), Biomass conversion processes for energy and fuels. Plenum Publishing Corp., New York. 22. Mclnerney, M. J., M. P. Bryant, R. B. Hespell , and J. W. Costerton. 1981. Syntrophomonas wolfei gen, nov. sp. nov. , an anaerobic, syntrophic, fatty acid-oxidizing bacterium. Appl. Environ. Microbiol . 41 : 1029-1039. 23. Mclnerney, M. J., M. P. Bryant, and N. Pfenning. 1979. Anaerobic bacterium that degrades fatty acids in syntrophic association with methanogens. Arch. Microbiol. 122:129-135.

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62 24. Mclnerney, M. J., R. I. Mackie, and M. P. Bryant. 1981. Syntrophic association of a butyrate-degrading bacterium and Methanosarcina enriched from bovine rumen fluid. Appl . Environ. Microbiol. 41:826-828. 25. Mink, R. W. , and P. R. Dugan. 1977. Tentative identification of methanogenic bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33:713-717. 26. Mountfort, D. 0., and M. P. Bryant. 1982. Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge. Arch. Microbiol. 133:249-256. 27. Scheifinger, C. C. , B. Lineham, and M. J. Wolin. 1975. H2 production by Selenomonas ruminantium in the absence and presence of methanogenic bacteria. Appl. Environ. Microbiol. 29:480-483. 28. Schlegel, H. G. , and K. Schneider. 1979. Introductory report: Distribution and physiological role of hydrogenases in microorganisms, p. 15-44. In H. G. Schlegel and K. Schneider (ed.), Hydrogenases: Their catalytic activity, structure and function. E. Goltze KG, Gottingen. 29. Smith, P. H. 1980. Studies of methanogenic bacteria in sludge. EPA-600/2-80-093. Research report, U.S. Government Printing Office, Washington, D.C. 30. Stadtman, T. C. , and H. A. Barker. 1951. Studies on the methane fermentation. VIII. Tracer experiments on the fatty acid oxidation by methane bacteria. J. Bacterid. 61:67-80. 31. Thauer, R. K. , K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic anaerobic bacteria. Bacterid. Rev. 41:100. 32. Varel, V. H., A. G. Hashimoto, and Y. R. Chen. 1980. Effect of temperature and retention time on methane production from beef cattle waste. Appl. Environ. Microbiol. 40:217-222. 33. Wolin, M. J. 1982. Hydrogen transfer in microbial communities, p. 323-356. ]n A. T. Bull and J. H. Slater (ed.), Microbial interactions and communities, vol. 1. Academic Press, London. 34. Wolin, M. J., and T. L. Miller. 1982. Interspecies hydrogen transfer: 15 years later. ASM News 48:561-565. 35. Zinder, S. H., and R. A. Mah. 1979. Isolation and characterization of a thermophilic strain of Methanosarcina unable to use H2-CO2 for methanogenesis. Appl. Environ. Microbiol. 38:9961008.

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BIOGRAPHICAL SKETCH John Michael Henson, son of John T. and Martha J. Henson, was born on August 11, 1952, in Greer, South Carolina. After graduating from Greer Senior High School in June 1970, he attended Furman University, Greenville, South Carolina. In 1971 he transferred to the University of South Carolina, Columbia, where in August 1975 he received the degree of Bachelor of Science with a major in biology. In 1976 he entered the graduate program at Clemson University, Clemson, South Carolina, and in 1978 received the degree of Master of Science with a major in microbiology. That fall he joined the faculty at Presbyterian College, Clinton, South Carolina, and taught general biology, microbiology, and cell biology. In 1980 he moved to Gainesville, Florida, and entered the graduate program in the Department of Microbiology and Cell Science at the University of Florida. On January 4, 1975, he married Ellen Adams Dobson. They have two children, Martha Anne ("Marne"), 6, and Jonathan Adams, 16 months. Henson served in the U.S. Naval Reserve from 1972 to 1978 as a field medical service technician attached to the U.S. Marine Corps Reserve. 63

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Paul H. Smith, Chairman Professor of Microbiology and Cell Science I certify that I conforms to acceptable adequate, in scope and Doctor of Philosophy. have read this study and that in my opinion it standards of scholarly presentation and is fully quality, as a dissertation for the degree of Arnold S. Bleiweis Professor of Microbiology and Cell Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Roge^ A. Nordstedt Associate Professor of Agricultural Engineering This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 1933 Dean /^College of Agriculture Dean for Graduate Studies and Research