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University Microfilms International 300 N. ZEEB RD., ANN ARBOR, Ml 48106

8222068

Corder, Robert Elwin

THE DEVELOPMENT OF AN INTEGRATED ANAEROBIC SYSTEM FOR THE CULTIVATION AND CHARACTERIZATION OF METHANOGENIC

The Ohio State University PH.D. 1982

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University Microfilms International

THE DEVELOPMENT OF AN INTEGRATED ANAEROBIC

SYSTEM FOR THE CULTIVATION AND CHARACTERIZATION

OF METHANOGENIC BACTERIA

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate School of

The Ohio State University

By

Robert Elwin Corder, B.S., M.S.

******

The Ohio State University

1982

Reading Committee: Approved By:

Dr. James I. Frea Dr. Robert M. Pfister Dr. Chester I. Randles Dr. Melvin S. Rheins Advisor Department of Microbiologyiologv ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. J.I. Frea, for his assistance during my graduate career. I also thank Dr. C.I. Randles,

Dr. M.S. Rheins, and Dr. R.M. Pfister for critiqueing this dissertation.

Financial support from the Department of Microbiology, The Ohio State

University, and the State of Ohio was greatly appreciated and was vital to the completion of this work.

Mr. Paul Hamilton supplied the agarose gel depicting the restriction analysis of different . Dr. L.A. Hook supplied the antibiotic sensitivities for the different producing cocci.

Discussions with Dr. Hook and Mr. Hamilton were helpful and greatly appreciated.

Finally, and most importantly, I thank my parents, Jack and Mary

Corder, and my wife, Ruth Ann. Without their unwavering emotional support and constant encouragement, this dissertation would not have been possible.

ii VITA

July 14, 1953...... Born, Cincinnati, Ohio

197 5 ...... B.S., University of Cincinnati, Cincinnati, Ohio

1976 to 1978...... Teaching Associate, Department of Microbiology, The Ohio State University, Columbus, Ohio

1978...... M.S., The Ohio State University, Columbus, Ohio

1978 to 1982...... Teaching Associate, Department of Microbiology, The Ohio State University, Columbus, Ohio

FIELDS OF INTEREST

Anaerobic physiology, microbial physiology, , and biomass conversion.

iii TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... ii

VITA...... iii

LIST OF TABLES...... v

LIST OF FIGURES...... vii

INTRODUCTION...... 1

LITERATURE REVIEW...... 3

MATERIALS AND METHODS...... 28

Preparation of a Defined Medium...... 28 Preparation of the Reducing Agent...... 33 Transfer and Maintenance of Cultures...... 33 Use of the Anaerobic Chamber...... 34 Growth of Methanogens on Plates...... 38 Isolation of New Methanogens from Sediments...... 44 Determination of Dry Weight...... 45 Serological Analysis of the Olentangy and Delta Isolates...... 46 Determination of Methane...... 46 Determination of Formate Utilization by Methanogens...... 47 Determination of Requirements of Methanogens 48 Sources of Known Strains of Methanogens...... 48 Electron Microscopy of the Delta and Olentangy Isolates.. 49

RESULTS...... 50

Development of a Defined Growth Medium...... 64 Isolation of New Methanogens...... 92 Characterization of the Olentangy River Coccus and the Mississippi River Delta Coccus...... 95

DISCUSSION...... 119

SUMMARY...... 141

LITERATURE CITED...... 143 iv LIST OF TABLES

Table No. Title Page

1 Vitamin requirements for Methanobrevibacter smithii strain PS, pre-grown on and thiamine. Determinations were performed in 18 x 150 mm aluminum seal tubes in tripli­ cate. Methane determinations were performed immediately after stationary phase was achieved...... 79

2 Vitamin requirements for Methanobrevibacter smithii PS pre-grown on thiamine only and incubated for 48 hours after stationary phase was achieved. Determinations were performed in 18 x 150 mm aluminum seal tubes in triplicate...... 82

3 Comparative growth characteristics of four­ teen methanogens grown on defined medium. (A) g(h) is the generation time in hours for the different organisms. (B) equals the nMoles of methane produced per mg dry weight of cells. All determinations were performed in triplicate...... 86

4 Vitamin requirements for Methanobacterium bryantii strain M.o.H. Determinations were performed in triplicate in 18 x 150 mm aluminum seal tubes...... b8

5 Vitamin requirements for Methanospirillum hungatei strain J.F. Determinations were performed in triplicate in 18 x 150 mm aluminum seal tubes...... 91

New methanogenic isolates, isolated using a defined medium...... 94

v Table No. Title Page

7 Physiological characterization of the Olentangy isolate. Determinations were performed in triplicate in 18 x 150 mm aluminum seal tubes. + means all ten vitamins were added; Na acetate was used in a concentration of 0.25%; yeast extract, 0.2%; methanol, 10%, formate, 1%; and Na-butyrate, 0.25%. N.G. = no growth. g(h) = generation time in hours. O.D. = optical density at 580 n m ...... 108

8 Physiological characterization of the Delta isolate. Determinations were performed in triplicate in 18 x 150 mm aluminum seal tubes. All ten vitamins were added or were deleted as indicated. g(h) = generation time in hours O.D. = optical density at 580 nm ...... 110

9 Cross-reactivity of formalized whole cells of Olentangy and Delta isolate with anti­ serum S probes by indirect immunofluorescence (IIF)...... 113

10 Antibiotic sensitivities of the Olentangy, Delta, and Cuyahoga isolates and Methanococcus maripaludis and Methanococcus vannielii. (-) indicates no zone of inhibition, a blank means the sensitivity was not determined, and the numbers 1, 10, or 100 indicate the lowest concentration (ug) of antibiotic which produced a zone of inhibition around an antibiotic impregnated filter-paper disc. The experimental antibodies were new antibiotics, not yet com­ mercially available, provided by Eli Lily and Company, Indianapolis, Ind. (Dr. L.A. Hook, The Ohio State University, provided the table of antibiotic sensitivities.)...... 139

vi LIST OF FIGURES

Figure Title Page

1 A scheme for the reduction of C0„ to CH^. Wolfe (106)...... 14

2 Proposed autotrophic CO2 assimilation pathway in Methanobacterium thermoautotrophicum. Fuchs jet al. (31) and Fuchs and Stupperich (35)...... 23

3 Gassing manifold A. 3-way ball valve (Swagelock, Scioto Valve, Columbus, Ohio) B. 1 inch capped copper pipe C. Nupro 1/4 valves D. Pressure/vacuum gauge E. Exhaust port F. Gas feed to anaerobe chamber Inset: Sterile gassing probe a. Cotton-filled 2 ml syringe barrel (Becton-Dickenson, New Jersey) b. B-D No. 3081 Luer-lok tip c. B-D No. 3096 Luer-lok tip d. 6 mm diameter Tygon tubing...... 31

4 Forma Scientific (Marietta, Ohio) model 1024/1030 anaerobic chamber. Copper gas lines feed either the anaerobe jar (A) or the working space of the chamber (B)...... 36

5 An aluminum kettle with lid (A) for safe autoclaving of one liter bottles. (B) The wire mesh cage prevents other bottles from being broken if one bottle bursts...... 40

6 Oxoid model HP 11 anaerobe jar: (A) replace­ ment 10 x 60 mm, stainless steel, metric hex- head bolt; (B) pressure/vacuum gauge; (C) pressure relief valve. Inset: (a) Milton No. 2-698 female air chuck; (b) Cajon No.B-4-TA-1-4 adapter; (c) 1/4 in copper tube fitted with Swagelok connector and rubber hose (handle); (d) Schraeder valve...... 43

vii Figure Title Page

7 Evolution of culture flasks for methanogens. A. A 125 ml Erlenmeyer flask with a modi­ fied stopper through which a cut-off Hungate tube is inserted, tied in place ■with stringJ B. A 500 ml round bottom flask with a modified stopper wired in place with copper wire. C. A 125 ml Wheaton (Wheaton Glass, Vineland, N.J.) stoppered with a thick butyl stopper (Bellco Glass, Vineland, N.J.) held in place by an aluminum seal (2). D. An 18 x 150 mm aluminum seal tube (Bellco Glass, Vineland, N.J.) stoppered with a thick butyl stopper. E. A one liter Wheaton bottle fitted with a stopper through which a cut-off 18 x 150 mm aluminum seal tube has been inserted. F. A Bellco pyrex one liter bottle fitted with a stopper through which a cut-off aluminum seal tube has been inserted...... 52

8 A plywood incubation box (A) for the incuba­ tion of serum bottles on their side, (B) the incubation of tubes in racks, and (C) the incubation of one liter bottles on their side...... 56

9 (A) A quartz-glass oxygen scrubber filled with copper turnings (Sargent-Welch, Cincin­ nati, Ohio); (B) inlet; (C) outlet...... 59

10 An all copper oxygen scrubber filled with copper turnings (A) and furnace (B). Inlet (C) and outlet (D) are 1/4 inch copper tubing. Connections (E) are made with brass Swagelock 1/4 inch unions (Scioto Valve, Columbus, Ohio) 61

11 Methanobacterium bryantii strain M.o.H. growing on a defined medium...... 66

12 barkerii strain MS growing on a defined medium...... 68

13 Methanococcus vannielii growing on defined medium...... 70

viii Title Page

Methanobrevibacter smithii strain PS growing on defined medium...... 72

Methanobacterium formicicum strain RC growing on defined medium...... 74

Colonial morphology of the Olentangy isolate growing on a defined medium...... 97

Phase contrast photomicrograph of the Olentangy isolate. Cells were fixed in 1% glutaraldehyde. .. . 99,

Electron micrographs of the Olentangy isolate (A) fixed with 1% glutaraldehyde and (B) not fixed in glutaraldehyde and stained with 2% uranylacetate. The bar represents 1 um ...... 101

Colonial morphology of the Delta isolate growing on a defined medium with 3% NaCl and 20 mM MgCl2 ...... 104

(A) Phase contrast photomicrograph of the Delta isolate. Cells were fixed in 1% glutaraldehyde. (B) Electron micrograph of the Delta isolate. Cells were fixed with 1% glutaraldehyde but were not negatively stained. The bar represents 1 um ...... 106

Temperature optima of the Olentangy and Delta isolates. Determinations were performed in triplicate in 18 x 150 mm aluminum seal tubes without shaking...... 116

NaCl optima for the Olentangy and Delta iso­ lates. Determinations were performed in triplicate in aluminum seal tubes at 37°C. Absorbance was measured at 580 nm ...... 118

Restriction analysis of DNA from the (A) Olentangy isolate, (B) the Delta isolate, (C) Msp. hungatei strain JF, (D) Me. vannielii, and (E) Me. maripaludis, using Hind III (tracks 1-5) and Bam HI (tracks 6-10) (BRL, Bethesda, Md.). Paul Hamilton (personal communication)...... 135 INTRODUCTION

Methane producing bacteria are unique microorganisms classified as

Archaebacteria (102). Research involving these organisms is important,

rot only because of their possible utilization as a source of methane

g is, but information concerning these organisms may aid in the under-

st mding of evolutionary relatedness among the bacteria. Archaebacteria

ar< believed to be very ancient on the evolutionary scale.

However, methanogens are difficult to study because of their

extieme sensitivity to oxygen. As a result, relatively few labora­

tories have worked with pure cultures of methanogenic bacteria. How­

ever, the development of reliable anaerobic techniques have enabled

many researchers to begin to study these organisms (2, 5). This

dissertation involves the development and description of an improved

integrated anaerobic system through which these organisms may be

isolated and grown in liquid culture and on solid medium under defined

growth ccaditions. The system that has been developed permits the

study of rethanogens with relative ease.

A defined medium was developed which was selective for methanogens

and facilitated the isolation of new methanogens from different eco­

systems. Tha protocols developed for the cultivation of the methanogens

greatly simplified the routine handling of these fastidious anaerobes.

With these developments, methanogens may be studied in a more conventional manner and the increased understanding of methanogenic physiology should aid in the interpretation of the roles that methano­ gens play in different ecosystems. LITERATURE REVIEW

Leeuwenhoek, in 1680 (21), was the first to demonstrate that

"animalcules" could exist and develop without air, or at any rate, in a highly rarified atmosphere. However, Pasteur was credited with dis­ covering anaerobiosis 181 years later (84) and introduced the terms aerobes and anaerobes to denote microorganisms which require oxygen to grow and microorganisms which grow without the presence of oxygen.

Pasteur also described the butyric acid fermentation of an anaerobic sporeforming bacillus now known as Clostridium butyricum.

Methanogenic bacteria are the most extreme of the anaerobes and with the development of improved techniques has come greater under­ standing of these unusual organisms. For decades, methanogens remained uninvestigated simply because they were difficult to cultivate. Culti­ vation of methanogens has undergone a drastic evolution in the last ten years. Barker (105), in 1940, utilized a pyrogallol-carbonate tube containing alkaline pyrogallol for the absorption of oxygen from the microbial culture. Standard aseptic techniques were used to carry out serial dilutions of an enrichment culture through a melted agar medium in cotton plugged tubes. usually was added to the medium as the reducing agent. When the agar solidified, a layer of vaspar

(paraffin wax and oil) could be added (optional). The sterile cotton plug could be cut in half and the sterile portion pushed down into the tube. The other half of the cotton plug was then inserted

after pyrogallol and sodium carbonate were added to this cotton plug.

A solid black stopper then was inserted firmly into the tube. Methano­

gens grew slowly in these tubes, taking four or more weeks to produce

observable colonies.

Hungate developed a procedure for preparing pre-reduced media

so that by the use of gassing probes, oxygen free gases, and cysteine

sulfide as a reducing agent, the medium could be prepared, sterilized, and aseptically dispensed into tubes at a low Eh without being exposed to oxygen (9).

The Hungate procedure employs sterile solid black rubber stoppers.

These same procedures were employed in the transfer and sub-culture of organisms. A Hungate roll tube was created by solidifying inoculated agar medium on the inner surface of a tube as the tube is rolled, pro­ viding a thin layer of medium; after incubation colonies may be picked easily. Organisms may be cultivated readily in liquid medium. Use of these tubes produced quantitative results in which the numbers of organisms were several logs higher than the numbers found by use of standard anaerobic techniques.

Manipulation of the rubber stoppers in a sterile manner and to prevent oxygen from entering the tube requires a great deal of skill.

With a heavy gas such as 100% CO2 , maintenance of anaerobiosis was not too difficult. However, for the cultivation of methanogens, requiring a mixture of H2 and CO2 , the procedure is much more difficult.

The sealed tube with the pressurized atmosphere was developed by

Balch (2). To prepare such a tube, the medium was boiled, degassed and sealed by standard Hungate procedures; it was then transferred into an anaerobic.chamber (1) where it was dispensed into tubes or vials.

The tubes were stoppered, and each stopper was crimped into place. The tubes were then transferred outside the chamber whereby means of a gassing manifold, the atmosphere was replaced by a mixture of H? and

CO2 (80:20) at 2.0 atmospheres pressure (2). Basically, Balch combined the chamber of Freter to a modified Wolin-Miller tube (56), in which a thick black rubber stopper that could maintain internal pressure of the tube after numerous penetrations by a syringe needle was substi­ tuted for the original closure.

The common use of syringes is an extension of the method described

1 by Schnellen and Hungate (104). For use, the syringe, a 1.0 ml GlasPak syringe with a 22 ga. needle, was inserted into a sterile gassing probe through which sterile was passed. The piston was moved back and forth several times to expel air. The syringe was filled with the gas mixture and held upright until the needle was inserted into the flamed rubber stopper of the culture tube.

Cultures may be scaled up from test tubes to vials or bottles; a

200 ml culture may be grown in a 1 liter bottle under a pressurized atmosphere (5). The use of a pressurized atmosphere has been extended to a modification of the procedure developed by Edwards and McBride

(24) for the growth of methanogens in petri dish cultures. In this procedure, plates were streaked in an anaerobic chamber and were loaded into a pressure cooker or cylinder which was then sealed, transferred outside the chamber where the atmosphere was evacuated, and replaced with H2 :C02 (80:20) at a pressure of 2 atm. (2). The ultra low oxygen chamber of Edwards and McBride consisted of

using an anaerobic glove box containing an inner chamber with separate

gas-flushing facilities. The inner chamber was flushed with to

promote the growth of methanogens and further lower the internal oxygen

concentration. The chamber was not constructed so that a positive pressure could be maintained.

Methanogens were the first organisms to be identified as being archaebacteria by Woese (103). Further investigation has demonstrated

that Kalobacterium, Halococcus, Thermoplasma, and Sulfolobus can also be considered archaebacteria (103).

These organisms have been classified as archaebacteria due pri­ marily to the work of Woese. The 16S ribosomal ENAs from different

species of methanogens were characterized in terms of the oligonucleo­

tides produced by T-^ RNase digestion. Comparative analysis of the sequence homologies of these oligonucleotides to those oligonucleotides obtained from other procaryotes and eucaryotes led to the conclusion that methanogens are only distantly related to other typical bacteria.

In fact, on the basis of sequence homology, methanogens show no more relationship to the ordinary bacteria than they do to eucaryotic cells

(103).

The name "archaebacteria" was used to connote antiquity. Some of their properties are suggestive of a kind of bacteria one might expect to have dominated the early Archaen ecology. Various archaebacterial niches which appear extreme in terms of modern terrestrial conditions, would have been much more common on a warm planet with a reducing atmosphere some 3-4 billion years ago. Also, methanogenesis was peculiarly well suited to the projected primitive atmosphere of this

planet, a mixture of gases rich in carbon dioxide and hydrogen (103).

Both gram positive and gram negative walls are known among the

archaebacteria (30). However, archaebacterial walls lack diaminopi-

melic acid and muramie acid (i.e., peptidoglycan) (40). Within the

archaebacterial group, wall structures are extremely varied. Methano­

gens alone exhibit four distinct wall types (40, 43).

All of the major archaebacterial lines (except of course

Thermoplasma) contain at least one genus whose wall is a simple,

regular (largely) proteinaceous covering.

30S and 50S ribosomal subunits containing 16S and 23S rRNAs again

seem to be the extent of the specific resemblance between the archae­

bacterial and eubacterial translation mechanisms (103). Ribosomal RNA

sequences are not specifically related between the two bacterial

groups (30). The archaebacterial 5S RNA secondary structure resembles

its typical bacterial counterparts in the same three segments (the

'molecular stalk,' the 'turned helix,' and the 'common arm base') that

are also found in the eucaryotic 5S RNAs (30). However, in the remain­

ing region of secondary structures, the so-called 'procaryote loop,'

the archaebacteria 5S RNA resembles typical bacterial counterparts no more than do the eucaryotic 5S RNA (103).

Lipid comparisons, too, underscore both the uniqueness of archae­ bacteria and the extent of evolutionary convergence at the molecular

level. Archaebacteria have lipid analogs for the major lipid groups

found in other organisms — glycolipids, phospholipids, etc. Ether

links replace ester links, and straight carbon chains are replaced by polyisoprenoid (branched) chains (54). In other words, archaebacteria

seem to contain classes of lipids with the same gross physical proper­

ties common to other organisms. Yet, in molecular structure and mode of biochemical synthesis (5), the archaebacterial lipids are completely unrelated to those of other organisms.

Over the past several decades, a complacency has developed towards the idea that all organisms employ a certain universal core of bio­ chemical pathways. However, what little experience there is with archaebacterial intermediates appears to challenge the concept of universal intermediary , at least the extent of such univer­ sality. Not only do archaebacteria appear to use novel pathways in constructing cell walls and synthesizing lipids, but the methanogens, at least, exhibit a spectrum of unique coenzymes (5). Moreover, attempts to demonstrate some of the more usual cofactors in methanogens have failed. Clearly a comprehensive examination of the metabolic pathways in the archaebacteria is needed. Given the possible antiquity of their ancestry, the intermediary metabolic patterns of archaebacteria may provide further insights linking intermediary metabolism to pre- biotic chemistry.

Methanogens are basically chemoautotrophs in their metabolism, and molecular hydrogen is a favorite substrate of practically all methano­ gens presently in pure culture. For the anaerobic oxidation of hydrogen, carbon dioxide serves as the electron acceptor. No substrate- level phosphorylation has been observed in methanogens (70), and it has been assumed that during the oxidation of hydrogen and the subsequent transport of electrons and protons, a charge separation occurs across a membrane, with electron transport phosphorylation being a source of

ATP (106).

Hydrogenase activity has been detected in whole cells or cell extracts of methanogens. A hydrogenase-containing fraction from extracts of Mb. thermoautotrophicum was studied recently by Gunsalus

(37). Cells were broken by passage through a French pressure cell.

After centrifugation for two hours at 150,000 x g, 75-80% total hydro­ genase was easily solubilized without the use of membrane disrupting procedures (detergents, EDTA). These observations do not rule out the possibility of finding membrane-bound hydrogenase in the 150,000 x g pellet. The soluble hydrogenase exhibited properties common to most hydrogenases in that a variety of compounds served as electron donors.

An apparent for the reduction of of 25 uM was observed, whereas for methyl viologen the ^ was 1.5 mM with a normal velocity of

140 umol of methyl viologen being reduced per minute per milligram of protein. Unexpectedly, carbon monoxide produced only an 11% inhibition of hydrogenase activity when added at a partial pressure of 0.2 to the assay mixtures or when preincubated with the . The temperature optimum was 60°C and the pH optimum was 7.0. It has an estimated molecular weight of greater than 500,000 daltons.

Tzeng jet jal. (95) has studied the hydrogenase of Methanobrevi­ bacter smithii strain PS. Cells were broken by sonication, and hydro­ genase activity was found in the supernatant after centrifugation at

22,000 x g for 30 min. In the presence of hydrogen, a variety of electron acceptors could be reduced by crude extracts (FMN, FAD, methyl viologen, benzyl viologen, Fe^+ , and coenzyme F ^ q )* Eirich (25) 10

studied the hydrogenase of Methanobacterium bryantii strain M.o.H. and

found the for reduction of coenzyme F ^ q to ^ u^'

Cheeseman (10) was the first to observe that cells of a

(Mb. M.o.H.) were highly fluorescent when exposed to oxygen. When

these cells were placed under a hydrogen atmosphere, the fluorescent

gradually disappeared. The fluorescent factor was purified by Cheese­

man and was found to have a strong absorbance at 420 nm. The compound

was thus designated F^g*

Tzeng ^ jil. (95, 96), working with extracts of Mbr. smithii PS,

demonstrated that F ^ q was a l°w potential electron carrier and so

that compound was designated coenzyme F^g* Crude extracts of this

organism readily reduced NADP in the presence of hydrogen. Coenzyme

F ^ q was required for the reduction of NADP. The rates of hydrogen

uptake by the system was proportional to the concentration of F420

added. An apparent Km for F ^ g was calculated to be 5 x 10-^ M. The

system was specific for NADP. NAD was not reduced. A similar F ^ q -

dependent NADP-linked hydrogenase system has been found in Mb. bryantii

M.o.H. and in Mb. thermoautotrophlcum.

Tzeng ^t al. (96) have also determined that coenzyme F ^ q is the

primary electron acceptor for formate dehydrogenase. F ^ g that was

reduced by formate also served as an electron donor for the formation

of hydrogen via hydrogenase or participated in the reduction of NADP via NADP oxidoreductase. A similar formate hydrogenlyase system has been found in Methanospirillum hungatei (106).

The structure of coenzyme F ^ g fron> Mb* bryantii M.o.H. was studied

by Eirich (25). F ^ g was fractionated from whole cells, and a yield of 11

160 mg/kg of wet cells was obtained. The proposed structure for the 1 13 chromophore was derived by use of UV-visible spectrometry, H and C

NMR spectrometry, quantitative elemental analyses of F^ an

ments, and by comparison of these data with similar analytical data.

The spectrum of identical to that of synthetic 8-hydroxy-5-

deazoriboflavin (106) but is shifted 3-6 nm; this shift is believed to

be due to the 7-CH^ group on the latter compound. Evidence indicates

that F ^ q f-s a low-potential electron carrier (a derivative of

8-hydroxy-7-demethyl-5-deaza-FMN) that participates in two-electron

transfer reactions.

The F^20-^^-n^ec^ hydrogenase system of Mb. bryantii M.o.H. was used

by Eirich (25) to assay for F ^ q f-n other methanogens. Levels in

different methanogens were found to be (in mg F^o/kg wet cells) : Mb.

thermoautotrophicum, 324; Mb. formicicum, 206; Msp. hungatei, 319; M g .

marisnigri R 1 , 120; Mb. AZ, 306; Mbr. ruminantium Ml, 6; and M s .

barker!, 16.4.

F420 ^as keen found only in methanogens. When F^2q from various methanogens was subjected to electrophoresis, the electrophoretic mobility was about the same for each isolate (5.9 cm to the anode) with

the exception of Ms. barkeri (which was 7.5). This is an interesting

observation since Ms. barkeri is a physiologically distinctive methano-

gen.

Extracts of Mb. thermoautotrophicum were examined by Zeikus al.

(Ill) and by Fuchs et al. (33) for enzymic activity of several oxido-

reductases. Good activity was detected (nmol/min/mg of protein) for pyruvate dehydrogenase, 175; alpha-ketoglutarate dehydrogenase, 100; 12

fumarate reductase, 360; malate dehydrogenase, 240; and glyceraldehyde-

3-phosphate dehydrogenase, 100. Coenzyme F/42q was f°un^ to t>e the

electron acceptor for pyruvate dehydrogenase and for alpha-ketoglutarate dehydrogenase. Fumarate reductase did not couple with coenzyme F420’

NAD, or NADP. Menaquinone was not found in this organism. NADP was the preferred electron acceptor for glyceraldehyde-3-phosphate dehydrogenase, but malate dehydrogenase was most active with NAD.

These are believed to be involved in synthetic reactions.

McBride and Wolfe (56) discovered coenzyme M, HS-CI^C^-SOg

(HS-CoM), and Taylor and Wolfe (89) determined its structure. CoM was found to be a required for the growth of Mbr. ruminantium

Ml. This organism is also utilized as an assay for CoM. With the discovery of CoM, it became possible to study the methylreductase system and to investigate how methanogens make methane.

Figure 1 depicts the current concept of the mechanism by which

CO2 is reduced to CH^.

The most thoroughly studied area of this scheme is reaction V, the

CH^-SCoM methylreductase. The conversion of the methyl groups of

2-(methylthio)ethanesulfonic acid to methane involves a complex series of events. At one time, it was thought that N^-methyltetrahydrofolate and were involved in methanogenesis at the methyl level

(since these compounds could donate a methyl group for reduction); there is at present no evidence for their involvement as material com­ ponents of the methylreductase system (100).

The CHg-S-CoM methylreductase system of Mb. thermoautotrophicum was studied by Gunsalus and Wolfe (38) and was resolved into three Figure 1. A scheme for the reduction of CO2 to CH^.

Wolfe (106).

13 14

3 E (Methyl) CH3-S-C0M \XCOOH \ (Carboxy). ' \^YH2* I y \Hydrogenote HS-CoM X CDR Factor I Ku i / ' H , 0

(Hydroxymethyl) HOCH2-S-C0M x/XCHO (For my I)

Hvdrooenose

Figure 1, components: (a) a hydrogenase complex of high molecular weight; (b) a small, heat-stable dialyzable coenzyme, and (c) an oxygen stable protein. When ATP and Mg+^ were added to these components under a hydrogen atmosphere, the methyl group of CH3-S-C0M was reduced stoichiometrically to methane (38). Wolin ^t aJL. (104) demonstrated that ATP was required for methane formation and Gunsalus and Wolfe (38) have shown this ATP requirement to be catalytic, one mole of ATP catalyzing the formation of 15 moles of methane. While studying the

CH^-S-CoM methylreductase, Gunsalus found that when carbon dioxide was added to the standard reaction mixture for the methylreductase assay, the rate of methane formation was stimulated 30*-fold and the total amount of methane formed was 12-fold higher. This phenomenon is referred to as the RPG effect. From this, Wolfe (105) concluded that the terminal reaction of carbon dioxide reduction in methanogens is coupled to the first, the activation of carbon dioxide. Romesser and

Wolfe (72) refined this system further. They separated carbon dioxide stimulation of the CH^-S-CoM methylreductase from net reduction of carbon dioxide to methane. Results of their experiments indicated that carbon dioxide is an effector for the methylreductase, stimulating the rate of the reaction by a mechanism that remains to be elucidated.

They also discovered a heat-stable, dialyzable cofactor about which little is known at present, designated CDR. When this cofactor was removed from cell extracts, reaction V was stimulated by the addition of carbon dioxide, but there was only a stoichiometric conversion of

CHg-S-CoM to methane. The RPG effect was abolished. However, upon addition of the factor back to the reaction mixture, the RPG effect was 16

restored. The factor was not required for the conversion of HO-CI^-S-

CoM or formaldehyde to methane. Therefore, the CDR factor is believed

to function prior to the aldehyde level of oxidation.

Conversion of acetate to methane attracted interest when it was

reported that carbon dioxide was not a major precursor of methane in

sewage sludge and that acetate was converted to methane (106). Stadt- man and Barker (86), Pine and Barker (67), and Pine and Vishniac (68) documented, by the use of isotopically-labeled acetate, that methane was formed from methyl carbon of acetate and carbon dioxide was formed from the carboxyl group. In addition, when CD^COOH was used as a substrate, CD^H was formed, an indication that the deuterium atoms remained with the carbon atom. The methyl group was reduced intact to methane. So far, only Methanosarcina and Mb. soehngenii have been shown to grow on acetate and to convert it to methane in pure culture

(6, 15, 52, 53, 79, 103). It has been reported that Methanosarcina requires adaptation in order to grow on acetate (52), however,

Scherer and Sahm (75) have reported that the adaptation is not required for growth on methanol, acetate, or hydrogen-carbon dioxide if the correct sulfide concentration is utilized during the culturing of the organism.

Smith and Mah (79) have demonstrated that methanol is the pre­ ferred substrate for Methanosarcina and Hippe _et _al. (40) have reported that Methanosarcina can also utilize methylamines for methane formation.

Acetate is also utilized as a carbon source by a number of different methanogens. Mbr. smithii PS requires acetate for growth, 17 and 60% of its cellular protein is acetate derived. Mb. thermoauto- trophicum do&s not require acetate; however, when acetate is supplied ft in the growth^medium, about 60% of its cellular carbon is derived from V, acetate. Obet^.iesV et al. (61) have demonstrated the presence of acetate thiokiftase in Mb. thermoautotrophicum. The specific activity v of acetate thio^.inase was high (100 nmol/min/mg of protein) in cells V growing with limited and was low in cells growing exponentially V (2 nm/min/mg of protein). This corresponded with their finding that * cells growing linearly in the presence of acetate assimilated the monocarboxylic aj:id in high amounts, whereas exponentially growing cells did not. j’his finding indicates that acetate thiokinase and free acetate are not involved in autotrophic CO2 f i x a t i o nin Mb. thermoauto­ trophicum. 1 Since no sublitrate-level ATP synthesis has been found so far in extracts of methanogens and since anaerobic hydrogen oxidation is a common property of methanogens in pure culture, it has been assumed for many years that ATP synthesis must take place via electron transport phosphorylation. Roberton (70) was the first to study ATP pool sizes in whole cells of a methanogen. Roberton demonstrated that the pool size increased as hydrogen was oxidized and carbon dioxide was reduced to methane (70). When hydrogen was replaced with other gases or by air, the ATP pool size decreased. Addition of uncouplers of electron transport phosphorylation to the cell suspension caused a decrease in pool size.

Doddema et al. (20, 22) have studied the ATPase and ATP synthesis in Mb. thermoautotrophicum. Extracts of this organism showed that the ATPase activity was in the supernatant fraction after centrifugation at 10,000 x g for 10 min. However, after the extract was centrifuged at 14,000 x g for 3 h, the ATPase activity was found in the pellet and could be stored at -90°C for a year without loss of activity. The 2 j optimal pH for the enzyme was 8 at 65-70°C. The reaction was Mg dependent, and a ratio of ATP:Mg of 0.5 yielded optimal activity.

Under these conditions the Km for ATP was 2 mM. Other divalent cations n | o I 2"f“ 2+ o.j (Mn , Co , Cu , and Zn ) replaced Mg to a significant extent.

TES (N-tris-(hydroxymethyl)methyl-2-amino-ethanesulfonate), Tris,

(tris-(hydroxymethyl)aminomethane), and diethanolamine buffers were inhibitory. The enzyme complex also hydrolyzed GTP and UTP, but ADP and AMP were not hydrolyzed. Hydrolysis of ATP was inhibited by DCCD

(N,N'dicyclohexylocarbodiimide). ATP synthesis in whole cells was stimulated by an artificially imposed protonmotive force. The internal pH of the cells was found to be 7.6. By lowering the external pH, an increase in intracellular ATP was observed, the shift from pH 7.5 to

3.0 being most effective in generating ATP. When valinomycin, at a concentration of 20 uM, was added, ATP formation also was observed and potassium ions were extruded from the cells.

It has been proposed recently (106) that fumarate reductase might be coupled to electron transport phosphorylation in methanogens. This proposal suggested that a fumarate-succinate cycle could be coupled to methylreductase. In light of the work of Gunsalus (37), this system seems highly unlikely to operate at the methylreductase level. When this proposal was tested by Fuchs _et _al. (33), (U-^C, 2, 3-^H)- succinate was found to be incorporated into cell material with a loss 19

of only 30% of the tritium, and H was not released into water in

sufficient amounts. It therefore appears that the function of the

fumarate reductase of Mb. thermoautotrophicum is to synthesize succinate

and not to catabolically oxidize succinate to fumarate.

Factor F43Q is a low molecular weight compound with an absorption

maximum at 430 nm and is present in all methanogenic bacteria (110).

It is probably the of methyl coenzyme M reductase (21)

which catalyzes the reduction of methyl CoM to methane. Diekert .et al.

(17) and Whitman and Wolfe (100) have shown F ^ q to contain ,

which explains why methanogens are dependent upon this transition

metal for growth. The mass/mol nickel was determined to be 1500 and

CE430 to be near 2300 cm-^ (mol/Ni)-^. Labelling studies with (^C)

succinate (18) and (^C)-aminolevulinic acid (4 -ALA) (19) indicated

that factor F ^ q has a nickel tetrapyrrol structure; 8 mol -ALA/mol

Ni are incorporated into the factor. The arrangement of the tetra-

pyrrole is probably macrocyclic (as in the porphyrins) rather than

linear (as in bile pigments) because nickel does not dissociate from

the factor, neither in strong acids (6 N HC1) nor under alkaline condi­

tions (pH 13) (19). The detailed structure of F43Q is not known.

Chlorophylls, , sirohemes, and vitamin are macrocyclic

tetrapyrroles of biological importance known to date. The four tetra-

pyrroles are either derived from protoporphyrin IX (chlorophylls, hemes,

and cytochromes) or from sirohydrochlorin (siroheme and vitamin

Both protoporphyrin IX and sirohydrochlorin are synthesized from uroporphyrinogen III, the former by six consecutive decarboxylations,

including oxidative decarboxylation, the latter by two reductive 20 methylations with 5-adenosyl methionine. Uroporphyrinogen III is formed from 8- & -ALA (19).

Methanogenic bacteria have been shown to contain cytochromes (51).

This indicates that these organisms synthesize uroporphyrinogen III from & -ALA. It is therefore reasonable to assume that factor F.is ... 430 derived from uroporphyrinogen III as are the other tetrapyrroles of biological importance.

From the presence of corrinoids, it further has been deduced (41) that methanogens can synthesize sirohydrochlorin, which contains two methionine-derived methyl groups. The finding that factor F ^ q con­ tains two methyl groups introduced from methionine suggests that the nickel tetrapyrrole could be derived from sirohydrochlorin as are siroheme and vitamin

Fuchs and Stupperich (35) have investigated the autotrophic CO^ fixation in Mb. thermoautotrophicum. Enzyme studies by Zeikus jet al.

(Ill) and labelling studies by Fuchs _et _al. (32, 33) indicated the presence of three CC^ assimilating reactions, i.e., pyruvate synthase, phosphoenolpyruvate carboxylase, and the alpha-ketoglutarate synthase reaction. Daniels and Zeikus (15) drew similar conclusions utilizing short term labelling experiments.

Pyruvate synthase catalyzing the reductive carboxylation of acetyl CoA to pyruvate was proposed to be a key enzyme in the autotrophic carbon fixation pathway of Mb. thermoautotrophicum. The proposal included the assumption that acetyl CoA was not formed from pyruvate. Acetyl CoA synthesis via a reductive tricarboxylic acid cycle has been excluded (32). It was left unexplained how 21

hexosephosphates and pentosephosphates were synthesized from CC^.

In 1980 Fuchs and Stupperich (35) demonstrated that acetyl CoA

is the precursor of pyruvate. It was shown that starting from acetyl

CoA plus CO2, not only dicarboxylic acids are synthesized via pyruvate, but also hexoses and pentoses. The results provide evidence that in

Mb. thermoautotrophicum a novel autotrophic CO2 fixation pathway is operating in which acetyl CoA is a central intermediate. Figure 2

shows the pathway that was proposed.

These experiments were done with long term labelling. Stupperich 14 and Fuchs (88) then studied CO^ fixation and the C labelling pattern of alanine in Mb. thermoautotrophicum, using pulse labelling techniques.

Incorporation of ^C0 by an exponentially growing culture was

followed. After a 2s incubation, most of the radioactivity of the ethanol-soluble fraction was present in the amino acids alanine, glutamate, glutamine, and aspartate, whereas phosphorylated compounds were only weakly labelled. The percentage of the total radioactivity fixed, which was contained in the principal early labelled amino acid alanine, increased in the first 20s and only then decreased, indicating that alanine is derived from primary products of CO2 fixation.

The labelling patterns of alanine produced during various incuba­ tion times were determined by degradation. After a 2s ^ C pulse, 61% of the radioactivity was located in C-l, 23% in C-2, and 16% in C-3.

The results were consistent with the proposed autotrophic CO2 assimila­ tion pathway shown in Figure 2.

■^CC>2 and (2-^C) acetate pulse labelling studies on whole cell suspensions by Daniels and Zeikus (15) demonstrated some discrepancies Figure 2 Proposed autotrophic CC^ assimilation pathway

in Methanobacterium thermoautotrophicum.

Fuchs _et_al. (31) and Fuchs and Stupperich (35).

22 23

GALACTOSE NH} ^HEXOSE'P —* PENTOSE'P^ PIBOSE

TRIOSE-P

I sis ASPARTATE

*»OXALOACETATE PHOSPHOENOLPYRUVATE / MALATE 2 ATP 4 FUMARATE P Y R U V A T E ^ ! ALANINE | 4 SUCCINATE Factor 420, ATP^

_SUCC INYL- CoA ACETYL- CoA a c e t y l ( ^ r ~ X ree,or 4K"<. ^ ~ o i- KETOGLUTARATE A T P 7

GLU TAMA TE

Figure 2. 24

with the proposed pathway. Following a brief incubation with ^CC^,

alanine was found as the most strongly labelled compound. However,

following incubation with (^C)acetate for up to 45s, succinate and

two other unidentified compounds, but not alanine, as expected, were

reported to contain 95% of the label incorporated from (^C) acetate.

This result would exclude the proposed pathway. Stupperich and Fuchs

(88) tested commercially available sodium (2-^C)-acetate and sodium-

(U-^C)acetate for purity and found them to contain significant amounts

of three labelled impurities which presumably arose through decomposi­

tion. One of these compounds was identified as succinate which had

been reported to be one of the three early labelled compounds in Mb.

thermoautotrophicum pulse labelled with isotopic acetate (15). Due to

the similarities between the described early labelled products and the

impurities in the isotopic acetate, Stupperich and Fuchs (88) proposed

that the apparent discrepancy between the data presented by them and

that of Daniels and Zeikus could be due to the contamination of the

label used by Daniels and Zeikus by succinate and the two other unknown compounds.

The exact nutrient requirements of methanogenic bacteria have not been well defined. Bryant et al. (9) investigated Mb. bryantii strain

M.o.H., Mbr. smithii PS, and Mbr. ruminantium M-l. These investigators determined that Mbr. smithii PS required acetate for growth and syn­ thesized 60% of its cellular carbon from acetate, Mb. bryantii M.o.H. could utilize CO2 as its main carbon source but was stimulated by acetate, biotin, vitamin and folic acid. Mbr. ruminantium M-l required -methyl butyrate for growth and an unidentified factor for 25 growth (later determined to be mercaptoethane sulfonate) (56, 89, 90).

They also determined that all of the organisms required NH^ as the main source of cellular nitrogen.

Other investigators have further refined the growth requirements of some specific methanogens, particularly Mb. thermoautotrophicum

(66, 77, 93). Schonheit at al. (76) determined that Mb. thermoauto­ trophicum required nickel, cobalt, and for growth. They could not demonstrate a need for copper, manganese, zinc, calcium, aluminum, or boron, all of which are normally added to methanogen medium as described by Zeikus and Wolfe (108). For the formation of

1.0 gram of cells (dry weight) approximately 150 nmol N i C ^ , 20 nmol

Co C^, and 20 nmol Na2Mo0^ were required.

Scherer and Sahm (74) investigated the trace element and vitamin requirements of Ms. barkeri. Growth of Ms. barkeri on methanol as an energy source was found to be dependent on cobalt and molybdenum. In —6 —7 the presence of 10 M Co and 5 x 10 M Mo optimal growth occurred.

It was also demonstrated that nickel and selenium, each in a concentra- -7 tion of 10 M, stimulated growth while B, Cr, Cu, Mn, and Pb had no influence. It was also demonstrated that riboflavin was the only required vitamin for growth in a defined medium.

Patel et al. (63) investigated the optimal levels of sulfate and iron required for the cultivation of Mb. bryantii strain M.o.H. Msp. hungatei GPI and a strain of Methanobacterium formicicum. They found that the 4.0 mM of sulfate normally incorporated in the laboratory media was above the optimum level, while the iron content normally incorpo­ rated (0.01-0.8 mM) was below the optimum requirement for these 26 methanogens. These tests were made in synthetic media containing acetate, carbon dioxide, and hydrogen as carbon and energy sources, and a cysteine-sodium sulfide solution as reducing agent. The optimum sulfate requirement of the organisms was determined to be in the range of 0.16-0.52 mM. Absence of sulfate in the medium decreased growth | | significantly. The optimum level of Fe was in the range of 0.3-0.9 mM.

Jones and Stadtman (43) demonstrated that Methanococcus vannielii required selenium for growth and was also stimulated by the addition of

100 uM sodium tungstate. The selenium dependent formate dehydrogenase of Me. vannielii was isolated from organisms grown in the presence of

(^Se)selenite. The formate dehydrogenase was found to contain selenocysteine (44).

The influence of sulfide and containing compounds on the growth of different methanogens has also been investigated. Various organic sulfides and inorganic sulfides were studied in respect to their effect on growth and methane production of Methanobacterium strain AZ by Wellinger and Wuhrmann (99). In mineral, sulfide-free medium, cysteine regulated the specific rate of methane production

(optimum concentration = 5 x 10^ mole/1). A supplement of sulfide

(10 ^ mole/1) caused an additional stimulation. Coenzyme M or could be substituted for cysteine when sulfide was present.

Growth was stimulated by CoM and glutathione to the same extent as with cysteine in sulfide containing media.

Scherer and Sahm (75) investigated the influence of sulfur- containing compounds on the growth of M£. barkeri. Optimal growth of Ms. barkeri occurred in a defined medium containing methanol when 2.5-

4.0 mM sodium sulfide was added, giving a concentration of 0.04-0.06 mM dissolved sulfide. When the sulfide concentration was too low for optimal growth (e.g., 0.1 mM added) the addition of the redox resin 'Serdoxit' acted as a sulfide reservoir and caused a significant stimulation of growth. It was also demonstrated that iron sulfide, zinc sulfide or L-methionine could also act as sulfur sources while the addition of sodium sulfate to sulfide-depleted media failed to restore growth. The amino acid L-cysteine (0.85 mM) stimulated growth but could not replace Na2S.

Under optimal cysteine and sulfide concentrations, the generation time of Ms. barkeri was about 7-9 h during growth on methanol, giving a growth yield of about 0.14 g/g methanol consumed. Different Ms. barkeri strains were also able to grow under these conditions on acetate (30-50 h doubling time) without a significant lag-phase and with complete substrate consumption even though the inoculum was grown on ^-CO^. When methanol and acetate were present as a mixture in the medium, both were used simultaneously.

This literature review was an attempt to review the current literature limited to the anaerobic cultivation techniques and basic physiology of methanogenic bacteria. Hopefully, this review placed the study of methanogenic bacteria in the proper perspective of being a rapidly developing area with much to be learned. MATERIALS AND METHODS

Preparation of Media

The basic medium was a defined medium having the following composition:

grams/liter

K2HP04 0.3

kh2po4 0.3

Na acetate 2.5

NH,SO. 0.3 4 4 NH. Cl 2.7 4 Na bicarbonate 5.0

FeSO. 0.01 4 NiCl2 0.00024

NaCl 0.61

NaMo04 0.00024

CoCl2 0.001

Cysteine 0.125

Na2S 0.125

MgCl2 0.2

MgS04 0.13

Stock vitamins solution* 10 mls/1

28 29

Stock vitamin solution contains (mg/1) biotin, 2.0; folic acid,

2.0; pyridoxine hydrochloride, 10.0; thiamine hydrochloride, 5.0;

riboflavin, 5.0; nicotinic acid, 5.0; DL-calcium pantothenate, 5.0;

vitamin Bj^, 0.1; p-aminobenzoic acid, 5.0; , 5.0.

All chemicals were of reagent grade and were purchased from either

Matheson, Coleman and Bell, Norwood, 0., Allied Chemical, Morristown,

N.J., Fisher Scientific Company, Fair Lawn, N.J., or Mallinckrodt,

Paris, Ky. The vitamins were purchased from Sigma Chemical Co., St.

Louis, Mo.

These constituents were added to double glass distilled water, and

the water was adjusted to 6.9. The medium was then boiled and dis­

pensed under a stream of nitrogen using a Cornwall pipettor (Becton-

Dickinson, N.J.) into Wheaton serum bottles (Wheaton Glass, N.J.), and

the bottles were stoppered with flanged butyl rubber stoppers (Bellco

Glass, Vineland, N.J.). The stoppers were held in place by aluminum

crimp seals which were fixed in place by a hand crimper tool (Pierce

Chemicals).

The atmosphere over the medium in the bottle was then nitrogen and

had to be replaced with 80% H2 :20% CO^. This was accomplished by use

of the gassing manifold shown in Figure 3. The manifold was pressur­

ized with H2 :C02 and each gassing probe was fitted with a 22 ga. x 1 in disposable needle. The needle was inserted through the rubber stopper of the bottle, the valves were opened, and the bottle was pressurized to 8 psi with H£:C02. The atmosphere was exchanged by repeated evacuation and repressurization of the bottle by using the three-way ball valve on the gassing manifold. A vacuum pump was used to pull a Figure 3. Gassing Manifold

A. 3-way ball valve (Swagelock, Scioto Valve, Columbus, Ohio) B. 1 inch capped copper pipe C. Nupro 1/4 valves D. Pressure/vacuum gauge E. Exhaust port F. Gas feed to anaerobe chamber

Inset: Sterile gassing probe

a. Cotton-filled 2 ml syringe barrel (Becton-Dickinson, New Jersey) b. B-D No. 3081 Luer-lok tip c. B-D No. 3096 Luer-lok tip d. 6 mm diameter Tygon tubing

30 31

Figure 3. 32 vacuum on the gassing manifold and the serum bottles when the ball valve was in the up position. When the ball valve was in the down position, the manifold and bottles were pressurized. The bottle was evacuated and repressurized a minimum of three times to change the head-space atmosphere from nitrogen to I^CC^. By observing the attached vacuum/pressure gauge (Fig. 3), the evacuation and repressuri- zation procedure could be monitored. When the gauge indicated a vacuum of 30 in Hg, the bottles were repressurised, and when the pressure read

8 psi, the bottles were re-evacuated. Six bottles or tubes could be evacuated and pressurized simultaneously using the gassing manifold.

After the atmosphere had been exchanged, the bottles were ready to be autoclaved. The bottles were placed in metal pans containing about an inch of water and autoclaved for 20 minutes at 121°C and 20 psi.

The water in the pan was used so that when the bottles were removed from the autoclave the temperature change was not too rapid, which could result in the rupture of the bottles.

After the bottles had cooled, sterile reducing agent was added to each bottle. The reducing agent consisted of cysteine-sulfide and was added in the amount of 1 ml/100 mis, or 0.2 ml/20 mis, by use of a syringe.

The bottles were then pressurized to 43-45 psi using the gassing manifold. The gas was sterilized by passage through the sterile gassing probe shown in Figure 3. The medium was reduced within an hour and was stored at room temperature. 33

Preparation of the Reducing Agent

Reducing agent was prepared by first dissolving 2.5 g cysteine-

HC1 in 100 mis of distilled water (105). This solution was adjusted to pH 11 by using 10% sodium hydroxide. Into a second 100 mis of distilled water, 2.5 g of sodium sulfide was added. The reducing agent was kept alkaline to prevent the loss of sulfide as hydrogen sulfide gas. The two separate solutions were added together, boiled and dispensed under'a stream of nitrogen into aluminum sealed bottles

(Wheaton) and sealed with Bellco flanged stoppers. Individual bottles of reducing agent were autoclaved and stored in the refrigerator until needed.

Transfer and Maintenance of Culture

Cultures were maintained in Wheaton serum bottles containing 20 mis of medium and incubated at 37°C. The cultures were placed in a specially constructed box in a horizontal position and shaken.

Exceptions to this method of incubation were Mb. thermoautotrophicum, which was incubated at 65°C, and Me. vannielii and Methanococcus maripaludis, which were incubated at 37°C without shaking due to their mechanical fragility.

Cultures were transferred as required. Most organisms were transferred every 48 hours. Methanococcus deltae strain RC was trans­ ferred every 24 hours. The transfer procedure was as follows: the stoppers were flame sterilized, and the bottle of fresh medium and the bottles with the grown culture were aseptically pressurized to 43-45 psi using a sterile gassing probe and sterile needle. Then, using a GlasPak (Becton Dickinson) 1 ml disposable syringe fitted with a 22 ga. x i in needle (Becton-Dickinson), a portion of the head gas from the

grown culture was removed and expelled from the syringe. This served to fill the syringe with and to expel any oxygen present in the

syringe. After the gas had been expelled, the syringe was reinserted through the stopper, the bottle was tilted upward, and 1.0 ml of culture was withdrawn and injected into the fresh bottle of medium.

Two mis (10%) was the usual inoculum size. Methanococcus deltae strain RC was an exception; only 0.4 mis were usually transferred due to its fast growth rate. Larger inocula resulted in overgrown cultures in 24 hours, and autolysis was a problem of stationary phase cultures resulting in lowered viability. Also, Mb. thermoautotrophicum and Mb. formicicum strain RC required only a l ml inoculum due to their fast growth rate as well. After transferring, the freshly inoculated cul­ tures were re-incubated and the grown cultures were stored. All of the cocci and Mb. formicicum were stored at room temperature.

Refrigeration of the cultures resulted in increased lysis of the cells.

The other methanogens were stored in the refrigerator. Mbr. smithii strain PS retained its viability much better in the refrigerator than at room temperature.

Use of the Anaerobic Chamber

The Forma Scientific (Marietta, Ohio) anaerobic chamber model 1024 was utilized for the anaerobic incubation of petri plates and for other manipulations which required an anaerobic atmosphere (Fig. 4). The chamber was utilized as described in the operation manual; however, Figure 4. Forma Scientific (Marietta, Ohio) model

1024/1030 anaerobic chamber. Copper gas

lines feed either the anaerobe jar (A)

or the working space of the chamber (B).

35 36

Figure 4. 37 whenever an item was taken into the chamber, the automatic interlock purge system was cycled twice to minimize oxygen contamination when entering the chamber.

The catalyst and dessicant wafers were replaced weekly. These wafers were regenerated by baking in an oven at 160°C overnight.

Since a significant amount of was released in the chamber, because of its use as a reducing agent in the methanogen medium, activated charcoal wafers were placed in front of the catalyst wafers in an attempt to absorb the b^S. The H^S will bind to the Pt-Pd catalyst irreversibly and poison the catalyst rendering it ineffective. To further decrease the l^S content in the chamber, solutions of zinc acetate were placed in the chamber and the atmosphere inside the chamber was pumped through the solution by the use of aquarium air pumps. The zinc acetate trapped the l^S as ZnS producing a white precipitate, reducing the H^S concentration in the chamber.

The anaerobic chamber was also modified by the installation of inlet and outlet valves. The inlet valve originated from the gassing manifold, and the outlet valve carried exhausting gas back to an exhaust fume hood. With this system, various manipulations could be performed inside of the chamber, such as purging with a gas different

(e.g., 80% 1^:20% CO2) from the atmosphere in the chamber (e.g., 85%

N2 :10% 1^:5% CO2) or a vacuum could be pulled and anaerobic filter sterilization could be performed. These gassing inlets and outlets greatly increased the versatility of the tasks that could be carried on inside the chamber. 38

Growth of Methanogens on Plates

The medium used to grow methanogens on plates was as already described with the exception that 2% agar was added to solidify the medium. The medium was prepared, boiled in an Erlenmeyer flask, and then dispensed under nitrogen into a one liter bottle (Wheaton Glass,

Millville, N.J.) (5) fitted with a stopper through which a bottomless aluminum seal tube had been placed. The bottle was sealed with a gray silicone stopper which was crimped in place with an aluminum seal. The atmosphere was purged with and the reducing agent was added.

A positive pressure of 1-2 psi was left on the bottle before auto- claving. The bottle was placed in a wire mesh cage and placed in a covered aluminum cooker (Fig. 5) with water covering the bottom of the cooker. The cooker was then placed in the autoclave and heated for

20 minutes, 18 psi, at 121°C. The wire mesh and cooker were used for safety's sake. After several autoclavings, the bottles were weakened and tended to explode unexpectedly upon removal from the autoclave.

After autoclaving, the bottled medium was cooled to approximately

60°C and taken inside the anaerobic chamber. Once inside, the bottle was de-capped, using a de-capper tool (Pierce Chemical Co.,

"dekapitator"). The gray stopper was then aseptically removed, using a disposable needle. The gray stopper was used specifically because of its ease in removal once inside the chamber. The thick Bellco stoppers were difficult to extricate in a sterile manner. The plates were then poured and allowed to solidify and dry overnight. The plas­ tic petri plates had to be in the chamber and exposed to the Figure 5. An aluminum kettle with lid (A) for

safe autoclaving of one liter bottles.

(B) The wire mesh cage prevents other

bottles from being broken if one bottle

bursts.

39 40

Figure 5. 41

atmosphere a minimum of 24 hours before the plates were poured. This

period removed any oxygen which was dissolved in the plastic. After

the poured plates were dry, they were placed in a BBL (BBL, Baltimore,

Md.) anaerobe jar containing a plate with 3 mis of cysteine-sulfide

reducing agent. The reducing agent in the plate released l^S which

absorbed into the agar forming FeS, turning the plates a dark gray and

helped to pre-reduce the agar surface before the organisms were added.

Methanogens require H^S for growth.

After the plates had been treated with the reducing agent (the

process lasted approximately three hours) methanogens were streaked

onto the agar surface. The loop used was platinum-iridium since

nickel-chromium loops formed oxidation products on their surface.

These oxidation products could kill small inocula of methanogens

through the oxidation of these organisms. The incinerator used to

sterilize the loop was a hot coil incinerator manufactured by Coy (Coy

Manufacturing Co., Ann Arbor, Mi.). After the methanogens were

streaked onto the agar, the plates were transferred into Oxoid anaerobe

jars (Oxoid Ltd., Hampshire, England) shown in Figure 6.

Using the Schraeder valves located on the lid, the jar was purged

with 80% ^ 120% CO^ using the inlet and outlet lines and the valve

adapters connected to the inlet and outlet (Fig. 6). After purging

for approximately 30 seconds, the exhaust line was removed and the jar was pressurized to 12 psi and incubated in the anaerobic chamber

incubator at 37°C. Growth could be followed both visually and by monitoring the pressure gauge. A drop in pressure indicated the use Figure 6. Oxoid model HP 11 anaerobe jar: (A) replacement

10 x 60 mm, stainless steel, metric hex-head

bolt; (B) pressure/vacuum gauge; (C) pressure

relief valve. Inset: (a) Milton No. 2-698 female

air chuck; (b) Cajon No. B-4-TA-1-4 adapter; (c)

1/4 in copper tube fitted with Swagelok connector

and rubber hose (handle); (d) Schraeder valve.

42 Figure 6. 44 of and the growth of methanogens. Depending upon the organism, good growth was achieved in 4 to 14 days.

Isolation of New Methanogens from Sediments

To investigate the utility of the medium proposed, attempts were made to isolate new methanogenic organisms from the environment.

Sediment was obtained from several sources; the Olentangy River, the Mississippi River Delta, the Cuyahoga River, a clam flat in Maine, and the Tennessee River in Alabama. The procedure followed was rela­ tively simple. A bottle of the sterile defined medium in the anaerobic chamber was unstoppered, a scoop of sediment added, and the bottle re-stoppered. The bottle was brought outside the chamber, purged with 80% 1^:20% CO2 and pressurized to 43-45 psi. The bottle was then incubated at 37°C with shaking, or at 65°C.

Daily monitoring was done by re-pressurizing the bottles to 43-45 psi. If a vacuum was apparent (demonstrated visually by a disturbance of the liquid as the re-pressurizing gas rushed in to equalize the pressure), the bottle was subcultured. Subculturing was repeated as needed, i.e., when a substantial vacuum was created. Subculturing resulted in a highly enriched methanogenic culture. This culture could be purified in two different ways, dilution to extinction and streaking for isolation. The Delta isolate and the Olentangy isolate were isolated by both methods. Mb. thermoautotrophicum was isolated by dilution to extinction in Bellco aluminum seal tubes.

Isolated colonies which developed on the streak plates were picked and re-inoculated back into liquid medium. This was achieved with a 45 sterile syringe fitted with a blunted 18 ga. needle. The isolated colony was stabbed with the needle and drawn up into the needle with the syringe. The needle was inserted through the stopper of a tube or bottle of sterile medium and rinsed from the needle by drawing the medium into the syringe and squirting it back into the vessel.

The purity of the isolates was checked by inoculating a fully grown culture into enriched media: cooked meat medium, trypticase soy broth with 0.4% glucose, and the defined medium with yeast extract and trypticase (0.2% each) added, with nitrogen in the head-space. The media used for purity tests were pre-reduced with cysteine-sulfide reducing agent. The test media were incubated at 37°C or 65°C for at least 72 hours and checked visually and microscopically for growth.

Determination of Dry Weight

The dry weights of cultures were determined to correlate total dry weight to the optical density observed using a Bausch and Lomb

Spectronic 20 at 580 nm. Cultures were spun*"down and resuspended in defined medium without resazurin to avoid any interference by the dye.

The suspension was diluted to an optical density of 0.9 at 580 nm using

18 mm Bellco aluminum seal tubes as cuvettes. Twenty milliliter aliquots were filtered through predried and weighed 47 mm, 0.45 urn pore size membrane filters (Gelman Sciences, Ann Arbor, Mi.). The filters were washed with 30 mis of 20 mM phosphate buffer, pH 3. The buffer was adjusted to pH 3 in order to dissolve any precipitated FeS.

After filtering, the filters were placed in preweighed aluminum pans and dried at 65°C to constant weight. 46

Serological Analysis of the Olentangy and Delta Isolates

M.J. Wolin and E. Conway de Macario, State of New York Department of Health, Office of Public Health in Albany, New York kindly performed the comparative serologies of the Olentangy and Delta isolates. The procedure was described by Conway de Macario et al. (12, 13, 14).

The immunological studies of the Olentangy and Delta isolates were performed using the indirect immunofluorescence (IIF) technique. The

S probe, which is the highest dilution of an antiserum which gave, by

IIF a maximum reaction (+4) with the immunizing (homologous) strain, was used. Twenty three S probes were used.

The cells were initially prepared for immunological finger-printing as follows: A 20 ml culture was grown to stationary phase, centrifuged at 4500 rpm for 20 minutes, the supernatant decanted, and the pellet resuspended in 5 mis of 4% formalin. The Delta isolate was resuspended in 4% formalin and 4% NaCl in order to maintain its cellular integrity.

Determination of Methane

A Varian gas chromatograph Model 2720, equipped with a flame ionization detector, was used to quantify methane. The glass column was 6 feet x 1/4 inch, packed with Porapak Q (Waters Associates). The o o injector temperature was 150 C, the detector 250 C, and the column was

110°C. The carrier gas was nitrogen, and the flow was 30 mls/min.

Ultra High Purity (99.97%) methane (Matheson Gas, Joliet, 111.) was used as the standard. 47

Unknown samples were removed from the growth vessels and Injected into the gas chromatograph using a 100 ul Pressure-lok syringe

(Precision Sampling, Baton Rcuge, La.)* The sample size was 40 ul.

Determination of Formate Utilization by Methanogens

A variety of isolates were tested for the ability to utilize formate. The medium was different from that used for the general maintenance of methanogens in several respects. The buffer system in this medium was 30 mM PIPES (piperazine-N, N'-bis 2 ’ethanesulfonic acid) (Sigma Chemical Co., St. Louis, Mo.). This replaced the Na- bicarbonate in the other medium. Romesser j|t al. (71) found that bicarbonate inhibited the utilization of formate by Methanogenium spp. and that the use of PIPES buffer allowed formate utilization. The medium also contained 15 g/1 of Na-formate. Resazurin was left out and phenol red (0.001%) was added as a pH indicator. The head-space was nitrogen instead of H2:C02- Utilization of formate was obvious due to the change in the phenol red indicator from yellow to purple. The starting pH was 7.0. Formate utilizing organisms produced an alkaline reaction as they grew. The pH of the culture was readjusted to neutrality using 10% sterile, anaerobic formic acid to allow continued growth. Gas chromatographic analysis of the head-space confirmed methane production, and turbidity was indicative of growth. If an organism did not utilize formate, no growth would occur and the pH indicator would remain yellow. 48

Determination of Vitamin Requirements of Methanogens

Vitamin requirements were determined by utilizing the defined synthetic medium and adding different vitamins alone or in combination.

Bellco aluminum seal anaerobe tubes (Bellco Glass, Vineland, N.J.) were used. All glassware was acid cleaned and baked at 160°C to remove contaminating traces of organics and contaminating vitamins. Test organisms were pre-grown in thiamine only, biotin only, or a vitamin free medium, to dilute out the other vitamins and to reduce or minimize vitamin carryover. Growth was followed by measuring the absorbance at

580 nm on a Bausch and Lomb Spectronic 20 spectrophotometer. After all tubes stopped growing, total methane production was determined using gas chromatography. Tubes were pressurized to 45 psi with 80%

H 2 :20% CO^ before inoculation and each time the optical density was measured.

Sources of the Known Strains of Methanogens

Methanobrevibacter smlthii strain PS, Methanosarcina barkeri strain M.o.H. and Methanospirillum hungatei strain JF were obtained from M.P. Bryant, University of Illinois. Methanosarcina barkeri strain 227, Methanobacterium thermoautotrophicum strain &H and

Methanococcus vannielii were obtained from R.S. Wolfe, University of

Illinois. Methanobacterium formicicum strain MF1 was obtained from

John Robinson, Queen Mary’s College, London, England, and Methanococcus maripaludis was obtained from J. Jones, University of Illinois. The confirmation of the identity of these different methanogens was based primarily on cellular morphology. When morphologies were

similar, such as with Mb. bryantii M.o.H. and Mb. formicicum, the ability to utilize formate was the differentiating characteristic, Mb.

formicicum being formate positive.

The two Methanosarcina strains can be differentiated by their appearance under the phase contrast microscope. Cells of strain MS appeared phase bright and refractile, and the cells of strain 227 appeared dark.

Electron Microscopy of the Delta and Olentangy Isolates

The Olentangy and the Delta isolates were suspended in 1% glutaraldehyde and were applied to 3 mm formvar coated copper grids.

The Olentangy isolate was negatively stained with 2% uranyl acetate.

The Delta isolate was not stained. The grids were viewed and photo­ graphed using a Philips 300 electron microscope. RESULTS

The manipulation of extreme anaerobes such as methanogens requires

the development of unique skills, techniques, and equipment in order to

handle these organisms in a reproducible and convenient manner. In

order to perform experiments with these organisms and study their

physiological characteristics in pure culture, many techniques were

developed in this laboratory and many were adapted, and in some cases

improved upon, from procedures published in the literature and used in

other methane laboratories.

The evolution of the vessels in which the methanogens were grown

is illustrated in Figure 7. Cultures were grown routinely in 125 ml

Erlenmeyer flasks fitted with a stopper through which a cut off Hungate

type tube was inserted. The stopper was tied in.place with heavy

twine. When autoclaved, which must be done closed, the breakage of

this type of flask was as high as 50% with, many times, 50% of the

remaining flasks being oxidized. To make ten flasks of medium was an

accomplishment.

The cut off Hungate tube inserted through the stopper was sealed

with a thin butyl stopper which allowed penetration by a syringe

needle to permit inoculation and additions to the culture. The thinness

of the stopper, however, would not allow pressurization over 10 psi without leakage occurring through needle holes in the stopper.

50 Figure 7. Evolution of culture flasks for methanogens.

A. A 125 ml Erlenmeyer flask with a modified stopper through which a cut-off Hungate tube is inserted, tied in place with string.

B. A 500 ml round bottom flask with a modified stopper wired in place with copper wire.

C. A 125 ml Wheaton (Wheaton Glass, Vineland, N.J.) stoppered with a thick butyl stopper (Bellco Glass, Vineland, N.J.) held in place by an aluminum seal (2).

D. An 18 x 150 mm aluminum seal tube (Bellco Glass, Vineland, N.J.) stoppered with a thick butyl stopper.

E. A one liter Wheaton bottle fitted with a stopper through which a cut-off 18 x 150 mm aluminum seal tube has been inserted.

F. A Bellco pyrex one liter bottle fitted with a stopper through which a cut-off aluminum seal tube has been inserted.

51 52

Figure 7. 53

The round-bottomed flasks in Figure 7 replaced the Erlenmeyer

flasks. These flasks still had the same stopper design as the Erlen­ meyer flasks, but on these flasks the stopper was wired in place with

copper wire. These flasks were much stronger than the Erlenmeyers due to their round design and were much more capable of surviving auto- claving without exploding. These were, however, somewhat cumbersome and difficult to incubate and took up a lot of storage space. Also, just as with the Erlenueyer flasks, 10 psi was the maximum pressure that could be used due to the thinness of the stopper.

Finally, these flasks were replaced by 50, 100, and 125 ml Wheaton serum bottles, described by Miller and Wolin (57), which were sealed by crimping an aluminum seal over the stopper. The stopper used was a thick stopper developed by Balch (2) and made by Bellco. These stoppers allowed the serum bottles to be pressurized up to 45 psi. Also, these stoppers could be penetrated numerous times without leaking. Growing methanogens in these bottles under 80% H2 :20% CO2 at higher pressures allowed better growth, faster generation times, and less maintenance time devoted to re-gassing the cultures.

Bellco also makes aluminum seal tubes (18 x 150 mm). These tubes can be pressurized up to 45 psi and turbidity can be measured directly in these tubes using a Spectronic 20 (Bausch and Lomb) spectrophotometer fitted with an 18 mm cuvette adaptor.

Using these tubes and bottles sealed with a thick butyl stopper, held in place by an aluminum crimp seal, allowed for good growth of the methanogens. In addition, these tubes and bottles were conveniently stored, could be incubated easily, and large numbers of bottles 54 containing fresh medium could be easily made. Using the serum bottles,

100 bottles of medium could be made conveniently at one time, whereas using the Erlenmeyer or round-bottom flasks, 15 flasks was the limit to the number of flasks which could be prepared at any one time. The serum bottles survived autoclaving very well, so that the loss of time and material due to breakage in the autoclave was very small.

A special box was constructed for incubating these bottles as well as one liter bottles. This box is shown in Figure 8 and was affixed to a floor shaker incubator. The compartments were built so that over 200

125 ml serum bottles could be incubated on their side at one time.

This provided efficient agitation for the growth of the methanogenic bacteria.

For larger cultures of 100 to 300 mis, one liter bottles were used. These bottles are shown in Figure 7. These bottles were con­ structed as described in the Materials and Methods and were developed by Balch (2). Wheaton and Bellco one liter bottles were used. Although the Wheaton bottles were suggested by Balch ^t ail. (5), Bellco bottles were preferred for several reasons. The Bellco bottles were made of

Pyrex glass as opposed to soft glass. Also, Bellco bottles have a lower, wider profile, so that when equal volumes were in each type of bottle the Bellco bottles provided a larger surface area. Lastly, being made of Pyrex, they were less likely to explode in the autoclave, an occurrence not uncommon to the Wheaton bottles.

Figure 5 shows the kettle and wire screens in which these bottles were placed prior to autoclaving. The kettle and wire cages were used to protect each bottle in the event that if one bottle exploded during Figure 8. A plywood incubation box (A) for the

incubation of serum bottles on their

side, (B) the incubation of tubes in

racks, and (C) the incubation of one

liter bottles on their side.

55 Figure 8. 57 autoclaving the remaining bottles would not be damaged and as protec­

tion for the individual removing the bottles from the autoclave. A

couple of inches of water was usually added to the kettle before auto­ claving to provide a relatively slow cool-down period.

To grow methanogenic bacteria, a source of gas is required.

Nitrogen is required for the Hungate technique and is required as a substrate for the organisms. Commercially available gases have trace amounts of oxygen which can inhibit the growth of methanogens.

These trace amounts of oxygen were removed by passage of the gas over hot copper turnings contained in a tube. Originally, a commercially available (Sargent-Welch, Cincinnati, Ohio) gas scrubber tube and oven were used. This system is shown in Figure 9. The tube was made of quartz glass. To make connections, rubber tubing was used. This pre­ sented problems because at high flow rates of ^ 1^2 and high pressures the tubing became very hot, would melt, and eventually rupture. Since high flow rates and pressures up to 45 psi were required, the glass tube was replaced with a one inch copper tube, filled with copper turnings, having 1/4 inch copper tubing as both inlet and outlet.

Therefore, using Swagelok unions, strong secure connections could be made to and from the scrubber. This scrubber not only allowed the use of high pressures and fast flow rates, but it was also unbreakable, unlike the quartz glass scrubber. The all copper scrubber is shown in

Figure 10.

In order to perform the Hungate technique, multiple gassing probes were required. To expedite the task of making numerous bottles of anaerobic medium, several gassing outlets were also required. An all Figure 9 (A) A quartz-glass oxygen scrubber filled

with copper turnings (Sargent-Welch,

Cincinnati, Ohio); (B) inlet; (C) outlet.

58 Figure 9. Figure 10. An all copper oxygen scrubber filled with

copper turnings (A) and furnace (B).

Inlet (C) and outlet (D) are 1/4 inch

copper tubing. Connections (E) are made

with brass Swagelock 1/4 inch unions (Scioto

Valve, Columbus, Ohio).

60 61

Figure 10. 62

copper gassing manifold was designed and constructed in order to pro­ vide multiple gassing outlets and to deliver gas at high pressures.

This manifold was shown in Figure 3 (Materials and Methods). Using

this manifold, up to six bottles could be gassed simultaneously and pressurized up to 45 psi. By way of the three-way ball valve, the manifold could be switched from pressure to vacuum. With this con­ venient valve, the head-space of a bottle was easily withdrawn and replaced by simply turning the valve. The manifold also had a port which led to the anaerobic chamber through which mixed gases were delivered to jars stored in the chamber. This port was also used for vacuum and anaerobic filter sterilization which could be done inside the chamber.

A sterile gassing probe was designed to aseptically deliver anaerobic gas to bottles of culture medium. A 2 cc glass syringe was filled with cotton and capped with a butyl stopper through which a female Luer-lok fitting was placed. The male Luer-lok fitting was located at the end of the tubing coming from cnc of the gassing ports of the gassing manifold. Using this Luer-lok system, the gas probe was easily and securely attached or detached from the gassing line with a simple twist. When the sterile probe was not required, it could be replaced with either 22 ga. dispo needles or 4 1/2 inch bent 18 ga. cannulae used during the Hungate technique for out-gassing bottles or tubes. The 22 ga. needles were used during the exchange of head-space gas prior to autoclaving (i.e., was exchanged for I^rCC^).

To cultivate methanogens on agar plates, the plates had to be pre­ pared and streaked anaerobically and incubated under and 80% 1^:20% CO2 63

atmosphere. At first, BBL plastic anaerobe jars were used to incubate

the plates under Each jar was fitted with a plexiglass lid with two stoppered holes. These holes provided a means by which the jar could be purged with mixed gas. These jars were filled with plates

inside the Forma anaerobic chamber and then taken out to be purged with I^rCC^, but the jars could hold only about 5 psi without leaking.

When the jars were placed back into the chamber for incubation through

the interlock, where the vacuum was pulled, often much of the would leak from the jars. This system gave erratic growth results on plates.

To remedy this problem, Oxoid anaerobe jars were purchased. These jars had a thick metal lid, a very large clamp to hold it in place, two

Schraeder valves, a pressure-vacuum gauge, and a pressure relief valve.

These jars were very convenient to use. The jars could be purged using the Schraeder valves, could be pressurized to 12 psi, and growth could be monitored both visually and by observing the drop in pressure as H2:C02 was utilized. The pressure relief valve had to be blocked with a hollowed out stopper due to slight leakage at 12 psi. These jars were always used, gassed, and incubated inside of the anaerobic chamber. Another modification of the jars was the replacement of the knurled knob tightening bolt with a shorter bolt with a hex head. This was done to make the jars shorter so that they could be stacked two- high in the large incubator of the anerobe chamber.

The Forma anaerobe chamber was an improvement over the previous chamber in the laboratory. The first chamber was all vinyl, did not have an incubator, and had a very high rate of gas loss due to diffusion. 64

The Forma anaerobic chamber was modified by the addition of gas lines from the gassing manifold so that mixed gas could be used to purge the Oxoid anaerobe jars. This modification is shown in Figure 4.

The jars were gassed using female air-chunks obtained from a local automotive parts store (Bucks Auto Supply) for about one-fifth the cost if purchased from Oxoid. Filling the anaerobe jars was similar to filling a bicycle tire.

In essence, the system used in this laboratory for growing methano- genic bacteria on plates was a hybridization of the Ultra low oxygen chamber of Edwards and McBride (24) and the steel cylinder (or pressure cooker) of Balch et a^. (5).

Figures 11, 12, 13, 14, and 15 illustrate the growth of methano- gens on plates using this system. Growth was luxuriant and was com­ parable to the type of growth obtained with organisms which are much less fastidious. A plate containing several mis of reducing agent was routinely ad led to the jars. This allowed H^S to escape and saturate the agar plates, providing a highly reduced agar surface as well as providing I^S, a required nutrient for the methanogens. Growth was always better when was added.

Development of a Defined Growth Medium

The development of a defined growth medium was undertaken in order to grow a variety of methanogenic bacteria under carefully defined con­ ditions in order to study more effectively the physiology of methano­ genic bacteria. If specific growth requirements were defined, a better understanding of the methanogenic physiology would be possible. Figure 11. Methanobacterium bryantii strain M.o.H.

growing on a defined medium.

65 Figure 11. Figure 12. Me.hanosarcina barkerii strain MS

growing on a defined medium.

67 Figure 12. Figure 13. Methanococcus vannielii growing on

defined medium.

69 70

Figure 13. Figure 14. Methanobrevibacter smithii strain PS

growing on defined medium.

71 72

m .

Figure 14. Figure 15. Methanobacterium formicicum strain RC

growing on defined medium.

73 Figure 15 75

At first, all of the methanogens in the laboratory were grown in

one medium developed by Bryant (8). It contained yeast extract and

trypticase soy broth as well as several salts. This medium allowed the

good growth of methanogens but made the identification of trace nutrients impossible.

The point in developing a defined medium was to be able to grow methanogens under defined conditions. The resulting medium accomplished this, even though all organisms did not grow quite as well on the defined medium as on the enriched medium. For example, Mbr. smithii strain PS grew with a generation time of seven hours in the organic medium, but its generation time was 10 h in the defined medium.

Growth yields were similar (e.g., optical densities obtained were 1.1-

1.3 at 580 nm). Msp. hungatei also grew faster in the organic medium

(g = nine hours), whereas in the defined medium, the spirillum had a generation time of 13 h (R.E. Corder, M.S. Thesis, Ohio State

University, 1978). Growth yields for the spirillum were better in the organic medium, with an O.D. at 580 nm of 1.0 as compared to 0.8 in the defined medium. Other organisms such as Mb. thermoautotrophicum (108),

Me. vanniellii (5), and Me. voltae (55) were also stimulated by the addition of organics. However, the Olentangy coccus and the Cuyahoga coccus were not stimulated by the addition of organics. No stimulation was observed whether 0.2% yeast extract was absent or present.

Generation times with and without yeast extract were 11.0 and 11.6 h, and methane production was 1.2 and 1.3, respectively. Under condi­ tions when cell mass was the most important consideration, the addition of yeast extract and trypticase was helpful in producing a maximal yield. The first defined medium tried was a modification of the medium of

Balch et al. (5) and contained the following (g/1): KH2PO^, 0.3;

K2HP04 , 0.3; NaHC03 , 5.0; ( N H ^ S O ^ , 0.3; NaCl, 0.61; Na acetate, 2.5;

MgS04 -7H20, 0.13; CaCl2 *2H20, 0.008; nitriloacetate, 0.015; CoCl2 ,

0.001; ZnS04 , 0.001; CuS04 ‘5H20, 0.0001; A1K(S04)2, 0.0001; H3B03,

0.0001; Na2Mo04*2H20, 0.0001; MnS04'2H20, 0.005; as well as the following vitamins (ug/1) : folic acid, 20; pyridoxine hydrochloride,

100; thiamine hydrochloride, 50; riboflavin, 50; nicotinic acid, 50; calcium pantothenate, 50; vitamin B^2 , 1; and para-amino-benzoic acid,

50. At this time, biotin and lipoic acid were not available and so were omitted.

When Mbr. smithii strain PS was transferred from the organic medium to this defined medium, the first transfer grew well, but on following transfers, growth became less and less until no growth occurred.

This indicated that the dilution of an essential nutrient was taking place. Biotin and lipoic acid were obtained. When these two vitamins were added to the defined medium, growth was good and was easy to maintain through many transfers. However, the generation time and cell yield were not very close to that obtained in organic medium

(approx. seven hours in organic medium) in that overnight the culture would grow to maximum turbidity whereas on the defined medium, it would take three to five days. To improve the medium, it was decided to add more ammonia and iron, as was found necessary by Taylor and Pirt (93) in order to grow Mb. thermoautotrophicum in a defined medium without encountering ammonium and iron limitations. The amount of ammonium 77

chloride was increased to 2.7 g/1, and the FeSO^ was increased to

0.007 g/1. These additions enhanced growth greatly. The cultures had

a generation time of approximately ten hours and grew to turbidities

comparable to those obtained in the organic medium. Therefore, the

essential requirements provided by the organic supplement, yeast

extract, and trypticase were primarily ammonium, iron, and vitamins.

Knowing this, it was possible to further define the growth requirements

of Mbr. ruminantium as well as other methanogens which would grow in

this medium.

The vitamin requirements of Mbr. smithii were determined using the

process of elimination as well as the information obtained when com­

posing the medium originally that either biotin, lipoic acid, or both

were required for good growth.

Table 1 shows one of the experiments performed to demonstrate the

vitamin requirements of Mbr. smithii.

These experiments were carried out in 18 x 150 mm Bellco aluminum

seal anaerobe tubes containing 5 mis of medium containing the indicated

vitamin(s). The inoculum was grown up in the defined medium containing

biotin and thiamine. This type of experiment was performed repeatedly,

using inocula grown without vitamins, with thiamine only, and with

thiamine and biotin. The results obtained were essentially the same.

When biotin and thiamine were present, the maximum growth yield was

obtained; when one or both of these vitamins were omitted, growth was

inhibited by at least 50%. What is interesting is that although the

optical densities of the culture without both vitamins was much lower,

the total amount of methane produced was not as markedly influenced. Table 1. Vitamin requirements for Methanobrevibacter

smithii strain PS, pre-grown on biotin and

thiamine. Determinations were performed in

18 x 150 mm aluminum seal tubes in triplicate.

Methane determinations were performed imme­

diately after stationary phase was achieved.

78 79

Table 1.

Max. 0. D. mM CH4 Vitamins added 580 nm produced

Biotin 0.2 1.0

Biotin and thiamine 0.73 1.8

All vitamins 0.73 1.8

No vitamins 0.17 0.8

All except biotin 0.26 1.2

All except biotin and thiamine 0.15 0.8 80

The methane determinations shown in Table 1 were performed

immediately after the stationary phase was achieved by the growing

cultures. Table 2 shows another experiment in which the incubation

went 48 hours past the stationary phase of the growing cultures. It

can be seen that the growth response to biotin and thiamine was the

same as in Table I, but that the differences in total methane was

almost unnoticeable. It appears that the effect of the vitamins was to

stimulate cell production and did not have an observable effect on the

production of methane by the organism.

This defined medium was developed to this point using Mbr. smithii

strain PS as the test organism. The only other methanogen which grew

fairly well in this medium was Ms. barkeri strain M.S. Msp. hungatei

and Mb. bryantii strain M.o.H. grew poorly. Growth of the spirillum was sporadic and Mb. bryantii strain M.o.H. required 10 to 14 days to

reach a turbidity of 0.8 at 580 nm.

Further improvement in the medium was sought to aleviate diffi­

culties involved in isolating a methanogenic coccus from the Olentangy

River.

The coccus was eventually isolated using serial dilutions. This

technique will be discussed later. Once the organism was isolated in pure culture, growth became erratic. While culturing the organism with

a minor contaminant, the culture grew to full turbidity every 72 hours

and was routinely transferred at these intervals. However, when the

coccus was obtained in pure culture, growth became sporadic and trans­

fers did not always grow. The organism became very difficult to maintain in culture. Table 2. Vitamin requirements for Methanobrevibacter

smithii PS pre-grown on thiamine only and

incubated for 48 hours after stationary phase

was achieved. Determinations were performed

in 18 x 150 mm aluminum seal tubes in

triplicate.

81 82

Table 2.

Max. O.D. mM CH^ Vitamins added______580 nm______produced

Biotin 0.16 1.2

Biotin and thiamine 0.75 1.4

All vitamins 0.75 1.4

No vitamins 0.11 1.3

All except biotin 0.11 1.3

All except biotin and thiamine 0.14 1.3 83

- j - j - Because Patel al. (63) found that the optimal Fe concentra­ tion for methanogens was in the range of 0.3 to 0.9 mM, it was decided to increase the amount of iron in the medium. The FeSO^^^O concen­ tration was increased from 0.007 g/1 to 0.01 g/1. It was not possible to add the amount of iron to the medium as suggested by Patel et al.

(63) because of the occurrence of a black precipitate when the recom­ mended amount of iron was added. The amount used, 0.01 g/1, was the maximum amount that could be added such that on addition of reducing agent little or no precipitation occurred. Any decrease in the amount of reducing agent added also resulted in a reduction of growth. This effect was, presumably, due to a sulfide limitation, since methanogens require sulfide for good growth (99, 110).

Nevertheless, with the increase in iron from 0.007 g/1 to 0.01 g/1, the problem with the pure culture of the methanogenic coccus was solved.

The organism began growing more reproducibly, had a generation time of

11 h, and could be routinely transferred every 48 hours.

The increase in iron also greatly stimulated Msp. hungatei and

Mb. bryantii strain M.o.H. Mb. bryantii grew with a generation time of six hours and reached maximum turbidity within 48 hours. The spirillum also grew much faster and was completely grown within 72 hours. These results were reproducible.

Therefore, a defined medium had been developed which would grow a diverse number of methanogens. Short generation times and high optical densities were obtainable using this medium. However, the medium was still complex in that it contained ten different vitamins as well as a complex assemblage of trace metals, including Al, B, Mn, Ca, Co, Zn, 84

Cu, Mg, Fe, Mo, and Ni. The goal was to eliminate as many of the superfluous components as possible. A paper by Schonheit ^t jil. (76) described the trace metal requirements of Mb. thermoautotrophicum.

These investigators demonstrated no requirement for Al, B, Ca, Cu, Mn, or Zn. However, the organism required Co, Mo, Ni, and Mg. Based on these findings, Al, B, Ca, Cu, Mn, and Zn were deleted from the medium.

No change in the growth characteristics of the methanogens was dis­ cerned. Since Mb. thermoautotrophicum did not require these metals, it seemed reasonable that the other methanogens did not require these metals in the medium.

In fact, to date, fourteen different methanogens have been grown on this medium with good results. The growth characteristics of the methanogens in this medium are shown in Table 3. The only methanogens tested which would not grow in this basic medium were organisms which had been previously reported to have special requirements. Mbr. ruminantium Ml required CoM and o( -methyl butyrate (9), and Me. voltae required leucine, isoleucine, and pantothenic acid (10). When leucine and isoleucine were added, Me. voltae grew in the defined medium. Mbr. ruminantium Ml also grew in the defined medium when CoM and oi -methyl butyrate was added, but it grew slowly when compared to its growth in a medium containing yeast extract and trypticase soy broth.

Since Mb. bryantii M.o.H. and Msp. hungatei JF grew well in the medium, the vitamin requirements for these two organisms were deter­ mined. Table 4 shows the data obtained when Mb. bryantii M.o.H. was grown on the defined medium containing different combinations of Table 3. Comparative growth characteristics of

fourteen methanogens grown on defined

medium. (A) g(h) is the generation time

in hours for the different organisms.

(B) CH^ equals the mMoles of methane

produced per mg dry weight of cells.

All determinations were performed in

triplicate.

85 86

Table 3.

Max. O.D. bY 8 (h)3 580 nm CH.

Methanobrevibacter smithii PS 10 1.1 0.7

Methanobacterlum thermoautotrophicum AH 1.6 1.1 1.4

Methanobacterium thermoautotrophicum RC 3.0 1.0 1.2

Methanobacterium formicicum MFl 6.0 1.2 0.9

Methanobacterium formicicum RC 3.8 1.3 0.9

Methanosarcina barker! MS 7.0 - 0.4

Methanosarcina barkerii 227 7.6 - 0.4

Methanococcus vannielii 16 0.7 0.6

Methanospirillum hungatei JF 13.0 0.8 -

Methanococcus maripaludis 3.0 0.9 0.6

Cuyahoga River coccus 11.6 0.9 1.0

Olentangy River coccus 11.0 0.9 1.0

Mississippi River Delta coccus 2.0 0.9 0.7

Methanobacterium bryantii M.o.H. 6.0 1.0 0.7 Table 4. Vitamin requirements for Methanobacterium

bryantii strain M.o.H. Determinations were

performed in triplicate in 18 x 150 mm

aluminum seal tubes.

87 88

Table 4.

Max. O.D. mM CH^ Vitamins added 580 nm produced

Biotin 0.74 1.3

Biotin and thiamine 0.76 1.3

All vitamins 0.60 1.2

No vitamins 0.20 0.9

All except biotin 0.30 1.0

All except biotin and thiamine 0.20 0.8 89 vitamins. The inoculum was pre-grown on thiamine. It was obvious that when biotin was present, good growth occurred, and when biotin was absent, growth was inhibited. Unlike Mbr. smithii PS, which required biotin and thiamine for optimal growth, Mb. bryantii M.o.H. required only biotin. However, as the Mbr. smithii PS, the main effect of the addition of biotin was in the production of biomass and not the pro­ duction of methane. For example, when all vitamins were absent, the maximum O.D. was only 0.2, but 0.9 mMoles of methane were produced.

However, the addition of biotin resulted in an O.D. of 0.74 and 1.3 mMoles of methane. Table 5 shows the results of growing Msp. hungatei

JF in the defined medium with different combinations of vitamins. No clear-cut effects could be discerned. The swirling manner in which the organism grew in liquid culture made the measurement of optical density difficult. But, the organism was maintained in a vitamin-free medium through multiple transfers. The only noticeable effect of the addition of vitamins was a slightly faster growth rate and slightly greater turbidity. The organism could be maintained in a vitamin-free medium, unlike Mbr. smithii PS and Mb. bryantii M.o.H.

With the determination of the vitamin requirements for Mbr. smithii PS, Mb. bryantii M.o.H. and M s p . hungatei, a more complete picture of the vitamin requirements of the main groups of methanogens was obtained. Mb. thermoautotrophicum, Me. vannielii, the Olentangy coccus, the Cuyahoga coccus, and the Delta coccus did not require vitamins. On the other hand, other researchers have determined the vitamin requirements of other methanogens. Scherer and Sahm (74) demonstrated that Ms. barkeri required riboflavin, while Whitman et al. Table 5. Vitamin requirements for Methanospirillum

hungatei strain J.F. Determinations were

performed in triplicate in 18 x 150 mm aluminum

seal tubes.

90 Table 5.

M a x . O.D. Vitamin(s) 580 nm

Thiamine only 0.53

No vitamins 0.58

Biotin only 0.45

Biotin and Thiamine 0.50

All ten 0.56

Lipoic only 0.43

Lipoic and Thiamine 0.45

Biotin and Lipoic 0.49

No Biotin, no Thiamine, no Lipoic 0.44

No Biotin, no Thiamine 0.54 92

(101) demonstrated a requirement of pantothenic acid by Me. voltae.

Isolation of New Methanogens

The preceding experiments indicated the utility of the defined

medium as a routine culture medium as well as for the study of growth

requirements and comparative characteristics. The medium has also

proved to be an excellent medium for the enrichment and isolation of a wide variety of methanogens from different environments. Table 6 lists

the different methanogens isolated and their sources. All were

isolated using the same basic defined medium. All three Maine methano­

gens were isolated from the same sediment sample. Penobscot Bay is a

clam flat on the coast of Maine and is a marine environment. However, none of the three isolates had a requirement for a high salt concen­

tration.

Other methanogens were seen in enrichments, but no attempts were made to isolate them, including spirilla and brevibacters. Usually,

Mb. formicicum would over-grow other types of methanogens. Fur

instance, in early enrichments, a spirillum or a Mb. bryantii may have

initially dominated, but on subsequent transfers strains of Mb. formicicum tended to out-grow the other methanogenic species and then predominate.

Numerous sites were surveyed for methanogens, such as a flooded corn field, a sewage digestor, river sediment and sediment from a salt water cove. All locations resulted in methanogenic activity when sedi­ ments from these sites were incubated in the defined medium. Table 6 New methanogenic isolates, isolated

using a defined medium.

93 Table 6.

Formate Isolate ______Source______Temperature Utilization

Methanosarcina barker! Penobscot Bay (Me.) 37° C -

Methanobacterium thermoautotrophicum Cuyahoga River (Ohio) 65°C -

Methanobacterium formicicum Cuyahoga River (Ohio) 37°C +

Methanobact erium formicicum Tennessee River (Ala.) 37°C +

Methanobacterium formicicum Ohio River (Ohio) 37°C +

Methanobacterium formicicum Olentangy River (Ohio) 37°C +

Olentangy coccus Olentangy River (Ohio) 37°C -

Cuyahoga coccus Cuyahoga River (Ohio) 37°C -

Delta coccus Mississippi River delta (La.) 37°C +

Maine coccus Penobscot Bay (Me.) 37°C -

Methanobacterium spp. Penobscot Bay (Me.) 37°C +

Methanobacterium spp. Cuyahoga River (Ohio) 37°C +

Methanobacterium bryantii Ohio River (Ind.) 37°C 95

In each case, at least one methanogen was isolated and sometimes

as many as three. Isolation of these organisms was a relatively simple matter when the enrichments were streaked for isolation and single

colonies were picked. Using the defined medium, colonies which grew well almost invariably proved to be methanogenic organisms when picked

off the agar surface and grown in liquid medium. Contaminants grew poorly on the medium and generally appeared as a haze on the surface

of the agar. With experience, colonies of methanogens were easily

recognized.

Characterization of the Olentangy River Coccus and the Mississippi

River Delta Coccus

The Olentangy coccus was isolated from sediment from the Olentangy

River using the isolation techniques already described. The organism was purified both by dilution to extinction and by streaking for

isolation. The organism was an irregular coccus occuring singly or

in pairs, with a cellular diameter of 1.0 to 1.5 um. It was non- motile, and flagella were not seen in negative stains viewed with an

electron microscope. On agar medium, the organism formed convex, mucoid yellow colonies up to 1.5 cm in diameter (Fig. 16).

Figure 17 is a phase contrast photomicrograph of the organism.

The cells were centrifuged into a pellet and fixed in 1% glutaralde- hyde. This step was necessary due to the fact that the cells were fragile and ruptured easily when resuspended in distilled water.

Figure 18a is an electron micrograph of the isolate, demonstrating its spherical morphology. These cells were also fixed with 1% Figure 16. Colonial morphology of the Olentangy

isolate growing on a defined medium.

96 Figure 16. Figure 17. Phc.se contrast photomicrograph of the

Olentangy Isolate. Cells were fixed

in 1% glutaraldehyde.

98 §k.* **f<*f J^;-V'*. -Vt *,;•:•'{* i H P K & k S & ’A # ■. - * *4 * ; ^ ^ - *> ' * % • & ■ • * **• ••; •* •. * ’& * vf

^ - f e :

Figure 17. Figure 18. Electron micrographs of the Olentangy

isolate (A) fixed with 1% glutaraldehyde

and (B) not fixed in glutaraldehyde and

stained with 2% uranyl acetate. The bar

represents 1 urn.

100 101

Figure 18. 102 glutaraldehyde. Figure 18b is the same organism, not fixed with glutaraldehyde. The spreading and flattening of the cell was due to the rupturing of the cells.

The Mississippi Delta isolate was also an irregular coccus occurring singly or in pairs and had a cellular diameter of 1.0 to 1*5 um. The colonies appeared greenish yellow, convex, often with dark centers (Figure 19). These colonies were much less mucoid than the colonies of the Olentangy isolate. The coccus was non-motile and did not exhibit flagella in negatively stained preparations when viewed with the electron microscope. Figure 20a is a phase contrast photo­ micrograph of the Delta isolate. The organism appeared dark and almost refractile under the microscope. Figure 20b is an electron micrograph of the organism. The irregularity seen was probably due to the fixa­ tion of the organism in glutaraldehyde, resulting in the observed sharp edges and corners. However, this preparation was unstained. No heavy metal salt was applied to give contrast. It was obvious that the cells were very electron dense. A possible explanation for their opacity may be that they concentrated metals from the growth medium,

Fe, Mo, Co, and Ni inside of the cells, producirg this electron dense effect.

Table 7 shows a physiological characterization of the Olentangy coccus. The organism required acetate for growth, was not stimulated by vitamins or yeast extract, and did not utilize methanol or formate for growth and methanogenesis.

Table 8 shows a physiological characterization of the Delta isolate. This organism grew very rapidly with a generation time of Figure 19. Colonial morphology of the Delta isolate

growing on a defined medium with 3% NaCl

and 20 mM MgC^.

103 Figure 19. Figure 20. (A) Phase contrast photomicrograph of

the Delta isolate. Cells were fixed

in 1% glutaraldehyde.

(B) Electron micrograph of the Delta

isolate. Cells were fixed with 1%

glutaraldehyde but were not negatively

stained. The bar represents 1 urn.

105 4*

M* . _»* I

1 u m

Figure 20. Table 7. Physiological characterization of the Olentangy

isolate. Determinations were performed in

triplicate in 18 x 150 mm aluminum seal tubes. i + vitamins means all ten vitamins were added;

Na acetate was used in a concentration of 0.25%;

yeast extract, 0.2%; methanol, 10%, formate, 1%;

and Na-butyrate, 0.25%.

N.G. = no growth.

g(h) = generation time in hours.

O.D. = optical density at 5'80 nm.

107 Table 7.

Head-space Total Substrates gas F i n d O.D. g(h) CH,, (mM)

1. Na Acetate + Vitamins +H2:C02 0.67 10.9 1.2

2. Na Acetate No Vitamins +H2 :C02 0.67 11.0 1.2

3. Na Acetate + Vitamins + Yeast Extract +H2 :C02 0.65 11.6 1.3

4. Na Acetate + Vitamins + Methanol +H2:C02 N.G. ——

5. Na Acetate + Vitamins + Methanol ~H2 :C02 N.G. — —

6. Na Acetate + Vitamins + Na Butyrate +H2:C02 0.60 12.6 1.2

7. Na Acetate + Vitamins + Na Formate -H2:C°2 N.G. — —

8. Methanol + Vitamins +H2 :C02 N.G. —— 1 9. Methanol + Vitamins -H2 :C02 N.G. ——

10. Na Butyrate + Vitamins +H2 :C02 0.10 17.0 0.1

11. Na Formate + Vitamins +H2 :C02 N.G. ——

12. Na Formate + Vitamins -H2 :CO2 N.G. — —

N.G. = No Growth g(h) = Generation time in hours Table 8. Physiological characterization of the Delta

isolate. Determinations were performed in

triplicate in 18 x 150 mm aluminum seal tubes.

All ten vitamins were added or were deleted as

indicated.

g(h) = generation time in hours.

O.D. = optical density at 580 nm.

109 110

Table 8.

Head-space Final Total Growth Conditions______Gas______O.D.______g(h)____ CH^ (mM)

3% NaCl, 20 mM MgCl2 , plus acetate & vitamins H2 :C02 0.94 2.0 1.1

3.5% NaCl, 1 mM MgCl2 , plus acetate & vitamins H2 :C02 0.85 2.4 1.0

3.5% NaCl, 1 mM MgCl2 , no acetate, no vitamins H2 :C02 0.86 3.0. 1.0

Methanol (0.5%) N2 0.06 — 0.0

Methylamine (0.5%) N2 0.08 — 0.0 Ill

only 2 h. It did not require acetate, vitamins, or a high concentration

of MgC^. Me. maripaludis, which appeared very similar to this

organism, had a requirement of at least 15 mM M g C ^ (J. Jones, personal

communication). The Delta isolate did not grow on methanol or methyl-

amine as a sole carbon and energy source, but unlike the Olentangy

coccus it did utilize formate.

Since there are very few known methane-producing cocci and taxo­ nomic criteria among the methanogens was very limited, it was very difficult to immediately place these isolates into their proper genus and species classification. In order to help in the assignment to a

specific group, formalinized samples of the organisms were sent to

Dr. M.J. Wolin, Albany, N.Y. Dr. Wolin had a bank of specific anti­ bodies to a great number of known methanogens. By using this bank of antibodies and grading cross-reactivity among the methanogens from +4 for a homologous antigen-antibody reaction to a +1 for little reaction, he was able to place unknown methanogens into their correct genus by means of how much cross-reaction occurred as described recently by

Conway de Macario j^t al. (12, 13, 14). The results of this serological typing are shown in Table 9.

These results indicated that the Delta isolate was probably a typical Methanococcus, only related to Me. vannielii, and was probably a new species. The result with the Olentangy coccus, however, was difficult to explain. The organism was obviously a coccus and not a brevibacter. The fact that there was a +4 homologous reaction with the

Mbr. smithii strain ALI and no reaction with strain PS or any other brevibacters indicated some serological determinant was common between Table 9. Cross-reactivity of formalinized whole

cells of Olentangy and Delta isolate with

antiserum S probes by indirect immuno­

fluorescence (IIF).

112 113

Table 9.

Test Organism Cross Reaction

Olentangy Isolate Methanobrevibacter smithii ALI, 4+

Delta Isolate Methanococcus vannielii, 1+

No Cross-Reactivity With:

Methanobrevibacter smithii PS

Methanobrevibacter ruminantium Ml

Methanobrevibacter arboriphilus DH1

Methanobrevibacter arboriphilus AZ

Methanococcus voltae PSv

Methanococcus mazei MC 6

Methanosarcina barkeri MS

Methanosarcina barkeri R1M3

Methanosarcina barkeri 227

Methano sarcina barkeri W

Methanogenium marisnigri JRlm

Methanogenium cariaci JRlc

Methanomicrobium mobile BP

Methanobacterium bryantii M.o.H.

Methanobacterium bryantii M.o.H.G.

Methanobacterium thermoautotrophicum GC1

Methanobacterium thermoautotrophicum A H

Methanobacterium formicicum MF

Methanospirillum hungatei JF1

Methanosarcina TM1 114 these two organisms. The Olentangy coccus may represent a new genus within the family Methanobacteriaceae since no cross-reactivity was seen with Methanococcus spp. or Methanogenium spp. (M.J. Wolin, personal communication).

Figure 21 demonstrates that the temperature optimum for both organisms was 37°C. Figure 22 shows the NaCl optimum for each organism.

The Olentangy isolate's NaCl optimum was 1%. This optimum was due to the stabilizing effect of the salt. When no salt was added to the medium, the organism grew as well as it did with 1% NaCl but lysed when it reached stationary phase. The 1% concentration was the maximum con­ centration which did not inhibit growth but still stabilized the cells during stationary phase, permitting them to continue growth and methanogenesis.

The NaCl optimum of the Delta isolate was from 3 to 4%. This salt requirement made this organism one of the few true marine methanogens.

Me. vannielii was isolated from the San Francisco Bay but does not require high concentrations of NaCl (86). Me. voltae does require high salt concentrations (4% optimal), but Me. maripaludis, isolated from a marine environment, requires high concentrations of M g C ^ (15 mM) for growth and not high concentrations of NaCl (100). Figure 21. Temperature optima of the Olentangy and

Delta isolates. Determinations were

performed in triplicate in 18 x 150 mm

aluminum seal tubes without shaking.

115 116

m

ro'

eo Temporatura

co n

uiu 08S •ouoqjotqy

Figure 21. Figure 22. NaCl optima for the Olentangy and Delta

isolates. Determinations were performed

in triplicate in aluminum seal tubes at 37°C.

Absorbance was measured at 580 nm.

117 118

«n

■co

eo m co

uiu 08? •siicqjosqy

Figure 22. DISCUSSION

The study of methanogenic bacteria has been a technically diffi­ cult proposition for most microbiologists. As a result, the number of laboratories working with methanogens is relatively small. Great strides have been made in solving the technical difficulties in the past several years (2, 57, 63, 74), and with an increase in interest by medical laboratories in anaerobic bacteria, commercial systems for dealing with these organisms are becoming available. As delineated in the Materials and Methods and Results sections, a large portion of this dissertation has dealt with the development of new techniques, apparati, and the modification of commercially available equipment for the easy, economical, reproducible, and controlled cultivation of methanogenic bacteria. The techniques described should be extremely helpful, not only to researchers new to methanogens but also to those who have experience in the field. One of the primary hindrances to innovations in methanogen cultivation is the uncertainty whether or not commercially available systems (e.g., Oxoid anaerobic jars, Forma anaerobe chambers) are applicable to methanogens. Due to the cost involved in purchasing these pieces of equipment without knowing their worth, few investigators are willing to commit to new equipment. The results demonstrated by the growth of the many different methanogens on plates demonstrated that the system described herein is an excellent 120

system for the cultivation of methanogenic bacteria.

The Oxoid jars operated superbly and were extremely convenient

when modified by blocking the relief valve and the replacement of the

knurled knob with a bolt to shorten the jar. This system was far

superior to that of Balch ^t _al. (2) who used a steel cylinder with end

plates bolted in place and only one port through which a vacuum was

pulled and then pressurized. The cylinders were loaded inside an

anaerobe chamber and then pressurized and incubated outside of the

chamber. To check for growth on plates, the cylinder had to be taken

back into the chamber. The Oxoid jars are polycarbonate and are

transparent, and when used in conjunction with a Forma anaerobe chamber

with incubator, need never be taken out of the chamber. Growth on

plates could be seen inside the jar. Also, the oxidation state of the

plates within the jars could be easily determined when resazurin was

included in the agar medium. When working with an all-steel cylinder,

the plates cannot be seen and accidental contamination by oxygen cannot

be determined without taking the cylinder back into the chamber and

opening it up. For the reasons of convenience as well as the commer­

cial availability of Oxoid anaerobe jars (steel cylinder incubation

chambers are not commercially available and must be custom made),

these jars were a great improvement over the old systems. The two

other incubation systems described in the literature were pressure

cookers (24) which had the same drawbacks as the steel cylinders and

the Ultra low oxygen chamber of Edwards and McBride (23). The ULOC was an incubated plastic box constructed inside of an anaerobe chamber

and purged with H2 :C02« It did not have the capability of being 121

pressurized. Pressurization of incubating plates with helped a

great deal since methanogens utilized so rapidly. For example,

actively growing plates of methanogens, when pressurized to 12 psi

pulled a vacuum of 10 in of Hg in 18 hours. This indicated, in order

to get the best growth on plates, excess must be supplied, and

atmospheric pressures of ^rCC^ were not sufficient to obtain maximal

growth. The addition of reducing agent to the anaerobe jars and the

pre-reduction of agar plates by exposure to l^S produced by the reducing

agent produced much faster and denser growth on solid media. The

reducing agent could be replaced with pure gaseous H^S, but due to its

toxicity, it was felt that it was more controllable and safer to allow

the gradual release of I^S from the cysteine-sulfide reducing agent.

Better growth of methanogens on plates has not been demonstrated in the

literature up to this point.

The design and construction of the gassing manifold and its inte­

gration with the anaerobe chamber has proven to be a boone. The mani­

fold was similar to the one designed by Balch et al_. (5) but was

simpler in design, more versatile, and allowed much higher gas flow

rates. This was due to the larger diameter tubing used in the con­

struction of the manifold. Where Balch et; _al. utilized 1/8 inch tubing

to connect all of their metering valves in series, 1 inch copper tubing was used instead for the installation of the 1/4 inch shut-off valves.

The 1 inch tubing allowed a pressurized reservoir to be built up behind

the Nupro shut-off valves. This reservoir effect allowed all of the

lines to be opened at once without a drastic drop in flow rates from

one end to the other. Therefore, exchanging the atmospheres on six 122 bottles at one time was relatively quick, easy, and uniform. Another improvement in the gassing manifold system was the use of Luer-lok connections in order to remove the sterile gassing probe and replace it with large gassing cannulae or with regular needles. The Balch et al. manifold did not have this capability since the sterile probes were not exchangeable with other types of gassing needles. An extra circuit was added to their gassing manifold to provide the flexibility of using the manifold for the Hungate technique (110).

The gassing manifold was also connected to the anaerobe chamber, and this made the system all the more versatile. The gassing manifold could be used for the Hungate techniques, exchanging head-space atmos­ pheres, sterile pressurization of media bottles up to 45 psi, filter sterilization outside and inside of the anaerobe chamber (by virtue of the ancillary vacuum pump), and purging and pressurization of the anaerobe jars inside of the anaerobe chamber.

The development of a defined medium also greatly simplified the cultivation and maintenance of the methanogens. The medium was designed to grow as great a variety of methanogens as possible, as simply as possible. All components were added with a purpose. This studied approach was relatively unusual when growing methanogens since many researchers tended to add extremely complex materials such as sewage sludge supernatant (53), yeast extract and trypticase (81), rumen fluid (5, 81, 108, 110), and a wide variety of volatile fatty acids. These additions were made, of course, because the growth requirements for methanogens were unknown and the difficulty encountered with anaerobic techniques was enough to worry about without being 123

concerned over whether all growth requirements were being supplied.

So far, it has usually turned out that of these complex nutrients,

the methanogens have required very simple compounds, such as CoM

(2-mercapto-ethanesulfonic acid) and e<-methyl butyrate from rumen

fluid and sludge supernatant (90), leucine and isoleucine from

trypticase for Me. voltae (101), and vitamins from yeast extract for

such organisms as Ms. barkeri (74). On the whole, however, most of the methanogens presently in pure culture have not been shown to require unusual compounds or cofactors for growth, but the complex organics are

still added (5). The defined medium was composed with the objective of adding only components which the methanogens actually required for growth.

K^HPO^ and KH^PO^ were added to provide phosphate and potassium and some buffering capacity. Sodium acetate was added as a carbon source. Several methanogens, such as Mbr. smithii PS, require acetate

(9), and while other methanogens did not require acetate, such as Mb. thermoautotrophicum, they did utilize acetate and incorporate it into cell carbon (31). Ammonium sulfate and ammonium chloride were added to provide nitrogen in the form of ammonium which methanogens require (9).

Both types of ammonium were used because methanogens require ammonium in relatively high concentrations (93) but also require sulfate (63).

Patel et a^. (63) indicated that the optimum sulfate concentration for the growth of Mb. bryantii M.o.H. and Msp. hungatei was in the range of

0.16 to 0.52 mM. An absence of sulfate drastically reduced growth.

Sodium bicarbonate was added to the medium to provide buffering. The bicarbonate did not provide a source of CO2 for the growth of the 124 methanogens. The active species of carbon dioxide fixation has been

shown to be CO2 (34). When Mbr. smithii strain PS was grown under 100%

H2 with 5 g/1 sodium bicarbonate in the medium, it was unable to grow.

Free carbon dioxide must be provided for growth to occur (or as formate

for formate utilizing methanogens). Ferrous sulfate was added as a

source of iron. This proved to be one of the most critical additions

to the defined medium when used in the concentration of 0.01 g/1.

Although Patel et al. (63) and Taylor and Pirt (93) demonstrated a need for increased iron in the medium, many investigators normally added only 0.003 g/1 (5) because yeast extract and trypticase are used and probably contain significant amounts of iron. When these were omitted from the medium, more iron had to be added. As stated in the Results section, 0.007 g/1 was originally used because good growth of Mb. smithii PS occurred at this concentration and very little black FeS precipitate occurred when reducing agent was added. However, growth of Mb. bryantii strain M.o.H. and Msp. hungatei was very slow and Mb. bryantii strain BCF (a laboratory isolate) would not grow in this medium. Also, the Olentangy isolate grew very erratically in the medium once in pure culture. However, when the iron concentration was increased to 0.01 g/1, Mb. bryantii M.o.H. grew with a generation time of 6.0 h and Msp. hungatei had a generation time of 13 h, Mb. bryantii

BCF was able to grow very well in this medium, and the growth of the

Olentangy isolate became much more predictable and consistent.

An interesting observation was that the Olentangy isolate did not require additional iron for growth before it was finally purified.

When the organism was still contaminated by another organism, the coccus 125

grew very consistently and predictably. Only after purification did

growth become erratic. This could be because the contaminant was pro­

viding cofac'.ors to the coccus or was providing iron to the organism

in a form which it could more effectively utilize. The Olentangy

coccus may have a relatively inefficient means of assimilating iron and

therefore required excessive amounts or different forms of iron to grow

optimally. This could also be true of the other methanogens which were

so highly stimulated by the increase in iron concentration.

Nickel chloride was added because it has been shown to be a

constituent of factor 100), a unique cofactor foundonly

in methanogens. Therefore, nickel is required for growth. Although nickel was probably present as a contaminant in sufficient concentration

for the growth of most methanogens, there may be some species of methanogen which require elevated nickel for optimal growth. In fact, when the Olentangy coccus was inoculated into medium which had not been supplemented with nickel, growth was very poor. When nickel was added to the medium and the coccus inoculated into the medium, the organism grew. Thus, the Olentangy coccus was not only sensitive to

iron concentrations, but also may be sensitive to the nickel content of the medium as well.

Sodium chloride was added to the medium for a supplemental source of sodium and for tonicity. Perski jst al. (66) have demonstrated that

Mb. thermoautotrophicum required sodium for growth and for CC>2 reduction to methane and that sodium had a specific function in the energy metabolism of the organism. 126

Cobalt chloride was added to the medium because it has been shown

to be required by methanogens (76) and it also has long been known

that methanogens contain a large amount of corrinoid compounds (49).

Kenealy and Zeikus (48), for example, have proposed that corrinoid

compounds have a role in the acetate metabolism of Ms. barkeri.

Magnesium sulfate and magnesium chloride were also added because

magnesium has been shown to be a required element for methanogens.

In this defined medium the M g C ^ content was higher (0.2 g/1) than in most other media published in the literature (5). This was because

increased magnesium was helpful in the stabilization of the new methane producing cocci. While the cocci still lysed after extended

storage, they lasted much longer than when the magnesium concentration was only 0.05 g/1. At least 15 mM magnesium was required for the optimal growth of Me. maripaludis (J. Jones, personal communication).

Routinely, 20 mM magnesium was added to the defined medium along with

3% NaCl to grow both Me. maripaludis and the Delta isolate. While the

Delta isolate did not require high magnesium, as shown in Table 8 in the Results section, the magnesium helped to stabilize the cells and

therefore remain viable and intact on the shelf much longer (up to six weeks).

Cysteine and sodium sulfide were added as reducing agents.

Methanogens require sulfide for growth (99), and cysteine has also been shown to be stimulatory for growth. Scherer and Sahm (75) found that optimal growth of Ms. barkeri occurred when 0.04 to 0.06 mM of dissolved sulfide was present in the medium and no lag was observed when cells grown on methanol were transferred to an acetate medium. 127

Wellinger and Wuhrman (99) found that for Methanobacterium strain AZ, in mineral sulfide-free medium, cysteine regulated the specific rate of methane production (optimum concentration = 4 x 10 ^ M/1). When sul­ fide was added (10-^ M/1) an additional stimulation was observed.

The vitamin solution added was as described by Wolin et jal. (104).

This is the least defined portion of the medium. Since the vitamin requirements for all of the methanogens have not been determined and the medium was used to grow twenty different methanogens, convenience dictated that all of the vitamins be included in the medium until vitamin requirements were determined for more methanogens. Scherer and Sahm (74) have determined that riboflavin was the only vitamin required by Ms. barkeri for growth in a defined medium. Bryant et a l .

(9) did some work with the vitamin requirements of Mb. bryantii M.o.H.

Although their results were not definitive, deletion of biotin, folic acid, or vitamin resulted in depressed growth. They made no attempt to determine the vitamin requirements of Mbr. smithii PS.

Whitman et al. (101) have recently determined the vitamin requirements for Me. voltae. Pantothenic acid was found to be the only vitamin required for optimal growth.

This medium proved to be of great utility in the everyday maintenance of cultures and the further study of growth requirements.

The medium did not contain many of the constituents added to the medium used by other researchers for the growth of methanogens (5, 14,

42, 53, 110). No requirement was demonstrated for boron, aluminum, zinc, copper, manganese, or calcium by Schoenheit jelt al. (76). So far, only Me. voltae has been demonstrated to require calcium (101). Me. 128

voltae also required MgS04, NaCl, CaC^, NiC^, and was

stimulated by cobalt. If other methanogens required such metals as

calcium, copper, zinc, manganese, boron, and aluminum, they must

obtain them from other reagents which may be contaminated with trace

amounts of these metals.

This defined medium made it possible to study the vitamin

requirements of Mbr. smithii PS, Mb. bryantii M.o.H., and Msp. hungatei

JF. As described in the Results section, Mbr. smithii PS required

biotin and thiamine for optimal growth. Mb. bryantii, however,

required only biotin. Biotin serves as a coenzyme for a class of

enzymes whose over-all function is the fixation and transfer of CC^.

Reactions which are catalyzed by biotin include propionyl CoA carboxy­

lase, acetyl CoA carbosylase, and methylmalonyl-oxaloacetate trans­

carboxylase (78). Stupperich and Fuchs (88) have examined CO2 fixation

patterns in Mb. thermoautotrophicum and have found evidence for a

pathway which starts with the formation of acetyl CoA from CO2 via one

carbon unit. The data are further consistent with the conversion of

acetyl CoA plus CO2 to pyruvate, phosphoenolpyruvate, and dicarboxylic

acids, catalyzed by pyruvate synthase, phosphoenolpyruvate synthetase,

phosphoenolpyruvate carboxylase, and enzymes of an incomplete reductive

tricarboxylic acid cycle. If similar reactions are occurring in Mb.

bryantii M.o.H. and Mbr. smithii PS, biotin could be playing a very

important role in the fixation of CO2 into cellular carbon. This would

explain why growth was very poor when biotin was left out of the medium. These organisms were probably unable to produce their own

biotin, and the growth that does occur was probably due to contaminating 129

quantities of biotin or by substitution of another cofactor for biotin with a much lower substrate affinity.

The biologically functional form of thiamine is thiamine pyro­ phosphate. The reactions in which TPP is involved include those cata­

lyzed by pyruvate dehydrogenase, pyruvate decarboxylase, and trans- ketolase and is involved in valine synthesis (78). Thus, thiamine deals mainly in reactions involving cellular metabolism and synthesis. The loss of this cofactor can therefore result in decreased growth, as occurs with Mbr. smithii PS. Although determining that biotin and thiamine were important cofactors for these organisms was interesting and will help in the future when CO^ fixation pathways are studied in these organisms, the fact that methanogenesis was relatively unaffected by the retarded growth was very surprising. It had been generally believed that growth and methanogenesis were directly related to each other. That is, if the cells were producing methane it was assumed that they were growing. The results in Tables 1, 2, and 4, however, demonstrated that methanogenesis by Mbr. smithii PS and Mb. bryantii M.o.H. occurred at rates which belied the amount of growth which occurred. With an extension of the incubation period, methane production was almost identical among the different vitamin combina­ tions whereas the optical densities ranged from 0.11 to 0.75. This was an interesting phenomenon and may indicate that the pathways involved in methane production are quite separate from those pathways involved in the production of cell carbon. This suggested that when

CO2 was taken up and converted to methane, this pathway was not involved in the production of cell carbon. In fact, the methane 130

production system and the cell carbon production system may have com­

pletely different mechanisms for fixation. This phenomenon could

be of potential use in studying the different systems of methane pro­

duction and CO2 fixation into cellular carbon. By growing cells in a

complete medium, then removing vitamins by centrifuging the cells out

and washing them in a vitamin free buffer, methane production reactions

could be studied independently from those reactions involved in pro­

ducing cellular mass, using labelled carbon dioxide.

The defined medium was not only very useful in defining growth

requirements, but also was an excellent enrichment medium for the

isolation of methanogens from the environment. Simply by adding sedi­

ment obtained from different anaerobic environments to the defined

medium, incubating for 3 to 14 days and repetitively transferring the

enrichments, methanogens predominated and were easily isolated.

Isolation was achieved through dilution to extinction and by streaking

for isolation on defined medium. Table 6 lists the many different

methanogens which were isolated using the defined medium. As can be

seen, a wide variety of methanogens are represented. No special

selective procedures were used to select one type of methanogen over

another. The only selective pressure used other than the defined

medium was to incubate some enrichments at 65°C to select for the

thermophilic methanogen Mb. thermoautotrophicum. The isolation of

different methanogens depended primarily on the source of the sediment.

The sediment from Penobscot Bay, Me., a marine clam flat, yielded

three distinctly different methanogens, a Methanosarcina spp., a

Methanobacterium spp., and a methane-producing coccus. All three of 131

these organisms were isolated by repeated transfers and streaking for

isolation. Although these organisms were isolated from a marine

environment, none required increased amounts of NaCl or magnesium.

Mb. formicicum was the most prominent or easiest methanogen to isolate

from the environment. In fact, sometimes in the attempts to isolate

an uncommon methanogen, such as a coccus, brevibacter, or spirillum,

the "formicicum" types tended to over-grow the other methanogens. In

other enrichments, a succession of methanogens was observed where

cocci, brevibacters, and spirilla occurred early in the enrichment with

the Methanobacterium types finally predominating. The initial enrich­ ments probably reflected the natural population more accurately, with

the Mb. formicicum strains being selected for by the defined medium.

A puzzling question is: Why is one methanogen favored over another in

the environment when their growth requirements and substrates are so

similar? More most be learned about the physiology of individual methanogens and a careful study of environmental conditions must be

undertaken before this question can be answered.

Interestingly, four different methane-producing cocci were iso­

lated from four different sites. Cocci have not been routinely iso­

lated by other researchers and certainly not from fresh water sources using a defined medium. Me. vannielii, Me. voltae, and Me. maripaludis are the only known Methanococci described in the literature. Methano­

genium marisnigri and Methanogenium cariaci are cocci but differ from

the methanococci in several respects (71). They have a high G + C con­

tent and grow at a temperature optimum of 25°C (71). All of these

coccus-shaped methanogens were isolated from marine environments. Only 132

Me. vannielii does not require NaCl or Mg in high concentrations (86).

Because of the unusual characteristics of the Olentangy coccus, in that it was a fresh water organism, non-motile, and unable to utilize formate for growth and methanogenesis, as well as not needing organic supplements or vitamins, further characterization was done.

The Delta isolate was also characterized because it too seemed unique.

It did not require vitamins, acetate, or organic supplements for growth

(as Me. voltae does), and it grew with a very fast generation time of only 2 h.

The Olentangy coccus appeared to belong to a heretofore unde­ scribed methanogenic genus. The immunological fingerprint in Table 9 shows that the Olentangy coccus had a 4+ reaction with Mbr. smithii ALI but not with Mbr. smithii PS. Dr. M.J. Wolin has stated (personal communication) that they have checked many brevibacter strains and all cross-reacted with both strains. The Olentangy coccus was the only organism they had seen that reacted with only one strain, There was no cross-reaction at all with Me. voltae, Me. vannielii, Mg. marisnigri, or Mj*. cariaci. This may indicate that the Olentangy coccus belonged to the Methanobacteriaceae family instead of the Methanococcaceae family. In order to definitely assign the organism to its proper family and genus, 16S RNA oligonucleotide catalogueing may have to be done. Also, knowing the 1% G + C content would be helpful in assigning the organism a place in the taxonomic scheme of the other methanogens.

The percentage of G + C was not definitely known; however, preliminary determinations tended to indicate that it was in the range of the

Methanogeniums (51.2 to 61.2). Some genetic characterization was done on these two isolates. The

Olentangy isolate was screened for plasmids on agarose gels and on

cesium chloride gradients. No evidence for plasmids was found. The

Delta isolate was also screened for plasmids on agarose gels as with

the Olentangy coccus. None were found. The agarose gels were done by

Paul Hamilton (a graduate student in the Department of Microbiology,

Ohio State University). In the course of Mr. Hamilton's research

concerning the cloning of methanogen DNA, it was noticed that DNA from

the Olentangy isolates was not restricted by the restriction enzyme

Hind III, whereas DNA from the Delta isolate was restricted. Figure

23 is a photograph of an agarose gel showing the digestion patterns

obtained when the DNA of five different methanogens was subjected to digestion by Hind III and Bam HI. It is obvious that Msp. hungatei

strain JF DNA and the Olentangy isolate DNA were resistant to Hind III but were cut by Bam HI. On the other hand, the Delta isolate, Me. vannielii, and Me. maripaludis were all cut by Hind III and Bam HI.

This parameter placed the Delta isolate with the other cocci and

separated the Olentangy coccus from the other Methanococci. So far, of those tested, only Msp. hungatei and the Olentangy isolate were resistant to Hind III digestion. The Hind III restriction enzyme recognizes a six-base DNA sequence in order to restrict. It would be almost statistically impossible for this sequence not to occur many times in the genome. This presented the strong likelihood of a modifi­ cation of the DNA in order to make the DNA resistant to restriction by

Hind III. Figure 23. Restriction analysis of DNA from the (A)

Olentangy isolate, (B) the Delta isolate,

(C) Msp. hungatei strain JF, (D) M e . vgara-elii,

and (E) Me. maripaludis, using Hind III

(tracks 1-5) and Bam HI (tracks 6-10) (BRL,

Bethesda, Md.). Paul Hamilton (personal

communication).

134 Figure 23. 136

The Cuyahoga coccus appeared to be a different strain of the

Olentangy coccus. Neither required vitamins or yeast extract for growth; both required acetate; they had very similar generation times

(11.6 and 11.0 h); their colonial morphologies were identical; and both were resistant to Hind III restriction. The Olentangy coccus was sensitive to monensin, and the Cuyahoga coccus was resistant.

Interestingly, neither utilized formate for growth and methanogenesis, but both turned the pH indicator in the formate test medium, phenol red, colorless. The organisms appeared to reduce the indicator. When the bottle was opened, oxidized, and acidified, the color returned, indicating the organisms were not decomposing the indicator. No other methanogen (except one) tested in the penol red formate medium turned the indicator colorless (i.e., M e . vannielii, M b . formicicum,

Mb. bryantii, Mb. thermoautotrophicum). The only other methanogen which turned the phenol red colorless was the Maine coccus. This coccus was not well characterized, but it did grow at a rate similar to the other cocci, and its cellular and colonial morphologies were similar to those of the Cuyahoga and Olentangy cocci (as far as being very mucoid on a plate), although the colonies were greener than those of the Olentangy and Cuyahoga cocci. To determine the relatedness among these organisms, more testing must be done.

The Delta isolate was distinctly different from the other newly isolated cocci in being a very fast grower, and it had a requirement for at least 3% NaCl. The immunological fingerprint of this isolate

(Table 9) indicated that it was related to Me. vanielii and it shared several characteristics common to the other Methanococci. The Delta 137

coccus was able to utilize formate for growth and methanogenesis.

However, it did not require acetate, amino acids, or vitamins for growth as did Me. voltae, and it did not require a high magnesium con­

tent as did Me. maripaludis. Me. vannielii was physiologically similar

to the Delta isolate but did not have an elevated NaCl requirement, was motile, mechanically fragile, and produced greenish translucent colonies on solid media.

Me. maripaludis appeared to be the coccus most similar to the

Delta isolate. The colonial morphologies of the two organisms were very similar in being greenish-yellow, opaque, and convex with entire edges. The G + C contents of the two organisms would further aid in the separation of these two organisms. The percentage of G + C of Me. maripaludis was reported as 34% (Abstract of the Annual Meeting of the

American Society for Microbiology, 1981). The percentage of G + C of the Delta isolate has not been determined. Until this information is obtained, designation of the Delta isolate as a new species of

Methanococcus may be premature. At this point, it is quite certain that the Delta isolate is different from the other Methanococci already described, but the G + C determination will be the final confirmation.

As shown in Table 10 (compiled by L.A. Hook), all of the cocci tested were extremely sensitive to metronidazole. This was very interesting since it has been proposed that the mode of action of metronidazole was by acting as an electron 'sink' and thus inhibiting hydrogenase systems as well as other electron transfer mechanisms by competing for electrons (69). Metronidazole has also been reported to accept electrons specifically from ferredoxin or flavodoxin (11, 39, 69) Table 10. Antibiotic sensitivities of the Olengangy, Delta,

and Cuyahoga isolates and Methanococcus maripaludis

and Methanococcus vannielii. (-) indicates no zone

of inhibition, a blank means the sensitivity was

not determined, and the numbers 1, 10, or 100

indicate the lowest concentration (ug) of antibiotic

which produced a zone of inhibition around an anti­

biotic impregnated filter-paper disc. The experi­

mental antibiotics were new antibiotics, not yet

commercially available, provided by Eli Lily and

Company, Indianapolis, Ind. (Dr. L.A. Hook, Ohio

State University, provided the table of antibiotic

sensitivities.)

138 139

Table 10.

co •H •H T3 •rH 3 rH rH CO CO •H 3 3 •H 3 3 M ■u CO 3 3 3 P> e •P t-H 3 O (U CO CO rH CO 4J 3 3 o M CO o o M rH o CJ M Ph o o o 6 0 CO u u 3 3 H o o 60 3 3 3 O 4J 3 CO 3 X s ■u .3 r C 3 (0 rH ■u 4J Antibiotic rH CO (0 3 3 O n s s U

Actinomycin -- 100 — -- -- Adriamycin 10 10 10 10 100 Anthelmycin ------Anthelvencin -- 100 ------Bleomycin -- -- 100 -- -- Cefotaxime ------Cefoxitin ------Cephaloridine ------Efrapeptin 10 10 100 100 100 Echinocandin -- 100 -- -- Hygromycin ------Leucinostatin 1 1 1 10 10 Metronidazole 1 1 1 ]. 1 Monensin 1 1 1 I -- Mycophenolic Acid -- -- 100 -- -- Neomycin ------Pleuromutilin -- 10 -- 10 Pyrollnitrin 100 10 100 100 Rutamycin -- 10 -- -- Sinefungin ------Tobramycin ------Tunicamycin -- 10 -- -- Experimental #1 (A2UIA) -- 10 100 — -- Experimental #2 (A2315) -- -- 1 1 -- Experimental #3 (A7413) 100 1 -- 10 Experimental #4 (A21978C) -- -- 1 -- -- Experimental #5 (A23187) ------Experimental #6 (A41030A) 100 100 ------Experimental #7 (G418) ------and to be a selective inhibitor of the nitrogenase activity in the cyanobacterium Anabaena (39). This was interesting in that if metronidazole does indeed accept electrons specifically from ferre- doxins or flavodoxins, the extreme sensitivity shown by these methano- gens to this antibiotic may indicate that methanogens may possess ferredoxins or flavodoxins. Neither ferredoxins nor flavodoxins have ever been shown to occur in methanogens (110). Metronidazole could be a useful tool for studying electron transport phenomena in methanogens. SUMMARY

An integrated system for the anaerobic cultivation of methano- genic bacteria was developed. With the use of the Forma anaerobic chamber, Oxoid anaerobe jars, a gassing manifold, serum bottles, and a defined medium, the isolation and cultivation of methanogens has been greatly simplified.

Iron was highly stimulatory for the growth of Mb. bryantii strain

M.o.H., Msp. hungatei strain JF, and Mb. bryantii strain BCF. The use of 0.01 g/1 of ferrous sulfate proved to be of great importance when isolating new methanogens from the environment.

Vitamin requirements for Mbr. smithii strain PS and Mb. bryantii strain M.o.H. were determined. Mbr. smithii required thiamine and biotin for optimal growth and methanogenesis while Mb. bryantii strain

M.o.H. required only thiamine. These vitamins appeared to be involved in the production of biomass and not methane.

Using the defined medium, Mb. formicicum, Mb. thermoautotrophicum,

Mb. bryantii M.o.H., Ms. barkeri RC, Methanobacterium spp., as well as four different methane-producing cocci, were isolated. All four of the< new coccus isolates were previously undescribed methanogens.

The Olentangy coccus was an irregular coccus occurring singly or in pairs with a cellular diameter of 1.0 to 1.5 um. It was non-motile and had no flagella. On solid medium it formed large, mucoid, yellow, 142

shiny, convex colonies. The preferred substrate was Formate did not support growth. Acetate was required for growth. Yeast extract, trypticase and vitamins were not required nor stimulatory.

The NaCl optimum for this organism was 1%, and its temperature optimum was 37°C. It was serologically unique in reacting with a single strain of Mbr. smithii ALI but not with other methanogens. Its DNA was resistant to restriction by Hind III, indicating the possibility of modification of its DNA. Other cocci tested were restricted.

The Delta coccus was an irregular coccus occurring singly or in pairs, with a cellular diameter of 1.0 to 1.5 urn. It was non-motile and had no flagella. On solid medium it formed greenish-yellow, shiny, convex colonies with dark centers. Its preferred substrates were

^ 5 002 and formate. Acetate, yeast extract, trypticase, and vitamins were not required for growth. Its NaCl optimum was 3.5 to 4.0%, and its temperature optimum was 37°C. The organism reacted weakly (l+.t with

Me. vannielii but not with other methanogens.

The organism appears to belong to the genus Methanococcus and its proposed name is Methanococcus deltae sp. nov. LITERATURE CITED

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