APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1981, p. 413-4200099-2240/81/090413-08$02.00/0

Vol. 42, No. 3

Conversion of Cellulose to Methane and Carbon Dioxide byTriculture of Acetivibrio cellulolyticus, Desulfovibrio sp., and

Methanosarcina barkeritVICTORIA M. LAUBE* AND STANLEY M. MARTIN

Division ofBiological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada KJA OR6

Received 2 March 1981/Accepted 2 June 1981

The fermentation of cellulose by monocultures ofAcetivibrio cellulolyticus andcocultures of A. cellulolyticus-Methanosarcina barkeri, A. cellulolyticus-Desul-fovibrio sp., and A. cellulolyticus-M. barkeri-Desulfovibrio sp. was studied. Themonoculture produced ethanol, acetate, H2, and C02. More acetate and lessethanol was formed by the cocultures than by the monoculture. Acetate was

utilized by M. barkeri in coculture with A. cellulolyticus after a lag period,whereas ethanol was metabolized by the sulfate reducer only under conditions oflow H2 partial pressure, i.e., when cocultured with A. celluloyticus-M. barkeri or

when grown together with the methanogen. Only the three-component culturecarried out the rapid conversion of cellulose to C02 and methane. Furthermore,this culture hydrolyzed the most cellulose-85% of that initially present. Thisamount was increased to 90% by increasing the population of M. barkeri in thetriculture. Methane production was also increased, and a quicker fermentationrate was achieved.

The anaerobic degradation of urban refuse,largely cellulose (26), to methane and carbondioxide, is of current interest in terms of renew-able energy recovery and pollution-load reduc-tion. Although such fermentations are used ex-tensively for waste treatment, they are not wellunderstood (28) and are plaqued by lack of reli-ability (17).The technique of culture enrichment has been

used in an attempt to clarify some of the micro-biological and biochemical aspects of cellulosedegradation to methane and carbon dioxide (11,13, 15, 16). A more fundamental approach toinvestigating the process has also been taken bycoculturing pure cultures of the microorganismsinvolved (18, 30). In a more recent coculturestudy (12), Acetivibrio cellulolyticus, a cellulosedegrader whose fermentation products are H2,carbon dioxide, acetate, and ethanol (14, 24),and Methanosarcina barkeri, a methanogen ca-pable of utilizing acetate, as well as H2 and C02,for methane production (20, 31), were used. Inthis case, a 4- to 6-week adaptation period wasrequired before M. barkeri utilized acetate. Thiswas followed by a further 2- to 3-week lag periodbefore acetate utilization in the coculture com-menced.

In an attempt to make the coculture fermen-tation more efficient, a sulfate reducer, recently

t National Research Council of Canada no. 19603.

isolated from a cellulose-enrichment culture (R.Latta, unpublished), was included in the A. cel-lulolyticus-M. barkeri culture system. In thispaper we examine the conversion of cellulose tomethane and carbon dioxide by this triculture.

MATERIALS AND METHODSOrganisms and growth conditions. A. cellulo-

lyticus (NRCC 2248; ATCC 33288), M. barkeri (NRCC2240, DSM 800; the type strain of the species), and asulfate reducer tentatively identified as Desulfovibriogigas (NRCC 2242) (R. Latta, unpublished) were usedin this study.A defined medium which had been shown to pro-

mote good methane production from cellulose by amixed culture of anaerobes (16) was used for mostexperiments, as well as for the maintenance of workingstock cultures of A. cellulolyticus. The Eh indicator,resazurin (1 mg/liter), was added to the basal mediumwhich contained vitamins and minerals. After adjust-ment of the pH to 7.2 with 3 N NaOH, the preparationof the medium was continued under an atmosphere of20% C02-80% N2 by the Hungate technique (8). Cys-teine HCI (250 mg/liter) and Na2S.9H20 (250 mg/liter) (7) were added as reducing agents, and 50-mivolumes of prereduced medium were dispensed intogassed 160-ml serum vials containing 0.25 g of CF-11cellulose powder (Whatman). The vials, stopperedwith butyl rubber stoppers and sealed with aluminumcaps, were autoclaved at 15 lb/in2 for 15 min. Oncecooled to room temperature, they were inoculatedwith 4-day-old cultures of the desired microorganisms(2% [vol/vol] inoculum of each culture, unless other-

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414 LAUBE AND MARTIN

wise indicated) and then flushed for about 2.5 minwith a 20% C02-80% N2 gas mixture. The headspace ofthe vials was equilibrated, after being flushed, beforethe incubation of the vials at 35°C with shaking.

Desulfovibrio sp. and M. barkeri were maintained(4- to 6- day transfers) in the synthetic medium de-scribed by Patel et al. (25) and prepared as above.When ethanol utilization was to be determined, thismedium was prepared without acetate. Filter-steri-lized anaerobically prepared acetate and ethanol wereadded to the autoclaved medium. After inoculation,vials were flushed with 20% C02-80% H2 (unless oth-erwise indicated), equilibrated, and incubated asabove.

Analytical methods. Duplicate vials were ana-lyzed at designated times, and all experiments wererepeated at least twice. H2, C02, and CH4 in theheadspace of the vials were analyzed chromatograph-ically by the method of van Huyssteen (29), all valuesbeing corrected for headspace pressure. In the case ofC02, adjustments were made to take into account theeffect of the bicarbonate equilibrium at the appropri-ate pH. The pH was measured immediately afteruncapping the vials. The contents of the vials werethen centrifuged for 20 min at 15,000 x g, and thesupernatants were analyzed for volatile acids and al-cohols by the method of Ackman (1). Reducing sugarsin the supernatants were measured colorimetricallyusing the dinitrosalicylic acid reagent (22) with glucoseas standard. The pellets, suspended in distilled water,were sonicated, treated with 8% formic acid, and thenfiltered through preweighed, 0.45-nm-pore-size, mem-brane filters (Millipore Corp., Bedford, Mass.). Thefilters were dried at 60°C to constant weight, andresidual cellulose was determined by difference (30).

RESULTSCellulose fermentation. Figure 1A shows

the time course of cellulose utilization in mono-and coculture. After an initial lag period of about

A100_

D 80 _ ,

60 .

o 40 v

uj20

24 h, cellulose utilization in the A. cellulolyticusculture was essentially linear up to 4 days. Sol-ubilization then continued at a reduced rate andleveled off at 78% degradation. The lag in thetwo cocultures was similar to that observed inthe monoculture, whereas the lag of the A. cel-lulolyticus-Desulfovibrio sp.-M. barkeri culturewas significantly shorter. All of the coculturesappeared to initially degrade cellulose more rap-idly than the monoculture, with lower rates de-veloping after about 3 days. The triculture hy-drolyzed the greatest amount of cellulose-ap-proximately 85%.

Cellulose degradation was accompanied by alarge decrease in culture pH (Fig. 1B), the pHdrop being more pronounced in the coculturesthan in the monoculture. As the fermentationproceeded, pH values, except those of the tricul-ture, continued to drop, eventually reachingabout pH 5.2. In contrast, the pH of the tricul-ture began to rise after 3 days and by 12 daysleveled off at pH 6, probably because of theutilization of acetate (Fig. 2D).

Cellulose fermentation products. Theprogress of product formation from cellulose isshown in Fig. 2. The main end products formedby the monoculture (Fig. 2A)-carbon dioxide,hydrogen, ethanol, and acetate-were producedat constant rates for about 6 days and thenleveled off. The A. cellulolyticus-Desulfovibriosp. coculture (Fig. 2B) produced the same endproducts as the monoculture, but the maximumamounts of carbon dioxide and hydrogen pro-duced were significantly lower, probably a resultof carbon fixation coupled with sulfate reductionby Desulfovibrio sp. In addition, more acetateand less ethanol was produced, a trend also

8 12 16 0 4 8DAYS DAYS

12 16

FIG. 1. Fermentation of cellulose by A. cellulolyticus (V), A. cellulolyticus and Desulfovibrio sp. (0), A.cellulolyticus and M. barkeri (-), and A. cellulolyticus, Desulfovibrio sp., and M. barkeri (A). (A) Cellulosedegraded; (B) culture pH.

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TRICULTURE CONVERSION OF CELLULOSE TO METHANE 415

E 50 w CARBON DIOXIDE E 50

50 O2E

0- IN,.40 o_o_o-o°OHYDROGEN E40 / -o¢ CAROM DIOXIDE |

E 30- W/ ,3 o- .A ACETATE>ETH

E 50 - C E 10 - D IM ETHANOL

REDU INGSUGARS-_

0 4 8 12 16 0 4 8 12 16DAYS DAYS

xMETHANE

E 50 C E 50- DxCACA3 DIOXIDE

E 40 E 40 !

0))0' 61) 4/ 2

CARBNDYIDE'Z 30- 30

E./.020-A&& CETATE ~ 0IEhNL~R

1010 I-0I .~, examETHANO1XREDUCING KIGARS RV-ACETATE

0e 48O DR2GE0 4' '60812

DAYS DAYSFIG. 2. Products of cellulose fermentation by (A) A. cellulolyticus, (B) A. cellulolyticus and Desulfovibrio

sp., (C) A. cellulolyticus, and M. barkeri, (D) A. cellulolyticus, Desulfovibrio sp., and M. barkeri.

found in the other cocultures.The A. cellulolyticus-M. barkeri coculture

(Fig. 2C) and the triculture (Fig. 2D) each pro-duced methane in addition to the products foundin the other cultures (Fig. 2A and B). Hydrogen,however, peaked at about 4 days and then de-creased until none was detectable in the head-space at 7 days. The fate of acetate was similarto that of hydrogen in the triculture (Fig. 2D),whereas acetate remained at a nearly constantlevel for 2 weeks in the A. cellulolyticus-M.barkeri culture (Fig. 20). Methane productionin the A. cellulolyticus-Desulfovibrio sp.-M.barkeri culture had two rates-an initial lowerrate probably reflecting the conversion of H2 andC02 to CH4 and a subsequent higher rate, prob-ably resulting from acetate metabolism. The rateof methane formation in the A. cellulolyticus-M. barkeri culture was initially similar to thelower rate mentioned above but then decreased.Since very little, if any, acetate disappeared fromthis culture within the 2-week interval examinedand the C02 level decreased, the methane prob-

ably originated from H2 and C02.The ethanol produced from ceilulolysis was

metabolized by the triculture. No significantamounts of ethanol were utilized by any of theother cultures.Low levels of reducing sugars were present in

all of the cultures when the fermentations werenear completion. This was probably due to con-tinued cellulase activity in the supernatant aftergrowth of A. cellulolyticus had ceased. Thesugars produced were not utilized and conse-quently accumulated in the medium.Carbon recoveries in the mono- and cocultures

were acceptable (77 to 90%) as were the oxida-tion/reduction indices for the A. cellulolyticusculture and the triculture (Table 1). However,the A. cellulolyticus-Desulfovibrio sp. coculturehad a somewhat high oxidation/reduction valuewhich could be the result of the presence of areduced compound, lacking in carbon, beingformed during the fermentation but not meas-ured, e.g., H2S. The high oxidation/reductionvalue of the A. cellulolyticus-M. barkeri culture

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416 LAUBE AND MARTIN

could be partially a result of high C02 valuesresulting from the oxidation of the methyl groupof acetate for the production of reducing powerwhen no exogenous H2 is present.Ethanol utilization. The sulfate reducer, the

only bacterium of the three known to utilizeethanol (5), was grown in medium containingboth acetate and ethanol (Table 2). When the

TABLE 1. Products of cellulose fermentation by A.cellulolyticus in mono- and coculturea

Product of cellulose fermentation(mmol of product per 100 mmnol of "an-hydroglucose equivalents"b fermented)

A. cellu-Product A. cellu- A cel lolyti-

A. cel- lolyti- lulolyti cus-M.lulolyti- cus-De- lolYti- barkeri-

cus sulfovi cus-M. Desulfo.brio sp. bre vibrio

sp.Acetate 54.0 60.8 74.1 0Ethanol 78.9 40.0 44.0 0Methane 64.8 208.4H2 166.4 100.4 0 0C02 211.2 159.2 162.1 202.7Reducing sugars 7.5 15.6 12.5 14.3Cellulose (CF-li) 195.0 202.5 175.1 210.0degraded (mg)'

O/R balanced 1.30 1.76 1.49 0.97Carbon recovery 0.87 0.77 0.90 0.83

aAfter 2 weeks of incubation.bMolecular weight of 162.' Cellulose initially present was 250 mg/50 ml of medium.d Calculated by the method of Wood (33). O/R, Oxidation/

reduction.

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headspace atmosphere was 80% H2-20% C02,Desulfovibrio sp. utilized little if any ethanol.However, approximately 30 mmol ofH2 per liter,1 mmol of acetate per liter, and some C02 (resultnot shown) was used during 8 days of incubation.Since sulfate reducers are capable of obtainingenergy from the reduction of sulfate with gas-eous H2 and of utilizing acetate and C02 as solecarbon sources, growth of Desulfovibrio sp. un-der an initial atmosphere of 80% H2-20% C02most probably occurred with the substrates asenergy and carbon sources. Although the pro-duction of H2S was not determined, it was evi-dent that significant amounts were present inthe cultures.

In contrast, under an atmosphere of 80% N2-20% C02, Desulfovibrio sp. used ethanol andproduced acetate and H2. Although significantamounts of ethanol were used, it seemed thatthe H2 which accumulated inhibited the utiliza-tion of all of the ethanol. Thus, an attempt wasmade to maintain low H2 levels by either flush-ing the headspace of the vials with N2-CO2 every2 days during growth of Desulfovibrio sp. or byculturing the bacterium with M. barkeri. Moreethanol was oxidized by Desulfovibrio sp. whenthe headspace was flushed than when it was not(Table 2). Hydrogen did, however, accumulatebetween purges and probably inhibited the totaloxidation of ethanol. In contrast, all of theethanol was used by 8 days when Desulfovibriosp. was cocultured with M. barkeri under an

atmosphere of either H2-Co2 or N2-CO2. When

TABLE 2. Utilization of ethanol andproduction of acetate by Desulfovibrio sp. and cocultures ofDesulfovibrio sp. and M. barkeri

Initial atmosphere Incubation AmMa H2present inCulture 8020) time headspace

(80%*20%) (days) Ethanol Acetate (mmol)

Desulfovibrio sp. H2-CO2b 2 NDC ND ND4 -1.0 -1.9 ND6 ND ND ND8 -0.4 -1.0 2.37

Desulfovibrio sp. N2-CO2 2 -9.3 6.6 0.054 -11.4 8.7 0.226 -13.3 11.4 0.258 -12.6 10.6 ND

Desulfovibrio sp. N2-CO2d 8 -16.3 14.8 0.38Desulfovibrio sp. and M. barkeri H2-CO2b 2 -2.4 -1.0 0.41

4 -10.8 5.9 0.026 -19.3 4.2 08 -19.3 13.1 0

Desulfovibrio sp. and M. barkeri N2-CO2 2 -10.9 6.1 0.014 -16.0 10.6 06 -19.7 9.1 08 -19.7 14.0 0

a Initial concentrations of acetate and ethanol were 18.2 and 19.8 mM, respectively.b Initial concentration of H2 in the 110-ml headspace was 3.9 mmol.c ND, Not determined.d The headspace was flushed with N2-CO2 for 2.5 min every 2 days and then equilibrated.

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TRICULTURE CONVERSION OF CELLULOSE TO METHANE 417

H2 was initially present in the headspace, lessethanol was used at the beginning of incubationthan when the culture atmosphere was initiallydevoid of H2. During this initial time, energy forgrowth of Desulfovibrio sp. was most probablyobtained from sulfate reduction. This reactionto some extent, as well as the utilization of H2for reducing CO2 to methane by M. barkeri,eventually allowed for the oxidation of ethanolby Desulfovibrio sp. The H2, produced concom-itantly, was then removed by the methanogen.In contrast, no lag in ethanol utilization wasevident when the coculture was grown under N2-C02. M. barkeri handled the H2 produced fromethanol utilization by Desulfovibrio sp., andthere was no gaseous hydrogen accumulation.This allowed Desulfovibrio sp. to more quicklyoxidize the ethanol.Rate-limiting step. In an attempt to deter-

mine which step in the triculture fermentationwas initially rate limiting, the tricultures wereprepared with a fivefold increase in inoculumsize of each organism separately and all threetogether. Substrate utilization and product for-mation were followed; cellulose degradation andmethane production are shown in Fig. 3.The initial rate of cellulose degradation in-

creased in all of the cultures when comparedwith the control. The highest rate of cellulolysisand the greatest total degradation of cellulose(90%) occurred when all three inocula were in-creased simultaneously. The initial rate of meth-ane production (Fig. 3B) was greater and thefinal methane yields were higher than in thecontrol when the M. barkeri inocula were in-

creased. In the other cultures, including the con-trol, initial methane production was slow al-though the rate of methane production in theculture with only a higher Desulfovibrio sp. in-oculum eventually did increase. The finalamount of CH4 produced by this latter culturewas comparable to the control. The triculturewith an increased A. cellulolyticus inoculum,however, had a lower yield than the control. Itwas also noted that the fermentation proceededto completion much faster in the cultures withincreased M. barkeri inocula; i.e., H2, acetate,and ethanol were utilized by 7 days.

DISCUSSIONThe results demonstrate a three-organism fer-

mentation of cellulose to CH4 and C02. Thethree bacteria are representative of the threegeneral bacterial types proposed by Bryant (4),and their involvement in the fermentation issummarized in Fig. 4. This is the first demon-stration, to our knowledge, of a coculture ofthree bacteria capable of degrading cellulose toCH4 and C02 in less than 2 weeks.H2 plays a critical role in the outcome of the

fermentations. The shift from ethanol predomi-nance in the A. cellulolyticus culture to acetatepredominance in the cocultures supports thehypothesis that hydrogen-utilizing organismscan cause a shift in the electron flow and aconcomitant change in the fermentation endproducts (9). Desulfovibrio sp. obtains energyfor growth and maintenance from the reductionof sulfate, as shown by the following equation:S042- + 4H2 -- H2S + 2H20 + 20H- (AG"' =

o~~~~~~~~bw~~ E

O0 400 1 8 O2 6 1 4 1

4 _L

ao60- £ 30w ~~~~~~~~~E

degradation;(B)effeE-40 20

20 10 /

0 2 6 10 14 IS 0 2 6 (0 14 18DAYS DAYS

FIG. 3. Effect of a fivefold increase in either the A. cellulolyticus (V), Desulfovibrio sp. (@), or M. barkeri(U) inoculum in the triculture, and an increase in all three inocula (A); control (0). (A) Effect on cellulosedegradation; (B) effect on methane production.

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418 LAUBE AND MARTIN

FIG. 4. Scheme showing the relationships of the three bacteria effecting the cellulose-to-methane fermen-tation.

-152.2 kJ/mol) (27). Similarly, M. barkeri ob-tains energy from the reduction of C02: C02 +4H2 -* CH4 + 2H20 (AG0' = -131 kJ/mol) (27).Thus, the use of H2 by these bacteria wouldresult in the channelling of the electrons in theA. cellulolyticus fermentation to H2 productionand away from ethanol. Such shifts have beenpreviously reported and were attributed to H2utilization by methanogens and other bacteriagrowing in coculture with carbohydrate fer-menters. Ben-Bassat et al. (3) showed that ace-tate, as opposed to ethanol, became the mainproduct of the saccharide fermentation of Ther-moanaerobium brockii when grown in the pres-ence of Methanobacterium thermoautotroph-icum. Similarly, acetate production was favoredover ethanol in cocultures of Ruminococcus al-bus and Vibrio succinogenes growing on glucose(10) and in the cellulose-degrading coculture ofClostridium thermocellum and Methanobacte-rium thermoautotrophicum (30).A unifying concept, termed interspecies H2

transfer, has been formulated by various inves-tigators to explain the biochemical basis of theelectron shifts in cocultures (3, 10, 18, 30). Theyhave proposed that the major part of the re-duced nicotinamide adenine dinucleotide(NADH) formed during saccharide catabolismby the oxidation of glyceraldehyde-3-phosphate

goes directly to the production of H2 and nico-tinamide adenine dinucleotide (NAD) when thepartial pressure of H2 is low. The removal of H2by the H2-utilizing bacteria present in the cocul-ture makes the reaction NADH + H+ -- NAD++ H2 energetically more favorable (32). With H2removal proceeding, more pyruvate becomesavailable and can be oxidized to acetate and C02via acetyl coenzyme A with the generation of 1mol of adenosine 5'-triphosphate per mol of ace-tate formed. This adenosine 5'-triphosphate gainby A. cellulolyticus could partially account forthe initial increased cellulose degradation in thecocultures.The partial pressure of H2 was also a factor in

ethanol utilization by Desulfovibrio sp. Whenthe partial pressure of H2 was decreased,whether by changing the headspace gas to anN2-C02 mixture (with and without intermitentflushing) and/or by introducing an efficient H2utilizer, M. barkeri, ethanol became the pre-ferred energy substrate for growth of Desulfo-vibrio sp. It is known that, with the removal ofH2, the reaction CH3CH20H + H20 =CH3CO0-H+ + 2H2 (AG"' = +19.2 kJ/mol) (27)becomes energetically more feasible. For exam-ple, with a methanogen present, the above re-action becomes 2 CH3CH20H + HCO3-= 2CH3C00 + H+ + CH4 + H20 (AG0' = -116.2

D SP (ACETOGENIC, H2-DUCING)SIL

"II ACTATE I

H2-PQUIN)TP

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TRICULTURE CONVERSION OF CELLULOSE TO METHANE 419

kJ/mol) (5) calculated from Thauer et al. (27).The acetate-to-ethanol ratio (ca. 1) (Table 2)obtained when Desulfovibrio sp. was grown un-der N2-CO2 fits well into the stoichiometry ofthe above equation of ethanol utilization, theslightly lower acetate values being in agreementwith the literature. Badziong et al. (2) reportedthat approximately 2 mM acetate can go intocell carbon. The hydrogen detected in the head-space, however, was significantly lower thanwould be expected. A possible explanation forthis is that, as H2 accumulated, ethanol utiliza-tion was inhibited and H2 was used for energyproduction via sulfate reduction (3.98 mM sul-fate present in medium) as well as for reducingpower in biosynthesis.The stoichiometric conversion of ethanol to

acetate was not evident when Desulfovibrio sp.was cultured with M. barkeri; acetate utilizationby the methanogen probably accounted for thelower amounts of acetate detected. A low H2partial pressure as prerequisite for ethanol uti-lization was previously reported. D. vulgaris re-quired the presence of Methanobacterium for-micicum before ethanol was used (5). Similarly,ethanol metabolism by the S organism isolatedfrom the Methanobacillus omelianskii cocul-ture required the presence of Methanobacte-rium strain MOH (6).When Desulfouibrio sp. and A. cellulolyticus

were cocultured, the conditions created by A.cellulolyticus were not conducive to ethanol uti-lization by Desulfovibrio sp. Little ifany ethanolwas used since H2 and ethanol were producedconcurrently from cellulose. Energy was mostprobably obtained by Desulfovibrio sp. from thereduction of sulfate (1.73 mM present in me-dium). In contrast, the low partial pressure of H2in the three-component culture, a condition cre-ated for the most part by M. barkeri, allowedfor ethanol utilization by Desulfovibrio sp. TheH2 and most of the acetate produced were usedby M. barkeri.As with ethanol utilization, H2 concentration

played a role in acetate metabolism. When H2,C02, and acetate were present, the gases wereutilized preferentially, and only when most hadbeen converted to CH4 was acetate readily usedby M. barkeri (19). We found a similar trend inthe triculture: H2 disappeared and then acetatewas metabolized. In the A. cellulolyticus-M. bar-keri culture, however, acetate was not immedi-ately utilized after the disappearance of H2. A 2-to 3-week lag period in acetate utilization wasobserved by us and was previously reported (12).This could be explained by an initial rapid deg-radation of cellulose by A. cellulolyticus broughtabout by the removal of H2 by M. barkeri. It hasbeen demonstrated that the periodic removal of

H2 from A. cellulolyticus cultures increases therate of cellulose degradation (G. B. Patel, per-sonal communication). The faster fermentationcreated an acetate overload and a drasticallydecreased culture pH. Although the methanefermentation proceeds in the pH range of 5 to 8,values below pH 6 are very restrictive (21). Thus,M. barkeri probably grew very slowly underthese conditions. The methanogen populationwas built up slowly over a period of about 2weeks by using acetate. With acetate being uti-lized, the pH began to rise, and eventually, witha more amicable pH and a larger methanogenicpopulation, acetate was used rapidly. A differentsituation prevailed in the triculture. With theDesulfovibrio sp. present, initial conditions wereconducive to good growth and cellulose degra-dation. The sulfate reducer could have acted toeither produce a stimulatory product such assulfide or to remove an inhibitor, e.g., ethanol.In the former case, even though sulfide waspresent in the medium (0.8 mM added sulfide),it is required in large amounts by both M. bar-keri (23) and A. cellulolyticus (G. B. Patel, per-sonal communication). Thus, in the case of M.barkeri, with better growth, a larger populationwas available to utilize acetate quickly. A similarexplanation of a larger population of M. barkeribeing capable of metabolizing acetate quicklywas proposed by Mah et al. (19). The higherrate of acetate utilization in the presence of H2and C02 than in the presence of acetate alonewas partially attributed by them to a more rapidinitial growth of M. barkeri on H2 and C02. Inaddition to a larger population of M. barkeribeing available for acetate conversion, fasteracetate utilization in the triculture could also bea result of H2 production by Desulfovibrio sp.Although H2 is not required for acetate metab-olism (20), low levels produced by Desulfovibriosp. during ethanol oxidation could have allowedfor the more efficient utilization of acetate. Thislow level of H2 could possibly have alleviatedthe necessity for M. barkeri to produce reducingequivalents by the oxidation of methyl groupsduring acetate metabolism.The population concept is supported by the

results obtained when the inocula of the threeconstituents of the coculture were increased.When only the methanogen was increased, thepH did not drop as drastically, there was lessaccumulation of acetate, ethanol, and H2, andthe fermentation was completed in 7 days. Theseresults were also seen when all three inoculawere increased simultaneously. Quicker andmore efficient utilization of acetate and H2 by alarger methanogenic population indicated thatthe methanogenic step of the culture fermenta-tion was initially rate limiting. The excess of

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420 LAUBE AND MARTIN

both methanogenic substrates during the initialstages of the fermentation and no change in thefermentation pattern when either A. cellulo-lyticus or Desulfovibrio sp. inocula were in-creased lend support to this. Methanogenesishas generally been found to be the slow step ofmost methane fermentations (4), although thereare reports of the cellulose hydrolysis step beingrate limiting (26, 30).The results presented here emphasize the in-

terdependence of the microbial processes in-volved in the cellulose-to-methane fermentation.Optimization and further investigations of thethree-component system are now in progess andshould lead to further elucidation of the inter-actions involved in the coculture.

ACKNOWLEDGMENTS

We thank C. R. MacKenzie, I. J. McDonald, and G. B.Patel for their comments on the manuscript.

LITERATURE CITED1. Ackman, R. G. 1972. Porous polymer bead packings and

formic acid vapor in the GLC of volatile fatty acids. J.Chromatogr. Sci. 10:560-565.

2. Badziong, W., R. K. Thauer, and T. G. Zeikus. 1978.Isolation and characterization of Desulfovibrio growingon hydrogen plus sulfate as the sole energy source.Arch. Microbiol. 16:41-49.

3. Ben-Bassat, A., R. Lamed, T. K. Ng, and J. G. Zeikus.1980. Metabolic control of microbial fuel productionduring thermophilic fermentation of biomass, p. 275-294. In J. W. White, W. McGrew, and M. R. Sutton(ed.), Symposium papers, energy from biomass andwastes IV. Institute of Gas Technology, Chicago.

4. Bryant, M. P. 1979. Microbial methane production-the-oretical aspects. J. Anim. Sci. 48:193-201.

5. Bryant, M. P., L. L. Campbell, C. A. Reddy, and M. R.Crabill. 1977. Growth of Desulfovibrio in acetate andethanol media low in sulphate in association with H2-utilizing methanogenic bacteria. Appl. Environ. Micro-biol. 33:1162-1169.

6. Bryant, M. P., E. A. Wolin, M. J. Wolin, and R. S.Wolfe. 1967. Methanobacillus omelianskii, a symbioticassociation of two species of bacteria. Arch. Microbiol.59:20-31.

7. Holdeman, L. V., and W. E. C. Moore (ed.). 1975.Anaerobe laboratory manual, 3rd ed. Anaerobe Labo-ratory, Virginia Polytechnic Institute and State Univer-sity, Blacksburg, Va.

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