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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 1027-10340099-2240/78/0035-1027$02.00/0Copyright X) 1978 American Society for Microbiology

Vol. 35, No. 6

Printed in U.S.A.

Effect of Sulfur-Containing Compounds on AnaerobicDegradation of Cellulose to Methane by Mixed Cultures

Obtained from Sewage SludgetA. W. KHAN* AND T. M. TROTTIER

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

Received for publication 25 October 1977

Tests were made to determine the effects of inorganic and organic sulfur sourceson the degradation of cellulose to methane in a chemically defined medium withsulfur-poor inoculum prepared from sewage sludge. The results show that a sulfursource of about a 0.85 mM concentration is essential for the degradation ofcellulose to CH4. However, the production of CH4 from C02 and H2 provided inthe headspace occurred with 0.1 mM sulfate or sulfide. At a 9 mM concentration,all inorganic sulfur compounds other than sulfate inhibited both cellulose degra-dation and methane formation, and this inhibition increased in the order thiosul-fate < sulfite < sulfide < H2S. It appears that the degradation of cellulose to CH4in a sulfate-free medium by inoculum maintained in a low-sulfur medium isinhibited because of the lack of availability of sulfur for growth of bacteria andsynthesis of cell materials and sulfur-containing cofactors involved in cellulosedegradation and methanogenesis. The reduction of methanogenesis by higherlevels of sulfate probably occurs as a result of stimulation of reactions convertingacetate and H2 to end products other than CH4.

Cellulose, one of the Earth's most abundantbiopolymers, is increasingly recognized as animportant potential energy source. One methodfor converting and upgrading the energy fromcellulose is by producing methane through an-aerobic digestion. Although methane formationfrom sewage sludge, which contains a substantialamount of cellulose, has been known for a longtime, the fundamental biochemistry and micro-biology of the process and the role played byvarious elements in the process are still poorlyunderstood (28). For example, the literature onanaerobic fermentation indicates the necessity,as well as the undesirability, of the presence ofsulfate and sulfides for degradation of solids tomethane. In the anaerobic digestion of waste,the presence of sulfides has been shown to pre-cipitate toxic heavy metals such as chromium,cobalt, copper, iron, nickel, and zinc (14, 15). Inrumen microorganisms, inorganic sulfur hasbeen implicated in the synthesis of sulfur-con-taining organic compounds which are incorpo-rated into microbial proteins (9, 10). For thecultivation of anaerobes, sulfur-containing com-pounds have been employed to maintain a re-ducing environment essential for the growth ofthese organisms (6), and they form an integralpart in ferredoxin (29) and other compoundsinvolved in electron transport systems (20), aswell as coenzyme M, which is involved in the

t Issued as National Research Council of Canada no. 16716.

methyl group transfer reactions in methanogen-esis (16, 26). On the other hand, sulfides havebeen shown to be toxic to many anaerobes atfairly low concentrations (17, 34) and sulfateshave been shown to inhibit methanogenesis (7,19, 34). This multiple role of inorganic sulfurnecessitated work to determine the effect ofvarious sulfur-containing compounds on cellu-lose degradation and methane formation bymixed cultures. This paper reports the effects ofa variety of sulfur-containing compounds on cel-lulose degradation to methane and the require-ments for sulfur-containing compounds to opti-mize this process.

MATERLALS AND METHODSPreparation of inoculum and medium. The ef-

fluents from a municipal sludge digester were dilutedwith an equal volume of medium and grown in a 10-liter fermentor (Sovirel, Lovallois-Perret, France) heldat 35°C under anaerobic conditions. This fermentorwas fed once a day and maintained using a sulfur-freechemically defined medium under the "repeated-fed-batch culture" technique (25) for 8 to 10 weeks. Themedium contained (mg/liter): tissue paper pulp (5,-000), NH4HCO3 (3,000), K2HPO4, (450), KH2PO4 (450),Na2CO3 (320), NH4Cl (180), MgCl2 6H20 (165),CaCl2 2H20 (120), NaCl (55), FeCI3 (12), MnCl2 4H20(5), CoCl2 6H20 (1), ZnCl2 (0.8), CuCl2 2H20 (0.1),H3BO3 (0.1), Na2MoO4 2H20 (0.1), pyridoxine HCI(0.1), thiamine HCI (0.05), riboflavin (0.05), nicotinicacid (0.05), biotin (0.02), folic acid (0.02), and vitamin

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1028 KHAN AND TROTTIER

B12 (0.005). The composition of this salt-vitamin mix-ture was adapted from the work of Bryant and Robin-son (5), Holdeman and Moore (13), and Wolin et al.(36) by replacing all the cations present as sulfate withtheir respective chlorides on an equimolar basis. Thefeeding rate and retention time were 1.0 g of celluloseper liter per week and 16 to 20 weeks, respectively.This incubation procedure was followed principally toreduce the concentration of sulfur-containing materi-als present in the inoculum obtained from the sludgedigester.

Further removal of sulfur-containing compoundsfrom this inoculum was carried out by removing thesupernatant and washing the cells. Portions of thefermenter liquid from the 10-liter fermentor werepassed through six layers of cheese cloth to removeundegraded cellulose and transferred to 160-ml serumvials under anaerobic conditions (50 ml/vial). Thevials were sealed, and the headspace in each vial wasflushed and pressurized to 20 lb/in2 with a 20%C02-0% H2 mixture before centrifuging at 650 x g for30 min. Higher speeds were not used because of theinability of the vials to withstand any greater centrif-ugal forces and the lack of centrifuge equipment forstrict anaerobic work. However, the inoculum thusobtained metabolized cellulose to CH4 effectively andwas found suitable for comparative studies (Table 1).The supernatant from each vial was removed with ahypodermic syringe and was replaced, on a volumetricbasis, by the sulfate-free salt-vitamin mixture. Thecells were resuspended in this solution and used as aninoculum without any dilution. All preparations werecarried out under an 80% H2-20% CO2 mixture at 350C.

Culture conditions. Experiments were carried outin 160-ml serum vials (22) containing 250 mg of ho-mogenized tissue paper in 50 ml of sulfur-free salt-vitamin mixture. This medium was supplemented withthe desired amount of sulfur by anaerobically injectingaqueous solutions of Na2SO4, Na2SO3, Na2S, H2S, or amixture of cysteine and methionine, which was pre-pared and bottled under the H2-CO2 mixture. Thisstep was necessary to prevent the oxidation of some ofthese compounds, especially Na2S. To test the effectsof low oxidation-reduction potentials on cellulosebreakdown to CH4, limited tests were also made byadding titanium-citrate buffer (37), in 80-ml/liter con-centrations, as a reducing agent. The headspace of allthe vials was flushed with 80% H2-20% C02 mixture.This gas mixture was selected for two reasons: (i) tomaintain an anaerobic atmosphere and (ii) to deter-mine the effects of these sulfur-containing compoundson methanogenesis occurring as a result of CO2 reduc-tion. The rate and extent of CH4 formation fromheadspace gases also provided information on anypossible batch-to-batch variation in the activity ofmethanogens present in the inoculum. For the calcu-lation of results on a cellulose degradation basis, thevolume and composition of the gas were corrected forconversion of headspace gas to CH4 by using stoichi-ometry 4H2 + C02 -* CH4 + 2H20. Since in mixedcultures the molar ratio of H2 conversion to CH4formed range from 5:1 to 10:1 (33) rather than thetheoretical ratio of 4:1, the amount of CH4 formedfrom cellulose breakdown might be higher than cal-culated, and values for CH4 formed from headspace

gas might be correspondingly lower. All vials wereincubated at 350C with shaking. Results are averagesfor at least three tests carried out on different occa-sions. All tests were run in duplicate.Redox potential (Eh) was measured with a plati-

num-calomel electrode combination. Under the testconditions, Eh values of the medium stayed below-350 mV. Addition of titanium-citrate buffer main-tained this value below -550 mV. The pH of themedium generally stayed between 6.7 and 7.0. A dropin pH below 6.7 was taken as failure of acid utilization.Methods of analysis. Analyses were made to de-

termine gas composition, volatile acids, cellulosebreakdown, and sulfate and sulfide content of thefermenter liquids. Gas composition was determined bythe method of van Huyssteen (31) by using a PorapakT column (¼4 inch by 10 feet [ca. 6.35 mm by 304.8cm]) on a Hewlett-Packard 5710A gas chromatographequipped with a model 3380A integrator. The columnwas held at 750C, and the thermal conductivity detec-tor was held at 1500C. Helium was used as a carriergas at a flow rate of 40 ml/min. Volatile acids weredetermined by the method of Ackman (1) by using aHewlett-Packard 5721A chromatograph equippedwith an automatic sampler, a model 3380A integrator,and a Chromosorb 101 column (% inch by 5.5 feet [ca.3.18 mm by 167.64 cm]). The column was held at1800C, and the single flame ionization detector washeld at 3500C. Helium was used as a carrier gas at aflow rate of 35 ml/min. Samples were prepared byadding 0.5 ml of sample to 0.5 mil of an internalstandard containing 2,000 mg of isobutyric acid perliter, and the chromatograph was calibrated with astandard containing 2,000 mg each of acetic, propionic,butyric, and isobutyric acids per liter. This equipmentwas able to detect and quantitate concentrations of 6to 10 mg/liter and higher of acetic, propionic, andbutyric acids at less than a 2% error. Low quantities ofpropionic or butyric acids, occasionally detected, wereequated 1:1 (on a molar basis) with acetic acid asacetate equivalents for the purpose of tabulation.The total contents of each vial were centrifuged at

6,000 x g for 20 min. The sediment was used for thedetermination of cellulose, whereas the supernatantwas used for the estimation of sulfates and sulfides.The sediment was suspended in 8% formic acid to lysethe cells (32) and then recentrifuged. The pellet wasdissolved in 72% H2SO4, and the cellulose content wasdetermined by anthrone reagent (12). In some cases,these estimations were checked by the dichromate-oxidation procedure (2) used for the determination ofchemical oxygen demand. For the measurement ofpure cellulose and cellulose in fermenter liquids, theoverall accuracy of anthrone reagent was better than5% and the accuracy of the dichromate-oxidation pro-cedure was better than 2%. For the estimation ofsulfates, the supernatant was treated with a uraniumacetate solution to remove proteinaceous materials,and sulfates were precipitated as benzidine sulfate,washed free of benzidine, and determined colorimet-rically by using sodium ,B-naphthaquinone-4-sulfonate(11). Sulfide content was estimated by a titrimetricmethod (2). In view of the absence of the characteristicblack iron sulfide precipitate in the sulfur-depletedfermenter liquids and the unavailability ofprecipitated

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CELLULOSE DEGRADATION BY SULFUR SOURCES 1029

sulfides to anaerobes (14, 15), the insoluble sulfidecontents were not determined.

RESULTS

Removal of supernatant to reduce the contentof sulfate and other soluble sulfur-containingcompounds present in the inoculum lowered thecellulolytic activity by about 30%, but had littleor no effect on methanogenesis occurring fromthe reduction of CO2 or from cellulose that wasdegraded (Table 1). Return of supernatant tothe washed cells or prolonged incubation ofwashed inoculum in sulfate-containing mediumrestored the cellulolytic activity almost to theoriginal level. Since the removal of soluble sul-fates and sulfur-containing compounds is essen-tial in studying the effects of these compoundson cellulose breakdown to CH4, washed cellshaving somewhat lower cellulolytic activity wereused in this study. Even this washing proceduredid not remove all the sulfates and sulfides, sincethe amount of these compounds in washed in-oculum varied between 0.10 and 0.15 mM ex-

pressed as sulfur. However, in the presence ofsulfate, the washed inoculum regained the lostactivity during 3 weeks of incubation (Table 1).Removal of sulfate, as well as other sulfur-

containing compounds from the medium, de-pressed the cellulose degradation by over 80%and reduced methane formation from cellulose,but did not affect its ability to convert H2 andCO2 present in the gas phase to CH4 (Table 2).This depression continued during incubationtime for up to 4 weeks, but the addition of sulfateto the medium restored the ability of this cultureto degrade cellulose. In the initial stages of fer-mentation (1 to 2 weeks of incubation), rapiddegradation of cellulose caused the accumula-tion of volatile acids up to a 10 mM concentra-tion (expressed as acetic acid) in the medium,

and the yields of CH4 were low. However, thisaccumulation of acids did not affect methano-genesis, since during subsequent incubation pe-

riods the methane yields increased to about 80%of the theoretical value based on stoichiometry,n C6HI206 -. 3n CH4 + 3n CO2. Absence or

presence of 0.85 to 1.75 mM sulfate had no effecton the conversion of H2 and CO2 present in theheadspace to CH4 by this culture. In these tests,the formation ofCH4 from headspace gas startedwithin 1 h after incubation and was completedwithin 1 day. At these concentrations, sulfatealso appears to stimulate CH4 formation fromcellulose breakdown products, since the removalof sulfate also reduced CH4 yields substantially.

Little or no change in Eh during 3 to 4 weeksof incubation indicates that the medium con-

tained in serum vials under an atmosphere of80% H2 and 20% CO2 maintained a reducingenvironment in the absence of cysteine-Na2S(Table 3). Addition of Ti-citrate buffer (37) alsohelped in maintaining the Eh of the mediumbelow -550 mV, but inhibited both cellulosedegradation and conversion of acids to CH4.However, this buffer did not affect the conver-

sion of headspace gases (H2 and C02) to CH4.The inhibition may have occurred as a result ofacid formation from citrate or by Ti itself. Littleor no degradation of cellulose in these testssuggests that the accumulation of volatile acidsin the medium occurred as a result of a break-down of citrate. Despite this inhibition, additionof sulfate to the medium containing Ti-citratebuffer improved the degradation of cellulose andCH4 formation. These results further demon-strate the necessity of a sulfur-containing source

in cellulose degradation by mixed cultures. Sincein these tests the Eh stayed below -300 mV, therequired value for some anaerobes (7, 19), theloss of cellulolytic activity in sulfur-free medium

TABLE 1. Effect of washing cells on cellulose degradation and methane formation by mixed culturea

Incubation Cellulose break- Volatile CH4formed from cellulose' CH, formedCulture time down b acidsC from headspace

(week) (mM) (mM) mM mol/mol of gaseglucose (mM)

Without washing 1 17.2 18 28.0 1.6 18.0After washing 1 11.9 16 18.8 1.6 18.0After washing + 1 17.6 18 28.9 1.6 18.0

supernatantfWithout washing 3 23.7 <1 58.7 2.5 18.0After washing 3 20.8 1 47.0 2.3 18.0

a Inoculum was maintained in a sulfur-free medium for 8 weeks before using. Medium used in these testscontained 1.75 mM sulfate and all the essential minerals and vitamins.

b Expressed as glucose equivalents.' Residual volatile acids expressed as acetic acid equivalents.d Corrected for headspace gas.eHeadspace gas, 80% H2 + 20% C02; 110 ml/vial. Calculations based on stoichiometry, 4H2 + CO2 -s CH4 +

2H20, at 25°C.f After washing the cells, a volume of supernatant equivalent to that removed was added.

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TABLE 2. Effect of added sulfate and length of incubation on cellulose degradation and methane formationby mixed culturea

Gas formed from cellulose" CH4 formedIncubation Cellulose from head-

Additions time breakdownb Total CH C 1of space gas'd(week) (mM) (mM) (mM) glucose) (mM)

None (control)e 1 1.6 19.6 0 0 18.0Sulfate 1 13.7 59.8 18.8 1.4 18.0None (control)' 2 2.7 31.2 0.9 0.3 18.0Sulfate 2 18.4 85.7 33.6 1.8 18.0None (control)e 3 4.1 33.0 3.5 0.9 18.0Sulfate 3 21.1 107.1 50.9 2.4 18.0None (control)e 4 5.3 40.1 6.3 1.2 18.0Sulfate 4 22.2 113.4 53.6 2.4 18.0

aInoculum, washed cells. Where indicated, Na2SO4 was added to increase sulfur content of medium to 1.75mM.

' Expressed as glucose equivalents.'Corrected for headspace gas.d Headspace gas, 80% H2 + 20% C02; 110 ml/vial. Calculations based on stoichiometry, 4H2 + CO2 -s CH4 +

2H20, at 25°C.e Inoculated medium used as control contained 0.1 mM sulfur in the form of sulfate and sulfide, and all

essential minerals and vitamins.

TABLE 3. Effect of sulfate and titanium-citrate buffer on maintaining reducing environment, cellulosedegradation, and methane formnation by mixed culture'

Eh(mV) Cellulose break- Volatile acids" CH formed'Additions" down' (mM) (m)4

Initial After incubation (mM) (mM) (mM)

None (control) -437 -463 1.7 <1 19.0Sulfate -456 -450 10.1 12 34.1Ti-citrate -550 -550 1.1 28 19.8Sulfate + Ti-citrate -545 -545 3.1 31 23.3

a Incubation time, 1 week.b Inoculated medium used as control contained 0.1 mM sulfur in the form of sulfide and sulfate. Na2SO4

added to increase sulfur content of medium to 1.75 mM, where indicated.'Expressed as glucose equivalents.d Total acids, expressed as acetic acid equivalents.'Methane formed from headspace gas is included.

does not appear to occur as a result of a rise inEh.

Addition of 1.5 to 1.7 mM sulfur in the formof sulfate, sulfite, thiosulfate, sulfide, or aminosulfur as cysteine and methionine was equallyeffective in restoring the ability of the culture todegrade cellulose to CH4 (Table 4). Sulfate, thi-osulfate, and sulfite are known to act as electronsinks in anaerobic metabolism, and competewith methanogens for hydrogen (4). This com-petition would not occur where sulfide or cys-teine and methionine were present. Since thenature of sulfur-containing compounds did notaffect cellulose degradation to CH4, sulfur ap-pears to be an essential nutrient for the mixedculture present in sewage sludge.

Presence of sulfate of up to a 0.85 mM con-centration appears necessary for anaerobic deg-radation of cellulose by mixed culture. Concen-trations of up to 1.75 mM had little or no inhib-

itory effect on cellulose degradation to CH4, buthigher concentrations adversely affected the ra-tio of cellulose degraded to methane formedafter 3 weeks of incubation (Fig. 1). However,this microbial ecosystem tolerated sulfate con-centrations of up to 12 mM with no more thana 50% loss in the efficiency of conversion ofcellulose to methane. Since there is a generalagreement that sulfate is reduced to sulfide inmixed cultures (27), the inhibition occurring asa result of higher concentrations of sulfate notedafter 3 weeks of incubation (Fig. 1), but not after1 week of incubation (Table 5), may be due tothe accumulation of sulfide in the medium. InLake Mendota sediments, a sulfate concentra-tion of 1.56 mM has been shown to cause inhi-bition (19) ofmethanogenesis, whereas a concen-tration of 10mM caused complete inhibition (7).In freshwater sediments, 0.5 to 1.0 mM sulfateconcentrations caused complete inhibition (34).



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TABLE 4. Effect of various sulfur-containingcompounds in low concentrations on cellulosedegradation and methane formation by mixed

culturesaCH4 formed from

Cellulose cellulosec

Sulfur source content break-(mM) (MM) mM of glu-

cose

No addition 0.1 2.4 2.6 1.1Na2SO4 0.8 19.1 26.6 1.4Na2SO4 1.7 18.7 23.4 1.3Na2SO3 0.8 16.6 26.1 1.2Na2SO3 1.7 17.9 19.3 1.1Na2S203 0.8 19.6 21.2 1.1Na2S203 1.7 17.0 19.0 1.1Na2S 0.8 20.4 23.2 1.1Na2S 1.7 21.3 22.4 1.0Cysteine + 1.7 21.6 17.7 0.8

methio-nine

a Incubation time, 1 week.bExpressed as glucose equivalents.c Corrected for headspace gas.

-0U,0

0'

0

0

E

0Ez0

2LLwz

Iw

2.4

2.0

1.6

1.2

0.81

0.41

2 4 6 8 10 12SULFATE CONCENTRATION (mM)

FIG. 1. Effect of sulfate concentration in the me-dium on methane formation from cellulose. Incuba-tion time, 3 weeks. Sulfate added as Na2SO4.

TABLE 5. Effect ofhigh concentration ofsome inorganic sulfur compounds on cellulose degradation andmethane formation by mixed cultures'

Cellulose break- CH4 formed from cellulose" CH formed fromSulfur source Sulfur content down(mM) (mM) mM mol/mol of glu- (mM)

cose (M

No addition 0.1 2.7 3e 1.1 18"Na2SO4 3.5 18.9 24f 1.3 18'

5.0 18.4 21f 1.1 18'9.0 17.2 19' 1.1 18'18.0 16.1 17' 1.1 18'

Na2SO3 3.5 18.9 18' 0.9 18'5.0 18.9 13e 0.7 18'9.0 3.6 <le 4e

18.0 1.4 <1e <leNa2S203 3.5 17.7 19f 1.1 18'

5.0 20.0 18f 0.9 18f9.0 6.7 <le 16f

18.0 3.1 <1' 12fNa2S 3.5 19.8 20' 1.0 18'

5.0 18.9 19' 1.0 18'9.0 2.5 <le 14e

18.0 0.3 <1e 12'

'Incubation time, 1 week.b Expressed as glucose equivalents.'Corrected for CH4 formed from headspace gas.dHeadspace gas, 80% H2 + 20% C02, 110 cm3/vial. Calculations based on stoichiometry, 4H2 + CO2 CH4

+ 2H20, at 25°C.etf Significantly different at P < 0.01.

Since little or no information is available on theamount of sulfate present in sewage sludge or onthe effects of sulfate on cellulose degradation bythe microbial ecosystem that exists in sewagesludge, direct comparison cannot be made. How-ever, the results obtained after 3 weeks of incu-bation (Fig. 1) agree generally with those re-ported by Cappenberg (7), MacGregor and Kee-

ney (19), and Winfrey and Zeikus (34), indicatingthat at higher concentrations sulfate inhibitsmethanogenesis.At higher concentrations, the extent of inhi-

bition of cellulose degradation and methanogen-esis depended on the nature of the inorganicsulfur (Table 5). At a 9 mM concentration, theinhibitory effect on cellulose degradation in-

I I I

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creased in the following order, sulfate < thiosul-fate < sulfite < sulfide. At this level, all inorganicsulfur compounds, except sulfate, lowered cel-lulose degradation, completely inhibited meth-ane formation from acids, and even reduced theamount of CH4 formed from headspace gases.For the conversion of H2 and C02 present in theheadspace to CH4, sulfite had maximum inhibi-tory effect, whereas sulfate had the least. Theseresults agree with the findings of Winfrey andZeikus (34), indicating that sulfide in higherconcentrations (10 mM) inhibits methanogene-sis, but differ from those obtained with co-cul-tures (4), indicating that Methanobacteriumstrain M.o.H. in association with desulfovibriocan tolerate sulfide levels of up to 20 mM. Thesedifferences may be due to metabolic dependenceand competition that exists in mixed cultures as

well as to differences in experimental design.At 1.75 mM concentrations, H2S inhibited

methanogenesis, reduced cellulose degradation,and caused the accumulation of volatile acids inthe medium as compared to similar levels ofsulfate (Table 6). These inhibitory effects ofH2Sincreased at higher concentrations, and the in-hibition was almost 100% at 10 mM. At thislevel, the formation of methane from C02 andH2 provided in the headspace was also totallyinhibited.

DISCUSSIONThe results indicate that the presence of sulfur

in a defined medium is essential for cellulosedegradation to CH4 by a mixed culture obtainedfrom sewage sludge and that more than 0.1 mMsulfur is not required for the reduction of C02 toCH4 when H2 is available. For cellulose degra-dation to CH4, sulfur contents of up to 0.8 mMin the form of sulfate, thiosulfate, sulfite, sulfide,or cysteine and methionine were equally effec-tive. In concentrations higher than 1.75 mM, the

inhibition of cellulose degradation to CH4 de-pended on the type of sulfur-containing com-pound and increased in the following order, sul-fate < thiosulfate < sulfite < sulfide < H2S.Among these, sulfite and H2S had the greatestinhibitory effect on methanogenesis. The hightolerance for sulfate by the ecosystem reportedhere is probably due to the presence of H2 in theinitial stages of fermentation and presence ofacetate after the start of cellulose degradation,both of which have been shown to prevent sul-fate inhibition of methanogenesis (33). All meth-anogenic bacteria so far studied utilize sulfide as

a sulfur source and some also use cysteine, butnone have yet been found to utilize other formsof sulfur. Sulfide is essential as a sulfur sourcefor at least three species (38). In medium con-taining sulfate as a sulfur source, sulfide formedfrom the reduction of sulfate (27) appears toserve as a sulfur source for the methanogens inmixed cultures. However, similar studies havenot been made on cellulose-degrading anaer-obes. The results reported here are important indeveloping a medium for optimal degradation ofcellulose to methane.The initial low yields of methane from washed

cells in medium containing sulfur appears tooccur as a result of the removal of stimulatorycompounds present in the supernatant (3, 18, 24,26, 30) rather than because of destruction ofmethanogens during the removal ofsupernatant,rise in Eh (19), or toxicity due to the presence ofheavy metals in the absence of sulfide (14). Thisis indicated by an increase in CH4 yields fromcellulose breakdown to about 80% of the theo-retical value, after an increased incubation time.The initial low yields of CH4 do not appear to bedue to a lower number of methanogens presentin the inoculum or to a longer doubling time.Had this been the case, the utilization of head-space H2 and CO2, of which all known methan-

TABLE 6. Effect of dissolved H2S in medium on cellulose degradation and methane formationby mixed culture'

Cellulosebreak- CH4 formed from:Sulfur content Cellulose reak Volatile acids"

Sulfur source (mM) (MM) (mM) Cellulose' Headspace gas'(MM) (MM)

No addition 0.10 3 3 3f 18Na2SO4 1.75 19 18 34g 18H2S 1.75 16 22 18f 18H2S 5.25 12 20 12f 18H2S 10.50 <0.1 <if <1

a Incubation time, 2 weeks.b Expressed as glucose equivalents.'Total acids expressed as acetic acid equivalents.d Corrected for headspace gas.eHeadspace gas, 80% H2 + 20% CO2, 110 cm3/vial. Calculations based on stoichiometry 4H2 + CO2 -. CH42H20, at 250C.f" Significantly different at P < 0.01.



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ogens use to derive energy (35), would haveoccurred at a much slower rate. In these tests,the formation of CH4 from headspace gases be-gan within 1 h after inoculation and was com-pleted within 1 day. The increase in the fattyacid content of the medium during week 1 ofincubation may indicate a slow initial growthrate of fatty acid-catabolizing organisms. Thisslow rate of initial growth may be due to thepresence ofH2 in the headspace during the initial16 to 24 h of incubation. Rise in Eh is not afactor, as the Eh of the samples containing lowconcentrations (0.8 mM) of sulfur salts was be-low -300 mV, a value at which methanogenesishas been known to proceed effectively (7, 19).Toxicity due to the presence of heavy metals(14) does not appear to affect these tests, asthese metals were present in essential minimumquantities and a doubling of these quantities didnot affect the inoculum performance. However,the importance of controlling the Eh of themedium in which small amounts of inocula andno reducing atmosphere are used (19, 23) andremoving heavy metals as their sulfides fromcomplex substrates such as sewage sludge (14,15) is well established.The inhibitory effects of high levels of sulfate,

sulfide, and H2S on methanogenesis by mixedcultures are well known (4, 7, 19, 34). In sensitiv-ity towards high levels of these compounds, themicrobial ecosystem that exists in sewage sludgeis comparable to the microbial ecosystem fromlake sediments (7, 19, 34), as well as to co-cul-tures containing sulfate-reducing and methane-producing bacteria (4). In sulfate-rich medium,poor methanogenesis, in spite of excellent deg-radation of cellulose, appears to occur as a resultofstimulation ofacetate metabolizing non-meth-anogens (33) and lack of availability of H2 (4, 21,34). The utilization of acetate in the presence ofsulfate via the reaction S042- + CH3C000HS- + 2HC03- has a AG0' of -47.3J, as com-pared to the conversion CH3C00 + H20 =CH4 + CH03 which has a AG01 of -31.OJ (27).Due to this more favorable change in free en-ergy, the growth of nonmethanogenic organismswith sulfate reduction may be selectively en-hanced. The presence of H2-using, sulfate-reduc-ing bacteria in the mixed cultures could furthercurtail the availability of H2 for methanogenesis(4).

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

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

2. American Public Health Association. 1965. Standardmethods for the examination of water and waste waterincluding bottom sediments and sludges, 12th ed., p.510. American Public Health Association, New York.

3. Brill, W. J., and K S. Wolfe. 1966. Acetaldehyde oxi-

dation by methanobacillus-a new ferredoxin-depend-ent reaction. Nature (London) 212:253-255.

4. Bryant, M. P., L L Campbell, C. A. Reddy, and M. R.Crabili. 1977. Growth of desulfovibrio in lactate orethanol media low in sulfate in association with Hrutilizing methanogenic bacteria. Appl. Environ. Micro-biol. 33:1162-1169.

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