APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1978, p. 186-197

0099-2240/78-0036/0186$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 36, No. 1 Printed in U.S.A.

Methanogenesis from Acetate: Enrichment Studies LARRY BARESI,' ROBERT A. MAH,1 * DAVID M. WARD,t AND ISAAC R. KAPLAN2 Division of Environmental and Nutritional Sciences, School of Public Health,' and Department of Earth and Space Sciences,2 University of California at Los Angeles, Los Angeles, California 90024 Received for publication 11 January 1978

An acetate enrichment culture was initiated by inoculating anaerobic sludge from a mesophilic methane digestor into a mineral salts medium with calcium acetate as the sole carbon and energy source. This enrichment was maintained indefinitely by weekly transfer into medium of the same composition. A study of this enrichment disclosed an unexpected age-dependent inhibition of methanogenesis by H2 and formate which apparently differed from the inhibition by chloroform and benzyl viologen. This age-dependent inhibition indicated that microbial interactions of the mixed enrichment population may play a regulatory role in methane formation. Furthermore, stimulation of methanogenesis in the acetate enrichment by addition of yeast extract showed a nutrient limitation which indicated that syntrophic interactions leading to formation of growth factors may also occur. A model is presented to illustrate the possible interrelationships between methanogenic and nonmethanogenic bacteria in their growth and formation of methane and carbon dioxide from acetate.

Methane and carbon dioxide are the terminal fermentation products of organic compounds in anaerobic environments such as sediments of fresh and salt water, waste treatment digestors, flooded soils, and the gastrointestinal tract of humans and animals. Labeling studies indicate that two major pathways for methane formation may exist in nature: (i) carbon dioxide reduction by H2 to form CH4 and water; (ii) acetate conversion by reduction of the methyl group to methane and oxidation of the carboxyl group to C02 (2, 6, 18, 20, 21). In either case, carbohydrates, organic nitrogen compounds, and lipids are dissimilated by a variety of pathways to form the acetate, the CO2, and the H2 used in these two pathways. In digestors, acetate is the major carbon precursor of methane and may account for approximately 70% of the total methane formed (11, 19), whereas in lake sediments from 53% (4) to 70% (7) methane may come from acetate. In contrast, only 5.6% of the methane is formed from acetate in the bovine rumen (17). This difference may be attributed to the difference in end-product (i.e., acetate) removal in the rumen versus non-gastrointestinal habitats and/or to the higher turnover of cell material. Because of the importance of acetate in methane formation, we focused our studies on an elective enrichment culture in which acetate served as the sole source of organic carbon and energy. Various types of acetate enrichments t Present address: Department of Microbiology, Montana State University, Bozeman, MT 59715.

have been reported (1, 6, 12, 18, 20); the conditions used in the present enrichment were modified from the study reported by Barker (1). The enrichment is maintained by weekly transfer and contains methanogenic and nonmethanogenic bacteria (13, 25; R. A. Mah, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, Q28, p. 195; D. M. Ward, I. R. Kaplan, and R. A. Mah, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, Q27, p. 195; L. Baresi and R. A. Mah, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, Q29, p. 195). The present work describes the effect of certain known methanogenic substrates and inhibitors on the acetate enrichment. Based on these and related studies, a model is presented to describe the interrelationships which may exist among the methanogenic and nonmethanogenic bacteria in the metabolism of acetate. MATERIALS AND METHODS Enrichment system. The enrichment medium contained per liter of tap water: NH4Cl, 1 g; K2HPO4 * 3H20, 0.4 g; MgCl2 * 6H20, 0.1 g; CaCO3, 100 g (J. T. Baker precipitated calcium carbonate, USP powder, lot 425952); calcium acetate, 20 g; and 0.1% resazurin (Eastman Kodak Co., Rochester, N.Y.), 1 ml. This salt solution was the same as that described by Barker (1), except for the addition of resazurin and standardization of the quantity of CaCO3. The medium was prepared, stored, and used according to the anaerobic methods of Hungate (10) under a 100% N2 atmosphere. After it was cooled to room temperature, the pH was anaerobically adjusted to pH 6.5 to 6.6 with concentrated HCl. The medium was then dispensed in 100-ml portions into 250-nil boiling flasks, stoppered under 100% N2, and autoclaved at 120°C 186

VOL. 36, 1978

and 20 lb/in2 for 15 min. Before inoculation, 3 ml of a sterile anaerobic solution containing 1% Na2S * 6H20 and 5% Na2CO3 was added to each flask. The final pH of the medium after inoculation was 7.0 to 7.1. The enrichment was initiated by inoculation with 7% anaerobic digesting sludge obtained from a mesophilic digestor at the Hyperion waste treatment facility in El Segundo, Calif. After stabilization of the enrichment as judged by methane production, cultures were routinely transferred at weekly intervals by introducing 20 ml of a week-old enrichment culture into 100 ml of fresh medium. The enrichments were incubated without shaking at 37°C, and the rate of gas production was reproducible at each transfer. Anaerobic procedures. The strict anaerobic methods developed by Hungate (10) were used throughout these investigations. Culture vessels and all media were outgassed with various percentages of N2, H2, or CO2 before, during, and after addition of solutions or inocula as described in the text. Traces of 02 in the gases were removed by passing the gas through a reduced copper column heated to ca. 350°C. Additions and withdrawals of all samples and nutrients were made with sterile outgassed syringes and hypodermic needles. Enumeration media. Viable counts of the enrichment were made with medium 2 (25), which contains the following components: tap water, 1 liter, NH4CI, 1 g; K2HPO4 * 3H20, 0.4 g; MgCl2 * 6H20, 0.1 g; Trypticase (BBL), 2 g; yeast extract (Difco), 2 g; NaHCO3, 1.5 g; cysteine. HCI, 0.5 g; DL-2-methylbutyric acid, 0.1 g; Na2S * 9H20, 0.3 g; calcium acetate, 1 g; and 0.1% resazurin, 1 ml. A 100% N2 atmosphere was used. For solid media, 2% agar was added. Again, the anaerobic procedures of Hungate (10) were employed through-out the preparation, storage, and use of these media. Decimal order dilutions of the inoculum were made by serially diluting 0.5-ml portions into culture tubes containing 4.5 ml of liquid medium. Agar media held at 45°C were then inoculated at the appropriate dilutions, solidified as roll tubes, and incubated at 370C. Analyses of gases. CH4, H2, and CO2 were analyzed by gas chromatography, using either a Loenco AD 2000 or a Varian Aerograph A-90P gas chromatograph, both equipped with thermal conductivity detectors.

Gas concentrations as low as 10.7 nM methane and 2.68 nM hydrogen were detected by injection of a 0.3ml sample obtained with a hypodermic needle and syringe into the Loenco gas chromatograph. Separation of constituent gases was achieved by using either a copper column (12.2 m by 2 mm) packed with Porapak Q (80 to 100 mesh; Applied Science Laboratories, State College, Pa.) or a copper column (4.57 m by 2 mm) packed with Molecular Sieve 5A (60 to 80 mesh; Applied Science Laboratories). The injector, detector, and column were operated at 1000C. The detector current was set at 65 mA. Nitrogen prepurified was employed as the carrier gas at a flow rate of 25 ml/min. Gas samples of 0.5 or 1.0 ml collected with a hypodermic needle and syringe were injected into the Varian Aerograph A-90P gas chromatograph, and separation of constituent gases was accomplished by using

METHANOGENESIS FROM ACETATE

187

an aluminum column (OD, 3.65 m by 6.4 mm) packed with acid-washed, 60- to 80-mesh, chromatographicgrade, activated charcoal (Applied Science Laboratories) (24). The injector and detector were operated at 60°C, and the column was operated at 183°C. The detector current was set at 280 mA. Helium was employed as the carrier gas at a flow rate of 60 ml/min. Gas concentrations as low as 150 nM H2, 2.7 nM CH4, and 8.9 nM CO2 could be detected with good separation of all gases, using 1-ml samples. A Teflon column (OD, 30.5 cm by 6.4 mm) packed with 10- to 20-mesh Drierite (A. H. Thomas, Philadelphia, Pa.) was introduced between the injector port and the column to absorb water from the incoming sample. In addition to the Drierite column, 20- to 30mesh Ascarite (A. H. Thomas) packed in a Teflon column (OD, 22.9 cm by 6.4 mm) was used whenever the column containing Molecular Sieve 5A was used to adsorb C02; it was inserted after the Drierite column and before the molecular sieve column. Radioactivity. The sodium salt of [2-'4C]acetate (specific activity, 56.5 mCi/mmol) was purchased from International Chemical and Nuclear Corp., Irvine, Calif. Radioactivity in the gaseous fermentation products were determined by the method of Nelson and Zeikus (16). Effluent from the Varian Aerograph gas chromatograph was fed directly into a Packard 894 gas proportional counter, where the gases were combusted to CO2 and water; radioactivity was quantified by a multiplicative ion collection device. The following factors were found to be optimal for the proportional counter: a time constant of 2 s and propane (chemically pure grade; Matheson Gas Products, Cucamonga, Calif.) used as the quench gas at a flow rate of 6 ml/min. The culture was prepared for gas sampling by titrating with 6 M H3PO4 or cold 10% trichloroacetic acid to ca. pH 3 to minimize the solubility of the gases. Assimilation of [2-'4C]acetate into cell material was measured by determining the radioactivity of an acidified culture sample after collecting the cells on a 0.45t,m membrane filter (Metricel, type GA-6; Gelman Inst. Co., Ann Arbor, Mich.). The filters were placed in scintillation vials and dried overnight in a vacuum oven at 60°C. Samples were counted in a liquid scintillation counter after the addition of 10 ml of scintillation fluid [0.4% 2,5-diphenyloxazole and 0.01% 1,4bis-[2-(5-phenyloxazolyl)]benzene in toluene]. Analysis of organic acids. Samples to be analyzed for volatile fatty acids were acidified to ca. pH 3 with 0.1 N HCI and centrifuged. A Hamilton 7105 syringe was used to inject 4-pl supernatant samples into a Varian Aerograph (series 200 B) gas chromatograph equipped with a flame ionization detector, a glass injector insert, and a 1-mV Sargent Welch recorder. The gas-solid chromatographic procedures reported by Carlsson (8) for analysis of volatile fatty acids were used. A glass column (OD, 1.82 m by 6 mm; ID, 4 mm) was packed with 80- to 100-mesh Chromosorb 101 (Applied Science Laboratories) and conditioned overnight at 250°C. Operating conditions were: column temperature, 200°C; injector temperature, 215°C; detector temperature, 200°C; carrier gas, helium at a flow rate of 94 ml/min, H2 at a flow rate of 19 ml/min, and 02 at a flow rate of 86 ml/min.

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-~ o_

APPL. ENVIRON. MICROBIOL.

RESULTS Development and description of the

ace-

tate enrichment. The present acetate enrichment was similar to that described by Barker (1) in which calcium acetate served as the sole organic compound with all other nutrient requirements met by the addition of inorganic salts. It differed only by the incorporation of a standard quantity of CaCO3 and by the choice of a rubberstoppered boiling flask with a standard 100% N2 gas headspace above the culture liquid. It was essential to shake the enrichment thor-

oughly when removing an inoculum since the organisms were associated with the CaCO3 substratum. An inoculum taken from the crystalclear supernatant liquid above the CaCO3 will not produce an active fermentation. The present

enrichment has been maintained since 15 May 1974 and has a reproducible rate of methane production. The typical gas production rate of the acetate enrichment is shown in Fig. 1. Within the first week, approximately one-fourth of the maximum amount of methane was already produced. The average composition of the gas at this time

was 45% methane-22% carbon dioxide. The quantity of C02 measured should equal the quantity of CH4 produced, assuming acetate is converted to equimolar amounts of C02 and CH4. However, the measured C02 was less than this theoretical value because of the formation of the bicarbonate and carbonate salts of calcium made available from the conversion of calcium acetate to CH4 and C02. Hydrogen was never detectable except in cultures whose rates of methane and gas production were abnormally decreased. Methane was produced at a linear kinetic rate (Fig. 1) of 7.95 ,mol/ml per day throughout the culture period. The pH of the culture at the time of inoculation was 7.0 to 7.1; over a period of 4 days, the pH gradually dropped to 6.1 to 6.2. The pH remained at 6.1 for an additional 15 days before increasing to pH 6.35 to 6.4 for the remainder of the culture period. Measurements of the oxidation-reduction potential showed an initial decrease between 0 and 7 days from -350 to -400 mV, followed by a gradual rise to -150 mV. The rate of incorporation of [2-14C]acetate (1.4 pCi/120 ml of culture) into the cell fraction

7

11 6

sso

(4~~~~1

'. . 13i

_4 10

so

0

2

4

1

3

10 1 2 1 4 16 DA Y S

I'

20 22 24

-211

X

-3803

2'1 23''36

FIG. 1. Gas production and substrate utilization kinetics in the Barker-type acetate enrichment. Symbols:

0, methane production; U, acetate utilization; A, gas production; 0, sulfate disappearance; O, pH; and A,

Eh. Appropriate measurements are expressed as milliliters, micromoles, or micrograms per milliliter of culture.

VOL. 36, 1978

was arithmetic and occurred in parallel to methane production. Only 1.9% of the added label was incorporated. However, 93% of the label was converted to methane, as measured by the loss of label from the culture supernatant. After incubation of the enrichment for 24 h, methane was exclusively labeled (i.e., C02 was not labeled). Microbiology of the enrichment culture. The occurrence in high numbers of a Methanosarcina and its isolation from an acetate enrichment were first reported by Schnellen (Ph.D. thesis, Technische Hoogeschool, Delft, Holland, 1947). Microscopic examination of the present enrichment after dissolving the CaCO3 by acid hydrolysis (Fig. 2) revealed many clusters of an organism also morphologically identified as a Methanosarcina. In addition, other small rods, vibrios, and occasionally cocci were always visible. When a sample of the enrichment was serially diluted and inoculated into enumeration media (see above), methane was the only recognizable gas (except C02) produced at low dilutions. Hydrogen was the only gas produced at high dilutions, and a mixture of H2 and CH4 gases was produced at dilutions of 1o-3 to 10-5. On extended incubation (3 months or longer), the presence or absence of CH4 or H2 was mutually exclusive. This indicated that there were probably fewer colony-forming units of methanogenic bacteria compared with nonmethanogenic bacteria present in the enrichment, although more complicated hypotheses involving interactions among these bacteria may be proposed as an explanation for these findings. When dilutions made in solid medium were compared with those in liquid medium (Fig. 3), methane was usually detected at higher dilutions in the liquid medium. In liquid medium, nutrients are freely available to the mixed population of bacteria introduced in the inoculum. If nutrient or other growth requirements (e.g., oxidation-reduction potential) produced by one organism were needed by another organism, the availability of such factors is favored in liquid rather than in the solid medium, where each bacterium is spatially fixed. Interactions of this type between two or more organisms may still occur in the higher liquid dilutions but not in solid media, where the same degree of interaction would be restricted by the fixed location of the organisms in the agar. Based on total colony counts, there were about 108 nonmethanogenic bacteria per ml. This number far exceeded the methanogenic count (Fig. 3). However, methane-producing bacteria were always detected at higher concentrations in liquid than in solid media. These findings support the hypothesis that nutritional and/or physical-chemical inter-

METHANOGENESIS FROM ACETATE

189

actions are important in the formation of methane extinction dilutions of mixed cultures. Experiments on the acetate enrichment. Various methanogenic substrates and inhibitors known to affect the metabolism of pure cultures of methanogens were tested for their effect on the methanogenesis of acetate by the enrichment culture. Organic nitrogen and carbon and energy sources were also examined because of the nutrient-deficient conditions of the acetatefed enrichment. (i) Effect of hydrogen. Because of the preferential metabolism of H2-CO2 by pure cultures of Methanosarcina isolated from the enrichment, hydrogen was tested as a substrate in the acetate enrichment by replacing the 100% N2 atmosphere with 100% H2 (CO2 was provided by addition of Na2CO3). Figure 4 shows that H2 had an inhibitory effect on the enrichment when introduced at various culture ages. Six pairs of enrichment vessels were inoculated at zero time. After 1, 2, 4, 7, and 9 days, the N2 atmosphere of each successive pair was replaced by 100% H2. Methane formation was monitored throughout the experiment (which lasted 19 days). In the 4-, 7-, and 9-day flasks, flushing with 100% H2 resulted in immediate and complete inhibition of methane production. In the 1- and 2-day cultures, a lag in methane production was followed by a decreased rate of methane formation, indicating that the formation of methane was only partially inhibited. Control vessels flushed with 100% N2 at the same tinme were not inhibited. Table 1 summarizes the percent inhibition observed in the 1- and 9-day flasks during the first 5 days after H2 outgassing. In the 9-dayold culture, inhibition after 3 days in the presence of H2 was 96.9% and increased to 100% after 5 and 7 days. No recovery from methane inhibition occurred in this or other later-stage cultures. In the 1-day-old culture, inhibition was 100% after 1 day and decreased to 62% after 5 days and 37% after 7 days as the culture gradually recovered. All of the methane ultimately produced in the early-stage cultures was essentially accounted for by the acetate added since the quantity of methane formed (data not shown in Fig. 4) was approximately the same as that of the unflushed control. In addition, little or no H2 was taken up even after 30 days (data not shown in Fig. 4) of incubation. The cultures were therefore more sensitive to H2 inhibition at later stages (4, 7, and 9 days) than at early stages (1 and 2 days) of growth. The inhibition apparently affected both the conversion of acetate to methane and carbon dioxide as well as the known ability of methanogens to convert H2 and C02 to methane and water. The inhibition by H2 led to the hypothesis

190

BARESI ET AL.

APPL. ENVIRON. MICROBIOL.

FIG. 2. Acetate enrichment. Phase-contrast photomicrograph, using a Zeiss photomicroscope. x500. (A) Untreated field; (B) same field after addition of I N HCI (note marker indicated by arrow).

191

METHANOGENESIS FROM ACETATE

VOL. 36, 1978

6 10

-

10

-

--2 °

Io

SOLID

-

5

10

Is

MEDIUM

AD

2'5

3lo

WE EKS

ID'

a

s1

tlOUID MEDIUM

DAYS

FIG. 3. Dilution studies on the acetate enrichment into liquid and solid medium: highest dilution showing detectable H2 or CH4 versus week first detected. Symbols: U, hydrogen; *, methane.

FIG. 4. Inhibition of acetate enrichment by 100% H2. On days 1 (E), 2 (A), 4 (A), 7 (), and 9 (0), the 100% N2 atmosphere was replaced by 100% H2; unaltered control (0).

that some soluble intermediate formed from H2

TABLE 1. Hydrogen inhibition of the acetate enrichmenta

might serve as an inhibitor of the methane-forming reaction. This was tested by introducing serial twofold dilutions of 2 ml of culture fluid from a hydrogen-inhibited 8-day-old culture directly into duplicate, uninhibited 8-day-old acetate enrichments (Table 2). In the control series containing 2 ml of culture fluid from an uninhibited culture or 2 ml of distilled water containing dissolved H2 or 02, methane production was not significantly inhibited. Inhibition in the experimental tubes occurred at two-, four-, and eightfold dilutions. This indicated that the inhibitory factor was soluble and was quickly diluted out. When an 8-day culture was examined immediately after flushing with 100% H2, no inhibition was found after the culture liquid was similarly diluted. However, since Fig. 4 shows no further increase in methane formation at 2 days after H2 outgassing, the inhibitor must have been formed

after outgassing. (ii) Effect of formate. Sensitivity to formate was also tested in the enrichment system since it was a possible soluble intermediate of H2-C02 metabolism. Sodium formate was tested at concentrations ranging from 6 to 240 mM (Table 3).

CCH.

Sampling time (h)

inhibition (%)

1-Day cultures

9-Day cultures

100 24 97 72 83 62 100 120 37 100 168 a These data were calculated from Fig. 4.

TABLE 2. Dilution of culture fluid from an H2inhibited 8-day enrichment Methane inhibition after 48 h (%) Dilution

(fold)

Uninhibited culture fluid

Hydro-

hibited culture fluid

water

water

66 21 6 0 0

0 0 0 0 0

1 0 0 0 0

2 1 0 0 0

H2-in-

soon

2 4 8 16 32

Oxy-

gen-

gen-

satu-

satu-

rated

rated

192

ENVIRON. MICROBIOL. BARESI SAPPL. ET AL.

TABLE 3. Formate inhibition of acetate enrichment Sodium formate

(MM)

2-Day culture H2 proCH4 inhi- duction bition (%) 20 ml)

(tmol/

6 30 60

Tr (stimulated 20%) 0.03 0 120 0.03 9 180 0.01 45 240 a At 2 h after formate addition.

9-Day culture H2production tion (%) 20 ml)

inCH4 hibi-

(pmol/

94

0.5a 0.6

95

0.5

92

95

0.5

bAt 6 h after formate addition.

A concentrated stock solution of 6 M sodium formate was injected at volumes sufficient to produce the final formate concentrations reported. Both early (2-day) and late (9-day) cultures were examined after 20 h of incubation in the presence of formate. The results showed that the older cultures were again more sensitive to inhibition than the younger cultures. The 9-dayold cultures were severely inhibited (92 to 95%) at all formate concentrations. Further incubation of these vessels for 1 month did not reverse the inhibition. A corresponding inhibition of methane production did not occur in the 2-dayold culture. At the highest formate concentration tested, only 45% inhibition occurred, and no inhibition was seen at the lower formate concentrations. However, continued incubation of the 2-day culture for 5 days did result in some inhibition at all formate concentrations, including the lowest concentration, 6 mM, which now exhibited an 18% inhibition. In no case was the degree of inhibition as great in the young (2-day) cultures as in the older (>9-day) cultures. H2 was detectable in all cultures inhibited with formate. However, the detectable quantity of H2 was low compared with the amount of formate added, and there was no stoichiometric relationship between the two (Table 3). (iii) Effects of other inhibitors. Because benzyl viologen and chloroform are known inhibitors of methanogenesis, they were examined for their effects on the acetate enrichment. Concentrated stock solutions of benzyl viologen or chloroform were injected to give the appropriate concentrations reported in Tables 4 and 5. Table 4 shows the effect of benzyl viologen on methane inhibition of 2- versus 9-day enrichment cultures. At 4.4 ,IM, benzyl viologen inhibited methane production by more than 90% regardless of culture age. Some inhibition occurred at the lower concentrations of benzyl viologen tested, but the amount of inhibition was probably not

TABLE 4. Benzyl viologen inhibition of the acetate enrichment after 48 h 9-Day culture

2-Day culture

Benzyl viologen

(gM)

CH4 inhibition (%)

H2 production

8 0.0088 8.6 0.044 11 0.088 6 0.44 2 0.88 96 4.4 8.8 a Trace after 14 days. bTrace after 2 days. 'Trace after 7 days.

(tLlmoll 20 ml)

CH4 inhibition (%)

H2 production (,umol/ 20 ml)

0 0 0

4 0 93 86

Oa Oa

OC

0 ob

0.07 0.08

TABLE 5. Chloroform inhibition of the acetate enrichment after 48 h

OHOIM

CHC13

2-Day culture (CH4 inhibition

9-Day culture H2 production tion

CH4 inhibi-

[%]) 0 2.5 0.67 9.5 12.3 3.35 22.4 59.8 6.7 87.2 13.4 98.3 99.5 20.1 97.5 26.8 a Trace after 48 h. b Amount determined after 22 h.

(umol/20 mlnin) 0 0 0 Oa

0.3

0.12b

significant. Thus, benzyl viologen inhibition does not appear to be affected by culture age in contrast to the striking dependency of inhibition by H2 or formate on culture age. Chloroforn inhibited methanogenesis in a manner similar to benzyl viologen (Table 5). Some inhibition occurred at the lower concentrations tested, but at 20.1 ,uM chloroform, more than 90% inhibition occurred regardless of culture age. (iv) Effect of glucose. Glucose was tested as a substrate in the acetate enrichment because

glucose-utilizing nonmethanogens were among the more numerous bacteria present (26). The addition of 0.1% glucose inhibited methanogenesis after an initial burst of gas production. Inhibition may have been related to a change in the bacterial population of the acetate enrichment. Further investigations on this observation were not pursued. (v) Yeast extract. The only acetate-using methanogen thus far successfully demonstrated and isolated from the enrichment is a Methanosarcina. In pure culture, this organism may be

193

VOL. 36, 1978

METHANOGENESIS FROM ACETATE

stimulated by the addition of complex organic nutrients such as yeast extract (L. Baresi, unpublished data). In the mixed culture of bacteria, which makes up the stable acetate enrichment, organic nutrients are limiting since acetate is the only organic compound added. To test the effect of complex organic nutrients on the acetate enrichment, yeast extract (YE) was added at concentrations ranging from 0.01 to 0.5%. This was done by injecting the appropriate volumes of a 10% YE stock solution into the enrichments. The incubation times varied for each YE con-

centration (Fig. 5) because of the differing times for the fernentation to go to completion. Figure 5 and Table 6 show the dramatic increase in the rate of methane production in medium containing 0.1 and 0.5% YE. The control and 0.01% YE had the same rate, indicating no stimnulation at the lower concentrations. The total amount of gas (i.e., methane) produced, with one exception, was approximately the same as the control regardless of the amount of YE added. The one exception in which the methane yield was slightly higher was probably due to variation in

2 2 o-

200-

130-

160-

140-

120-

X

100-

a

oo-

a '

|

40

20'

L

i

i4

'

i2

if

20

'

24

'

2'

3

oAV S

FIG. 5. Methane production rates by acetate enrichment with added YE at following concentrations: 0.5% (-), 0.1% (A), 0.05% (0), 0.01% (0), and 0.0% (0).

TABLE 6. Rate of methane production in acetate enrichment at varying YE concentrations Expt 3 Expt 2 Expt 1 YE (%

0.000 0.001 0.01 0.05 0.1 0.5

Rate (ml/day)

Total ml of gas produced

34.3

782

39.1 62.6 71 89.2

798 914 752 728

(ml/day)

Total ml of gas produced

(R/ay) (ml/day)

Total ml of gas produced

30.7

680

32.7

750

30.7 36.4

690 780

65.8

720

37.1 83.9

790 650

Rae

194

APPL. ENVIRON. MICROBIOL.

BARESI ET AL.

total culture volume (Table 6). Thus, these data show that although the rate of methanogenesis is dependent on higher YE concentrations, the quantity of methane is directly dependent on acetate and not on YE. In addition to YE, sludge supernatant from an active anaerobic digestor and 2-methylbutyric acid were both tested for stimulatory effects. Sludge supernatant was chosen because it may contain growth factors stimulatory to anaerobes (14), whereas 2-methylbutyric acid is a known growth factor for some pure cultures of methanogens. Neither of these factors exhibited any stimulatory effect on the enrichment. The effect of H2 inhibition on methanogenesis was reexamined in the acetate enrichment in the presence of YE. Enrichment cultures containing 0.1% YE were flushed with 100% H2 at 2 and 9 days after inoculation. After 24, 48, and 120 h, flasks were analyzed for methane. The results (Table 7) again showed that the older 9-day cultures were more sensitive to H2 inhibition than were the younger 2-day cultures. However, the sensitivity of these enrichment cultures to hydrogen inhibition with added YE (Table 7) may have differed slightly from the normal nutrient-deficient enrichments (Table 1). DISCUSSION Investigations on methanogenic enrichment cultures with calcium acetate as the sole organic carbon source in a mineral salts solution containing calcium carbonate as a substratum were first reported by Sohngen (Ph.D. thesis Technische Hoogeschool, Delft, Holland, 1906) and amplified in the studies of Barker (1) on methane-producing bacteria. These enrichment methods were modified in the present studies by adopting a routine transfer protocol in which the quantity of calcium carbonate added and the gaseous headspace were standardized (see above). Under these conditions the acetate enrichment culture was maintained indefinitely, and the predictable gas production rates indicated that a stable population of bacteria was present. The restrictive conditions ofthis enrichment limited the mixture of bacteria to those which depended on acetate directly or indirectly as a source of carbon and/or energy. TABLE 7. Hydrogen inhibition of the acetate enrichment containing 0.1% YE Time after H. outgassing

24 48 120

CH, inhibition (%) 2-Day culture 9-Day culture

10.7 20 19

95.7 95.6 84.9

Gas production in the acetate enrichment was linear with time (Fig. 1). For reasons discussed below, it was assumed that rates of gas production reflected the rates of growth. Arithmetic growth kinetics are evident in pure cultures when some essential nutrient factor is limiting (22). In the acetate enrichment, reasons for the observed arithmetic rate may be complicated by the presence of a mixed population of organisms; therefore, one simple limiting factor may not be responsible. Because of the probable nutrient interdependency of the organisms, a succession of limiting factors may exist, although the net result is still an arithmetic function. Substrates known to be utilized by pure cultures of methane bacteria and/or which may serve as additional electron donors for methanogenesis were tested to determine their effect on the arithmetic rate of gas production in these enrichment cultures. H2 was a likely energy source because it was produced by pure cultures of nonmethanogenic isolates obtained from this enrichment (25), and because it was rapidly utilized by pure cultures of a Methanosarcina also isolated from this enrichment (R. A. Mah, unpublished data). However, the addition of H2 by outgassing the N2 headspace with 100% H2 led to an inhibition of the acetate enrichment, as evidenced by a cessation of methane production. The degree of inhibition varied with the age of the enrichment culture. The less sensitive younger (9-day) cultures. The age-dependent inhibition by H2 and formate probably indicated a change in the composition of the enrichment population during normal growth of the culture. In fact, some culture evidence (L. Baresi, unpublished data) indicated a rapid growth and decline of sulfatereducing bacteria by the time the culture was 7 days old. As the culture grew, a succession of populations likely developed. Older cultures may contain organisms that produced soluble compounds from H2 and formate to inhibit methanogenesis. The effect of H2 on acetate conversion to methane and CO2 in the enrichment is more complicated than is the H2 effect on ethanol fermentation exhibited by Methanobacterium strain MOH and "S" organism reported by Bryant et al. (5), since H2 (and formate) inhibited methanogenesis completely in the present mixed culture. Yet, a pure culture of Methanosarcina, the only methanogenic organism that we have been able to isolate from the enrichment, utilized H2 and produced methane via CO2 reduction. Nonetheless, this latter reaction was also inoperative in our enrichment. Thus, the formation of methane from acetate in mixedculture fermentations is likely subject to complex regulatory reactions involving as yet unknown soluble factors produced by members of the mixed bacterial population. The age-independent inhibition by benzyl viologen and chloroform suggested that these latter two inhibitors may have acted directly on the methanogenic fraction of the mixed bacterial population present in the acetate enrichment. The concentration of benzyl viologen necessary for inhibition of the enrichment (4.4 ,uM) was approximately the same as that required for inhibition of methanogenesis (5.2 ,uM) in Methanobacillus omelianskii (26). In contrast to the observations on chloroform inhibition of methanogenesis reported by previous workers (3, 23), only trace amounts of H2 were detected, in agreement with previous findings by Chynoweth and Mah (9). This difference in quantity of H2 may be due to differences in the primary origin of methane (acetate versus CO2 reduction route). In the routine maintenance of the enrichment, acetate served as the only organic compound. The arithmetic growth rate and low cell yield of the culture probably reflected a nutrient-limited condition as discussed earlier. However, addition

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of glucose as an extra source of carbon and energy caused an inhibition of methanogenesis, perhaps because of an undesirable shift in the composition of the balanced bacterial population or end products formed from the metabolism of glucose. Sludge supernatant from an anaerobic digestor and 2-methylbutyrate were also not stimulatory. However, YE increased the rate of methanogenesis more than twofold, although the total yield of methane remained proportional to the amount of acetate added. Hence, organic or inorganic constituents of YE may satisfy certain nutrient restrictions of the enrichment and stimulate the rate of methanogenesis as well as the rate of growth. Under these conditions, the cell yield may be dependent on acetate solely as an energy source since the methane formed was equivalent to the acetate present. H2 outgassing of acetate enrichment cultures containing YE yielded results similar to the inhibition of the normal nutrient-limited enrichments. The similarity in age sensitivity to inhibition by H2 may reflect a population balance comparable to the routine enrichment. On the basis of these and other studies on the acetate enrichment, we propose a working hypothesis (Fig. 6) to describe the bacterial interactions involved during methanogenesis from acetate. The methanosarcinae occupy a key position since they may be the chief (if not sole) metabolizer of acetate in our enrichment. In this capacity, Methanosarcina serves as a "primary producer" of all substrates and growth factors required for the development of the nonmethanogenic associates in the enrichment. There is no evidence that energy is obtained from acetate by any route other than methanogenesis since addition of [2-'4C]acetate to the enrichment leads to recovery of label in "4CH4 with no 14CO2 produced. The nonmethanogenic members of the enrichment utilize organic carbon sources such as carbohydrates (glucose) and amino acids, which must ultimately originate from acetate metabolism by the Methanosarcina. In addition, growth factors such as vitamin B12 and possibly other B vitamins are also produced by the Methanosarcina to supply requirements of nutritionally fastidious organisms (25) known to be present. The interaction between the Methanosarcina and its dependent associates may vary from a nutritionally exacting requirement for a specific growth factor, as illustrated by the satellite bacterium (Fig. 6, no. 1), to the requirements of other undescribed fastidious heterotrophs (Fig. 6, no. 2), to the general requirement for organic carbon and energy sources as shown by Eubacterium limosum (F. Palmer, M. S. thesis, University of California at Los Angeles, Los Angeles, 1977) (Fig. 6, no. 3). (E. limosum

196

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BARESI ET AL.

AA's H2

GF Eh

Amino Acids H ydrogen Growth Factors Oxtidation- reduction pot en t ial Not Yet Determined

GF

AA's

3

N2 Eh

(ubacterium limosum NON- FASTIDIOUS HETE ROTROPH

FIG. 6. Model of acetate enrichment.

may utilize formate or H2 and CO2 to produce acetate [N. V. Hamlett and B. A. Blaylock, Abstr. Annu. Meet. Am. Soc. Microbiol. 1969, P207, p. 149], but neither of these compounds is present in substrate quantities in the enrichment.) Although the Methanosarcina grows in pure culture on acetate, it does so more slowly than on other methanogenic substrates such as methanol or H2-CO2 (13). In the presence of added organic nutrients, growth may be greatly stimulated. In the acetate enrichment, the nonmethanogenic bacteria may serve to maintain a low oxidation-reduction potential and to interact

syntrophically with the Methanosarcina by producing factors such as vitamins, amino acids, fatty acids, or even trace amounts of H2, all of which benefit the Methanosarcina by stimulating its growth. Addition of YE to supply nutrient factors to the normal nutrient-deficient enrichment culture stimulated methanogenesis from acetate. Thus, in addition to the regulatory factors which may exert an effect via H2 metabolism on the overall rate of methanogenesis, syntrophic interactions also play an important role in the vigor of this enrichment. A better understanding of the ecology of this methanogenic system and the growth stimulatory and ratecontrolling factors during methanogenesis from acetate is essential to improve rates of methanogenesis and decomposition of organic compounds in waste treatment systems and in furthering our knowledge of aquatic sedimentary (i.e., non-gastrointestinal) systems in general. ACKNOWLEDGMENTS We thank Daisy A. Kuhn for the phase-contrast photomicrographs of the acetate enrichment and Michael Smith and Thomas Glass for discussions of our results. We also thank Allyson Braun and Alan Miller for technical assistance.

This work was supported by a grant (4-445930-57008) from the Southern California Edison Co. LITERATURE CITED 1. Barker, H. A. 1936. Studies upon the methane-producing bacteria. Arch. Mikrobiol. 7:420-438. 2. Barker, H. A. 1943. Studies on the methane fermentation. VI. The influence of carbon dioxide concentration on the rate of carbon dioxide reduction by molecular hydrogen. Proc. Natl. Acad. Sci. U.S.A. 29:184-190. 3. Bauchop, T. 1967. Inhibition of rumen methanogenesis by methane analogues. J. Bacteriol. 94:171-175. 4. Belyaev, S. S., and M. V. Ivanov. 1975. A radioisotope method for determining the rate of bacterial methane formation. Microbiology (USSR) 44:140-142. 5. Bryant, M. P., E. A. Wolin, M. J. Wolin, and R. S. Wolfe. 1967. Methanobacillus omelianskii, a symbiotic association of two species of bacteria. Arch. Mikrobiol. 59:20-31. 6. Buswell, A. M., and F. W. Solo. 1948. The mechanism of methane fermentation. J. Am. Chem. Soc. 70:1778-1780. 7. Cappenberg, Th. E., and R. A. Prins. 1974. Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. III. Experiments with 14C-labeled substrates. Antonie van Leeuwenhoek J. Microbiol. Serol. 40:457-469. 8. Carlsson, J. 1973. Simplified gas chromatographic procedure for identification of bacterial metabolic products. Appl. Microbiol. 25:287-289. 9. Chynoweth, D. P., and R. A. Mah. 1971. Volatile acid formation in sludge digestion. Adv. Chem. Ser. 105:41-54. 10. Hungate, R. E. 1968. A roil tube method for cultivation of strict anaerobes, p. 117-132. In R. Norris and D. W. Ribbons (ed.), Methods in microbiology, Vol. 3B. Academic Press Inc., London. 11. Jeris, J. S., and P. L. McCarty. 1965. The biochemistry of the methane fermentation using C14 tracers. J. Water Poilut. Control Fed. 37:178-192. 12. Lawrence, A. W., and P. L. McCarty. 1969. Kinetics of methane fermentation in anaerobic treatment. J. Water Poilut. Control Fed. 41:1-17. 13. Mah, R. A., M. R. Smith, and L. Baresi. 1978. Studies on an acetate-fermenting strain of Methanosarcina. Appl. Environ. Microbiol. 35:1174-1184. 14. Mah, R. A., and C. Sussman. 1967. Microbiology of anaerobic sludge fermentation. I. Enumeration of the nonmethanogenic anaerogic bacteria. Appl. Microbiol.

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21. Stadtman, T. C., and H. A. Barker. 1951. Studies on the methane fermentation. IX. The origin of methane in the acetate and methanol fermentations by Methanosarcina. J. Bacteriol. 61:81-86. 22. Stanier, R. Y., M. Doudoroff, and J. Ingram. 1976. The microbiol world, p. 871. Prentice-Hall, Inc., Englewood Cliffs, N.J. 23. Thiel, P. G. 1969. The effect of methane analogues on methanogenesis in anaerobic digestion. Water Res. 3:215-223. 24. Thornsberry, L. W. 1971. Isothermal gas chromatographic separation of carbon dioxide, carbon oxysulfide, hydrogen sulfide, carbon disulfide, and sulfur dioxide. Anal. Chem. 43:452-453. 25. Ward, D. M., R. A. Mah, and I. R. Kaplan. 1978. Methanogenesis from acetate: a nonmethanogenic bacterium from an anaerobic acetate enrichment. Appl. Environ. Microbiol. 35:1185-1192. 26. Wolin, E. A., R. S. Wolfe, and M. J. Wolin. 1964. Viologen dye inhibition of methane formation by Methanobacillus omelianskii. J. Bacteriol. 87:993-998.