Amino acid fermentation and hydrogen transfer in mixed cultures *

Transcription

1 FEMS Microbiology Ecology 31 (1985) Published by Elsevier FEC SUMMARY Amino acid fermentation and hydrogen transfer in mixed cultures * (Amino acid fermentation; sulfate reduction; methanogenesis; acetate reduction) Henk J. Nanninga and Jan C. Gottschal Department of Microbiology. Unit~ersity of Groningen, Kerklaan NN Haren, The Netherlands. The degradation of the following amino acids was investigated in mixed cultures obtained from a waste water purification plant: aspartate, glutamate, serine, alanine, valine and leucine. Inhibition of sulfate-reducing bacteria in these mixed cultures by molybdate was found to inhibit amino acid degradation. The degradation of serine, alanine, valine and leucine was accelerated considerably by active sulfate reduction. The fermentation of aspartate and glutamate was not stimulated by the presence of sulfate-reducing bacteria. The existence of species which are able to ferment valine and leucine by coupling their oxidation to the reduction of exogenous acetate to butyrate was demonstrated. 2. INTRODUCTION Because of the ubiquitous occurrence of proteins in almost any ecosystem, the turnover of amino acids is a very important microbiological process. Current knowledge of the processes responsible for the anaerobic mineralization of amino * Dedicated to Professor N. Pfennig on the occasion of his both birthday. Received 3 July 1985 Revision received and accepted 11 July 1985 acids in anoxic environments is still far from complete. Although one or more fermentation pathways in isolated species have been described for most amino acids [1,2], the degradation routes in bacterial communities are largely unknown. It is to be expected that, as in the case of anaerobic breakdown of carbohydrates, alcohols and fatty acids [3-7], hydrogen transfer reactions could play a crucial role. Indeed, hydrogen has been shown to be involved in amino acid fermentation in several ways. In some cases, hydrogen or reducing equivalents required for hydrogenation reactions can be obtained by the uptake of molecular hydrogen, or may be generated from one amino acid for the reduction of another, in a Stickland reaction [1,8]. The fermentation of aspartate by Campylobacter sp. does not require hydrogen, but uptake of molecular hydrogen results in an increased growth rate, enhanced succinate production and diminished acetate production [9]. The degradation of amino acids such as glutamate, alanine, valine and leucine can yield molecular hydrogen [1A0]. Such hydrogen-yielding fermentations might in some cases be inhibited when hydrogen accumulates. For example, accumulation of hydrogen strongly interferes with the growth of Acidaminobacter hydrogenoformans when fermenting glutamate, and also affects the nature of the fermentation products [11]. Fermentation of alanine and a /85/$03.30,~ 1985 Federation of European Microbiological Societies

2 262 number of other amino acids by this organism was completely dependent on a low hydrogen partial pressure, and occurred only in mixed culture with hydrogenotrophs. On the other hand, Clostridium cochlearium, when fermenting glutamate, appears almost unaffected by elevated hydrogen partial pressures (H.J. Laanbroek and H.J. Nanninga, unpublished results). Since hydrogen is consumed in some amino acid fermentations and produced in others, interactions based on hydrogen transfer may develop between certain amino acid-fermenting species. In addition, other physiological types of bacteria which either donate or accept hydrogen may equally well be involved in such interspecies interactions. In particular, sulfate-reducing bacteria, of which several species possess a high affinity for hydrogen [12-15], may affect the degradation of amino acids. The first reactor of the purification plant of the AVEBE potato-starch factory (De Krim, The Netherlands) is an example of an environment in which amino acid degradation and sulfate reduction occur simultaneously [16]. In the present paper it will be shown that sulfate-reducing bacteria do indeed affect the fermentation of some amino acids to a considerable extent. Furthermore, evidence is presented indicating the importance of acetate as an electron sink in the fermentation of the amino acids leucine and valine. 3. MATERIALS AND METHODS 3.1. Organisms Desulfovibrio desulfuricans strain DK81 was isolated from the anaerobic purification plant of a potato-starch factory (AVEBE, De Krim, The Netherlands). Details concerning this strain will be published elsewhere (Nanninga and Gottschal, in preparation). Methanospirillum hungatei (DSM- 864) was obtained from the Deutsche Sammlung von Mikroorganismen, G6ttingen, F.R.G. Campylobacter sp. (DSM806) and Clostridium cochlearium were obtained from Dr. H.J. Laanbroek. These two species had previously been isolated from a similar anaerobic purification plant in a potato-starch factory [9,17] Media and cultivation The screw-cap bottles used for all batch culture experiments had a volume of 600 ml and initially contained 200 ml bicarbonate-buffered (ph 7) basal medium supplied with one or more carbon and energy sources. The gas phase initially present consisted of 80% N 2 and 20% CO 2, unless otherwise indicated. Gases were freed of oxygen by passage over hot copper filings. The bottles were incubated in a gyratory shaker at 120 rev./min and 35 C. The basal medium had the following composition (g/l): NaCI, 1.2; MgCI 2-6H20, 0.4; KC1, 0.3; CaCI 2.2H20, 0.15; NHaCI, 0.3; KH2PO 4, 0.2; NaHCO 3, 2.35; Na2S.9H20, 0.3; sodium acetate, 0.2; yeast extract, 0.025; casamino acids, 0.025; resazurin, 0.001; trace element solution (1 ml/l) and vitamin solution (1 ml/l). In some cases, in which amino acids served as the energy- and nitrogen-source, NHaCI was omitted. The trace element solution contained per 1:4 ml 12.5 N HC1; 2000 mg FeCI 2 4H20; 70 mg ZnC12; 100 mg MnCI 2-4H20; 62 mg H3BO3; 190 mg CoCI 2.6H20; 17 mg CuCI 2.2H20; 24 mg NiCI 2.6H20; 36 mg Na2MoQ-2H20; 39 mg Na2SeO 3-5H20 and 49 mg Na2WO 4 2H20. The vitamin solution contained (mg/1): biotin, 10; nicotinic acid, 100; B-amino benzoic acid, 50; thiamine 100; pantothenic acid, 50; pyridoxamine, 150 and cobalamin, 50. The following stock solutions were sterilized separately and added aseptically to the medium: vitamins; trace elements; NaHCO 3, 84 g/l; Na2S. 9H20, 100 g/l; sodium acetate, 82 g/l; yeast extract, 50 g/l; and casamino acids, 50 g/1. The vitamin and trace element solutions were filter-sterilized (0.2/.tm) and other components were autoclaved. The concentrated NaHCO 3 solution was sterilized under a CO 2 atmosphere. All substrates were added from sterile 1 M stock solutions which had been autoclaved. Only lactate stock solutions were filter-sterilized (0.2 ~m); valine and leucine were autoclaved together with their salts. Only L-forms of lactate and amino acids were used. The media were inoculated with 5 ml fluid from the first reactor of an anaerobic purification plant of the potato-starch factory [16], unless otherwise stated. In some cases, the batch cultures were also

3 inoculated with a 5-ml suspension of actively growing cultures of D. desulfuricans DKB1 or M. hungatei, with absorbances at 660 nm of and , respectively. Both species were pre-grown in basal medium under an atmosphere of 80% H 2 and 20% CO 2. The medium of D. desulfuricans DK81 contained 2.5 mm lactate and 10 mm sulfate, and in the medium of M. hungatei the amount of yeast extract was raised to 0.25 g/ Chemical analyses Both volatile and non-volatile short chain fatty acids were analysed with a Packard 437 gas chromatograph equipped with a flame ionisation detector, connected to a Packard 604 integrator. Glass columns (2 m long; 2 mm inner diameter) were filled with Chromosorb WAW, mesh , coated with 10% SP-1000 and 3% H~PO 4 (Chrompack Nederland B.V., Middelburg, The Netherlands). The flow rate of the carrier gas (nitrogen) was 50 ml/min. The temperature at the injection port, column and detector was 175, 120 and 175 C, respectively. The flow rates of hydrogen and air were 30 and 200 ml/min, respectively. Volatile fatty acids were determined after diethyl ether extraction [18]. Isovalerate (3-methylbutyrate) was not separated from 2-methylbutyrate. In the results, isovalerate may therefore represent either of these compounds. Lactate and succinate were determined after methylation and chloroform extraction, using malonic acid as internal standard [18]. Hydrogen and methane in the gas phase of the cultures were analysed with a Pye Unicam 104 gas chromatograph by thermal conductivity [18]. The detection limits for hydrogen and methane were 0.001% and 0.01% (v/v), respectively. Alcohols were analyzed with a Packard 427 gas chromatograph [19]. Sulfide was determined by the method of Pachmayr [20]. Formate was analyzed according to Lang and Lang [21]. For ammonium a colorimetric assay based on the Berthelot reaction was used [22] Miscellaneous Absorbances of cultures were measured in a 1-cm cuvette in a Vitatron colorimeter at 660 nm. 263 Lactate was obtained from Fluka (Buchs, Switzerland). All other chemicals were of analytical grade. 4. RESULTS 4.1. Sulfate reduction and amino acid fermentation When untreated samples from the first reactor of the purification plant were incubated anaerobically at 35 C for periods of up to 80 h, the accumulation of ammonium, hydrogen sulfide and fatty acids was observed, indicating the fermentation of proteins and amino acids. Addition of sodium molybdate (5-20 mm) to the samples almost completely prevented the accumulation of ammonium and hydrogen sulfide. Furthermore, molecular hydrogen accumulated in these samples. Since molybdate is a known inhibitor of sulfate reduction [13,23-26], this result was taken as a preliminary indication that a considerable amount of the amino acids was degraded via hydrogenproducing reactions, which depended on the hydrogen-scavenging capacity of the sulfidogens. The number and activity of methanogens in these reactor samples was very low [16] and apparently insufficient to prevent hydrogen accumulation. A possible alternative explanation for the observed effect of molybdate would be a direct inhibition of the amino acid-fermenting bacteria. Therefore experiments were performed with pure cultures of a Campylobacter sp., fermenting aspartate, and two glutamate-fermenting species, Clostridium cochlearium and an as yet unidentified strain isolated from the reactor fluid, producing ammonium, acetate, propionate and CO~ from glutamate (Nanninga, Drent and Gottschal, in preparation). Sodium molybdate (final concentration 20 ram) was added to batch cultures of these species containing mm sulfide, during the mid-exponential growth phase. The growth rate of the two glutamate-fermenting species was not affected by this addition. The Campylobacter sp. reacted differently: aspartate fermentation continued (although at a linear rate) but the increase in absorbance ceased abruptly. Another species, Acidaminobacter hydrogenoformans, was unaffected by molybdate (20 mm) while fermenting glutamate (A.J.M. Stams, personal communication).

4 264 y- 20 f 15 z 5 o ' ' 1'0 ' 20 ' 3to t.'o ' so 6'0 ' time (h) Fig. 1. Release of ammoniun during the fermentation of aspartate in a batch culture containing 20 mm aspartate (0 O) and in a batch culture supplied with aspartate (20 mm), sulfate (10 mm) and D. desulfuricans DK8I (0 0) Fermentation of aspartate and glutamate The data in Figs. 1 and 2 show the results of the fermentation of aspartate and glutamate in 200-ml batch cultures (basal medium) inoculated with 5 ml reactor fluid in the presence and absence of sulfate. The fermentation rate of aspartate was similar under both conditions, but the presence of sulfate and D. desulfuricans DK81 resulted in a prolonged lag phase (Fig. 1). After depletion of all aspartate, acetate ( mm) and propionate ( mm) were the only acid end products present. Furthermore, succinate was detected transtently (up to 5 ram) during the active growth 2O I: gl0-4" "I- Z s 0 'k + - S~ D desutf) i i 11 i i i i i i ; SI i t,0 0 time (h) Fig. 2. Release of ammonium during the fermentation of glutamate in a batch culture containing 20 mm glutamate (0 O) and in a batch culture supplied with glutamate (20 mm), sulfate (10 ram) and D. desulfuricans DK81 (o o). phase. The partial hydrogen pressure (ph2) in the gas phase of both cultures always remained below 10 4 atm. The presence of sulfate-reducing bacteria did not appear to affect the rate of glutamate fermentation (Fig. 2). The fatty acids detected after glutamate depletion ( mm acetate, mm propionate and mm butyrate) were similar in both cultures and the ph 2 in the gas phase of both cultures remained below 10-4 atm. In both the aspartate- and glutamate-amended cultures, the presence of sulfate-reducing bacteria and sulfate did not result in significant sulfate reduction. Less than 0.6 mmol sulfate per 1 was reduced to hydrogen sulfide in these cultures. 2O 15 S 0,~0 2 i 10_3 ~-, io I i I I I I I i I I I I I I i i i i pch~ (*SO~*D desutf ) i d i i i ~ i L 30 t~ time (h) Fig. 3.The ph 2, pch 4 and the release of ammonium during the fermentation of serine in a batch culture containing 20 mm serine (O O) and in a batch culture supplied with serine (20 mm), sulfate (10 mm) and D. desulfuricans DK81 (o o).

5 Fermentation of serine and alanine A shorter lag phase and an accelerated fermentation of serine were observed in the culture to which sulfate and D. desulfuricans DK81 had been added (Fig. 3). The specific rate of increase of the ammonium concentration of this culture amounted to 0.19/h, vs. 0.09/h in the culture without added sulfate. The fatty acids detected in this latter culture after complete degradation of serine were formate, acetate and butyrate (4.8, 10.1 and 3.1 mm, respectively). Furthermore, hydrogen accumulated in the gas phase (Fig. 3). In the presence of sulfate, the same acid end products were formed (3.5 mm formate, 13.6 mm acetate and 3.6 mm butyrate), but hydrogen did not accumulate and up to 3 mm of the sulfate was reduced to hydrogen sulfide. The fermentation of alanine in a sulfateamended culture proceeded with a shorter lag phase and was stimulated to the same extent by active sulfate reduction as observed with serine fermentation. Final concentrations of acetate, propionate and butyrate were 8.0, 8.0 and 0.5 mm, respectively. No hydrogen could be detected in the gas phase in the course of the experiment and 2 mm sulfide had been formed. In the absence of sulfate a slightly different fermentation pattern was observed: acetate, propionate and butyrate (6.0, 11.0 and 0.5 mm final concentration) accumulated, but hydrogen also accumulated to a partial pressure of atm Fermentation of leucine and oaline In the course of the fermentation of leucine two distinct stages were observed, separated by a lag phase (Fig. 4). Neither the presence of a methanogenic population (M. hungatei) nor of a sulfate-reducing population (D. desulfuricans DK81) affected the length of the initial, short (approx. 50 h) active fermentation period. The presence of either of these organisms, however, considerably shortened the length of the observed lag period to about half of that in their absence. Due to sulfate reduction (a total of 10 mmol/l) and methanogenesis (a total of 11 mmol/1) the ph 2 remained very low in these cultures. In the absence of these added hydrogen-consuming populations the initially high ph 2 (Fig. 4) began to fall rapidly only 120 h after the start of the experiment, apparently due to the gradual development of a methanogenic population present in low numbers in the inoculum. The resumption of leucine fermentation in this culture coincided with the moment the ph 2 fell below 10-4 atm. From those fatty acids formed during the degradation of leucine only isovalerate was quantitatively important ( mm final concentration). The fermentation of valine followed a pattern very similar to that described for leucine, but with two major differences. The lag phase in the culture E 10,-4" -t- Z E =- f._ o ~ /. ( H hungatei ) ( S T D desulf) i I I I, I I i p I ~ ~ pch~, (*M hungatei) pch& ~ H 2 \ \ pch/~ ( S0~*D desulf ) \\~ /- ph 2 (*S~ Ddesulf) ' ~ - - ph 2 {*Hhungatei) 300 ~0 500 time Fig. 4. The ph 2, pch 4 and the release of ammonium during the fermentation of leucine in 3 different media: 20 mm leucine (O O); 20 mm leucine+ 10 mm sulfate+ D. desulfuricans DK81 (O O); 20 mm leucine + M. hungatei (A I,). (h)

6 266 containing valine in the absence of added hydrogen-consuming populations was about 50 h shorter, and isobutyrate, not isovalerate, constituted the major acid end-product ( mm final concentration). The fermentation of leucine during the first stage, which proceeded despite a relatively high ph 2 (Fig. 4), was investigated further. It was found that in this period a small quantity of acetate initially present was reduced to butyrate. Depletion of acetate coincided with the end of the first fermentation state. In order to decide whether this reduction of acetate was accomplished by the leucine-fermenting species itself or by a second species reducing acetate with hydrogen produced by the leucine-fermenting species (hydrogen transfer), the following growth experiments were carried out. Samples (5 ml) from a mixed culture containing a leucine-degrading population were incubated in batch cultures with a basal medium (containing 2 mm acetate) to which the following additions were made: 20 ~ 15 E g ~ 10 o ~ s c o u A E 3O 20 (a) ammonium P i ; I i p p I I I \ / acetate (a) 20 mm leucine + 30 mm acetate (gas phase: 80% N2: 20% CO2) (b) 20 mm leucine +2 mm acetate (gas phase: 80% N2: 20% CO2) (c) 30 mm acetate (gas phase: 80% H2: 20% CO 2 ) In the cultures amended according to (a), leucine was fermented without a noticeable lag phase (Fig. 5). The ph 2 varied between 10 4 and 10-3 atm. After an incubation period of 90 h, 13.5 mmol/l leucine had been fermented and the amount of acetate had decreased by 10.6 mmol/1. The detected products were (mmol/l): ammonium, 13.5; isovalerate, 13.5; butyrate, 9.3; and methane, 3.1. Under the culture conditions as specified under (b) the fermentation of leucine and the formation of butyrate stopped abruptly when acetate was depleted (Fig. 5). Here, after 90 h, 4.0 mmol leucine was fermented per litre whereas acetate had been metabolized completely. The detected products were (mmol/1): ammonium, 4.0; isovalerate, 4.0; butyrate, 2.1; and methane, 1.3. In (b) - amrnonpum isova[er ai'e i I ~ I I P ~ I i P butyrate g ~a bufyrate i i i i i i i i ~ i h i 0 20 z~o firne (h) 1 0 acetale J i J i L i i i i i 20 / fime (h) Fig. 5. The fermentation of leucine in the presence of 32 mm acetate (a) and in the presence of 4.2 mm acetate (b).

7 the third experiment (c) no reduction of acetate to butyrate was observed, and methane was produced instead. The methane produced (approx. 20 mmol/l medium after 90 h incubation) was mainly formed from H2/CO2, as in this period the amount of acetate decreased with only 1 mmol/l. Thus, the fermentation of leucine does not necessarily depend on hydrogen-scavenging species such as most sulfidogens and methanogens, as long as sufficient acetate is available to serve as an electron acceptor. In parallel experiments with valine, the same principle was shown to apply. 5. DISCUSSION Molybdate is widely used as a specific inhibitor of sulfate reduction. This technique has proved particularly useful in studying the influence of hydrogen transfer on the flow of carbon in anaerobic ecosystems [7,13,24]. Indeed, in many cases, the inhibition of sulfate reduction was shown to result in a significantly altered pattern of anaerobic mineralization [13, 23-25, 27, 28]. However, in many cases, it has not been unequivocally established whether this was due to some extent to a direct effect of molybdate on the fermentative bacterial populations. Moreover, it may be so that molybdate-inhibited sulfate-reducing species are capable of metabolizing substrates potentially suitable for fermentative species. This may apply to some marine sediments, from which amino acidmetabolizing sulfate reducers have been isolated [29]. However, attempts to isolate similar sulfate reducers from the purification plant have failed (H.J. Nanninga, unpublished results). In the present study, we have presented some evidence that the growth of an aspartate-fermenting Campylobatter sp. was indeed hampered by the presence of molybdate. Whether this was due to toxic complex formation by the reaction of molybdate with free hydrogen sulfide [30] remains to be established. Although some glutamate-metabolizing species were unaffected by molybdate, results obtained with mixed cultures treated with this inhibitor must be interpreted with some caution. The above experiments with alanine, serine, valine and leucine revealed that the rate of fermen- 267 tation of these amino acids was significantly enhanced by the presence of hydrogen-consuming species such as D. desulfuricans DK81. In fact, fermentation of leucine and valine hardly proceeded in the presence of hydrogen at partial pressures > 10-4 atm in the absence of acetate. Once the hydrogen pressure had reached values of 10 4 atm, both leucine and valine were readily fermented, apparently in accordance with the following overall equations: ieucine + 3H20 ---, isovalerate- +NH~ + HCOf + 2H: + H + (1) valine + 3H20 ~ isobutyrate- +NH~- + HCO; + 2H 2 + H + (2) Eqns. (1) and (2) also agree with results obtained with other crude or defined mixed cultures fermenting leucine and valine [10,11,31]. Moreover, it has been reported that in samples from an anaerobic digestor, the degradation of leucine and valine was accelerated by an active methanogenic population [10]. The conversion of leucine according to Eqn. (1) is thermodynamically favourable (32). For example, the AG' of this reaction at a ph 2 of 1, 10-2 and 10 4 atm is -14.2, and k J/reaction, respectively. These values are valid under the following conditions: 25 C, 15 mm leucine, 15 mm isovalerate, 20 mm NH4 ~, and 30 mm bicarbonate. Given these data, one would not at first sight expect such a strong dependence of leucine fermentation on ph2. This apparent contradiction may be explained by assuming the following conversions to be involved [311: leucine + H20 --, a-ketoisocaproate + NH 3 + Hz a-ketoisocaproate + CoA isovaleryl-coa + CO 2 + H 2 isovaleryl-coa + ADP + Pi --, isovalerate + ATP + CoA In this sequence of reactions, the first step is likely to depend strongly on a low ph 2 in analogy with the conversion of other amino acids to the corresponding a-ketoacid and H2, e.g., alanine to pyruvate or glutamate to a-ketoglutarate, as discussed in relation to the mixed culture fermenta-

8 268 tions carried out by Acidaminobacter hydrogenoforroans [11]. Apparently, both sulfate-reducing bacteria and methanogens were capable of maintaining a sufficiently low ph 2 to favour leucine fermentation. Interestingly, there appears to be yet another way to ferment leucine, namely by coupling its oxidation to the reduction of exogenous acetate (Fig. 5). The stoichiometry of this reaction appears to be: 2 acetate-+ H++ 2H 2 ~ butyrate + 2H20 (3) The AG' of this reaction at ph and 10 4 atm is and k J/reaction, respectively, at 25 C and with acetate and butyrate concentrations of 10 mm (calculated from data in [32]). The thermodynamics of this reaction imply that as long as acetate is available, leucine fermentation can proceed in the presence of relatively high hydrogen partial pressures (see also Fig. 4). The reduction of acetate to butyrate is not really a new phenomenon. Clostridium tyrobutyricum and Butyribacterium rettgeri (= Eubacterium limosum) are examples of bacteria fermenting lactate only in the presence of exogenous acetate [33]. Clostridium kluyveri also requires acetate when metabolizing ethanol [33,34]. Furthermore, Eubacterium limosum required external acetate when fermenting valine and isoleucine [35]. In this latter organism, acetate was probably also used as an electron acceptor, as some butyrate was detected in these cultures. But to our knowledge, the leucine fermentation reported here is the first example demonstrating clearly the acetate reduction during an amino acid fermentation (Fig. 5). The conversion of 1 mol of leucine yields 1 mol isovalerate + 2 mol H 2. This amount of H 2 can be used to reduce 2 mol of acetate to 1 mol of butyrate. The experiment shown in Fig. 5 does not exactly fit this stoichiometry, as about 30% of the reducing equivalents produced were metabolized by methanogens. At present it is very difficult to estimate the importance of acetate as an electron acceptor in anaerobic habitats. It would seem rather likely that particularly in sulfate-depleted, low-ph environments, where neither sulfidogens nor methanogens thrive, acetate would be a fairly ob- vious alternative electron acceptor for certain fermentations. Finally, although the results described above were obtained with mixed cultures, we have isolated in pure culture a leucine-fermenting species which possesses exactly those fermentative capabilities described above (Nanninga and Gottschal, in preparation). This strain, a rod-shaped, strictly anaerobic, endospore-forming organism unable to perform dissimilatory sulfate reduction, was tentatively identified as a Clostridium sp. ACKNOWLEDGEMENTS Thanks are due to T.A. Hansen and H. Veldkamp for valuable discussions and for reading the manuscript. REFERENCES [1 ] Barker, H.A. (1961) Fermentation of nitrogenous organic compounds, in The Bacteria (Gunsalus, I.C. and Stannier, R.Y., Eds.), Vol. 2, pp Academic Press, London. [2] Barker, H.A. (1981) Amino acid degradation by anaerobic bacteria. Ann. Rev. Biochem. 50, [3] Wolin M.J. (1982) Hydrogen transfer in microbial communities, in Microbial Interactions and Communities (Bull, A.T. and Slater, J.H., Eds.), Vol. 1, pp Academic Press, London. [4] Archer, D.B. (1983) The microbial basis of process control in methanogenic fermentation of soluble wastes. Enzyme Microbial Technol. 5, [5] Gujer, W. and Zehnder, A.J.B. (1983) Conversion processes in anaerobic digestion. Water Sci. Technol. 15, [6] Zehnder, A.J.B. and Koch, M.E. (1983) Thermodynamic and kinetic interactions of the final steps in anaerobic digestion. Proc. Eur. Symp. on Anaerobic Waste Water Treatment, Noordwijkerhout, The Netherlands, pp [7] Nedwell, D.B. (1984) The input and mineralization of organic carbon in anaerobic aquatic sediments. Adv. Microbial Ecol. 7, [8] Nisman, B. (1954) The Stickland reaction. Bact. Rev. 1, [9] Laanbroek, H.J., Stal, L.J. and Veldkamp, H. (1978) Utilization of hydrogen and formate by Campylobacter sp. under aerobic and anaerobic conditions. At'ch. Microbiol. 119, " [10] Nagase, M. and Matsuo, T. (1982) Interactions between amino-acid-degrading bacteria and methanogenic bacteria in anaerobic digestion. Biotechnol. Bioeng. 24,

9 269 [11] Stams, A.J.M. and Hansen, T.A. (1984) Fermentation of glutamate and other compounds by Acidaminobacter hydrogenoformans gen. nov., sp. nov., an obligate anaerobe isolated from black mud. Studies with pure cultures and mixed cultures with sulfate-reducing and methanogenic bacteria. Arch. Microbiol. 137, [12] Kristjansson, J.K., Sch/Snheit, P. and Thauer, R.K. (1982) Different K~ values for hydrogen of methanogenic bacteria and sulfate-reducing bacteria: an explanation for the apparent inhibition of methanogens by sulfate. Arch. Microbiol. 131, [13] Lovley, D.R., Dwyer, D.F. and Klug, M.J. (1982) Kinetic analysis of competition between sulfate reducers and methanogens for hydrogen in sediments. Appl. Env. Microbiol. 43, [14] Robinson, J.A. and Tiedje, J.M. (1984) Competition between sulfate-reducing and methanogenic bacteria for H 2 under resting and growing conditions. Arch. Microbiol. 137, [15] Widdel, F. and Pfennig N. (1984) Dissimilatory sulfate- or sulfur-reducing bacteria, In Bergey's Manual of Systematic Bacteriology (Krieg, N.R. and Holt, J.G., Eds.), Vol. 1, pp Williams and Wilkins, Baltimore. [16] Nanninga, H.J. and Gottschal, J.C. (1985) Anaerobic purification of waste water from a potato-starch producing factory. Water Research, in press. [17] Laanbroek, H.J., Smit, A.J., Klein, Klein Nulend, G. and Veldkamp H. (1979) Competition for L-glutamate between specialised and versatile Clostridium species. Arch. Microbiol. 120, [18] Laanbroek, H.J., Geerligs, H.J., Peijnenburg, A.A.C.M. and Siesling, J. (1983) Competition for L-lactate between Desulfovibrio, Veillonella, and Acetobacterium species isolated from anaerobic intertidal sediments. Microb. Ecol. 9, (19] Laanbroek, H.J., Geerligs, H.J., Sijtsma, L. and Veldkamp H. (1984) Competition for sulfate and ethanol among Desulfobacter, Desulfobulbus, and Desulfovibrio species isolated from intertidal sediments. Appl. Env. Microbiol. 47, [20] Tr~per, H.G. and Schlegel, H.G. (1964) Sulphur metabolism in Thiorhodaceae, 1. Quantitative measurements of growing cells of Chromatium okenii. Antonie van Leeuwenhoek J. Microbiol. Serol. 30, [21] Lang, E., Lang, H. (1972) Spezifische Farbreaktion zum direkten Nachweis der Ameisens~.ure. Z. Anal. Chem. 260, [22] Richterich, R. (1965) Klinische Chemie. Akademische Verlagsgesellschaft, Frankfurt, FRG. [23] Smith, R.L. and Klug, M.J. (1981) Electron donors utilized by sulfate-reducing bacteria in eutrophic lake sediments. Appl. Env. Microbiol. 42, [24] Sorensen, J., Christensen, D. and Jorgensen, B.B. (1981) Volatile fatty acids and hydrogen as substrates for sulfate reducing bacteria in anaerobic marine sediments. Appl. Env. Microbiol. 42, [25] Banat, I.M., Lindstrom, E.B., Nedwell, D.B. and Balba, M.T. (1981) Evidence for coexistence of two distinct functional groups of sulfate-reducing bacteria in salt marsh sediment. Appl. Env. Microbiol. 42, [26] Postgate, J.R. (1984) The Sulfate-Reducing Bacteria, 2nd ed. Cambridge University Press, London. [27] Lovley, D.D. and Klug, M.J. (1983) Sulfate reducers can outcompete methanogens at freshwater sulfate concentrations. Appl. Env. Microbiol. 45, [28] Winfrey, M.R. and Ward, D.M. (1983) Substrates for sulfate reduction and methane production in intertidal sediments. Appl. Env. Microbiol. 45, [29] Stams, A.J.M., Hansen, T.A. and Skyring, G.W. (1985) Utilization of amino acids as energy substrates by two marine Desulfouibrio strains. FEMS Microbiol. Ecol. 31, [30] Wolin, M.J. and Miller, T.L. (1980) Molybdate and sulfide inhibit H 2 and increase formate production from glucose by Ruminococcus albus. Arch. Microbiol. 124, [31] Russell, J.B. and Jeraci, J.L (1984) Effect of carbon monoxide on fermentation of fiber, starch, and amino acids by mixed rumen microorganisms in vitro. Appl. Env. Microbiol. 48, [32] Thauer, R.K., Jungermann K. and Decker, K. (1977) Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41, [33] Barker, H.A. (1957) Bacterial Fermentations, Wiley, New York. [34] Kenealy, W.R. and Waselefsky, D.M. (1985) Studies on the substrate range of CIostridium kh(vueri; the use of propanol and succinate. Arch. Microbiol. 141, [35] Genthner, B.R.S., Davies, C.L. and Bryant, M.P. (1981) Features of rumen and sewage sludge strains of Eubacteriurn limosum, a methanol- and Hz-COz-utilizing species. Appl. Env. Microbiol. 42,