2,3-Butanediol production from starch by engineered Klebsiella pneumoniae G31-A

2,3-Butanediol (2,3-BD) is an organic compound, which is widely used as a fuel and fuel additive and applied in chemical, food, and pharmaceutical industries. Contemporary strategies for its economic synthesis include the development of microbial technologies that use starch as cheap and renewable feedstock. The present work encompasses the metabolic engineering of the excellent 2,3-BD producer Klebsiella pneumoniae G31. In order to perform direct starch conversion into 2,3-BD, the amyL gene encoding quite active, liquefying α-amylase in Bacillus licheniformis was cloned under lac promoter control in the recombinant K. pneumoniae G31-A. The enhanced extracellular over-expression of amyL led to the highest extracellular amylase activity (68 U/ml) ever detected in Klebsiella. The recombinant strain was capable of simultaneous saccharification and fermentation (SSF) of potato starch to 2,3-BD. In SSF batch process by the use of 200 g/l starch, the amount of total diols produced was 60.9 g/l (53.8 g/l 2,3-BD and 7.1 g/l acetoin), corresponding to 0.31 g/g conversion rate. The presented results are the first to show successful starch conversion to 2,3-BD by K. pneumoniae in a one-step process.     

2,3-Butanediol production from xylose and other hemicellulosic components by Bacillus polymyxa

In the xylose fermentation of Bacillus polymyxa strain 9035, best 2,3-butanediol yields were obtained with 1.0 % yeast extract, 4–6 % xylose, shaking at 125 rpm and incubation at 30°C. Under these conditions, mannose, galactose, L-arabinose, cellobiose, starch and glucose were readily metabolized and yielded significant amounts of diol. Diol production from xylan was also demonstrated. In addition, the screening of a number of B. polymyxa strains on xylose revealed that only strains 9031-1 and 9035 used xylose extensively and produced significant amounts of diol. The latter strain proved best under scaled-up conditions.NRCC #22775     

2,3-Butanediol production from glucose by Klebsiella pneumoniae in a cell recycle system

Kinetics of 2,3-butanediol production by Klebsiella pneumoniae from glucose was studied in a cell recycle system with total recycle of biomass. Under these conditions productivity greater than batch or continuous system were obtained. However, when the cell concentration in the bioreactor built up to 40 g l⁻¹, the production of 2,3-butanediol started decreasing. The coefficient of mass transfer for oxygen decreased significantly and the viscosity increased rapidly after this cell concentration was reached. The increase in viscosity was partially due to production of polysaccharides. This appears at high cell concentration, due to severe oxygen limitation, when the organism may switch from 2,3-butanediol to polysaccharide production.     

2,3-Butanediol production by Enterobacter aerogenes in continuous culture: role of oxygen supply

Summary The influence of oxygen on growth and production of 2,3-butanediol and acetoin by Enterobacter aerogenes was studied in continuous culture. At all dilution rates (D) studied cell mass increased steadily with increasing oxygen uptake rate (OUR), hence the micro-aerobic cultivation was energy-limited. The biomass yield on oxygen increased with D which suggests that cells need more energy for maintenance functions at lower D. At each D an optimal OUR giving highest volumetric productivity for the sum of butanediol and acetoin was found. The optimal OUR increased with D. The occurrence of optimal OURs results from the various effects of O₂ on growth and specific productivity. The latter was found to be a linear function of the specific OUR irrespective of D. At optimal OUR the cells proved to have equal specific OURs and equal specific productivities representing a fixed relationship between fermentative and respiratory metabolism. The product yield based on glucose and corrected for biomass formation was 80%. A product concentration as high as 43 g/l was obtained at D =0.1 h^–1 while the volumetric productivity was the highest at D =0.28 h^–1 (5.6 g/l and hour). The findings further indicate that growth and product generation are obviously non-associated phenomena. Hence, high productivities may be achievable by cell recycling and cell immobilisation systems. Offprint requests to: W.-D. Deckwer     

代谢工程改造多粘芽孢杆菌及合成光学纯(R,R)-2,3-丁二醇

(R,R)-2,3-BD是一种重要的四碳平台化合物,在液晶材料、高附加值手性化合物,尤其是不对称合成光学纯药物等方面有天然优势.将来源于多粘芽孢杆菌(Paenibacillus polymyxa)DSM 365的α-乙酰乳酸合成酶(α-acetolactate synthase)基因 alsS、α-乙酰乳酸脱羧酶(α-acetolactate decarboxylase)基因alsD和(R,R)-2,3-丁二醇脱氢酶(2,3-butanediol dehydrogenase)基因R,R-bdh与表达载体pMA5连接,导入多粘芽孢杆菌P.polymyxa DSM 365中加强(R,R)-2,3-丁二醇的主代谢途径,构建可高效合成(R,R)-2,3-丁二醇的多粘芽孢杆菌工程菌株DM-5.利用工程菌株DM-5补料分批发酵60 h,(R,R)-2,3-丁二醇产量达54.91 g/L,得率为0.52 g/g,生产强度为0.92 g·L-1·h-1,与野生菌株相比(R,R)-2,3-丁二醇产量增加19.66％,且副产物甲醇浓度不变,乙醇、乙偶姻积累下降.本研究结果表明,在多粘芽孢杆菌中过量表达关键基因alsS、alsD和R,R-bdh 能够显著提高(R,R)-2,3-丁二醇的产量和生产强度,为多粘芽孢杆菌的代谢工程改造和工业化生产(R,R)-2,3-丁二醇提供参考.     

2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii

The acetogenic bacterium Acetobacterium woodii is able to reduce CO₂ to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD⁺-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO₂ to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.     

Antifungal and Root Surface Colonization Properties of GFP-Tagged Paenibacillus brasilensis PB177

Summary This study evaluates the potential of Paenibacillus brasilensis strain PB177 to inhibit phytopathogenic fungi commonly causing maize diseases and to colonize maize plants. In vitro assays demonstrated antagonistic activity against the fungal pathogens, Fusarium moniliforme and Diplodia macrospora. The PB177 strain was tagged with the gfp gene, encoding the green fluorescent protein (GFP) and GFP-tagged bacteria were detected attached to maize roots by stereo- and confocal microscopy. The GFP-tagged bacteria were also used to treat maize seeds before challenging the seeds with two phytopathogenic fungi. The results demonstrated that the bacterial cells are mobilized to the maize roots in the presence of the fungal pathogens. The ability of P. brasilensis PB177 to inhibit fungal growth in vitro and its capability of colonization of maize roots in vivo suggest a potential application of this strain as a biological control agent. This is the first report on the successful introduction of the GFP marker gene into a P. brasilensis strain, enabling the direct observation of these promising plant growth promoting bacteria on maize roots in situ.     

2,3-Butanediol production by Enterobacter aerogenes: selection of the optimal conditions and application to food industry residues

Optimum values of temperature, pH, and starting substrate concentration are experimentally determined for 2,3-butanediol production by Enterobacter aerogenes through three set of batch fermentations of synthetic glucose solutions. The results of tests carried out at variable temperature show an optimum of 39 °C and are used to estimate, for both fermentation and thermal inactivation, the activation enthalpies (7.19 and 23.6 kJ mol^у) and the related entropies (т.32 and т.27 kJ mol^у K^у). An optimum pH value of 6.0 is evidenced from batch runs at variable pH, whose results are also used to make reasonable hypotheses on the reaction controlling the metabolic pathway which leads to butanediol. The fermentability of different food industry wastes, namely starch hydrolysate, both raw and decoloured molasses, and whey, is finally checked.     

Separation of Racemic frommeso-2,3-Butanediol

2,3-Butanediol containing less than 3% of themesoform has been obtained from samples containing up to 50% of themesoform. The diacetate was obtained by esterification with acetic anhydride in the presence of traces of sulfuric acid as a catalyst and was then purified. When the diacetate was held at 4°C, crystals of racemic 2,3-butanediol diacetate formed, and these were separated by filtration. The diacetate was then transformed back to 2,3-butanediol by transesterification with methanol in the presence of sodium methylate as a catalyst. The resulting 2,3-butanediol contained less than 3% of themesoform. For an original batch of 2,3-butanediol containing 50%dland 50%meso,this method can isolate up to 70% of the racemate content. If the original 2,3-butanediol contains too muchmesoform, racemic 2,3-butanediol diacetate does not crystallize, but 2,3-butanediol containing up to 60% of themesoform can be enriched up to 70% racemate by distillation.     

Production of an antimicrobial substance against Cryptococcus neoformans by Paenibacillus brasilensis Sa3 isolated from the rhizosphere of Kalanchoe brasiliensis

An antifungal substance produced by Paenibacillus brasilensis strain Sa3 was preliminary characterized and showed to be stable after treatment with different enzymes and organic solvents and at a wide range of pH, and presented a molecular weight between 3 and 10 kDa. In vitro antagonism of this strain towards Cryptococcus neoformans was investigated by optical and electronic microscopic analyses and a fungicidal effect on C. neoformans was observed. Ultrastructural analysis showed intense changes on the fungus when it was paired cultured with strain Sa3, mainly the detachment of the capsule from the cell wall and the presence of altered organelles in the cytoplasm. This novel antifungal substance produced by P. brasilensis Sa3 may represent a new insight in antifungal therapy mainly against emergent fungi. Also, prospective studies on rhizobacteria of plants as Kalanchoe brasiliensis may offer a potential source for the discovery of bioactive compounds with medical value.     

Klebsiella pneumoniae CICC10011发酵产2,3-丁二醇的工艺研究

2,3-丁二醇应用广泛,是一种潜在的平台化合物,可以用于替代传统平台化合物-四碳烃。基于能源安全及绿色环保的需求,生物炼制制备2,3-丁二醇受到人们的青睐。与化学法相比,生物炼制制备2,3-丁二醇具有明显的优势。因此,开发合适的2,3-丁二醇发酵工艺是实验室研究的重点。针对菌种Klebsiella pneumoniae CICC10011,研究人员对菌种发酵产2,3-丁二醇的性质进行了初步考察,并通过控制不同的发酵条件,研究了pH、通空气量和转速在发酵过程中对菌种代谢的影响,从而确定了菌种发酵产2,3-丁二醇的工艺条件。发酵过程中,pH、通空气量以及转速均采用两段调控。在前12h菌种生长阶段,控制pH 6.8,通空气量1.0vvm,转速400r/min,转发酵之后控制发酵条件为pH 6.0,通空气量0.5vvm,转速300r/min。     

Enhancement of (R,R)-2,3-butanediol production from xylose by Paenibacillus polymyxa at elevated temperatures

Conversion of xylose to (R,R)-2,3-butanediol by Paenibacillus polymyxa in anaerobic batch and continuous cultures was increased by 39% and 52%, respectively, by increasing the growth temperatures from 30 to 39 °C. There was no effect of temperature when glucose was used as substrate. 39 mM (R,R)-2,3-butanediol, 65 mM ethanol, and 47 mM acetate were obtained from 100 mM xylose after 24 h batch culture at 39 °C. With 100 mM glucose and 100 mM xylose used together in a batch culture at 39 °C, all xylose was consumed after 24 h and 82 mM (R,R)-2,3-butanediol, 124 mM ethanol and 33 mM acetate were produced.     

Improved Production of 2,3-Butanediol in Bacillus amyloliquefaciens by Over-Expression of Glyceraldehyde-3-Phosphate Dehydrogenase and 2,3-butanediol Dehydrogenase

Background

Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.

Methodology/Principal Findings

In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD⁺ to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD⁺. In this study, to improve 2,3-BD production, we first over-produced NAD⁺-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.

Conclusions/Significance

Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.     

Medium optimization and proteome analysis of (R,R)-2,3-butanediol production by Paenibacillus polymyxa ATCC 12321

Paenibacillus polymyxa can produce the (R,R)-stereoisomer of 2,3-butanediol (2,3-BDL) which is industrially very useful. Two important factors affecting (R,R)-BDL production by P. polymyxa ATCC 12321, medium composition, and addition of acetic acid to the culture were investigated in this study with accompanying comparative proteomic analysis. For this purpose, a simple control strategy of O₂ supply was applied on the basis of an optimized basal medium: after a short period of batch cultivation with relatively high O₂ supply, the culture is switched into strong O₂ limitation, thereby promoting BDL formation. Three parallel fed-batch cultures starting from the same batch culture in an early stationary phase were then comparatively studied: the first one was running as control with the only change of O₂ supply; the second was, in addition, supplemented with 0.5 g/L yeast extract; and the third one was further added with 6 g/L acetate. Proteomic analyses of the three fed-batch cultures identified more than 86 proteins involved primarily in the central carbon metabolism, amino acid biosynthesis, energy metabolism, and stress responses. The examination of expression patterns of selected proteins, especially combined with fermentation data, gave valuable insights into the metabolic regulation of P. polymyxa under the different given conditions. Based on the proteomic analysis and further medium optimization studies, methionine was identified as one major growth-limiting factor in the basal medium and explains well the effect of yeast extract. Acetic acid was found to trigger the so far less studied acetone biosynthesis pathway in this organism. The latter is suggested in turn to enhance the switch from acidogenesis to solventogenesis. Thus, these findings extended our knowledge about BDL formation in P. polymyxa and provided useful information for further strain and process optimization.     

2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.     

Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing,Low-Alcohol Saccharomyces cerevisiae

Engineered Saccharomyces cerevisiae strains overexpressing GPD1, which codes for glycerol-3-phosphate dehydrogenase, and lacking the acetaldehyde dehydrogenase Ald6 display large-scale diversion of the carbon flux from ethanol toward glycerol without accumulating acetate. Although GPD1 ald6 strains have great potential for reducing the ethanol contents in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1, coding for native NADH-dependent Bdh1, and the engineered gene BDH1_221,222,223, coding for an NADPH-dependent Bdh1 enzyme with the amino acid changes 221 EIA 223 to 221 SRS 223, in a glycerol-overproducing wine yeast. We have shown that both the amount of Bdh1 and the NADH availability limit the 2,3-butanediol dehydrogenase reaction. During wine fermentation, however, the major limiting factor was the level of synthesis of Bdh1. Consistent with this finding, the overproduction of native or engineered Bdh1 made it possible to redirect 85 to 90% of the accumulated acetoin into 2,3-butanediol, a compound with neutral sensory characteristics. In addition, the production of diacetyl, a compound causing off-flavor in alcoholic beverages, whose production is increased in glycerol-overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to that in the control cells, was not further modified in BDH1 cells. Overall, rerouting carbons toward glycerol and 2,3-butanediol represents a new milestone in the engineering of a low-alcohol yeast with desirable organoleptic features, permitting the decrease of the ethanol contents in wines by up to 3°.A large number of quality wines produced by modern winemaking practices, which favor harvesting fully ripened grapes, frequently contain an excessive ethanol content. This tendency is observed in the majority of the world''s wine-producing areas, and reducing the alcohol levels in wines has become a major concern of the wine industry. Consequently, numerous attempts have been made to engineer Saccharomyces cerevisiae yeast strains with reduced ethanol yields, which would offer faster and less expensive biological alternatives to the current physical processes available for the production of low- and reduced-alcohol wines (29). The biological approaches used so far are all based on diverting sugar metabolism toward by-products other than ethanol by metabolic engineering strategies (7, 8, 18, 19, 21, 22, 30). However, these strategies have so far not satisfied the need to obtain a significant reduction in the ethanol yield without causing the accumulation of undesirable secondary products and/or without affecting yeast physiology. Among these various advances, an efficient strategy is based on the rerouting of the carbon flux toward the production of glycerol. This polyol is a relatively neutral compound from an olfactory perspective, and it is has been demonstrated previously to contribute positively to wine quality through enhanced sweetness and viscosity (27). In yeast, glycerol plays a major role as an osmolyte under osmotic stress conditions and also functions as an essential redox sink in the absence of oxygen, when the reoxidation of excess cytosolic NADH is required (1, 2, 42, 44, 46). This compound is formed by the reduction of dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase (encoded by GPD1 and GPD2), followed by dephosphorylation by glycerol-3-phosphatase, which exists as two isoforms: Gpp1 and Gpp2p (see Fig. ​Fig.1).1). By overexpressing GPD1 or GPD2, the production of glycerol has been greatly enhanced, making it possible to decrease the ethanol yield as the result of carbon diversion and reduced NADH availability for the alcohol dehydrogenase reaction. This strategy has been used previously to reduce the ethanol yields in wine and brewer''s yeast (4, 7, 23, 25, 26, 31). For wine, it was shown that these glycerol-overproducing strains have the potential to reduce the ethanol content by 1 to 2°. Nevertheless, major modifications in the production levels of other metabolites, in particular acetate and acetoin (23, 31), which are undesirable at high concentrations in wine, are generated. The production of acetate in glycerol-overproducing wine yeasts has been reduced to a normal level by the deletion of ALD6, coding for an acetaldehyde dehydrogenase (4, 30). The major problem of the accumulation of acetoin, which was shown to accumulate at several grams per liter in commercial GPD1 ald6 wine yeast strains (4), remains to be overcome. At usual concentrations in wine, which vary from undetectable levels to 80 mg/liter (32, 33, 40), this compound has no negative organoleptic influence. However, at concentrations higher than its threshold level (around 150 mg/liter [11]), acetoin can confer an unpleasant buttery flavor on wines. In contrast, the reduced form of acetoin, 2,3-butanediol (2,3-BD), has neutral sensory qualities (data not shown). It is found in wines at concentrations ranging from 0.2 to 3 g/liter (14, 41).Open in a separate windowFIG. 1.Schematic representation of metabolic pathways implicated in our design strategy for a low-alcohol yeast. GP, glycerol phosphatase, encoded by GPP1 and GPP2; GPDH, glycerol phosphate dehydrogenase, encoded by GPD1 and GPD2; PDC, pyruvate decarboxylase, encoded by PDC1, PDC5, and PDC6; ACDH, acetaldehyde dehydrogenase, encoded by ALD4, ALD5, and ALD6; ADH, alcohol dehydrogenase, encoded by ADH1; BDH, Bdh1, encoded by BDH1 (other BDHs exist; however, no other identified gene has been associated with BDH activity); ALS, acetolactate synthase, encoded by ILV2; DS, diacetyl synthetase; DR, diacetyl reductase; G3P, glycerol-3-phosphatase; DHAP, dihydroxyacetone phosphate; acetyl CoA, acetyl coenzyme A; TPP, thiamine PP_i.Bdh1, encoded by BDH1, is the only identified enzyme in yeast catalyzing the reduction of acetoin into 2,3-BD (12). This enzyme has strict stereospecificity for the OHs of carbons in R configuration and acts preferentially as a reductase rather than as a dehydrogenase (11, 12). It is essentially responsible for the formation of (2R,3R)-2,3-BD and part of meso-2,3-BD from (3R)-acetoin and (3S)-acetoin, respectively.The accumulation of acetoin in strains engineered for glycerol overproduction has been attributed to several factors (4). On one hand, it was assumed that the amount of Bdh1 is a rate-limiting factor in the conversion of acetoin into 2,3-BD. On the other hand, it is possible that the Bdh1 reaction is limited by the low level of available NADH since this coenzyme is preferentially used for glycerol synthesis in these strains.The aim of the present study was to investigate in detail the metabolic prerequisites for reducing accumulated acetoin in S. cerevisiae overproducing glycerol and exhibiting reduced acetate formation by promoting the conversion of acetoin into the compound 2,3-BD, which has neutral sensory characteristics. In this study, we first determined the role of Bdh1 in the reduction of acetoin into 2,3-BD during wine fermentation. Next, we studied the impact of the overproduction of NADH-dependent Bdh1 or an engineered NADPH-dependent form of Bdh1 in a model wine yeast, V5, overexpressing GPD1 and lacking ALD6 during fermentation in synthetic must with various sugar concentrations. The NADPH-dependent Bdh1 has been obtained previously by the replacement of three amino acids involved in the NADH binding domain, resulting in the complete reversal of the coenzyme specificity from NADH to NADPH (6). The effects on the growth and fermentation properties of the engineered strains and the levels of by-products and key aromatic compounds formed by the strains were determined.     

Methane production by the sulfate-reducing bacterium Desulfosarcina variabilis

The sulfate-reducing bacterium Desulfosarcina variabilis VKM B-1694 was found to produce up to 1.62 mumol methane per mg protein when grown on different substrates. The role of methanogenesis and the physicochemical factors determining this process in sulfate-reducing bacteria are discussed.