Lactate is mainly fermented to butyrate by human intestinal microfloras but inter-individual variation is evident

AIM: To assess the role of lactate as a precursor for butyrate biosynthesis in human colonic microflora. METHODS AND RESULTS: Three human faecal microfloras were incubated in vitro with media supplemented with 30 mmol l(-1) unenriched or 13C-enriched lactate. Lactate metabolism and short-chain fatty acid (SCFA) production were quantified. Lactate conversion to butyrate was investigated by gas chromatography-mass spectrometry and the pathways involved were identified by 13C nuclear magnetic resonance spectroscopy. All human faecal microfloras rapidly and completely fermented lactate, yielding approx. 19 mmol l(-1) total SCFAs. However, the SCFA composition varied markedly between microfloras. Butyrate was the main end-product for two microfloras but not for the third (60 and 61%vs 27% of the net concentration of SCFA produced respectively). The latter was typified by its ability to produce propionate as a major product (37%), and valerate (3%). 13C-Labelling showed that butyrate was produced through the acetyl-CoA pathway and that the three microfloras possessed significant differences in their metabolic pathways for lactate consumption. CONCLUSIONS: In contrast to the ruminal microflora, the human intestinal microflora can utilize both d- and l-lactate as precursors for butyrate synthesis. Inter-individual variation is found. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that the butyrogenic capability of colonic prebiotics could be related to lactate availability. These findings will direct the development of selection strategies for the isolation of new butyrate-producing bacteria among the lactate-utilizing bacteria present in the human intestinal microfloras.     

Increases in microbial nitrogen production and efficiency in vitro with three inhibitors of ruminal methanogenesis

It was hypothesized that the addition of crotonic acid or 3-butenoic acid would relieve constraints in digestibility observed when methane formation is inhibited by lumazine, propynoic acid, or ethyl 2-butynoate. In six incubations, one of the three methanogenesis inhibitors, at three different concentrations, was combined with either crotonic acid or 3-butenoic acid at two different concentrations. A mixture of buffer and ruminal fluid (4:1) was incubated with grass hay in Erlenmeyer flasks for 72 h. Initial concentrations were 0, 0.6, and 1.2 mmol/L for lumazine; 0, 2, and 4 mmol/L for propynoic acid; and 0, 4, and 8 mmol/L for ethyl 2-butynoate. 15Nitrogen (N) incorporation was used as a microbial marker. All three methanogenesis inhibitors decreased proteolysis. Propynoic acid and ethyl 2-butynoate at 8 mmol/L also decreased the digestibility of organic matter and neutral detergent fibre. However, all three inhibitors of methanogenesis increased the production of microbial N through an improvement of synthetic efficiency. Crotonic acid and 3-butenoic acid were generally ineffective in compensating digestibility decreases caused by the inhibitors of methanogenesis. It is of interest to elucidate the mechanisms by which these compounds increased the efficiency of microbial N production. Lumazine and the addition of low levels of ethyl 2-butynoate could potentially benefit animal production by lowering methane emissions, decreasing ruminal proteolysis, and increasing microbial N production without affecting organic matter digestibility.     

Effects of volatile fatty acids,ammonium and agitation on thermophilic methane production from biogas plant sludge in lab-scale experiments

The effects of different volatile fatty acids (VFA, formate, acetate, propionate and butyrate), ammonium (NH (4) (+)) and agitation on methane (CH(4)) production were determined in 120-mL serum bottles. We showed that the addition of formate did not lead to an inhibition of methanogenesis until a concentration of 120 mmol/L. A complete inhibition of methanogenesis was detected in variants containing 360 mmol/L formate or propionate until day 3 but the production started afterwards within next 2 days. This might indicate a kind of adaptation to the higher volatile fatty acid concentrations. Increasing NH (4) (+) concentrations led to higher initial CH(4) production, with an optimum at 120 mmol/L. The addition of 720 mmol/L NH (4) (+) led to a complete inhibition until day 3; subsequently, CH(4) production started again on day 5 though it was still significantly lower compared to the other variants. Finally, also the speed of agitation showed significant effects on methanogenesis. The CH(4) production from complex carbon sources was most favourable at a moderate agitation of 150 rpm of the lab-scale serum bottles. A lower or higher speed brought about a distinct reduction of CH(4) production.     

Hydrogenotrophic methanogenesis by moderately acid-tolerant methanogens of a methane-emitting acidic peat

The emission of methane (1.3 mmol of CH(4) m(-2) day(-1)), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH(4) (0.4 to 1.7 micro mol g [dry wt] of soil(-1) day(-1)) under anoxic conditions. At in situ pH, supplemental H(2)-CO(2), ethanol, and 1-propanol all increased CH(4) production rates while formate, acetate, propionate, and butyrate inhibited the production of CH(4); methanol had no effect. H(2)-dependent acetogenesis occurred in H(2)-CO(2)-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H(2) that were subsequently consumed. The accumulation of H(2) was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H(2); these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H(2)-utilizing methanogens. A total of 10(9) anaerobes and 10(7) hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H(2)-CO(2)-supplemented, fatty acid-enriched defined medium. CH(4) production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH(4)-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H(2) that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H(2) is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Effects of select nitrocompounds on in vitro ruminal fermentation during conditions of limiting or excess added reductant

Ruminal methane (CH(4)) production results in the loss of up to 12% of gross energy intake and contributes nearly 20% of the United States' annual emission of this greenhouse gas. We report the effects of select nitrocompounds on ruminal fermentation after 22 h in vitro incubation (39 degrees C) with or without additions of hydrogen (H(2)), formate or both. In incubations containing no added reductant, CH(4) production was inhibited 41% by 2-nitro-1-propanol (2NPOH) and >97% by 3-nitro-1-propionic acid (3NPA), nitroethane (NE) and 2-nitroethanol (2NEOH) compared to non-treated controls and H(2) did not accumulate. With formate as the sole added reductant, nitro-treatment reduced CH(4) production by >99% and caused 42% to complete inhibition of formate catabolism compared to controls, and the accumulation of H(2) increased slightly. Nitro-treatment decreased CH(4) production 57-98% from that of controls when supplied H(2) or formate plus H(2). Formate catabolism was decreased 42-84% from that in controls by all nitro-treatments except 3NPA with both formate and H(2). Greater than 97% of the added H(2) was catabolized within controls; >84% was catabolized in nitro-treated incubations. Acetate, propionate and butyrate accumulations were unaffected by nitro-treatment irregardless of reductant; however, effects on ammonia and branched chain fatty acid accumulations varied. These results suggest that nitro-treatment inhibited formate dehydrogenase/formate hydrogen lyase and hydrogenase activity.     

短链脂肪酸丁酸抑制动脉粥样硬化形成及其分子机制

本研究通过观察丁酸对动脉粥样硬化斑块形成以及肠道组织结构和功能的影响,探讨丁酸防治动脉粥样硬化的效应及可能机制.选取8周龄雄性载脂蛋白E基因敲除(apolipoprotein E-knockout,ApoE-/-)小鼠,随机分成对照组(高脂高胆固醇饲料+饮水中给予200 mmol/L氯化钠,n = 10)和丁酸组(高脂...     

Use of some novel alternative electron sinks to inhibit ruminal methanogenesis

Several compounds were evaluated in vitro as alternative electron sinks to ruminal methanogenesis. They were incubated with ruminal fluid, buffer mixture, and finely ground alfalfa hay for 24 h, at 0, 6, 12, and 18 mM initial concentrations. The propionate enhancer oxaloacetic acid, the butyrate enhancer beta-hydroxybutyrate, and the butyrate unsaturated analog 3-butenoic acid were ineffective in decreasing methanogenesis. Nevertheless, beta-hydroxybutyrate increased apparent fermentation of the alfalfa hay substrate from 58.0 to 63.4%, and 3-butenoic acid seemed to increase it from 62.0 to 73.7%. Almost all of added oxaloacetic acid disappeared during the incubation, while only between 30.3 and 53.4% of beta-hydroxybutyrate disappeared. The butyrate enhancers acetoacetate and crotonic acid, and the butyrate unsaturated analog 2-butynoic acid, decreased methanogenesis by a maximum of 18,9 and 9%, respectively. Crotonic acid at 18 mM initial concentration seemed to increase the substrate apparent fermentation from 57.0 to 68.2%. Between 78.6 and 100% of acetoacetate disappeared during the incubation. The propionate unsaturated analog propynoic acid, and the unsaturated ester ethyl 2-butynoate, decreased methanogenesis by a maximum of 76 and 79%, respectively. Less than 5% of propynoic acid disappeared. The substrate apparent fermentation was decreased by propynoic acid from 62.0 to 57.4%, and seemed to have been decreased by ethyl 2-butynoate from 62.0 to 29.3%. More accurate measurements of the disappearance of some of the compounds studied are needed to better understand how they are metabolized and how they affect fermentation.     

Quantitative evaluation of ruminal methane and carbon dioxide formation from formate through C-13 stable isotope analysis in a batch culture system

Methane produced from formate is one of the important methanogensis pathways in the rumen. However, quantitative information of CH₄ production from formate has been rarely reported. The aim of this study was to characterize the conversion rate (CR) of formic acid into CH₄ and CO₂ by rumen microorganisms. Ground lucerne hay was incubated with buffered ruminal fluid for 6, 12, 24 and 48 h. Before the incubation, ¹³C-labeled H¹³COOH was also supplied into the incubation bottle at a dose of 0, 1.5, 2.2 or 2.9 mg/g of DM substrate. There were no interactions (P>0.05) between dose and incubation time for all variables evaluated. When expressed as an absolute amount (ml in gas sample) or a relative CR (%), both ¹³CH₄ and ¹³CO₂ production quadratically increased (P<0.01) with the addition of H¹³COOH. The total ¹³C (¹³CH₄ and ¹³CO₂) CR was also quadratically increased (P<0.01) when H¹³COOH was added. Moreover, formate addition linearly decreased (P<0.031) the concentrations of NH₃-N, total and individual volatile fatty acids (acetate, propionate and butyrate), and quadratically decreased (P<0.014) the populations of protozoa, total methanogens, Methanosphaera stadtmanae, Methanobrevibacter ruminantium M1, Methanobrevibacter smithii and Methanosarcina barkeri. In summary, formate affects ruminal fermentation and methanogenesis, as well as the rumen microbiome, in particular microorganisms which are directly or indirectly involved in ruminal methanogenesis. This study provides quantitative verification for the rapid dissimilation of formate into CH₄ and CO₂ by rumen microorganisms.     

Butyrate inhibits proliferation-induced Proliferating Cell Nuclear Antigen expression (PCNA) in rat vascular smooth muscle cells

Arterial injury-induced vascular smooth muscle cell (VSMC) proliferation in intima is the important etiologic factor in vascular proliferative disorders such as atherosclerosis, hypertension and restenosis after balloon angioplasty. Butyrate, a naturally occurring short chain fatty acid, is produced by bacterial fermentation of dietary fiber and by mammary glands of certain mammals. Studies have shown that butyrate at millimolar concentrations, which are physiological, induces growth arrest, differentiation and apoptosis. We examined the effect of physiological concentrations of butyrate on rat VSMC proliferation and proliferation-induced PCNA expression to determine anti-atherogenic potential of butyrate. Butyrate concentrations, closer to physiological range, exhibited antiproliferative effects on both serum-induced proliferation of serum-starved quiescent VSMCs and actively proliferating non-confluent VSMCs. Treatment of serum-starved quiescent VSMCs with 1-8 mmol/l concentration of butyrate caused a concentration-dependent decrease in serum-induced VSMC proliferation and cell proliferation-associated increase in total cellular proteins and RNA levels. Similarly, exposure of actively growing VSMCs to 5 mmol/l butyrate resulted in the inhibition of cell proliferation and proliferation-induced increase in cellular proteins and RNA levels. Furthermore, cellular morphology was significantly altered. Analysis of cell cycle regulatory proteins indicated that levels of PCNA, an excellent marker for cell proliferation, was significantly altered by butyrate both in actively proliferating and serum-induced quiescent VSMCs. These observations suggest that butyrate exhibits potential antiatherogenic capability by inhibiting VSMC proliferation and proliferation-associated increase in PCNA expression and thus merits further investigations regarding therapeutic significance of butyrate in vascular proliferative disorders.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6 could grow at a NaCl concentration in the medium of 30-230 g/l, with an optimum at 80-100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H2 + CO2 were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H2 + CO2 or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37 degrees C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the subclass Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.     

Optimization of glutamate concentration and pH for H production from volatile fatty acids by Rhodopseudomonas capsulata

AIMS: This study attempted to employ response surface methodology (RSM) to evaluate the effects of glutamate concentration and pH on H(2) production from volatile fatty acids by Rhodopseudomonas capsulata. METHODS AND RESULTS: A mixture of acetate, propionate and butyrate was used as a carbon source for the H(2) production by R. capsulata. The H(2) yield and H(2) production rate were strongly affected by the glutamate concentration, pH and their interaction. The predicted maximum H(2) yield of 0.534 was obtained when glutamate concentration and pH were 6.56 mmol l(-1) and 7.29 respectively. On the contrary, the maximum H(2) production rate of 18.72 ml l(-1) h(-1) was achieved at a glutamate concentration of 7.01 mmol l(-1) and pH 7.31. CONCLUSIONS: Taking H(2) yield and H(2) production rate together into account, a glutamate concentration of 6.56-7.01 mmol l(-1) and pH of 7.29-7.31 should be selected for H(2) production from a mixture of acetate, propionate and butyrate by R. capsulata. SIGNIFICANCE AND IMPACT OF THE STUDY: The RSM was a useful tool for maximizing H(2) production by photosynthetic bacteria (PSB).     

Effects of several inhibitors on pure cultures of ruminal methanogens

AIMS: To examine the effects of five inhibitors of methanogenesis, 2-bromoethanesulphonate (BES), 3-bromopropanesulphonate (BPS), lumazine, propynoic acid and ethyl 2-butynoate, on CH4 production of the ruminal methanogens Methanobrevibacter ruminantium, Methanosarcina mazei and Methanomicrobium mobile. METHODS AND RESULTS: Methanogens were grown in MS medium including 25% (v/v) clarified ruminal fluid. Methane production was measured after 4 and 6 days of incubation. Methanobrevibacter ruminantium was the most sensitive species to BES, propynoic acid and ethyl 2-butynoate. Methanosarcina mazei was the least sensitive species to those chemical additives, and Mm. mobile was intermediate. BPS failed to inhibit any of the methanogens. All three species were almost completely inhibited by 50- and 100%-lumazine saturated media, but the inhibition was somewhat lower with a 25%-lumazine saturated media. CONCLUSIONS: There were important differences among species of methanogens regarding their sensitivity to the different inhibitors. In general, Ms. mazei was the most resistant to inhibitors, Mb. ruminantium the least resistant, and Mm. mobile was intermediate. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences among methanogens regarding their resistance to chemical inhibitors should be considered when designing strategies of inhibition of ruminal methanogenesis, as selection of resistant species may result.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

Effect of select nitrocompounds on ruminal fermentation; an initial look at their potential to reduce economic and environmental costs associated with ruminal methanogenesis

Methane production by ruminal microbes during the digestion of feedstuffs is an inefficient process resulting in losses of 2-12% of the gross energy consumed by ruminants. Presently, we report the effect of three inhibitors on ruminal methane production in vitro. Mixed populations of ruminal microbes collected from cannulated cows maintained on an alfalfa hay:corn diet (50:50) were incubated at 39 degrees C for 24 h under a 100% carbon dioxide gas phase in closed tubes with 72 mM added sodium formate. Cultures were supplemented with 12 mM 2-nitropropanol, nitroethane or nitroethanol (experiment 1) or with 2, 12 or 24 mM nitroethane or a combination of 12 mM nitroethane and 4 mM nitroethanol (experiment 2). Control cultures containing no added nitrocompound were incubated simultaneously with treated incubations. Methane concentrations were reduced (P<0.05) from those measured in control incubations (27.6 +/- 2.1 and 17.7 +/- 0.8 micromol/ml; mean +/- SD for experiments 1 and 2, respectively) by at least 57% and as much as 94% in the nitrocompound supplemented incubations. By comparison, the widely fed methane inhibitor, monensin, typically reduces ruminal methane production by about 33%. Concentrations of volatile fatty acids and ammonia that accumulated in the nitrocompound supplemented incubations were not markedly affected compared to those produced by control cultures despite the reductions in methane produced. Hydrogen accumulated only slightly in cultures supplemented with the nitrocompounds. These results demonstrate that 2-nitropropanol, nitroethane and nitroethanol inhibit ruminal methane production. Further research is warranted to determine the mechanisms responsible for this inhibition and to see if these inhibitors can be used in practical application to reduce economic and environmental costs associated with ruminal methanogenesis.     

Effect of temperature on anaerobic ethanol oxidation and methanogenesis in acidic peat from a northern wetland

The effects of temperature on rates and pathways of CH4 production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68 degrees N). We monitored the production of CH4 and CO2 over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH3F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25 degrees C (2.3 micromol CH4.g [dry weight](-1) . day(-1)), but the activity was relatively high even at 4 degrees C (0.25 micromol CH4. g [dry weight](-1) . day(-1)). The theoretical lower limit for methanogenesis was calculated to be at -5 degrees C. The optimum temperature for growth as revealed by real-time PCR was 25 degrees C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H2 sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

The new facultatively chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium Desulfovermiculus halophilus gen. nov., sp. nov., isolated from an oil field

The new mesophilic, chemolithoautotrophic, moderately halophilic, sulfate-reducing bacterium strain 11-6, could grow at a NaCl concentration in the medium of 30–230 g/l, with an optimum at 80–100 g/l. Cells were vibrios motile at the early stages of growth. Lactate, pyruvate, malate, fumarate, succinate, propionate, butyrate, crotonate, ethanol, alanine, formate, and H₂/CO₂ were used in sulfate reduction. Butyrate was degraded completely, without acetate accumulation. In butyrate-grown cells, a high activity of CO dehydrogenase was detected. Additional growth factors were not required. Autotrophic growth occurred, in the presence of sulfate, on H₂/CO₂ or formate without other electron donors. Fermentation of pyruvate and fumarate was possible in the absence of sulfate. Apart from sulfate, sulfite, thiosulfate, and elemental sulfur were able to serve as electron acceptors. The optimal growth temperature was 37°C; the optimum pH was 7.2. Desulfoviridin was not detected. Menaquinone MK-7 was present. The DNA G+C content was 55.2 mol %. Phylogenetically, the bacterium represented a separate branch within the cluster formed by representatives of the family Desulfohalobiaceae in the class Deltaproteobacteria. The bacterium was assigned to a new genus and species, Desulfovermiculus halophilus gen. nov., sp. nov. The type strain is 11-6^T (= VKM B-2364), isolated from the highly mineralized formation water of an oil field.     

Formation and Fate of Fermentation Products in Hot Spring Cyanobacterial Mats

The fate of representative fermentation products (acetate, propionate, butyrate, lactate, and ethanol) in hot spring cyanobacterial mats was investigated. The major fate during incubations in the light was photoassimilation by filamentous bacteria resembling Chloroflexus aurantiacus. Some metabolism of all compounds occurred under dark aerobic conditions. Under dark anaerobic conditions, only lactate was oxidized extensively to carbon dioxide. Extended preincubation under dark anaerobic conditions did not enhance anaerobic catabolism of acetate, propionate, or ethanol. Acetogenesis of butyrate was suggested by the hydrogen sensitivity of butyrate conversion to acetate and by the enrichment of butyrate-degrading acetogenic bacteria. Accumulation of fermentation products which were not catabolized under dark anaerobic conditions revealed their importance. Acetate and propionate were the major fermentation products which accumulated in samples collected at temperatures ranging from 50 to 70°C. Other organic acids and alcohols accumulated to a much lesser extent. Fermentation occurred mainly in the top 4 mm of the mat. Exposure to light decreased the accumulation of acetate and presumably of other fermentation products. The importance of interspecies hydrogen transfer was investigated by comparing fermentation product accumulation at a 65°C site, with naturally high hydrogen levels, and a 55°C site, where active methanogenesis prevented significant hydrogen accumulation. There was a greater relative accumulation of reduced products, notably ethanol, in the 65°C mat.     

Methanogenesis and methanogenic pathways in a peat from subarctic permafrost

Few studies have dealt so far with methanogenic pathways and populations in subarctic and arctic soils. We studied the effects of temperature on rates and pathways of CH4 production and on the relative abundance and structure of the archaeal community in a mildly acidic peat from a permafrost region in Siberia (67 degrees N). We monitored the production of CH4 and CO2 over time and measured the consumption of Fe(II), ethanol and volatile fatty acids. All experiments were performed with and without specific inhibitors [2-bromoethanesulfonate (BES) for methanogenesis and CH3F for acetoclastic methanogenesis]. The optimum temperature for methanogenesis was between 26 degrees C and 28 degrees C [4.3 micromol CH4 (g dry weight)(-1) day(-1)], but the activity was high even at 4 degrees C [0.75 micromol CH4 (g dry weight)(-1) day(-1)], constituting 17% of that at 27 degrees C. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Acetoclastic methanogenesis accounted for about 70% of the total methanogenesis. Most 16S rRNA gene sequences clustered with Methanosarcinales, correlating with the prevalence of acetoclastic methanogenesis. In addition, sequences clustering with Methanobacteriales were recovered. Fe reduction occurred in parallel to methanogenesis. At lower and higher temperatures Fe reduction was not affected by BES. Because butyrate was consumed during methanogenesis and accumulated when methanogenesis was inhibited (BES and CH3F), it is proposed to serve as methanogenic precursor, providing acetate and H2 by syntrophic oxidation. In addition, ethanol and caproate occurred as intermediates. Because of thermodynamic constraints, homoacetogenesis could not compete with hydrogenotrophic methanogenesis.     

Production of gamma-linolenic acid by disrupted mycelia of Mortierella isabellina

AIMS: To optimize the production of linolenic acid by disrupted mycelia of Mortierella isabellina. METHODS AND RESULTS: Effects of incubation conditions such as incubation time, pH of reaction mixture, concentration of Mg2+ or malate and incubation temperature on production of linolenic acid were studied. The production of gamma-linolenic acid reached 224 mg g-1 dry cells when the reaction mixture was composed of 1.0 g (dry mycelial mass) of disrupted mycelia of M. isabellina, 50 ml (50 mmol l(-1)) potassium phosphate buffer supplemented with 0.312 mmol l(-1) of Mg2+ and 10 mmol l(-1) of malate, pH 7.0 and incubated at 5 degrees C for 1 day. CONCLUSIONS: Incubation temperature, concentration of Mg2+ and malate showed major effects on the increased linolenic acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights conditions for increasing gamma-linolenic acid production by cell-free mycelia of M. isabellina and an insight into rapidly gaining high production of polyunsaturated fatty acids.