Relative importance of trophic group concentrations during anaerobic degradation of volatile fatty acids

Although obligate syntrophic reactions cannot proceed without hydrogenotrophs, it has been unclear from the literature whether potential improvements are achievable with higher concentrations of hydrogenotrophs. In this study, the relative importance of formate-/H(2)-utilizing and acetate-utilizing trophic groups in the anaerobic degradation of butyrate and propionate was assessed by adding various proportions of these enriched cultures to a mixed anaerobic seed inoculum. The improvement resulting from the additional acetate-utilizing cultures was much greater than with formate/H(2) utilizers. Furthermore, formate/H(2) utilizers did not improve propionate utilization significantly, suggesting the importance of optimum utilization of hydrogenotrophic capacity. During most of the volatile fatty acid (VFA) degradation period, the system responded with characteristic hydrogen levels to maintain the Gibbs free energy of oxidation approximately constant for both butyrate (-6 kJ) and propionate (-14 kJ). These free-energy values were independent of methanogenic activity, as well as the volume of the seed inoculum and the VFA concentrations present. By comparing the experimental results with kinetic and mass transfer models, it was postulated that the diffusional transfer of reducing equivalents was the major limiting factor for efficient VFA degradation. Therefore, for optimum utilization of the hydrogenotrophs, low acetate concentrations are vital to enable the system to respond with higher formate/H(2) levels, thus leading to improved transfer of reducing equivalents. Due to the small number of propionate utilizers (and hence their limited surface area) and low bulk liquid concentrations, the additional formate/H(2) utilizers were of minimal use for improving the degradation rate further. The butyrate degradation rates strongly correlated with the cumulative activity of hydrogenotrophs and acetotrophs over the experimental range studied, indicating the need to model obligate syntrophic reactions as a dependent function of methanogenic activity.     

Metabolic properties and kinetics of methanogenic granules

Two types of mesophilic methanogenic granules (R- and F-granules) were developed on different synthetic feeds containing acetate, propionate and butyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H₂-CO₂ were related to the feed composition used for their development. These granules performed a reversible reaction between H₂ production from formate and formate synthesis from H₂ plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and butyrate during butyrate or isobutyrate degradation. Valerate and 2-methylbutyrate were produced and consumed during propionate-butyrate degradation. The respective apparent K _m (mm) for various substrates in disrupted R- and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an optimum temperature range from 40 to 50° C for H₂-CO₂ and formate utilization and 40° C for acetate, propionate and butyrate utilization and a similar optimum pH. Correspondence to: J. G. Zeikus     

Response surface methodology analysis of anaerobic syntrophic degradation of volatile fatty acids in an upflow anaerobic sludge bed reactor inoculated with enriched cultures

Anaerobic oxidation of volatile fatty acids (VFAs) as the key intermediates is restricted thermodynamically. Presently, enriched acetogenic and methanogenic cultures were used for syntrophic anaerobic digestion of VFAs in an upflow anaerobic sludge bed reactor fed with acetic, propionic, and butyric acids at maximum concentrations of 5.0, 3.0, and 4.0 g/L, respectively. Interactive effects of propionate, butyrate and acetate were analyzed. Hydraulic retention time (HRT) and acetate oxidizing syntrophs and methanogen (hydrogenotrophs) to syntrophic bacteria (propionate- and butyrate-oxidizing bacteria) population ratio (M/A) were investigated as key microbiological and operating variables of VFA anaerobic degradations. M/A did not affect the size distribution and had little effect on extracellular polymer contents of the granules. Granular sludge with close spatial microbial proximity enhanced syntrophic degradation of VFAs compared to other cultures, such as suspended cultures. Optimum conditions were found to be propionate = 1.93 g/L, butyrate = 2.15 g/L, acetate = 2.50 g/L, HRT = 22 h, and M/A = 2.5 corresponding to maximum VFA removal and biogas production rate. Results of verification experiments and predicted values from fitted correlations were in close agreement at the 95% confidence interval. Granules seemed to be smaller particles and less stable in construction with an irregular fractured surface compared to the original granules.     

Sulfate reduction in methanogenic bioreactors

Abstract: In the anaerobic treatment of sulfate-containing wastewater, sulfate reduction interferes with methanogenesis. Both mutualistic and competitive interactions between sulfate-reducing bacteria and methanogenic bacteria have been observed. Sulfate reducers will compete with methanogens for the common substrates hydrogen, formate and acetate. In general, sulfate reducers have better growth kinetic properties than methanogens, but additional factors which may be of importance in the competition are adherence properties, mixed substrate utilization, affinity for sulfate of sulfate reducers, relative numbers of bacteria, and reactor conditions such as pH, temperature and sulfide concentration. Sulfate reducers also compete with syntrophic methanogenic consortia involved in the degradation of substrates like propionate and butyrate. In the absence of sulfate these methanogenic consortia are very important, but in the presence of sulfate they are thought to be easily outcompeted by sulfate reducers. However, at relatively low sulfate concentrations, syntrophic degradation of propionate and butyrate coupled to HZ removal via sulfate reduction rather than via methanogenesis may become important. A remarkable feature of some sulfate reducers is their ability to grow fermentatively or to grow in syntrophic association with methanogens in the absence of sulfate.     

Methanogenesis at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H₂/CO₂ as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H₂/CO₂-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.     

Anaerobic degradation of volatile fatty acids at different sulphate concentrations

The effect of sulfate on the anaerobic breakdown of mixtures of acetate, propionate and butyrate at three different sulfate to fatty acid ratios was studied in upflow anaerobic sludge blanket reactors. Sludge characteristics were followed with time by means of sludge activity tests and by enumeration of the different physiological bacterial groups. At each sulfate concentration acetate was completely converted into methane and CO₂, and acetotrophic sulfate-reducing bacteria were not detected. Hydrogenotrophic methanogenic bacteria and hydrogenotrophic sulfate-reducing bacteria were present in high numbers in the sludge of all reactors. However, a complete conversion of H₂ by sulfate reducers was found in the reactor operated with excess sulfate. At higher sulfate concentrations, oxidation of propionate by sulfate-reducing bacteria became more important. Only under sulfate-limiting conditions did syntrophic propionate oxidizers out-compete propionate-degrading sulfate reducers. Remarkably, syntrophic butyrate oxidizers were well able to compete with sulfate reducers for the available butyrate, even with an excess of sulfate. Correspondence to: A. Visser     

The genome of Syntrophorhabdus aromaticivorans strain UI provides new insights for syntrophic aromatic compound metabolism and electron flow

How aromatic compounds are degraded in various anaerobic ecosystems (e.g. groundwater, sediments, soils and wastewater) is currently poorly understood. Under methanogenic conditions (i.e. groundwater and wastewater treatment), syntrophic metabolizers are known to play an important role. This study explored the draft genome of Syntrophorhabdus aromaticivorans strain UI and identified the first syntrophic phenol‐degrading phenylphosphate synthase (PpsAB) and phenylphosphate carboxylase (PpcABCD) and syntrophic terephthalate‐degrading decarboxylase complexes. The strain UI genome also encodes benzoate degradation through hydration of the dienoyl‐coenzyme A intermediate as observed in Geobacter metallireducens and Syntrophus aciditrophicus. Strain UI possesses electron transfer flavoproteins, hydrogenases and formate dehydrogenases essential for syntrophic metabolism. However, the biochemical mechanisms for electron transport between these H₂/formate‐generating proteins and syntrophic substrate degradation remain unknown for many syntrophic metabolizers, including strain UI. Analysis of the strain UI genome revealed that heterodisulfide reductases (HdrABC), which are poorly understood electron transfer genes, may contribute to syntrophic H₂ and formate generation. The genome analysis further identified a putative ion‐translocating ferredoxin : NADH oxidoreductase (IfoAB) that may interact with HdrABC and dissimilatory sulfite reductase gamma subunit (DsrC) to perform novel electron transfer mechanisms associated with syntrophic metabolism.     

Perturbation of syntrophic isobutyrate and butyrate degradation with formate and hydrogen

The effect of formate and hydrogen on isomerization and syntrophic degradation of butyrate and isobutyrate was investigated using a defined methanogenic culture, consisting of syntrophic isobutyrate-butyrate degrader strain IB, Methanobacterium formicicum strain T1N, and Methanosarcina mazeii strain T18. Formate and hydrogen were used to perturb syntrophic butyrate and isobutyrate degradation by the culture. The reversible isomerization between isobutyrate and butyrate was inhibited by the addition of either formate or hydrogen, indicating that the isomerization was coupled with syntrophic butyrate degradation for the culture studied. Energetic analysis indicates that the direction of isomerization between isobutyrate and butyrate is controlled by the ratio between the two acids, and the most thermodynamically favorable condition for the degradation of butyrate or isobutyrate in conjunction with the isomerization is at almost equal concentrations of isobutyrate and butyrate. The degradation of isobutyrate and butyrate was completely inhibited in the presence of a high hydrogen partial pressure (>2000 Pa) or a measurable level of formate (10 muM or higher). Significant formate (more than 1 mM) was detected during the perturbation with hydrogen (17 to 40 kPa). Resumption of butyrate and isobutyrate degradation was related to the removal of formate. Energetic analysis supported that formate was another electron carrier, besides hydrogen, during syntrophic isobutyrate-butyrate degradation by this culture. (c) 1996 John Wiley & Sons, Inc.     

Diffusion of the Interspecies Electron Carriers H2 and Formate in Methanogenic Ecosystems and Its Implications in the Measurement of Km for H2 or Formate Uptake

We calculated the potential H₂ and formate diffusion between microbes and found that at H₂ concentrations commonly found in nature, H₂ could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H₂ concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H₂ concentration at the cell surface of H₂-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 μm from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the K_m for H₂ or formate.     

A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in Syntrophomonas wolfei

In syntrophic conversion of butyrate to methane and CO₂, butyrate is oxidized to acetate by secondary fermenting bacteria such as Syntrophomonas wolfei in close cooperation with methanogenic partner organisms, e.g., Methanospirillum hungatei. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO₂ reduction, in order to transfer these electrons to the methanogenic partner via hydrogen and/or formate.In the present study, all prominent membrane-bound and soluble proteins expressed in S. wolfei specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases.Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in S. wolfei. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further via a b-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.     

Microbial composition and characterization of prevalent methanogens and acetogens isolated from syntrophic methanogenic granules

The microbial species composition of methanogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (10¹²/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (10¹¹/g dry weight), but low numbers of hydrolytic-fermentative bacteria (10⁹/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospirillum hungatei strain BD, and a non-filamentous, bamboo-shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading sporeforming oval-shaped species, strain PT, and a propionate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophically with an H₂-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H₂, formate, acetate and butyrate but they were involved in propionate degradation.Correspondence to: M. K. Jain     

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.     

典型煤层水微生物产甲烷潜力及其群落结构研究

内蒙古自治区二连盆地、海拉尔盆地是我国重要的煤层气产区,其中生物成因煤层气是煤层气的重要来源,但复杂物质转化产甲烷相关微生物群落结构及功能尚不清楚。【目的】研究煤层水中的微生物代谢挥发性脂肪酸产甲烷的生理特征及群落特征。【方法】以内蒙古自治区二连盆地和海拉尔盆地的四口煤层气井水作为接种物,分别添加乙酸钠、丙酸钠和丁酸钠厌氧培养;定期监测挥发性脂肪酸降解过程中甲烷和底物的变化趋势,应用高通量测序技术,分析原始煤层气井水及稳定期产甲烷菌液的微生物群落结构。【结果】除海拉尔盆地H303煤层气井微生物不能代谢丙酸外,其他样品均具备代谢乙酸、丙酸和丁酸产生甲烷的能力,其生理生态参数存在显著差异,产甲烷延滞期依次是乙酸丁酸丙酸;最大比产甲烷速率和底物转化效率依次是丙酸乙酸丁酸。富集培养后,古菌群落结构与煤层气井水的来源显著相关,二连盆地优势古菌为氢营养型产甲烷古菌Methanocalculus (相对丰度13.5%–63.4%)和复合营养型产甲烷古菌Methanosarcina (7.9%–51.3%),海拉尔盆地的优势古菌为氢营养型产甲烷古菌Methanobacterium(24.3%–57.4%)和复合营养型产甲烷古菌Methanosarcina(29.6%–66.5%);细菌群落则与底物类型显著相关,硫酸盐还原菌Desulfovibrio(12.0%–41.0%)、互营丙酸氧化菌Syntrophobacter(39.6%–75.5%)和互营丁酸菌Syntrophomonas(8.5%–21.9%)分别在乙酸钠、丙酸钠和丁酸钠处理组显著富集。【结论】煤层气井水微生物可降解挥发性脂肪酸(乙酸、丙酸和丁酸)并具有产甲烷潜力;乙酸可能被古菌直接代谢产甲烷,而丙酸和丁酸通过互营细菌和产甲烷古菌代谢产甲烷。Desulfovibrio、Syntrophobacter和Syntrophomonas分别在乙酸、丙酸和丁酸代谢过程中发挥了重要作用。这些结果为煤层气生物强化开采提供了一定的微生物资源基础。     

Anaerobic Degradation of Normal- and Branched-Chain Fatty Acids with Four or More Carbons to Methane by a Syntrophic Methanogenic Triculture

Syntrophic degradation of normal- and branched-chain fatty acids with 4 to 9 carbons was investigated with a mesophilic syntrophic isobutyrate-butyrate-degrading triculture consisting of the non-spore-forming, syntrophic, fatty acid-degrading, gram-positive rod-shaped strain IB, Methanobacterium formicicum T1N, and Methanosarcina mazei T18. This triculture converted butyrate and isobutyrate to methane and converted valerate and 2-methylbutyrate to propionate and methane. This triculture also degraded caproate, 4-methylvalerate, heptanoate, 2-methylhexanoate, caprylate, and pelargoate. During the syntrophic conversion of isobutyrate and butyrate, a reversible isomerization between butyrate and isobutyrate occurred; isobutyrate and butyrate were isomerized to the other isomeric form to reach nearly equal concentrations and then their concentrations decreased at the same rates. Butyrate was an intermediate of syntrophic isobutyrate degradation. When butyrate was degraded in the presence of propionate, 2-methylbutyrate was synthesized from propionate and isobutyrate formed from butyrate. During the syntrophic degradation of valerate, isobutyrate, butyrate, and 2-methylbutyrate were formed and then degraded. During syntrophic degradation of 2-methylbutyrate, isobutyrate and butyrate were formed and then degraded.     

Energetics of syntrophic fatty acid oxidation

Abstract: Fatty acids are key intermediates in methanogenic degradation of organic matter in sediments as well as in anaerobic reactors. Conversion of butyrate or propionate to acetate, (CO₂), and hydrogen is endergonic under standard conditions, and becomes possible only at low hydrogen concentrations (10^-4-10^-5 bar). A model of energy sharing between fermenting and methanogenic bacteria attributes a maximum amount of about 20 kJ per mol reaction to each partner in this syntrophic cooperation system. This amount corresponds to synthesis of only a fraction (one-third) of an ATP to be synthesized per reaction. Recent studies on the biochemistry of syntrophic fatty acid-oxidizing bacteria have revealed that hydrogen release from butyrate by these bacteria is inhibited by a protonophore or the ATPase inhibitor DCCD ( N , N '-dicyclohexyl carbodiimide), indicating that a reversed electron transport step is involved in butyrate or propionate oxidation. Hydrogenase, butyryl-CoA dehydrogenase, and succinate dehydrogenase acitivities were found to be partially associated with the cytoplasmic membrane fraction. Also glycolic acid is degraded to methane and CO₂ by a defined syntrophic coculture. Here the most difficult step for hydrogen release is the glycolate dehydrogenase reaction ( E '₀=−92 mV). Glycolate dehydrogenase, hydrogenase, and ATPase were found to be membrane-bound enzymes. Membrane vesicles produced hydrogen from glycolate only in the presence of ATP; protonophores and DCCD inhibited this hydrogen release. This system provides a suitable model to study reversed electron transport in interspecies hydrogen transfer between fermenting and methanogenic bacteria in methanogenic biomass degradation.     

Anaerobic Degradation of Propionate by a Mesophilic Acetogenic Bacterium in Coculture and Triculture with Different Methanogens

A mesophilic acetogenic bacterium (MPOB) oxidized propionate to acetate and CO₂ in cocultures with the formate- and hydrogen-utilizing methanogens Methanospirillum hungatei and Methanobacterium formicicum. Propionate oxidation did not occur in cocultures with two Methanobrevibacter strains, which grew only with hydrogen. Tricultures consisting of MPOB, one of the Methanobrevibacter strains, and organisms which are able to convert formate into H₂ plus CO₂ (Desulfovibrio strain G11 or the homoacetogenic bacterium EE121) also degraded propionate. The MPOB, in the absence of methanogens, was able to couple propionate conversion to fumarate reduction. This propionate conversion was inhibited by hydrogen and by formate. Formate and hydrogen blocked the energetically unfavorable succinate oxidation to fumarate involved in propionate catabolism. Low formate and hydrogen concentrations are required for the syntrophic degradation of propionate by MPOB. In triculture with Methanospirillum hungatei and the aceticlastic Methanothrix soehngenii, propionate was degraded faster than in biculture with Methanospirillum hungatei, indicating that low acetate concentrations are favorable for propionate oxidation as well.     

The nexus of syntrophy‐associated microbiota in anaerobic digestion revealed by long‐term enrichment and community survey

Anaerobic digestion (AD) processes are known to effectively convert organic waste to CO₂ and CH₄, but much of the microbial ecology remains unclear. Specifically, we have limited insights into symbiotic syntroph and methanogen (‘syntrophy’) acid degradation, although they are essential for preventing process deterioration. Also, we often observed many uncharacterized or uncultivated organisms, but poorly understood their role(s) in relation to syntrophy. To define syntrophy‐associated populations, this study enriched methanogenic communities with propionate, butyrate, benzoate, acetate, formate and H₂ from two different inocula over 3 years. 16S pyrotag analysis revealed core populations of known syntrophs (six clades) and methanogens (nine clades) associated with acid degradation, and evidence for substrate‐ and/or inoculum‐dependent specificity in syntrophic partnerships. Based on comprehensive re‐evaluation of publically available microbial community data for AD, the known syntrophs and methanogens identified were clearly representatives of the AD‐associated syntrophs and methanogens. In addition, uncultivated clades related to Bacteroidetes, Firmicutes, Actinobacteria and Chloroflexi were ubiquitously found in AD and enrichments. These organisms may be universally involved in AD syntrophic degradation, but only represented <23% of the yet‐to‐be‐cultivated organisms (89 of 390 clades). Thus, the contribution of these uncultured organisms in AD remains unclear and warrants further investigation.     

Thermophilic anaerobic degradation of butyrate by a butyrate-utilizing bacterium in coculture and triculture with methanogenic bacteria

We studied syntrophic butyrate degradation in thermophilic mixed cultures containing a butyrate-degrading bacterium isolated in coculture with Methanobacterium thermoautotrophicum or in triculture with M. thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic bacterium. Butyrate was beta-oxidized to acetate with protons as the electron acceptors. Acetate was used concurrently with its production in the triculture. We found a higher butyrate degradation rate in the triculture, in which both hydrogen and acetate were utilized, than in the coculture, in which acetate accumulated. Yeast extract, rumen fluid, and clarified digestor fluid stimulated butyrate degradation, while the effect of Trypticase was less pronounced. Penicillin G, d-cycloserine, and vancomycin caused complete inhibition of butyrate utilization by the cultures. No growth or degradation of butyrate occurred when 2-bromoethanesulfonic acid or chloroform, specific inhibitors of methanogenic bacteria, was added to the cultures and common electron acceptors such as sulfate, nitrate, and fumarate were not used with butyrate as the electron donor. Addition of hydrogen or oxygen to the gas phase immediately stopped growth and butyrate degradation by the cultures. Butyrate was, however, metabolized at approximately the same rate when hydrogen was removed from the cultures and was metabolized at a reduced rate in the cultures previously exposed to hydrogen.     

Metabolic resistance of a psychrotolerant VFA-oxidizing microbial community from an anaerobic bioreactor to changes in the cultivation temperature

A psychrotolerant microbial consortium from a low-temperature anaerobic EGSB bioreactor was grown separately on acetate, propionate, butyrate, and H₂/CO₂ at 30 and 10°C in glass flasks. In the course of the experiments, the cultivation temperature was changed at different time intervals. The initial rates of substrate utilization were higher at 30 than at 10°C. However, the microbial consortium was found to be well adapted to low temperatures; when grown at 10°C for 1.5–5 months, the rates of butyrate, propionate, and H₂/CO₂ utilization increased steadily. When grown at 30°C for 1.5–2.5 months, this consortium retained its ability to degrade VFA and H₂/CO₂ at 10°C. However, after long-term (150 days) cultivation at 10°C, its ability to utilize the substrates at 30°C decreased. In the consortium grown in the acetate-containing medium, a Methanosaeta-like methanogen was predominant; in media with propionate and butyrate, besides VFA-degrading bacteria, acetoclastic Methanosaeta-like and hydrogenotrophic Methanospirillum-like methanogenic archaea prevailed. A Methanospirillum-like strain predominated in the H₂/CO₂-containing medium. The Methanospirillum strain of this microbial community was presumably psychrotolerant. A method based on changes in the cultivation temperature is of practical interest and can be used to start up new bioreactors.     

Butyrate oxidation by Syntrophospora bryantii in co-culture with different methanogens and in pure culture with pentenoate as electron acceptor

Syntrophospora bryantii degraded butyrate in co-culture with methanogens that can use both H₂ and formate for growth, but not in co-culture with methanogens that metabolize only H₂, suggesting that in suspended cultures formate may be a more important electron carrier in the syntrophic degradation of butyrate than H₂. Syntrophic butyrate oxidation was inhibited by the addition of 20 mm formate or the presence of 130 kPa H₂. In the absence of methanogens, S. bryantii is able to couple the oxidation of butyrate to acetate with the reduction of pentenoate to valerate. Under these conditions, up to 300 Pa H₂ was measured in the gas phase and up to 0.3 mm formate in the liquid phase. S. bryantii was unable to grow syntrophically with the aceticlastic methanogen Methanothrix soehngenii. However in triculture with Methanospirillum hungatei and Methanothrix soehngenii, S. bryantii degraded butyrate faster than in a biculture with only M. hungatei. Hydrogenase and formate dehydrogenase activities were demonstrated in cell-free extracts of S. bryantii.