Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions

The basis for the outcome of competition between sulfidogens and methanogens for H₂ was examined by comparing the kinetic parameters of representatives of each group separately and in co-culture. Michaelis-Menten parameters (V _max and K _m) for four methanogens and five sulfate-reducing bacteria were determined from H₂-depletion data. Further, Monod growth parameters (_max, K _s, Y _H2) for Desulfovibrio sp. G11 and Methanospirillum hungatei JF-1 were similarly estimated. H₂ K _m values for the methanogenic bacteria ranged from 2.5 M (Methanospirillum PM1) to 13 M for Methanosarcina barkeri MS; Methanospirillum hungatei JF-1 and Methanobacterium PM2 had intermediate H₂ K _m estimates of 5 M. Average H₂ K _m estimates for the five sulfidogens was 1.2 M. No consistent difference among the V _max estimates for the above sulfidogens (mean=100 nmol H₂ min^-1 mg^-1 protein) and methanogens (mean=110 nmol H₂ min^-1 mg^-1 protein) was found. A two-term Michaelis-Menten equation accurately predicted the apparent H₂ K _m values and the fate of H₂ by resting co-cultures of sulfate-reducers and methanogens. Half-saturation coefficients (K _s) for H₂-limited growth of Desulfovibrio sp. G11 (2–4 M) and Methanospirillum JF-1 (6–7 M) were comparable to H₂ K _m estimates obtained for these organisms. Maximum specific growth rates for Desulfovibrio sp. G11 (0.05 h^-1) were similar to those of Methanospirillum JF-1 (0.05–0.06 h^-1); whereas G11 had an average yield coefficient 4 x that of JF-1. Calculated _max and V _max/K _m values for the methanogens and sulfidogens studied predict that the latter bacterial group will process more H₂ whether these organisms are in a growing or resting state, when the H₂ concentration is in the first-order region.     

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.     

High rate performance and characterization of granular methanogenic sludges in upflow anaerobic sludge blanket reactors fed with various defined substrates

High rate granular methanogenic fermentations were performed in one-phase upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewaters containing starch, sucrose, ethanol, and butyrate plus propionate. All granules formed showed high settling velocities which enabled high cell mass retention and accommodation of high loading rates. The maximum COD removal rates (g COD/l-reactor·d) obtained after 500-d operations were 7.6 for starch, 10.5 for sucrose, 32.1 for ethanol, and 42.6 for butyrate-propionate. Long-term growth on various defined substrates altered the population of bacterial trophic groups and overall characteristics of granules. The starch- and sucrose-grown granules were characterized by larger size and more abundant extracellular polymeric substances (EPS) than the ethanol- or fatty acids-grown granules. The fatty acids-grown granules contained a considerable amount of inorganic salts (ash content: 56 to 63%) but a small amount of EPS, and showed a denser ultrastructure than the other three types of granules. The granules grown on ethanol under slightly acidic conditions showed the lowest specific gravity and volatile suspended solids (VSS) density as well as ash content among all of the granules. As aceticlastic methanogens, Methanothrix spp. were predominant in the starch-, sucrose-, and fatty acids-grown granules, whereas comparable numbers of Methanosarcina spp. were observed only in the ethanol-grown granules. The populations of hydrogenotrophic methanogens were the largest of all bacterial trophic groups in the respective granules. The data confirm that the prevalence of Methanothrix spp. and high methanogenic activity for H₂ are general characteristics of methanogenic granucles and that EPS and inorganic deposits contribute chemically to the enhancement of structural stability and mechanical strength of granules.     

Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri

Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H₂-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H₂ as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H₂, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H₂ transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H₂ or electrons derived from DIET for CO₂ reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.     

Photoproduction of H2 from acetate by syntrophic cocultures of green sulfur bacteria and sulfur-reducing bacteria

The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H₂ and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H₂ production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H₂ was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H₂ production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m^-2 · s^-1), in sulfide-reduced mineral medium (2 mM Na₂S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H₂ (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H₂ production was 1300 ml H₂ per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates     

Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge

An anaerobic, motile, gram-negative, rod-shaped bacterium is described which degrades benzoate in coculture with an H₂-utilizing organism and in the absence of exogenous electron acceptors such as O₂, SO ₄ ⁼ or NO ₃ ^- . The bacterium was isolated from a municipal primary, anaerobic sewage digestor using anaerobic roll-tube medium with benzoate as the main energy source and in syntrophic association with an H₂-utilizing sulfate-reducing Desulfovibrio sp. which cannot utilize benzoate or fatty acids apart from formate as energy source. The benzoate utilizer produced acetate (3 mol/mol of substrate degraded) and presumably CO₂ and H₂, or formate from benzoate. In media without sulfate and with Methanospirillum hungatei (a methanogen that utilizes only H₂–CO₂ or formate as the energy source) added, 3 mol of acetate and 0.7 mol of methane were produced per mol of benzoate and CO₂ was probably formed. Low numbers of Desulfovibrio sp. were present in the methanogenic coculture and a pure coculture of the benzoate utilizer with M. hungatei was not obtained. The generation times for growth of the sulfate-reducing and methanogenic cocultures were 132 and 166h, respectively. The benzoate utilizer did not utilize other common aromatic compounds, C ₃ ^- –C₇ monocarboxylic acids, or C₄-C₆ dicarboxylic acids for growth, nor did it appear to use SO ₄ ⁼ , NO ₃ ^- or fumarate as alternative electron acceptors. Addition of H₂ inhibited growth and benzoate degradation.     

Growth of Desulfovibrio in Lactate or Ethanol Media Low in Sulfate in Association with H2-Utilizing Methanogenic Bacteria

In the analysis of an ethanol-CO₂ enrichment of bacteria from an anaerobic sewage digestor, a strain tentatively identified as Desulfovibrio vulgaris and an H₂-utilizing methanogen resembling Methanobacterium formicicum were isolated, and they were shown to represent a synergistic association of two bacterial species similar to that previously found between S organism and Methanobacterium strain MOH isolated from Methanobacillus omelianskii. In lowsulfate media, the desulfovibrio produced acetate and H₂ from ethanol and acetate, H₂, and, presumably, CO₂ from lactate; but growth was slight and little of the energy source was catabolized unless the organism was combined with an H₂-utilizing methanogenic bacterium. The type strains of D. vulgaris and Desulfovibrio desulfuricans carried out the same type of synergistic growth with methanogens. In mixtures of desulfovibrio and strain MOH growing on ethanol, lactate, or pyruvate, diminution of methane produced was stoichiometric with the moles of sulfate added, and the desulfovibrios grew better with sulfate addition. The energetics of the synergistic associations and of the competition between the methanogenic system and sulfate-reducing system as sinks for electrons generated in the oxidation of organic materials such as ethanol, lactate, and acetate are discussed. It is suggested that lack of availability of H₂ for growth of methanogens is a major factor in suppression of methanogenesis by sulfate in natural ecosystems. The results with these known mixtures of bacteria suggest that hydrogenase-forming, sulfate-reducing bacteria could be active in some methanogenic ecosystems that are low in sulfate.     

Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain ΔH) in a fed-batch fermentor

During growth of Methanobacterium thermoautotrophicum in a fed-batch fermentor, the cells are confronted with a steady decrease in the concentration of the hydrogen energy supply. In order to investigate how the organism responds to these changes, cells collected during different growth phases were examined for their methanogenic properties. Cellular levels of the various methanogenic isoenzymes and functionally equivalent enzymes were also determined. Cells were found to maintain the rates of methanogenesis by lowering their affinity for hydrogen: the apparent K _m ^H2 decreased in going from the exponential to the stationary phase. Simultaneously, the maximal specific methane production rate changed. Levels of H₂-dependent methenyl-tetrahydromethanopterin dehydrogenase (H₂-MDH) and methyl coenzyme M reductase isoenzyme II (MCR II) decreased upon entry of the stationary phase. Cells grown under conditions that favored MCR II expression had higher levels of MCR II and H₂-MDH, whereas in cells grown under conditions favoring MCR I, levels of MCR II were much lower and the cells had an increased affinity for hydrogen throughout the growth cycle. The use of thiosulfate as a medium reductant was found to have a negative effect on levels of MCR II and H₂-MDH. From these results it was concluded that M. thermoautotrophicum responds to variations in hydrogen availability and other environmental conditions (pH, growth temperature, medium reductant) by altering its physiology. The adaptation includes, among others, the differential expression of the MDH and MCR isoenzymes.     

Growth characteristics of an obligately psychrophilic Vibrio sp.

The growth characteristics of an obligately psychrophilic Vibrio sp. have been studied in a chemostat with glucose or lactose as the limiting substrate over a temperature range 0–23°C. Vibrio AF-1 has an optimum growth temperature of 15°C and maximum growth temperature which is dependent upon the carbon source. On glucose growth ceases at 20°C whereas on lactose growth continues to 23°C. Growth rate is also a function of the carbon source provided. When grown on glucose, fructose, sucrose, maltose and galactose _max values of 0.046 h^-1 at 15°C were recorded whereas on lactose, mannose, ribose and xylose _max values of 0.020 h^-1 were obtained. Substrate affinities (K _s ) for the 9 sugars also fall into 2 divisions as for _max and are temperature dependent. Those sugars which support a high growth rate have highest K _s values at 0°C whereas these which give a low growth rate show maximum affinities at 15°C. Vibrio AF-1 produces the maximum cell yield (0.6 g/g sugar consumed) at temperature <8°C irrespective of the carbon source utilised and correlated with maximum rates of sugar uptake and minimum O₂ consumption. Maintenance energy determination on glucose grown cells show that at 2° C 2% of the carbon input is used for maintenance whereas at 20°C the requirement increases to 10% of the carbon input.     

Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for the apparent inhibition of methanogenesis by sulfate

Desulfovibrio vulgaris (Marburg) and Methanobrevibacter arboriphilus (AZ) are anaerobic sewage sludge bacteria which grow on H₂ plus sulfate and H₂ plus CO₂ as sole energy sources, respectively. Their apparent K_s values for H₂ were determined and found to be approximately 1 M for the sulfate reducing bacterium and 6 M for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of H₂ consumption by D. vulgaris was five times that of M. arboriphilus, when the hydrogen supply was rate limiting. The apparent inhibition of methanogenesis was of the same order as expected from the different K_s values for H₂. Difference in substrate affinities can thus account for the inhibition of methanogenesis from H₂ and CO₂ in sulfate rich environments, where the H₂ concentration is well below 5 M.     

Energetics of syntrophic ethanol oxidation in defined chemostat cocultures

The ethanol-oxidizing, proton-reducing Pelobacter acetylenicus was grown in chemostat cocultures with either Acetobacterium woodii, Methanobacterium bryantii, or Desulfovibrio desulfuricans. Stable steady state conditions with tightly coupled growth were reached at various dilution rates between 0.02 and 0.14 h^-1. Both ethanol and H₂ steady state concentrations increased with growth rate and were lower in cocultures with the sulfate reducer < methanogen < homoacetogen. Due to the higher affinity for H₂, D. desulfuricans outcompeted M. bryantii, and this one A. woodii when inoculated in cocultures with P. acetylenicus. Cocultures with A. woodii had lower H₂ steady state concentrations when bicarbonate reduction was replaced by the energetically more favourable caffeate reduction. Similarly, cocultures with D. desulfuricans had lower H₂ concentrations with nitrate than with sulfate as electron acceptor. The Gibbs free energy (G) available to the H₂-producing P. acetylenicus was independent of growth rate and the H₂-utilizing partner, whereas the G available to the latter increased with growth rate and the energy yielding potential of the H₂ oxidation reaction. The critical Gibbs free energy (G_c), i.e. the minimum energy required for H₂ production and H₂ oxidation, was-5.5 to-8.0 kJ mol^-1 H₂ for P. acetylenicus,-5.1 to-6.3 kJ mol^-1 H₂ for A. woodii,-7.5 to-9.1 kJ mol^-1 H₂ for M. bryantii, and-10.3 to-12.3 kJ mol^-1 H₂ for D. desulfuricans. Obviously, the potentially available energy was used more efficiently by homoacetogens > methanogens > sulfate reducers.     

Degradation of Acetaldehyde and Its Precursors by Pelobacter carbinolicus and P. acetylenicus

Pelobacter carbinolicus and P. acetylenicus oxidize ethanol in syntrophic cooperation with methanogens. Cocultures with Methanospirillum hungatei served as model systems for the elucidation of syntrophic ethanol oxidation previously done with the lost “Methanobacillus omelianskii” coculture. During growth on ethanol, both Pelobacter species exhibited NAD⁺-dependent alcohol dehydrogenase activity. Two different acetaldehyde-oxidizing activities were found: a benzyl viologen-reducing enzyme forming acetate, and a NAD⁺-reducing enzyme forming acetyl-CoA. Both species synthesized ATP from acetyl-CoA via acetyl phosphate. Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent acetaldehyde: ferredoxin oxidoreductases and formate dehydrogenase. Tungsten limitation resulted in slower growth and the expression of a molybdenum-dependent isoenzyme. Putative comproportionating hydrogenases and formate dehydrogenase were expressed constitutively and are probably involved in interspecies electron transfer. In ethanol-grown cocultures, the maximum hydrogen partial pressure was about 1,000 Pa (1 mM) while 2 mM formate was produced. The redox potentials of hydrogen and formate released during ethanol oxidation were calculated to be E_H2 = -358±12 mV and E_HCOOH = -366±19 mV, respectively. Hydrogen and formate formation and degradation further proved that both carriers contributed to interspecies electron transfer. The maximum Gibbs free energy that the Pelobacter species could exploit during growth on ethanol was −35 to −28 kJ per mol ethanol. Both species could be cultivated axenically on acetaldehyde, yielding energy from its disproportionation to ethanol and acetate. Syntrophic cocultures grown on acetoin revealed a two-phase degradation: first acetoin degradation to acetate and ethanol without involvement of the methanogenic partner, and subsequent syntrophic ethanol oxidation. Protein expression and activity patterns of both Pelobacter spp. grown with the named substrates were highly similar suggesting that both share the same steps in ethanol and acetalydehyde metabolism. The early assumption that acetaldehyde is a central intermediate in Pelobacter metabolism was now proven biochemically.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H2 on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H₂ and CO₂ for methanogenesis, degraded lactate, with the production of acetate and CH₄. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H₂-CO₂ and acetate for methanogenesis, lactate was stoichiometrically degraded to CH₄ and presumably to CO₂. During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH₄. Later, acetate was degraded to CH₄ and presumably to CO₂. In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H₂ was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H₂, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH₄ from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H₂-CO₂ as the methanogenic substrate produced CH₄ from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H₂ produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Effects of Organic Acid Anions on the Growth and Metabolism of Syntrophomonas wolfei in Pure Culture and in Defined Consortia

The effects of organic acid anions on the growth of Syntrophomonas wolfei was determined by varying the initial concentration of the acid anion in the medium. The addition of 15 mM acetate decreased the growth rate of a butyrate-catabolizing coculture containing Methanospirillum hungatei from 0.0085 to 0.0029 per hour. Higher initial acetate concentrations decreased the butyrate degradation rate and the yield of cells of S. wolfei per butyrate degraded. Inhibition was not due to the counter ion or the effect of acetate on the methanogen. Initial acetate concentrations above 25 mM inhibited crotonate-using pure cultures and cocultures of S. wolfei. Benzoate and lactate inhibited the growth of S. wolfei on crotonate in pure culture and coculture. Lactate was an effective inhibitor of S. wolfei cultures at concentrations greater than 10 mM. High concentrations of acetate and lactate altered the electron flow in crotonate-catabolizing cocultures, resulting in the formation of less methane and more butyrate and caproate. The inclusion of the acetate-using methanogen, Methanosarcina barkeri, in a methanogenic butyrate-catabolizing coculture increased both the yield of S. wolfei cells per butyrate degraded and the efficacy of butyrate degradation. Butyrate degradation by acetate-inhibited cocultures occurred only after the addition of Methanosarcina barkeri. These results showed that the metabolism of S. wolfei was inhibited by high levels of organic acid anions. The activity of acetate-using methanogens is important for the syntrophic degradation of fatty acids when high levels of acetate are present.     

The induction and repression of benzene and catechol oxidizing capacity of Pseudomonas putida ML2 studied in perturbed chemostat culture

The oxidation of catechol, an intermediate in benzene catabolism, was studied using transient variations in dissolved oxygen tension (DOT) when a succinate limited steady state culture of Pseudomonas putida ML2 was perturbed with a pulse of another substrate. A model was developed and tested for the effect of fluctuations in oxidizing enzyme activity on DOT. It was found that the rate of induction of catechol oxidizing enzymes was independent of dilution rate up to a relative growth rate /_max of 0.75. Only at higher dilution rates was catabolite repression observed.Abbreviations DOT dissolved oxygen tension - K _L a gas transfer coefficient - specific growth rate - _max maximum specific growth rate - K_s substrate saturation constant     

Variation of carbon isotope fractionation in hydrogenotrophic methanogenic microbial cultures and environmental samples at different energy status

Methane is a major product of anaerobic degradation of organic matter and an important greenhouse gas. Its stable carbon isotope composition can be used to reveal active methanogenic pathways, if associated isotope fractionation factors are known. To clarify the causes that lead to the wide variation of fractionation factors of methanogenesis from H₂ plus CO₂ (), pure cultures and various cocultures were grown under different thermodynamic conditions. In syntrophic and obligate syntrophic cocultures thriving on different carbohydrate substrates, fermentative bacteria were coupled to three different species of hydrogenotrophic methanogens of the families Methanobacteriaceae and Methanomicrobiaceae. We found that C‐isotope fractionation was correlated to the Gibbs free energy change (ΔG) of CH₄ formation from H₂ plus CO₂ and that the relation can be described by a semi‐Gauss curve. The derived relationship was used to quantify the average ΔG that is available to hydrogenotrophic methanogenic archaea in their habitat, thus avoiding the problems encountered with measurement of low H₂ concentrations on a microscale. Boreal peat, rice field soil, and rumen fluid, which represent major sources of atmospheric CH₄, exhibited increasingly smaller , indicating that thermodynamic conditions for hydrogenotrophic methanogens became increasingly more favourable. Vice versa, we hypothesize that environments with similar energetic conditions will also exhibit similar isotope fractionation. Our results, thus, provide a mechanistic constraint for modelling the ¹³C flux from microbial sources of atmospheric CH₄.     

青藏高原三个盐碱湖的产甲烷菌群和产甲烷代谢途径分析

【目的】分析青藏高原不同类型盐碱湖中的优势产甲烷菌群和优势产甲烷代谢途径。【方法】以不同盐度和植被类型的公珠错、昆仲错和无植被的兹格塘错的沉积物为研究对象,通过高通量测序和q PCR定量古菌16S r RNA多样性分析优势古菌类群;模拟原位盐浓度及p H,比较不同产甲烷底物(甲醇、三甲胺、乙酸和H_2/CO_2)富集沉积物的产甲烷速率,分析其优势产甲烷菌代谢类型。通过添加产甲烷抑制剂(2-溴乙烷磺酸盐),检测沉积物中产甲烷底物积累,确定不同盐碱湖中主要的产甲烷途径。【结果】昆仲错的优势菌群包括甲基/乙酸型的甲烷八叠球菌科(Methanosarcinaceae,11%),乙酸型的甲烷鬃菌科(Methanosaetaceae,7.9%)和氢型甲烷菌甲烷杆菌目(Methanomicrobiales,7.4%);公珠错和兹格塘错的优势菌群为甲烷鬃菌科(Methanosaetaceae)分别占15%和15.3%,及甲烷杆菌属(Methanobacterium)和甲基型的甲烷叶菌属(Methanolobus)。公珠错和昆仲错分别以乙酸和甲醇产甲烷速率最高,而兹格塘错从不同底物产甲烷速率无差异。抑制甲烷产生后,公珠错主要积累乙酸,昆仲错主要积累甲醇;兹格塘错不仅甲烷排放低,也无产甲烷物质显著积累。【结论】昆仲错沉积物中的甲烷主要来自甲醇,公珠错中的甲烷主要来自乙酸,而兹格塘错产甲烷和底物积累不活跃。因而推测高原盐碱湖主要的产甲烷途径和菌群可能与周围植被类型的相关性更高,而与盐度的直接相关性较低。     

Production of methane and hydrogen by anaerobic ciliates containing symbiotic methanogens

Rates of methane production by three anaerobic ciliates containing symbiotic methanogens (the marine Metopus contortus and Plagiopyla frontata, and the limnic Metopus palaeformis) were quantified. Hydrogen production by normal (containing active symbionts), aposymbiotic and BES-treated cells was also measured in the case of the marine species. Methanogenesis was closely coupled to host metabolism and growth; at maximum ciliate growth rates (20°C) each methanogen produced about 1 fmol CH₄ per hour corresponding to about 7, 4 and 0.35 pmol per ciliate per hour for M. contortus, P. frontata and M. palaeformis, respectively. Normal cells produced traces of H₂. Hydrogen production by BES-treated or aposymbiotic cells accounted for 75 and 45% of the methane production of normal M. contortus and P. frontata cells, respectively. However, it is possible that hydrogen production was partly inhibited in the absence of methanogens. Theoretical considerations suggest that hydrogen transfer is significant to the metabolism of larger anaerobic ciliates. Ciliates with methanogens produced CH₄ under microaerobic conditions due to their ability to maintain an anoxic intracellular environment at low external oxygen tensions. Methanogenesis was still detectable at a pO₂ of 0.63 kPa (3 %atm sat).     

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.