从宏基因组推测扎龙湿地未培养甲烷古菌Rice Cluster II的代谢途径与可能的盐碱适应性

【背景】湿地是重要的甲烷排放源,因为其中栖息着各种产甲烷古菌。已知未培养甲烷古菌Rice Cluster Ⅱ (RCⅡ)类群广泛分布于低温酸性和北方泥炭藓湿地、淡水湿地及草本沼泽等环境,但它们在低温盐碱湿地中的分布及代谢途径尚未知。【目的】分析扎龙盐碱湿地未培养甲烷古菌RCⅡ类群的多样性、推测产甲烷代谢途径及其潜在的盐碱适应机制。【方法】16S rRNA基因扩增子测序分析扎龙湿地土壤中甲烷古菌群组成;构建16S rRNA基因克隆文库分析扎龙湿地土壤RCⅡ的多样性;宏基因组分析推测RCⅡ古菌编码的产甲烷途径及与耐盐碱相关物质的合成基因。【结果】16SrRNA基因高通量测序发现未培养甲烷古菌的RCⅡ类群占扎龙盐碱湿地总甲烷古菌的13.280%±0.019%;系统发育学分析表明该湿地的RCⅡ由3个分支组成;宏基因组分析组装了2个优势的未培养RCⅡ的基因组,均含完整的氢还原二氧化碳产甲烷途径的基因,并编码海藻糖的转运与合成基因。【结论】扎龙盐碱湿地土壤富含未培养RCⅡ甲烷古菌,推测它们通过氢还原二氧化碳产甲烷,利用细胞内高的海藻糖适应盐碱环境。     

Colonization of rice roots with methanogenic archaea controls photosynthesis‐derived methane emission

The methane emitted from rice fields originates to a large part (up to 60%) from plant photosynthesis and is formed on the rice roots by methanogenic archaea. To investigate to which extent root colonization controls methane (CH₄) emission, we pulse‐labeled rice microcosms with ¹³CO₂ to determine the rates of ¹³CH₄ emission exclusively derived from photosynthates. We also measured emission of total CH₄ (¹²⁺¹³CH₄), which was largely produced in the soil. The total abundances of archaea and methanogens on the roots and in the soil were analysed by quantitative polymerase chain reaction of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase respectively. The composition of archaeal and methanogenic communities was determined with terminal restriction fragment length polymorphism (T‐RFLP). During the vegetative growth stages, emission rates of ¹³CH₄ linearly increased with the abundance of methanogenic archaea on the roots and then decreased during the last plant growth stage. Rates of ¹³CH₄ emission and the abundance of methanogenic archaea were lower when the rice was grown in quartz‐vermiculite with only 10% rice soil. Rates of total CH₄ emission were not systematically related to the abundance of methanogenic archaea in soil plus roots. The composition of the archaeal communities was similar under all conditions; however, the analysis of mcrA genes indicated that the methanogens differed between the soil and root. Our results support the hypothesis that rates of photosynthesis‐driven CH₄ emission are limited by the abundance of methanogens on the roots.     

The pterin lumazine inhibits growth of methanogens and methane formation

The pterin compound lumazine [2, 4-(1H, 3H)-pteridinedione] inhibited the growth of several methanogenic archaea completely at a concentration of ≤ 0.6 mM and was bacteriocidal for Methanobacterium thermoautotrophicum strain Marburg. In contrast, growth of two non-methanogenic archaea, several eubacteria, and one eukaryote was not strongly affected at much higher concentrations. In washed-cell suspensions, methanogenesis from H₂ and CO₂ by Mb. thermoautotrophicum or from H₂ and methanol by Methanosarcina barkeri was inhibited by addition of lumazine. In cell-free extracts of Mb. thermoautotrophicum, H₂-driven methane production from CO₂ or CH₃-S-CoM was completely inhibited by 0.6 mM lumazine. The results suggest that the compound may be useful in probing the methanogenesis pathway or in selecting against methanogens. Received: 30 January 1996 / Accepted 15 May 1996     

Biogenic methane production in formation waters from a large gas field in the North Sea

Methanogenesis was investigated in formation waters from a North Sea oil rimmed gas accumulation containing biodegraded oil, which has not been subject to seawater injection. Activity and growth of hydrogenotrophic methanogens was measured but acetoclastic methanogenesis was not detected. Hydrogenotrophic methanogens showed activity between 40 and 80°C with a temperature optimum (ca. 70°C) consistent with in situ reservoir temperatures. They were also active over a broad salinity range, up to and consistent with the high salinity of the waters (90 g l⁻¹). These findings suggest the methanogens are indigenous to the reservoir. The conversion of H₂ and CO₂ to CH₄ in methanogenic enrichments was enhanced by the addition of inorganic nutrients and was correlated with cell growth. Addition of yeast extract also stimulated methanogenesis. Archaeal 16S rRNA gene sequences recovered from enrichment cultures were closely related to Methanothermobacter spp. which have been identified in other high-temperature petroleum reservoirs. It has recently been suggested that methanogenic oil degradation may be a major factor in the development of the world’s heavy oils and represent a significant and ongoing process in conventional deposits. Although an oil-degrading methanogenic consortium was not enriched from these samples the presence and activity of communities of fermentative bacteria and methanogenic archaea was demonstrated. Stimulation of methanogenesis by addition of nutrients suggests that in situ methanogenic biodegradation of oil could be harnessed to enhance recovery of stranded energy assets from such petroleum systems.     

Wo lebten die ersten Zellen – und wovon?

How, and where, did the first cells on Earth grow? The last universal common ancestor of all cells (Luca) was long considered as the common ancestor of bacteria, archaea and eukaryotes. New trees of life have a host for the origin of mitochondria (of eukaryotes) branching within the archaea, making Luca the common ancestor of bacteria and archaea. New comparative genomic investigations have reconstructed Luca's microbial ecology. The 355 protein families that trace back to Luca by phylogenetic criteria describe Luca as anaerobic, CO₂ ‐ and N₂ ‐fixing, H₂ ‐dependent and thermophilic. Luca's biochemistry was replete with FeS clusters and radical reaction mechanisms, its cofactors reveal an essential role for transition metals in its metabolism. Luca lived in an anaerobic geochemical active environment rich in H₂ , CO₂ and iron. This lifestyle is similar to modern acetogens (bacteria) and methanogens (archaea), the physiologically most ancient microbes.     

Higher-level classification of the Archaea: evolution of methanogenesis and methanogens

We used a phylogenetic approach to analyze the evolution of methanogenesis and methanogens. We show that 23 vertically transmitted ribosomal proteins do not support the monophyly of methanogens, and propose instead that there are two distantly related groups of extant archaea that produce methane, which we have named Class I and Class II. Based on this finding, we subsequently investigated the uniqueness of the origin of methanogenesis by studying both the enzymes of methanogenesis and the proteins that synthesize its specific coenzymes. We conclude that hydrogenotrophic methanogenesis appeared only once during evolution. Genes involved in the seven central steps of the methanogenic reduction of carbon dioxide (CO(2)) are ubiquitous in methanogens and share a common history. This suggests that, although extant methanogens produce methane from various substrates (CO(2), formate, acetate, methylated C-1 compounds), these archaea have a core of conserved enzymes that have undergone little evolutionary change. Furthermore, this core of methanogenesis enzymes seems to originate (as a whole) from the last ancestor of all methanogens and does not appear to have been horizontally transmitted to other organisms or between members of Class I and Class II. The observation of a unique and ancestral form of methanogenesis suggests that it was preserved in two independent lineages, with some instances of specialization or added metabolic flexibility. It was likely lost in the Halobacteriales, Thermoplasmatales and Archaeoglobales. Given that fossil evidence for methanogenesis dates back 2.8 billion years, a unique origin of this process makes the methanogenic archaea a very ancient taxon.     

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.     

Identification of Methanogenic archaea in the Hyporheic Sediment of Sitka Stream

Methanogenic archaea produce methane as a metabolic product under anoxic conditions and they play a crucial role in the global methane cycle. In this study molecular diversity of methanogenic archaea in the hyporheic sediment of the lowland stream Sitka (Olomouc, Czech Republic) was analyzed by PCR amplification, cloning and sequencing analysis of the methyl coenzyme M reductase alpha subunit (mcrA) gene. Sequencing analysis of 60 clones revealed 24 different mcrA phylotypes from hyporheic sedimentary layers to a depth of 50 cm. Phylotypes were affiliated with Methanomicrobiales, Methanosarcinales and Methanobacteriales orders. Only one phylotype remains unclassified. The majority of the phylotypes showed higher affiliation with uncultured methanogens than with known methanogenic species. The presence of relatively rich assemblage of methanogenic archaea confirmed that methanogens may be an important component of hyporheic microbial communities and may affect CH₄ cycling in rivers.     

Dependence of the specific growth rate of methanogenic mutualistic cocultures on the methanogen

Methanogenesis from ethanol was studied in batch cocultures of a proton-reducing acetogenic Desulfovibrio sp. together with one of eight methanogenic bacteria representing five genera. A mathematical model of mutualistic cocultures predicts an equalisation in the specific growth rates of the two species which defines a specific growth rate for the coculture. At non-limiting ethanol concentrations the model predicts that the specific growth rate of the coculture is dependent upon the K _s (H₂) of the methanogen and its maximum specific growth rate in axenic culture when utilising H₂ as the energy source. We demonstrate experimentally that with methanogens known to have similar K _s (H₂) values, the specific growth rates of methanogenic mutualistic cocultures are dependent upon the maximum specific growth rates of the methanogens.     

Stable Carbon Isotope Fractionation by Methylotrophic Methanogenic Archaea

In natural environments methane is usually produced by aceticlastic and hydrogenotrophic methanogenic archaea. However, some methanogens can use C₁ compounds such as methanol as the substrate. To determine the contributions of individual substrates to methane production, the stable-isotope values of the substrates and the released methane are often used. Additional information can be obtained by using selective inhibitors (e.g., methyl fluoride, a selective inhibitor of acetoclastic methanogenesis). We studied stable carbon isotope fractionation during the conversion of methanol to methane in Methanosarcina acetivorans, Methanosarcina barkeri, and Methanolobus zinderi and generally found large fractionation factors (−83‰ to −72‰). We further tested whether methyl fluoride impairs methylotrophic methanogenesis. Our experiments showed that even though a slight inhibition occurred, the carbon isotope fractionation was not affected. Therefore, the production of isotopically light methane observed in the presence of methyl fluoride may be due to the strong fractionation by methylotrophic methanogens and not only by hydrogenotrophic methanogens as previously assumed.     

Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture

Carbon materials have been reported to facilitate direct interspecies electron transfer (DIET) between bacteria and methanogens improving methane production in anaerobic processes. In this work, the effect of increasing concentrations of carbon nanotubes (CNT) on the activity of pure cultures of methanogens and on typical fatty acid‐degrading syntrophic methanogenic coculture was evaluated. CNT affected methane production by methanogenic cultures, although acceleration was higher for hydrogenotrophic methanogens than for acetoclastic methanogens or syntrophic coculture. Interestingly, the initial methane production rate (IMPR) by Methanobacterium formicicum cultures increased 17 times with 5 g·L^?1 CNT. Butyrate conversion to methane by Syntrophomonas wolfei and Methanospirillum hungatei was enhanced (～1.5 times) in the presence of CNT (5 g·L^?1), but indications of DIET were not obtained. Increasing CNT concentrations resulted in more negative redox potentials in the anaerobic microcosms. Remarkably, without a reducing agent but in the presence of CNT, the IMPR was higher than in incubations with reducing agent. No growth was observed without reducing agent and without CNT. This finding is important to re‐frame discussions and re‐interpret data on the role of conductive materials as mediators of DIET in anaerobic communities. It also opens new challenges to improve methane production in engineered methanogenic processes.     

Quantitative Determination of H2-Utilizing Acetogenic and Sulfate-Reducing Bacteria and Methanogenic Archaea from Digestive Tract of Different Mammals

Total number of bacteria, cellulolytic bacteria, and H₂-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogens were not detected in some samples of each animal. Archaea methanogens were the largest H₂-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 × 10⁴ and 4 × 10⁴ cells ml⁻¹ respectively).     

Formate production and utilization by methanogens and by sewage sludge consortia — interference with the concept of interspecies formate transfer

Pure cultures of H₂/CO₂- and formate-utilizing methanogens or mixed consortia of sewage sludge generated some formate from H₂/CO₂ at H₂ partial pressure in the gas phase above 200 kPa. At decreasing H₂ partial pressure the formate was taken up again and converted to methane. If methanogenesis was inhibited by bromoethanesulphonic acid (BESA) or a high redox potential (–180 to –200 mV), formate-utilizing methanogens produced high amounts of formate from H₂/CO₂. No formate was excreted by the species, which could only utilize H₂/CO₂ for methanogenesis. In contrast, H₂ formation from formate was observed in cultures of Methanobacterium thermoformicicum and M. formicicum. Measurable amounts were, however, only formed if its immediate utilization for methane production was inhibited by BESA. In the light of the data on formate formation from H₂/CO₂ and its re-utilization by all formate-utilizing methanogens, the concept of interspecies formate transfer of Thiele and Zeikus should be reconsidered. In pure cultures of methanogens or complex ecosystems with excess H₂, formate formation seemed to serve more as a means of disposal of surplus reducing power than for H₂ transfer. Correspondence to: J. Winter     

Metabolic interactions between anaerobic bacteria in methanogenic environments

In methanogenic environments organic matter is degraded by associations of fermenting, acetogenic and methanogenic bacteria. Hydrogen and formate consumption, and to some extent also acetate consumption, by methanogens affects the metabolism of the other bacteria. Product formation of fermenting bacteria is shifted to more oxidized products, while acetogenic bacteria are only able to metabolize compounds when methanogens consume hydrogen and formate efficiently. These types of metabolic interaction between anaerobic bacteria is due to the fact that the oxidation of NADH and FADH₂ coupled to proton or bicarbonate reduction is thermodynamically only feasible at low hydrogen and formate concentrations. Syntrophic relationships which depend on interspecies hydrogen or formate transfer were described for the degradation of e.g. fatty acids, amino acids and aromatic compounds.     

Decreasing ammonia inhibition in thermophilic methanogenic bioreactors using carbon fiber textiles

Ammonia accumulation is one of the main causes of the loss of methane production observed during fermentation. We investigated the effect of addition of carbon fiber textiles (CFT) to thermophilic methanogenic bioreactors with respect to ammonia tolerance during the process of degradation of artificial garbage slurry, by comparing the performance of the reactors containing CFT with the performance of reactors without CFT. Under total ammonia-N concentrations of 3,000 mg L⁻¹, the reactors containing CFT were found to mediate stable removal of organic compounds and methane production. Under these conditions, high levels of methanogenic archaea were retained at the CFT, as determined by 16S rRNA gene analysis for methanogenic archaea. In addition, Methanobacterium sp. was found to be dominant in the suspended fraction, and Methanosarcina sp. was dominant in the retained fraction of the reactors with CFT. However, the reactors without CFT had lower rates of removal of organic compounds and production of methane under total ammonia-N concentrations of 1,500 mg L⁻¹. Under this ammonia concentration, a significant accumulation of acetate was observed in the reactors without CFT (130.0 mM), relative to the reactors with CFT (4.2 mM). Only Methanobacterium sp. was identified in the reactors without CFT. These results suggest that CFT enables stable proliferation of aceticlastic methanogens by preventing ammonia inhibition. This improves the process of stable garbage degradation and production of methane in thermophilic bioreactors that include high levels of ammonia.     

Suppression of methanogenesis by dissimilatory Fe(III)-reducing bacteria in tropical rain forest soils: implications for ecosystem methane flux

Tropical forests are an important source of atmospheric methane (CH₄), and recent work suggests that CH₄ fluxes from humid tropical environments are driven by variations in CH₄ production, rather than by bacterial CH₄ oxidation. Competition for acetate between methanogenic archaea and Fe(III)‐reducing bacteria is one of the principal controls on CH₄ flux in many Fe‐rich anoxic environments. Upland humid tropical forests are also abundant in Fe and are characterized by high organic matter inputs, steep soil oxygen (O₂) gradients, and fluctuating redox conditions, yielding concomitant methanogenesis and bacterial Fe(III) reduction. However, whether Fe(III)‐reducing bacteria coexist with methanogens or competitively suppress methanogenic acetate use in wet tropical soils is uncertain. To address this question, we conducted a process‐based laboratory experiment to determine if competition for acetate between methanogens and Fe(III)‐reducing bacteria influenced CH₄ production and C isotope composition in humid tropical forest soils. We collected soils from a poor to moderately drained upland rain forest and incubated them with combinations of ¹³C‐bicarbonate, ¹³C‐methyl labeled acetate (¹³CH₃COO^?), poorly crystalline Fe(III), or fluoroacetate. CH₄ production showed a greater proportional increase than Fe²⁺ production after competition for acetate was alleviated, suggesting that Fe(III)‐reducing bacteria were suppressing methanogenesis. Methanogenesis increased by approximately 67 times while Fe²⁺ production only doubled after the addition of ¹³CH₃COO^?. Large increases in both CH₄ and Fe²⁺ production also indicate that the two process were acetate limited, suggesting that acetate may be a key substrate for anoxic carbon (C) metabolism in humid tropical forest soils. C isotope analysis suggests that competition for acetate was not the only factor driving CH₄ production, as ¹³C partitioning did not vary significantly between ¹³CH₃COO^? and ¹³CH₃COO^?+Fe(III) treatments. This suggests that dissimilatory Fe(III)‐reduction suppressed both hydrogenotrophic and aceticlastic methanogenesis. These findings have implications for understanding the CH₄ biogeochemistry of highly weathered wet tropical soils, where CH₄ efflux is driven largely by CH₄ production.     

Anaerobic 2-Propanol Degradation in Anoxic Paddy Soil and the Possible Role of Methanogens in Its Degradation

The anaerobic degradation of 2-propanol in anoxic paddy soil was studied with soil cultures and a 2-propanol-utilizing methanogen. Acetone was the first and the major intermediate involved in the methanogenic degradation of 2-propanol. Analyses with a methanogenesis inhibitor, bacteria antibiotics, and the addition of H₂ to the gas phase revealed that 2-propanol oxidation to acetone directly occurred using 2-propanol-utilizing methanogens, but not with H₂-producing syntrophic bacteria, for which the removal of acetone is required for complete 2-propanol oxidation. The 2-propanol-utilizing strain IIE1, which is phylogenetically closely related to Methanoculleus palmolei, was isolated from paddy soil, and the potential role of the strain in 2-propanol degradation was investigated. 2-Propanol is one of the representative fermentation intermediates in anaerobic environments. This is the first report on the anaerobic 2-propanol degradation process.     

Metabolism of methanogens

Methanogenic archaea convert a few simple compounds such as H₂ + CO₂, formate, methanol, methylamines, and acetate to methane. Methanogenesis from all these substrates requires a number of unique coenzymes, some of which are exclusively found in methanogens. H₂-dependent CO₂ reduction proceeds via carrier-bound C₁ intermediates which become stepwise reduced to methane. Methane formation from methanol and methylamines involves the disproportionation of the methyl groups. Part of the methyl groups are oxidized to CO₂, and the reducing equivalents thereby gained are subsequently used to reduce other methyl groups to methane. This process involves the same C₁ intermediates that are formed during methanogenesis from CO₂. Conversion of acetate to methane and carbon dioxide is preceeded by its activation to acetyl-CoA. Cleavage of the latter compound yields a coenzyme-bound methyl moiety and an enzyme-bound carbonyl group. The reducing equivalents gained by oxidation of the carbonyl group to carbon dioxide are subsequently used to reduce the methyl moiety to methane. All these processes lead to the generation of transmembrane ion gradients which fuel ATP synthesis via one or two types of ATP synthases. The synthesis of cellular building blocks starts with the central anabolic intermediate acetyl-CoA which, in autotrophic methanogens, is synthesized from two molecules of CO₂ in a linear pathway.