Molecular evolution of methanogens based on their metabolic facets

The information provided by completely sequenced genomes of methanogens can yield insights into a deeper molecular understanding of evolutionary mechanisms.This review describes the advantages of using...     

In silico description of cobalt and nickel assimilation systems in the genomes of methanogens

Methanogens are a diverse group of organisms that can live in a wide range of environments. Herein, cobalt and tungsten assimilation pathways have proposed to be established in the genomes of Methanococcus maripaludies C5 and Methanosarcina mazei Go1, respectively. All of the proteins involved in the proposed pathways were identified from public domain databases and then complied manually to reconstruct the pathways. The function of proteins with unknown function was assigned by a combined prediction approach. Totally, 17 proteins were identified to cobalt transport and assimilation processes whereas 7 proteins reported to tungsten assimilation system. Phylogenetic analysis of this study revealed that heavy metal transporter of methanogens could be evolved from closely related members in the different genera of methanogens. Nevertheless, genes encoding for metal resistance proteins could be originated from thermophilic and sulfur reducing bacteria. Many metalloenzymes in methanogens were very unique to the species of methanogens. It implied that these metal ions were utilized to produce the precursors for energy driven processes of methanogens. This study suggested that in combination of systems models and evolutionary inference can only correlate metabolic fluxes and physiological changes in methanogens. In silico models of this study will provide insights to design experiments for heavy metal assimilation processes of methanogens growing under heavy metal-rich environments and or in a laboratory condition.     

New perspectives on anaerobic methane oxidation

Anaerobic methane oxidation is a globally important but poorly understood process. Four lines of evidence have recently improved our understanding of this process. First, studies of recent marine sediments indicate that a consortium of methanogens and sulphate-reducing bacteria are responsible for anaerobic methane oxidation; a mechanism of 'reverse methanogenesis' was proposed, based on the principle of interspecies hydrogen transfer. Second, studies of known methanogens under low hydrogen and high methane conditions were unable to induce methane oxidation, indicating that 'reverse methanogenesis' is not a widespread process in methanogens. Third, lipid biomarker studies detected isotopically depleted archaeal and bacterial biomarkers from marine methane vents, and indicate that Archaea are the primary consumers of methane. Finally, phylogenetic studies indicate that only specific groups of Archaea and SRB are involved in methane oxidation. This review integrates results from these recent studies to constrain the responsible mechanisms.     

The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review

Among different conversion processes for biomass, biological anaerobic digestion is one of the most economic ways to produce biogas from various biomass substrates. In addition to hydrolysis of polymeric substances, the activity and performance of the methanogenic bacteria is of paramount importance during methanogenesis. The aim of this paper is primarily to review the recent literature about the occurrence of both acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of particulate biomass to methane (not wastewater treatment), while this review does not cover the activity of the acetate oxidizing bacteria. Both acetotrophic and hydrogenotrophic methanogens are essential for the last step of methanogenesis, but the reports about their roles during this phase of the process are very limited. Despite, some conclusions can still be drawn. At low concentrations of acetate, normally filamentous Methanosaeta species dominate, e.g., often observed in sewage sludge. Apparently, high concentrations of toxic ionic agents, like ammonia, hydrogen sulfide (H₂S) and volatile fatty acids (VFA), inhibit preferably Methanosaetaceae and especially allow the growth of Methanosarcina species consisting of irregular cell clumps, e.g., in cattle manure. Thermophilic conditions can favour rod like or coccoid hydrogenotrophic methanogens. Thermophilic Methanosarcina species were also observed, but not thermophilic Methanosaetae. Other environmental factors could favour hydrogentrophic bacteria, e.g., short or low retention times in a biomass reactor. However, no general rules regarding process parameters could be derivated at the moment, which favours hydrogenotrophic methanogens. Presumably, it depends only on the hydrogen concentration, which is generally not mentioned in the literature.     

Sulfate Reducers Can Outcompete Methanogens at Freshwater Sulfate Concentrations

Acetate and hydrogen metabolism by sulfate reducers and methanogens in the profundal sediments of an oligotrophic lake were examined. Inhibition of sulfate reduction with molybdate stimulated methane production from both hydrogen and acetate. Molybdate did not stimulate methane production in sediments that were preincubated to deplete the sulfate pool. Sulfate reduction accounted for 30 to 81% of the total of terminal metabolism proceeding through sulfate reduction and methane production in Eckman grab samples of surface sediments. The ability of sulfate reducers to effectively compete with methanogens for acetate was related to the sulfate reducers' lower half-saturation constant for acetate metabolism at in situ sulfate concentrations. Processes other than sulfate reduction and methanogenesis consumed hydrogen at elevated hydrogen partial pressures and prevented a kinetic analysis of hydrogen uptake by sulfate reducers and methanogens. The demonstration that sulfate reducers can successfully compete with methanogens for hydrogen and acetate in sediments at in situ sulfate concentrations of 60 to 105 μM extends the known range of sediment habitats in which sulfate reduction can be a dominant terminal process.     

Detection and Quantitation of Methanogens by Enzyme-Linked Immunosorbent Assay

Antisera to two methanogenic bacteria, Methanosarcina barkeri and a Methanobacterium sp., were raised in rabbits and used to develop an enzyme-linked immunosorbent assay (ELISA) method. ELISA was shown to be a sensitive technique, detecting as little as 4 ng of methanogen protein. The specificities of the antisera toward other methanogens were evaluated, and it was found that the antisera recognized species of the same genus as the immunizing species, but gave very little cross-reaction with methanogens of different genera. ELISA was used to estimate the growth of methanogens in pure culture. In natural environments and in anaerobic digesters methanogens exist as part of a mixed bacterial community, so the possibility of using ELISA to quantitate methanogens in mixed cultures was examined. The two antisera gave very little reaction in ELISA when non-methanogenic bacteria were used as antigens and ELISA was used to quantitate methanogens in an acetate enrichment culture. I conclude that the ELISA is a useful method for quantitating methanogens in defined mixed cultures, but has limited applicability to more complex systems.     

Analysis of Methanogen Diversity in the Rumen Using Temporal Temperature Gradient Gel Electrophoresis: Identification of Uncultured Methanogens

A temporal temperature gradient gel electrophoresis (TTGE) method was developed to determine the diversity of methanogen populations in the rumen. Tests with amplicons from genomic DNA from 12 cultured methanogens showed single bands for all strains, with only two showing apparently comigrating bands. Fingerprints of methanogen populations were analyzed from DNA extracted from rumen contents from two cattle and four sheep grazing pasture. For one sheep, dilution cultures selective for methanogens were grown and the culturable methanogens in each successive dilution examined by TTGE. A total of 66 methanogen sequences were retrieved from bands in fingerprints and analyzed to reveal the presence of methanogens belonging to the Methanobacteriales, the Methanosarcinales, and to an uncultured archaeal lineage. Twenty-four sequences were most similar to Methanobrevibacter ruminantium, five to Methanobrevibacter smithii, four to Methanosphaera stadtmanae, and for three, the nearest match was Methanimicrococcus blatticola. The remaining 30 sequences did not cluster with sequences from cultured archaea, but when combined with published novel sequences from clone libraries formed a monophyletic lineage within the Euryarchaeota, which contained two previously unrecognized clusters. The TTGE bands from this lineage showed that the uncultured methanogens had significant population densities in each of the six rumen samples examined. In cultures of dilutions from one rumen sample, TTGE examination revealed these methanogens at a level of at least 10⁵ g⁻¹. Band intensities from low-dilution cultures indicated that these methanogens were present at similar densities to Methanobrevibacter ruminantium-like methanogens, the sole culturable methanogens in high dilutions (10⁶–10⁻¹⁰ g⁻¹). It is suggested that the uncultured methanogens together with Methanobrevibacter spp. may be the predominant methanogens in the rumen. The TTGE method presented in this article provides a new opportunity for characterizing methanogen populations in the rumen microbial ecosystem.     

Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC-I

Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33°56'N, 102°52'E) region and under the annual temperature around 1°C. Previous studies indicated that Zoige wetland was one of the CH₄ emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales , could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10⁷ cells per gram of soil, accounting for about 30% of the total Archeae . By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO₂, methanol or trimethylamine was determined to increase to 10⁸ cells ml⁻¹, but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30°C and 15°C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae , and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.     

Methyl coenzyme M reductase (<Emphasis Type="Italic">mcrA</Emphasis>) gene based phylogenetic analysis of methanogens population in Murrah buffaloes (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>)

The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.     

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane

Hydrogenotrophic methanogens can use gaseous substrates, such as H₂ and CO₂, in CH₄ production. H₂ gas is used to reduce CO₂. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH₄ production from CO₂ and H₂. CO₂ and H₂ were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5–5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH₄ production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH₄ production process. The results show that acidic operation of a CH₄ production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.     

Soil type links microbial colonization of rice roots to methane emission

Most of the methane (CH₄) emission from rice fields is derived from plant photosynthates, which are converted to CH₄. Rice cluster I (RC-1) archaea colonizing the rhizosphere were found to be the methanogens responsible for this process. Hence, RC-1 methanogens seem to play a crucial role in emission of the greenhouse gas CH₄. We determined the community composition and activity of methanogens colonizing the roots of eight different rice cultivars after growth on both Italian rice soil and river bank soil, which contained different communities of methanogenic archaea. The community composition was analyzed by terminal restriction fragment length polymorphism and cloning/sequencing of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase. When grown on rice field soil, the methanogenic community of the different rice cultivars was always dominated by RC-1 methanogens. In contrast, roots were colonized by Methanomicrobiales when grown on river bank soil, in which RC-1 methanogens were initially not detectable. Roots colonized with Methanomicrobiales compared with RC-1 exhibited lower CH₄ production and CH₄ emission rates. The results show that the type of methanogens colonizing rice roots has a potentially important impact on the global CH₄ cycle.     

嗜酸产甲烷菌及其在厌氧处理中的应用

产甲烷菌在自然界碳素循环过程中发挥着重要作用.酸性泥炭沼泽环境中存在着多种未知的产甲烷古菌,其中嗜酸产甲烷菌因其特殊的生长代谢特征近年来引起学者的广泛关注.若将嗜酸产甲烷菌应用于高浓度有机废物或废水的厌氧消化过程中,可从本质上克服因酸积累造成的产甲烷抑制,减少运行成本,扩展厌氧消化处理技术的应用范围.本文综述了嗜酸产甲烷菌的富集分离培养方法、生理生化特性、代谢特征及相关分子生物学研究等内容,并对其在厌氧处理中的应用前景进行了分析和展望,提出了未来研究的方向.     

闽江口芦苇沼泽湿地土壤产甲烷菌群落结构的垂直分布

应用PCR-RFLP技术及测序分析对闽江口芦苇湿地土壤产甲烷菌群落结构的垂直分布特征进行了研究。在构建的6个克隆文库中,每个克隆文库随机挑选100个克隆进行菌落PCR验证,共得到591个阳性克隆。PCR产物经限制性内切酶MspⅠ进行RFLP分析后得到37个不同的分类操作单元(OTUs)。对37个克隆子进行了序列测定,与GenBank数据库中的序列进行比对,最近相似性在91%—99%之间。RFLP分析和系统发育分析表明,闽江口芦苇湿地土壤中产甲烷菌群落包括3大类群:甲烷杆菌目(Methanobacteriales)、甲烷微菌目(Methanomirobiales)和甲烷八叠球菌目(Methanosarcinales)。不同土壤深度中产甲烷菌群落的分布呈现出不同的特征。土壤表层(0—10 cm)优势产甲烷菌类群为Methanoregula,约占76%;10—20 cm土层主要的产甲烷菌类群为Methanolinea和Methanoregula,分别约占23%和29%;20—30cm土层优势的产甲烷菌类群为Methanolinea,约占66%。Shannon指数(H’)和Simpson多样性指教(D)表明,10—20cm土层产甲烷菌多样性高于土壤表层(0—10 cm)和20—30 cm土层。37个测序OTUs中有26个OTUs属于不可培养的产甲烷菌序列,表明闽江口芦苇湿地土壤中存在大量不可培养的产甲烷菌。     

嗜冷产甲烷菌及其在废水厌氧处理中的应用

嗜冷产甲烷菌对于自然界的碳素循环具有非常重要的意义,近年来引起了国内外学者的广泛关注.利用嗜冷产甲烷菌实现低温厌氧生物处理过程,可从本质上突破低温厌氧工艺的技术瓶颈,进而大大拓展厌氧生物处理技术的应用范围并降低废水处理的成本.本文针对研究者广泛关注的热点问题,从分离培养及生理生化特性、适冷机制和分子生物学研究几个方面,对嗜冷产甲烷菌的研究进展进行了全面的综述,并对其在低温厌氧生物处理技术中的应用前景进行了分析和展望.     

Gene Order Phylogeny and the Evolution of Methanogens

Methanogens are a phylogenetically diverse group belonging to Euryarchaeota. Previously, phylogenetic approaches using large datasets revealed that methanogens can be grouped into two classes, “Class I” and “Class II”. However, some deep relationships were not resolved. For instance, the monophyly of “Class I” methanogens, which consist of Methanopyrales, Methanobacteriales and Methanococcales, is disputable due to weak statistical support. In this study, we use MSOAR to identify common orthologous genes from eight methanogen species and a Thermococcale species (outgroup), and apply GRAPPA and FastME to compute distance-based gene order phylogeny. The gene order phylogeny supports two classes of methanogens, but it differs from the original classification of methanogens by placing Methanopyrales and Methanobacteriales together with Methanosarcinales in Class II rather than with Methanococcales. This study suggests a new classification scheme for methanogens. In addition, it indicates that gene order phylogeny can complement traditional sequence-based methods in addressing taxonomic questions for deep relationships.     

Phylogenetic analysis of methanogens from the bovine rumen

Background  

Interest in methanogens from ruminants has resulted from the role of methane in global warming and from the fact that cattle typically lose 6 % of ingested energy as methane. Several species of methanogens have been isolated from ruminants. However they are difficult to culture, few have been consistently found in high numbers, and it is likely that major species of rumen methanogens are yet to be identified.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Anaerobic microbial counts of different potential anaerobic inocula

Sludges of various origin were analysed on the basis of microbial counts of anaerobes in order to investigate potential and alternative sources for inoculating anaerobic digestors. Sludges from neutralization and oil and fat separation tanks of a sugar cane factory, and from a stabilisation pond of a purified terephthalic-acid-producing plant were found to be adequate potential inocula. Activated sludges were found to have both hydrogenotrophic and acetoclastic methanogens and obligate hydrogen-producing acetogens (OHPA). In all sludges tested, hydrogenotrophic methanogens were the dominant strict anaerobes. The ratio between most probable number counts of acetoclastic methanogens and OPHA seems to be different between dispersed sludges and granular sludges. Correspondence to: J. P. Guyot     

The effect of methanogen growth on mineral substrates: will Ni markers of methanogen‐based communities be detectable in the rock record?

Methanogens, thought to be present on early Earth, have a high requirement for Ni, suggesting that Ni utilization could be a potential biosignature for methanogens if enhanced Ni extraction from surrounding minerals accompanies methanogenic growth. To test the potential for such Ni extraction from minerals by methanogens, Ni release from Ni‐containing silicate glass was measured in Ni‐free growth medium in the presence of the methanogen Methanothermobacter thermoautotrophicus (average pH ∼7.0) and observed to be higher than an abiotic control (average pH ∼6.8). However, batch dissolution experiments and a siderophore assay indicate that cell exudates such as siderophores, low molecular weight organic acids, or lysates accompanying cell death are not responsible for the observed increase in Ni release rate. In addition, scanning electron microscopy (SEM) shows little to no evidence of direct microbe–mineral interactions such as biofilms or pitting. Instead, comparison with abiotic experiments suggests that changes in pH due to CO₂ uptake may be responsible for enhanced dissolution in the presence of metabolizing cells. These results document that methanogens may not preferentially extract Ni from surrounding minerals although they may indirectly affect mineral reaction rates that are pH sensitive. Thus identifiable Ni biosignatures may not exist in the rock record to document the presence of methanogens on early Earth or Mars.     

模拟增温对青海湖鸟岛土壤产甲烷菌群落特征的影响

甲烷生成与产甲烷菌群落关系密切,探究温度升高对产甲烷菌群落的影响可以预测产甲烷菌群落特征对气候变暖的响应.以青海湖鸟岛为研究对象,通过高通量测序方法开展研究.结果表明,基于97％的序列相似度聚类,共得到697个操作分类单元.鸟岛土壤产甲烷菌群落以甲烷微菌目(Methanomicrobiales)、甲烷八叠球菌目(Met...