Methanotrophy - Environmental, Industrial and Medical Applications.

Aerobic methanotrophs are an intriguing group of microbes with the singular ability to consume methane as their sole source of carbon and energy. As such, methanotrophs are receiving increased attention to control methane emissions to limit future climate change. Methanotrophs have a wide range of other applications, including pollutant remediation and methane valorization (e.g. conversion of methane to protein, bioplastics, and biodiesel amongst other products). Methanotrophs also produce a novel copper-binding compound, methanobactin, that has significant potential for the treatment of copper-related human pathologies. Here we provide an overview of aerobic methanotrophy, describe current and future applications of these unique microbes, as well as discuss various strategies one can consider to better realize the opportunities these microbes present.

Diversity of Methane Cycling Microorganisms in Soils and Their Relation to Oxygen.

Microorganisms are important players in the global methane cycle. Anaerobic methanogenic archaea are largely responsible for methane production, while aerobic methanotrophic bacteria, as well as anaerobic methanotrophic bacteria and archaea, are involved in methane oxidation. In anoxic wetland soils, methanogens produce methane, while methanotrophs act as a filter and reduce methane emissions. In the predominantly oxic upland soils, aerobic methanotrophs oxidize atmospheric methane. This review gives an overview of the diversity of methanogenic and methanotrophic microorganisms, highlights recent discoveries and provides information concerning their occurrence in soils. Recent findings indicate that the methanogenic and methanotrophic lifestyles are more widespread in microorganisms than previously thought, and that the metabolic versatility of some methane-cycling organisms is broader than known from well characterized cultivated organisms. It also turned out that the control of methanogenic and methanotrophic bacteria by oxygen is more complex than previously thought. The implications this finding may have for the life of these microorganisms in soils and on soil methane fluxes is discussed.

Metagenomic Approaches Unearth Methanotroph Phylogenetic and Metabolic Diversity.

Methanotrophic microorganisms utilize methane as an electron donor and a carbon source. To date, the capacity to oxidize methane is restricted to microorganisms from three bacterial and one archaeal phyla. Most of our knowledge of methanotrophic metabolism has been obtained using highly enriched or pure cultures grown in the laboratory. However, many methanotrophs currently evade cultivation, thus metagenomics provides a complementary approach for gaining insight into currently unisolated microorganisms. Here we synthesize the studies using metagenomics to glean information about methanotrophs. We complement this summary with an analysis of methanotroph marker genes from 235 publically available metagenomic datasets. We analyze the phylogenetic and environmental distribution of methanotrophs sampled by metagenomics. We also highlight metabolic insights that methanotroph genomes assembled from metagenomes are illuminating. In summary, metagenomics has increased methanotrophic foliage within the tree of life, as well as provided new insights into methanotroph metabolism, which collectively can guide new cultivation efforts. Lastly, given the importance of methanotrophs for biotechnological applications and their capacity to filter greenhouse gases from a variety of ecosystems, metagenomics will continue to be an important component in the arsenal of tools needed for understanding methanotroph diversity and metabolism in both engineered and natural systems.

CH4 flux and methanogen community dynamics from five common emergent vegetations in a full-scale constructed wetland.

To investigate the effects of emergent plants on CH4 efflux and elucidate the key factors responsible for these effects, annual monitoring of CH4 emissions and methanogen community dynamics in a full-scale constructed wetland (CW) was conducted. Five emergent plants (Typha orientalis, Cyperus alternifolius, Arundo domax, Iris pseudacorus, and Thalia dealbata) commonly used in CWs were selected for investigation. The greatest CH4 flux (annual mean 19.4 mg m-2 h-1) was observed from I. pseudacorus, while the lowest CH4 flux (7.1 mg m-2 h-1) was observed from Thalia dealbata. The CH4 flux from five emergent plants showed marked seasonal variation. Total nitrogen (TN) and total phosphorous (TP) were weakly correlated with CH4 emissions, whereas total carbon (TC) and root biomass of plants were positively correlated with CH4 emissions. Quantitative real-time PCR (q-PCR) analysis indicated that the gene abundance of eubacterial 16S rRNA, particulate methane monooxygenase (pmoA) and methyl coenzyme M reductase (mcrA) significantly differed among plant species. Differences in TC, root biomass, and dissolved oxygen (DO) caused by plant species were potential factors responsible for differences in methanogens, methanotrophs, and CH4 emissions. Methanobacteriaceae, Methanoregulaceae, Methanomicrobiaceae, and Methanosarcinaceae were the dominant families of methanogens. The pathways of methanogenesis from the five emergent plants differed, with the main pathway being hydrogenotrophic, while both hydrogenotrophic and acetotrophic methanogens were involved in A. domax. Redundancy analysis (RDA) further indicated that emergent plant types had a profound influence on the methanogenic communities. Taken together, these results suggest emergent plant species can significantly influence CH4 fluxes in CW through microbial communities, biochemical pathways for methanogenesis, TC, and DO. Furthermore, plant species in CWs should be considered an important factor in evaluating greenhouse gases emission. Finally, it is necessary to effectively manage CWs vegetation to maximize their environmental benefits. Graphical abstract ᅟ.

Community structure of the metabolically active rumen bacterial and archaeal communities of dairy cows over the transition period.

Dairy cows experience dramatic changes in host physiology from gestation to lactation period and dietary switch from high-forage prepartum diet to high-concentrate postpartum diet over the transition period (parturition +/- three weeks). Understanding the community structure and activity of the rumen microbiota and its associative patterns over the transition period may provide insight for e.g. improving animal health and production. In the present study, rumen samples from ten primiparous Holstein dairy cows were collected over seven weeks spanning the transition period. Total RNA was extracted from the rumen samples and cDNA thereof was subsequently used for characterizing the metabolically active bacterial (16S rRNA transcript amplicon sequencing) and archaeal (qPCR, T-RFLP and mcrA and 16S rRNA transcript amplicon sequencing) communities. The metabolically active bacterial community was dominated by three phyla, showing significant changes in relative abundance range over the transition period: Firmicutes (from prepartum 57% to postpartum 35%), Bacteroidetes (from prepartum 22% to postpartum 18%) and Proteobacteria (from prepartum 7% to postpartum 32%). For the archaea, qPCR analysis of 16S rRNA transcript number, revealed a significant prepartum to postpartum increase in Methanobacteriales, in accordance with an observed increase (from prepartum 80% to postpartum 89%) in relative abundance of 16S rRNA transcript amplicons allocated to this order. On the other hand, a significant prepartum to postpartum decrease (from 15% to 2%) was observed in relative abundance of Methanomassiliicoccales 16S rRNA transcripts. In contrast to qPCR analysis of the 16S rRNA transcripts, quantification of mcrA transcripts revealed no change in total abundance of metabolically active methanogens over the transition period. According to T-RFLP analysis of the mcrA transcripts, two Methanobacteriales genera, Methanobrevibacter and Methanosphaera (represented by the T-RFs 39 and 267 bp), represented more than 70% of the metabolically active methanogens, showing no significant changes over the transition period; minor T-RFs, likely to represent members of the order Methanomassiliicoccales and with a relative abundance below 5% in total, decreased significantly over the transition period. In accordance with the T-RFLP analysis, the mcrA transcript amplicon sequencing revealed Methanobacteriales to cover 99% of the total reads, dominated by the genera Methanobrevibacter (75%) and Methanosphaera (24%), whereas the Methanomassiliicoccales order covered only 0.2% of the total reads. In conclusion, the present study showed that the structure of the metabolically active bacterial and archaeal rumen communities changed over the transition period, likely in response to the dramatic changes in physiology and nutritional factors like dry matter intake and feed composition. It should be noted however that for the methanogens, the observed community changes were influenced by the analyzed gene (mcrA or 16S rRNA).

Changes in microbial community structures due to varying operational conditions in the anaerobic digestion of oxytetracycline-medicated cow manure.

Management of manure containing veterinary antibiotics is a major concern in anaerobic treatment systems because of their possible adverse effects on microbial communities. Therefore, the aim of study was to investigate how oxytetracycline (OTC) influences bacteria and acetoclastic and hydrogenotrophic methanogens under varying operational conditions in OTC-medicated and non-medicated anaerobic cow manure digesters. Concentrations of OTC and its metabolites throughout the anaerobic digestion were determined using ultraviolet-high-performance liquid chromatography (UV-HPLC) and tandem liquid chromatography-mass spectrometry (LC/MS/MS), respectively. Fluorescent in situ hybridization, denaturing gradient gel electrophoresis, cloning, and sequencing analyses were used to monitor changes in microbial community structures. According to the results of analytical and molecular approaches, operating conditions highly influence active microbial community dynamics and associate with biogas production and elimination of OTC and its metabolites during anaerobic digestion of cow manure in the presence of an average initial concentration of 2.2 mg OTC/L. The impact of operating conditions has a drastic effect on acetoclastic methanogens than hydrogenotrophic methanogens and bacteria.

Abundance and potential metabolic activity of methanogens in well-aerated forest and grassland soils of an alpine region.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

Effects of freezing storage on the DNA extraction and microbial evaluation from anaerobic digested sludges.

BACKGROUND: The anaerobic digestion is one of the most spread renewable energy technology. The input biomasses included various environmental problematic wastes such as sludge coming from wastewater treatment plant (WWTP) and organic fraction of municipal solid waste (OFMSW). As biomolecular procedures have become important tools for the microbial characterisation of anaerobic samples coming from the reactors, it is crucial sampling and extracting properly DNA in order to employ such types of techniques. The current study is aimed to evaluate how freezing temperature and length of storage at -20 °C influence both the extracted DNA yield and microbial community quantifications from digested sludge samples collected at full-scale plants. RESULTS: From WWTP sludge samples, we observed a reduction of DNA concentration comparing fresh and stored samples for 10 days at -20 °C (ANOVA test p < 0.0001), with an estimated DNA loss of approximately 65% for such types of samples, however the methanogen communities can be assessed respecting the fresh conditions. From OFMSW sludge samples, we observed a reduction in extracted DNA (-90%), after 120 frozen days, while microbial communities are determined respecting the fresh conditions within 2 months of frozen storage. CONCLUSIONS: The remarkable effect of frozen storage on sludge samples suggests as the better procedure to perform the DNA extraction from fresh sample. On the other hand it is not generally possible, so approximately 2 months of storage at -20 °C appears to be suitable time at which DNA concentrations remain sufficient to perform coherent microbial characterization through quantitative qRT-PCR.

Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods.

Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥ 91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6-V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to 16S rRNA gene references produced taxonomic identification to Order level including 2-3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19-35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10-18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6-V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.

Finding a robust strain for biomethanation: anaerobic fungi (Neocallimastigomycota) from the Alpine ibex (Capra ibex) and their associated methanogens.

Anaerobic fungi occupy the rumen and digestive tract of herbivores, where they play an important role in enzymatic digestion of lignocellulosic and cellulosic substrates, i.e. organic material that their hosts are unable to decompose on their own. In this study we isolated anaerobic fungi from a typical alpine herbivore, the Alpine ibex (C. ibex). Three fungal strains, either as pure culture (ST2) or syntrophic co-culture with methanogens (ST3, ST4) were successfully obtained and morphologically characterised by different microscopy- and staining-techniques and by rDNA ITS gene sequencing. The isolated fungi were identified as Neocallimastix frontalis (ST2) and Caecomyces communis (ST3 and ST4). We introduce a novel field of application for lactofuchsin-staining, combined with confocal laser scanning microscopy. This approach proved as an effective method to visualize fungal structures, especially in the presence of plant biomass, generally exhibiting high autofluorescence. Moreover, we could demonstrate that fungal morphology is subject to changes depending on the carbon source used for cultivation. Oxygen tolerance was confirmed for both, C. communis-cultures for up to three, and for the N. frontalis-isolate for up to 12 h, respectively. With PCR, FISH and an oligonucleotide microarray we found associated methanogens (mainly Methanobacteriales) for C. communis, but not for N. frontalis.

The 16S rRNA and mcrA gene based comparative diversity of methanogens in cattle fed on high fibre based diet.

In the present study, the diversity of rumen methanogens in crossbred Karan Fries cattle was determined by constructing 16S rRNA and mcrA (methyl coenzyme-M reductase α subunit) gene libraries using specific primers. All thirteen OTUs or phylotypes from 16S rRNA library clustered with order Methanobacteriales, twelve of which aligned with Methanobrevibacter spp., whereas one OTU resemble with Methanosphaera stadtmanae. Out of eighteen OTUs identified from mcrA gene library, fifteen clustered with order Methanobacteriales, two resemble with Methanomicrobiales and remaining one grouped with Methanosarcinales. These results revealed that Methanobrevibacter phylotype was predominantly present in Karan Fries crossbred cattle fed on high fibrous diet containing wheat straw. Compared to 16S rRNA gene, mcrA gene OTUs clustered in three orders providing better insights of rumen methanogens diversity in cattle.

McrA gene and 16S rRNA gene in the phylogenetic analysis of methanogens in the rumen of faunated and unfaunated cattle.

The influence of rumen protozoa on the composition of rumen methanogens was studied by using seven growing Holstein cattle divided into two groups: four faunated and three unfaunated. 16S ribosomal RNA gene (rDNA) and methyl coenzyme-M reductase (MCR) α subunit (mcrA) gene clonal libraries were constructed. The results of each analysis showed that Methanobacteriales was dominant in the rumen of both groups. By mcrA gene analysis, 22.1% of unfaunated clones were classified into unfaunated group 1, which was not detected from faunated cattle. The 16S rRNA gene analysis showed that the number of operational taxonomic units was higher in unfaunated than faunated cattle, suggesting the diversity of methanogens tended to be higher by the removal of protozoa. The results of the LIBSHUFF program indicated that the 16S rRNA gene and mcrA gene clone libraries for the faunated group differed from those for the unfaunated group (P = 0.001). It was suggested that the presence of protozoa strongly affected the composition of rumen methanogens.

Integrated biogas upgrading and hydrogen utilization in an anaerobic reactor containing enriched hydrogenotrophic methanogenic culture.

Biogas produced by anaerobic digestion, is mainly used in a gas motor for heat and electricity production. However, after removal of CO(2) , biogas can be upgraded to natural gas quality, giving more utilization possibilities, such as utilization as autogas, or distant utilization by using the existing natural gas grid. The current study presents a new biological method for biogas upgrading in a separate biogas reactor, containing enriched hydrogenotrophic methanogens and fed with biogas and hydrogen. Both mesophilic- and thermophilic anaerobic cultures were enriched to convert CO(2) to CH(4) by addition of H(2) . Enrichment at thermophilic temperature (55°C) resulted in CO(2) and H(2) bioconversion rate of 320 mL CH(4) /(gVSS h), which was more than 60% higher than that under mesophilic temperature (37°C). Different dominant species were found at mesophilic- and thermophilic-enriched cultures, as revealed by PCR-DGGE. Nonetheless, they all belonged to the order Methanobacteriales, which can mediate hydrogenotrophic methanogenesis. Biogas upgrading was then tested in a thermophilic anaerobic reactor under various operation conditions. By continuous addition of hydrogen in the biogas reactor, high degree of biogas upgrading was achieved. The produced biogas had a CH(4) content, around 95% at steady-state, at gas (mixture of biogas and hydrogen) injection rate of 6 L/(L day). The increase of gas injection rate to 12 L/(L day) resulted in the decrease of CH(4) content to around 90%. Further study showed that by decreasing the gas-liquid mass transfer by increasing the stirring speed of the mixture the CH(4) content was increased to around 95%. Finally, the CH(4) content around 90% was achieved in this study with the gas injection rate as high as 24 L/(L day).

Methanogen diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis.

The methanogenic communities in buffalo rumen were characterized using a culture-independent approach of a pooled sample of rumen fluid from three adult Surti buffaloes. Buffalo rumen is likely to include species of various methanogens, so 16S rDNA sequences were amplified and cloned from the sample. A total of 171 clones were sequenced to examine 16S rDNA sequence similarity. About 52.63% sequences (90 clones) had ≥ 90% similarity, whereas, 46.78% of the sequences (81 clones) were 75-89% similar to 16S rDNA database sequences, respectively. Phylogenetic analyses were also used to infer the makeup of methanogenic communities in the rumen of Surti buffalo. As a result, we distinguished 23 operational taxonomic units (OTUs) based on unique 16S rDNA sequences: 12 OTUs (52.17%) affiliated to Methanomicrobiales order, 10 OTUs (43.47%) of the order Methanobacteriales and one OTU (4.34%) of Methanosarcina barkeri like clone, respectively. In addition, the population of Methanomicrobiales and Methabacteriales orders were also observed, accounting 4% and 2.17% of total archea. This study has revealed the largest assortment of hydrogenotrophic methanogens phylotypes ever identified from rumen of Surti buffaloes.

Diversity of bovine rumen methanogens In vitro in the presence of condensed tannins, as determined by sequence analysis of 16S rRNA gene library.

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.

Methanogenic community development in anaerobic granular bioreactors treating trichloroethylene (TCE)-contaminated wastewater at 37 °C and 15 °C.

Four expanded granular sludge bed (EGSB) bioreactors were seeded with a mesophilically-grown granular sludge and operated in duplicate for mesophilic (37 °C; R1 & R2) and low- (15°; R3 & R4) temperature treatment of a synthetic volatile fatty acid (VFA) based wastewater (3 kg COD m(-3) d(-1)) with one of each pair (R1 & R3) supplemented with increasing concentrations of trichloroethylene (TCE; 10, 20, 40, 60 mg l(-1)) and one acting as a control. Bioreactor performance was evaluated by % COD removal efficiency and % biogas methane (CH(4)) content. Quantitative Polymerase Chain Reaction (qPCR) was used to investigate the methanogenic community composition and dynamics in the bioreactors during the trial, while specific methanogenic activity (SMA) and toxicity assays were utilized to investigate the activity and TCE/dichloroethylene (DCE) toxicity thresholds of key trophic groups, respectively. At both 37 °C and 15 °C, TCE levels of 60 mg l(-1) resulted in the decline of % COD removal efficiencies to 29% (Day 235) and 37% (Day 238), respectively, and in % biogas CH(4) to 54% (Day 235) and 5% (Day 238), respectively. Despite the inhibitory effect of TCE on the anaerobic digestion process, the main drivers influencing methanogenic community development, as determined by qPCR and Non-metric multidimensional scaling analysis, were (i) wastewater composition and (ii) operating temperature. At the apical TCE concentration both SMA and qPCR of methanogenic archaea suggested that acetoclastic methanogens were somewhat inhibited by the presence of TCE and/or its degradation derivatives, while competition by dechlorinating organisms may have limited the availability of H(2) for hydrogenotrophic methanogenesis. In addition, there appeared to be an inverse correlation between SMA levels and TCE tolerance, a finding that was supported by the analysis of the inhibitory effect of TCE on two additional biomass sources. The results indicate that low-temperature anaerobic digestion is a feasible approach for the treatment of TCE-containing wastewater.

[Analysis of methanogenic community of anaerobic granular sludge in a full-scale UASB treating avermectin wastewater].

Methanogens is considered to be important functional microbial population in anaerobic granular sludge. Fluorescence in situ hybridization was used to analyze methanogens of anaerobic granular sludge in a full-scale UASB treating avermectin wastewater. The results indicated that the distribution forms of methanogens, Methanobacteriales and Methanosarcinales were identical on the surface and inner face of granular sludge samples in different formation stages (with different diameters) , although the relative abundances of these methanogens were different. The relative abundances of methanogens on the inner face were larger than these on the surface of granular sludge samples. The relative abundances of Methanobacteriales were larger than these of Methanosarcinales. The relative abundances of methanogens in all granular sludge samples ranged from (25.50 +/- 8.63)% to (48.67 +/- 8.87)%. The maximum relative abundances of methanogens were obtained in mature granular sludge with diameter of 1.0-2.0 mm, (47.08 +/- 8.26)% on the surface and (48.67 +/- 8.87)% on the inner face, respectively. The avermectin residue in wastewater showed possible inhibition effect on methanogens. The maximum specific methanogenic activities of granular sludge samples ranged from 1.311 to 1.562 g/(g x d), varying as same as COD removal ratios and relative abundances of methanogens, implying the strong correlation of methanogens with bioactivity of granular sludge.

[Diversity analysis of anaerobic fungi in the co-cultures with or without methanogens by amplified ribosomal intergenic spacer analysis].

OBJECTIVE: A molecular-based approach for anaerobic fungal community analysis was developed. The diversity of anaerobic fungi in the co-cultures with or without methanogens was analyzed by amplified ribosomal intergenic spacer analysis. METHODS: Co-cultures of anaerobic fungi and methanogens were obtained from rumen digesta using anaerobic fungal medium and the addition of penicillin and streptomycin and ampicillin alternatively and then subcultured 15 times by transferring cultures every 3 d separately for each replicate. At the end of the third subcultures, the co-cultures were inoculated to another bottles adding with chloramphenicol to obtain fungal cultures without methanogens. Total DNA from the original rumen digesta and subcultured co-cultures and fungal cultures was used for amplified ribosomal intergenic spacer analysis. RESULTS: The diversity of anaerobic fungi decreased corresponding with the subculture of the co-cultures. The anaerobic fungi represented by 354-375 and 425-438 bp in the amplified ribosomal intergenic spacer analysis profiles were not deteched in the co-cultures after the second subcultures and the anaerobic fungi represented by 383, 389-391 and 413-418 bp were dominant along with the subcultures. The community of anaerobic fungi was different in the co-cultures with or without methanogens. The anaerobic fungi represented by 383.51, 391.44 and 413.55 bp in the amplified ribosomal intergenic spacer analysis profiles were dominant in the co-cultures with methanogens, while the anaerobic fungi represented by 415.80, 425.66, 437.46 and 438.47 bp were dominant in the co-cultures without methangens. CONCLUSION: The molecular-based approach amplified ribosomal intergenic spacer analysis was suitable for analysis of anaerobic fungi in the environmental samples. The diversity of anaerobic fungi decreased along with the subculture of the co-cultures and the anaerobic fungal community became stable after the 4th subculture of the co-cultures. The anaerobic fungal community was different in the co-cultures with or without methanogens.

Methanogens in biogas production from renewable resources--a novel molecular population analysis approach.

The population structure of thermo- and mesophilic biogas reactors digesting maize silage as the sole substrate was investigated employing a novel, highly degenerated PCR-primer pair targeting mcrA/mrtA coding for the key enzyme of methanogens. No sequence affiliating with Methanococcales, Methanopyrales, ANME-, rice or fen soil clusters was detected. Direct MeA PCR-cloning results indicated that Methanobacteriales were the most important methanogens in the thermophilic reactors. 57% and 80% of the analysed sequences affiliated with this order, 14% and 20% with Methanosarcinaceae and 0% and 29% with Methanomicrobiales. Methanomicrobiales dominated in the mesophilic reactors at the given conditions, 69% and 84% of the sequences recovered from direct MeA primed cloning affiliated with this order, 31% and 0% with Methanosarcinaceae and 0% and 16% with Methanobacteriales. No sequence affiliating with Methanosaetaceae was found. MeA primed PCR-single-strand conformation polymorphism indicated that population fluctuations occurred. According to sequence analysis of excised bands, Methanosarcinaceae dominated and Methanobacteriales were significantly represented in the thermophilic fermenter. Only 1 Methanosaetaceae sequence was found. Hydrogenotrophs appear to have a much higher and obligate acetoclastic methanogens a much lower importance than previously thought in biogas production from renewable resources.

Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge.

Methanogens play a critical role in the decomposition of organics under anaerobic conditions. The methanogenic consortia in saturated wetland soils are often subjected to large temperature fluctuations and acidic conditions, imposing a selective pressure for psychro- and acidotolerant community members; however, methanogenic communities in engineered digesters are frequently maintained within a narrow range of mesophilic and circumneutral conditions to retain system stability. To investigate the hypothesis that these two disparate environments have distinct methanogenic communities, the methanogens in an oligotrophic acidic fen and a mesophilic anaerobic digester treating municipal wastewater sludge were characterized by creating clone libraries for the 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes. A quantitative framework was developed to assess the differences between these two communities by calculating the average sequence similarity for 16S rRNA genes and mcrA within a genus and family using sequences of isolated and characterized methanogens within the approved methanogen taxonomy. The average sequence similarities for 16S rRNA genes within a genus and family were 96.0 and 93.5%, respectively, and the average sequence similarities for mcrA within a genus and family were 88.9 and 79%, respectively. The clone libraries of the bog and digester environments showed no overlap at the species level and almost no overlap at the family level. Both libraries were dominated by clones related to uncultured methanogen groups within the Methanomicrobiales, although members of the Methanosarcinales and Methanobacteriales were also found in both libraries. Diversity indices for the 16S rRNA gene library of the bog and both mcrA libraries were similar, but these indices indicated much lower diversity in the 16S digester library than in the other three libraries.