Anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens' has a pleomorphic life cycle.

'Candidatus Methanoperedens' are anaerobic methanotrophic (ANME) archaea with global importance to methane cycling. Here meta-omics and fluorescence in situ hybridization (FISH) were applied to characterize a bioreactor dominated by 'Candidatus Methanoperedens nitroreducens' performing anaerobic methane oxidation coupled to nitrate reduction. Unexpectedly, FISH revealed the stable co-existence of two 'Ca. M. nitroreducens' morphotypes: the archetypal coccobacilli microcolonies and previously unreported planktonic rods. Metagenomic analysis showed that the 'Ca. M. nitroreducens' morphotypes were genomically identical but had distinct gene expression profiles for proteins associated with carbon metabolism, motility and cell division. In addition, a third distinct phenotype was observed, with some coccobacilli 'Ca. M. nitroreducens' storing carbon as polyhydroxyalkanoates. The phenotypic variation of 'Ca. M. nitroreducens' probably aids their survival and dispersal in the face of sub-optimal environmental conditions. These findings further demonstrate the remarkable ability of members of the 'Ca. Methanoperedens' to adapt to their environment.

Borgs are giant genetic elements with potential to expand metabolic capacity.

Anaerobic methane oxidation exerts a key control on greenhouse gas emissions1, yet factors that modulate the activity of microorganisms performing this function remain poorly understood. Here we discovered extraordinarily large, diverse DNA sequences that primarily encode hypothetical proteins through studying groundwater, sediments and wetland soil where methane production and oxidation occur. Four curated, complete genomes are linear, up to approximately 1 Mb in length and share genome organization, including replichore structure, long inverted terminal repeats and genome-wide unique perfect tandem direct repeats that are intergenic or generate amino acid repeats. We infer that these are highly divergent archaeal extrachromosomal elements with a distinct evolutionary origin. Gene sequence similarity, phylogeny and local divergence of sequence composition indicate that many of their genes were assimilated from methane-oxidizing Methanoperedens archaea. We refer to these elements as 'Borgs'. We identified at least 19 different Borg types coexisting with Methanoperedens spp. in four distinct ecosystems. Borgs provide methane-oxidizing Methanoperedens archaea access to genes encoding proteins involved in redox reactions and energy conservation (for example, clusters of multihaem cytochromes and methyl coenzyme M reductase). These data suggest that Borgs might have previously unrecognized roles in the metabolism of this group of archaea, which are known to modulate greenhouse gas emissions, but further studies are now needed to establish their functional relevance.

Cold Seeps on the Passive Northern U.S. Atlantic Margin Host Globally Representative Members of the Seep Microbiome with Locally Dominant Strains of Archaea.

Marine cold seeps are natural sites of methane emission and harbor distinct microbial communities capable of oxidizing methane. The majority of known cold seeps are on tectonically active continental margins, but recent discoveries have revealed abundant seeps on passive margins as well, including on the U.S. Atlantic Margin (USAM). We sampled in and around four USAM seeps and combined pore water geochemistry measurements with amplicon sequencing of 16S rRNA and mcrA (DNA and RNA) to investigate the microbial communities present, their assembly processes, and how they compare to communities at previously studied sites. We found that the USAM seeps contained communities consistent with the canonical seep microbiome at the class and order levels but differed markedly at the sequence variant level, especially within the anaerobic methanotrophic (ANME) archaea. The ANME populations were highly uneven, with just a few dominant mcrA sequence variants at each seep. Interestingly, the USAM seeps did not form a distinct phylogenetic cluster when compared with other previously described seeps around the world. Consistent with this, we found only a very weak (though statistically significant) distance-decay trend in seep community similarity across a global data set. Ecological assembly indices suggest that the USAM seep communities were assembled primarily deterministically, in contrast to the surrounding nonseep sediments, where stochastic processes dominated. Together, our results suggest that the primary driver of seep microbial community composition is local geochemistry-specifically methane, sulfide, nitrate, acetate, and ammonium concentrations-rather than the geologic context, the composition of nearby seeps, or random events of dispersal. IMPORTANCE Cold seeps are now known to be widespread features of passive continental margins, including the northern U.S. Atlantic Margin (USAM). Methane seepage is expected to intensify at these relatively shallow seeps as bottom waters warm and underlying methane hydrates dissociate. While methanotrophic microbial communities might reduce or prevent methane release, microbial communities on passive margins have rarely been characterized. In this study, we investigated the Bacteria and Archaea at four cold seeps on the northern USAM and found that despite being colocated on the same continental slope, the communities significantly differ by site at the sequence variant level, particularly methane-cycling community members. Differentiation by site was not observed in similarly spaced background sediments, raising interesting questions about the dispersal pathways of cold seep microorganisms. Understanding the genetic makeup of these discrete seafloor ecosystems and how their microbial communities develop will be increasingly important as the climate changes.

Structure of an ancestral ADP-dependent kinase with fructose-6P reveals key residues for binding, catalysis, and ligand-induced conformational changes.

ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFKs), specific glucokinases (ADP-GKs), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, none exhibits fructose-6-phosphate (F6P) at the active site. Using an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with F6P at its active site. Key residues for sugar binding and catalysis were identified by alanine scanning, D36 being a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding because its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semiclosed, and closed).

Genomic and transcriptomic insights into methanogenesis potential of novel methanogens from mangrove sediments.

BACKGROUND: Methanogens are crucial to global methane budget and carbon cycling. Methanogens from the phylum Euryarchaeota are currently classified into one class and seven orders, including two novel methanogen taxa, Methanofastidiosa and Methanomassiliicoccales. The relative importance of the novel methanogens to methane production in the natural environment is poorly understood. RESULTS: Here, we used a combined metagenomic and metatranscriptomic approach to investigate the metabolic activity of methanogens in mangrove sediments in Futian Nature Reserve, Shenzhen. We obtained 13 metagenome-assembled genomes (MAGs) representing one class (Methanofastidiosa) and five orders (Methanomassiliicoccales, Methanomicrobiales, Methanobacteriales, Methanocellales, and Methanosarcinales) of methanogens, including the two novel methanogens. Comprehensive annotation indicated the presence of an H2-dependent methylotrophic methanogenesis pathway in Methanofastidiosa and Methanomassiliicoccales. Based on the functional gene analysis, hydrogenotrophic and methylotrophic methanogenesis are the dominant pathways in mangrove sediments. MAG mapping revealed that hydrogenotrophic Methanomicrobiales were the most abundant methanogens and that methylotrophic Methanomassiliicoccales were the most active methanogens in the analyzed sediment profile, suggesting their important roles in methane production. CONCLUSIONS: Partial or near-complete genomes of two novel methanogen taxa, Methanofastidiosa and Methanomassiliicoccales, in natural environments were recovered and analyzed here for the first time. The presented findings highlight the ecological importance of the two novel methanogens and complement knowledge of how methane is produced in mangrove ecosystem. This study implies that two novel methanogens play a vital role in carbon cycle. Video Abstract.

Presence of diverse nitrate-dependent anaerobic methane oxidizing archaea in sewage sludge.

AIM: The aim of this study was to explore the community diversity and abundance of nitrate-dependent anaerobic methane oxidizing archaea, Candidatus Methanoperedens nitroreducens, in sewage sludge from wastewater treatment plants. METHODS AND RESULTS: Seasonal sampling of the sewage sludge was carried out from two wastewater treatment plants (WWTPs) located in the northern and southern parts of China. Through amplicon sequencing using our newly designed primers, a large number of Candidatus Methanoperedens nitroreducens-like (M. nitroreducens) archaeal sequences (638 743) were generated. These sequences were assigned into 742 operational protein units (OPUs) at 90% cut-off level and classified as Group B member of M. nitroreducens archaea in the phylogenetic tree. More than 80% of the OPUs were not shared between these two WWTPs, showing the M. nitroreducens-like archaeal community in each WWTP was unique. Quantitative PCR assays also confirmed the presence of M. nitroreducens-like archaea and revealed a higher abundance in autumn and winter than other seasons, indicating that the environmental attributes in these seasons might favour the growth of this archaea. Further redundancy analysis revealed that volatile solid and pH were the significant environmental attributes (P < 0·05) in shaping the M. nitroreducens-like archaeal community based on variance inflation factor selection and Monte Carlo permutation test. CONCLUSIONS: The results confirmed the presence of diverse M. nitroreducens-like archaea in sewage sludge using Illumina-based mcrA gene sequencing and quantitative PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study revealed the ecological characteristics of M. nitroreducens-like archaea in sewage sludge that improved our understanding of nitrate-dependent anaerobic methane oxidation process and may be the basis for future application of M. nitroreducens-like archaea for new nitrogen removal in WWTPs.

Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle.

Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8-11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.

Bridging human chaperonopathies and microbial chaperonins.

Chaperonins are molecular chaperones that play critical physiological roles, but they can be pathogenic. Malfunctional chaperonins cause chaperonopathies of great interest within various medical specialties. Although the clinical-genetic aspects of many chaperonopathies are known, the molecular mechanisms causing chaperonin failure and tissue lesions are poorly understood. Progress is necessary to improve treatment, and experimental models that mimic the human situation provide a promising solution. We present two models: one prokaryotic (the archaeon Pyrococcus furiosus) with eukaryotic-like chaperonins and one eukaryotic (Chaetomium thermophilum), both convenient for isolation-study of chaperonins, and report illustrative results pertaining to a pathogenic mutation of CCT5.

Active pathways of anaerobic methane oxidation across contrasting riverbeds.

Anaerobic oxidation of methane (AOM) reduces methane emissions from marine ecosystems but we know little about AOM in rivers, whose role in the global carbon cycle is increasingly recognized. We measured AOM potentials driven by different electron acceptors, including nitrite, nitrate, sulfate, and ferric iron, and identified microorganisms involved across contrasting riverbeds. AOM activity was confined to the more reduced, sandy riverbeds, whereas no activity was measured in the less reduced, gravel riverbeds where there were few anaerobic methanotrophs. Nitrite-dependent and nitrate-dependent AOM occurred in all sandy riverbeds, with the maximum rates of 61.0 and 20.0 nmol CO2 g-1 (dry sediment) d-1, respectively, while sulfate-dependent and ferric iron-dependent AOM occurred only where methane concentration was highest and the diversity of AOM pathways greatest. Diverse Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria and Candidatus Methanoperedens nitroreducens (M. nitroreducens)-like archaea were detected in the sandy riverbeds (16S rRNA gene abundance of 9.3 × 105 to 1.5 × 107 and 2.1 × 104 to 2.5 × 105 copies g-1 dry sediment, respectively) but no other known anaerobic methanotrophs. Further, we found M. oxyfera-like bacteria and M. nitroreducens-like archaea to be actively involved in nitrite- and nitrate/ferric iron-dependent AOM, respectively. Hence, we demonstrate multiple pathways of AOM in relation to methane, though the activities of M. oxyfera-like bacteria and M. nitroreducens-like archaea are dominant.

Response of the Anaerobic Methanotroph "Candidatus Methanoperedens nitroreducens" to Oxygen Stress.

"Candidatus Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of "Ca Methanoperedens nitroreducens" could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a "Ca Methanoperedens nitroreducens" culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, "Candidatus Methanoperedens nitroreducens Verserenetto." RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of "Ca Methanoperedens nitroreducens" revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of "Ca Methanoperedens nitroreducens Verserenetto" to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion.IMPORTANCE "Candidatus Methanoperedens nitroreducens" is an anaerobic archaeon which couples the reduction of nitrate to the oxidation of methane. This microorganism is present in a wide range of aquatic environments and man-made ecosystems, such as paddy fields and wastewater treatment systems. In such environments, these archaea may experience regular oxygen exposure. However, "Ca Methanoperedens nitroreducens" is able to thrive under such conditions and could be applied for the simultaneous removal of dissolved methane and nitrogenous pollutants in oxygen-limited systems. To understand what machinery "Ca Methanoperedens nitroreducens" possesses to counteract the oxidative stress and survive, we characterized the response to oxygen exposure using a multi-omics approach.

Different clusters of Candidatus 'Methanoperedens nitroreducens'-like archaea as revealed by high-throughput sequencing with new primers.

The newly discovered Candidatus 'Methanoperedens nitroreducens' (M. nitroreducens), mediating nitrate-dependent anaerobic oxidation of methane, is an important microorganism in linking carbon and nitrogen cycles. In order to explore the diversity of M. nitroreducens-like archaea in various environmental niches with advanced high-throughput sequencing, new primers based on alpha subunit of methyl-coenzyme M reductase gene were designed. The PCR results demonstrated that the new primers could effectively detect M. nitroreducens-like archaea from an enrichment culture dominated by M. nitroreducens as well as samples collected from a natural freshwater lake and a full-scale wastewater treatment plant (WWTP). By high-throughput sequencing, more than 30,000 M. nitroreducens-like sequences were obtained. Phylogenetic analysis of these sequences along with published sequences showed that M. nitroreducens-like archaea could be divided into three sub-branches (named as Group A, Group B and Group C in this study). Clear geographical difference was observed, with Group A and Group B dominating samples in Queensland (Australia) and in European ecosystems, respectively. Further quantitative PCR revealed that the M. nitroreducens-like archaea were more abundant in WWTP than the freshwater lake. The study provided a large number of sequences for M. nitroreducens-like archaeal communities, thus expanded our understanding on the ecological diversity of M. nitroreducens-like archaea.

Metabolic capability and in situ activity of microorganisms in an oil reservoir.

BACKGROUND: Microorganisms have long been associated with oxic and anoxic degradation of hydrocarbons in oil reservoirs and oil production facilities. While we can readily determine the abundance of microorganisms in the reservoir and study their activity in the laboratory, it has been challenging to resolve what microbes are actively participating in crude oil degradation in situ and to gain insight into what metabolic pathways they deploy. RESULTS: Here, we describe the metabolic potential and in situ activity of microbial communities obtained from the Jiangsu Oil Reservoir (China) by an integrated metagenomics and metatranscriptomics approach. Almost complete genome sequences obtained by differential binning highlight the distinct capability of different community members to degrade hydrocarbons under oxic or anoxic condition. Transcriptomic data delineate active members of the community and give insights that Acinetobacter species completely oxidize alkanes into carbon dioxide with the involvement of oxygen, and Archaeoglobus species mainly ferment alkanes to generate acetate which could be consumed by Methanosaeta species. Furthermore, nutritional requirements based on amino acid and vitamin auxotrophies suggest a complex network of interactions and dependencies among active community members that go beyond classical syntrophic exchanges; this network defines community composition and microbial ecology in oil reservoirs undergoing secondary recovery. CONCLUSION: Our data expand current knowledge of the metabolic potential and role in hydrocarbon metabolism of individual members of thermophilic microbial communities from an oil reservoir. The study also reveals potential metabolic exchanges based on vitamin and amino acid auxotrophies indicating the presence of complex network of interactions between microbial taxa within the community.

Ecological and genomic profiling of anaerobic methane-oxidizing archaea in a deep granitic environment.

Recent single-gene-based surveys of deep continental aquifers demonstrated the widespread occurrence of archaea related to Candidatus Methanoperedens nitroreducens (ANME-2d) known to mediate anaerobic oxidation of methane (AOM). However, it is unclear whether ANME-2d mediates AOM in the deep continental biosphere. In this study, we found the dominance of ANME-2d in groundwater enriched in sulfate and methane from a 300-m deep underground borehole in granitic rock. A near-complete genome of one representative species of the ANME-2d obtained from the underground borehole has most of functional genes required for AOM and assimilatory sulfate reduction. The genome of the subsurface ANME-2d is different from those of other members of ANME-2d by lacking functional genes encoding nitrate and nitrite reductases and multiheme cytochromes. In addition, the subsurface ANME-2d genome contains a membrane-bound NiFe hydrogenase gene putatively involved in respiratory H2 oxidation, which is different from those of other methanotrophic archaea. Short-term incubation of microbial cells collected from the granitic groundwater with 13C-labeled methane also demonstrates that AOM is linked to microbial sulfate reduction. Given the prominence of granitic continental crust and sulfate and methane in terrestrial subsurface fluids, we conclude that AOM may be widespread in the deep continental biosphere.

ADP-dependent phosphofructokinases from the archaeal order Methanosarcinales display redundant glucokinase activity.

The genome of Methanosarcinales organisms presents both ADP-dependent glucokinase and phosphofructokinase genes. However, Methanococcoides burtonii has a truncate glucokinase gene with a large deletion at the C-terminal, where the catalytic GXGD motif is located. Characterization of its phosphofructokinase annotated protein shows that is a bifunctional enzyme able to supply the absence of the glucokinase activity. Moreover, kinetic analyses of the phosphofructokinase annotated enzyme from, Methanohalobium evestigatum demonstrated that this enzyme is also bifunctional. The high conservation of the active site residues of all the enzymes from the order Methanosarcinales suggest that they should be bifunctional, as was previously reported for the ADP-dependent kinases from Methanococcales, highlighting the redundancy of the glucokinase activity in this archaeal group. The presence of active glycolytic enzymes would be important when glycogen storage of these organisms needs to be degraded to be used as energy source. Kinetic and structural information allows us to establish a substrate specificity signature that identifies specific GK or PFK, and bifunctional enzymes in this family.

Methane-metabolizing microbial communities in sediments of the Haima cold seep area, northwest slope of the South China Sea.

Cold seeps are widespread chemosynthetic ecosystems in the deep-sea environment, and cold seep microbial communities of the South China Sea are poorly constrained. Here we report on the archaeal communities, particularly those involved in methane metabolization, in sediments of a newly discovered cold seep (named 'Haima') on the northwest slope of the South China Sea. Archaeal diversity, abundance and distribution were investigated in two piston cores collected from a seep area (QDN-14B) and a non-seep control site (QDN-31B). Geochemical investigation of the QDN-14B core identified an estimated sulfate-methane transition zone (Estimated SMTZ) at 300-400 cm below sea floor (cmbsf), where a high abundance of anaerobic methane-oxidizing archaea (ANME) occurred, as revealed by analysis of the 16S rRNA gene and the gene (mcrA) encoding the α-subunit of the key enzyme methyl-coenzyme M reductase. ANME-2a/b was predominant in the upper and middle layers of the estimated SMTZ, whereas ANME-1b outcompeted ANME-2 in the sulfate-depleted bottom layers of the estimated SMTZ and the methanogenic zone. Fine-scale phylogenetic analysis further divided the ANME-1b group into three subgroups with different distribution patterns: ANME-1bI, ANME-1bII and ANME-1bIII. Multivariate analyses indicated that dissolved inorganic carbon and sulfate may be important factors controlling the composition of the methane-metabolizing community. Our study on ANME niche separation and interactions with other archaeal groups improves our understanding of the metabolic diversity and flexibility of ANME, and the findings further suggest that ANME subgroups may have evolved diversified/specified metabolic capabilities other than syntrophic anaerobic oxidation of methane coupled with sulfate reduction in marine sediments.

Identification of Pelomyxa palustris Endosymbionts.

Pelomyxa palustris is a giant anaerobic/microaerobic amoeba, characterized by a number of exceptional cytological and physiological features, among them the presumed absence of energy producing organelles and the presence of endosymbiotic bacteria. These endosymbionts have been previously distinguished as: a large rectangular-shaped Gram-variable rod with a central cleft; a slender Gram-negative rod; and a slender Gram-positive rod. Using DNA extracted from P. palustris cysts, we have obtained three SSU rRNA gene sequences. We have determined that these sequences are affiliated to three different prokaryotic genera: Methanosaeta (a methanogenic archaea), Syntrophorhabdus (a syntrophic Gram-negative bacteria) and Rhodococcus (an aerobic chemoorganotrophic Gram-positive bacteria). To our knowledge, it is the first time that Syntrophorhabdus has been described as an endosymbiont in association with a methanogen. Strikingly, no traces of Methanobacterium formicicum could be detected, despite this methanogen had allegedly been isolated from trophozoites of P. palustris. It seems that the host and the endosymbionts have established a multipartite syntrophic consortium resembling to some extent those found in sewage treatment plants.

Metagenomic analysis reveals the contribution of anaerobic methanotroph-1b in the oxidation of methane at the Ulleung Basin, East Sea of Korea.

We have previously identified a sulfate methane transition zone (SMTZ) within the methane hydrate-bearing sediment in the Ulleung Basin, East Sea of Korea, and the presence of ANME-1b group in the sediment has been shown by phylogenetic analysis of a 16S rRNA gene. Herein, we describe taxonomic and functional profiling in the SMTZ sample by metagenomic analysis, comparing with that of surface sediment. Metagenomic sequences of 115 Mbp and 252 Mbp were obtained from SMTZ and surface sediments, respectively. The taxonomic profiling using BLASTX against the SEED within MG-RAST showed the prevalence of methanogens (19.1%), such as Methanosarcinales (12.0%) and Methanomicrobiales (4.1%) predominated within the SMTZ metagenome. A number of 185,200 SMTZ reads (38.9%) and 438,484 surface reads (62.5%) were assigned to functional categories, and methanogenesis-related reads were statistically significantly overrepresented in the SMTZ metagenome. However, the mapping analysis of metagenome reads to the reference genomes, most of the sequences of the SMTZ metagenome were mapped to ANME-1 draft genomes, rather than those of methanogens. Furthermore, the two copies of the methyl-coenzyme M reductase gene (mcrA) segments of the SMTZ metagenome were clustered with ANME-1b in the phylogenetic cluster. These results indicate that ANME-1b reads were miss-annotated to methanogens due to limitation of database. Many of key genes necessary for reverse methanogenesis were present in the SMTZ metagenome, except for N 5,N 10-methenyl-H4MPT reductase (mer) and CoB-CoM heterodisulfide reductase subunits D and E (hdrDE). These data suggest that the ANME-1b represents the primary player the anaerobic methane oxidation in the SMTZ, of the methane hydrate-bearing sediment at the Ulleung Basin, East Sea of Korea.

Effect of domestication on microorganism diversity and anaerobic digestion of food waste.

To accomplish the rapid start-up and stable operation of biogas digesters, an efficient inoculum is required. To obtain such an inoculum for food waste anaerobic digestion, we domesticated dairy manure anaerobic digestion residue by adding food waste every day. After 36 days, the pH and biogas yield stabilized signifying the completion of domestication. During domestication, the microbial communities in the inocula were investigated by constructing 16S rDNA clone libraries. We evaluated the effect of the domesticated inoculum by testing batch food waste anaerobic digestion with a non-domesticated inoculum as a control. The pH and methane yield of the digestion systems were determined as measurement indices. Domestication changed the composition and proportion of bacteria and archaea in the inocula. Of the bacteria, Clostridia (49.3%), Bacteroidales (19.5%), and Anaerolinaceae (8.1%) species were dominant in the seed sludge; Anaerolinaceae (49.0%), Clostridia (28.4%), and Bacteroidales (9.1%), in domestication sludge. Methanosaeta was the dominant genus in both of the seed (94.3%) and domestication (74.3%) sludge. However, the diversity of methanogenic archaea was higher in the domestication than in seed sludge. Methanoculleus, which was absent from the seed sludge, appeared in the domestication sludge (21.7%). When the domesticated inoculum was used, the digestion system worked stably (organic loading rate: 20 gVS/L; methane yield: 292.2 ± 9.8 mL/gVS; VS = volatile solids), whereas the digestion system inoculated with seed sludge failed to generate biogas. The results indicate that inoculum domestication ensures efficient and stable anaerobic digestion by enriching the methanogenic strains.

Activation of CO2-reducing methanogens in oil reservoir after addition of nutrient.

Nutrient addition as part of microbial enhanced oil recovery (MEOR) operations have important implications for more energy recovery from oil reservoirs, but very little is known about the in situ response of microorganisms after intervention. An analysis of two genes as biomarkers, mcrA encoding the key enzyme in methanogenesis and fthfs encoding the key enzyme in acetogenesis, was conducted during nutrient addition in oil reservoir. Clone library data showed that dominant mcrA sequences changed from acetoclastic (Methanosaetaceae) to CO2-reducing methanogens (Methanomicrobiales and Methanobacteriales), and the authentic acetogens affiliated to Firmicutes decreased after the intervention. Principal coordinates analysis (PCoA) and Jackknife environment clusters revealed evidence on the shift of the microbial community structure among the samples. Quantitative analysis of methanogens via qPCR showed that Methanobacteriales and Methanomicrobiales increased after nutrient addition, while acetoclastic methanogens (Methanosaetaceae) changed slightly. Nutrient treatment activated native CO2-reducing methanogens in oil reservoir. The high frequency of Methanobacteriales and Methanomicrobiales (CO2-reducers) after nutrient addition in this petroleum system suggested that CO2-reducing methanogenesis was involved in methane production. The nutrient addition could promote the methane production. The results will likely improve strategies of utilizing microorganisms in subsurface environments.