Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes.

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na+). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as Nε-acetyl-ß-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments. IMPORTANCE Using genome-centric metagenomics, we discovered a new Methanothrix harundinacea subspecies that appears to be a halotolerant acetoclastic methanogen with the flexibility for adaptation in the anaerobic digestion process both at low (5 g/L Na+) and high salinity conditions (20 g/L Na+). Annotation of the recovered M. harundinacea genome revealed salinity stress-related functions, including the modification of EPS glycoconjugates and the production of compatible solutes. This is the first study reporting these genomic features within a Methanothrix sp., a milestone further supporting previous studies that identified M. harundinacea as a key-driver in anaerobic granulation under high salinity stress.

Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

The Structure of the Dimeric State of IscU Harboring Two Adjacent [2Fe-2S] Clusters Provides Mechanistic Insights into Cluster Conversion to [4Fe-4S].

IscU serves as a scaffold for the de novo assembly of a [2Fe-2S] cluster prior to its delivery to recipient protein. It has also been proposed that on one dimer of bacterial IscU, two [2Fe-2S] clusters can be converted into a single [4Fe-4S] cluster. However, lack of structural information about the dimeric state of IscU has hindered our understanding of the underlying mechanisms. In this study, we determine the X-ray crystal structure of IscU from the thermophilic archaeon Methanothrix thermoacetophila and demonstrate a dimer structure of IscU in which two [2Fe-2S] clusters are facing each other in close proximity at the dimer interface. Our structure also reveals for the first time that Asp40 serves as a fourth ligand to the [2Fe-2S] cluster with three Cys ligands in each monomer, consistent with previous spectroscopic data. We confirm by EPR spectroscopic analysis that in solution two adjacent [2Fe-2S] clusters in the wild-type dimer are converted to a [4Fe-4S] cluster via reductive coupling. Furthermore, we find that the H106A substitution abolishes the reductive conversion to the [4Fe-4S] cluster without structural alteration, suggesting that His106 is functionally involved in this process. Overall, these findings provide a structural explanation for the assembly and conversion of Fe-S clusters on IscU and highlight a dynamic process that advances via association and dissociation of the IscU dimer.

Methyl-compounds driven benthic carbon cycling in the sulfate-reducing sediments of South China Sea.

Methane is a potent greenhouse gas; methane production and consumption within seafloor sediments has generated intense interest. Anaerobic oxidation of methane (AOM) and methanogenesis (MOG) primarily occur at the depth of the sulfate-methane transition zone or underlying sediment respectively. Methanogenesis can also occur in the sulfate-reducing sediments through the utilization of non-competitive methylated compounds; however, the occurrence and importance of this process are not fully understood. Here, we combined a variety of data, including geochemical measurements, rate measurements and molecular analyses to demonstrate the presence of a cryptic methane cycle in sulfate-reducing sediments from the continental shelf of the northern South China Sea. The abundance of methanogenic substrates as well as the high MOG rates from methylated compounds indicated that methylotrophic methanogenesis was the dominant methanogenic pathway; this conclusion was further supported by the presence of the methylotrophic genus Methanococcoides. High potential rates of AOM were observed in the sediments, indicating that methane produced in situ could be oxidized simultaneously by AOM, presumably by ANME-2a/b as indicated by 16S rRNA gene analysis. A significant correlation between the relative abundance of methanogens and methanotrophs was observed over sediment depth, indicating that methylotrophic methanogenesis could potentially fuel AOM in this environment. In addition, higher potential rates of AOM than sulfate reduction rates at in situ methane conditions were observed, making alternative electron acceptors important to support AOM in sulfate-reducing sediment. AOM rates were stimulated by the addition of Fe/Mn oxides, suggesting AOM could be partially coupled to metal oxide reduction. These results suggest that methyl-compounds driven methane production drives a cryptic methane cycling and fuels AOM coupled to the reduction of sulfate and other electron acceptors.

A CUGGU/UUGGU-specific MazF homologue from Methanohalobium evestigatum.

MazF is a sequence-specific endoribonuclease or mRNA interferase, which cleaves RNA at a specific sequence. Since the expression of a specific gene or a group of specific genes can be regulated by MazF, expanding the repertoire of recognition sequences by MazF mRNA interferases is highly desirable for biotechnological and medical applications. Here, we identified a gene for a MazF homologue (MazFme) from Methanohalobium evestigatum, an extremely halophilic archaeon. In order to suppress the toxicity of MazFme to the E. coli cells, the C-terminal half of the cognate antitoxin MazEme was fused to the N-terminal end of MazFme. Since the fusion of the C-terminal half of MazEme to MazFme was able to neutralize MazFme toxicity, the MazEme-MazFme fusion protein was expressed in a large amount without any toxic effects. After purification of the MazEme, the free MazFme RNA cleavage specificity was determined by primer extension and synthetic ribonucleotides, revealing that MazFme is a CUGGU/UUGGU-specific endoribonuclease.

Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis.

Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an 'all-or-none' pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry.

Metatranscriptomic Evidence for Direct Interspecies Electron Transfer between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils.

The possibility that Methanothrix (formerly Methanosaeta) and Geobacter species cooperate via direct interspecies electron transfer (DIET) in terrestrial methanogenic environments was investigated in rice paddy soils. Genes with high sequence similarity to the gene for the PilA pilin monomer of the electrically conductive pili (e-pili) of Geobacter sulfurreducens accounted for over half of the PilA gene sequences in metagenomic libraries and 42% of the mRNA transcripts in RNA sequencing (RNA-seq) libraries. This abundance of e-pilin genes and transcripts is significant because e-pili can serve as conduits for DIET. Most of the e-pilin genes and transcripts were affiliated with Geobacter species, but sequences most closely related to putative e-pilin genes from genera such as Desulfobacterium, Deferribacter, Geoalkalibacter, and Desulfobacula, were also detected. Approximately 17% of all metagenomic and metatranscriptomic bacterial sequences clustered with Geobacter species, and the finding that Geobacter spp. were actively transcribing growth-related genes indicated that they were metabolically active in the soils. Genes coding for e-pilin were among the most highly transcribed Geobacter genes. In addition, homologs of genes encoding OmcS, a c-type cytochrome associated with the e-pili of G. sulfurreducens and required for DIET, were also highly expressed in the soils. Methanothrix species in the soils highly expressed genes for enzymes involved in the reduction of carbon dioxide to methane. DIET is the only electron donor known to support CO2 reduction in Methanothrix Thus, these results are consistent with a model in which Geobacter species were providing electrons to Methanothrix species for methane production through electrical connections of e-pili.IMPORTANCEMethanothrix species are some of the most important microbial contributors to global methane production, but surprisingly little is known about their physiology and ecology. The possibility that DIET is a source of electrons for Methanothrix in methanogenic rice paddy soils is important because it demonstrates that the contribution that Methanothrix makes to methane production in terrestrial environments may extend beyond the conversion of acetate to methane. Furthermore, defined coculture studies have suggested that when Methanothrix species receive some of their energy from DIET, they grow faster than when acetate is their sole energy source. Thus, Methanothrix growth and metabolism in methanogenic soils may be faster and more robust than generally considered. The results also suggest that the reason that Geobacter species are repeatedly found to be among the most metabolically active microorganisms in methanogenic soils is that they grow syntrophically in cooperation with Methanothrix spp., and possibly other methanogens, via DIET.

Unexpected competitiveness of Methanosaeta populations at elevated acetate concentrations in methanogenic treatment of animal wastewater.

Acetoclastic methanogenesis is a key metabolic process in anaerobic digestion, a technology with broad applications in biogas production and waste treatment. Acetoclastic methanogenesis is known to be performed by two archaeal genera, Methanosaeta and Methanosarcina. The conventional model posits that Methanosaeta populations are more competitive at low acetate levels (<1 mM) than Methanosarcina and vice versa at higher acetate concentrations. While this model is supported by an extensive body of studies, reports of inconsistency have grown that Methanosaeta were observed to outnumber Methanosarcina at elevated acetate levels. In this study, monitoring of anaerobic digesters treating animal wastewater unexpectedly identified Methanosaeta as the dominant acetoclastic methanogen population at both low and high acetate levels during organic overloading. The surprising competitiveness of Methanosaeta at elevated acetate was further supported by the enrichment of Methanosaeta with high concentrations of acetate (20 mM). The dominance of Methanosaeta in the methanogen community could be reproduced in anaerobic digesters with the direct addition of acetate to above 20 mM, again supporting the competitiveness of Methanosaeta over Methanosarcina at elevated acetate levels. This study for the first time systematically demonstrated that the dominance of Methanosaeta populations in anaerobic digestion could be linked to the competitiveness of Methanosaeta at elevated acetate concentrations. Given the importance of acetoclastic methanogenesis in biological methane production, findings from this study could have major implications for developing strategies for more effective control of methanogenic treatment processes.

Effect of ammonia on methane production pathways and reaction rates in acetate-fed biogas processes.

In order to understand the correlation between ammonia and methanogenesis metabolism, methane production pathways and their specific rates were studied at total ammonium nitrogen (TAN) concentrations of 0.14-9 g/L in three methanogenic sludges fed with acetate, at both mesophilic and thermophilic conditions. Results showed that high levels of TAN had significant inhibition on methanogenesis; this could, however, be recovered via syntrophic acetate oxidation (SAO) coupled with Hydrogenotrophic Methanogenesis (HM) performed by acetate oxidizing syntrophs or through Acetoclastic Methanogenesis (AM) catalyzed by Methanosarcinaceae, after a long lag phase >50 d. Free ammonia (NH3) was the active component for this inhibition, of which 200 mg/L is suggested as the threshold for the pathway shift from AM to SAO-HM. Methane production rate via SAO-HM at TAN of 7-9 g/L was about 5-9-fold lower than that of AM at TAN of 0.14 g/L, which was also lower than the rate of AM pathway recovered at TAN of 7 g/L in the incubations with a French mesophilic inoculum. Thermophilic condition favored the establishment of the SAO-catalyzing microbial community, as indicated by the higher reaction rate and shorter lag phase. The operational strategy is thus suggested to be adjusted when NH3 exceeds 200 mg/L.

Microbial community dynamics during the early stages of plant polymer breakdown in paddy soil.

We used paddy soil slurries amended with rice straw to identify the microbial populations involved in the methanogenic breakdown of plant polymers. Rice straw greatly stimulated microbial activity over the 28-day incubation period. On day 7, the transient peak concentration of acetate (24 mM) coincided with the onset of increased methane production. Microbial 16S rRNA transcript numbers increased by one to two orders of magnitude, but not the 16S rRNA gene copy numbers. Using metatranscriptomic rRNA, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae and Pseudomonadaceae were identified to be the most abundant and the most dynamic bacterial groups. Changes in methanogen rRNA and mRNA abundances corresponded well with methanogenic activity. Acetate determined the abundance ratio between Methanosarcinaceae and Methanosaetaceae. Methanocellaceae dominated hydrogenotrophic methanogenesis. Transcript levels of mRNA families involved in plant polymer breakdown increased slightly with time. Glycosyl hydrolase (GH) transcripts involved in cellulose and chitin breakdown were predominantly expressed by the Firmicutes, whereas those involved in hemicellulose breakdown exhibited more diverse taxonomic sources, including Acidobacteria, Bacteriodetes and Chloroflexi. Taken together, we observed strong population dynamics and the expression of taxonomically diverse GH families, suggesting that not only Firmicutes, but also less abundant groups play a major functional role in the decomposition of rice straw.

Biomethane production and microbial community response according to influent concentration of molasses wastewater in a UASB reactor.

This study aimed to investigate the interaction between methane production performance and active microbial community dynamics at different loading rates by increasing influent substrate concentration. The model system was an upflow anaerobic sludge blanket (UASB) reactor using molasses wastewater. The active microbial community was analyzed using a ribosomal RNA-based approach in order to reflect active members in the UASB system. The methane production rate (MPR) increased with an increase in organic loading rate (OLR) from 3.6 to 5.5 g COD·L(-1)·day(-1) and then it decreased with further OLR addition until 9.7 g COD·L(-1)·day(-1). The UASB reactor achieved a maximum methane production rate of 0.48 L·L(-1)·day(-1) with a chemical oxygen demand (COD) removal efficiency of 91.2 % at an influent molasses concentration of 16 g COD·L(-1) (OLR of 5.5 g COD·L(-1)·day(-1)). In the archaeal community, Methanosarcina was predominant irrespective of loading rate, and the relative abundance of Methanosaeta increased with loading rate. In the bacterial community, Firmicutes and Eubacteriaceae were relatively abundant in the loading conditions tested. The network analysis between operation parameters and microbial community indicated that MPR was positively associated with most methanogenic archaea, including the relatively abundant Methanosarcina and Methanosaeta, except Methanofollis. The most abundant Methanosarcina was negatively associated with Bifidobacterium and Methanosaeta, whereas Methanosaeta was positively associated with Bifidobacterium.

Characterization of the COD removal, electricity generation, and bacterial communities in microbial fuel cells treating molasses wastewater.

The chemical oxygen demand (COD) removal, electricity generation, and microbial communities were compared in 3 types of microbial fuel cells (MFCs) treating molasses wastewater. Single-chamber MFCs without and with a proton exchange membrane (PEM), and double-chamber MFC were constructed. A total of 10,000 mg L(-1) COD of molasses wastewater was continuously fed. The COD removal, electricity generation, and microbial communities in the two types of single-chamber MFCs were similar, indicating that the PEM did not enhance the reactor performance. The COD removal in the single-chamber MFCs (89-90%) was higher than that in the double-chamber MFC (50%). However, electricity generation in the double-chamber MFC was higher than that in the single-chamber MFCs. The current density (80 mA m(-2)) and power density (17 mW m(-2)) in the double-chamber MFC were 1.4- and 2.2-times higher than those in the single-chamber MFCs, respectively. The bacterial community structures in single- and double-chamber MFCs were also distinguishable. The amount of Proteobacteria in the double-chamber MFC was 2-3 times higher than those in the single-chamber MFCs. For the archaeal community, Methanothrix (96.4%) was remarkably dominant in the single-chamber MFCs, but Methanobacterium (35.1%), Methanosarcina (28.3%), and Methanothrix (16.2%) were abundant in the double-chamber MFC.

Direct interspecies electron transfer accelerates syntrophic oxidation of butyrate in paddy soil enrichments.

Syntrophic interaction occurs during anaerobic fermentation of organic substances forming methane as the final product. H2 and formate are known to serve as the electron carriers in this process. Recently, it has been shown that direct interspecies electron transfer (DIET) occurs for syntrophic CH4 production from ethanol and acetate. Here, we constructed paddy soil enrichments to determine the involvement of DIET in syntrophic butyrate oxidation and CH4 production. The results showed that CH4 production was significantly accelerated in the presence of nanoFe3 O4 in all continuous transfers. This acceleration increased with the increase of nanoFe3 O4 concentration but was dismissed when Fe3 O4 was coated with silica that insulated the mineral from electrical conduction. NanoFe3 O4 particles were found closely attached to the cell surfaces of different morphology, thus bridging cell connections. Molecular approaches, including DNA-based stable isotope probing, revealed that the bacterial Syntrophomonadaceae and Geobacteraceae, and the archaeal Methanosarcinaceae, Methanocellales and Methanobacteriales, were involved in the syntrophic butyrate oxidation and CH4 production. Among them, the growth of Geobacteraceae strictly relied on the presence of nanoFe3 O4 and its electrical conductivity in particular. Other organisms, except Methanobacteriales, were present in enrichments regardless of nanoFe3 O4 amendment. Collectively, our study demonstrated that the nanoFe3 O4 -facilitated DIET occurred in syntrophic CH4 production from butyrate, and Geobacter species played the key role in this process in the paddy soil enrichments.

Temperature impacts differentially on the methanogenic food web of cellulose-supplemented peatland soil.

The impact of temperature on the largely unresolved intermediary ecosystem metabolism and associated unknown microbiota that link cellulose degradation and methane production in soils of a moderately acidic (pH 4.5) fen was investigated. Supplemental [(13) C]cellulose stimulated the accumulation of propionate, acetate and carbon dioxide as well as initial methane production in anoxic peat soil slurries at 15°C and 5°C. Accumulation of organic acids at 15°C was twice as fast as that at 5°C. 16S rRNA [(13) C]cellulose stable isotope probing identified novel unclassified Bacteria (79% identity to the next cultured relative Fibrobacter succinogenes), unclassified Bacteroidetes (89% identity to Prolixibacter bellariivorans), Porphyromonadaceae, Acidobacteriaceae and Ruminococcaceae as main anaerobic degraders of cellulose-derived carbon at both 15°C and 5°C. Holophagaceae and Spirochaetaceae were more abundant at 15°C. Clostridiaceae dominated the degradation of cellulose-derived carbon only at 5°C. Methanosarcina was the dominant methanogenic taxa at both 15°C and 5°C. Relative abundance of Methanocella increased at 15°C whereas that of Methanoregula and Methanosaeta increased at 5°C. Thaumarchaeota closely related to Nitrosotalea (presently not known to grow anaerobically) were abundant at 5°C but absent at 15°C indicating that Nitrosotalea sp. might be capable of anaerobic growth at low temperatures in peat.

Proteomic insights into the temperature responses of a cold-adaptive archaeon Methanolobus psychrophilus R15.

Cold-adaptive methanogens contribute significantly to methane emission from the cold area, while the cold-adaptive mechanisms used by Archaea remain elusive. Methanolobus psychrophilus R15, a cold-adaptive methanogen isolated from a Tibetan plateau wetland, grows at 0-25 °C and optimally at 18 °C when isolated; however, it grows optimally at 30 °C after culturing at 18 °C for several years. Aiming to gain insights into the protein profiles that are involved in optimal growth and cold adaptation of this methanogen, here, we performed a comparative proteomic study using 2D DIGE on the cultures grown at 30, 18 and 4 °C. 1439 protein spots (3167 ORFs annotated in the R15 genome) were detected, and 202 of 322 differentially expressed protein spots were identified by MALDI-TOF/TOF. The protein abundance of most enzymes involved in methanogenesis, energy conservation and central metabolism were increased at 30 °C, while most ribosome proteins were decreased at 30 °C. Proteasome and ROS scavengers increased expressions at 4 °C, suggesting more aberrant proteins and ROS formed at lower temperatures. Different from the cold-adaptive Methanococcoides burtonii, some chaperones were increased at 4 °C, implying that protein folding was impaired at cold in this psychrophilic archaeon. This study indicates that diverse cold-adaptive mechanisms can be used by different methanogenic Archaea.

Determination of methanogenic pathways through carbon isotope (δ13C) analysis for the two-stage anaerobic digestion of high-solids substrates.

This study used carbon isotope (δ(13)C)-based calculations to quantify the specific methanogenic pathways in a two-stage experimental biogas plant composed of three thermophilic leach bed reactors (51-56 °C) followed by a mesophilic (36.5 °C) anaerobic filter. Despite the continuous dominance of the acetoclastic Methanosaeta in the anaerobic filter, the methane (CH4) fraction derived from carbon dioxide reduction (CO2), fmc, varied significantly over the investigation period of 200 days. At organic loading rates (OLRs) below 6.0 gCOD L(-1) d(-1), the average fmc value was 33%, whereas at higher OLRs, with a maximum level of 17.0 gCOD L(-1) d(-1), the fmc values reached 47%. The experiments allowed for a clear differentiation of the isotope fractionation related to the formation and consumption of acetate in both stages of the plant. Our data indicate constant carbon isotope fractionation for acetate formation at different OLRs within the thermophilic leach bed reactors as well as a negligible contribution of homoacetogenesis. These results present the first quantification of methanogenic pathway (fmc values) dynamics for a continually operated mesophilic bioreactor and highlight the enormous potential of δ(13)C analysis for a more comprehensive understanding of the anaerobic degradation processes in CH4-producing biogas plants.

Long-Term Incubation Reveals Methanogenic Biodegradation of C5 and C6 iso-Alkanes in Oil Sands Tailings.

iso-Alkanes are major components of petroleum and have been considered recalcitrant to biodegradation under methanogenic conditions. However, indigenous microbes in oil sands tailings ponds exposed to solvents rich in 2-methylbutane, 2-methylpentane, 3-methylpentane, n-pentane, and n-hexane produce methane in situ. We incubated defined mixtures of iso- or n-alkanes with mature fine tailings from two tailings ponds of different ages historically exposed to different solvents: one, ~10 years old, receiving C5-C6 paraffins and the other, ~35 years old, receiving naphtha. A lengthy incubation (>6 years) revealed iso-alkane biodegradation after lag phases of 900-1800 and ~280 days, respectively, before the onset of methanogenesis, although lag phases were shorter with n-alkanes (~650-1675 and ~170 days, respectively). 2-Methylpentane and both n-alkanes were completely depleted during ~2400 days of incubation, whereas 2-methylbutane and 3-methylpentane were partially depleted only during active degradation of 2-methylpentane, suggesting co-metabolism. In both cases, pyrotag sequencing of 16S rRNA genes showed codominance of Peptococcaceae with acetoclastic (Methanosaeta) and hydrogenotrophic (Methanoregula and Methanolinea) methanogens. These observations are important for predicting long-term greenhouse-gas emissions from oil sands tailings ponds and extend the known range of hydrocarbons susceptible to methanogenic biodegradation in petroleum-impacted anaerobic environments.

Treatment of domestic wastewater with an anaerobic ceramic membrane bioreactor (AnCMBR).

In this study, a ceramic membrane with a pore size of 80 nm was incorporated into an anaerobic membrane bioreactor for excellent stability and integrity. Chemical oxygen demand (COD) removal efficiencies by biodegradation reached 78.6 ± 6.0% with mixed liquor suspended solids (MLSS) of 12.8 ± 1.2 g/L. Even though the total methane generated was 0.3 ± 0.03 L/g CODutilized, around 67.4% of it dissolved in permeate and was lost beyond collection. As a result, dissolved methane was 2.7 times of the theoretical saturating concentration calculated from Henry's law. When transmembrane pressure (TMP) of the ceramic membrane reached 30 kPa after 25.3 d, 95.2% of the total resistance was attributed to the cake layer, which made it the major contributor to membrane fouling. Compared to the mixed liquor, cake layer was rich in colloids and soluble products that could bind the solids to form a dense cake layer. The Methanosarcinaceae family preferred to attach to the ceramic membranes.

Glycine betaine as a direct substrate for methanogens (Methanococcoides spp.).

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.

Methanolobus use unspecific methyltransferases to produce methane from dimethylsulphide in Baltic Sea sediments.

BACKGROUND: In anoxic coastal and marine sediments, degradation of methylated compounds is the major route to the production of methane, a powerful greenhouse gas. Dimethylsulphide (DMS) is the most abundant biogenic organic sulphur compound in the environment and an abundant methylated compound leading to methane production in anoxic sediments. However, understanding of the microbial diversity driving DMS-dependent methanogenesis is limited, and the metabolic pathways underlying this process in the environment remain unexplored. To address this, we used anoxic incubations, amplicon sequencing, genome-centric metagenomics and metatranscriptomics of brackish sediments collected along the depth profile of the Baltic Sea with varying sulphate concentrations. RESULTS: We identified Methanolobus as the dominant methylotrophic methanogens in all our DMS-amended sediment incubations (61-99%) regardless of their sulphate concentrations. We also showed that the mtt and mta genes (trimethylamine- and methanol-methyltransferases) from Methanolobus were highly expressed when the sediment samples were incubated with DMS. Furthermore, we did not find mtsA and mtsB (methylsulphide-methyltransferases) in metatranscriptomes, metagenomes or in the Methanolobus MAGs, whilst mtsD and mtsF were found 2-3 orders of magnitude lower in selected samples. CONCLUSIONS: Our study demonstrated that the Methanolobus genus is likely the key player in anaerobic DMS degradation in brackish Baltic Sea sediments. This is also the first study analysing the metabolic pathways of anaerobic DMS degradation in the environment and showing that methylotrophic methane production from DMS may not require a substrate-specific methyltransferase as was previously accepted. This highlights the versatility of the key enzymes in methane production in anoxic sediments, which would have significant implications for the global greenhouse gas budget and the methane cycle. Video Abstract.