Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii.

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.

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.

Methane production by Methanothrix thermoacetophila via direct interspecies electron transfer with Geobacter metallireducens.

Methanothrix is widely distributed in natural and artificial anoxic environments and plays a major role in global methane emissions. It is one of only two genera that can form methane from acetate dismutation and through participation in direct interspecies electron transfer (DIET) with exoelectrogens. Although Methanothrix is a significant member of many methanogenic communities, little is known about its physiology. In this study, transcriptomics helped to identify potential routes of electron transfer during DIET between Geobacter metallireducens and Methanothrix thermoacetophila. Additions of magnetite to cultures significantly enhanced growth by acetoclastic methanogenesis and by DIET, while granular activated carbon (GAC) amendments impaired growth. Transcriptomics suggested that the OmaF-OmbF-OmcF porin complex and the octaheme outer membrane c-type cytochrome encoded by Gmet_0930, were important for electron transport across the outer membrane of G. metallireducens during DIET with Mx. thermoacetophila. Clear differences in the metabolism of Mx. thermoacetophila when grown via DIET or acetate dismutation were not apparent. However, genes coding for proteins involved in carbon fixation, the sheath fiber protein MspA, and a surface-associated quinoprotein, SqpA, were highly expressed in all conditions. Expression of gas vesicle genes was significantly lower in DIET- than acetate-grown cells, possibly to facilitate better contact between membrane-associated redox proteins during DIET. These studies reveal potential electron transfer mechanisms utilized by both Geobacter and Methanothrix during DIET and provide important insights into the physiology of Methanothrix in anoxic environments. IMPORTANCE Methanothrix is a significant methane producer in a variety of methanogenic environments including soils and sediments as well as anaerobic digesters. Its abundance in these anoxic environments has mostly been attributed to its high affinity for acetate and its ability to grow by acetoclastic methanogenesis. However, Methanothrix species can also generate methane by directly accepting electrons from exoelectrogenic bacteria through direct interspecies electron transfer (DIET). Methane production through DIET is likely to further increase their contribution to methane production in natural and artificial environments. Therefore, acquiring a better understanding of DIET with Methanothrix will help shed light on ways to (i) minimize microbial methane production in natural terrestrial environments and (ii) maximize biogas formation by anaerobic digesters treating waste.

Glycol/glycerol-fed electrically conductive aggregates suggest a mechanism of stimulating direct interspecies electron transfer in methanogenic digesters.

The possibility of stimulating direct interspecies electron transfer (DIET) within aggregates of methanogenic digesters respectively with ethanol, glycol, and glycerol as a primary substrate was investigated to better understand the mechanisms of alcohol compounds stimulating DIET. Aggregates fed with ethanol, glycol, and glycerol were electrically conductive (10.4-19.4 uS/cm), with a temperature dependence of metallic-like conductivity. Close examination of transmission electron microscope images observed the potential interspecies connected networks assembled by filaments within these aggregates. Further investigations via metatranscriptomics found that, genes for electrically conductive pili (e-pili) (Log2FPKM, 9.39-10.96) and c-type cytochromes (8.90-9.64) were highly expressed within aggregates. Glycerol-fed aggregates exhibited the highest gene expression for e-pili, while glycol-fed aggregates exhibited the highest gene expression for c-type cytochromes. Methanothrix species were dominant and metabolically active within aggregates. Genes encoding the enzymes involved in carbon dioxide reduction were highly expressed in Methanothrix species, suggesting that they participated in DIET. In addition, transcript abundance of genes encoding alcohol dehydrogenase and NADH-quinone oxidoreductase in alcohol dehydrogenation closely associated with NADH/NAD+ transformation within glycol- and glycerol-fed aggregates was generally higher than that within ethanol-fed aggregates. These results, and the fact that NADH/NAD+ transformation was very linked to the ATP synthesis complex that further supported the formation of extracellular electrical connection components, e-pili and membrane-bound multi-heme c-type cytochromes (MHCs), provided a possibility that alcohol compounds comprised of hydroxy groups could stimulate DIET and more hydroxy groups comprised were better for this stimulation.

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.

Activity-based, genome-resolved metagenomics uncovers key populations and pathways involved in subsurface conversions of coal to methane.

Microbial metabolisms and interactions that facilitate subsurface conversions of recalcitrant carbon to methane are poorly understood. We deployed an in situ enrichment device in a subsurface coal seam in the Powder River Basin (PRB), USA, and used BONCAT-FACS-Metagenomics to identify translationally active populations involved in methane generation from a variety of coal-derived aromatic hydrocarbons. From the active fraction, high-quality metagenome-assembled genomes (MAGs) were recovered for the acetoclastic methanogen, Methanothrix paradoxum, and a novel member of the Chlorobi with the potential to generate acetate via the Pta-Ack pathway. Members of the Bacteroides and Geobacter also encoded Pta-Ack and together, all four populations had the putative ability to degrade ethylbenzene, phenylphosphate, phenylethanol, toluene, xylene, and phenol. Metabolic reconstructions, gene analyses, and environmental parameters also indicated that redox fluctuations likely promote facultative energy metabolisms in the coal seam. The active "Chlorobi PRB" MAG encoded enzymes for fermentation, nitrate reduction, and multiple oxygenases with varying binding affinities for oxygen. "M. paradoxum PRB" encoded an extradiol dioxygenase for aerobic phenylacetate degradation, which was also present in previously published Methanothrix genomes. These observations outline underlying processes for bio-methane from subbituminous coal by translationally active populations and demonstrate activity-based metagenomics as a powerful strategy in next generation physiology to understand ecologically relevant microbial populations.

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.

Transition of microbial communities and degradation pathways in anaerobic digestion at decreasing retention time.

Tuning of operational variables is a common practice to control the anaerobic digestion process and, in advanced applications, to promote the accumulation of fermentation products. However, process variables are interrelated. In this study, the hydraulic retention time (HRT) was decoupled from the organic loading rate (OLR) in order to isolate the effect of HRT as a selective pressure on: process performance, metabolic rates (hydrolytic, acetogenic, and methanogenic) and the microbial community. Four mesophilic anaerobic digesters were subjected to a sequential decrease in HRT from 15 to 8, 4 and 2 days while keeping the OLR constant at chemical oxygen demand of 1 gCOD L r-1 d-1. The results showed that HRT alone was insufficient to washout methanogens from the digesters, which in turn prevented the accumulation of volatile fatty acids (VFA). Methanosaeta was the dominant genus in the four digesters at all HRTs. Metabolic rates showed that process performance was controlled by hydrolysis, with a clear shift in acetogenic rates, from butyrate and propionate degradation to ethanol degradation at 4 and 2d HRT. The change in acetogenic pathways was attributed to a shift in the fermentation pathways co-current with changes in fermentative bacteria. At 2d HRT, biofilm was formed on the walls and paddles of the digesters, probably as a survival strategy. Most of the taxa in the biofilm were also present in the digester media. Overall, it is the combination of HRT with other operational parameters which promotes the washout of methanogens and the accumulation of VFA.

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.

Production of methylmercury by methanogens in mercury contaminated estuarine sediments.

Anaerobic bacteria are known to produce neurotoxic methylmercury [MeHg] when elemental mercury [Hg(0)] is provided as the sole mercury source. In this study, we examined the formation of MeHg in anaerobic incubations of sediment collected from the San Jacinto River estuary (Texas, USA) amended with aqueous Hg(0) to investigate the microbial communities involved in the conversion of Hg(0) to MeHg. The results show that the addition of the methanogen inhibitor 2-bromoethanesulfonate (BES) significantly decreased MeHg production. The mercury methylation gene, hgcA, was detected in these sediments using archaeal specific primers, and 16S rRNA sequencing showed that a member of the Methanosarcinaceae family of methanogens was active. These results suggest that methanogenic archaea play an underappreciated role in the production of MeHg in estuarine sediments contaminated with Hg(0).

Enhanced glycosylation of an S-layer protein enables a psychrophilic methanogenic archaeon to adapt to elevated temperatures in abundant substrates.

Adaptation to higher temperatures would increase the environmental competitiveness of psychrophiles, organisms that thrive in low-temperature environments. Methanolobus psychrophilus, a cold wetland methanogen, 'evolved' as a mesophile, growing optimally at 30 °C after subculturings, and cells grown with ample substrates exhibited higher integrity. Here, we investigated N-glycosylation of S-layer proteins, the major archaeal envelope component, with respect to mesophilic adaptation. Lectin affinity enriched a glycoprotein in cells grown at 30 °C under ample substrate availability, which was identified as the S-layer protein Mpsy_1486. Four N-glycosylation sites were identified on Mpsy_1486, which exhibited different glycosylation profiles, with N94 only found in cells cultured at 30 °C. An N-linked glycosylation inhibitor, tunicamycin, reduced glycosylation levels of Mpsy_1486 and growth at 30 °C, thus establishing a link between S-layer protein glycosylation and higher temperature adaptation of the psychrophilic archaeon M. psychrophilus.

The effect of introduction of chicken manure on the biodiversity and performance of an anaerobic digester

Background: Ammonium stress is a prime limiting phenomenon that occurs during methane formation from poultry manure. It is caused by elevated ammonium nitrogen concentrations that result from substrate decomposition. The amounts of methane formed depend on the activity of methanogenic microbes. Results: During the research reported in this paper, the response of a mesophilic consortium inhabiting a biogas reactor to rising load of poultry manure was investigated. The taxonomic composition of bacterial population was mostly typical, however syntrophic bacteria were not detected. This absence resulted in limitation of succession of some methanogenic microorganisms, especially obligate hydrogenotrophs. The methanogenic activity of the consortium was totally dependent on the activity of Methanosaeta. Inhibition of methanoganesis was noticed at ammonium nitrogen concentration of 3.68 g/L, total cessation occurred at 5.45 g/L. Significant amounts of acetic acid in the fermentation pulp accompanied the inhibition. Conclusions: The effectiveness of the consortium was totally dependent on the metabolic activity of the acetoclastic Methanoseata genus and lack of SAOB did not allow hydrogenotrophic methanogens to propagate and lead to cessation of biogas production at an elevated ammonium concentration at which acetoclastic methanogens were inhibited.

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.

Deltaproteobacteria (Pelobacter) and Methanococcoides are responsible for choline-dependent methanogenesis in a coastal saltmarsh sediment.

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13C2-choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13C2-choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment.

Long-term succession in a coal seam microbiome during in situ biostimulation of coalbed-methane generation.

Despite the significance of biogenic methane generation in coal beds, there has never been a systematic long-term evaluation of the ecological response to biostimulation for enhanced methanogenesis in situ. Biostimulation tests in a gas-free coal seam were analysed over 1.5 years encompassing methane production, cell abundance, planktonic and surface associated community composition and chemical parameters of the coal formation water. Evidence is presented that sulfate reducing bacteria are energy limited whilst methanogenic archaea are nutrient limited. Methane production was highest in a nutrient amended well after an oxic preincubation phase to enhance coal biofragmentation (calcium peroxide amendment). Compound-specific isotope analyses indicated the predominance of acetoclastic methanogenesis. Acetoclastic methanogenic archaea of the Methanosaeta and Methanosarcina genera increased with methane concentration. Acetate was the main precursor for methanogenesis, however more acetate was consumed than methane produced in an acetate amended well. DNA stable isotope probing showed incorporation of 13C-labelled acetate into methanogenic archaea, Geobacter species and sulfate reducing bacteria. Community characterisation of coal surfaces confirmed that methanogenic archaea make up a substantial proportion of coal associated biofilm communities. Ultimately, methane production from a gas-free subbituminous coal seam was stimulated despite high concentrations of sulfate and sulfate-reducing bacteria in the coal formation water. These findings provide a new conceptual framework for understanding the coal reservoir biosphere.

Methanogenic degradation of tetramethylammonium hydroxide by Methanomethylovorans and Methanosarcina.

This study evaluated the methanogens responsible for methanogenic degradation of tetramethylammonium hydroxide (TMAH) in a continuous flow bioreactor. The enriched methanogens attained an estimated maximum specific TMAH degradation rate and half-saturation constant of 39.5 mg TMAH/gVSS/h and 820 mg/L, following the Monod-type kinetic expression for methanogenic TMAH degradation. Presence of sulfide more than 20 mg/L significantly extended lag period and slowed down specific TMAH degradation rates. The results of terminal restriction fragment length polymorphism (T-RFLP), cloning/sequencing, and quantitative real-time PCR analyses targeting on the methyl coenzyme M reductase alpha subunit (mcrA) genes retrieved from the bioreactor and batch experiments indicated that Methanomethylovorans species were the dominant methanogens responsible for methanogenic degradation of TMAH. The isolated TMAH-degrading methanogen from the bioreactor, however, was identified closely related to Methanosarcina mazei. It is likely that a very low TMAH environment in the bioreactor favored the growth of Methanomethylovorans hollandica, while the much higher TMAH in the isolation growth medium proliferated Methanosarcina mazei.

The performance and membrane fouling rate of a pilot-scale anaerobic membrane bioreactor for treating antibiotic solvent wastewater under different cross flow velocity.

The performance of a pilot-scale anaerobic membrane bioreactor (AnMBR) for treating antibiotic solvent wastewater under different cross flow velocities (CFV) was investigated. Effects of mixed liquid suspended solids (MLSS), colloid total organic carbon (TOC) and CFV on membrane fouling rate (RMF) were also explored in this paper. Throughout 341 days of experiment, the average total removal rate of N, N-Dimethylformamide (DMF) was 98.5% which hardly affected by the variation of CFV, and the compliance rate of DMF was 92% according to the Chinese standard (<25 mg/L). However, the relevant high total removal rate of M-cresol (MC) was achieved as 97.5%, the content of effluent failed to meet the national level emission standard (<0.1 mg/L). The biogas yield and the methane content of the biogas increased gradually with the increase of CFV, and the average methane content were over 70%. There were four kinds of methanogens in AnMBR, Methanosaeta spp was the largest methanogenic community, with an area of 45-70% of the archae. There was a linear relationship between colloid TOC and RMF at different MLSS concentrations. Then a universal mathematical model for the changes of RMF with influence factors was established. The result showed that model well fitted the laboratory data. It is suggested that the model proposed could reflect and manage the membrane fouling of AnMBR treating antibiotic solvent wastewater.

Anaerobic digestion of pig manure supernatant at high ammonia concentrations characterized by high abundances of Methanosaeta and non-euryarchaeotal archaea.

We examined the effect of ammonium and temperature on methane production in high rate upflow anaerobic sludge bed reactors treating pig manure supernatant. We operated four reactors at two ammonium concentrations ('low' at 1.9, 'high' at 3.7 g L-1, termed LA and HA reactors, respectively) and at variable temperatures over 358 days. Archaeal and bacterial communities were characterized by Illumina sequencing of 16S rRNA amplicons. Ammonium was a major selective factor for bacterial and archaeal community structure. After ~200 days of adaptation to high ammonium levels, acetate and propionate removal and methane production improved substantially in HA reactors. Aceticlastic Methanosaeta was abundant and positively correlated to methane yield in the HA reactors, whereas Methanosarcina was more abundant in LA reactors. Furthermore, a group of monophyletic OTUs that was related to Thaumarchaeota in phylogenetic analysis was highly abundant in the archaeal communities, particularly in the HA reactors. The most abundant bacterial OTU in LA reactors, representing Syntrophomonadaceae, was also positively correlated to methane yield in the HA reactors, indicating its importance in methane production under ammonia stress. In conclusion, efficient methane production, involving aceticlastic methanogenesis by Methanosaeta took place in the reactors at free ammonia concentrations as high as 1 g L-1.

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.