Regulation of nitrite-dependent anaerobic methane oxidation bacteria by available phosphorus and microbial communities in lake sediments of cold and arid regions.

As global anthropogenic nitrogen inputs continue to rise, nitrite-dependent anaerobic methane oxidation (N-DAMO) plays an increasingly significant role in CH4 consumption in lake sediments. However, there is a dearth of knowledge regarding the effects of anthropogenic activities on N-DAMO bacteria in lakes in the cold and arid regions. Sediment samples were collected from five sampling areas in Lake Ulansuhai at varying depth ranges (0-20, 20-40, and 40-60 cm). The ecological characterization and niche differentiation of N-DAMO bacteria were investigated using bioinformatics and molecular biology techniques. Quantitative PCR confirmed the presence of N-DAMO bacteria in Lake Ulansuhai sediments, with 16S rRNA gene abundances ranging from 1.72 × 104 to 5.75 × 105 copies·g-1 dry sediment. The highest abundance was observed at the farmland drainage outlet with high available phosphorus (AP). Anthropogenic disturbances led to a significant increase in the abundance of N-DAMO bacteria, though their diversity remained unaffected. The heterogeneous community of N-DAMO bacteria was affected by interactions among various environmental characteristics, with AP and oxidation-reduction potential identified as the key drivers in this study. The Mantel test indicated that the N-DAMO bacterial abundance was more readily influenced by the presence of the denitrification genes (nirS and nirK). Network analysis revealed that the community structure of N-DAMO bacteria generated numerous links (especially positive links) with microbial taxa involved in carbon and nitrogen cycles, such as methanogens and nitrifying bacteria. In summary, N-DAMO bacteria exhibited sensitivity to both environmental and microbial factors under various human disturbances. This study provides valuable insights into the distribution patterns of N-DAMO bacteria and their roles in nitrogen and carbon cycling within lake ecosystems.

Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense (Ca. M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca. M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca. M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca. M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca. M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

Methane flux at the water-gas interface is influenced by complex interactions among phytoplankton, phosphorus inputs and methane-functional bacteria: A microcosm systems study.

Phytoplankton affect carbon cycling and emissions in eutrophic reservoirs dramatically, but our knowledge about carbon emissions response to phytoplankton bloom and phosphorus enrichment is rather limited. Here we performed a microcosm experiment with five treatments to investigate how phytoplankton blooms and phosphorus addition will impact the carbon emissions and the methane-functional bacterial community. During the 43-day incubation, the CH4 and CO2 flux at the water-air interface in the five water columns fluctuated between 7.536 and 16.689 µmol and between 2788.501 and 4142.726 µmol, respectively. The flux of CH4 and CO2 during phytoplankton decay was 1.542 to 10.397 times and 4.203 to 8.622 times higher, respectively, compared to that during phytoplankton growth. Furthermore, exogenous phosphorus increases bloom biomass of phytoplankton and subsequent CH4 production, even with low nitrogen concentrations. The addition of 1 mg KH2PO4 resulted in a conservative increase of 0.0715 µmol in CH4 emission and 11.911 µmol in CO2 emission in the water column, respectively, compared to the in-situ water column. High throughput sequencing determined that hydrogenotrophic Methanoregula dominated methanogens (MPB) and Methylocystaceae dominated methanotrophs (MOB) in the sediment. Phosphorus inhibited the relative abundance of Methanoregula after incubation, resulting in a significant decrease. Real-time quantitative polymerase chain reaction indicated that the absolute abundance of MPB and MOB (i.e., the mcrA gene and the pmoA gene) in the sediments ranged from 5.1354E+06 to 6.3176E+07 copies·g-1 and 1.1656E+06 to 9.5056E+06 copies·g-1, respectively. The mcrA gene showed a preference for sediments with high organic carbon content. The effect of eutrophication on CH4 emissions is closely related to nutrient load and distinct niche of methane-functional bacteria.

Syntrophic entanglements for propionate and acetate oxidation under thermophilic and high-ammonia conditions.

Propionate is a key intermediate in anaerobic digestion processes and often accumulates in association with perturbations, such as elevated levels of ammonia. Under such conditions, syntrophic ammonia-tolerant microorganisms play a key role in propionate degradation. Despite their importance, little is known about these syntrophic microorganisms and their cross-species interactions. Here, we present metagenomes and metatranscriptomic data for novel thermophilic and ammonia-tolerant syntrophic bacteria and the partner methanogens enriched in propionate-fed reactors. A metagenome for a novel bacterium for which we propose the provisional name 'Candidatus Thermosyntrophopropionicum ammoniitolerans' was recovered, together with mapping of its highly expressed methylmalonyl-CoA pathway for syntrophic propionate degradation. Acetate was degraded by a novel thermophilic syntrophic acetate-oxidising candidate bacterium. Electron removal associated with syntrophic propionate and acetate oxidation was mediated by the hydrogen/formate-utilising methanogens Methanoculleus sp. and Methanothermobacter sp., with the latter observed to be critical for efficient propionate degradation. Similar dependence on Methanothermobacter was not seen for acetate degradation. Expression-based analyses indicated use of both H2 and formate for electron transfer, including cross-species reciprocation with sulphuric compounds and microbial nanotube-mediated interspecies interactions. Batch cultivation demonstrated degradation rates of up to 0.16 g propionate L-1 day-1 at hydrogen partial pressure 4-30 Pa and available energy was around -20 mol-1 propionate. These observations outline the multiple syntrophic interactions required for propionate oxidation and represent a first step in increasing knowledge of acid accumulation in high-ammonia biogas production systems.

Class III hybrid cluster protein homodimeric architecture shows evolutionary relationship with Ni, Fe-carbon monoxide dehydrogenases.

Hybrid cluster proteins (HCPs) are Fe-S-O cluster-containing metalloenzymes in three distinct classes (class I and II: monomer, III: homodimer), all of which structurally related to homodimeric Ni, Fe-carbon monoxide dehydrogenases (CODHs). Here we show X-ray crystal structure of class III HCP from Methanothermobacter marburgensis (Mm HCP), demonstrating its homodimeric architecture structurally resembles those of CODHs. Also, despite the different architectures of class III and I/II HCPs, [4Fe-4S] and hybrid clusters are found in equivalent positions in all HCPs. Structural comparison of Mm HCP and CODHs unveils some distinct features such as the environments of their homodimeric interfaces and the active site metalloclusters. Furthermore, structural analysis of Mm HCP C67Y and characterization of several Mm HCP variants with a Cys67 mutation reveal the significance of Cys67 in protein structure, metallocluster binding and hydroxylamine reductase activity. Structure-based bioinformatics analysis of HCPs and CODHs provides insights into the structural evolution of the HCP/CODH superfamily.

The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus ΔH.

This study is a continuation by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. For this, we expanded our system for genetic modification of M. thermautotrophicus ΔH using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain. IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH.

Impact of carbon monoxide on performance and microbial community of extreme-thermophilic hydrogenotrophic methanation in horizontal rotary bioreactor.

A novel horizontal rotary bioreactor was developed for upgrading biogas from coke oven gas at extreme-thermophilic condition. The introduction of CO decreased the outlet methane content from 80% to 50% due to insufficient H2. This hindrance was overcome by increasing the proportion of incoming hydrogen, coupled with a prolonged gas retention time from 24 to 72 h, leading to a restoration of methane content to 91.6%. Notably, CO and CO2 exhibited a competitive relationship to hydrogen, which was determined by their contents. The substitution of Methanothermobacter for Methanobacterium as the dominant genus was observed at 70 °C, with relative abundance exceeding 98%. Incorporation of CO increased bacteria diversity and fostered a syntrophic relationship between the bacterial community and M. thermautotrophicus. This study provides both theoretical basis and practical support for biogas upgrading from coke oven gas using a biofilm reactor, thus aiding its future industrialization prospects.

Identification and Characterization of HEPN-MNT Type II TA System from Methanothermobacter thermautotrophicus ΔH.

Toxin-antitoxin (TA) systems are widespread in bacteria and archaea plasmids and genomes to regulate DNA replication, gene transcription, or protein translation. Higher eukaryotic and prokaryotic nucleotide-binding (HEPN) and minimal nucleotidyltransferase (MNT) domains are prevalent in prokaryotic genomes and constitute TA pairs. However, three gene pairs (MTH304/305, 408/409, and 463/464) of Methanothermobacter thermautotropicus ΔH HEPN-MNT family have not been studied as TA systems. Among these candidates, our study characterizes the MTH463/MTH464 TA system. MTH463 expression inhibited Escherichia coli growth, whereas MTH464 did not and blocked MTH463 instead. Using site-directed MTH463 mutagenesis, we determined that amino acids R99G, H104A, and Y106A from the R[ɸX]4-6H motif are involved with MTH463 cell toxicity. Furthermore, we established that purified MTH463 could degrade MS2 phage RNA, whereas purified MTH464 neutralized MTH463 activity in vitro. Our results indicate that the endonuclease toxin MTH463 (encoding a HEPN domain) and its cognate antitoxin MTH464 (encoding the MNT domain) may act as a type II TA system in M. thermautotropicus ΔH. This study provides initial and essential information studying TA system functions, primarily archaea HEPN-MNT family.

Anaerobic co-digestion of three commercial bio-plastic bags with food waste: Effects on methane production and microbial community structure.

The emergence of bioplastic bags as a replacement for traditional petroleum-based plastic bags is promising for their simultaneous anaerobic digestion with food waste. In this study, the degradation of three bioplastic bags is evaluated during anaerobic co-digestion with food waste under mesophilic/thermophilic conditions, and the results indicated PBAT/PLA/starch > PLA > PBAT for methane production rate. The PBAT/PLA/starch mixture produced 23.4 ml/g of methane at 55 °C, and the cumulative methane production increased by 28.4 % compared to the control. In addition, the lag time before methane production was reduced by one to four days when anaerobic co-digestion was performed under thermophilic conditions, and the conversion of the bioplastics improved by 9.11-11.2 %. Microscopy further showed obvious physical degradation of the PBAT/PLA/starch material. The FTIR analysis showed that the characteristic peaks of the material at 3320, 2957, and 934 cm-1 decreased significantly after anaerobic fermentation. The biodegradability of the polymer decreased with an increase in the content of the crystalline area in the structure. The addition of a comonomer reduced the crystallinity of the polymer. In addition, the biodegradability was increased by adjusting the hydrolysis reaction and microbial activity of the polymer surface. An analysis of the structural features of the microbial communities revealed that Archaea exhibited different biodiversity at distinct temperatures. In particular, under thermophilic conditions, the relative abundance of Methanothermobacter was 56.0 %, and it plays an important role in the anaerobic degradation of PBAT/PLA/starch materials, while bacterial communities showed smaller differences. Overall, the bioplastic was able to be co-digested anaerobically with food waste to produce renewable energy. This study provides a plan for the practical application of biodegradable plastic bag collection for the combined treatment of food waste in anaerobic digesters. It provides a theoretical basis for modifications of bioplastic and domestication of anaerobic microorganisms.

An Interdomain Conjugation Protocol for Plasmid-DNA Transfer into Methanothermobacter thermautotrophicus ΔH.

Methanogenic archaea of the order Methanobacteriales are widespread in anaerobic environments and play pivotal roles in microbial communities. The family of Methanobacteriaceae encompasses mesophilic and thermophilic hydrogenotrophic species. Mesophilic species are found in various natural and anthropogenic environments (e.g., are associated with the microbiome in animals and humans). Thermophilic species can be found in thermally active bogs and warm sulfuric springs, but also in anthropogenic environments, such as wastewater treatment plants and anaerobic digesters. Recently, genetic tools for Methanothermobacter thermautotrophicus ΔH, as the first representative of this order of methanogenic archaea, were successfully implemented. This protocol describes the methods for interdomain conjugational DNA transfer from Escherichia coli to M. thermautotrophicus ΔH with shuttle-vector plasmid DNA, which allows the genetic manipulation of this microbe, and provides a basis for the development of further genetic methods for this and potentially other representatives of Methanobacteriales.

The effect of 3-nitrooxypropanol, a potent methane inhibitor, on ruminal microbial gene expression profiles in dairy cows.

BACKGROUND: Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H2) + carbon dioxide and from H2 + methanol or methylamines. The methanogenic substrates are provided by non-methanogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbiome and to determine the fate of H2 that accumulates less than expected under inhibited methanogenesis. RESULTS: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methanosphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H2 concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H2-producing bacteria to regulate the amount of H2 production. No previously reported alternative H2 sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway. CONCLUSION: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H2 metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. Video Abstract.

Integrating metagenomic binning with flux balance analysis to unravel syntrophies in anaerobic CO2 methanation.

BACKGROUND: Carbon fixation through biological methanation has emerged as a promising technology to produce renewable energy in the context of the circular economy. The anaerobic digestion microbiome is the fundamental biological system operating biogas upgrading and is paramount in power-to-gas conversion. Carbon dioxide (CO2) methanation is frequently performed by microbiota attached to solid supports generating biofilms. Despite the apparent simplicity of the microbial community involved in biogas upgrading, the dynamics behind most of the interspecies interaction remain obscure. To understand the role of the microbial species in CO2 fixation, the biofilm generated during the biogas upgrading process has been selected as a case study. The present work investigates via genome-centric metagenomics, based on a hybrid Nanopore-Illumina approach the biofilm developed on the diffusion devices of four ex situ biogas upgrading reactors. Moreover, genome-guided metabolic reconstruction and flux balance analysis were used to propose a biological role for the dominant microbes. RESULTS: The combined microbiome was composed of 59 species, with five being dominant (> 70% of total abundance); the metagenome-assembled genomes representing these species were refined to reach a high level of completeness. Genome-guided metabolic analysis appointed Firmicutes sp. GSMM966 as the main responsible for biofilm formation. Additionally, species interactions were investigated considering their co-occurrence in 134 samples, and in terms of metabolic exchanges through flux balance simulation in a simplified medium. Some of the most abundant species (e.g., Limnochordia sp. GSMM975) were widespread (~ 67% of tested experiments), while others (e.g., Methanothermobacter wolfeii GSMM957) had a scattered distribution. Genome-scale metabolic models of the microbial community were built with boundary conditions taken from the biochemical data and showed the presence of a flexible interaction network mainly based on hydrogen and carbon dioxide uptake and formate exchange. CONCLUSIONS: Our work investigated the interplay between five dominant species within the biofilm and showed their importance in a large spectrum of anaerobic biogas reactor samples. Flux balance analysis provided a deeper insight into the potential syntrophic interaction between species, especially Limnochordia sp. GSMM975 and Methanothermobacter wolfeii GSMM957. Finally, it suggested species interactions to be based on formate and amino acids exchanges. Video Abstract.

Factors shaping the abundance and diversity of the gut archaeome across the animal kingdom.

Archaea are common constituents of the gut microbiome of humans, ruminants, and termites but little is known about their diversity and abundance in other animals. Here, we analyse sequencing and quantification data of archaeal and bacterial 16S rRNA genes from 250 species of animals covering a large taxonomic spectrum. We detect the presence of archaea in 175 animal species belonging to invertebrates, fish, amphibians, birds, reptiles and mammals. We identify five dominant gut lineages, corresponding to Methanobrevibacter, Methanosphaera, Methanocorpusculum, Methanimicrococcus and "Ca. Methanomethylophilaceae". Some archaeal clades, notably within Methanobrevibacter, are associated to certain hosts, suggesting specific adaptations. The non-methanogenic lineage Nitrososphaeraceae (Thaumarchaeota) is frequently present in animal samples, although at low abundance, but may have also adapted to the gut environment. Host phylogeny, diet type, fibre content, and intestinal tract physiology are major drivers of the diversity and abundance of the archaeome in mammals. The overall abundance of archaea is more influenced by these factors than that of bacteria. Methanogens reducing methyl-compounds with H2 can represent an important fraction of the overall methanogens in many animals. Together with CO2-reducing methanogens, they are influenced by diet and composition of gut bacteria. Our results provide key elements toward our understanding of the ecology of archaea in the gut, an emerging and important field of investigation.

A thermophilic phosphatase from Methanothermobacter marburgensis and its application to in vitro biosynthesis.

Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis, was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli, and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 °C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D-tagatose 6-phosphate, and was relatively specific for this substrate than for D-glucose 1-phosphate, D-glucose 6-phosphate, and D-fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D-tagatose in the future.

Combining a robust thermophilic methanogen and packing material with high liquid hold-up to optimize biological methanation in trickle-bed reactors.

The hydrogen gas-to-liquid mass transfer is the limiting factor in biological methanation. In trickle-bed reactors, mass transfer can be increased by high flow velocities in the liquid phase, by adding a packing material with high liquid hold-up or by using methanogenic archaea with a high methane productivity. This study developed a polyphasic approach to address all methods at once. Various methanogenic strains and packings were investigated from a microbial and hydrodynamic perspective. Analyzing the ability to produce high-quality methane and to form biofilms, pure cultures of Methanothermobacter performed better than those of the genus Methanothermococcus. Liquid and static hold-up of a packing material and its capability to facilitate attachment was not attributable to a single property. Consequently, it is recommended to carefully match organism and packing for optimized performance of trickle-bed reactors. The ideal combination for the ORBIT-system was identified as Methanothermobacter thermoautotrophicus IM5 and DuraTop®.

A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH.

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable ß-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different ß-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

Syngas biomethanation: effect of biomass-gas ratio, syngas composition and pH buffer.

The concept of syngas biomethanation is attractive, however, it still needs improvement in optimizing the operational conditions. In the present study, syngas fermentations under different carbon monoxide (CO), carbon dioxide (CO2) and hydrogen (H2) compositions were conducted under two different biomass-gas ratio (BGR) systems. The results showed that high BGR enhanced the CO consumption rate, achieving a 60% enhancement with CO as the sole substrate. Stoichiometric H2 addition could successfully convert all the CO and CO2 to pure methane, however, higher H2 partial pressure might decline the CO consumption due to pH inhibition from consumption of bicarbonate. Microbial analysis showed different syngas composition could affect the bacteria community, while, archaea community was only slightly affected with Methanothermobacter as the dominant methanogen. This study provided strategy for efficient syngas biomethanation and deeper insight into effect of H2 addition on CO conversion under different BGR systems.

Characteristics of in-situ hydrogen biomethanation at mesophilic and thermophilic temperatures.

In-situ hydrogen biomethanation is a promising technology to upgrade biogas. The efficiency of biomethanation relies on various parameters, e.g. gas supplement, temperature and hydrogenotrophic methanogens. Therefore, it is important to investigate the characteristics of in-situ hydrogen biomethanation under different conditions. In this study, two experiments (lasted for 91 days and 105 days) were carried out to investigate the impacts of feeding gas and operating conditions on performances of reactors and microorganisms. During the whole experiment, no obvious fluctuation of pH and limitation of gas-liquid mass transfer were found. Results showed that the hydrogenotrophic methanogenesis performed better at thermophilic condition, while the dominant archaea genera at mesophilic and thermophilic temperature was determined to be Methanobacterium and Methanothermobacter, respectively. The highest CH4 content (greater than 90%) was obtained when H2 and CO2 was feeding at ratio of 4:1 and Methanothermobacter was dominant. These findings can provide useful information for promoting hydrogen biomethanation.

Long-term in situ bioelectrochemical monitoring of biohythane process: Metabolic interactions and microbial evolution.

Microbial stability and evolution are a critical aspect for biosensors, especially in detecting dynamic and emerging anaerobic biohythane production. In this study, two upflow air-cathode chamber microbial fuel cells (UMFCs) were developed for in situ monitoring of the biohydrogen and biomethane reactors under a COD range of 1000-6000 mg/L and 150-1000 mg/L, respectively. Illumina MiSeq sequencing evidenced the dramatic shift of dominant microbial communities in UMFCs from hydrolytic and acidification bacteria (Clostridiaceae_1, Ruminococcaceae, Peptostreptococcaceae) to acetate-oxidizing bacteria (Synergistaceae, Dysgonomonadaceae, Spirochaetaceae). In addition, exoelectroactive bacteria evaluated from Enterobacteriaceae and Burkholderiaceae to Desulfovibrionaceae and Propionibacteriaceae. Especially, Hydrogenotrophic methanogens (Methanobacteriaceae) were abundant at 93.41% in UMFC (for monitoring hydrogen reactor), which was speculated to be a major metabolic pathway for methane production. Principal component analysis revealed a similarity in microbial structure between UMFCs and methane bioreactors. Microbial network analysis suggested a more stable community structure of UMFCs with 205 days' operation.

Analysis of a Methanogen and an Actinobacterium Dominating the Thermophilic Microbial Community of an Electromethanogenic Biocathode.

Electromethanogenesis refers to the bioelectrochemical synthesis of methane from CO2 by biocathodes. In an electromethanogenic system using thermophilic microorganisms, metagenomic analysis along with quantitative real-time polymerase chain reaction and fluorescence in situ hybridization revealed that the biocathode microbiota was dominated by the methanogen Methanothermobacter sp. strain EMTCatA1 and the actinobacterium Coriobacteriaceae sp. strain EMTCatB1. RNA sequencing was used to compare the transcriptome profiles of each strain at the methane-producing biocathodes with those in an open circuit and with the methanogenesis inhibitor 2-bromoethanesulfonate (BrES). For the methanogen, genes related to hydrogenotrophic methanogenesis were highly expressed in a manner similar to those observed under H2-limited conditions. For the actinobacterium, the expression profiles of genes encoding multiheme c-type cytochromes and membrane-bound oxidoreductases suggested that the actinobacterium directly takes up electrons from the electrode. In both strains, various stress-related genes were commonly induced in the open-circuit biocathodes and biocathodes with BrES. This study provides a molecular inventory of the dominant species of an electromethanogenic biocathode with functional insights and therefore represents the first multiomics characterization of an electromethanogenic biocathode.