Achieving completeness of multipartite bacterial genomes has been a difficult task, especially in rhizobia. In this study, we performed a deep bioinformatic analysis of the newly re-sequenced genome of Rhizobium favelukesii LPU83T. This strain was isolated from acid soils in Argentina and is capable of nodulating several leguminous plants, although it is unable to fix nitrogen efficiently in any of them. Oxford Nanopore sequencing allowed us to completely assemble the symbiotic plasmid of the strain, pRfaLPU83b, and we discovered that it harbors three intact prophages and a high density of insertion sequences (ISs). These characteristics show why it is often so difficult to complete the symbiotic plasmids of rhizobial strains and the importance of having long-read sequencing methods. Upon detailed analysis of this replicon, we identified a complete conjugation system with gene structure consistent with quorum sensing-associated systems that may have contributed to the genetic mosaic structure of the strain. Furthermore, we identified in the symbiotic plasmid of R. favelukesii LPU83T a large proportion of the symbiotic genes previously identified as essential for Biological Nitrogen Fixation (BNF) in symbiosis with alfalfa, with a high percentage of identity with respect to those of Sinorhizobium meliloti 2011. Among the determinants related to BNF, we found genes encoding the HrrP and SapA peptidases in the LPU83 genome, previously described and related to the degradation of nodule-specific cysteine-rich peptides. These peptides are essential for bacteroid differentiation and, therefore, efficient BNF. Our results show that despite having these genes, they are not directly responsible for the inefficient BNF phenotype of LPU83.

Plasmid conjugative transfer (CT) is a major mechanism of horizontal gene transfer in bacteria, facilitating genome evolution and dissemination of adaptive traits. Due to the energetic cost of CT, its regulation becomes an important process to ensure energetic balance within cells. In Rhizobium favelukesii, the plasmid pLPU83a belongs to group I-C of rhizobial plasmids, which require the transcriptional regulator TraR for CT. In well-characterized systems, TraR typically activates conjugative genes in response to quorum-sensing (QS) signals such as acyl-homoserine lactones. However, pLPU83a does not respond to these signals, raising questions about how TraR is regulated in this system. This study addresses the function of RcgA and RcgR, two proteins encoded upstream of traR on pLPU83a, whose function has previously been associated with CT modulation. Through proteomic, transcriptomic, and microscopy approaches, we show that RcgR acts as a repressor of CT, inhibiting traR expression and, therefore, the transcription of genes involved in CT, thereby reducing plasmid transfer rate. In contrast, RcgA is essential for CT but does not affect the expression of CT genes; it is localized at the membrane and may play a structural role in the mating pair formation system. Functional assays revealed that the repression facilitated by RcgR is independent of the anti-activator TraM and that TraR is essential for transfer even in the absence of RcgR. These findings locate RcgA and RcgR as key elements of a new circuit that modulates rhizobial plasmid conjugation and propose a novel mechanism of TraR control in systems uncoupled from QS signaling.IMPORTANCEPlasmid transfer is a central mechanism of gene exchange in bacteria, enabling the spread of traits with ecological and evolutionary relevance. Rhizobium favelukesii is a soil bacterium that carries multiple plasmids, including pLPU83a, which serves as a model to study conjugative transfer. This plasmid requires the transcriptional regulator TraR for transfer but-unlike classical systems-lacks the cognate gene that encodes the AHL synthase typically involved in quorum-sensing regulation. In previous work, two novel proteins encoded on pLPU83a, RcgA and RcgR, were identified as key elements in this regulatory system. Here, we further characterized their roles: RcgR represses the transcription of traR and, consequently, that of all conjugative genes, while RcgA is essential for transfer and localizes to the membrane, suggesting a structural function. These results provide mechanistic insight into how plasmid transfer is regulated in systems uncoupled from quorum sensing, highlighting alternative layers of control in bacterial conjugation.

Beneficial microbial consortia provide an eco-friendly alternative to conventional inorganic fertilizers and can serve as a complementary management tool for enhancing soil fertility and crop productivity. This study aimed to assess the impact of microbial consortia application on the indigenous maize rhizosphere microbiome under different fertilization regimes in organically managed fields in Germany. Three experimental microbial consortia (MC_B, MC_C, MC_C_AMF) and one commercial product (Micosat F) were tested in combination with three fertilization levels (unfertilized, 110 kg nitrogen ha- 1, and 200 kg nitrogen ha- 1) in a split plot design. The diversity, composition and functional potential of the maize rhizosphere microbiome were analyzed at different maize growth stages. Fertilization levels exerted a stronger influence than microbial consortia, significantly shaping community composition and functional traits of the indigenous soil microbiome. Increasing fertilization intensity altered the abundance of specific plant growth-promoting (PGP)-determinants, either stimulating or suppressing potential PGP bacteria. In contrast, microbial consortia application did not impact PGP-associated abundance profiles. Overall, the results indicate that multifunctional microbial consortia can act as effective biofertilizers in sustainable maize cultivation without compromising resident microbiome diversity, thereby reducing long-term ecological risks on natural biodiversity.

The metagenome analysis of complex environments with thousands of datasets, such as those in the Sequence Read Archive, requires substantial computational resources for it to be completed within a reasonable time frame. Efficient use of infrastructure is essential, and analyses must be fully reproducible with publicly available workflows to ensure transparency. Here, we introduce the Metagenomics-Toolkit, a scalable, data-agnostic workflow that automates the analysis of short and long metagenomic reads from Illumina and Oxford Nanopore Technology devices, respectively. The Metagenomics-Toolkit provides standard features such as quality control, assembly, binning, and annotation, along with unique capabilities including plasmid identification, recovery of unassembled microbial community members, and discovery of microbial interdependencies through dereplication, co-occurrence, and genome-scale metabolic modeling. Additionally, the Metagenomics-Toolkit includes a machine learning-optimized assembly step that adjusts peak RAM usage to match actual requirements, reducing the need for high-memory hardware. It can be executed on user workstations and includes optimizations for efficient cloud-based cluster execution. We compare the Metagenomics-Toolkit with five widely used metagenomics workflows and demonstrate its capabilities on 757 sewage metagenome datasets to investigate a possible sewage core microbiome. The Metagenomics-Toolkit is open source and available at https://github.com/metagenomics/metagenomics-tk.

Publicly available metagenomics datasets are crucial for ensuring the reproducibility of scientific findings and supporting contemporary large-scale studies. However, submitting a comprehensive metagenomics dataset is both cumbersome and time-consuming. It requires including sample information, sequencing reads, assemblies, binned contigs, metagenome-assembled genomes (MAGs), and appropriate metadata. As a result, metagenomics studies are often published with incomplete datasets or, in some cases, without any data at all. subMG addresses this challenge by simplifying and automating the data submission process, thereby encouraging broader and more consistent data sharing. subMG streamlines the process of submitting metagenomics study results to the European Nucleotide Archive (ENA) by allowing researchers to input files and metadata from their studies in a single form and automating downstream tasks that otherwise require extensive manual effort and expertise. The tool comes with comprehensive documentation as well as example data tailored for different use cases and can be operated via the command-line or a graphical user interface (GUI), making it easily deployable to a wide range of potential users. By simplifying the submission of genome-resolved metagenomics study datasets, subMG significantly reduces the time, effort, and expertise required from researchers, thus paving the way for more numerous and comprehensive data submissions in the future. An increased availability of well-documented and FAIR data can benefit future research, particularly in meta-analyses and comparative studies.

ABSTRACT Environmental soil samples enriched with xanthan gum led to the discovery and isolation of a novel strain of motile Paenarthrobacter ilicis in Bielefeld, Germany, which possesses a previously uncharacterized xanthan utilization gene region on its sole plasmid. This bacterium is the first member of the Paenarthrobacter genus to feature the ability to degrade xanthan. Growth experiments elucidated the optimal growth conditions with regard to pH (7.0) and temperature (28°C–30°C). The genome of Paenarthrobacter ilicis strain 6C consists of two replicons, namely a chromosome with 4,049,144 nucleotides (62.8% GC content) and a plasmid of 147,742 base pairs (61.8% GC content). This novel Paenarthrobacter ilicis strain carries 3,806 predicted coding regions, approximately half (51%) of which were verified with proteome analysis. Cultures grown with either glucose or xanthan as a carbon source were compared in relation to protein abundances at four growth stages and time points with regard to both intracellular and extracellular protein fractions. The proteome analysis (data are available via ProteomeXchange with identifier PXD063987) was undertaken using an LC-ESI-MS-MS shotgun proteomics approach, and the proteins were identified and quantified via label-free quantification using MaxQuant 2.6.6. This proteomics investigation exposed significant differences in protein abundances with regard to time and condition. Among the most highly enriched proteins seen when feeding exclusively on xanthan as a carbon source were enzymes encoded on a xanthan degradation plasmid (pPANIL_6C) with a xanthan utilization region coding for the PL8 xanthan lyase, GH38-, GH3-, GH9-family proteins, and several sugar transporters. These proteins constitute key components of a prospective xanthan degradation pathway.IMPORTANCEA novel Paenarthrobacter isolate was sequenced and characterized by proteome analysis to provide the first clear look at a novel genus in the realm of xanthan-degrading microorganisms. This research provides additional groundwork for the ongoing characterization of Paenarthrobacter, as well as widening the understanding of xanthan-degrading microorganisms. For the first time, a xanthan degradation region was identified on a plasmid 1 kb directly downstream from a mobilization gene (mobF), posing the question of whether this metabolic capacity can be shared through horizontal gene transfer. Overall, this research expands the current knowledge base regarding Paenarthrobacter biology, as well as microbial xanthan degradation and utilization.

The yield and productivity of biogas plants depend on the degradation performance of their microbiomes. The spatial separation of the anaerobic digestion (AD) process into a separate hydrolysis and a main fermenter should improve cultivation conditions of the microorganisms involved in the degradation of complex substrates like lignocellulosic biomass (LCB) and, thus, the performance of anaerobic digesters. However, relatively little is known about such two-stage processes. Here, we investigated the process performance of a two-stage agricultural AD over one year, focusing on chemical and technical process parameters and metagenome-centric metaproteomics. Technical and chemical parameters indicated stable operation of the main fermenter but varying conditions for the open hydrolysis fermenter. Matching this, the microbiome in the hydrolysis fermenter has a higher dynamic than in the main fermenter. Metaproteomics-based microbiome analysis revealed a partial separation between early and common steps in carbohydrate degradation and primary fermentation in the hydrolysis fermenter but complex carbohydrate degradation, secondary fermentation, and methanogenesis in the main fermenter. Detailed metagenomics and metaproteomics characterization of the single metagenome-assembled genomes showed that the species focus on specific substrate niches and do not utilize their full genetic potential to degrade, for example, LCB. Overall, it seems that a separation of AD in a hydrolysis and a main fermenter does not improve the cleavage of complex substrates but significantly improves the overall process performance. In contrast, the remaining methanogenic activity in the hydrolysis fermenter may cause methane losses.

The current focus on renewable energy in global policy highlights the importance of methane production from biomass through anaerobic digestion (AD). To improve biomass digestion while ensuring overall process stability, microbiome-based management strategies become more important. In this study, metagenomes and metaproteomes were used for metagenomically assembled genome (MAG)-centric analyses to investigate a full-scale biogas plant consisting of three differentially operated digesters. Microbial communities were analyzed regarding their taxonomic composition, functional potential, as well as functions expressed on the proteome level. Different abundances of genes and enzymes related to the biogas process could be mostly attributed to different process parameters. Individual MAGs exhibiting different abundances in the digesters were studied in detail, and their roles in the hydrolysis, acidogenesis and acetogenesis steps of anaerobic digestion could be assigned. Methanoculleus thermohydrogenotrophicum was an active hydrogenotrophic methanogen in all three digesters, whereas Methanothermobacter wolfeii was more prevalent at higher process temperatures. Further analysis focused on MAGs, which were abundant in all digesters, indicating their potential to ensure biogas process stability. The most prevalent MAG belonged to the class Limnochordia; this MAG was ubiquitous in all three digesters and exhibited activity in numerous pathways related to different steps of AD.

Abstract Background For a sustainable production of food, research on agricultural soil microbial communities is inevitable. Due to its immense complexity, soil is still some kind of black box. Soil study designs for identifying microbiome members of relevance have various scopes and focus on particular environmental factors. To identify common features of soil microbiomes, data from multiple studies should be compiled and processed. Taxonomic compositions and functional capabilities of microbial communities associated with soils and plants have been identified and characterized in the past few decades. From a fertile Loess–Chernozem-type soil located in Germany, metagenomically assembled genomes (MAGs) classified as members of the phylum Thaumarchaeota/Thermoproteota were obtained. These possibly represent keystone agricultural soil community members encoding functions of relevance for soil fertility and plant health. Their importance for the analyzed microbiomes is corroborated by the fact that they were predicted to contribute to the cycling of nitrogen, feature the genetic potential to fix carbon dioxide and possess genes with predicted functions in plant-growth-promotion (PGP). To expand the knowledge on soil community members belonging to the phylum Thaumarchaeota, we conducted a meta-analysis integrating primary studies on European agricultural soil microbiomes. Results Taxonomic classification of the selected soil metagenomes revealed the shared agricultural soil core microbiome of European soils from 19 locations. Metadata reporting was heterogeneous between the different studies. According to the available metadata, we separated the data into 68 treatments. The phylum Thaumarchaeota is part of the core microbiome and represents a major constituent of the archaeal subcommunities in all European agricultural soils. At a higher taxonomic resolution, 2074 genera constituted the core microbiome. We observed that viral genera strongly contribute to variation in taxonomic profiles. By binning of metagenomically assembled contigs, Thaumarchaeota MAGs could be recovered from several European soil metagenomes. Notably, many of them were classified as members of the family Nitrososphaeraceae, highlighting the importance of this family for agricultural soils. The specific Loess-Chernozem Thaumarchaeota MAGs were most abundant in their original soil, but also seem to be of importance in other agricultural soil microbial communities. Metabolic reconstruction of Switzerland_1_MAG_2 revealed its genetic potential i.a. regarding carbon dioxide (CO $$_2$$ 2 ) fixation, ammonia oxidation, exopolysaccharide production and a beneficial effect on plant growth. Similar genetic features were also present in other reconstructed MAGs. Three Nitrososphaeraceae MAGs are all most likely members of a so far unknown genus. Conclusions On a broad view, European agricultural soil microbiomes are similarly structured. Differences in community structure were observable, although analysis was complicated by heterogeneity in metadata recording. Our study highlights the need for standardized metadata reporting and the benefits of networking open data. Future soil sequencing studies should also consider high sequencing depths in order to enable reconstruction of genome bins. Intriguingly, the family Nitrososphaeraceae commonly seems to be of importance in agricultural microbiomes.

An anaerobic bacterial strain, designated strain M3/9T, was isolated from a laboratory-scale biogas fermenter fed with maize silage supplemented with 5 % wheat straw. Cells were straight, non-motile rods, which stained Gram-negative. Optimal growth occurred between 30 and 40°C, at pH 7.5-8.5, and up to 3.9 % (w/v) NaCl was tolerated. When grown on peptone from casein and soymeal, strain M3/9T produced mainly acetic acid, ethanol, and isobutyric acid. The major cellular fatty acids of the novel strain were C16 : 0 and C16 : 0 DMA. The genome of strain M3/9T is 3757  330 bp in size with a G+C content of 38.45 mol%. Phylogenetic analysis allocated strain M3/9T within the family Lachnospiraceae with Clostridium colinum DSM 6011T and Anaerotignum lactatifermentans DSM 14214T being the most closely related species sharing 57.86 and 56.99% average amino acid identity and 16S rRNA gene sequence similarities of 91.58 and 91.26 %, respectively. Based on physiological, chemotaxonomic and genetic data, we propose the description of a novel species and genus Anaeropeptidivorans aminofermentans gen. nov., sp. nov., represented by the type strain M3/9T (=DSM 100058T=LMG 29527T). In addition, an emended description of Clostridium colinum is provided.

Rhizobia are Gram-negative bacteria that are able to establish a nitrogen-fixing symbiotic interaction with leguminous plants. Rhizobia genomes usually harbor several plasmids which can be transferred to other organisms by conjugation. Two main mechanisms of the regulation of rhizobial plasmid transfer have been described: quorum sensing (QS) and the rctA/rctB system. Nevertheless, new genes and molecules that modulate conjugative transfer have recently been described, demonstrating that new actors can tightly regulate the process. In this work, by means of bioinformatics tools and molecular biology approaches, two hypothetical genes are identified as playing key roles in conjugative transfer. These genes are located between conjugative genes of plasmid pRfaLPU83a from Rhizobium favelukesii LPU83, a plasmid that shows a conjugative transfer behavior depending on the genomic background. One of the two mentioned genes, rcgA, is essential for conjugation, while the other, rcgR, acts as an inhibitor of the process. In addition to introducing this new regulatory system, we show evidence of the functions of these genes in different genomic backgrounds and confirm that homologous proteins from non-closely related organisms have the same functions. These findings set up the basis for a new regulatory circuit of the conjugative transfer of plasmids. IMPORTANCE Extrachromosomal DNA elements, such as plasmids, allow for the adaptation of bacteria to new environments by conferring new determinants. Via conjugation, plasmids can be transferred between members of the same bacterial species, different species, or even to organisms belonging to a different kingdom. Knowledge about the regulatory systems of plasmid conjugative transfer is key in understanding the dynamics of their dissemination in the environment. As the increasing availability of genomes raises the number of predicted proteins with unknown functions, deeper experimental procedures help to elucidate the roles of these determinants. In this work, two uncharacterized proteins that constitute a new regulatory circuit with a key role in the conjugative transfer of rhizobial plasmids were discovered.

The microbial biogas network is complex and intertwined, and therefore relatively stable in its overall functionality. However, if key functional groups of microorganisms are affected by biotic or abiotic factors, the entire efficacy may be impaired. Bacteriophages are hypothesized to alter the steering process of the microbial network. In this study, an enriched fraction of virus-like particles was extracted from a mesophilic biogas reactor and sequenced on the Illumina MiSeq and Nanopore GridION sequencing platforms. Metagenome data analysis resulted in identifying 375 metagenome-assembled viral genomes (MAVGs). Two-thirds of the classified sequences were only assigned to the superkingdom Viruses and the remaining third to the family Siphoviridae, followed by Myoviridae, Podoviridae, Tectiviridae, and Inoviridae. The metavirome showed a close relationship to the phage genomes that infect members of the classes Clostridia and Bacilli. Using publicly available biogas metagenomic data, a fragment recruitment approach showed the widespread distribution of the MAVGs studied in other biogas microbiomes. In particular, phage sequences from mesophilic microbiomes were highly similar to the phage sequences of this study. Accordingly, the virus particle enrichment approach and metavirome sequencing provided additional genome sequence information for novel virome members, thus expanding the current knowledge of viral genetic diversity in biogas reactors.

Research on biogas-producing microbial communities aims at elucidation of correlations and dependencies between the anaerobic digestion (AD) process and the corresponding microbiome composition in order to optimize the performance of the process and the biogas output. Previously, Lachnospiraceae species were frequently detected in mesophilic to moderately thermophilic biogas reactors. To analyze adaptive genome features of a representative Lachnospiraceae strain, Anaeropeptidivorans aminofermentans M3/9T was isolated from a mesophilic laboratory-scale biogas plant and its genome was sequenced and analyzed in detail. Strain M3/9T possesses a number of genes encoding enzymes for degradation of proteins, oligo- and dipeptides. Moreover, genes encoding enzymes participating in fermentation of amino acids released from peptide hydrolysis were also identified. Based on further findings obtained from metabolic pathway reconstruction, M3/9T was predicted to participate in acidogenesis within the AD process. To understand the genomic diversity between the biogas isolate M3/9T and closely related Anaerotignum type strains, genome sequence comparisons were performed. M3/9T harbors 1,693 strain-specific genes among others encoding different peptidases, a phosphotransferase system (PTS) for sugar uptake, but also proteins involved in extracellular solute binding and import, sporulation and flagellar biosynthesis. In order to determine the occurrence of M3/9T in other environments, large-scale fragment recruitments with the M3/9T genome as a template and publicly available metagenomes representing different environments was performed. The strain was detected in the intestine of mammals, being most abundant in goat feces, occasionally used as a substrate for biogas production.

Rhizoctonia solani AG1-IB of the phylum Basidiomycota is known as phytopathogenic fungus affecting various economically important crops, such as bean, rice, soybean, figs, cabbage and lettuce. The isolates 1/2/21 and O8/2 of the anastomosis group AG1-IB originating from lettuce plants with bottom rot symptoms represent two less aggressive R. solani isolates, as confirmed in a pathogenicity test on lettuce. They were deeply sequenced on the Illumina MiSeq system applying the mate-pair and paired-end mode to establish their genome sequences. Assemblies of obtained sequences resulted in 2092 and 1492 scaffolds, respectively, for isolate 1/2/21 and O8/2, amounting to a size of approximately 43 Mb for each isolate. Gene prediction by applying AUGUSTUS (v. 3.2.1.) yielded 12,827 and 12,973 identified genes, respectively. Based on automatic functional annotation, genes potentially encoding cellulases and enzymes involved in secondary metabolite synthesis were identified in the AG1-IB genomes. The annotated genome sequences of the less aggressive AG1-IB isolates were compared with the isolate 7/3/14, which is highly aggressive on lettuce and other vegetable crops such as bean, cabbage and carrot. This analysis revealed the first insights into core genes of AG1-IB isolates and unique determinants of each genome that may explain the different aggressiveness levels of the strains.

There are almost 9500 biogas plants in Germany, which are predominantly operated with energy crops and residues from livestock husbandry over the last two decades. In the future, biogas plants must be enabled to use a much broader range of input materials in a flexible and demand-oriented manner. Hence, the microbial communities will be exposed to frequently varying process conditions, while an overall stable process must be ensured. To accompany this transition, there is the need to better understand how biogas microbiomes respond to management measures and how these responses affect the process efficiency. Therefore, 67 microbiomes originating from 49 agricultural, full-scale biogas plants were taxonomically investigated by 16S rRNA gene amplicon sequencing. These microbiomes were separated into three distinct clusters and one group of outliers, which are characterized by a specific distribution of 253 indicative taxa and their relative abundances. These indicative taxa seem to be adapted to specific process conditions which result from a different biogas plant operation. Based on these results, it seems to be possible to deduce/assess the general process condition of a biogas digester based solely on the microbiome structure, in particular on the distribution of specific indicative taxa, and without knowing the corresponding operational and chemical process parameters. Perspectively, this could allow the development of detection systems and advanced process models considering the microbial diversity.

A growing body of evidence demonstrates the potential of various microbes to enhance plant productivity in cropping systems although their successful field application may be impaired by several biotic and abiotic constraints. In the present work, we aimed at developing multifunctional synthetic microbial consortia to be used in combination with suitable bioactive compounds for improving crop yield and quality. Plant growth-promoting microorganisms (PGPMs) with different functional attributes were identified by a bottom-up approach. A comprehensive literature survey on PGPMs associated with maize, wheat, potato and tomato, and on commercial formulations, was conducted by examining peer-reviewed scientific publications and results from relevant European projects. Metagenome fragment recruitments on genomes of potential PGPMs represented in databases were also performed to help identify plant growth-promoting (PGP) strains. Following evidence of their ability to coexist, isolated PGPMs were synthetically assembled into three different microbial consortia. Additionally, the effects of bioactive compounds on the growth of individually PGPMs were tested in starvation conditions. The different combination products based on microbial and non-microbial biostimulants (BS) appear worth considering for greenhouse and open field trials to select those potentially adoptable in sustainable agriculture.

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

In the thermophilic biogas-producing microbial community, the genus Methanothermobacter was previously described to be frequently abundant. The aim of this study was to establish and analyze the genome sequence of the archaeal strain Methanothermobacter wolfeii SIV6 originating from a thermophilic industrial-scale biogas fermenter and compare it to related reference genomes. The circular chromosome has a size of 1,686,891 bases, featuring a GC content of 48.89%. Comparative analyses considering three completely sequenced Methanothermobacter strains revealed a core genome of 1494 coding sequences and 16 strain specific genes for M. wolfeii SIV6, which include glycosyltransferases and CRISPR/cas associated genes. Moreover, M. wolfeii SIV6 harbors all genes for the hydrogenotrophic methanogenesis pathway and genome-centered metatranscriptomics indicates the high metabolic activity of this strain, with 25.18% of all transcripts per million (TPM) belong to the hydrogenotrophic methanogenesis pathway and 18.02% of these TPM exclusively belonging to the mcr operon. This operon encodes the different subunits of the enzyme methyl-coenzyme M reductase (EC: 2.8.4.1), which catalyzes the final and rate-limiting step during methanogenesis. Finally, fragment recruitment of metagenomic reads from the thermophilic biogas fermenter on the SIV6 genome showed that the strain is abundant (1.2%) within the indigenous microbial community. Detailed analysis of the archaeal isolate M. wolfeii SIV6 indicates its role and function within the microbial community of the thermophilic biogas fermenter, towards a better understanding of the biogas production process and a microbial-based management of this complex process.

Members of the genera Proteiniphilum and Petrimonas were speculated to represent indicators reflecting process instability within anaerobic digestion (AD) microbiomes. Therefore, Petrimonas mucosa ING2-E5AT was isolated from a biogas reactor sample and sequenced on the PacBio RSII and Illumina MiSeq sequencers. Phylogenetic classification positioned the strain ING2-E5AT in close proximity to Fermentimonas and Proteiniphilum species (family Dysgonomonadaceae). ING2-E5AT encodes a number of genes for glycosyl-hydrolyses (GH) which are organized in Polysaccharide Utilization Loci (PUL) comprising tandem susCD-like genes for a TonB-dependent outer-membrane transporter and a cell surface glycan-binding protein. Different GHs encoded in PUL are involved in pectin degradation, reflecting a pronounced specialization of the ING2-E5AT PUL systems regarding the decomposition of this polysaccharide. Genes encoding enzymes participating in amino acids fermentation were also identified. Fragment recruitments with the ING2-E5AT genome as a template and publicly available metagenomes of AD microbiomes revealed that Petrimonas species are present in 146 out of 257 datasets supporting their importance in AD microbiomes. Metatranscriptome analyses of AD microbiomes uncovered active sugar and amino acid fermentation pathways for Petrimonas species. Likewise, screening of metaproteome datasets demonstrated expression of the Petrimonas PUL-specific component SusC providing further evidence that PUL play a central role for the lifestyle of Petrimonas species.