Microbiome Diversity and Community-Level Change Points within Manure-based small Biogas Plants

Theuerl S, Klang J, Hülsemann B, Mächtig T, Hassa J. Microbiome Diversity and Community-Level Change Points within Manure-based small Biogas Plants. Microorganisms. 2020;8(8).Efforts to integrate biogas plants into bioeconomy concepts will lead to an expansion of manure-based (small) biogas plants, while their operation is challenging due to critical characteristics of some types of livestock manure. For a better process understanding, in this study, three manure-based small biogas plants were investigated with emphasis on microbiome diversity. Due to varying digester types, feedstocks, and process conditions, 16S rRNA gene amplicon sequencing showed differences in the taxonomic composition. Dynamic variations of each investigated biogas plant microbiome over time were analyzed by terminal restriction fragment length polymorphism (TRFLP), whereby nonmetric multidimensional scaling (NMDS) revealed two well-running systems, one of them with a high share of chicken manure, and one unstable system. By using Threshold Indicator Taxa Analysis (TITAN), community-level change points at ammonium and ammonia concentrations of 2.25 g L-1 and 193 mg L-1 or volatile fatty acid concentrations of 0.75 g L-1were reliably identified which are lower than the commonly reported thresholds for critical process stages based on chemical parameters. Although a change in the microbiome structure does not necessarily indicate an upcoming critical process stage, the recorded community-level change points might be a first indication to carefully observe the process

Metagenome, metatranscriptome, and metaproteome approaches unraveled compositions and functional relationships of microbial communities residing in biogas plants

The production of biogas by anaerobic digestion (AD) of agricultural residues, organic wastes, animal excrements, municipal sludge, and energy crops has a firm place in sustainable energy production and bio-economy strategies. Focusing on the microbial community involved in biomass conversion offers the opportunity to control and engineer the biogas process with the objective to optimize its efficiency. Taxonomic profiling of biogas producing communities by means of high-throughput 16S rRNA gene amplicon sequencing provided high-resolution insights into bacterial and archaeal structures of AD assemblages and their linkages to fed substrates and process parameters. Commonly, the bacterial phyla Firmicutes and Bacteroidetes appeared to dominate biogas communities in varying abundances depending on the apparent process conditions. Regarding the community of methanogenic Archaea, their diversity was mainly affected by the nature and composition of the substrates, availability of nutrients and ammonium/ammonia contents, but not by the temperature. It also appeared that a high proportion of 16S rRNA sequences can only be classified on higher taxonomic ranks indicating that many community members and their participation in AD within functional networks are still unknown. Although cultivation-based approaches to isolate microorganisms from biogas fermentation samples yielded hundreds of novel species and strains, this approach intrinsically is limited to the cultivable fraction of the community. To obtain genome sequence information of non-cultivable biogas community members, metagenome sequencing including assembly and binning strategies was highly valuable. Corresponding research has led to the compilation of hundreds of metagenome-assembled genomes (MAGs) frequently representing novel taxa whose metabolism and lifestyle could be reconstructed based on nucleotide sequence information. In contrast to metagenome analyses revealing the genetic potential of microbial communities, metatranscriptome sequencing provided insights into the metabolically active community. Taking advantage of genome sequence information, transcriptional activities were evaluated considering the microorganism’s genetic background. Metaproteome studies uncovered enzyme profiles expressed by biogas community members. Enzymes involved in cellulose and hemicellulose decomposition and utilization of other complex biopolymers were identified. Future studies on biogas functional microbial networks will increasingly involve integrated multi-omics analyses evaluating metagenome, transcriptome, proteome, and metabolome datasets. © 2018, The Author(s)

Genetic potential of the biocontrol agent pseudomonas brassicacearum (Formerly P. trivialis) 3Re2-7 unraveled by genome sequencing and mining, comparative genomics and transcriptomics

The genus Pseudomonas comprises many known plant-associated microbes with plant growth promotion and disease suppression properties. Genome-based studies allow the prediction of the underlying mechanisms using genome mining tools and the analysis of the genes unique for a strain by implementing comparative genomics. Here, we provide the genome sequence of the strain Pseudomonas brassicacearum 3Re2-7, formerly known as P. trivialis and P. reactans, elucidate its revised taxonomic classification, experimentally verify the gene predictions by transcriptome sequencing, describe its genetic biocontrol potential and contextualize it to other known Pseudomonas biocontrol agents. The P. brassicacearum 3Re2-7 genome comprises a circular chromosome with a size of 6,738,544 bp and a GC-content of 60.83%. 6267 genes were annotated, of which 6113 were shown to be transcribed in rich medium and/or in the presence of Rhizoctonia solani. Genome mining identified genes related to biocontrol traits such as secondary metabolite and siderophore biosynthesis, plant growth promotion, inorganic phosphate solubilization, biosynthesis of lipo- and exopolysaccharides, exoproteases, volatiles and detoxification. Core genome analysis revealed, that the 3Re2-7 genome exhibits a high collinearity with the representative genome for the species, P. brassicacearum subsp. brassicacearum NFM421. Comparative genomics allowed the identification of 105 specific genes and revealed gene clusters that might encode specialized biocontrol mechanisms of strain 3Re2-7. Moreover, we captured the transcriptome of P. brassicacearum 3Re2-7, confirming the transcription of the predicted biocontrol-related genes. The gene clusters coding for 2,4-diacetylphloroglucinol (phlABCDEFGH) and hydrogen cyanide (hcnABC) were shown to be highly transcribed. Further genes predicted to encode putative alginate production enzymes, a pyrroloquinoline quinone precursor peptide PqqA and a matrixin family metalloprotease were also found to be highly transcribed. With this study, we provide a basis to further characterize the mechanisms for biocontrol in Pseudomonas species, towards a sustainable and safe application of P. brassicacearum biocontrol agents.Fil: Nelkner, Johanna. Universitat Bielefeld. Center For Biotechnology; AlemaniaFil: Torres Tejerizo, Gonzalo Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Hassa, Julia. Universitat Bielefeld. Center For Biotechnology; AlemaniaFil: Lin, Timo Wentong. Universitat Bielefeld. Center For Biotechnology; AlemaniaFil: Witte, Julian. Universitat Bielefeld. Center For Biotechnology; AlemaniaFil: Verwaaijen, Bart. Universitat Bielefeld. Center For Biotechnology; AlemaniaFil: Winkler, Anika. Universitat Bielefeld. Center For Biotechnology; AlemaniaFil: Bunk, Boyke. Leibniz - Institute Dsmzgerman Collection Of Microorgani; AlemaniaFil: Spröer, Cathrin. Leibniz - Institute Dsmzgerman Collection Of Microorgani; AlemaniaFil: Overmann, Jörg. Leibniz - Institute Dsmzgerman Collection Of Microorgani; AlemaniaFil: Grosch, Rita. Leibniz - Institute of Vegetable and Ornamental Crops; AlemaniaFil: Pühler, Alfred. Universitat Bielefeld. Center For Biotechnology; AlemaniaFil: Schlüter, Andreas. Universitat Bielefeld. Center For Biotechnology; Alemani

The Role of Petrimonas mucosa ING2-E5AT in Mesophilic Biogas Reactor Systems as Deduced from Multiomics Analyses

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.Peer Reviewe

The role of petrimonas mucosa ING2-E5at in mesophilic biogas reactor systems as deduced from multiomics analyses

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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Effect of Long-Term Farming Practices on Agricultural Soil Microbiome Members Represented by Metagenomically Assembled Genomes (MAGs) and Their Predicted Plant-Beneficial Genes

Nelkner J, Henke C, Lin TW, et al. Effect of Long-Term Farming Practices on Agricultural Soil Microbiome Members Represented by Metagenomically Assembled Genomes (MAGs) and Their Predicted Plant-Beneficial Genes. Genes. 2019;10(6): 424.To follow the hypothesis that agricultural management practices affect structure and function of the soil microbiome regarding soil health and plant-beneficial traits, high-throughput (HT) metagenome analyses were performed on Chernozem soil samples from a long-term field experiment designated LTE-1 carried out at Bernburg-Strenzfeld (Saxony-Anhalt, Germany). Metagenomic DNA was extracted from soil samples representing the following treatments: (i) plough tillage with standard nitrogen fertilization and use of fungicides and growth regulators, (ii) plough tillage with reduced nitrogen fertilization (50%), (iii) cultivator tillage with standard nitrogen fertilization and use of fungicides and growth regulators, and (iv) cultivator tillage with reduced nitrogen fertilization (50%). Bulk soil (BS), as well as root-affected soil (RS), were considered for all treatments in replicates. HT-sequencing of metagenomic DNA yielded approx. 100 Giga bases (Gb) of sequence information. Taxonomic profiling of soil communities revealed the presence of 70 phyla, whereby Proteobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Thaumarchaeota, Firmicutes, Verrucomicrobia and Chloroflexi feature abundances of more than 1%. Functional microbiome profiling uncovered, i.a., numerous potential plant-beneficial, plant-growth-promoting and biocontrol traits predicted to be involved in nutrient provision, phytohormone synthesis, antagonism against pathogens and signal molecule synthesis relevant in microbe–plant interaction. Neither taxonomic nor functional microbiome profiling based on single-read analyses revealed pronounced differences regarding the farming practices applied. Soil metagenome sequences were assembled and taxonomically binned. The ten most reliable and abundant Metagenomically Assembled Genomes (MAGs) were taxonomically classified and metabolically reconstructed. Importance of the phylum Thaumarchaeota for the analyzed microbiome is corroborated by the fact that the four corresponding MAGs were predicted to oxidize ammonia (nitrification), thus contributing to the cycling of nitrogen, and in addition are most probably able to fix carbon dioxide. Moreover, Thaumarchaeota and several bacterial MAGs also possess genes with predicted functions in plant–growth–promotion. Abundances of certain MAGs (species resolution level) responded to the tillage practice, whereas the factors compartment (BS vs. RS) and nitrogen fertilization only marginally shaped MAG abundance profiles. Hence, soil management regimes promoting plant-beneficial microbiome members are very likely advantageous for the respective agrosystem, its health and carbon sequestration and accordingly may enhance plant productivity. Since Chernozem soils are highly fertile, corresponding microbiome data represent a valuable reference resource for agronomy in general.</jats:p

Genomics and prevalence of bacterial and archaeal isolates from biogas-producing microbiomes

Background: To elucidate biogas microbial communities and processes, the application of high-throughput DNA analysis approaches is becoming increasingly important. Unfortunately, generated data can only partialy be interpreted rudimentary since databases lack reference sequences. Results: Novel cellulolytic, hydrolytic, and acidogenic/acetogenic Bacteria as well as methanogenic Archaea originating from different anaerobic digestion communities were analyzed on the genomic level to assess their role in biomass decomposition and biogas production. Some of the analyzed bacterial strains were recently described as new species and even genera, namely Herbinix hemicellulosilytica T3/55T, Herbinix luporum SD1DT, Clostridium bornimense M2/40T, Proteiniphilum saccharofermentans M3/6T, Fermentimonas caenicola ING2-E5BT, and Petrimonas mucosa ING2-E5AT. High-throughput genome sequencing of 22 anaerobic digestion isolates enabled functional genome interpretation, metabolic reconstruction, and prediction of microbial traits regarding their abilities to utilize complex bio-polymers and to perform specific fermentation pathways. To determine the prevalence of the isolates included in this study in different biogas systems, corresponding metagenome fragment mappings were done. Methanoculleus bourgensis was found to be abundant in three mesophilic biogas plants studied and slightly less abundant in a thermophilic biogas plant, whereas Defluviitoga tunisiensis was only prominent in the thermophilic system. Moreover, several of the analyzed species were clearly detectable in the mesophilic biogas plants, but appeared to be only moderately abundant. Among the species for which genome sequence information was publicly available prior to this study, only the species Amphibacillus xylanus, Clostridium clariflavum, and Lactobacillus acidophilus are of importance for the biogas microbiomes analyzed, but did not reach the level of abundance as determined for M. bourgensis and D. tunisiensis. Conclusions: Isolation of key anaerobic digestion microorganisms and their functional interpretation was achieved by application of elaborated cultivation techniques and subsequent genome analyses. New isolates and their genome information extend the repository covering anaerobic digestion community members. © 2017 The Author(s)

Insights into Adaptations of Biogas-Producing Microbial Communities from Taxonomic Profiling, Metagenomically Assembled Genomes and Analysis of the Methanogenic Isolate Methanothermobacter wolfeii SIV6

Hassa J. Insights into Adaptations of Biogas-Producing Microbial Communities from Taxonomic Profiling, Metagenomically Assembled Genomes and Analysis of the Methanogenic Isolate Methanothermobacter wolfeii SIV6. Bielefeld: Universität Bielefeld; 2024.Biogas is an important renewable energy source in Germany. However, in the course of the German energy transition and for political and economic reasons, biogas plants must become more flexible with regard to varying residue-based substrates and thus changing process conditions, while ensuring a stable and efficient process. To meet these requirements, a deeper understanding of the complex biogas-producing microbial communities and the role of community members in the anaerobic digestion (AD) process is necessary, under consideration of the prevailing process conditions. Therefore, the aim of this thesis was to comprehensively analyse biogas-producing microbial communities with regard to their adaptations to the process conditions in differently operated large-scale agricultural digesters. For this purpose, three different approaches were applied to unravel certain types of adaptations of the microbial communities within biogas plants. Firstly, a 16S rRNA gene-based taxonomic profiling of 67 differently operated full-scale biogas digesters from 49 biogas plants revealed three marker microbiome clusters and one group of outliers, which could be explained by the prevailing process conditions of the respective biogas digesters. Moreover, taxa were identified which adapted to specific process conditions and were indicative for these marker microbiomes. In contrast, also resilient taxa were identified, which were process condition independent und thus might support stable process conditions within the analyzed 67 biogas digesters. Secondly, a deep metagenome- and metaproteome-based analysis of the microbial communities of three differently operated digesters of one biogas plant revealed taxonomic and functional adaptations of the whole microbiome and also the role of ten differentially and four evenly abundant metagenomically assembled genomes (MAGs) in the biogas process. Here, especially MAG 80, assigned as member of the class *Limnochordia*, was highly abundant and active in all three digesters suggesting an important role within the AD process, regardless of the prevailing process conditions. Moreover, for MAG 64, classified as the hydrogenotrophic archaeael species *Methanothermobacter wolfeii*, an important role for thermophilic biogas processes is assumed, as it was abundant under thermophilic process conditions and showed high expression of hydrogenotrophic methanogenesis enzymes. Finally, detailed genome-based analyses of the archaeal isolate *Methanothermobacter wolfeii* SIV6, in combination with *in situ* metatranscriptomics and metagenomics, revealed specific genome features as potential adaptations of this strain, that could explain its competitiveness under the thermophilic process conditions of the corresponding biogas digester. The high transcription of the hydrogenotrophic methanogenesis pathway of this strain indicated the important role of this organism in the final methanogenesis step of the thermophilic AD process

Microbiome diversity and community-level change points within manure-based small biogas plants

Efforts to integrate biogas plants into bioeconomy concepts will lead to an expansion of manure-based (small) biogas plants, while their operation is challenging due to critical characteristics of some types of livestock manure. For a better process understanding, in this study, three manure-based small biogas plants were investigated with emphasis on microbiome diversity. Due to varying digester types, feedstocks, and process conditions, 16S rRNA gene amplicon sequencing showed differences in the taxonomic composition. Dynamic variations of each investigated biogas plant microbiome over time were analyzed by terminal restriction fragment length polymorphism (TRFLP), whereby nonmetric multidimensional scaling (NMDS) revealed two well-running systems, one of them with a high share of chicken manure, and one unstable system. By using Threshold Indicator Taxa Analysis (TITAN), community-level change points at ammonium and ammonia concentrations of 2.25 g L-1 and 193 mg L-1 or volatile fatty acid concentrations of 0.75 g L-1were reliably identified which are lower than the commonly reported thresholds for critical process stages based on chemical parameters. Although a change in the microbiome structure does not necessarily indicate an upcoming critical process stage, the recorded community-level change points might be a first indication to carefully observe the process. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Genome Analyses and Genome-Centered Metatranscriptomics of Methanothermobacter wolfeii Strain SIV6, Isolated from a Thermophilic Production-Scale Biogas Fermenter

Hassa J, Wibberg D, Maus I, Pühler A, Schlüter A. Genome Analyses and Genome-Centered Metatranscriptomics of Methanothermobacter wolfeii Strain SIV6, Isolated from a Thermophilic Production-Scale Biogas Fermenter. Microorganisms. 2020;8(1): 13.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 proces