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Additional file 2:. Review history

Additional File 1:. Fig. S1-S4 and Tables S1-S8

Instructions for reproducing the comparisons of taxonomic profilers. (PDF 79 kb)

Supplementary information. Taxonomic profile based community design: BIOM format details, Reference database. De novo community design: Creation of the mapping file. Genome assembly metrics. Methods: iTol, Parameters, PICRUST, Configfile. OTU mapping file. (PDF 146 kb)

Supplementary tables and figures. (PDF 2601 kb)

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. Electronic supplementary material The online version of this article (10.1186/s13068-017-0947-1) contains supplementary material, which is available to authorized users.

Background Biofuel production from conversion of biomass is indispensable in the portfolio of renewable energies. Complex microbial communities are involved in the anaerobic digestion process of plant material, agricultural residual products and food wastes. Analysis of the genetic potential and microbiology of communities degrading biomass to biofuels is considered to be the key to develop process optimisation strategies. Hence, due to the still incomplete taxonomic and functional characterisation of corresponding communities, new and unknown species are of special interest. Results Three mesophilic and one thermophilic production-scale biogas plants (BGPs) were taxonomically profiled using high-throughput 16S rRNA gene amplicon sequencing. All BGPs shared a core microbiome with the thermophilic BGP featuring the lowest diversity. However, the phyla Cloacimonetes and Spirochaetes were unique to BGPs 2 and 3, Fusobacteria were only found in BGP3 and members of the phylum Thermotogae were present only in the thermophilic BGP4. Taxonomic analyses revealed that these distinctive taxa mostly represent so far unknown species. The only exception is the dominant Thermotogae OTU featuring 16S rRNA gene sequence identity to Defluviitoga tunisiensis L3, a sequenced and characterised strain. To further investigate the genetic potential of the biogas communities, corresponding metagenomes were sequenced in a deepness of 347.5 Gbp in total. A combined assembly comprised 80.3 % of all reads and resulted in the prediction of 1.59 million genes on assembled contigs. Genome binning yielded genome bins comprising the prevalent distinctive phyla Cloacimonetes, Spirochaetes, Fusobacteria and Thermotogae. Comparative genome analyses between the most dominant Thermotogae bin and the very closely related Defluviitogatunisiensis L3 genome originating from the same BGP revealed high genetic similarity. This finding confirmed applicability and reliability of the binning approach. The four highly covered genome bins of the other three distinct phyla showed low or very low genetic similarities to their closest phylogenetic relatives, and therefore indicated their novelty. Conclusions In this study, the 16S rRNA gene sequencing approach and a combined metagenome assembly and binning approach were used for the first time on different production-scale biogas plants and revealed insights into the genetic potential and functional role of so far unknown species. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0565-3) contains supplementary material, which is available to authorized users.

Additional file 4: Table S1. Genes encoding proteins involved in energy generation found in the reference genome of Defluviitoga tunisiensis L3 [90] and their presence or absence within the Thermotogae genome bin.

Additional file 1. Additional information is available at Biotechnology for Biofuels journal’s website as pdf document, Maus_et_al_v2016-08-03_additional_file_1.pdf. This file contains Additional information on methods as well as additional figures and tables as follows: (i) Additional information on methods for extraction of total microbial RNA. (ii) Additional information on methods for identification of isolates. (iii) Table S1. Statistics of quality-controlled metagenomes sequences data for the microbial community of the thermophilic production-scale biogas plant. (iv) Table S2. Statistics of taxonomically classified sequences obtained from the metagenome and metatranscriptome datasets. (v) Table S3. Number of 16S rRNA sequences and corresponding percentages of bacterial and archaeal genera within the analyzed metagenome and metatranscriptome datasets. The number of metatranscriptome-derived 16S rRNA sequences, i.e. 532,381, was normalized to the number of metagenome-derived sequences, i.e. 18,817. Only genera with at least 10 (normalized) sequences in one of the datasets were considered. ND = no sequences were determined in the dataset. (vi) Figure S1. Exemplary work-flow illustration of the different strategies for the isolation of cellulolytic bacteria. Isolation strategy (3): direct enrichment and isolation using different media. Isolation strategy (4): serial dilution (i.e. dilution to extinction) in liquid medium. Isolation strategy (5): direct plating on solid medium without previous enrichment in liquid culture. (vii) Figure S2. Relative abundance of the 25 most abundant families within the biogas microbial community of the thermophilic biogas plant as deduced from 16S rRNA gene sequences of combined metagenome datasets (>= 0.05 % of the sequences in the metagenome). Taxonomic assignments of the 16S rRNA gene sequences were obtained applying BLASTN against the RDP database applying MGX. (viii) Figure S3. Relative abundance of the 25 most abundant families within the biogas microbial community of the thermophilic biogas plant as deduced from 16S rRNA sequences of the metatranscriptome datasets (>= 0.05 % of the sequences in the metatranscriptome). Taxonomic assignments of the 16S rRNA sequences were obtained applying BLASTN against the RDP database applying MGX. (ix) Figure S4. Cultivable microbial species applying the isolation strategies (1)–(2) and corresponding cell counts in samples of substrate (swine manure), fermenter content (main fermenter), and digestate (second fermenter). The cultivation temperature was 37 or 50 °C (*), respectively. (x) Figure S5. Light- (a) and fluorescent (b) microscopic picture of Methanothermobacter marburgensis isolated from the thermophilic biogas plant. (xi) Figure S6. Phylogenetic tree of selected archaeal (a) and bacterial (b) isolates in relation to corresponding type species. The 16S rRNA gene sequences from closely related type species were obtained from the SILVA database [47]. The isolate T3/55T represents the type strain for a novel genus and species, namely Herbinix hemicellulosilytica, which was recently described by [35]. (xii) Figure S7. Fragment recruitment of metagenome sequences derived from the microbial community of the analyzed thermophilic biogas plant to the genomes of the strains Clostridium cellulosi str. DG5 (a), Herbinix hemicellulosilytica str. T3/55T (b) and Defluviitoga tunisiensis str. L3 (c). Only metagenome sequence reads with more than 75 % sequence identity (y-axis) to the corresponding genome were mapped. Percent identity (y-axis) of a mapped metagenome read was plotted against the mapping position on the genome sequence (x-axis) of H. hemicellulosilytica str. T3/55T or D. tunisiensis str. L3. The x-axis represents the extension (scale) of the C. cellulosi str. DG5, H. hemicellulosilytica str. T3/55T or D. tunisiensis str. L3 genome. Sequence coverage is visualized by gray scale intensity (right margin). Matching sequence read length is indicated by the diameter of the circle representing the mapping position (scale is given on the top right).

The vast majority of microbial species found in nature has yet to be grown in pure culture, turning metagenomics and – more recently – single cell genomics into indispensable methods to study the microbial dark matter. I developed, applied, and benchmarked genome assembly protocols for single cell and metagenome sequencing data to access microbial dark matter genomes. In the first part of my thesis, I propose new algorithms that naturally exploit the complementary nature of single cells and metagenomes to improve the quality of single cell assemblies. In the second part, I apply advanced metagenome assembly and binning techniques to untangle genomes from metagenomes, eventually reconstructing hundreds of near-complete genomes of process-relevant community members in the biogas microbiome.

Background The production of biogas takes place under anaerobic conditions and involves microbial decomposition of organic matter. Most of the participating microbes are still unknown and non-cultivable. Accordingly, shotgun metagenome sequencing currently is the method of choice to obtain insights into community composition and the genetic repertoire. Findings Here, we report on the deeply sequenced metagenome and metatranscriptome of a complex biogas-producing microbial community from an agricultural production-scale biogas plant. We assembled the metagenome and, as an example application, show that we reconstructed most genes involved in the methane metabolism, a key pathway involving methanogenesis performed by methanogenic Archaea. This result indicates that there is sufficient sequencing coverage for most downstream analyses. Conclusions Sequenced at least one order of magnitude deeper than previous studies, our metagenome data will enable new insights into community composition and the genetic potential of important community members. Moreover, mapping of transcripts to reconstructed genome sequences will enable the identification of active metabolic pathways in target organisms.