Identification and genome reconstruction of abundant distinct taxa in microbiomes from one thermophilic and three mesophilic production-scale biogas plants

Abstract

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 Defluviitoga tunisiensis 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.

Keywords: 16S rRNA gene; Anaerobic digestion; Biogas; Cloacimonetes (WWE1); Fusobacteria; Genome binning; Metagenomics; Microbial community; Spirochaetes; Thermotogae.

Fig. 1

Taxonomic profiles of the four biogas plants (BGPs) on phylum level, based on 16S rRNA gene amplicon sequencing. The respective relative abundances of the replicates for each BGP are shown. Four taxa, distinctly and abundantly present in one or two of the BGPs, were identified: Thermotogae, Fusobacteria, Spirochaetes and Cloacimonetes, highlighted with asterisks

Fig. 2

Partial phylogenetic trees of all available type strains with operational taxonomic units (OTUs) of Thermotogae (a), Fusobacteria, Spirochaetes and Cloacimonetes (b) taxa of the studied biogas plants and their closest non-type strain relatives embedded. Type strains are in black, OTUs are in red, their closest relatives in purple, their closest sequenced relatives in blue and their closest type strain relatives in green. 16S rRNA sequence tree construction was done using the ARB software [31]

Fig. 3

Venn diagrams showing the number of unique and shared genes between the five genome bins and their respective reference strains. Diagrams are shown for genome bins assigned to the phyla Thermotogae (206_Thermotogae, a), Fusobacteria (175_Fusobacteria, b), Spirochaetes (138_Spirochaetes, c) and Cloacimonetes (120_Cloacimonetes, 244_Cloacimonetes; d). Venn diagrams were redrawn manually, based on the original EDGAR output, with their areas drawn to scale