A pyrosequencing-based metagenomic study of methane-producing microbial community in solid-state biogas reactor

Abstract

Background: A solid-state anaerobic digestion method is used to produce biogas from various solid wastes in China but the efficiency of methane production requires constant improvement. The diversity and abundance of relevant microorganisms play important roles in methanogenesis of biomass. The next-generation high-throughput pyrosequencing platform (Roche/454 GS FLX Titanium) provides a powerful tool for the discovery of novel microbes within the biogas-generating microbial communities.

Results: To improve the power of our metagenomic analysis, we first evaluated five different protocols for extracting total DNA from biogas-producing mesophilic solid-state fermentation materials and then chose two high-quality protocols for a full-scale analysis. The characterization of both sequencing reads and assembled contigs revealed that the most prevalent microbes of the fermentation materials are derived from Clostridiales (Firmicutes), which contribute to degrading both protein and cellulose. Other important bacterial species for decomposing fat and carbohydrate are Bacilli, Gammaproteobacteria, and Bacteroidetes (belonging to Firmicutes, Proteobacteria, and Bacteroidetes, respectively). The dominant bacterial species are from six genera: Clostridium, Aminobacterium, Psychrobacter, Anaerococcus, Syntrophomonas, and Bacteroides. Among them, abundant Psychrobacter species, which produce low temperature-adaptive lipases, and Anaerococcus species, which have weak fermentation capabilities, were identified for the first time in biogas fermentation. Archaea, represented by genera Methanosarcina, Methanosaeta and Methanoculleus of Euryarchaeota, constitute only a small fraction of the entire microbial community. The most abundant archaeal species include Methanosarcina barkeri fusaro, Methanoculleus marisnigri JR1, and Methanosaeta theromphila, and all are involved in both acetotrophic and hydrogenotrophic methanogenesis.

Conclusions: The identification of new bacterial genera and species involved in biogas production provides insights into novel designs of solid-state fermentation under mesophilic or low-temperature conditions.

Figure 1

The electrophoresis results of DNA preparations based on the five methods from a biogas reactor sample. M, molecular marker Transplus 2 K. P, F, E, EY, S refer to the five protocols, respectively. Except for Protocol P, which was loaded with 5 μl of undiluted DNA solution, only 1 μl of undiluted DNA solution was loaded on the 0.8% agarose gels for the rest of the extracts.

Figure 2

Rarefaction curves (A) and the relative proportions of taxonomic classification of bacteria, archaea, eukaryota, and viruses (B) from the BE-1 and BEY libraries. For the rarefaction curves, the analysis was performed on the total (including bacteria, archaea, eukaryota, viruses and environmental sequences), archaeal, and bacterial taxa from the two libraries.

Figure 3

The statistics for the reads assigned (A), the number of reads/contigs for the 11 most prevalent phyla (B), and the number of reads/contigs of 15 most prevalent genera in bacteria and the 3 most prevalent genera in archaea (C) obtained in the taxonomic classification analysis using BLASTN/BLASTX tools against the GenBank NT/NR database with a E-value cutoff of 10^-5 based on total reads/contigs.

Figure 4

Statistics for the reads assigned to microbial genome sequences using BLASTN/BLASTX tools against the GenBank NT/NR database with an E-value cutoff of 10^-5 based on the total reads. The x-axis denotes the number of reads assigned to the 50 most prevalent microbial strain genomes.

Figure 5

Methanogenesis pathway predicted in our fermenter based on the KEGG analysis. The ellipses denote the substances involved in the reaction. The boxes show the enzymes involved in methanogenesis. The acetotrophic (green), the hydrogenotrophic (blue), and the shared pathways (grey) are all color-encoded and the color depth indicates the amount of reads assigned. The enzymes that are potentially associated with Anaerococcus and Psychrobacter in the methanogenesis pathway are shown in the yellow box.

Figure 6

Categorization of the biogas-fermenter metagenomic sequencing reads according to the Clusters of Orthologous Groups of proteins (COGs). The categories are abbreviated as follows: J, translation, ribosomal structure and biogenesis; A, RNA processing and modification; K, transcription; L, replication, recombination and repair; B, chromatin structure and dynamics; D, cell cycle control, cell division, chromosome partitioning; V, defense mechanisms; T, signal transduction mechanisms; M, cell wall/membrane/envelope biogenesis; N, cell motility; W, extracellular structures; U, intracellular trafficking, secretion, and vesicular transport; O, posttranslational modification, protein turnover, chaperones; C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; Q, secondary metabolites biosynthesis, transport and catabolism; R, general function prediction only; and S, function unknown.