Non-autotrophic methanogens dominate in anaerobic digesters

Abstract

Anaerobic digesters are man-made habitats for fermentative and methanogenic microbes, and are characterized by extremely high concentrations of organics. However, little is known about how microbes adapt to such habitats. In the present study, we report phylogenetic, metagenomic, and metatranscriptomic analyses of microbiomes in thermophilic packed-bed digesters fed acetate as the major substrate, and we have shown that acetoclastic and hydrogenotrophic methanogens that utilize acetate as a carbon source dominate there. Deep sequencing and precise binning of the metagenomes reconstructed complete genomes for two dominant methanogens affiliated with the genera Methanosarcina and Methanothermobacter, along with 37 draft genomes. The reconstructed Methanosarcina genome was almost identical to that of a thermophilic acetoclastic methanogen Methanosarcina thermophila TM-1, indicating its cosmopolitan distribution in thermophilic digesters. The reconstructed Methanothermobacter (designated as Met2) was closely related to Methanothermobacter tenebrarum, a non-autotrophic hydrogenotrophic methanogen that grows in the presence of acetate. Met2 lacks the Cdh complex required for CO₂ fixation, suggesting that it requires organic molecules, such as acetate, as carbon sources. Although the metagenomic analysis also detected autotrophic methanogens, they were less than 1% in abundance of Met2. These results suggested that non-autotrophic methanogens preferentially grow in anaerobic digesters containing high concentrations of organics.

Figure 1

Bioreactor operation and enrichment of methanogenic consortia. (a) Time course of OLR, HRT, COD in effluent, and biogas production rates in reactor 1. Data on the operation of reactor 2 are shown in Supplementary Fig. S2. (b) Relative abundances of major archaeal and bacterial families in reactor 1 (days 122 and 200) and reactor 2 (day 159) based on pyrosequenced 16S rRNA gene amplicons.

Figure 2

Neighbor-joining trees based on nucleotide sequences of mcrA genes showing phylogenetic relationships in the genus Methanosarcina (a) and Methanothermobacter (b). Bootstrap values (100 trials, only >50 are shown) are indicated at branching points. The bar indicates 5% sequence divergence. GI numbers are shown in parentheses.

Figure 3

Relative abundances of methanogens in reactor 1 based on RPKM values for mcrA genes.

Figure 4

Differential coverage plot for assembled contigs. DNA reads from BF and PF samples were assembled together, and the coverage values of these reads were plotted for each contig. Contigs were phylogenetically classified at the phylum level using PhyloPythiaS. Contig lengths correspond to bubble sizes. Representative bin-genomes are indicated by dotted ellipses.

Figure 5

Comparative genomics of reconstructed Methanosarcina (a,b) and Methanothermobacter (c,d) strains. (a,c) Overall genome comparisons with closely related isolates using BRIG. The homology regions with the genomes of Ms. thermophila TM-1 (BLASTN e-value ≤ 1e-9) and Mt. thermautotrophicus ∆H (e-value ≤ 1e-2) are indicated by colors. The first (inner-most) and second circles show the GC contents and GC-skew, respectively. The locations of some genes of interest are indicated by red boxes. (b,d) Venn diagrams showing peculiar and shared CDSs coded in Methanosarcina (b) and Methanothermobacter (d). Redundant genes were excluded from the analysis.

Figure 6

Reconstructed pathways for methanogenesis and associated metabolism in the four methanogens and their gene-expression profiles. Normalized expression levels (mRNA/DNA) and fold changes in the expression level (days 122/day 200) are shown as heatmaps. For an enzyme encoded by multiple subunit genes, average values for each gene are shown. Genes absent from each bin-genome are shown as blanks in heatmap boxes. The pathways are depicted according to previous reports^{, , , –}. Abbreviations: Fd_ox/Fd_red, oxidized and reduced ferredoxin; MP/MPH₂, oxidized and reduced methanophenazine; CoB-SH, coenzyme B; CoM-SH, coenzyme M; CoM-S-S-CoB, mixed disulfide of CoM-SH and CoB-SH; F₄₂₀/F₄₂₀H₂, oxidized and reduced Factor 420; H₄MPT, tetrahydromethanopterin; Fwd, tungsten formylmethanofuran dehydrogenase; Fmd, molybdenum formylmethanofuran dehydrogenase; Ftr, formylmethanofuran:H₄MPT formyltransferase; Mch, methenyl-H₄MPT cyclohydrolase; Mtd, F₄₂₀-dependent methylene-H₄MPT dehydrogenase; Mer, methylene-H₄MPT reductase; Mtr, methyl-H₄MPT: coenzyme M methyltransferase; Mcr, methyl-coenzyme M reductase; Hdr, heterodisulfide reductase; Ech, energy-converting hydrogenase; Frh, F₄₂₀-reducing hydrogenase; Mvh/Vhu/Vhc/Vht/Hya/Hyb, non F₄₂₀-reducing hydrogenase; AcsS, acetyl-CoA synthetase; Ack, acetate kinase; Pta, phosphotransacetylase; Cdh, CO dehydrogenase/acetyl-CoA synthase; MtaBC, methanol: 5-hydroxybenzimidazolylcobamide Co-methyltransferase; MtaA, methylcobalamin:coenzyme M methyltranferase; Ntp; proton or sodium-translocating ATPase.