Syntrophic acetate oxidation replaces acetoclastic methanogenesis during thermophilic digestion of biowaste

Abstract

Background: Anaerobic digestion (AD) is a globally important technology for effective waste and wastewater management. In AD, microorganisms interact in a complex food web for the production of biogas. Here, acetoclastic methanogens and syntrophic acetate-oxidizing bacteria (SAOB) compete for acetate, a major intermediate in the mineralization of organic matter. Although evidence is emerging that syntrophic acetate oxidation is an important pathway for methane production, knowledge about the SAOB is still very limited.

Results: A metabolic reconstruction of metagenome-assembled genomes (MAGs) from a thermophilic solid state biowaste digester covered the basic functions of the biogas microbial community. Firmicutes was the most abundant phylum in the metagenome (53%) harboring species that take place in various functions ranging from the hydrolysis of polymers to syntrophic acetate oxidation. The Wood-Ljungdahl pathway for syntrophic acetate oxidation and corresponding genes for energy conservation were identified in a Dethiobacteraceae MAG that is phylogenetically related to known SAOB. 16S rRNA gene amplicon sequencing and enrichment cultivation consistently identified the uncultured Dethiobacteraceae together with Syntrophaceticus, Tepidanaerobacter, and unclassified Clostridia as members of a potential acetate-oxidizing core community in nine full-scare digesters, whereas acetoclastic methanogens were barely detected.

Conclusions: Results presented here provide new insights into a remarkable anaerobic digestion ecosystem where acetate catabolism is mainly realized by Bacteria. Metagenomics and enrichment cultivation revealed a core community of diverse and novel uncultured acetate-oxidizing bacteria and point to a particular niche for them in dry fermentation of biowaste. Their genomic repertoire suggests metabolic plasticity besides the potential for syntrophic acetate oxidation. Video Abstract.

Keywords: 16S rRNA gene amplicons; Anaerobic digestion; Enrichment cultures; Metagenome; Syntrophic acetate-oxidizing bacteria.

Fig. 1

Taxonomic assignment of quality trimmed metagenome reads summarized on phylum level (a) based on total reads (outer ring) and 16S rRNA gene fragments (inner ring). Phylogeny of metagenome assembled genomes based on concatenated marker protein sequences and selected metabolic functions encoded in their genomes (b). Further information about contamination and completeness of the MAGs is provided in Additional file 1: Table S1. RAxML bootstrap support > 70% is indicated by open circles, and bootstrap support > 90% is indicated by closed circles. Multifurcations were introduced for branches with bootstrap support < 50%

Fig. 2

Number and groups of carbohydrate active enzymes identified in the MAGs. Major functions associated with selected groups are indicated. Glycosyl hydrolase families (GH) primarily include carbohydrate degrading enzymes

Fig. 3

Putative functions of the MAGs in the anaerobic digestion food web (a). The asterisk (MAG-R9) indicates a potential function that merely proposed in literature (see text for details). Wood-Ljungdahl pathway genes in MAGs of syntrophic and putatively syntrophic bacteria as well as the corresponding energy-conserving mechanisms (b). Hyd, [FeFe] hydrogenase group A3; Hdr, heterodisulfide reductase; NiFe H2ase, energy-conserving group 1a [NiFe] hydrogenase; Rnf, Na⁺/H⁺ translocating ferredoxin:NAD⁺ oxidoreductase; Q, quinone; ETF, electron transfer flavoprotein; ETF-Q OR, electron transfer flavoprotein/quinone oxidoreductase; FDH, formate dehydrogenase; Ftr, formate transporter; EM, electron mediator; CM, cytoplasmic membrane; THF, tetrahydrofolate. Genes involved in the Wood-Ljungdahl pathway are acetate kinase (1), phosphotransacetylase (2), acetyl-CoA synthase complex including corrinoid protein (CoFeSP) (3), carbon monoxide dehydrogenase (4), methyltransferase (5), methylene-THF reductase (6), methylene-THF dehydrogenase (7), methenyl-THF cyclohydrolase (8), formyl-THF synthethase (9), and formate dehydrogenase (10)

Fig. 4

Relative abundance of major bacterial and archaeal groups in acetate-fed enrichment cultures. The microbial community was either analyzed by 16S rRNA gene amplicon sequencing (iTags) or metagenome sequencing (Meta)

Fig. 5

Microbial community composition in nine full-scale thermophilic biowaste digesters. The archaeal fraction of total prokaryotes and the relative abundance of archaeal amplicons was revealed by 16S rRNA gene amplicon sequencing (a). Percentages of taxa relative to total bacterial amplicons are only shown for major groups that contributed ≥ 1% to all bacterial 16S rRNA sequences in at least one sample (b). Bray-Curtis dissimilarity based on OTUs clustered at 97% identity is depicted at the bottom