Elucidating Syntrophic Butyrate-Degrading Populations in Anaerobic Digesters Using Stable-Isotope-Informed Genome-Resolved Metagenomics

Abstract

Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in ¹³C after growing on [¹³C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [¹³C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (<95%) with any cultured representative species, indicating that it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for acetoclastic methanogenesis, indicating that it was enriched in ¹³C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to identify in situ interspecies metabolic cooperation within syntrophic consortia important to anaerobic waste treatment as well as global carbon cycling.IMPORTANCE Predicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in situ metabolic activities of uncultured species may enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in situ function of mixed microbiomes by combining stable-isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C₄ fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve "who is doing what" by obtaining confirmatory assimilation of the labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.

Keywords: anaerobic catabolic pathways; anaerobic digestion; metagenomics; methanogenesis; stable-isotope probing; syntrophy.

FIG 1

Relative 16S rRNA gene amplicon sequence abundances of the top 12 most abundant prokaryotic genera in heavy DNA from [¹³C]butyrate-amended microcosms and the [¹²C]butyrate-amended controls for the pulse-fed codigester (A) and the continuously fed codigester (B). Values for each biological duplicate are shown for each condition (¹²C or ¹³C) for both anaerobic digesters.

FIG 2

Phylogenomic tree showing the relationship of ¹³C-enriched Syntrophomonas BUT1 to other genomes available from the Syntrophomonadaceae family in the NCBI nr database (downloaded April 2018). The tree is based on a concatenated alignment of 139 bacterial single-copy marker genes (77) obtained using anvi’o (74). Open reading frames were predicted with Prodigal v.2.6.3 (70) and queried against sequences in a database of bacterial single-copy marker genes using HMMER v.2.3.2 (81). The tree was calculated using FastTree (82). The Clostridium ultunense genome was used as the outgroup.

FIG 3

Phylogenomic tree showing the relationship of the ¹³C-enriched Methanothrix BUT2 to other genomes within the order Methanosarcinales in the NCBI nr database (downloaded April 2018). The tree is based on a concatenated alignment of 162 archaeal single-copy marker genes (78) obtained using anvi’o (74). Open reading frames were predicted with Prodigal v.2.6.3 (70) and queried against sequences in a database of archaeal single-copy marker genes using HMMER v.2.3.2 (81). The tree was calculated using FastTree (82). The “Candidatus Methanoperedens nitroreducens” genome was used as the outgroup.

FIG 4

Cell diagram showing proposed metabolic pathways for anaerobic butyrate degradation in syntrophic cooperation between Syntrophomonas BUT1 and Methanothrix BUT2. The H₂/formate-utilizing methanogenic partner is shown for conceptual purposes but was not identified with [¹³C]DNA-SIP in this study due to its autotrophic growth in the microcosms. Dotted lines indicate the direction of electron flow. Details of predicted proteins are given in Data Sets S2 and S3. Enzyme abbreviations are as follows: Fd, ferredoxin; (Syntrophomonas BUT1) Acd, acyl-CoA dehydrogenase; Crt, enoyl-CoA hydratase; HbdH, 3-hydroxybutyryl-CoA dehydrogenase; AtoB, acetyl-CoA acetyltransferase; AckA, acetate kinase; Pta, phosphate acetyltransferase; EtfA, electron transfer flavoprotein A; EtfB, electron transfer flavoprotein B; EtfD, EtfAB:quinone oxidoreductase; HydABC, bifurcating [Fe-Fe] hydrogenase; HyaABC, [NiFe] hydrogenase; FdhA-HylBC, formate dehydrogenase (electron bifurcating); FdnGHI, formate dehydrogenase (membrane bound, quinone reducing); FixC, electron transfer flavoprotein dehydrogenase; FixX, FixABC-associated ferredoxin; (Methanothrix BUT2) Acs, acetyl-coenzyme A synthetase; CooS, carbon monoxide dehydrogenase; CdhA, acetyl-CoA decarbonylase/synthase complex; Mtr, methyltetrahydromethanopterin:CoM methyltransferase; McrABG, methyl-coenzyme M reductase; HdrED, coenzyme B-coenzyme M heterodisulfide reductase; FpoABDHIJKLMNO, F₄₂₀H₂ dehydrogenase.