Microbial dark matter ecogenomics reveals complex synergistic networks in a methanogenic bioreactor

Abstract

Ecogenomic investigation of a methanogenic bioreactor degrading terephthalate (TA) allowed elucidation of complex synergistic networks of uncultivated microorganisms, including those from candidate phyla with no cultivated representatives. Our previous metagenomic investigation proposed that Pelotomaculum and methanogens may interact with uncultivated organisms to degrade TA; however, many members of the community remained unaddressed because of past technological limitations. In further pursuit, this study employed state-of-the-art omics tools to generate draft genomes and transcriptomes for uncultivated organisms spanning 15 phyla and reports the first genomic insight into candidate phyla Atribacteria, Hydrogenedentes and Marinimicrobia in methanogenic environments. Metabolic reconstruction revealed that these organisms perform fermentative, syntrophic and acetogenic catabolism facilitated by energy conservation revolving around H2 metabolism. Several of these organisms could degrade TA catabolism by-products (acetate, butyrate and H2) and syntrophically support Pelotomaculum. Other taxa could scavenge anabolic products (protein and lipids) presumably derived from detrital biomass produced by the TA-degrading community. The protein scavengers expressed complementary metabolic pathways indicating syntrophic and fermentative step-wise protein degradation through amino acids, branched-chain fatty acids and propionate. Thus, the uncultivated organisms may interact to form an intricate syntrophy-supported food web with Pelotomaculum and methanogens to metabolize catabolic by-products and detritus, whereby facilitating holistic TA mineralization to CO2 and CH4.

Figure 1

Comparison of PhylopythiaS and manually curated metagenome binning results. Principal component analysis on tetramer frequency of metagenomic contigs (>1 kb) binned through (a) PhylopythiaS and (b) combined PhylopythiaS, Metawatt and manual curation are shown with taxonomic bins differentiated by color. Angled (top) and front-face (bottom) views are shown for plots with principal components (PC1 and PC2) and contig read coverage on the horizontal and vertical axes respectively. For the raw PhylopythiaS output, several bins had high read coverage ranges (highlighted with dotted outlines) indicating poor bin quality. Relative to (a) the raw output, (b) manual curation allowed clearer separation of metagenomic bins. Some bins were manually taxonomic reclassified based on 16S rRNA gene phylogeny and BLAST.

Figure 2

TA reactor biofilm microbial community bin and SAG phylogeny and key anaerobic energy conservation pathways. The 16S rRNA gene-based phylogenetic tree (bootstrap 1000: >90% black node, >70% gray node with black outline and >50% gray node) contains sequences from single-cell genomes, analyzed bins (bolded, blue if methanogenic, and red or turquoise based on identified energy conservation pathways detailed below), and related isolates and clones (gray) with phyla distinguished by alternating background color. Classification of target taxa and their proposed Candidatus names are shown to the right of the tree. For each taxon, the genome completeness of SAG (gray), binned draft genome (black), and pangenome (black with *) are shown as pie charts. The following right-hand columns indicate the presence (circle) or absence (blank) of specific genes related to general (turquoise) and syntroph-associated (red) energy conservation. General energy conservation includes electron-confurcating hydrogenase (ECHyd), Rhodobacter nitrogen fixation complex (Rnf) and electron-bifurcating formate dehydrogenase, membrane-bound hydrogenase (Mbh) with or without accessory formate dehydrogenase H (FdhH), whereas syntroph-associated pathways are heterodisulfide reductase-associated putative ion-translocating ferredoxin:NADH oxidoreductase (Hdr-Ifo) and electron-transfer-flavoprotein-oxidizing hydrogenase (FixABCX). The Hdr-Ifo column indicates presence of an Hdr-Ifo gene cassette (red circle), non-adjacent Ifo and Hdr genes (light red circle with red outline) and only Hdr genes (empty red circle).

Figure 3

Reverse electron transport and electron confurcation mechanisms relevant to acetogen, fermenter and syntroph energy conservation. (a) The Rhodobacter nitrogen fixation complex (Rnf) reverse electron transport (RET) generates reduced ferredoxin (Fd_red) for electron-confurcating hydrogenase (ECHyd) H₂ generation. (b) The heterodisulfide reductase (Hdr)-associated putative ion-translocating ferredoxin:NADH oxidoreductase (Hdr-Ifo) is thought to also perform RET-driven Fd_red generation to facilitate energy-conserving H₂ production through either ECHyd or putative Methanothermobacter-like electron-confurcating hydrogenase associated with the Hdr-Ifo cassette. (c) The electron-transfer-flavoprotein (ETF)-oxidizing hydrogenase complex (FixABCX) takes advantage of quinol oxidation-driven H₂ production. (d) Membrane-bound hydrogenases (Mbh) can conserve energy by extruding protons when performing Fd_red-oxidizing H₂ generation. This complex can be modulated by a formate dehydrogenase H (FdhH) to use formate (Fo) as an alternative electron donor. (e) An electron-bifurcating formate dehydrogenase (EBFdh) can facilitate energy-conserving formate metabolism; however, its reversibility remains unclear. (f) The NADH-dependent Fd_red:NADP⁺ oxidoreductase is an electron bi(con)furcating enzyme found in organisms that utilize NADP(H) as an electron carrier (that is, acetogenesis and acetate-degrading syntrophy).

Figure 4

Holistic carbon flux from TA to CH₄ and CO₂. TA degradation generates catabolic by-products and detrital compounds. Catabolic by-products are primarily methanogen-utilizable substrates (MUS) (that is, acetate and H₂) and also include butyrate. Detrital compounds consist of biological macromolecules such as lipids and protein. Syntrophy-related (red), fermentation-related (green), methanogenesis-related (blue) and acetogenesis-related (purple) taxa (name), substrates (box) and pathways (arrow) are shown. Taxa and substrates related to two processes are indicated by both colors. Syntrophic, fermentative and acetogenic secondary degraders and scavengers interact to metabolize these compounds to acetate and H₂ that are finally mineralized to CH₄ and CO₂ by methanogens (blue). In particular, syntroph-to-syntroph substrate transfer (secondary syntrophy, circle-headed line) may play an important role in completing degradation of TA and protein. For scavenging detrital compounds, exoenzyme-producing organisms (bolded) are necessary to hydrolyze macromolecules (dotted gray arrow).