Omics-based characterization of complex anaerobic metabolism in methanogenic wastewater treatment

Below the familiar oxygenated biosphere lay ecosystems teeming with “anaerobic” prokaryotes thriving in the absence of O2. As anaerobes exhaust compounds for favorable respiration (e.g., NO3- and SO42-), microorganisms resort to fermentation and respiration of H+ and CO2. Across Earth, microbial communities under such environmental conditions are estimated to annually mineralize 1~2 GT of organic carbon to CH4 and CO2, thereby driving a critical step in the global carbon cycle. Since the discovery that we can tame such “methanogenic” (methane-generating) microbial communities to convert society’s organic waste to CH4 as a recoverable fuel, this biotechnology has become an essential component of managing municipal and industrial waste and development of sustainable energy. Driven by the environmental and technological significance, research has found four major niches form metabolic interactions to facilitate methanogenic degradation of organic carbon: hydrolyzers, fermenters, syntrophs, and methanogens. Despite this defined general ecological structure, many organisms and metabolism in methanogenic ecosystems remain uncharacterized due to challenges in handling and cultivating anaerobes. To tackle this issue, we can employ rapidly developing sequencing technology to recover genomes for uncultivated organisms directly from the environment (“metagenomics”), obtain insight into their physiology, and ultimately uncover hitherto overlooked ecological and biochemical processes taking place in methanogenic natural ecosystems and engineered systems. In the series of studies presented in this dissertation, we use methanogenic wastewater treatment bioreactors as model ecosystems and implement cutting-edge bioinformatics with rigorous annotation of anaerobic metabolic capacities to investigate the ecological roles of uncultured syntrophs, methanogens, and organisms from other bacterial lineages. For syntrophs, we characterize novel aromatic compound degradation pathways and find that syntrophic catabolism and interactions are much more diverse and flexible than previously anticipated, opening new possibilities for ecological niches that syntrophs can exploit. In investigating methanogens, we successfully recover the first genomes for a methanogen-related Euryarchaeota class WSA2 found across various anaerobic environments and discover that they encode unique H2-oxidizing methyl-compound-reducing methanogenesis, suggesting that this may be a major process in both natural and engineered methanogenic environments. As for uncharacterized bacterial lineages, we acquire genomes for populations spanning 15 phyla, of which 5 are bacterial phyla with no cultured representatives (“candidate phyla”). We find that these organisms may contribute to novel syntrophic, fermentative, and acetogenic processes and form intricate metabolic interactions to facilitate complete mineralization of organic matter to in methanogenic ecosystems. Finally, to expand the application of the approach used throughout these studies, we compile the accumulated insight into genomics and complex metabolism and perform an unprecedentedly large-scale comparative genomics analysis on a bacterial phylum that contains both uncultivated lineages affiliated with methanogenic ecosystems and poorly understood lineages prevalent across Earth: Bacteroidetes. This reveals novel relationships between phylogeny, metabolism, and habitats and unnoticed ecological roles that Bacteroidetes can take in methanogenic environments, marine ecosystems, and even the human gastrointestinal tract. In total, we demonstrate that integration of metagenomics, comparative genomics, and strict annotation of metabolic capacity can effectively characterize the ecophysiology of uncultivated organisms and reveal novel ecological niches in methanogenic environments and beyond

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

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

Draft Genome Sequence of Syntrophorhabdus aromaticivorans Strain UI, a Mesophilic Aromatic Compound-Degrading Syntroph

Syntrophorhabdus aromaticivorans strain UI is a mesophilic bacterium capable of degrading aromatic substrates in syntrophic cooperation with a partner methanogen. The draft genome sequence is 3.7 Mb, with a G+C content of 52.0%

Methanogenic archaea use a bacteria-like methyltransferase system to demethoxylate aromatic compounds

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth’s diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H(2)), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C(1)-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F(420)H(2) re-oxidation) as found for methylotrophic methanogenesis

A hydrogen-dependent geochemical analogue of primordial carbon and energy metabolism

Hydrogen gas, H2, is generated by alkaline hydrothermal vents through an ancient geochemical process called serpentinization in which water reacts with iron containing minerals deep within the Earth's crust. H2 is the electron donor for the most ancient and the only energy releasing route of biological CO2 fixation, the acetyl-CoA pathway. At the origin of metabolism, CO2 fixation by hydrothermal H2 within serpentinizing systems could have preceded and patterned biotic pathways. Here we show that three hydrothermal minerals—greigite (Fe3S4), magnetite (Fe3O4) and awaruite (Ni3Fe)—catalyse the fixation of CO2 with H2 at 100°C under alkaline aqueous conditions. The product spectrum includes formate (up to 200 mM), acetate (up to 100 µM), pyruvate (up to 10 µM), methanol (up to 100 µM), and methane. The results shed light on both the geochemical origin of microbial metabolism and on the nature of abiotic formate and methane synthesis in modern hydrothermal vents

Elucidating syntrophic butyrate-degrading populations in anaerobic digesters using stable-isotope-informed genome-resolved metagenomics

<p>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 (&lt;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.</p

Elucidating syntrophic butyrate-degrading populations in anaerobic digesters using stable-isotope-informed genome-resolved metagenomics

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 (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.</p

Elucidation of the biodegradation pathways of bis(2-hydroxyethyl) terephthalate and dimethyl terephthalate under anaerobic conditions revealed by enrichment culture and microbiome analysis

With the globally rising usage of plastics, including polyethylene terephthalate (PET), the environmental risk that disposal of waste plastics to landfills and discharge of microplastics to the marine environment pose have also increased. For example, observation of animal ingestion of fragmented waste plastics (micro-and nano-plastics) has driven awareness for the need of proper environmental risk assessment. In evaluating the biodegradability of PET-derived byproducts and their precursors, most work has focused on hydrolytic enzymes and aerobic or-ganisms that possess such genes, but only few reports on biodegradation in the absence of oxygen (i.e., anaerobic) are available. Here, to elucidate the fate of PET-derived materials under anaerobic environments, a sludge -derived microbial community was cultured with bis(2-hydroxyethyl) terephthalate (BHET) as a model sub-strate for byproducts of PET degradation and dimethyl terephthalate (DMT) as a potential environmental pollutant discharged from the PET manufacturing process. Metagenome-and metabolome-informed microbiome analyses identified anaerobic BHET and DMT degradation pathways, uncultured organisms affiliated with Spi-rochaetota and Negativicutes predominant in the BHET-fed cultures, and Methanomethylovorans and Trepone-ma_G predominant in the DMT-fed cultures. Metagenomic analyses newly identified three BHET-degrading and two DMT-degrading enzymes from the genomes of Spirochaeota. In addition, the Negativicutes in the BHET enrichment cultures possessed genes for acetogenically metabolizing EG and/or ethanol. Overall, this study successfully established anaerobic BHET-and DMT-degrading microbial consortia and newly proposed these degradation mechanisms under anaerobic conditions. This study indicated that the cultivation, microbiome, and metabolome analyses can be powerful tools for elucidating consortia capable of degrading plastics-associated waste compounds and the relevant metabolic mechanisms

Microbial Community Analysis of Anaerobic Reactors Treating Soft Drink Wastewater

<div><p>The anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors containing methanogenic microbial consortia were applied to treat synthetic soft drink wastewater, which contains polyethylene glycol (PEG) and fructose as the primary constituents. The AP and HP reactors achieved high COD removal efficiency (>95%) after 80 and 33 days of the operation, respectively, and operated stably over 2 years. 16S rRNA gene pyrotag analyses on a total of 25 biofilm samples generated 98,057 reads, which were clustered into 2,882 operational taxonomic units (OTUs). Both AP and HP communities were predominated by <i>Bacteroidetes</i>, <i>Chloroflexi</i>, <i>Firmicutes</i>, and candidate phylum KSB3 that may degrade organic compound in wastewater treatment processes. Other OTUs related to uncharacterized <i>Geobacter</i> and <i>Spirochaetes</i> clades and candidate phylum GN04 were also detected at high abundance; however, their relationship to wastewater treatment has remained unclear. In particular, KSB3, GN04, <i>Bacteroidetes</i>, and <i>Chloroflexi</i> are consistently associated with the organic loading rate (OLR) increase to 1.5 g COD/L-d. Interestingly, KSB3 and GN04 dramatically decrease in both reactors after further OLR increase to 2.0 g COD/L-d. These results indicate that OLR strongly influences microbial community composition. This suggests that specific uncultivated taxa may take central roles in COD removal from soft drink wastewater depending on OLR.</p></div