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

Novel energy conservation strategies and behavior of Pelotomaculum schinkii driving syntrophic propionate catabolism

Under methanogenic conditions, short-chain fatty acids are common byproducts from degradation of organic compounds and conversion of these acids is an important component of the global carbon cycle. Due to the thermodynamic difficulty of propionate degradation, this process requires syntrophic interaction between a bacterium and partner methanogen; however, the metabolic strategies and behavior involved are not fully understood. In this study, the first genome analysis of obligately syntrophic propionate degraders (Pelotomaculum schinkii HH and P. propionicicum MGP) and comparison with other syntrophic propionate degrader genomes elucidated novel components of energy metabolism behind Pelotomaculum propionate oxidation. Combined with transcriptomic examination of P. schinkii behavior in co-culture with Methanospirillum hungatei, we found that formate may be the preferred electron carrier for P. schinkii syntrophy. Propionate-derived menaquinol may be primarily re-oxidized to formate, and energy was conserved during formate generation through newly proposed proton-pumping formate extrusion. P. schinkii did not overexpress conventional energy metabolism associated with a model syntrophic propionate degrader Syntrophobacter fumaroxidans MPOB (i.e., CoA transferase, Fix, and Rnf). We also found that P. schinkii and the partner methanogen may also interact through flagellar contact and amino acid and fructose exchange. These findings provide new understanding of syntrophic energy acquisition and interactions. This article is protected by copyright. All rights reserved.We thank Steven Aalvink for scanning electron microscopy analysis and WEMC for making the system available. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013) / ERC Grant Agreement n. [323009] and a Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Organisation for Scientific Research (NWO). This work was also supported by The Japan Society for the Promotion of Science with Grant-in-Aid for Scientific Research No. 18H03367 to MK Nobu and 17H05239 and 18H01576 to T Narihiro.info:eu-repo/semantics/publishedVersio

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

Metagenomic characterization of Candidatatus Defluviicoccus tetraformis TFO71, a tetrad-forming organism, predominant in an anaerobic-aerobic membrane bioreactor with deteriorated biological phosphorus removal

In an acetate-fed anaerobic-aerobic membrane bioreactor with deteriorated enhanced biological phosphorus removal (EBPR), Defluviicoccus-related tetrad-forming organisms (DTFO) were observed to predominate in the microbial community. Using metagenomics, a partial genome of the predominant DTFO, “Candidatus Defluviicoccus tetraformis TFO71,” was successfully constructed and characterized. Examining the genome confirmed the presence of genes related to the synthesis and degradation of glycogen and polyhydroxyalkanoate (PHA), which function as energy and carbon storage compounds. Both TFO71 and Candidatus Accumulibacter phosphatis (CAP) UW-1, a representative polyphosphate-accumulating organism (PAO), have PHA metabolism-related genes with high homology, but TFO71 has unique genes for PHA synthesis, gene regulation, and granule management. We further discovered genes encoding DTFO polyphosphate (polyP) synthesis, suggesting that TFO71 may synthesize polyP under untested conditions. However, TFO71 may not activate these genes under EBPR conditions because the retrieved genome does not contain inorganic phosphate transporters that are characteristic of PAOs (CAP UW-1, Microlunatus phosphovorus NM-1, and Tetrasphaera species). As a first step in characterizing EBPR-associated DTFO metabolism, this study identifies important differences between TFO and PAO that may contribute to EBPR community competition and deterioration

Recovery from terrestrial oil spills: the damage, soil microbial oil degradation, and plant revegetation

As a result of modern oil exploration, use, and transportation, many terrestrial ecosystems have been polluted by oil spills. This oil pollution significantly stresses the flora and soil microorganisms through both toxic effects and drastic environmental changes. In response to this ecosystem disturbance, hydrocarbon-degrading microorganisms rapidly respond to degrade the pollutants. During this natural bioremediation process, recent studies have found evidence for hydrocarbon-degrading microorganism community succession. As this microbial bioremediation proceeds, the microorganisms often exhaust the soils\u27 bioavailable nitrogen. While most flora seldom revegetate such a nitrogen poor soil, legumes often exploit this niche and dominate the oil-polluted soil

The euxinic zone anaerobic phototrophic microbial ecology of meromictic Green Lake as an analogue for Proterozoic ocean microbial ecology

During the Proterozoic, Earth\u27s atmosphere remained low in oxygen despite the advent of oxygenic primary production. The Canfield Ocean and Johnston model for the ancient Proterozoic ocean a stratified ocean with anoxygenic phototrophs dominating the primary production rather than oxygenic phototrophs. This model necessitates a stratified ocean with an oxic layer overlying a euxinic layer. Anaerobic anoxygenic photrophs such as purple and green sulfur bacteria can contribute significantly to the primary production if the euxinic zone reaches the photic zone. Fayetteville Green Lake (FGL) mimics this model\u27s stratified ocean environment. A fluorescence in-situ hybridization study on the bacterial community reveals that purples sulfur bacteria (PSB) dominate the chemocline at the oxic-euxinic interface. Under euxinic conditions, green sulfur bacteria tend to have many advantages over purple sulfur bacteria in competition. However, competition experiments in microcosm manipulation experiments show that the FGL purple sulfur bacteria may have the capability to perform micro-aerobic sulfur oxidation in dark conditions in addition to anoxygenic photosynthesis. The combination of dark aerobic and light anaerobic reactions could allow the FGL PSB to harness photic energy at day and chemical energy at night. The chemocline in both FGL and the Johnston Proterozoic ocean model is a unique environment where both oxic and euxinic conditions are available and the most photosynthetically active radiation is available in the euxinic zone. I infer that PSB may outcompete other anoxygenic phototrophs simply by energetic advantage through exploiting the chemocline\u27s unique chemical environment