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At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

FeGenie: a comprehensive tool for the identification of iron genes and iron gene neighborhoods in genomes and metagenome assemblies

Iron is a micronutrient for nearly all life on Earth. It can be used as an electron donor and electron acceptor by iron-oxidizing and iron-reducing microorganisms and is used in a variety of biological processes, including photosynthesis and respiration. While it is the fourth most abundant metal in the Earth’s crust, iron is often limiting for growth in oxic environments because it is readily oxidized and precipitated. Much of our understanding of how microorganisms compete for and utilize iron is based on laboratory experiments. However, the advent of next-generation sequencing and surge in publicly available sequence data has made it possible to probe the structure and function of microbial communities in the environment. To bridge the gap between our understanding of iron acquisition, iron redox cycling, iron storage, and magnetosome formation in model microorganisms and the plethora of sequence data available from environmental studies, we have created a comprehensive database of hidden Markov models (HMMs) based on genes related to iron acquisition, storage, and reduction/oxidation in Bacteria and Archaea. Along with this database, we present FeGenie, a bioinformatics tool that accepts genome and metagenome assemblies as input and uses our comprehensive HMM database to annotate provided datasets with respect to iron-related genes and gene neighborhood. An important contribution of this tool is the efficient identification of genes involved in iron oxidation and dissimilatory iron reduction, which have been largely overlooked by standard annotation pipelines. We validated FeGenie against a selected set of 28 isolate genomes and showcase its utility in exploring iron genes present in 27 metagenomes, 4 isolate genomes from human oral biofilms, and 17 genomes from candidate organisms, including members of the candidate phyla radiation. We show that FeGenie accurately identifies iron genes in isolates. Furthermore, analysis of metagenomes using FeGenie demonstrates that the iron gene repertoire and abundance of each environment is correlated with iron richness. While this tool will not replace the reliability of culture-dependent analyses of microbial physiology, it provides reliable predictions derived from the most up-to-date genetic markers. FeGenie’s database will be maintained and continually updated as new genes are discovered.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Editorial: Microbial C1 Metabolism and Biotechnology

Archaea and bacteria with the capacity to utilize one-carbon (C1) molecules as carbon and energy sources are widespread across the planet and are important players in many biogeochemical processes. As such, they occupy an array of diverse anaerobic and aerobic niches and perform unique metabolisms that enable the utilization of carbon dioxide (CO 2 ), carbon monoxide (CO), formate (HCOOH), methanol (CH 3 OH), and methane (CH 4 ) for growth. Several industrial processes have been developed that leverage these microbes for the targeted conversion of anthropogenic waste gases that contain C1 molecules, including natural gas (CH 4 ), anaerobic digestion-derived biogas (CH 4 and CO 2 ), industrial flue gas (CO 2 and CO), and syngas (CO 2 , CO, and H 2 ) (Figure 1). Further, there is increasing interest in electrochemical reduction of CO 2 to CO, CH 4 , and intermediates with increased solubility compared to gaseous C1 molecules (formate and methanol) using electrons derived from renewable sources like wind, solar, or hydrothermal systems to mitigate atmospheric greenhouse gas. Thus, C1-utilizing microbes will likely play an integral role in biotechnologies that are part of a sustainable, circular bioeconomy. However, fundamental knowledge gaps into the metabolism and physiology of these microbes exist that limit their utility as biocatalysts for carbon-efficient biomanufacturing. This special topic presents studies focused on the fundamental aspects of C1 metabolism in diverse microbial systems with the ability to convert anthropogenic greenhouse gases into valuable products.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A small number of point mutations confer formate tolerance in Shewanella oneidensis

ABSTRACT Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO 2 and clean electricity. However, limitations in electron transfer efficiency and gaps in understanding of electron transfer pathways in MES systems prevent full realization of this technology. Shewanella oneidensis could serve as an MES biocatalyst because it has a well-studied, efficient transmembrane electron transfer pathway. A key first step in MES in this organism could be CO 2 reduction to formate. However, we report that wild-type S. oneidensis does not tolerate high levels of formate. In this work, we created and characterized formate-tolerant strains of S. oneidensis for further engineering and future use in MES systems through adaptive laboratory evolution. Two different point mutations in a gene encoding a predicted sodium-dependent bicarbonate transporter and a DUF2721-containing protein separately confer formate tolerance to S. oneidensis . The mutations were further evaluated to understand their role in improving formate tolerance. We also show that the wild-type and mutant versions of the putative sodium-dependent bicarbonate transporter improve formate tolerance of Zymomonas mobilis , indicating the potential of transferring this formate tolerance phenotype to other organisms. IMPORTANCE Shewanella oneidensis is a bacterium with a well-studied, efficient extracellular electron transfer pathway. This capability could make this organism a suitable host for microbial electrosynthesis using CO 2 or formate as feedstocks. However, we report here that formate is toxic to S. oneidensis , limiting the potential for its use in these systems. In this work, we evolve several strains of S. oneidensis that have improved formate tolerance, and we investigate some mutations that confer this phenotype. The phenotype is confirmed to be attributed to several single point mutations by transferring the wild-type and mutant versions of each gene to the wild-type strain. Finally, the formate tolerance mechanism of one variant is studied using structural modeling and expression in another host. This study, therefore, presents a simple method for conferring formate tolerance to bacterial hosts.

Cross, Megan C. Gruenberg (ORCID:0000000291589900)↗
DOE OSTI2021

Nutrient Inputs Stimulate Mercury Methylation by Syntrophs in a Subarctic Peatland

Climate change dramatically impacts Arctic and subarctic regions, inducing shifts in wetland nutrient regimes as a consequence of thawing permafrost. Altered hydrological regimes may drive changes in the dynamics of microbial mercury (Hg) methylation and bioavailability. Important knowledge gaps remain on the contribution of specific microbial groups to methylmercury (MeHg) production in wetlands of various trophic status. Here, we measured aqueous chemistry, potential methylation rates (k meth ), volatile fatty acid (VFA) dynamics in peat-soil incubations, and genetic potential for Hg methylation across a groundwater-driven nutrient gradient in an interior Alaskan fen. We tested the hypotheses that (1) nutrient inputs will result in increased methylation potentials, and (2) syntrophic interactions contribute to methylation in subarctic wetlands. We observed that concentrations of nutrients, total Hg, and MeHg, abundance of hgcA genes, and rates of methylation in peat incubations (k meth ) were highest near the groundwater input and declined downgradient. hgcA sequences near the input were closely related to those from sulfate-reducing bacteria (SRB), methanogens, and syntrophs. Hg methylation in peat incubations collected near the input source (FPF2) were impacted by the addition of sulfate and some metabolic inhibitors while those down-gradient (FPF5) were not. Sulfate amendment to FPF2 incubations had higher k meth relative to unamended controls despite no effect on k meth from addition of the sulfate reduction inhibitor molybdate. The addition of the methanogenic inhibitor BES (25 mM) led to the accumulation of VFAs, but unlike molybdate, it did not affect Hg methylation rates. Rather, the concurrent additions of BES and molybdate significantly decreased k meth , suggesting a role for interactions between SRB and methanogens in Hg methylation. The reduction in k meth with combined addition of BES and molybdate, and accumulation of VFA in peat incubations containing BES, and a high abundance of syntroph-related hgcAsequences in peat metagenomes provide evidence for MeHg production by microorganisms growing in syntrophy. Collectively the results suggest that wetland nutrient regimes influence the activity of Hg methylating microorganisms and, consequently, Hg methylation rates. Our results provide key information about microbial Hg methylation and methylating communities under nutrient conditions that are expected to become more common as permafrost soils thaw.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

BETO 2021 Peer Review - Improving Formate Upgrading by Cupriavidus Necator 2.3.2.111

Formate can be generated by electrocatalytic reduction of CO2 and has been proposed as a soluble intermediate for the storage of carbon and energy. Biological systems capable of assimilating formate could enable conversion of formate generated from low-cost renewable energy and waste CO2 to myriad fuels and chemicals. To that end, the goal of this project is to develop the natural formatotroph Cupriavidus necator as a robust microbial chassis for efficient conversion of formate to value-added products. Using a combination of laboratory evolution and rational engineering, we have identified mutations and genetic deletions that improve its growth rate and biomass yield on formate by 1.2X. Using RNA-seq transcriptomics, we are elucidating the mechanisms that underly these improvements and anticipate this will contribute to our understanding of formate conversion. We have also engineered C. necator to convert formate to the potential polymer precursor 2-hydroxymuconate semialdehyde (2HMS) by introducing an exogenous pathway and deleting competing pathways and demonstrated production and robust growth using a pH-stat bioreactor. Final techno-economic analysis and life-cycle assessment on biological production of 2HMS from formate will enable the DOE and other stake holders to evaluate the economic and environmental impact of this project and related technologies.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2024

The Mobility of Mo during Microbially Mediated Ferrihydrite Phase Transformation

Molybdenum (Mo) is an essential nutrient for almost all organisms. However, at high concentrations, it can be toxic to animals and plants. This study investigated the interactions of Mo(VI) with iron oxyhydroxides during ferrihydrite bioreduction in the presence of the Fe(III)- reducing Geobacter sulfurreducens. Here, in this study, we showed that Mo concentration controlled ferrihydrite phase transformation, leading to Mo release. With the biotic reduction of ferrihydrite and Fe(II) production, Mo(VI) reduction and Mo(IV)O 2 formation were observed for the first time, which further immobilised Mo after surface adsorption of Mo(VI). At low Mo levels (Mo/Fe molar ratios of 1-2 %), sufficient Fe(II) adsorption onto ferrihydrite resulted in its transformation into magnetite nanoparticles (>80%, ~25 nm) which catalysed the reduction of Mo(VI) to form Mo(IV)O 2 and immobilised Mo. Contrastingly, at high Mo concentrations (Mo/Fe molar ratios of 5-10%), Mo(VI)O 4 2- adsorption onto ferrihydrite limited Fe(II) adsorption, subsequently less magnetite (<8-12%) formed while more goethite (~30- 50%, width & length > 15 & 100 nm, respectively) and siderite (~20-30%, width & length > 100 & 200 nm, respectively) with larger particle sizes formed instead, causing Mo(VI) release due to lower Mo adsorption. This study provides a comprehensive understanding of the interaction mechanisms among Geobacter sulfurreducens, Mo(VI), and iron oxyhydroxides, enabling predictions and controls of long-term Mo mobility and Fe mineral transformation under a variety of biogeochemical scenarios.

Geobacter sulfurreducens↗
DOE OSTI2020

Antimicrobial Properties of Corn Stover Lignin Fractions Derived from Catalytic Transfer Hydrogenolysis in Supercritical Ethanol with a Ru/C Catalyst

Converting lignin to value-added products at high yields provides an avenue for making ethanol biorefineries more profitable while reducing the carbon footprint of products generally derived from petroleum. In this study, corn stover lignin was depolymerized by catalytic transfer hydrogenolysis (CTH) in supercritical ethanol with a Ru/C catalyst. The lignin-derived bio-oil was then sequentially extracted utilizing hexane, petroleum ether, chloroform, and ethyl acetate as solvents in order of less polar to polar, and the subsequent bio-oils were characterized using GPC, GC/MS, and HSQC NMR. In this work, results show that the monomers in the bio-oil fractions contained primarily alkylated phenols, hydrogenated hydroxycinnamic acid derivatives, syringol and guaiacol-type lignins created from reductive cleavages of ether linkages, which were sequentially extracted into groups depending on the solvent polarity. The antimicrobial properties of the bio-oils were screened against Gram-positive (Bacillus subtilis, Lactobacillus amylovorus, and Staphylococcus epidermidis) and Gram-negative (Escherichia coli) bacteria and yeast (Saccharomyces cerevisiae) by examining microbial growth inhibition. Results show that CTH-derived bio-oils inhibited all tested organisms at concentrations less than 3 mg/mL. Total monomer concentration and the presence of specific monomers (i.e., syringyl propane) showed correlations to antimicrobial activity, likely due to cell death or membrane damage. This study provides insights into using sequential extraction to fractionate lignin-derived compounds and correlations between the properties of the extracted compounds and their antimicrobial activity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Microbe-iron interactions control lignin decomposition in soil

Lignin decomposition is critically linked to terrestrial carbon (C) cycle due to the enormous C mass of lignin and its importance in controlling overall rates of litter decomposition. Interactions between lignin and iron (Fe) minerals have been increasingly recognized as key mediators of lignin decomposition in experimental studies. However, we still lack a quantitative understanding of how Fe minerals interact with microbes to control lignin decomposition. Here, we leveraged experimental results from an incubation of Fe-rich soil, in which lignin decomposition rates were measured at aerobic conditions after four levels of pre-treated O2 availability, to examine microbe-Fe (MiFe) interactions in lignin decomposition with a MiFe model. We quantified how Fe redox cycling interacted with microbial activities to control lignin decomposition via data-model integration. Our results showed that the MiFe model with time-dependent growth and mortality functions better represented CO2 release from lignin decomposition (R2 ranging from 0.96 to 0.97) than models assuming either first-order or Michaelis-Menten kinetics. Reduction of Fe(III) to Fe(II) after pre-treatments with lower O2 availability stimu-lated the Fenton reaction to break down macro-molecular lignin into small molecules available to microbes. The small molecules of lignin and necromass bounded with oxidized Fe and were protected from decomposition. After 1-year incubation, the model implied that most of C stabilized with Fe minerals was derived from small molecular lignin C. Our quantitative analysis of microbe-Fe interactions sheds new light on lignin decomposition and preservation and helps improve model prediction of soil C persistence under global change.

Liao, Cuijuan↗
DOE OSTI2021

Fluorescence Assay for Tolyporphins Amidst Abundant Chlorophyll in Crude Cyanobacterial Extracts

Abstract Tolyporphins are distinctive tetrapyrrole natural products found singularly in a filamentous cyanobacterial‐microbial holobiont (termed HT‐58‐2) from Micronesia. The absorption and fluorescence features of tolyporphins resemble those of chlorophyll a , complicating direct analysis of culture samples. Treatment of the crude (unfractionated) organic extract (CH 2 Cl 2 /2‐propanol, 1:1) of HT‐58‐2 cultures with NaBH 4 in methanol causes reduction of the peripheral ketone auxochromes, whereupon tolyporphins (predominantly 7,17‐dioxobacteriochlorins) exhibit a bathochromic shift ( λ abs ˜ 676 → ˜ 700 nm) and chlorophyll a (a 13 1 ‐oxochlorin) exhibits a hypsochromic shift ( λ abs 665 → 634 nm). Fluorescence excitation spectroscopy (at 368 and 491 nm with λ em 710 nm) enabled detection of reduced tolyporphins amidst abundant reduced chlorophyll a (1:19 ratio), a detection sensitivity >5 times that without reduction. The resulting assay combines simple sample preparation from non‐axenic cultures at microscale quantities (2 mL, 2 μ m ), absence of any fractionation procedures, and fluorescence detection. Tolyporphins were readily detected in cultures of HT‐58‐2 at reasonable growth periods in the absence of environmental stressors, which was not possible previously.

Nguyen, Kathy‐Uyen↗
DOE OSTI2021

Anaerobic Microbial Metabolism of Dichloroacetate

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising “Candidatus Dichloromethanomonas elyunquensis” strain RM utilizes DCA as an energy source, and the transient formation of formate, H 2 , and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated “Candidatus Dichloromethanomonas elyunquensis” strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg -1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Urbanization and edge effects interact to drive mutualism breakdown and the rise of unstable pathogenic communities in forest soil

Temperate forests are threatened by urbanization and fragmentation, with over 20% (118,300 km 2 ) of U.S. forest land projected to be subsumed by urban land development. We leveraged a unique, well-characterized urban-to-rural and forest edge-to-interior gradient to identify the combined impact of these two land use changes—urbanization and forest edge creation—on the soil microbial community in native remnant forests. We found evidence of mutualism breakdown between trees and their fungal root mutualists [ectomycorrhizal (ECM) fungi] with urbanization, where ECM fungi colonized fewer tree roots and had less connectivity in soil microbiome networks in urban forests compared to rural forests. However, urbanization did not reduce the relative abundance of ECM fungi in forest soils; instead, forest edges alone led to strong reductions in ECM fungal abundance. At forest edges, ECM fungi were replaced by plant and animal pathogens, as well as copiotrophic, xenobiotic-degrading, and nitrogen-cycling bacteria, including nitrifiers and denitrifiers. Urbanization and forest edges interacted to generate new “suites” of microbes, with urban interior forests harboring highly homogenized microbiomes, while edge forest microbiomes were more heterogeneous and less stable, showing increased vulnerability to low soil moisture. When scaled to the regional level, we found that forest soils are projected to harbor high abundances of fungal pathogens and denitrifying bacteria, even in rural areas, due to the widespread existence of forest edges. Our results highlight the potential for soil microbiome dysfunction—including increased greenhouse gas production—in temperate forest regions that are subsumed by urban expansion, both now and in the future.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Microbial Diversity and Sulfur Cycling in an Early Earth Analogue: From Ancient Novelty to Modern Commonality

Life emerged and diversified in the absence of molecular oxygen. The prevailing anoxia and unique sulfur chemistry in the Paleo-, Meso-, and Neoarchean and early Proterozoic eras may have supported microbial communities that differ from those currently thriving on the earth’s surface. Zodletone spring in southwestern Oklahoma represents a unique habitat where spatial sampling could substitute for geological eras namely, from the anoxic, surficial light-exposed sediments simulating a preoxygenated earth to overlaid water column where air exposure simulates oxygen intrusion during the Neoproterozoic era. We document a remarkably diverse microbial community in the anoxic spring sediments, with 340/516 (65.89%) of genomes recovered in a metagenomic survey belonging to 200 bacterial and archaeal families that were either previously undescribed or that exhibit an extremely rare distribution on the current earth. Such diversity is underpinned by the widespread occurrence of sulfite, thiosulfate, tetrathionate, and sulfur reduction and the paucity of sulfate reduction machineries in these taxa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Optimized CRISPR Interference System for Investigating Pseudomonas alloputida Genes Involved in Rhizosphere Microbiome Assembly

Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe–microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/P m , LacI/P lac , and AraC/P BAD ) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three promoter systems in terms of how well they can precisely modulate gene expression, and we discuss the impact of environmental factors, such as media choice, on the success of CRISPRi. We demonstrate that CRISPRi is functional in bacteria colonizing the rhizosphere, with repression of essential genes leading to a 10–100-fold reduction in P. alloputida cells per root. Finally, we show that CRISPRi can be used to modulate microbe–microbe interactions. When the gene pvdH is repressed and P. alloputida is unable to produce pyoverdine, it loses its ability to inhibit other microbes in vitro. Furthermore, our design is amendable for future CRISPRi-seq studies and in multispecies microbial communities, with the different promoter systems providing a means to control the level of gene expression in many different environments.

Bacteria↗
DOE OSTI2022

Chemolithoautotroph distributions across the subsurface of a convergent margin

Subducting oceanic crusts release fluids rich in biologically relevant compounds into the overriding plate, fueling subsurface chemolithoautotrophic ecosystems. To understand the impact of subsurface geochemistry on microbial communities, we collected fluid and sediments from 14 natural springs across a ~200 km transect across the Costa Rican convergent margin and performed shotgun metagenomics. The resulting 404 metagenome-assembled genomes (MAGs) cluster into geologically distinct regions based on MAG abundance patterns: outer forearc-only (25% of total relative abundance), forearc/arc-only (38% of total relative abundance), and delocalized (37% of total relative abundance) clusters. In the outer forearc, Thermodesulfovibrionia, Candidatus Bipolaricaulia, and Firmicutes have hydrogenotrophic sulfate reduction and Wood-Ljungdahl (WL) carbon fixation pathways. In the forearc/arc, Anaerolineae, Ca. Bipolaricaulia, and Thermodesulfovibrionia have sulfur oxidation, nitrogen cycling, microaerophilic respiration, and WL, while Aquificae have aerobic sulfur oxidation and reverse tricarboxylic acid carbon fixation pathway. Transformation-based canonical correspondence analysis shows that MAG distribution corresponds to concentrations of aluminum, iron, nickel, dissolved inorganic carbon, and phosphate. While delocalized MAGs appear surface-derived, the subsurface chemolithoautotrophic, metabolic, and taxonomic landscape varies by the availability of minerals/metals and volcanically derived inorganic carbon. Furthermore, the WL pathway persists across all samples, suggesting that this versatile, energy-efficient carbon fixation pathway helps shape convergent margin subsurface ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Experimental evidence for recovery of mercury-contaminated fish populations

Anthropogenic releases of mercury (Hg) are a human health issue because the potent toxicant methylmercury (MeHg), formed primarily by microbial methylation of inorganic Hg in aquatic ecosystems, bioaccumulates to high concentrations in fish consumed by humans. Predicting the efficacy of Hg pollution controls on fish MeHg concentrations is complex because many factors influence the production and bioaccumulation of MeHg. Here we conducted a 15-year whole-ecosystem, single-factor experiment to determine the magnitude and timing of reductions in fish MeHg concentrations following reductions in Hg additions to a boreal lake and its watershed. During the seven-year addition phase, we applied enriched Hg isotopes to increase local Hg wet deposition rates fivefold. The Hg isotopes became increasingly incorporated into the food web as MeHg, predominantly from additions to the lake because most of those in the watershed remained there. Thereafter, isotopic additions were stopped, resulting in an approximately 100% reduction in Hg loading to the lake. The concentration of labelled MeHg quickly decreased by up to 91% in lower trophic level organisms, initiating rapid decreases of 38–76% of MeHg concentration in large-bodied fish populations in eight years. Although Hg loading from watersheds may not decline in step with lowering deposition rates, this experiment clearly demonstrates that any reduction in Hg loadings to lakes, whether from direct deposition or runoff, will have immediate benefits to fish consumers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin

Upgrading lignin, an underutilized component of biomass, is essential for the sustainability of biorefineries. Biocatalysis has considerable potential for upgrading lignin, but our lack of knowledge of relevant enzymes and pathways has limited its application. Herein, we describe a microbial pathway responsible for catabolizing alkylguaiacols, a major component of several industrial lignin streams. Catabolism is initiated by a cytochrome P450, with related P450s catalyzing the O-demethylation of different lignin-derived guaiacols and subsequent catabolism depending on the substitution pattern of the guaiacol. Importantly, the alkylguaiacol catabolic pathway enables bacterial growth on corn stover lignin produced by reductive catalytic fractionation. Overall, these insights greatly facilitate the engineering of P450s and bacteria to biocatalytically upgrade lignin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Synergistic interactions between anammox and dissimilatory nitrate reducing bacteria sustains reactor performance across variable nitrogen loading ratios

Anaerobic ammonium oxidizing (anammox) bacteria are utilized for high efficiency nitrogen removal from nitrogen-laden sidestreams in wastewater treatment plants. The anammox bacteria form a variety of competitive and mutualistic interactions with heterotrophic bacteria that often employ denitrification or dissimilatory nitrate reduction to ammonium (DNRA) for energy generation. These interactions can be heavily influenced by the influent ratio of ammonium to nitrite, NH 4 + :NO 2 - , where deviations from the widely acknowledged stoichiometric ratio (1:1.32) have been demonstrated to have deleterious effects on anammox efficiency. Thus, it is important to understand how variable NH 4 + :NO 2 - ratios impact the microbial ecology of anammox reactors. We observed the response of the microbial community in a lab scale anammox membrane bioreactor (MBR) to changes in the influent NH 4 + :NO 2 - ratio using both 16S rRNA gene and shotgun metagenomic sequencing. Ammonium removal efficiency decreased from 99.77 ± 0.04% when the ratio was 1:1.32 (prior to day 89) to 90.85 ± 0.29% when the ratio was decreased to 1:1.1 (day 89–202) and 90.14 ± 0.09% when the ratio was changed to 1:1.13 (day 169–200). Over this same timespan, the overall nitrogen removal efficiency (NRE) remained relatively unchanged (85.26 ± 0.01% from day 0–89, compared to 85.49 ± 0.01% from day 89–169, and 83.04 ± 0.01% from day 169–200). When the ratio was slightly increased to 1:1.17–1:1.2 (day 202–253), the ammonium removal efficiency increased to 97.28 ± 0.45% and the NRE increased to 88.21 ± 0.01%. Analysis of 16S rRNA gene sequences demonstrated increased relative abundance of taxa belonging to Bacteroidetes, Chloroflexi, and Ignavibacteriae over the course of the experiment. The relative abundance of Planctomycetes, the phylum to which anammox bacteria belong, decreased from 77.19% at the beginning of the experiment to 12.24% by the end of the experiment. Analysis of metagenome assembled genomes (MAGs) indicated increased abundance of bacteria with nrfAH genes used for DNRA after the introduction of lower influent NH 4 + :NO 2 - ratios. The high relative abundance of DNRA bacteria coinciding with sustained bioreactor performance indicates a mutualistic relationship between the anammox and DNRA bacteria. Understanding these interactions could support more robust bioreactor operation at variable nitrogen loading ratios.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Community Structure and Microbial Associations in Sediment-Free Methanotrophic Enrichment Cultures from a Marine Methane Seep

We report Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b (“Candidatus Methanocomedenaceae”) and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides, Anaerolineales, and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.

59 BASIC BIOLOGICAL SCIENCES↗