Biology subjects

Edwards, E. A.

Publications and source records attributed to Edwards, E. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Thermodynamically controlled biokinetics modelling of a lindane-degrading anaerobic enrichment culture

The anaerobic biotransformation of lindane ({gamma}-hexachlorocyclohexane), a persistent legacy pesticide and challenging subsurface contaminant was modelled using an integrated Monod biokinetics framework. Calibrated against batch experimental data, this model accounted for the dissolution of solid phase lindane and the thermodynamics of reactions involved. The study focused on a methanogenic, lindane-degrading enrichment culture (GT2) that used ethanol as the electron donor, where ethanol fermentation supplied hydrogen to support reductive dechlorination of lindane. A Dehalobacter strain was identified as the lindane-degrading organism, while a member of bacterial order Geobacterales carried out ethanol fermentation. First-order decay constants for lindane-degrading Dehalobacter were estimated from experimental data and showed variation across different growth conditions. The biokinetics model captured interactions among key microbial processes, namely lindane dechlorination, ethanol fermentation, hydrogenotrophic methanogenesis, and acetoclastic methanogenesis. Thirteen biokinetics parameters including maximum substrate utilization rates (k), half-velocity constants (Ks), and decay constants for these microbial processes were estimated through a Bayesian optimization approach. Global sensitivity analysis showed that eight parameters, primarily k and Ks, had significant linear impact on the model output, whereas responses to decay constants were more variable and potentially non-linear. This study proposed an effective modelling approach for characterizing syntrophic microbial growth within enrichment cultures and provides valuable biokinetics parameters estimates for real-world applications of anaerobic lindane bioremediation.

microbiology↗

bioRxiv2026

A novel virus lineage is abundant in metaviromes from Dehalococcoides-containing mixed cultures

Dehalococcoides mccartyi are obligately anaerobic organohalide-respiring bacteria that play important roles in the detoxification of chlorinated pollutants in groundwater and sediments. Despite having small genomes, they host a diverse set of mobile elements. Here we characterize a family of mobile elements, termed integrative and mobilizable element 1 or IME1, comprising 20 from Dehalococcoides and one from Dehalogenimonas alkenigignens. IME1s are 20,930 - 28,058 bp and are found both integrated in the genomes and as circular episomes. Bioinformatic characterization of IME1 encoded proteins revealed a highly conserved structure with 14 hierarchical orthologous groups (HOGs) found in all 21 IME1s. IME1s lack recognizable hallmark proteins of tailed bacterial viruses (or tailed phages) but encode proteins with similarities to those of filamentous bacterial viruses. In particular, one conserved HOG shows sequence similarity to the pI-like ATPase, the only conserved marker protein identified across filamentous bacterial viruses. Additionally, IME1s encode several small proteins with predicted transmembrane domains and signal peptides, another feature used to identify filamentous bacterial viruses. Both features are also found in budding archaeal viruses of various morphotypes. IME1s dominated metaviromes obtained from the Dehalococcoides-containing KB-1 mixed culture and electron micrographs of the corresponding viral fractions revealed abundant filamentous virus-like particles. We therefore propose that the IME1s represent a novel lineage of double stranded budding, likely filamentous, viruses. Database searches suggest IME1s are found in Dehalococcodia and other Chlorofexota but are so far restricted to this phylum.

microbiology↗

bioRxiv2026

Unraveling the metabolic interactions of a Dehalobacter-containinganaerobic mixed culture for bioremediation

Organohalide-respiring bacteria (OHRB), such as Dehalobacter, play key roles in the bioremediation of anoxic environments contaminated with chlorinated aromatic compounds. These obligate anaerobes rely on syntrophic interactions to obtain essential resources--hydrogen, acetate, and corrinoid cofactors--from acetogens and fermenters. However, the metabolic interactions enabling complete reductive dehalogenation of compounds like 1,2,4-trichlorobenzene (1,2,4-TCB) to benzene remain incompletely understood. In this study, we asked: (1) What are the key microbial taxa and their functional roles within a Dehalobacter-containing anaerobic microbial community detoxifying chlorinated benzenes? (2) How do syntrophic interactions enable complete dehalogenation of 1,2,4-TCB to benzene under anaerobic conditions? (3) Can genome-resolved metagenomics and genome-scale metabolic modeling elucidate the metabolic dependencies supporting organohalide respiration in complex consortia? To address these questions, we cultivated microbial communities in batch reactors using methanol as electron donor and either 1,2,4-TCB or monochlorobenzene (MCB) as electron acceptor. In active MCB-fed cultures, benzene increased from 0 to 62.3{micro}mol per bottle while MCB decreased from 88.3 to 22.0{micro}mol per bottle over 120 days, with this pattern repeating across multiple substrate additions. Using genome-resolved metagenomics to identify dominant taxa and select 12 high-quality metagenome-assembled genomes (MAGs) for modeling, we reconstructed genome-scale metabolic models (GEMs) to identify candidate metabolic interactions and predict syntrophic dependencies that may support organohalide respiration in these consortia. Community flux sampling predicted that methanol, H2, acetate, and CO2 formed the dominant exchange backbone of the modeled community, while also indicating competition for shared electron donors between the two Dehalobacter populations. Model-guided minimal-community analysis further identified a narrow dechlorinating core in which all feasible minimal consortia retained a Dehalobacter member together with Methanothrix. These results provide a modeling-informed framework for hypothesis generation and future experimental validation of anaerobic consortia relevant to bioremediation.

systems biology↗

bioRxiv2025

Modeling reveals metabolic basis of competition among Dehalobacter strains during tandem CF and DCM metabolism

SC05-UT is an anaerobic mixed microbial enrichment culture that reduces chloroform (CF) to dichloromethane (DCM) through reductive dechlorination, which it further mineralizes to carbon dioxide. This DCM mineralization yields electron equivalents that are used to reduce CF without addition of exogenous electron donor. By studying this self-feeding CF-amended culture and a DCM-amended enrichment culture (DCME), we previously found the genomic potential to perform both biodegradation steps in two distinct Dehalobacter strains: Dehalobacter restrictus SAD and Candidatus Dehalobacter alkaniphilus DAD. Though present in each enrichment culture, strain SAD is more abundant in CF-fed subculture SC05-UT, while strain DAD is more prominent in the DCM-fed subculture DCME. To understand if genomic differences between strains and impact their metabolic mechanisms, the genome of each strain was curated to reconstruct genome-scale metabolic models of each strain, which were then constrained based on thermodynamic and experimental conditions. We demonstrate that metabolic differences between the two strains may allow Dehalobacter strain DAD to outcompete strain SAD in the absence of CF, while strain SAD exhibits an advantage in the presence of CF. Additionally, we predict electron cycling methods to reconcile redox imbalances in the cell is required for tandem CF and DCM dechlorination. This work highlights the importance of hydrogen and amino acid exchange in these microbial communities and contributes to the growing body of work surrounding organohalide syntrophy. ImportanceChloroform and dichloromethane contaminate groundwater around the world but can be remediated by microbes capable of metabolizing these toxic compounds. Here we study two distinct strains of Dehalobacter and show that while both strains can degrade both chloroform and dichloromethane, differences in their genetic makeup allow each strain to thrive under different environmental conditions. This has implications for understanding the fate of halogenated methanes in the environment and the application of Dehalobacter for bioremediation of chlorinated compounds.

microbiology↗

bioRxiv2025

Dehalobacter converts 2,3-dichloroaniline to aniline via 2-chloroaniline in a methanogenic enrichment culture

12,3-dichloroaniline (2,3-DCA) has widespread use in chemical manufacturing and remains a persistent groundwater contaminant. To better understand the pathway and kinetics of its reductive dechlorination, we conducted a laboratory kinetic experiment using an anaerobic enrichment culture dominated by Dehalobacter. At an initial field-relevant concentration of 40 mg/L, complete and stoichiometric dechlorination of 2,3-DCA to aniline via 2-CA was achieved. The intermediates, 2-chloroaniline (2-CA) and 3-chloroaniline, were transiently formed in a ratio of 8:1. The growth yields of Dehalobacter on 2,3-DCA and 2-CA were 1.2 {+/-} 0.1 x 108 and 1.3 {+/-} 0.1 x 108 16S rRNA gene copies/mol chloride, respectively. The maximum specific growth rate for 2,3-DCA, max, was 0.18 {+/-} 0.03 day-{superscript 1} with a half-saturation constant, Ks, at 45 {+/-} 16 mg /L. The first-order decay constant for Dehalobacter when starved of chlorinated electron acceptors was estimated at 0.017 {+/-} 0.001 day-{superscript 1}. Lactate fermentation, acetogenesis from ethanol, syntrophic propionate oxidation, and hydrogenotrophic methanogenesis were observed during dechlorination. This work provides insights into the organohalide respiration of 2,3-DCA to aniline and advances the understanding of microbial interactions during anaerobic dechlorination. These results offer guidance for developing stable dechlorinating microbial ecosystems and key kinetic parameters for predictive modeling of groundwater 2,3-DCA fate and transport. 3 SynopsisThis study demonstrated stoichiometric dechlorination of 2,3-dichloroaniline to aniline, characterized syntrophic interactions in a mixed anaerobic dechlorinating culture, and determined growth yield, first-order decay and Monod constants for Dehalobacter. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/639126v3_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@5d59c4org.highwire.dtl.DTLVardef@b05a3aorg.highwire.dtl.DTLVardef@1f39f6borg.highwire.dtl.DTLVardef@1676753_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Wang, S. (2025) https://BioRender.com/f47t993

microbiology↗

bioRxiv2025

Dehalobacter dechlorinates dichloroanilines and contributes to the natural attenuation of dichloronitrobenzenes at a complex industrial site

The potential for bioremediation of dichloroanilines (2,3- and 3,4-DCA) and dichloronitrobenzenes (3,4-, 2,5- and 2,3-DCNB) was investigated using inocula from an industrial site in northeast of Brazil. Anaerobic biotransformation of these chlorinated compounds was observed in microcosms simulating site conditions, particularly when electron donor was added. To disentangle specific transformation reactions, sub-cultures from active anaerobic microcosms were enriched with individual DCA or DCNB isomers. In these single-compound enrichment cultures, DCNB isomers were reduced to the corresponding DCA. Each DCA isomer was stoichiometrically dechlorinated to the corresponding monochloroaniline (CA) isomers. Further dechlorination of CA to aniline was noted. The reduction of DCNB to DCA was sustained by known fermenters and possibly Desulfitobacterium, while Dehalobacter was clearly responsible for dechlorination of DCAs. These findings helped to attribute function to microbial community data retrieved from field samples to inform the conceptual site model and enable identification of bioactive zones and rate-limiting conditions at a complex site. More generally, these data broaden the understanding of the metabolic repertoire of Dehalobacter and provide a model for incorporating microbial community data into site remediation efforts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/638192v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1667948org.highwire.dtl.DTLVardef@a4a128org.highwire.dtl.DTLVardef@507067org.highwire.dtl.DTLVardef@103a155_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Short synopsis statementA diverse microbial community contributes to natural attenuation at a complex contaminated site. Notably, dichloroanilines produced by biologically-mediated nitroreduction of dichloronitrobenzenes were dechlorinated by Dehalobacter.

microbiology↗

bioRxiv2024

Identification of a Cluster of Benzene Activation Enzymes in a Strictly Anoxic Methanogenic Consortium

The Oil Refinery (OR) consortium is a model methanogenic enrichment culture used to study anaerobic benzene degradation. Over half of the cultures bacterial community consists of two closely related Desulfobacterota strains, designated ORM2a and ORM2b, whose mechanisms of benzene activation are unknown. Two new metagenomes, including a complete circularized metagenome-assembled genome (MAG) for ORM2a, enabled a thorough investigation of this cultures proteome. Among the proteins identified were Bam-like subunits of an ATP-independent benzoyl-CoA degradation pathway, as well as downstream {beta}-oxidation proteins yielding acetate. The most abundant proteins identified mapped to two ORM2a gene clusters of unknown function. Homologous and syntenic gene clusters were identified in genomes of ORM2b and a sulfate-reducing Pelotomaculum that also degrades benzene, as well as in nine contigs assembled from hydrothermal vent metagenomes. Extensive homology and structural predictions suggest that the first cluster - termed the "Magic" gene cluster - encodes for enzymes catalyzing the chemically challenging activation of benzene and subsequent transformation steps yielding benzoyl-CoA. The second ("Nanopod") gene cluster encodes a transmembrane complex that may facilitate benzene transport across the cell membrane. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of strict anaerobes specialized for benzene degradation, which we propose naming "Candidatus Anaerobenzenivorax". IMPORTANCEBenzene is a widespread, persistent and toxic pollutant that can accumulate in anoxic environments such as groundwater and sediments. Despite decades of study, the biochemical mechanisms by which benzene is activated under anaerobic conditions remain unproven. This study provides strong genetic and proteomic evidence for a new class of enzymes that initiate anaerobic benzene activation and proposes a preliminary model for their underlying biochemistry. These findings lay a foundation for future biochemical studies and expand our understanding of how microbes carry out extreme redox chemistry in the absence of oxygen.

microbiology↗

bioRxiv2024

High quality Bathyarchaeia MAGs from lignocellulose-impacted environments elucidate metabolism and evolutionary mechanisms.

The archaeal class Bathyarchaeia are widely and abundantly distributed in anoxic habitats. Metagenomic studies have suggested that they are mixotrophic, capable of CO2 fixation and heterotrophic growth, and involved in acetogenesis and lignin degradation. We analysed 35 Bathyarchaeia metagenome assembled genomes (MAGs), including the first complete circularized MAG (cMAG) of the Bathy-6 subgroup, from the metagenomes of three full scale pulp and paper mill anaerobic digesters and three laboratory methanogenic enrichment cultures maintained on pre-treated poplar. Thirty-three MAGs belong to the Bathy-6 lineage while two are from the Bathy-8 lineage. In our previous analysis of the microbial community in the pulp mill digesters, Bathyarchaeia were abundant and positively correlated to hydrogenotrophic and methylotrophic methanogenesis. Several factors likely contribute to the success of the Bathy-6 lineage compared to Bathy-8 in the reactors. The Bathy-6 genomes are larger than those of Bathy-8 and have more genes involved in lignocellulose degradation including carbohydrate-active enzymes (CAZymes) not present in the Bathy-8. Bathy-6 also share the Bathyarchaeal O-demethylase-system recently identified in Bathy-8. All the Bathy-6 MAGs had numerous membrane-associated pyrroloquinoline quinone-domain (PQQ) proteins that we suggest are involved in lignin modification or degradation, together with Radical-SAM and Rieske domain proteins, and AA2, AA3, and AA6-family oxidoreductases. We also identified a complete B12 synthesis pathway and a complete nitrogenase gene locus. Finally, comparative genomic analyses revealed that Bathyarchaeia genomes are dynamic and have interacted with other organisms in their environments through gene transfer to expand their gene repertoire.

genomics↗

bioRxiv2024

Deciphering Reductive Dehalogenase Specificity Through Targeted Mutagenesis of Chloroalkane Reductases

Reductive dehalogenases (RDases) are essential in the anaerobic degradation of various organohalide contaminants. This family of enzymes has broad sequence diversity, but high structural conservation. There have been few studies assessing how RDase peptide sequences affect their substrate selectivity. Here we focus on two chloroalkane RDases, CfrA and DcrA, which have 95% protein sequence identity but have diverged to hold distinct substrate preferences. CfrA will dechlorinate chloroform and 1,1,1-trichloroethane, whilst DcrA will dechlorinate 1,1-dichloroethane. We mutated several residues in the active site of CfrA to investigate a change in substrate preference and to identify which wild-type residues contribute the most to substrate specialization. We determined that no individual residue solely dictates substrate discrimination, but both Y80W and F125W mutations were needed to force CfrA to prefer 1,1-dichloroethane as a substrate. This double mutation also altered the transformation pathway of 1,1,2-trichloroethane from hydrogenolysis (forms 1,2-dichloroethane) to dihaloelimination (forms vinyl chloride). We use predictive protein models and substrate docking to predict what interactions are made between the enzyme and substrate to aid in selection. The residues of significance identified in this study are consistent with those identified from chloroethene RDases, suggesting residue locations with a particularly high impact on activity. ImportanceReductive dehalogenases play an integral role in the removal of chlorinated solvents from the environment. These enzymes have specificity towards different chlorinated compounds, and it is known that small natural changes in their peptide sequence can change their activity drastically. How these specific sequence variations influence activity is largely unknown. In this study, we demonstrate that mutating a few residues within the active site of CfrA--a chloroform and trichloroethane-specific dehalogenase--changes its substrate preference to dichloroethane. We determine that only two mutations are needed to disrupt the native activity, underscoring the nuances in substrate-structure relationships in reductive dehalogenases. Though we are still far from predicting function from the sequence, this knowledge can give some insight into engineering reductive dehalogenases for new target contaminants.

biochemistry↗

bioRxiv2024

Contrasting kinetics of highly similar chloroalkane reductive dehalogenases

Chloroform and trichloroethanes are pervasive groundwater contaminants for which bioremediation has been an effective treatment strategy. Reductive dehalogenase (RDase) enzymes from organohalide-respiring bacteria are essential for their remediation under anaerobic conditions. RDases are responsible for dehalogenating these chlorinated solvents, leading to their removal. This work explores the kinetic characteristics of three closely related Dehalobacter chloroalkane-reductases--TmrA, CfrA, and AcdA--and identifies differences between their activity on chloroform (CF), 1,1,1-trichloroethane (TCA), and 1,1,2-TCA. The side-by-side comparison of these enzymes has emphasized that TmrA and AcdA are specialized toward CF with both having 4-fold higher maximum specific activity (Vmax) on CF than 1,1,1-TCA, whereas, CfrA has very similar rates on both CF and 1,1,1-TCA. AcdA is the most sensitive to substrate inhibition by CF and 1,1,2-TCA, and inhibition by a common co-contaminant trichloroethene. Finally, the reduction of 1,1,2-TCA, which can produce both 1,2-dichloroethane and vinyl chloride, was assessed for each enzyme. Interestingly, each enzyme has a distinct preference for the major product it produces, indicating a favoured reaction pathway. Despite over 95% sequence identity, TmrA, CfrA, and AcdA exhibit substantial differences in kinetic behaviour, highlighting the importance of understanding such nuances for informed bioremediation strategies. SYNOPSISThree similar dechlorinating enzymes were found to have distinct reaction rates and levels of sensitivity to inhibition. These differences have implications for the enzymes use in bioremediation.

biochemistry↗

bioRxiv2023

Identification of functional genes in a chloroform and dichloromethane-degrading microbial culture

Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes such as methanogenesis. Anaerobic biotransformation of CF and DCM has been well documented but typically independently of one another. CF is the electron acceptor for certain organohalide-respiring bacteria that use reductive dehalogenases (RDases) to dechlorinate CF to DCM. In contrast, known DCM-degraders use DCM as their electron donor, which is oxidized using a series of methyltransferases and associated proteins encoded by the mec cassette to facilitate the entry of DCM to the Wood-Ljungdahl pathway. The SC05 culture is an enrichment culture sold commercially for bioaugmentation, that transforms CF via DCM to CO2. This culture has the unique ability to dechlorinate CF to DCM using electron equivalents provided by the oxidation of DCM to CO2. Here we use metagenomic and metaproteomic analysis to identify the functional genes involved in each of these transformations. Though 91 metagenome-assembled genomes were assembled, the genes for an RDase--named acdA--and a complete mec cassette were found to be encoded on a single contig belonging to Dehalobacter. AcdA and critical Mec proteins were also highly expressed by the culture. Heterologously-expressed AcdA dechlorinated CF and other chloroalkanes but had 100-fold lower activity on DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM, and subsequent mineralization of DCM using the mec cassette. ImportanceChloroform (CF) and dichloromethane (DCM) are regulated groundwater contaminants. A cost-effective approach to remove these pollutants from contaminated groundwater is to employ microbes that transform CF and DCM as part of their metabolism, thus depleting the contamination as the microbes continue to grow. In this work, we investigate bioaugmentation culture SC05, a mixed microbial consortium that effectively and simultaneously degrades both CF and DCM coupled to the growth of Dehalobacter. We identified the functional genes responsible for the transformation of CF and DCM in SC05. These genetic biomarkers provide a means to monitor the remediation process in the field.

microbiology↗

bioRxiv2023

Microbial community organization during anaerobic wastewater treatment

Amplicon sequencing data and operating data from anaerobic wastewater treatment plants from three Canadian pulp and paper mills were explored using correlation and network modularization approaches to study the microbial community organization and identify relationships between organisms and operating conditions. Each of the digesters contain two or three modules consisting of organisms that cover all trophic stages of anaerobic digestion. These modules are functioning independently from each other, and their relative abundance changes in response to varying operating conditions. The time delay between a change in digester operation and the change in the abundance of microorganisms was investigated using time-lagged operating parameters. This time delay ranged between two to four days and is likely influenced by the growth rates of the anaerobic microorganisms and the digester hydraulic retention time. Digester upsets due to plant shutdown periods and organic overload caused a drastic increase in the population of acetoclastic methanogens, acidogenic fermenters, and syntrophic acid degraders. As a response to impaired process conditions, the same Methanothrix amplicon sequence variant (ASV) dominated methanogenesis in the digesters of all three mills. The common characteristics of the organisms represented by this ASV should be further investigated for their role in alleviating the impact of digester upset conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/553022v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@140e214org.highwire.dtl.DTLVardef@baa227org.highwire.dtl.DTLVardef@ebd2b8org.highwire.dtl.DTLVardef@573194_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

A multifunctional Dehalobacter? Tandem chloroform and dichloromethane degradation in a mixed microbial culture

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world, which can be remediated through bioaugmentation. Although several strains of Dehalobacter restrictus can reduce CF to DCM, and multiple Peptococcaceae can ferment DCM, these processes cannot happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here we present a mixed microbial culture that can simultaneously metabolize CF and DCM to carbon dioxide and create an additional enrichment culture fed only DCM. Through species-specific qPCR, we find that a Dehalobacter strain grows both while CF alone and DCM alone are converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without addition of exogenous electron donor, and through electron balance calculations we show that DCM mineralization produces sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and understanding this mechanism informs strategies for culture maintenance and scale-up, and benefits contaminated sites where the culture is employed for remediation worldwide. SYNOPSISDechlorination of chloroform to dichloromethane and dichloromethane mineralization are performed concurrently by a Dehalobacter-containing mixed microbial community without provision of exogenous electron donor. TOC ART O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/552028v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@8e3002org.highwire.dtl.DTLVardef@f1d396org.highwire.dtl.DTLVardef@7a59dborg.highwire.dtl.DTLVardef@a179bc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Visualization of syntrophic benzene-fermenting Desulfobacterota ORM2 in a methanogenic enrichment culture using fluorescence in situ hybridization

Although benzene degradation under strictly anoxic conditions was first reported over 25 years ago, the mechanism for benzene activation in the absence of oxygen is still elusive. A major limitation has been the difficulty to grow anaerobic benzene-degrading enrichment cultures. Our laboratory has maintained a methanogenic enrichment culture for decades, harboring a benzene fermenter referred to as Desulfobacterota ORM2. Recent genomic analyses indicate that ORM2 is not affiliated with any characterized genus, but it is phylogenetically similar to several other known and predicted benzene degraders. Desulfobacterota ORM2 has a doubling time of approximately 30 days and often enters a long lag or decay phase after inoculation into sterile pre-reduced anaerobic medium. A specific fluorescent in situ hybridization (FISH) probe was used to observe Desulfobacterota ORM2 cells during this decay phase, revealing a rod-shaped cell of variable length with a tendency to associate with other cells, particularly methanogens. Microscopic and genomic analyses indicate that Desulfobacterota ORM2 may produce extracellular polymeric substances (EPS) that likely contribute to cell aggregation. The production of EPS may consume a significant amount of energy, perhaps contributing to the lag time before onset of growth of Desulfobacterota ORM2 post-inoculation. We observed little cell aggregation in a culture amended with very high concentrations of benzene (90-120 mg/L). This study visualized the cells of a novel clade within the Desulfobacterota for the first time, enabling monitoring of spatial organization within a methanogenic consortium and provides hints to improve the growth rate of ORM2. ImportanceA specific FISH probe was designed for the poorly characterized benzene fermenter Desulfobacterota ORM2. This probe was used to monitor changes in spatial organization in a methanogenic benzene-degrading enrichment culture. ORM2 cells were often found in cell aggregates, revealing a possible reason for the long lag phases observed after inoculation.

microbiology↗

bioRxiv2022

Candidatus Nealsonbacteria (OD1) in a methanogenic benzene-degrading enrichment culture is likely an ectosymbiotic biomass recycler

The Candidate Phyla Radiation (CPR) is a very large group of bacteria with no pure culture representatives, first discovered by metagenomic analyses. Within the CPR, candidate phylum Parcubacteria (previously referred to as OD1) within the candidate superphylum Patescibacteria is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the Candidate clade Nealsonbacteria. Because of its persistence over many years, we hypothesized that Ca. Nealsonbacteria DGGOD1a must serve an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10 fold) increase in the absolute abundance of Ca. Nealsonbacteria DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate Ca. Nealsonbacteria in biomass recycling. Fluorescent in situ hybridization and cryogenic transmission electron microscope images revealed that Ca. Nealsonbacteria DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other Ca. Nealsonbacteria found in anoxic environments.

microbiology↗

bioRxiv2022

Transient Oxygen Exposure Causes Profound and Lasting Changes to a Benzene-Degrading Methanogenic Community

We investigated the impact of oxygen on a strictly anaerobic, methanogenic benzene-degrading enrichment culture derived decades ago from oil-contaminated sediment. The culture includes a benzene fermenter from Deltaproteobacteria Candidate clade Sva0485 (referred to as ORM2) and methanogenic archaea. A relatively small one-time injection of air, simulating a small leak into a batch culture bottle, had no measurable impact on benzene degradation rates, although retrospectively, a tiny enrichment of aerobic taxa was detected. A subsequent 100 times larger injection of air stalled methanogenesis and caused drastic perturbation of the microbial community. A benzene-degrading Pseudomonas became highly enriched and consumed benzene and all available oxygen. Anaerobic benzene-degrading ORM2 cell numbers plummeted during this time; re-growth and associated recovery of methanogenic benzene degradation took almost one year. These results highlight the oxygen-sensitivity of this methanogenic culture and confirm that the mechanism for anaerobic biotransformation of benzene is independent of oxygen, fundamentally different from established aerobic pathways, and is carried out by distinct microbial communities. The study further highlights the importance of including microbial decay in characterizing and modelling and mixed microbial communities. SYNOPSISMethanogenic benzene degradation in a highly enriched anaerobic consortium was inhibited for a year after transient exposure to oxygen, causing mass decay of benzene-fermenting bacteria. GRAPHIC FOR ABSTRACT ART O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2022

Microbial Community Dynamics of a Sequentially Fed Anaerobic Digester Treating Solid Organic Waste

A 50 kg-scale high solids anaerobic digestor comprising six sequentially-fed leach beds with a leachate recirculation system and an upflow anaerobic sludge blanket reactor was operated at 37{degrees}C for 88 weeks. The feedstock contained a constant fibre fraction (a mix of cardboard, boxboard, newsprint, and fine paper) and varying proportions of food waste. Significantly enhanced co-digestion and methane production from the fibres was observed as the proportion of FW was increased. The most abundant 16S rRNA amplicon sequence variant (ASV), classified as Clostridium butyricum, was correlated with the amount of FW in the system and total methane yield. However, methane yield specifically from the fibre fraction was significantly correlated with organisms classified as Candidatus Roizmanbacteria and Spirochaetaceae. These ASVs together with ASVs classified as Anaerovorax and Methanoculleus correlated strongly to other ASVs in the microbial community, suggesting these are vitally important for ecosystem function and methane production. In addition, tracing the fate of microbes derived from incoming food waste helped to diagnose a poor batch of bulking agent. Table of Contents (TOC)/ Abstract Art O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/487789v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@2a191forg.highwire.dtl.DTLVardef@1d7497corg.highwire.dtl.DTLVardef@119a43aorg.highwire.dtl.DTLVardef@6d056e_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Heterologous expression of Dehalobacter spp. respiratory reductive dehalogenases in Escherichia coli

Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, primarily because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. In this work we outline a novel respiratory RDase expression system using Escherichia coli as the host. The overexpression of E. colis cobamide transport system, btu, and RDase expression under anaerobic conditions were established to be essential for the expression of active RDases from Dehalobacter - an obligate organohalide respiring bacterium. The expression system was validated on six RDase enzymes with amino acid sequence identities ranging from >30-95%. Dehalogenation activity was verified for each RDase by assaying cell-free extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of both the cobamide and iron-sulfur cluster cofactors was verified, and the specific activity of TmrA was found to be consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases. IMPORTANCEUnderstanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter, a key group of organisms used in bioremediation, in E. coli. This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.

microbiology↗