Global prevalence of organohalide-respiring bacteria dechlorinating polychlorinated biphenyls in sewage sludge.

BACKGROUND: Massive amounts of sewage sludge are generated during biological sewage treatment and are commonly subjected to anaerobic digestion, land application, and landfill disposal. Concurrently, persistent organic pollutants (POPs) are frequently found in sludge treatment and disposal systems, posing significant risks to both human health and wildlife. Metabolically versatile microorganisms originating from sewage sludge are inevitably introduced to sludge treatment and disposal systems, potentially affecting the fate of POPs. However, there is currently a dearth of comprehensive assessments regarding the capability of sewage sludge microbiota from geographically disparate regions to attenuate POPs and the underpinning microbiomes. RESULTS: Here we report the global prevalence of organohalide-respiring bacteria (OHRB) known for their capacity to attenuate POPs in sewage sludge, with an occurrence frequency of ~50% in the investigated samples (605 of 1186). Subsequent laboratory tests revealed microbial reductive dechlorination of polychlorinated biphenyls (PCBs), one of the most notorious categories of POPs, in 80 out of 84 sludge microcosms via various pathways. Most chlorines were removed from the para- and meta-positions of PCBs; nevertheless, ortho-dechlorination of PCBs also occurred widely, although to lower extents. Abundances of several well-characterized OHRB genera (Dehalococcoides, Dehalogenimonas, and Dehalobacter) and uncultivated Dehalococcoidia lineages increased during incubation and were positively correlated with PCB dechlorination, suggesting their involvement in dechlorinating PCBs. The previously identified PCB reductive dehalogenase (RDase) genes pcbA4 and pcbA5 tended to coexist in most sludge microcosms, but the low ratios of these RDase genes to OHRB abundance also indicated the existence of currently undescribed RDases in sewage sludge. Microbial community analyses revealed a positive correlation between biodiversity and PCB dechlorination activity although there was an apparent threshold of community co-occurrence network complexity beyond which dechlorination activity decreased. CONCLUSIONS: Our findings that sludge microbiota exhibited nearly ubiquitous dechlorination of PCBs indicate widespread and nonnegligible impacts of sludge microbiota on the fate of POPs in sludge treatment and disposal systems. The existence of diverse OHRB also suggests sewage sludge as an alternative source to obtain POP-attenuating consortia and calls for further exploration of OHRB populations in sewage sludge. Video Abstract.

[Characterization of Reductive Dechlorination of Chlorinated Ethylenes by Anaerobic Consortium].

Tetrachloroethylene （PCE） and trichloroethylene （TCE） are typical volatile halogenated organic compounds in groundwater that pose serious threats to the ecological environment and human health. To obtain an anaerobic microbial consortium capable of efficiently dechlorinating PCE and TCE to a non-toxic end product and to explore its potential in treating contaminated groundwater， an anaerobic microbial consortium W-1 that completely dechlorinated PCE and TCE to ethylene was obtained by repeatedly feeding PCE or TCE into the contaminated groundwater collected from an industrial site. The dechlorination rates of PCE and TCE were （120.1 ±4.9） µmol·ï¼ˆL·d）-1 and （172.4 ±21.8） µmol·ï¼ˆL·d）-1 in W-1， respectively. 16S rRNA gene amplicon sequencing and quantitative PCR （qPCR） showed that the relative abundance of Dehalobacter increased from 1.9% to 57.1%， with the gene copy number increasing by 1.7×107 copies per 1 µmol Cl- released when 98.3 µmol of PCE was dechlorinated to cis-1，2-dichloroethylene （cis-1，2-DCE）. The relative abundance of Dehalococcoides increased from 1.1% to 53.8% when cis-1，2-DCE was reductively dechlorinated to ethylene. The growth yield of Dehalococcoides gene copy number increased by 1.7×108 copies per 1 µmol Cl- released for the complete reductive dechlorination of PCE to ethylene. The results indicated that Dehalobacter and Dehalococcoides cooperated to completely detoxify PCE. When TCE was used as the only electron acceptor， the relative abundance of Dehalococcoides increased from （29.1 ±2.4）% to （7.7 ±0.2）%， and gene copy number increased by （1.9 ±0.4）×108 copies per 1 µmol Cl- released， after dechlorinating 222.8 µmol of TCE to ethylene. The 16S rRNA gene sequence of Dehalococcoides LWT1， the main functional dehalogenating bacterium in enrichment culture W-1， was obtained using PCR and Sanger sequencing， and it showed 100% similarity with the 16S rRNA gene sequence of D. mccartyi strain 195. The anaerobic microbial consortium W-1 was also bioaugmented into the groundwater contaminated by TCE at a concentration of 418.7 µmol·L-1. The results showed that （69.2 ±9.8）% of TCE could be completely detoxified to ethylene within 28 days with a dechlorination rate of （10.3 ±1.5） µmol·ï¼ˆL·d）-1. This study can provide the microbial resource and theoretical guidance for the anaerobic microbial remediation in PCE or TCE-contaminated groundwater.

Deep-sea in situ and laboratory multi-omics provide insights into the sulfur assimilation of a deep-sea Chloroflexota bacterium.

Chloroflexota bacteria are abundant and globally distributed in various deep-sea ecosystems. It has been reported based on metagenomics data that two deep-sea Chloroflexota lineages (the SAR202 group and Dehalococcoidia class) have the potential to drive sulfur cycling. However, the absence of cultured Chloroflexota representatives is a significant bottleneck toward understanding their contribution to the deep-sea sulfur cycling. In this study, we find that Phototrophicus methaneseepsis ZRK33 isolated from deep-sea sediment has a heterotrophic lifestyle and can assimilate sulfate and thiosulfate. Using combined physiological, genomic, proteomic, and in situ transcriptomic methods, we find that strain ZRK33 can perform assimilatory sulfate reduction in both laboratory and deep-sea conditions. Metabolism of sulfate or thiosulfate by strain ZRK33 significantly promotes the transport and degradation of various macromolecules and thereby stimulates the energy production. In addition, metagenomic results show that genes associated with assimilatory and dissimilatory sulfate reduction are ubiquitously distributed in the metagenome-assembled genomes of Chloroflexota members derived from deep-sea sediments. Metatranscriptomic results also show that the expression levels of related genes are upregulated, strongly suggesting that Chloroflexota bacteria may play undocumented roles in deep-sea sulfur cycling. IMPORTANCE: The cycling of sulfur is one of Earth's major biogeochemical processes and is closely related to the energy metabolism of microorganisms living in the deep-sea cold seep and hydrothermal vents. To date, some of the members of Chloroflexota are proposed to play a previously unrecognized role in sulfur cycling. However, the sulfur metabolic characteristics of deep-sea Chloroflexota bacteria have never been reported, and remain to be verified in cultured deep-sea representatives. Here, we show that the deep-sea Chloroflexota bacterium ZRK33 can perform sulfate assimilation in both laboratory and deep-sea conditions, which expands our knowledge of the sulfur metabolic potential of deep-sea Chloroflexota bacteria. We also show that the genes associated with assimilatory and dissimilatory sulfate reduction ubiquitously distribute in the deep-sea Chloroflexota members, providing hints to the roles of Chloroflexota bacteria in deep-sea sulfur biogeochemical cycling.

Metabolic Synergy of Dehalococcoides Populations Leading to Greater Reductive Dechlorination of Polychlorinated Biphenyls.

Polychlorinated biphenyls (PCBs) are dioxin-like pollutants that cause persistent harm to life. Organohalide-respiring bacteria (OHRB) can detoxify PCBs via reductive dechlorination, but individual OHRB are potent in dechlorinating only specific PCB congeners, restricting the extent of PCB dechlorination. Moreover, the low biomass of OHRB frequently leads to the slow natural attenuation of PCBs at contaminated sites. Here we constructed defined microbial consortia comprising various combinations of PCB-dechlorinating Dehalococcoides strains (CG1, CG4, and CG5) to successfully enhance PCB dechlorination. Specifically, the defined consortia consisting of strains CG1 and CG4 removed 0.28-0.44 and 0.23-0.25 more chlorine per PCB from Aroclor1260 and Aroclor1254, respectively, compared to individual strains, which was attributed to the emergence of new PCB dechlorination pathways in defined consortia. Notably, different Dehalococcoides populations exhibited similar growth when cocultivated, but temporal differences in the expression of PCB reductive dehalogenase genes indicated their metabolic synergy. Bioaugmentation with individual strains (CG1, CG4, and CG5) or defined consortia led to greater PCB dechlorination in wetland sediments, and augmentation with the consortium comprising strains CG1 and CG4 resulted in the greatest PCB dechlorination. These findings collectively suggest that simultaneous application of multiple Dehalococcoides strains, which catalyze complementary dechlorination pathways, is an effective strategy to accelerate PCB dechlorination.

Integrated Advanced Molecular Tools Predict In Situ cVOC Degradation Rates: Field Demonstration.

Chlorinated volatile organic compound (cVOC) degradation rate constants are crucial information for site management. Conventional approaches generate rate estimates from the monitoring and modeling of cVOC concentrations. This requires time series data collected along the flow path of the plume. The estimates of rate constants are often plagued by confounding issues, making predictions cumbersome and unreliable. Laboratory data suggest that targeted quantitative analysis of Dehalococcoides mccartyi (Dhc) biomarker genes (qPCR) and proteins (qProt) can be directly correlated with reductive dechlorination activity. To assess the potential of qPCR and qProt measurements to predict rates, we collected data from cVOC-contaminated aquifers. At the benchmark study site, the rate constant for degradation of cis-dichloroethene (cDCE) extracted from monitoring data was 11.0 ± 3.4 yr-1, and the rate constant predicted from the abundance of TceA peptides was 6.9 yr-1. The rate constant for degradation of vinyl chloride (VC) from monitoring data was 8.4 ± 5.7 yr-1, and the rate constant predicted from the abundance of TceA peptides was 5.2 yr-1. At the other study sites, the rate constants for cDCE degradation predicted from qPCR and qProt measurements agreed within a factor of 4. Under the right circumstances, qPCR and qProt measurements can be useful to rapidly predict rates of cDCE and VC biodegradation, providing a major advance in effective site management.

Genome-Resolved Metagenomics and Metatranscriptomics Reveal Insights into the Ecology and Metabolism of Anaerobic Microbial Communities in PCB-Contaminated Sediments.

Growth of organohalide-respiring bacteria such as Dehalococcoides mccartyi on halogenated organics (e.g., polychlorinated biphenyls (PCBs)) at contaminated sites or in enrichment culture requires interaction and support from other microbial community members. To evaluate naturally occurring interactions between Dehalococcoides and key supporting microorganisms (e.g., production of H2, acetate, and corrinoids) in PCB-contaminated sediments, metagenomic and metatranscriptomic sequencing was conducted on DNA and RNA extracted from sediment microcosms, showing evidence of both Dehalococcoides growth and PCB dechlorination. Using a genome-resolved approach, 160 metagenome-assembled genomes (MAGs), including three Dehalococcoides MAGs, were recovered. A novel reductive dehalogenase gene, distantly related to the chlorophenol dehalogenase gene cprA (pairwise amino acid identity: 23.75%), was significantly expressed. Using MAG gene expression data, 112 MAGs were assigned functional roles (e.g., corrinoid producers, acetate/H2 producers, etc.). A network coexpression analysis of all 160 MAGs revealed correlations between 39 MAGs and the Dehalococcoides MAGs. The network analysis also showed that MAGs assigned with functional roles that support Dehalococcoides growth (e.g., corrinoid assembly, and production of intermediates required for corrinoid synthesis) displayed significant coexpression correlations with Dehalococcoides MAGs. This work demonstrates the power of genome-resolved metagenomic and metatranscriptomic analyses, which unify taxonomy and function, in investigating the ecology of dehalogenating microbial communities.

Alleviating Chlorinated Alkane Inhibition on Dehalococcoides to Achieve Detoxification of Chlorinated Aliphatic Cocontaminants.

Cocontamination by multiple chlorinated solvents is a prevalent issue in groundwater, presenting a formidable challenge for effective remediation. Despite the recognition of this issue, a comprehensive assessment of microbial detoxification processes involving chloroethenes and associated cocontaminants, along with the underpinning microbiome, remains absent. Moreover, strategies to mitigate the inhibitory effects of cocontaminants have not been reported. Here, we revealed that chloroform exhibited the most potent inhibitory effects, followed by 1,1,1-trichloroethane and 1,1,2-trichloroethane, on dechlorination of dichloroethenes (DCEs) in Dehalococcoides-containing consortia. The observed inhibition could be attributed to suppression of biosynthesis and enzymatic activity of reductive dehalogenases and growth of Dehalococcoides. Notably, cocontaminants more profoundly inhibited Dehalococcoides populations harboring the vcrA gene than those possessing the tceA gene, thereby explaining the accumulation of vinyl chloride under cocontaminant stress. Nonetheless, we successfully ameliorated cocontaminant inhibition by augmentation with Desulfitobacterium sp. strain PR owing to its ability to attenuate cocontaminants, resulting in concurrent detoxification of DCEs, trichloroethanes, and chloroform. Microbial community analyses demonstrated obvious alterations in taxonomic composition, structure, and assembly of the dechlorinating microbiome in the presence of cocontaminants, and introduction of strain PR reshaped the dechlorinating microbiome to be similar to its original state in the absence of cocontaminants. Altogether, these findings contribute to developing bioremediation technologies to clean up challenging sites polluted with multiple chlorinated solvents.

Intermittent Oxygen Supply Facilitates Codegradation of Trichloroethene and Toluene by Anaerobic Consortia.

Biodegradation is commonly employed for remediating trichloroethene- or toluene-contaminated sites. However, remediation methods using either anaerobic or aerobic degradation are inefficient for dual pollutants. We developed an anaerobic sequencing batch reactor system with intermittent oxygen supply for the codegradation of trichloroethylene and toluene. Our results showed that oxygen inhibited anaerobic dechlorination of trichloroethene, but dechlorination rates remained comparable to that at dissolved oxygen levels of 0.2 mg/L. Intermittent oxygenation engendered reactor redox fluctuations (-146 to -475 mV) and facilitated rapid codegradation of targeting dual pollutants, with trichloroethene degradation constituting only 27.5% of the noninhibited dechlorination. Amplicon sequencing analysis revealed the predominance of Dehalogenimonas (16.0% ± 3.5%) over Dehalococcoides (0.3% ± 0.2%), with ten times higher transcriptomic activity in Dehalogenimonas. Shotgun metagenomics revealed numerous genes related to reductive dehalogenases and oxidative stress resistance in Dehalogenimonas and Dehalococcoides, as well as the enrichment of diversified facultative populations with functional genes related to trichloroethylene cometabolism and aerobic and anaerobic toluene degradation. These findings suggested that the codegradation of trichloroethylene and toluene may involve multiple biodegradation mechanisms. Overall results of this study demonstrate the effectiveness of intermittent micro-oxygenation in aiding trichloroethene-toluene degradation, suggesting the potential for the bioremediation of sites with similar organic pollutants.

Microbial Reductive Dechlorination by a Commercially Available Dechlorinating Consortium Is Not Inhibited by Perfluoroalkyl Acids (PFAAs) at Field-Relevant Concentrations.

Perfluoroalkyl acids (PFAAs) have been shown to inhibit biodegradation (i.e., organohalide respiration) of chlorinated ethenes. The potential negative impacts of PFAAs on microbial species performing organohalide respiration, particularly Dehalococcoides mccartyi (Dhc), and the efficacy of in situ bioremediation are a critical concern for comingled PFAA-chlorinated ethene plumes. Batch reactor (no soil) and microcosm (with soil) experiments, containing a PFAA mixture and bioaugmented with KB-1, were completed to assess the impact of PFAAs on chlorinated ethene organohalide respiration. In batch reactors, PFAAs delayed complete biodegradation of cis-1,2-dichloroethene (cis-DCE) to ethene. Maximum substrate utilization rates (a metric for quantifying biodegradation rates) were fit to batch reactor experiments using a numerical model that accounted for chlorinated ethene losses to septa. Fitted values for cis-DCE and vinyl chloride biodegradation were significantly lower (p < 0.05) in batch reactors containing ≥50 mg/L PFAAs. Examination of reductive dehalogenase genes implicated in ethene formation revealed a PFAA-associated change in the Dhc community from cells harboring the vcrA gene to those harboring the bvcA gene. Organohalide respiration of chlorinated ethenes was not impaired in microcosm experiments with PFAA concentrations of 38.7 mg/L and less, suggesting that a microbial community containing multiple strains of Dhc is unlikely to be inhibited by PFAAs at lower, environmentally relevant concentrations.

Oral metagenomes from Native American Ancestors reveal distinct microbial lineages in the pre-contact era.

OBJECTIVES: Limited studies have focused on how European contact and colonialism impacted Native American oral microbiomes, specifically, the diversity of commensal or opportunistically pathogenic oral microbes, which may be associated with oral diseases. Here, we studied the oral microbiomes of pre-contact Wichita Ancestors, in partnership with the Descendant community, The Wichita and Affiliated Tribes, Oklahoma, USA. MATERIALS AND METHODS: Skeletal remains of 28 Wichita Ancestors from 20 archeological sites (dating approximately to 1250-1450 CE) were paleopathologically assessed for presence of dental calculus and oral disease. DNA was extracted from calculus, and partial uracil deglycosylase-treated double-stranded DNA libraries were shotgun-sequenced using Illumina technology. DNA preservation was assessed, the microbial community was taxonomically profiled, and phylogenomic analyzes were conducted. RESULTS: Paleopathological analysis revealed signs of oral diseases such as caries and periodontitis. Calculus samples from 26 Ancestors yielded oral microbiomes with minimal extraneous contamination. Anaerolineaceae bacterium oral taxon 439 was found to be the most abundant bacterial species. Several Ancestors showed high abundance of bacteria typically associated with periodontitis such as Tannerella forsythia and Treponema denticola. Phylogenomic analyzes of Anaerolineaceae bacterium oral taxon 439 and T. forsythia revealed biogeographic structuring; strains present in the Wichita Ancestors clustered with strains from other pre-contact Native Americans and were distinct from European and/or post-contact American strains. DISCUSSION: We present the largest oral metagenome dataset from a pre-contact Native American population and demonstrate the presence of distinct lineages of oral microbes specific to the pre-contact Americas.

Sustained detoxification of 1,2-dichloroethane to ethylene by a symbiotic consortium containing Dehalococcoides species.

1,2-Dichloroethane (1,2-DCA) is a ubiquitous volatile halogenated organic pollutant in groundwater and soil, which poses a serious threat to the ecosystem and human health. Microbial reductive dechlorination has been recognized as an environmentally-friendly strategy for the remediation of sites contaminated with 1,2-DCA. In this study, we obtained an anaerobic microbiota derived from 1,2-DCA contaminated groundwater, which was able to sustainably convert 1,2-DCA into non-toxic ethylene with an average dechlorination rate of 30.70 ± 11.06 µM d-1 (N = 6). The microbial community profile demonstrated that the relative abundance of Dehalococcoides species increased from 0.53 ± 0.08% to 44.68 ± 3.61% in parallel with the dechlorination of 1,2-DCA. Quantitative PCR results showed that the Dehalococcoides species 16S rRNA gene increased from 2.40 ± 1.71 × 108 copies∙mL-1 culture to 4.07 ± 2.45 × 108 copies∙mL-1 culture after dechlorinating 110.69 ± 30.61 µmol of 1,2-DCA with a growth yield of 1.55 ± 0.93 × 108 cells per µmol Cl- released (N = 6), suggesting that Dehalococcoides species used 1,2-DCA for organohalide respiration to maintain cell growth. Notably, the relative abundances of Methanobacterium sp. (p = 0.0618) and Desulfovibrio sp. (p = 0.0001995) also increased significantly during the dechlorination of 1,2-DCA and were clustered in the same module with Dehalococcoides species in the co-occurrence network. These results hinted that Dehalococcoides species, the obligate organohalide-respiring bacterium, exhibited potential symbiotic relationships with Methanobacterium and Desulfovibrio species. This study illustrates the importance of microbial interactions within functional microbiota and provides a promising microbial resource for in situ bioremediation in sites contaminated with 1,2-DCA.

Genome-centric metagenomic insights into the role of Chloroflexi in anammox, activated sludge and methanogenic reactors.

BACKGROUND: The phylum Chloroflexi is highly abundant in a wide variety of wastewater treatment bioreactors. It has been suggested that they play relevant roles in these ecosystems, particularly in degrading carbon compounds and on structuring flocs or granules. Nevertheless, their function is not yet well understood as most species have not been isolated in axenic cultures. Here we used a metagenomic approach to investigate Chloroflexi diversity and their metabolic potential in three environmentally different bioreactors: a methanogenic full-scale reactor, a full-scale activated sludge reactor and a lab scale anammox reactor. RESULTS: Differential coverage binning approach was used to assemble the genomes of 17 new Chloroflexi species, two of which are proposed as new Candidatus genus. In addition, we recovered the first representative genome belonging to the genus 'Ca. Villigracilis'. Even though samples analyzed were collected from bioreactors operating under different environmental conditions, the assembled genomes share several metabolic features: anaerobic metabolism, fermentative pathways and several genes coding for hydrolytic enzymes. Interestingly, genome analysis from the anammox reactor indicated a putative role of Chloroflexi in nitrogen conversion. Genes related to adhesiveness and exopolysaccharides production were also detected. Complementing sequencing analysis, filamentous morphology was detected by Fluorescent in situ hybridization. CONCLUSION: Our results suggest that Chloroflexi participate in organic matter degradation, nitrogen removal and biofilm aggregation, playing different roles according to the environmental conditions.

Salinity determines performance, functional populations, and microbial ecology in consortia attenuating organohalide pollutants.

Organohalide pollutants are prevalent in coastal regions due to extensive intervention by anthropogenic activities, threatening public health and ecosystems. Gradients in salinity are a natural feature of coasts, but their impacts on the environmental fate of organohalides and the underlying microbial communities remain poorly understood. Here we report the effects of salinity on microbial reductive dechlorination of tetrachloroethene (PCE) and polychlorinated biphenyls (PCBs) in consortia derived from distinct environments (freshwater and marine sediments). Marine-derived microcosms exhibited higher halotolerance during PCE and PCB dechlorination, and a halotolerant dechlorinating culture was enriched from these microcosms. The organohalide-respiring bacteria (OHRB) responsible for PCE and PCB dechlorination in marine microcosms shifted from Dehalococcoides to Dehalobium when salinity increased. Broadly, lower microbial diversity, simpler co-occurrence networks, and more deterministic microbial community assemblages were observed under higher salinity. Separately, we observed that inhibition of dechlorination by high salinity could be attributed to suppressed viability of Dehalococcoides rather than reduced provision of substrates by syntrophic microorganisms. Additionally, the high activity of PCE dechlorinating reductive dehalogenases (RDases) in in vitro tests under high salinity suggests that high salinity likely disrupted cellular components other than RDases in Dehalococcoides. Genomic analyses indicated that the capability of Dehalobium to perform dehalogenation under high salinity was likely owing to the presence of genes associated with halotolerance in its genomes. Collectively, these mechanistic and ecological insights contribute to understanding the fate and bioremediation of organohalide pollutants in environments with changing salinity.

Effect of polyhydroxyalkanoates on the microbial reductive dechlorination of polychlorinated biphenyls and competing anaerobic respirations in a marine microbial culture.

The effect of polyhydroxyalkanoates (PHAs) with different composition on the reductive dechlorination activity of a polychlorinated biphenyls (PCBs) dechlorinating marine microbial community and on the activity of sulfate-reducing (SRB) and methanogenic bacteria (MB), were investigated in marine sediment microcosms and compared with the main monomer, 3-hydroxybutyric acid (3HB). Despite PHAs were fermented more slowly than 3HB, all electron donors stimulated constantly sulfate-reduction, methanogenesis and, only transiently, PCB reductive dechlorination. No relevant differences were observed with different compositions of PHAs. According to electron balances, the majority of the supplied electrons (50 %) were consumed by SRB and to less extent by MB (9-31 %), while a small percentage (0.01 %) was delivered to OHRB. In the studied conditions PHAs were confirmed as potential slow­hydrogen releasing compounds in marine environment but their fermentation rate was sufficiently high to mainly stimulate the competitors of organohalide respring bacteria for electron donors.

Continuous cultivation of Dehalococcoides mccartyi with brominated tyrosine avoids toxic byproducts and gives tight reactor control.

Dehalococcoides mccartyi strain CBDB1 is a strictly anaerobic organohalide-respiring bacterium with strong application potential to remediate aquifers and soils contaminated with halogenated aromatics. To date, cultivation of strain CBDB1 has mostly been done in bottles or fed-batch reactors. Challenges with such systems include low biomass yield and difficulties in controlling the growth conditions. Here, we report the cultivation of planktonic D. mccartyi strain CBDB1 in a continuous stirring tank reactor (CSTR) that led to high cell densities (∼8 × 108 cells mL-1) and dominance of strain CBDB1. The reactor culture received acetate, hydrogen, and the brominated amino acid D- or L-3,5-dibromotyrosine as substrates. Both D- and L-3,5-dibromotyrosine were utilized as respiratory electron acceptors and are promising for biomass production due to their decent solubility in water and the formation of a non-toxic debromination product, tyrosine. By monitoring headspace pressure decrease which is indicative of hydrogen consumption, the organohalide respiration rate was followed in real time. Proteomics analyses revealed that the reductive dehalogenase CbdbA238 was highly expressed with both D- and L-3,5-dibromotyrosine, while other reductive dehalogenases including those that were previously suggested to be constitutively expressed, were repressed. Denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes indicated that the majority of cells in the community belonged to the Dehalococcoides although the CSTR was operated under non-sterile conditions. Hence, tightly controlled CSTR cultivation of Dehalococcoides opens novel options to improve biomass production for bioaugmentation and for advanced biochemical studies.

Resilience of organohalide-detoxifying microbial community to oxygen stress in sewage sludge.

Organohalide pollutants are prevalent in the environment, causing harms to wildlife and human. Organohalide-respiring bacteria (OHRB) could detoxify these pollutants in anaerobic environments, but the most competent OHRB (i.e., Dehalococcoides) is susceptible to oxygen. This study reports exceptional resistance and resilience of sewage sludge microbial communities to oxygen stress for attenuation of structurally distinct organohalide pollutants, including tetrachloroethene, tetrabromobisphenol A, and polybrominated diphenyl ethers. The dehalogenation rate constant of these organohalide pollutants in oxygen-exposed sludge microcosms was maintained as 74-120% as that in the control without oxygen exposure. Subsequent top-down experiments clarified that sludge flocs and non-OHRB contributed to alleviating oxygen stress on OHRB. In the dehalogenating microcosms, multiple OHRB (Dehahlococcoides, Dehalogenimonas, and Sulfurospirillum) harboring distinct reductive dehalogenase genes (pceA, pteA, tceA, vcrA, and bdeA) collaborated to detoxify organohalide pollutants but responded differentially to oxygen stress. Comprehensive microbial community analyses (taxonomy, diversity, and structure) demonstrated certain resilience of the sludge-derived dehalogenating microbial communities to oxygen stress. Additionally, microbial co-occurrence networks were intensified by oxygen stress in most microcosms, as a possible stress mitigation strategy. Altogether the mechanistic and ecological findings in this study contribute to remediation of organohalide-contaminated sites encountering oxygen disturbance.

Sponge holobionts shift their prokaryotic communities and antimicrobial activity from shallow to lower mesophotic depths.

In this study, we used 16S rRNA gene amplicon sequencing to investigate prokaryotic community composition of the Caribbean sponges Xestospongia muta and Agelas sventres from three depth ranges: < 30 m (shallow), 30-60 m (upper mesophotic), and 60-90 m (lower mesophotic). The prokaryotic community in shallow samples of X. muta was enriched in Cyanobacteria, Chloroflexota, and Crenarchaeota compared to samples from mesophotic depths, while mesophotic samples of X. muta were enriched in Acidobacteriota. For A. sventres, relative abundance of Acidobacteriota, Chloroflexota, and Gammaproteobacteria was higher in shallow samples, while Proteobacteria and Crenarchaeota were enriched in mesophotic A. sventres samples. Antimicrobial activity was evaluated by screening crude extracts of sponges against a set of Gram-positive and Gram-negative bacteria, a yeast, and an oomycete. Antibacterial activities from crude extracts of shallow sponge individuals were generally higher than observed from mesophotic individuals, that showed limited or no antibacterial activities. Conversely, the highest anti-oomycete activity was found from crude extracts of X. muta individuals from lower mesophotic depth, but without a clear pattern across the depth gradient. These results indicate that sponge-associated prokaryotic communities and the antimicrobial activity of sponges change within species across a depth gradient from shallow to mesophotic depth.

Combined read- and assembly-based metagenomics to reconstruct a Dehalococcoides mccartyi genome from PCB-contaminated sediments and evaluate functional differences among organohalide-respiring consortia in the presence of different halogenated contaminants.

Microbial communities that support respiration of halogenated organic contaminants by Dehalococcoides sp. facilitate full-scale bioremediation of chlorinated ethenes and demonstrate the potential to aid in bioremediation of halogenated aromatics like polychlorinated biphenyls (PCBs). However, it remains unclear if Dehalococcoides-containing microbial community dynamics observed in sediment-free systems quantitatively resemble that of sediment environments. To evaluate that possibility we assembled, annotated, and analyzed a Dehalococcoides sp. metagenome-assembled genome (MAG) from PCB-contaminated sediments. Phylogenetic analysis of reductive dehalogenase gene (rdhA) sequences within the MAG revealed that pcbA1 and pcbA4/5-like rdhA were absent, while several candidate PCB dehalogenase genes and potentially novel rdhA sequences were identified. Using a compositional comparative metagenomics approach, we quantified Dehalococcoides-containing microbial community structure shifts in response to halogenated organics and the presence of sediments. Functional level analysis revealed significantly greater abundances of genes associated with cobamide remodeling and horizontal gene transfer in tetrachloroethene-fed cultures as compared to halogenated aromatic-exposed consortia with or without sediments, despite little evidence of statistically significant differences in microbial community taxonomic structure. Our findings support the use of a generalizable comparative metagenomics workflow to evaluate Dehalococcoides-containing consortia in sediments and sediment-free environments to eludicate functions and microbial interactions that facilitate bioremediation of halogenated organic contaminants.

Microbial community response to a bioaugmentation test to degrade trichloroethylene in a fractured rock aquifer, Trenton, N.J.

Bioaugmentation is a promising strategy for enhancing trichloroethylene (TCE) degradation in fractured rock. However, slow or incomplete biodegradation can lead to stalling at degradation byproducts such as 1,2-dichloroethene (cis-DCE) and vinyl chloride (VC). Over the course of 7 years, we examined the response of groundwater microbial populations in a bioaugmentation test where an emulsified vegetable oil solution (EOS®) and a dechlorinating consortium (KB-1®), containing the established dechlorinator Dehalococcoides (DHC), were injected into a TCE-contaminated fractured rock aquifer. Indigenous microbial communities responded within 2 days to added substrate and outcompeted KB-1®, and over the years of monitoring, several other notable turnover events were observed. Concentrations of ethene, the end product in reductive dechlorination, had the strongest correlations (P< .05) with members of Candidatus Colwellbacteria but their involvement in reductive dechlorination is unknown and warrants further investigation.DHC never exceeded 0.6% relative abundance of groundwater microbial communities, despite its previously presumed importance at the site. Increased concentrations of carbon dioxide, acetic acid, and methane were positively correlated with increasing ethene concentrations; however, concentrations of cis-DCE and VC remained high by the end of the monitoring period suggesting preferential enrichment of indigenous partial dechlorinators over bioaugmented complete dechlorinators. This study highlights the importance of characterizing in situ microbial populations to understand how they can potentially enhance or inhibit augmented TCE degradation.

Organohalide respiration potential in marine sediments from Aarhus Bay.

Organohalide respiration (OHR), catalysed by reductive dehalogenases (RDases), plays an important role in halogen cycling. Natural organohalides and putative RDase-encoding genes have been reported in Aarhus Bay sediments, however, OHR has not been experimentally verified. Here we show that sediments of Aarhus Bay can dehalogenate a range of organohalides, and different organohalides differentially affected microbial community compositions. PCE-dechlorinating cultures were further examined by 16S rRNA gene-targeted quantitative PCR and amplicon sequencing. Known organohalide-respiring bacteria (OHRB) including Dehalococcoides, Dehalobacter and Desulfitobacterium decreased in abundance during transfers and serial dilutions, suggesting the importance of yet uncharacterized OHRB in these cultures. Switching from PCE to 2,6-DBP led to its complete debromination to phenol in cultures with and without sulfate. 2,6-DBP debrominating cultures differed in microbial composition from PCE-dechlorinating cultures. Desulfobacterota genera recently verified to include OHRB, including Desulfovibrio and Desulfuromusa, were enriched in all microcosms, whereas Halodesulfovibrio was only enriched in cultures without sulfate. Hydrogen and methane were detected in cultures without sulfate. Hydrogen likely served as electron donor for OHR and methanogenesis. This study shows that OHR can occur in marine environments mediated by yet unknown OHRB, suggesting their role in natural halogen cycling.