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Blue holes are vertical water-filled openings in carbonate rock that exhibit complex morphology, ecology, and water chemistry. In this study, macroscopic microbial mat structures found in complete anoxic conditions in the Faanu Mudugau Blue Hole (Maldives) were studied by metagenomic methods. Such communities have likely been evolutionary isolated from the surrounding marine environment for more than 10,000 years since the Blue Hole formation during the last Ice Age. A total of 48 high-quality metagenome-assembled genomes (MAGs) were recovered, predominantly composed of the phyla Chloroflexota, Proteobacteria and Desulfobacterota. None of these MAGs have been classified to species level (<95% ANI), suggesting the discovery of several new microbial taxa. In particular, MAGs belonging to novel bacterial genera within the order Dehalococcoidales accounted for 20% of the macroscopic mat community. Genome-resolved metabolic analysis of this dominant microbial fraction revealed a mixotrophic lifestyle based on energy conservation via fermentation, hydrogen metabolism and anaerobic CO 2 fixation through the Wood-Ljungdahl pathway. Interestingly, these bacteria showed a high proportion of ancestral genes in their genomes providing intriguing perspectives on mechanisms driving microbial evolution in this peculiar environment. Overall, our results provide new knowledge for understanding microbial life under extreme conditions in blue hole environments., (© 2024 The Author(s). Environmental Microbiology Reports published by John Wiley & Sons Ltd.)

Much attention is placed on organohalide-respiring bacteria (OHRB), such as Dehalococcoides, during the design and performance monitoring of chlorinated solvent bioremediation systems. However, many OHRB cannot function effectively without the support of a diverse group of other microbial community members (MCMs), who play key roles fermenting organic matter into more readily useable electron donors, producing corrinoids such as vitamin B12, or facilitating other important metabolic processes or biochemical reactions. While it is known that certain MCMs support dechlorination, a metric considering their contribution to bioremediation performance has yet to be proposed. Advances in molecular biology tools offer an opportunity to better understand the presence and activity of specific microbes, and their relation to bioremediation performance. In this paper, we test the hypothesis that a specific microbial consortium identified within 16S ribosomal ribonucleic acid (rRNA) gene next generation sequencing (NGS) data can be predictive of contaminant degradation rates. Field-based data from multiple contaminated sites indicate that increasing relative abundance of specific MCMs correlates with increasing first-order degradation rates. Based on these results, we present a framework for computing a simplified metric using NGS data, the Microbial Community Structure Index, to evaluate the adequacy of the microbial ecosystem during assessment of bioremediation performance., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 °C), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95 % conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The archaeal mevalonate pathway is a recently discovered modified version of the eukaryotic mevalonate pathway. This pathway is widely conserved in archaea, except for some archaeal lineages possessing the eukaryotic or other modified mevalonate pathways. Although the pathway seems almost exclusive to the domain Archaea, the whole set of homologous genes of the pathway is found in the metagenome-assembled genome sequence of an uncultivated bacterium, Candidatus Promineifilum breve, of the phylum Chloroflexota. To prove the existence of the archaea-specific pathway in the domain Bacteria, we confirmed the activities of the enzymes specific to the pathway, phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, because only these two enzymes are absent in closely related Chloroflexota bacteria that possess a different type of modified mevalonate pathway. The activity of anhydromevalonate phosphate decarboxylase was evaluated by carotenoid production via the archaeal mevalonate pathway reconstituted in Escherichia coli cells, whereas that of phosphomevalonate dehydratase was confirmed by an in vitro assay using the recombinant enzyme after purification and iron-sulfur cluster reconstruction. Phylogenetic analyses of some mevalonate pathway-related enzymes suggest an evolutionary route for the archaeal mevalonate pathway in Candidatus P. breve, which probably involves horizontal gene transfer events.IMPORTANCEThe recent discovery of various modified mevalonate pathways in microorganisms, such as archaea and Chloroflexota bacteria, has shed light on the complexity of the evolution of metabolic pathways, including those involved in primary metabolism. The fact that the archaeal mevalonate pathway, which is almost exclusive to the domain Archaea, exists in a Chloroflexota bacterium provides valuable insights into the molecular evolution of the mevalonate pathways and associated enzymes. Putative genes probably involved in the archaeal mevalonate pathway have also been found in the metagenome-assembled genomes of Chloroflexota bacteria. Such genes can contribute to metabolic engineering for the bioproduction of valuable isoprenoids because the archaeal mevalonate pathway is known to be an energy-saving metabolic pathway that consumes less ATP than other mevalonate pathways do., Competing Interests: The authors declare no conflict of interest.

Rural waste accumulation leads to heavy metal soil pollution, impacting microbial communities. However, knowledge gaps exist regarding the distribution and occurrence patterns of bacterial communities in multi-metal contaminated soil profiles. In this study, high-throughput 16 S rRNA gene sequencing technology was used to explore the response of soil bacterial communities to various heavy metal pollution in rural simple waste dumps in karst areas of Southwest China. The study selected three habitats in the center, edge, and uncontaminated areas of the waste dump to evaluate the main factors driving the change in bacterial community composition. Pollution indices reveal severe contamination across all elements, except for moderately polluted lead (Pb); contamination severity ranks as follows: Mn > Cd > Zn > Cr > Sb > V > Cu > As > Pb. Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteriota predominate, collectively constituting over 60% of the relative abundance. Analysis of Chao and Shannon indices demonstrated that the waste dump center boasted the greatest bacterial richness and diversity. Correlation data indicated a predominant synergistic interaction among the landfill's bacterial community, with a higher number of positive associations (76.4%) compared to negative ones (26.3%). Network complexity was minimal at the dump's edge. RDA analysis showed that Pb(explained:46%) and Mn(explained:21%) were the key factors causing the difference in bacterial community composition in the edge area of the waste dump, and AK(explained:42.1%) and Cd(explained:35.2%) were the key factors in the center of the waste dump. This study provides important information for understanding the distribution patterns, co-occurrence networks, and environmental response mechanisms of bacterial communities in landfill soils under heavy metal stress, which helps guide the formulation of rural waste treatment and soil remediation strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

SAR202 bacteria in the Chloroflexota phylum are abundant and widely distributed in the ocean. Their genome coding capacities indicate their potential roles in degrading complex and recalcitrant organic compounds in the ocean. However, our understanding of their genomic diversity, vertical distribution, and depth-related metabolisms is still limited by the number of assembled SAR202 genomes. In this study, we apply deep metagenomic sequencing (180 Gb per sample) to investigate microbial communities collected from six representative depths at the Bermuda Atlantic Time Series (BATS) station. We obtain 173 SAR202 metagenome-assembled genomes (MAGs). Intriguingly, 154 new species and 104 new genera are found based on these 173 SAR202 genomes. We add 12 new subgroups to the current SAR202 lineages. The vertical distribution of 20 SAR202 subgroups shows their niche partitioning in the euphotic, mesopelagic, and bathypelagic oceans, respectively. Deep-ocean SAR202 bacteria contain more genes and exhibit more metabolic potential for degrading complex organic substrates than those from the euphotic zone. With deep metagenomic sequencing, we uncover many new lineages of SAR202 bacteria and their potential functions which greatly deepen our understanding of their diversity, vertical profile, and contribution to the ocean's carbon cycling, especially in the deep ocean., (© 2024. The Author(s).)

Hot spring environments encompass broad physicochemical ranges, in which temperature and pH account for crucial factors shaping hot spring microbial community and diversity. However, the presence of photosynthetic microbial mats adjacent to boiling hot spring vents, where fluid temperatures extend beyond photosynthetic capability, questions the microbial profiles and the actual temperatures of such adjacent mats. Therefore, this study aims to characterize thermophilic microbial communities at Pong Dueat Pa Pae hot spring using next-generation sequencing, including investigating hot spring mineralogy. Results suggest that Pong Dueat Pa Pae hot spring precipitates comprise mainly silica which also acts as the main preservative medium for microbial permineralization. Molecular results revealed the presence of cyanobacterial and Chloroflexi species in the thick, orange and green subaerial mats surrounding the vents, suggesting the mats would be at least 30 °C cooler than source vents despite constantly receiving geyser splashes. Bacterial abundance was considerably higher than archaeal (97.9% versus 2.1%). Cyanobacterial (mainly Synechococcus and Leptolygbya) and Chloroflexi species (mainly Roseiflexus) accounted for almost half (40.04%) of the bacterial community, while DHVEG-6 and Thaumarchaeota comprised dominant members (> 90%) of the archaeal fraction. This study updates and provides insights into thermophilic microbial community composition and mineralogy of hot springs in Thailand., (© 2024. The Author(s), under exclusive licence to Springer Nature Japan KK.)

In the next decades, the increasing material and energetic demand to support population growth and higher standards of living will amplify the current pressures on ecosystems and will call for greater investments in infrastructures and modern technologies. A valid approach to overcome such future challenges is the employment of sustainable bio-based technologies that explore the metabolic richness of microorganisms. Collectively, the metabolic capabilities of Chloroflexota , spanning aerobic and anaerobic conditions, thermophilic adaptability, anoxygenic photosynthesis, and utilization of toxic compounds as electron acceptors, underscore the phylum's resilience and ecological significance. These diverse metabolic strategies, driven by the interplay between temperature, oxygen availability, and energy metabolism, exemplify the complex adaptations that enabled Chloroflexota to colonize a wide range of ecological niches. In demonstrating the metabolic richness of the Chloroflexota phylum, specific members exemplify the diverse capabilities of these microorganisms: Chloroflexus aurantiacus showcases adaptability through its thermophilic and phototrophic growth, whereas members of the Anaerolineae class are known for their role in the degradation of complex organic compounds, contributing significantly to the carbon cycle in anaerobic environments, highlighting the phylum's potential for biotechnological exploitation in varying environmental conditions. In this context, the metabolic diversity of Chloroflexota must be considered a promising asset for a large range of applications. Currently, this bacterial phylum is organized into eight classes possessing different metabolic strategies to survive and thrive in a wide variety of extreme environments. This review correlates the ecological role of Chloroflexota in such environments with the potential application of their metabolisms in biotechnological approaches., Competing Interests: The authors declare no conflict of interest.

Alginate is derived from brown algae, which can be cultivated in large quantities. It can be broken down by alginate lyase into alginate oligosaccharides (AOSs), which exhibit a higher added value and better bioactivity than alginate. In this study, metagenomic technology was used to screen for genes that code for high-efficiency alginate lyases. The candidate alginate lyase gene alg169 was detected from Psychromonas sp. SP041, the most abundant species among alginate lyase bacteria on selected rotten kelps. The alginate lyase Alg169 was heterologously expressed in Escherichia coli BL21 (DE3), Ni-IDA-purified, and characterized. The optimum temperature and pH of Alg169 were 25 °C and 7.0, respectively. Metal ions including Mn 2+ , Co 2+ , Ca 2+ , Mg 2+ , Ni 2+ , and Ba 2+ led to significantly increased enzyme activity. Alg169 exhibited a pronounced dependence on Na + , and upon treatment with Mn 2+ , its activity surged by 687.57%, resulting in the highest observed enzyme activity of 117,081 U/mg. Bioinformatic analysis predicted that Alg169 would be a double-domain lyase with a molecular weight of 65.58 kDa. It is a bifunctional enzyme with substrate specificity to polyguluronic acid (polyG) and polymannuronic acid (polyM). These results suggest that Alg169 is a promising candidate for the efficient manufacturing of AOSs from brown seaweed.

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., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

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·（L·d） -1 and （172.4 ±21.8） μmol·（L·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 1.7×10 7 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 1.7×10 8 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）×10 8 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·（L·d） -1 . This study can provide the microbial resource and theoretical guidance for the anaerobic microbial remediation in PCE or TCE-contaminated groundwater.

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.

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.

Isolate studies have been a cornerstone for unraveling metabolic pathways and phenotypical (functional) features. Biogeochemical processes in natural and engineered ecosystems are generally performed by more than a single microbe and often rely on mutualistic interactions. We demonstrate the rational bottom-up design of synthetic, interdependent co-cultures to achieve concomitant utilization of chlorinated methanes as electron donors and organohalogens as electron acceptors. Specialized anaerobes conserve energy from the catabolic conversion of chloromethane or dichloromethane to formate, H2, and acetate, compounds that the organohalide-respiring bacterium Dehalogenimonas etheniformans strain GP requires to utilize cis-1,2-dichloroethenene and vinyl chloride as electron acceptors. Organism-specific qPCR enumeration matched the growth of individual dechlorinators to the respective functional (i.e. dechlorination) traits. The metabolite cross-feeding in the synthetic (co-)cultures enables concomitant utilization of chlorinated methanes (i.e. chloromethane and dichloromethane) and chlorinated ethenes (i.e. cis-1,2-dichloroethenene and vinyl chloride) without the addition of an external electron donor (i.e. formate and H2). The findings illustrate that naturally occurring chlorinated C1 compounds can sustain anaerobic food webs, an observation with implications for the development of interdependent, mutualistic communities, the sustenance of microbial life in oligotrophic and energy-deprived environments, and the fate of chloromethane/dichloromethane and chlorinated electron acceptors (e.g. chlorinated ethenes) in pristine environments and commingled contaminant plumes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

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., (© 2023 The Authors. American Journal of Biological Anthropology published by Wiley Periodicals LLC.)

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 H 2 , 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/H 2 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.

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.

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.

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.

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 × 10 8 copies∙mL -1 culture to 4.07 ± 2.45 × 10 8 copies∙mL -1 culture after dechlorinating 110.69 ± 30.61 μmol of 1,2-DCA with a growth yield of 1.55 ± 0.93 × 10 8  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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (© 2023. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

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., (© 2023. The Author(s).)

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 × 10 8 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 slow‑hydrogen releasing compounds in marine environment but their fermentation rate was sufficiently high to mainly stimulate the competitors of organohalide respring bacteria for electron donors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Hexachloro-1,3-butadiene (HCBD) as one of emerging persistent organic pollutants (POPs) poses potential risk to human health and ecosystems. Organohalide-respiring bacteria (OHRB)-mediated reductive dehalogenation represents a promising strategy to remediate HCBD-contaminated sites. Nonetheless, information on the HCBD-dechlorinating OHRB and their dechlorination pathways remain unknown. In this study, both in vivo and in vitro experiments, as well as quantum chemical calculation, were employed to successfully identify and characterize the reductive dechlorination of HCBD by Dehalococcoides. Results showed that some Dehalococcoides extensively dechlorinated HCBD to (E)-1,2,3-tri-CBD via (E)-1,1,2,3,4-penta-CBD and (Z,E)-1,2,3,4-tetra-CBD in a co-metabolic way. Both qPCR and 16S rRNA gene amplicon sequencing analyses suggested that the HCBD-dechlorinating Dehalococcoides coupled their cell growth with dechlorination of perchloroethene (PCE), rather than HCBD. The in vivo and in vitro ATPase assays indicated ≥78.89% decrease in ATPase activity upon HCBD addition, which suggested HCBD inhibition on ATPase-mediated energy harvest and provided rationality on the Dehalococcoides-mediated co-metabolic dechlorination of HCBD. Interestingly, dehalogenation screening of organohalides with the HCBD-dechlorinating enrichment cultures showed that debromination of bromodichloromethane (BDCM) was active in the in vitro RDase assays but non-active in the in vivo experiments. Further in vitro assays of hydrogenase activity suggested that significant inhibition of BDCM on the hydrogenase activity could block electron derivation from H 2 for consequent reduction of organohalides in the in vivo experiments. Therefore, our results provided unprecedented insight into metabolic, co-metabolic and RDase-active-only dehalogenation of varied organohalides by specific OHRB, which could guide future screening of OHRB for remediation of sites contaminated by HCBD and other POPs., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

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., (Published by Oxford University Press on behalf of FEMS 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Alkaline hot springs in Yellowstone National Park (YNP) provide a framework to study the relationship between photoautotrophs and temperature. Previous work has focused on studying how cyanobacteria (oxygenic phototrophs) vary with temperature, sulfide, and pH, but many questions remain regarding the ecophysiology of anoxygenic photosynthesis due to the taxonomic and metabolic diversity of these taxa. To this end, we examined the distribution of genes involved in phototrophy, carbon fixation, and nitrogen fixation in eight alkaline (pH 7.3-9.4) hot spring sites near the upper temperature limit of photosynthesis (71ºC) in YNP using metagenome sequencing. Based on genes encoding key reaction center proteins, geographic isolation plays a larger role than temperature in selecting for distinct phototrophic Chloroflexi, while genes typically associated with autotrophy in anoxygenic phototrophs, did not have distinct distributions with temperature. Additionally, we recovered Calvin cycle gene variants associated with Chloroflexi, an alternative carbon fixation pathway in anoxygenic photoautotrophs. Lastly, we recovered several abundant nitrogen fixation gene sequences associated with Roseiflexus, providing further evidence that genes involved in nitrogen fixation in Chloroflexi are more common than previously assumed. Together, our results add to the body of work on the distribution and functional potential of phototrophic bacteria in Yellowstone National Park hot springs and support the hypothesis that a combination of abiotic and biotic factors impact the distribution of phototrophic bacteria in hot springs. Future studies of isolates and metagenome assembled genomes (MAGs) from these data and others will further our understanding of the ecology and evolution of hot spring anoxygenic phototrophs. IMPORTANCE Photosynthetic bacteria in hot springs are of great importance to both microbial evolution and ecology. While a large body of work has focused on oxygenic photosynthesis in cyanobacteria in Mushroom and Octopus Springs in Yellowstone National Park, many questions remain regarding the metabolic potential and ecology of hot spring anoxygenic phototrophs. Anoxygenic phototrophs are metabolically and taxonomically diverse, and further investigations into their physiology will lead to a deeper understanding of microbial evolution and ecology of these taxa. Here, we have quantified the distribution of key genes involved in carbon and nitrogen metabolism in both oxygenic and anoxygenic phototrophs. Our results suggest that temperature >68ºC selects for distinct groups of cyanobacteria and that carbon fixation pathways associated with these taxa are likely subject to the same selective pressure. Additionally, our data suggest that phototrophic Chloroflexi genes and carbon fixation genes are largely influenced by local conditions as evidenced by our gene variant analysis. Lastly, we recovered several genes associated with potentially novel phototrophic Chloroflexi. Together, our results add to the body of work on hot springs in Yellowstone National Park and set the stage for future work on metagenome assembled genomes.

Chloroflexi members are ubiquitous and have been extensively studied; however, the evolution and metabolic pathways of Chloroflexi members have long been debated. In the present study, the evolution and the metabolic potentials of 17 newly obtained Chloroflexi metagenome-assembled genomes (MAGs) were evaluated using genome and horizontal gene transfer (HGT) analysis. Taxonomic analysis suggests that the MAGs of the present study might be novel. One MAG encodes genes for anoxygenic phototrophy. The HGT analysis suggest that genes responsible for anoxygenic phototrophy in the MAG might have been transferred from Proteobacteria/Chlorobi. The evolution of anaerobic photosynthesis, which has long been questioned, has now been shown to be the result of HGT events. An incomplete Wood-Ljungdahl pathway (with missing genes metF, acsE, fdh, and acsA) was reported in Dehalococcoidetes members. In the present study, MAGs that were not the Dehalococcoidetes members encode genes acsA, acsB, metF and acsE. The genes responsible for sulfate reduction (sat, cysC and sir), dissimilatory sulfite reductase (dsrA and dsrB), and aerobic and anaerobic carbon monoxide oxidation (coxSML and cooSF) were detected in the present study MAGs. The present study expands our knowledge of the possible metabolic potentials of the phylum Chloroflexi and clarifies the evolution of anaerobic photosynthesis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The deep sea harbors the majority of the microbial biomass in the ocean and is a key site for organic matter (OM) remineralization and storage in the biosphere. Microbial metabolism in the deep ocean is greatly controlled by the generally depleted but periodically fluctuating supply of OM. Currently, little is known about metabolic potentials of dominant deep-sea microbes to cope with the variable OM inputs, especially for those living in the hadal trenches-the deepest part of the ocean., Results: In this study, we report the first extensive examination of the metabolic potentials of hadal sediment Chloroflexi, a dominant phylum in hadal trenches and the global deep ocean. In total, 62 metagenome-assembled-genomes (MAGs) were reconstructed from nine metagenomic datasets derived from sediments of the Mariana Trench. These MAGs represent six novel species, four novel genera, one novel family, and one novel order within the classes Anaerolineae and Dehalococcoidia. Fragment recruitment showed that these MAGs are globally distributed in deep-sea waters and surface sediments, and transcriptomic analysis indicated their in situ activities. Metabolic reconstruction showed that hadal Chloroflexi mainly had a heterotrophic lifestyle, with the potential to degrade a wide range of organic carbon, sulfur, and halogenated compounds. Our results revealed for the first time that hadal Chloroflexi harbor pathways for the complete hydrolytic or oxidative degradation of various recalcitrant OM, including aromatic compounds (e.g., benzoate), polyaromatic hydrocarbons (e.g., fluorene), polychlorobiphenyl (e.g., 4-chlorobiphenyl), and organochlorine compounds (e.g., chloroalkanes, chlorocyclohexane). Moreover, these organisms showed the potential to synthesize energy storage compounds (e.g., trehalose) and had regulatory modules to respond to changes in nutrient conditions. These metabolic traits suggest that Chloroflexi may follow a "feast-or-famine" metabolic strategy, i.e., preferentially consume labile OM and store the energy intracellularly under OM-rich conditions, and utilize the stored energy or degrade recalcitrant OM for survival under OM-limited condition., Conclusion: This study expands the current knowledge on metabolic strategies in deep-ocean Chlorolfexi and highlights their significance in deep-sea carbon, sulfur, and halogen cycles. The metabolic plasticity likely provides Chloroflexi with advantages for survival under variable and heterogenic OM inputs in the deep ocean. Video Abstract., (© 2022. The Author(s).)

Thermomicrobium roseum DSM 5159 lipase (TrLip) is an enzyme with marked thermostability and excellent solvent resistance. However, TrLip reveals relatively high catalytic efficiency on short-chain substrates but poor activity against mid-long or long-chain fatty acids, which would limit its industrial application. In this study, ancestral sequence reconstruction (ASR), a common engineering tool for the evolutionary history of protein families, was employed to identify the natural evolutionary trends within 5 Å around the catalytic center. Two mutation libraries were constructed, one for the catalytic center and the other for the pocket flexibility. A total of 69 mutants were expressed and purified in the Escherichia coli expression system to determine the kinetic parameters, and W219G could significantly enhance the catalytic efficiency against substrates with 12-, 16- and 18-carbon side chains. In addition, the double mutant W219G/F265M could further catalyze the breakdown of the above three substrates up to 6.34-, 4.21- and 4.86-folds compared to the wild-type TrLip, while the initial pH and thermostability were maintained. Through bioinformatics analysis, the significantly enhanced catalytic efficiency against longer-side chain substrates should be associated with the reduction of steric hindrance. With the outstanding stability and the promoted activity, TrLip should be of great potential in chemical and food industry., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This study presents the isolation of a novel strain of Dehalococcoides mccartyi, NIT01, which can completely dechlorinate up to 4.0 mM of trichloroethene to ethene via 1,2-cis-dichroroethene and vinyl chloride within 25 days. Strain NIT01 dechlorinated chloroethenes (CEs) at a temperature range of 25-32 °C and pH range of 6.5-7.8. The activity of the strain was inhibited by salt at more than 1.3% and inactivated by 1 h exposure to 2.0% air or 0.5 ppm hypochlorous acid. The genome of NIT01 was highly similar to that of the Dehalococcoides strains DCMB5, GT, 11a5, CBDB1, and CG5, and all included identical 16S rRNA genes. Moreover, NIT01 had 19 rdhA genes including NIT01-rdhA7 and rdhA13, which are almost identical to vcrA and pceA that encode known dehalogenases for tetrachloroethene and vinyl chloride, respectively. We also extracted RdhAs from the membrane fraction of NIT01 using 0.5% n-dodecyl-β-d-maltoside and separated them by anion exchange chromatography to identify those involved in CE dechlorination. LC/MS identification of the LDS-PAGE bands and RdhA activities in the fractions indicated cellular expression of six RdhAs. NIT01-RdhA7 (VcrA) and NIT01-RdhA15 were highly detected and NIT01-RdhA6 was the third-most detected. Among these three RdhAs, NIT01-RdhA15 and NIT01-RdhA6 had no biochemically identified relatives and were suggested to be novel functional dehalogenases for CEs. The expression of multiple dehalogenases may support bacterial tolerance to high concentrations of CEs., (Copyright © 2021. Published by Elsevier Inc.)

" Candidatus Thermofonsia" represents a novel class within the phylum Chloroflexi . Metagenomic analysis reveals " Ca. Thermofonsia" harbors phototrophs outside the classically phototrophic Chloroflexia class. Unfortunately, the paucity of pure cultures limits further insights into their potential phototrophy. Here, we report the successful isolation of a " Ca. Thermofonsia" representative ( Phototrophicus methaneseepsis ZRK33) from a deep-sea cold seep. Using combined physiological, genomic, and transcriptomic methods, we further show the long-wavelength light (e.g., red and infrared light) could promote the growth of strain ZRK33 and upregulate the expression of genes associated with phototrophy. In particular, strain ZRK33 has a typical phototrophic lifestyle under both laboratory and deep-sea conditions. Strain ZRK33 also possesses the ability to fix inorganic carbon through the 3-hydroxypropionate bicycle in both laboratory and deep-sea in situ environments, and the combined autotrophic, phototrophic, and heterotrophic capabilities endow strain ZRK33 with a photomixotrophic lifestyle. Notably, the predicted genes associated with phototrophy broadly exist in the metagenomes of 27 deep-sea Chloroflexi members, strongly suggesting diverse phototrophic Chloroflexi members are distributed in various unexplored deep biospheres. IMPORTANCE The deep ocean microbiota represents the unexplored majority of global ocean waters. The phylum Chloroflexi is abundant and broadly distributed in various deep-sea ecosystems. It was reported that some members of " Candidatus Thermofonsia" clade 2 might possess phototrophs; however, the absence of cultured representatives is a significant bottleneck toward understanding their phototrophic characteristics. In the present study, we successfully isolated a representative of the novel class " Ca. Thermofonsia" from a deep-sea cold seep by using an enrichment medium constantly supplemented with rifampicin, allowing researchers to isolate more Chloroflexi members in the future. Importantly, outside the classically phototrophic Chloroflexia class, we discover a novel phototrophic clade within the phylum Chloroflexi and demonstrate the existence of phototrophic lifestyles in the deep sea. Thus, this study expands the range of phototrophic Chloroflexi and provides a good model to study the mechanism of phototrophy performed in the deep biosphere.

Polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) are notorious persistent organic pollutants. However, few organohalide-respiring bacteria that harbor reductive dehalogenases (RDases) capable of dehalogenating these pollutants have been identified. Here, we report reductive dehalogenation of penta-BDEs and PCBs by Dehalococcoides mccartyi strain MB. The PCE-pregrown cultures of strain MB debrominated 86.6 ± 7.4% penta-BDEs to di- to tetra-BDEs within 5 days. Similarly, extensive dechlorination of Aroclor1260 and Aroclor1254 was observed in the PCE-pregrown cultures of strain MB, with the average chlorine per PCB decreasing from 6.40 ± 0.02 and 5.40 ± 0.03 to 5.98 ± 0.11 and 5.19 ± 0.07 within 14 days, respectively; para -substituents were preferentially dechlorinated from PCBs. Moreover, strain MB showed distinct enantioselective dechlorination of different chiral PCB congeners. Dehalogenation activity and cell growth were maintained during the successive transfer of cultures when amended with penta-BDEs as the sole electron acceptors but not when amended with only PCBs, suggesting metabolic and co-metabolic dehalogenation of these compounds, respectively. Transcriptional analysis, proteomic profiling, and in vitro activity assays indicated that MbrA was involved in dehalogenating PCE, PCBs, and PBDEs. Interestingly, resequencing of mbrA in strain MB identified three nonsynonymous mutations within the nucleotide sequence, although the consequences of which remain unknown. The substrate versatility of MbrA enabled strain MB to dechlorinate PCBs in the presence of either penta-BDEs or PCE, suggesting that co-metabolic dehalogenation initiated by multifunctional RDases may contribute to PCB attenuation at sites contaminated with multiple organohalide pollutants.

Chlorinated ethanes, including 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA), are widespread groundwater contaminants. Enrichment cultures XR DCA and XR TCA derived from river sediment dihaloeliminated 1,2-DCA to ethene and 1,1,2-TCA to vinyl chloride (VC), respectively. The XR TCA culture subsequently converted VC to ethene via hydrogenolysis. Microbial community profiling demonstrated the enrichment of Geobacter 16S rRNA gene sequences in both the XR DCA and XR TCA cultures, and Dehalococcoides mccartyi ( Dhc ) sequences were only detected in the ethene-producing XR TCA culture. The presence of a novel Geobacter population, designated as Geobacter sp. strain IAE, was identified by the 16S rRNA gene-targeted polymerase chain reaction and Sanger sequencing. Time-resolved population dynamics attributed the dihaloelimination activity to strain IAE, which attained the growth yields of 0.93 ± 0.06 × 10 7 and 1.18 ± 0.14 × 10 7 cells per μmol Cl - released with 1,2-DCA and 1,1,2-TCA as electron acceptors, respectively. In contrast, Dhc growth only occurred during VC-to-ethene hydrogenolysis. Our findings discover a Geobacter sp. strain capable of respiring multiple chlorinated ethanes and demonstrate the involvement of a broader diversity of organohalide-respiring bacteria in the detoxification of 1,2-DCA and 1,1,2-TCA.

Complete dechlorination of trichloroethene (TCE) by Dehalococcoides mccartyi is catalyzed by reductive dehalogenases (RDases), which possess cobalamin as the crucial cofactor. However, virtually all D. mccartyi isolated thus far are corrinoid auxotrophs. The exogenous addition of commercially available cobalamin for TCE-contaminated site decontamination is costly. In this study, TCE reduction by a D. mccartyi-containing microbial consortium utilizing biosynthetic cobalamin generated by interior corrinoid-producing organisms within this microbial consortium was studied. The results confirmed that subcultures without exogenous cobalamin in the medium were apparently unaffected and were able to successively metabolize TCE to nonchlorinated ethene. The 2-bromoethanesulfonate and ampicillin resistance tests results suggested that ampicillin-sensitive bacteria rather than methanogenic archaea within this microbial consortium were responsible for biosynthesizing cobalamin. Moreover, relatively stable carbon isotopic enrichment factor (ɛ- carbon ) values of TCE were obtained regardless of whether exogenous cobalamin or selective inhibitors existed in the medium, indicating that the cobalamin biosynthesized by these organisms was absorbed and utilized by D. mccartyi for RDase synthesis and eventually participated in TCE reduction. Finally, the Illumina MiSeq sequencing analysis indicated that Desulfitobacterium and Acetobacterium in this microbial consortium were responsible for the de novo cobalamin biosynthesis to fulfill the requirements of D. mccartyi for TCE metabolism.

N-demethylases have been reported to remove the methyl groups on primary or secondary amines, which could further affect the properties and functions of biomacromolecules or chemical compounds; however, the substrate scope and the robustness of N-demethylases have not been systematically investigated. Here we report the recreation of natural evolution in key microdomains of the Thermomicrobium roseum sarcosine oxidase (TrSOX), an N-demethylase with marked stability (melting temperature over 100 °C) and enantioselectivity, for enhanced substrate scope and catalytic efficiency on -C-N- bonds. We obtained the structure of TrSOX by crystallization and X-ray diffraction (XRD) for the initial framework. The natural evolution in the nonconserved residues of key microdomains-including the catalytic loop, coenzyme pocket, substrate pocket, and entrance site-was then identified using ancestral sequence reconstruction (ASR), and the substitutions that accrued during natural evolution were recreated by site-directed mutagenesis. The single and double substitution variants catalyzed the N-demethylation of N-methyl-L-amino acids up to 1800- and 6000-fold faster than the wild type, respectively. Additionally, these single substitution variants catalyzed the terminal N-demethylation of non-amino-acid compounds and the oxidation of the main chain -C-N- bond to a -C=N- bond in the nitrogen-containing heterocycle. Notably, these variants retained the enantioselectivity and stability of the initial framework. We conclude that the variants of TrSOX are of great potential use in N-methyl enantiomer resolution, main-chain Schiff base synthesis, and alkaloid modification or degradation., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogen-releasing substrates can stimulate the reductive dechlorination of trichloroethene (TCE) mediated by organohalide-respiring bacteria (OHRB) at contaminated sites. However, how the substrate affects microbiome assembly and the accompanying influences on the growth of OHRB and reductive TCE dechlorination remains unclear. We evaluated the effects of microbial community structures and potential functions on the reductive dechlorination of TCE in three anaerobic reactors with acetate, soybean oil, or molasses as the substrate and no cobalamin or amino acid supplementation. The molasses-fed reactor exhibited superior performance and dechlorination of TCE loadings to ethene, and the oil-fed reactor exhibited a high growth rate of the key OHRB, Dehalococcoides. This finding suggests an effect of the substrate on reductive dechlorination and the growth of Dehalococcoides. The three reactors developed distinct microbial community structures and the predicted metagenomes were distinguished on the basis of vitamin and amino acid metabolisms as well as fermentation pathways. In addition to the diversified hydrogen-producing pathways, the molasses-induced microbiome exhibited high potential to synthesize the cobalamin, which may account for its high Dehalococcoides activity and thus effective dechlorination performance. The substrate dependence of microbiomes may provide insight into strategies of exogenous amino acid supplementation to benefit Dehalococcoides growth. This study adds novel insight into the interplay of hydrogen-releasing substrates and OHRB. The results may contribute to the development of tailored and cost-effective management for the reductive dechlorination of chlorinated solvents in bioremediation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported NOR subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported., (© 2021 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Core samples from bioretention cell media as well as surface stormwater sediment samples from seven urban areas were collected to assess the potential for biotransformation activity of polychlorinated biphenyls (PCBs). The presence of putative organohalide-respiring bacteria in these samples was studied. Based on extracted DNA, Dehalobacter, Dehalogenimonas and Dehalococcoides were detected. Other organohalide-respiring bacteria like Desulfitobacterium and Sulfurospirillum were not studied. Bacteria containing the genes encoding for biphenyl 2,3-dioxygenase (bphA) or 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC) were detected in 29 of the 32 samples. These genes are key factors in PCB aerobic degradation. Transcribed bacterial genes from putative organohalide-respiring bacteria as well as genes encoding for bphA and bphC were obtained from the microbial community, thus showing the potential of organohalide respiration of PCBs and aerobic PCB degradation under both aerobic and anaerobic conditions in the surface samples collected at the bioretention site. Presence and concentrations of 209 PCB congeners in the bioretention media were also assessed. The total PCB concentration ranged from 38.4 ± 2.3 ng/g at the top layer of the inlet to 11.6 ± 1.2 ng/g at 20-30 cm at 3 m from the inlet. These results provide documentation that bacteria capable of PCB transformation, including both anaerobic dechlorination and aerobic degradation, were present and active in the bioretention., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Climate change is altering the frequency and severity of drought events. Recent evidence indicates that drought may produce legacy effects on soil microbial communities. However, it is unclear whether precedent drought events lead to ecological memory formation, i.e., the capacity of past events to influence current ecosystem response trajectories. Here, we utilize a long-term field experiment in a mountain grassland in central Austria with an experimental layout comparing 10 years of recurrent drought events to a single drought event and ambient conditions. We show that recurrent droughts increase the dissimilarity of microbial communities compared to control and single drought events, and enhance soil multifunctionality during drought (calculated via measurements of potential enzymatic activities, soil nutrients, microbial biomass stoichiometry and belowground net primary productivity). Our results indicate that soil microbial community composition changes in concert with its functioning, with consequences for soil processes. The formation of ecological memory in soil under recurrent drought may enhance the resilience of ecosystem functioning against future drought events., (© 2021. The Author(s).)

A mesophilic filamentous anoxygenic phototrophic bacterium, designated M50-1, was isolated from a microbial mat of the Chukhyn Nur soda lake (northeastern Mongolia) with salinity of 5-14 g/L and pH 8.0-9.3. The organism is a strictly anaerobic phototrophic bacterium, which required sulfide for phototrophic growth. The cells formed short undulate trichomes surrounded by a thin sheath and containing gas vesicles. Motility of the trichomes was not observed. The cells contained chlorosomes. The antenna pigments were bacteriochlorophyll d and β- and γ-carotenes. Analysis of the genome assembled from the metagenome of the enrichment culture revealed all the enzymes of the 3-hydroxypropionate bi-cycle for autotrophic CO2 assimilation. The genome also contained the genes encoding a type IV sulfide:quinone oxidoreductase (sqrX). The organism had no nifHDBK genes, encoding the proteins of the nitrogenase complex responsible for dinitrogen fixation. The DNA G + C content was 58.6%. The values for in silico DNA‒DNA hybridization and average nucleotide identity between M50-1 and a closely related bacterium 'Ca. Chloroploca asiatica' B7-9 containing bacteriochlorophyll c were 53.4% and 94.0%, respectively, which corresponds to interspecies differences. Classification of the filamentous anoxygenic phototrophic bacterium M50-1 as a new 'Ca. Chloroploca' species was proposed, with the species name 'Candidatus Chloroploca mongolica' sp. nov., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Traditionally, filamentous fungi and actinomycetes are well-known cellulolytic microorganisms that have been utilized in the commercial production of cellulase enzyme cocktails for industrial-scale degradation of plant biomass. Noticeably, the Ktedonobacteria lineage (phylum Chloroflexi) with actinomycetes-like morphology was identified and exhibited diverse carbohydrate utilization or degradation abilities. In this study, we performed genome-wide profiling of carbohydrate-active enzymes (CAZymes) in the filamentous Ktedonobacteria lineage. Numerous CAZymes (153-290 CAZymes, representing 63-131 glycoside hydrolases (GHs) per genome), including complex mixtures of endo- and exo-cellulases, were predicted in 15 available Ktedonobacteria genomes. Of note, 4-28 CAZymes were predicted to be extracellular enzymes, whereas 3-29 CAZymes were appended with carbohydrate-binding modules (CBMs) that may promote their binding to insoluble carbohydrate substrates. This number far exceeded other Chloroflexi lineages and were comparable to the cellulolytic actinomycetes. Six multi-modular extracellular GHs were cloned from the thermophilic Thermosporothrix hazakensis SK20-1 T strain and heterologously expressed. The putative endo-glucanases of ThazG5-1, ThazG9, and ThazG12 exhibited strong cellulolytic activity, whereas the putative exo-glucanases ThazG6 and ThazG48 formed weak but observable halos on carboxymethyl cellulose plates, indicating their potential biotechnological application. The purified recombinant ThazG12 had near-neutral pH (optimal 6.0), high thermostability (60°C), and broad specificity against soluble and insoluble polysaccharide substrates. It also represented described a novel thermostable bacterial β-1,4-glucanase in the GH12 family. Together, this research revealed the underestimated cellulolytic potential of the Ktedonobacteria lineage and highlighted its potential biotechnological utility as a promising microbial resource for the discovery of industrially useful cellulases., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Microbial communities involving dehalogenating bacteria assist in bioremediation of areas contaminated with halocarbons. To understand molecular interactions between dehalogenating bacteria, we co-cultured Sulfurospirillum multivorans, dechlorinating tetrachloroethene (PCE) to cis-1,2-dichloroethene (cDCE), and Dehalococcoides mccartyi strains BTF08 or 195, dehalogenating PCE to ethene. The co-cultures were cultivated with lactate as electron donor. In co-cultures, the bacterial cells formed aggregates and D. mccartyi established an unusual, barrel-like morphology. An extracellular matrix surrounding bacterial cells in the aggregates enhanced cell-to-cell contact. PCE was dehalogenated to ethene at least three times faster in the co-culture. The dehalogenation was carried out via PceA of S. multivorans, and PteA (a recently described PCE dehalogenase) and VcrA of D. mccartyi BTF08, as supported by protein abundance. The co-culture was not dependent on exogenous hydrogen and acetate, suggesting a syntrophic relationship in which the obligate hydrogen consumer D. mccartyi consumes hydrogen and acetate produced by S. multivorans. The cobamide cofactor of the reductive dehalogenase-mandatory for D. mccartyi-was also produced by S. multivorans. D. mccartyi strain 195 dechlorinated cDCE in the presence of norpseudo-B 12 produced by S. multivorans, but D. mccartyi strain BTF08 depended on an exogenous lower cobamide ligand. This observation is important for bioremediation, since cofactor supply in the environment might be a limiting factor for PCE dehalogenation to ethene, described for D. mccartyi exclusively. The findings from this co-culture give new insights into aggregate formation and the physiology of D. mccartyi within a bacterial community.

One of the challenges to implementing the modeling of the biological reductive dechlorination (RD) process is the evaluation of biological parameters that represent the abundance/activity levels of the microorganisms involved in the biodegradation of chloroethenes. Here we report a combined analysis of kinetic and specific biomass parameters conducted on three dechlorinating consortia enriched on PCE, TCE and cis-1,2-DCE. In these consortia, Dehalococcoides mccartyi (Dhc) represented ≥70% of the bacterial population identified via 16S rRNA gene amplicon sequencing. Quantitative biomolecular methods were used to generate specific biomass parameters targeting either the Dhc population (16S rRNA genes or cells) or specific genes encoding RD process-involved reductive dehalogenases. The correlation factor between the abundance of active Dhc cells or tceA gene copies and maximum RD rates allowed to predict an increment of 7E+09 of active Dhc cells or 5E+09 tceA gene copies/L under controlled conditions. Diversely, the utilization of gene transcripts as biomass parameters for RD modeling did not provide reliable correlations with kinetic performances. This study provides valuable insights for further modeling of the RD process through the utilization of specific biomass parameters., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chloroethenes are common soil and groundwater pollutants. Their dechlorination is impacted by environmental factors, such as the presence of metal ions. We here investigated the effect of ferrous iron on bacterial reductive dechlorination of chloroethenes and on methanogen community. Reductive dechlorination of tetrachloroethene was assayed with a groundwater sample originally containing 6.3 × 103 copies mL-1 of Dehalococcoides 16S rRNA gene and 2 mg L-1 of iron. Supplementation with 28 mg L-1 of ferrous iron enhanced the reductive dechlorination of cis-dichloroethene (cis-DCE) and vinyl chloride in the presence of methanogens. The supplementation shortened the time required for complete dechlorination of 1 mg L-1 of tetrachloroethene to ethene and ethane from 84 to 49 d. Methanogens, such as Candidatus 'Methanogranum', Methanomethylovorans and Methanocorpusculum, were significantly more abundant in iron-supplemented cultures than in non-supplemented cultures (P < 0.01). Upon methanogen growth inhibition by 2-bromoethanesulfonate and in the absence of iron supplementation, cis-DCE was not dechlorinated. Further, iron supplementation induced 71.3% dechlorination of cis-DCE accompanied by an increase in Dehalococcoides 16S rRNA and dehalogenase vcrA gene copies but not dehalogenase tceA gene copies. These observations highlight the cooperative effect of iron and methanogens on the reductive dechlorination of chloroethenes by Dehalococcoides spp., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.)