MICROBIAL DEGRADATION OF ORGANIC POLLUTANTS IN ANAEROBIC ENVIRONMENTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1012785
• Project Start Date: May 1, 2017
• Project End Date: Apr 30, 2022
• Program Code: [(N/A)]- (N/A)

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Biochemistry & Microbiology

Non Technical Summary
A plethora of anthropogenic contaminants of aquatic sediments and soils pose a serious challenge for human populations. These different chemicals can enter the food chain through various routes, threaten human and environmental health and may ultimately accumulate in sediments and soils. Remediation of soils, sediments and groundwater contaminated with toxic chemicals is a continuing challenge, and the need for effective remediation, restoration and mitigation approaches is enhanced by climate change and increasing urbanization. Microbes play a key role in the ultimate fate of these contaminant through degradation and detoxification. The common theme of our research is in elucidating the "unusual appetites" of bacteria, particularly in their ability to biodegrade and/or detoxify anthropogenic, xenobiotic, chemicals. The research approach spans from answering fundamental questions on the physiology, ecology and diversity of bacteria involved in biodegradation of anthropogenic chemicals to applied research projects that address the environmental problems facing impacted industrialized sites. Our aim is to apply our understanding of microbial metabolic processes for development of bioremediation technologies for treatment of contaminated soils, sediments and groundwater. One of the fundamental challenges has been to elucidate the microorganism(s) responsible for degradation of pollutant chemicals in anaerobic environments.The overall goal of this project is to identify the microbial communities responsible for degradation of these chemicals in soils and sediments. The knowledge base will lay the foundation for understanding the fate of select anthropogenic pollutants in the environment and for the development of technologies for bioremediation of contaminated soils and sediments as well as tools for site assessment and bioremediation monitoring. Identification of the microorganisms mediating anaerobic degradation of different contaminant chemicals will provide bioindicators that allow for a more detailed assessment and monitoring of important microbial processes that can be used in combination with chemical monitoring. Demonstration of these methods will provide much needed illustrative examples to increase the use of these technologies and expand the suite of tools available for management of aquatic resources.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	4010	1100	100%

Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

biodegradation

bioremediation

organic pollutants

aquatic sediments

soil

environmental microbiology and ecology

Goals / Objectives
Pollutants including toxic, legacy industrial chemicals, such as polychlorinated biphenyls (PCBs), ubiquitous polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) continually enter the environment via anthropogenic and natural inputs, as well as current commercial manufacturing chemicals including the brominated flame retardants. Methyl tert-butyl ether (MTBE) used as a fuel oxygenate in gasoline is an intriguing groundwater pollutant and a synthetic chemical with no natural sources. As a consequence of several decades of extensive use, MTBE has become one of the most frequently detected groundwater contaminants in the United States. Most recently, pharmaceuticals and personal care products (PPCPs) have been recognized as emerging pollutants of aquatic environments and soils. PPCPs include a diverse array of thousands of chemical substances, including therapeutic drugs, fragrances, and cosmetics, which are released directly or indirectly to the environment, but their long-term fate is poorly understood.One of the fundamental challenges has been to elucidate the microorganism(s) responsible for degradation of pollutant chemicals in anaerobic environments. The overall goal of the proposed project is to identify and characterize microbial communities responsible for anaerobic degradation of diverse organic pollutants (e.g., PCCD/Fs, halogenated flame retardants, fuel oxygenates, PPCPs) and to develop technologies for stimulating in these activities for bioremediation of contaminated soils and sediments.The specific objectives of the study are to:1. Characterize, identify and quantify the activity of anaerobic dehalogenating bacteria and their dehalogenase genes/enzymes found in anoxic sediments using complementary cultivation-based and molecular methods.2. Identify and characterize novel anaerobic bacteria and their genes responsible for the degradation of MTBE in contaminated aquifers and sediments3. Determine the biodegradability and environmental fate of PPCPs and their metabolites and determine how the redox environment impacts the biodegradability of a suite of PPCP compounds in aquatic environments.These objectives will be accomplished through a suite of microcosm and enrichment culture studies using sediment and soil samples from several different contaminated locations. These studies will elucidate which bacterial communities are active in different environments and how their metabolic activities are impacted by the redox condition. Using complementary cultivation-based, molecular community analyses, and metagenomics and genomic approaches we will screen contaminant-degrading microbial communities and assess their activity at different sites and under different conditions. These studies will identify the active members of the community and through (meta)genomic analyses provide information on the key genes involved in degradation. The fundamental understanding of the degradation processes and their controls lay the foundation for development of remediation approaches and mitigation controls. Identification of the anaerobic bacteria and their genes responsible for degradation will be used for the development of tools for monitoring natural attenuation and enhancing biodegradation in the environment.Our work will examine three different pollutant classes and associated research questions:1. Anaerobic Dehalogenation of Chlorinated and Brominated Organic Compounds in Contaminated Sediments and Soils.The overall goal is to identify the microbial communities responsible for anaerobic reductive dehalogenation of organohalide compounds, including brominated flame retardants, polychlorinated dibenzo-p-dioxins, polychlorinated biphenyls and diverse pesticides. The knowledge base will lay to foundation for development of technologies for bioremediation of contaminated sediments. Enhancement of microbial dehalogenation in situ is an attractive remediation alternative that could potentially detoxify soils and sediments,2. Natural Attenuation and Enhanced Biodegradation of Methyl tert-Butyl Ether in Anoxic Aquifers.Our primary hypothesis is that long-term MTBE contamination will enrich for anaerobic microbial communities that are capable of utilizing this compound as a source of carbon and energy. By applying complementary approaches, we aim to identify the microorganisms that mediate MTBE degradation in anoxic subsurface environments, develop molecular monitoring tools, and elucidate strategies for enhancing bioremediation.3. Anaerobic Biodegradability of Pharmaceuticals and Personal Care Products in Aquatic Ecosystems.The overall research goal is to determine the biodegradability and environmental fate of pharmaceuticals and personal care products PPCPs and their metabolites. We postulate that the redox environment, and the availability of alternate electron acceptors, such as sulfate in estuarine sediments, will control the activity of microorganisms active in PPCP degradation. Our specific objectives are to determine how the redox environment impacts the biodegradability of a suite of PPCP compounds in anoxic estuarine sediments and to identify the bacterial community members active in anaerobic degradation of select PPCP compounds.

Project Methods
The project objectives will be accomplished through a suite of microcosm and enrichment culture studies using sediment and soil samples from several different contaminated locations. These studies will elucidate which bacterial communities are active in different environments and how their metabolic activities are impacted by the environmental conditions. A suite of complementary cultivation-based, molecular community analyses, and metagenomics and genomic approaches we be used to screen contaminant-degrading microbial communities and assess their activity at different sites and under different conditions.Study sites - Microcosms and Enrichment Cultures. A suite of microcosms and enrichments will be established using soils and sediment samples from several different contaminated locations, including sediments from the Hudson River, Raritan River, Hackensack River and the Meadowlands, and pristine sites such as the Jacques Cousteau Reserve. Microcosms and enrichment cultures will be established following previous protocols established in our laboratory and spiked with specific pollutant compounds and/or their mixtures.Characterization and identification of contaminant-degrading bacteria and their genes/enzymes. The microbial communities active in degradation /transformation will be characterized and key members identified using a suite of molecular approaches. Complementary molecular methods are under development for identifying and enumerating microorganisms active in contaminant degradation and for monitoring functional genes, e.g. reductive dehalogenase genes. These methods will be used to characterize and quantify active members of the community from select microcosms of interest. Identification of microorganisms that mediate degradation or transformation of the different chemicals will lay the foundation for developing bioindicators to monitor their activity in the environment.A key approach to identify active bacteria is stable isotope probing (SIP). A suite of variations to target metabolically active bacteria have been developed using isotope incorporation into macromolecules. SIP can be used to identify organisms utilizing a compound of interest for growth in environmental samples without having to obtain pure cultures. DNA-SIP is based on the incorporation of a 13C-labeled substrate into cellular nucleic acids, separation of 13C- from 12C-DNA by density gradient centrifugation, and identification of active populations using gene sequencing approaches. We have successfully applied this approach to identify bacteria involved in degradation of a variety of pollutant chemicals. We will combine the enrichment culture and SIP approaches with molecular community fingerprinting and (meta)genomic analysis to identify the microorganisms responsible for compound degradation and their other metabolic capabilities. We propose to first utilize the long read capability of the MinION to profile entire ribosomal operons from bacteria and archaea within our SIP microcosms. This will be accomplished by amplifying the target operon with domain-specific forward primers in the small ribosomal RNA subunit (16S) and domain-specific reverse primers within the large rRNA subunit (23S) using a High Fidelity Taq polymerase with proofreading capabilities. This approach will yield an amplicon containing both ribosomal subunits for phylogenetic assignment (4200 bp of sequence), plus the ITS region (500+ bp) containing species/strain information to distinguish various members within the microbial community. Isolation and characterization of novel bacteria. Culture-dependent techniques, including enrichment of consortia and isolation of pure cultures, provide a straightforward approach to identify the active microorganisms involved in contaminant degradation (at least those that can be cultured). Isolation of microbes responsible for biodegradation of the target substrate is the most direct method to identify active biodegraders. Initial microcosms will be used to establish enrichment cultures, where the specific contaminant is provided as the carbon or energy source. To promote the growth of anaerobic microorganisms, defined anaerobic media to provide nutrients and trace metals for microbial growth can be used in place of site water. To promote different redox conditions, exogenous electron acceptors such as nitrate and sulfate are amended to the microcosms/enrichment cultures. Once microcosms are established, the activity of the microbial community can be monitored by measuring the concentration of the targeted contaminants over time. Through sequential sub-culturing the degrader populations can be enriched and eventually isolated. Isolated bacteria will be characterized (taxonomy and physiology) and novel species described. The genomes of select isolates will be sequences for more detailed analysis of metabolic pathways.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Research results and new technology developments are being communicated at scientific and technical meetings and through peer-reviewed publications. New bioremediation technologies are of interest to site managers, industry, as well as state and federal regulatory agencies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students are working their Ph.D. theses as part of this project. Four undergraduate students were engaged in discovery-based learning. Three international visiting scientists/students are being trained in environmental microbiology techniques and approaches. How have the results been disseminated to communities of interest?Scientific publications and presentations. What do you plan to do during the next reporting period to accomplish the goals?We are continuing a suite of microcosms experiments to study the fate of chlorinated and brominated aromatic pollutants in anoxic sediments. We have also continuing on set of experiments to determine the biodegradability and environmental fate of pharmaceuticals and personal care products (PPCPs) and their metabolites in aquatic sediments.

Impacts
What was accomplished under these goals? Transcriptomic and proteomic response of the organohalide respiring bacterium Desulfoluna spongiiphila to growth with 2,6-dibromophenol as electron acceptor Organohalide compounds are widespread in the environment as a result of anthropogenic activities and natural production. Many organohalides are released into the environment through their use in industry and agriculture. It is now well recognized that microbial activities play important roles in a global organohalogen cycle as well as in the removal of organohalide contaminants. Organohalide respiration is a process in which microorganisms utilize halogenated compounds as electron acceptors in an anaerobic respiration for energy generation. The dehalogenated transformation products are frequently more amenable to further degradation, which may facilitate the complete removal of halogenated contaminants. Respiratory reductive dehalogenation is an important process in the overall cycling of both anthropogenic and natural organohalide compounds. Organohalide respiration is an important process in the global halogen cycle and for bioremediation. Widespread environmental contamination with organohalogen compounds and their harmful impacts to human and environmental health has been the driver for finding organisms that can degrade these compounds. Organohalide-respiring bacteria (OHRBs) of diverse phyla have been identified from various environments. Marine sponges produce a vast array of bioactive compounds as secondary metabolites, including diverse halogenated compounds that may enrich for dehalogenating bacteria. Desulfoluna spongiiphila strain AA1 was originally enriched and isolated from the marine sponge Aplysina aerophoba and can grow with both brominated compounds and sulfate as electron acceptors for respiration. An understanding of the overall gene expression and the protein production profile in response to organohalides is needed to identify the full complement of genes or enzymes involved in organohalide respiration. Elucidating the metabolic capacity of this sponge-associated bacterium lays the foundation for understanding how dehalogenating bacteria may control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. In this study, we compared the global transcriptomic and proteomic analysis of Desulfoluna spongiiphila strain AA1, an organohalide-respiring Desulfobacterota isolated from a marine sponge, with 2,6-dibromophenol or with sulfate as electron acceptor. The most significant difference of the transcriptomic analysis was the expression of one reductive dehalogenase gene cluster (rdh16), which was significantly upregulated with the addition of 2,6-dibromophenol. The corresponding protein, reductive dehalogenase RdhA16032 was detected in the proteome of the 2,6-dibromophenol treatment but not with sulfate only. There was no significant difference in corrinoid biosynthesis gene expression between the two treatments, indicating that the production of corrinoid in D. spongiiphila is constitutive or not specific for organohalide vs. sulfate respiration. Electron transporting proteins or mediators unique for reductive dehalogenation were not revealed in our analysis and we hypothesize that reductive dehalogenation may share electron transporting system with sulfate reduction. The metabolism of D. spongiiphila, predicted from transcriptomic and proteomic results, demonstrates high metabolic versatility, and provides insights into survival strategies of a marine sponge symbiont in an environment rich in organohalide compounds and other secondary metabolites.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Dam HT, Sun S, McGuinness L, Kerkhof LJ, H�ggblom MM (2019) Identification of a tetrachlorodibenzo-p-dioxin dechlorinating Dehalococcoides spp. by stable isotope probing. Environ. Sci. Technol. 53:14409-14419.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Gadkari P, McGuinness L, M�nnist� MK, Kerkhof LJ, H�ggblom MM (2020) Arctic tundra soil bacterial communities active at subzero temperatures detected by stable isotope probing. FEMS Microbiology Ecology 96:fiz192. https://doi.org/10.1093/femsec/fiz192
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Porter AW, Wolfson SJ, H�ggblom M, Young LY (2020) Microbial transformation of widely used pharmaceutical and personal care product compounds. F1000Research 9(F1000 Faculty Rev):130 doi: 10.12688/f1000research.21827.1
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Liu J, Adrian L, H�ggblom MM (2020) Transcriptomic and proteomic response of the organohalide respiring bacterium Desulfoluna spongiiphila to growth with 2,6-dibromophenol as electron acceptor. Applied and Environmental Microbiology 86:e02146-19.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Research results and new technology developments are being communicated at scientific and technical meetings and through peer-reviewed publications. New bioremediation technologies are of interest to site managers, industry, as well as state and federal regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students are working their Ph.D. theses as part of this project. 4 Undergraduate students were engaged in discovery-based learning. Three international visiting scientists/students are being trained in environmental microbiology techniques and approaches How have the results been disseminated to communities of interest?Scientific publications and presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals?We are continuing a suite of microcosms experiments to study the fate of chlorinated and brominated aromatic pollutants in anoxic sediments. We have also continuing on set of experiments to determine the biodegradability and environmental fate of pharmaceuticals and personal care products (PPCPs) and their metabolites in aquatic sediments.

Impacts
What was accomplished under these goals? Identification of a chlorodibenzo-p-dioxin dechlorinating Dehalococcoides mccartyi by stable isotope probing Polychlorinated dibenzo-p-dioxins (PCDDs) are released into the environment from a variety of both anthropogenic and natural sources. Historical deposition of PCDDs in the environment is mainly associated with large scale production, storage, utilization, and disposal of chlorinated herbicides and pesticides in which PCDDs are present as impurities. In addition, combustion of municipal solid waste has contributed to the overall input of PCDDs into the environment. PCDDs are highly hydrophobic, they bioaccumulate and biomagnify through the food chain, and can pose negative effects on human health. PCDDs are often found at substantially high concentrations in soil and sediment environments long after chemical production or municipal waste combustion. Decades after the peak of PCDD production they are still problematic and more efficient methods to remediate PCDD contamination are sought. While highly chlorinated dibenzo-p-dioxins are persistent under oxic conditions, in anoxic environments these organohalogens can be reductively dechlorinated to less chlorinated compounds that are then more amenable to subsequent aerobic degradation. Identifying the microorganisms responsible for dechlorination is an important step in developing bioremediation approaches. Stable isotope probing (SIP) analysis allows for linking microbial activity and phylogeny in complex environmental samples and has been successfully applied to identify bacteria involved in degradation of various contaminants. In this study, we demonstrated the use of a DNA-stable isotope probing (SIP) approach to identify the bacteria active in dechlorination of PCDDs in river sediments, with 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) as a model. Sediment enrichment cultures from the Hackensack River, NJ were assessed by using DNA-SIP with 13C-acetate as the carbon source to trace the activity of 1,2,3,4-TeCDD dechlorinating bacteria. In addition, pyrosequencing of reverse transcribed 16S rRNA of TeCDD dechlorinating enrichment cultures was used to reveal active members of the bacterial community not restricted only to acetate utilizing dechlorinating members. A set of operational taxonomic units (OTUs) responded positively to the addition of 1,2,3,4-TeCDD in SIP microcosms assimilating 13C-acetate as the carbon source. Analysis of bacterial community profiles of the 13C labeled heavy DNA fraction revealed that an OTU corresponding to Dehalococcoides mccartyi accounted for a significantly greater abundance in cultures amended with 1,2,3,4-TeCDD than in culture without 1,2,3,4-TeCDD. This implies the involvement of this Dehalococcoides mccartyi strain in reductive dechlorination of 1,2,3,4-TeCDD, and suggests the applicability of SIP for a robust assessment of bioremediation potential of organohalogen contaminated sites. Reductive dechlorination of hydrophobic polychlorinated pollutants, such as PCDDs, may play an important role in bioremediation of contaminated environments. The products of PCDD reductive dechlorination containing fewer chlorines are typically less toxic or nontoxic compared to PCDDs, in particular when dechlorination occurs at the 2,3,7,8-positions, and are more prone to aerobic degradation and eventually aromatic ring cleavage. Hence, reductive dechlorination is an important biological process for bioremediation of PCDD contaminated environments.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Liu J, H�ggblom MM (2018) Genome guided identification of organohalide-respiring Deltaproteobacteria from the marine environment. mBio 9:e02471-18.
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Sorokin DY, Merkel AY, H�ggblom MM (2019) Desulfurispirillum, In: Bergeys Manual of Systematics of Archaea and Bacteria. DOI: 10.1002/9781118960608.gbm01698

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Research results and new technology developments are being communicated at scientific and technical meetings and through peer-review publications. New bioremediation technologies are of interest to site managers, industry, as well as state and federal regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students are working their Ph.D. theses as part of this project. Four Undergraduate students were engaged in discovery-based learning. How have the results been disseminated to communities of interest?Scientific publications and presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals?We are continuing a suite of microcosms experiments to study the fate of chlorinated aromatic pollutants in anoxic sediments. We are continuing our studies to determine the biodegradability and environmental fate of pharmaceuticals and personal care products (PPCPs) and their metabolites in aquatic sediments.

Impacts
What was accomplished under these goals? Evaluation of Carbon Isotope Fractionation during Anaerobic Reductive Dehalogenation of Chlorinated and Brominated Benzenes Tools for evaluating and demonstrating in situ biodegradation are important for the assessment of bioremediation, in particular in the application of natural attenuation as a remediation option of contaminated groundwater plumes. Compound specific stable isotope analysis (CSIA) is a promising option for monitoring and quantification of in situ biodegradation of contaminants at polluted sites. In principle, the rate of biodegradation is reduced by the presence of the heavier isotope since in biological systems lighter isotopes are preferentially reacted. Hence, this results in the accumulation of the heavier isotope in the residual substrate (Hunkeler et al., 1999) and the lighter isotope is enriched in the product of degradation. This is referred to as compound specific stable isotope fractionation. Over the last decade CSIA has been applied to study degradation of various halogenated compounds. Most studies have focused on carbon stable isotope fractionation during the reductive dehalogenation process. Here, CSIA was used to determine microbial dehalogenation of chloro- and bromobenzenes in microcosms derived from Hackensack River sediments. Gas chromatography-isotope ratio mass spectrometry (GC-IRMS) was used to measure carbon isotope fractionation during reductive dehalogenation of hexachlorobenzene (HCB), pentachlorobenzene (PeCB), 1,2,3,5-tetrachlorobenzene (TeCB), 1,2,3,5-tetrabromobenzene (TeBB), and 1,3,5-tribromobenzene (TriBB). Strong evidence of isotope fractionation coupled to dehalogenation was not observed in the substrate, possibly due to the low solubilities of the highly halogenated benzene substrates and a dilution of the isotope signal. Nonetheless, we could measure a depletion of the δ13C value in the dichlorobenzene product during dechlorination of HCB, the sequential depletion and enrichment of δ13C value for trichlorobenzene in TeCB dechlorinating cultures, and the enrichment of δ13C during debromination of TriBB. This indicates that a measurable isotope fractionation occurred during reductive dehalogenation of highly halogenated chloro- and bromobenzenes in aquatic sediments. The limited solubilities of highly halogenated substrates may mask intrinsic isotope effects, causing low observed isotope effects. Thus, although more quantitative measurements will be needed, the data suggests that CSIA may have application for monitoring in situ microbial reductive dehalogenation of highly halogenated benzenes. Halogenated organic compounds, organohalogens, are important environmental chemicals. They have been indispensable to many industries and as a consequence of the extensive have heavily contaminated the environment. The toxic nature and environmental persistence of industrial organohalogens raised public concern. Little is known about their fate in estuarine systems, in particular under the anoxic conditions of the water column and sediment. Microbial metabolism is central in determining the ultimate fate of anthropogenic organohalides in the environment, with cleavage of the carbon-halogen bond being one of the critical steps in degradation of organohalides. Dehalogenation is important in reducing the toxicity of many organohalide pollutants and makes them more amenable to further biodegradation. Our research fills a fundamental gap in our knowledge and offers new approaches for monitoring microbial degradation processes in watersheds. Monitoring tools are key in gaining an understanding of how microbial processes, and thus remediation, are affected by different engineering approaches.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Sohn SY, Kuntze K, Nijenhuis I, H�ggblom MM (2018) Evaluation of carbon isotope fractionation during dehalogenation of chlorinated and brominated benzenes. Chemosphere 193:785-792. doi.org/10.1016/j.chemosphere.2017.11.089
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Atashgahi S, H�ggblom MM, Smidt H (2018) Organohalide respiration in pristine environments: implications for natural halogen cycle. Environmental Microbiology 20:934948. doi:10.1111/1462-2920.14016

Progress 05/01/17 to 09/30/17

Outputs
Target Audience:Research results and new technology developments are being communicated at scientific and technical meetings and through peer-review publications. New bioremediation technologies are of interest to site managers, industry, as well as state and federal regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students are working their Ph.D. theses as part of this project. Fourundergraduate students were engaged in discovery-based learning. How have the results been disseminated to communities of interest?Scientific publications and presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals?We are continuing a suite of microcosms experiments to study the fate of chlorinated aromatic pollutants in anoxic sediments. We have also initiated a new set of experiments to determine the biodegradability and environmental fate of pharmaceuticals and personal care products (PPCPs) and their metabolites in aquatic sediments.

Impacts
What was accomplished under these goals? 1. Reconstructed genomes of novel Dehalococcoides mccartyi strains from 1,2,3,4-tetrachlorodibenzo-p-dioxin-dechlorinating enrichment cultures reveal divergent reductive dehalogenase gene profiles. Polychlorinated dibenzo-p-dioxin (PCDD) contaminated sites are widespread and associated with a variety of anthropogenic sources. Even though PCDDs are persistent and toxic, they are biodegradable by certain microorganisms. PCDDs and other organohalide pollutants can serve as terminal electron acceptors for anaerobic respiration by specialized bacteria containing reductive dehalogenases (RdhA). These microorganisms, therefore, play an important role in bioremediation of PCDD contaminated sites. Reductive dechlorination in member of the genus Dehalococcoides is mediated by reductive dehalogenases as the key enzymes, and in some cases, PCDD dechlorination can support the growth of Dehalococcoides as the sole mode of energy conservation. In order to gain a better understanding of the physiology and potential activities of PCDD dechlorinating bacteria in their native sediments, we reconstructed draft genomes of two 1,2,3,4-TeCDD dechlorinating Dehalococcoides mccartyi strains from metagenomes of dehalogenating enrichment cultures established from contaminated river sediments. Two anaerobic enrichment cultures established using sediments collected from the PCDD polluted Hackensack (USA) and Kymijoki (Finland) rivers showed robust reductive dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD). In the study by Dam et al (2017) we report on the draft genome reconstructions of the two predominant Dehalococcoides strains from the metagenomes of these dehalogenating enrichment cultures. Furthermore, we gathered a complete list of reductive dehalogenases in the two predominant Dehalococcoides strains, and determined which are likely to be responsible for reductive dechlorination of PCDDs. The divergent rdhA gene profiles of the Dehalococcoides strains likely reflect their exposure to different organohalide compounds in their original habitats. Both draft genomes contained a full length rdhA gene with high sequence similarity to a reductive dehalogenase gene found in Dehalococcoides mccartyi CBDB1 known to reductively dechlorinate 1,2,3,4-tetrachlorobenzene, i.e. cbrA. This gene homologue might also be responsible for reductive dechlorination of 1,2,3,4-TeCDD in the enrichments and could be used as a biomarker to determine the potential for bioremediation of PCDD contaminated sediments. 2. Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila The marine environment is a major source of naturally occurring organohalides produced by algae, jellyfish, acorn worms and sponges. These natural sources of brominated compounds also appear to select for dehalogenating bacteria living within the host animal. Considering the extraordinary pumping capacity and abundant microbial communities of sponges, an understanding of the microbial processes that control the fate of organohalide compounds in sponges is needed in order to understand the role that these dehalogenating bacteria play within the animal and a marine organobromine cycle. Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. In the study by Liu et al (2017), we set out to identify the reductive dehalogenase genes in the sponge-associated bacterium D. spongiiphila strain AA1. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb. Three putative reductive dehalogenase (rdhA) genes involved in respiratory metabolism of organohalides were identified within the sequence. Conserved motifs found in respiratory reductive dehalogenases (a twin arginine translocation signal sequence and two iron-sulfur clusters) were present in all three putative AA1 rdhA genes. Transcription of one of the three rdhA genes was significantly upregulated during respiration of 2,6-dibromophenol and sponge extracts. Strain AA1 appears to have the ability to synthesize cobalamin, the key cofactor of most characterized reductive dehalogenase enzymes. The genome contains genes involved in cobalamin synthesis and uptake and can grow without cobalamin supplementation. Identification of this target gene associated with debromination lays the foundation for understanding how dehalogenating bacteria control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. In the sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration. Our findings represent an example of a respiratory debrominase and provide an avenue to explore the role of organohalide respiration in the marine halogen cycle.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu J, Lopez N, Ahn Y, Goldberg T, Bromberg Y, Kerkhof LJ, H�ggblom MM (2017) Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila. Environmental Microbiology Reports 9:537-549 DOI:10.1111/1758-2229.12556
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Dam HT, Vollmers J, Kaster AK, H�ggblom MM (2017) Reconstructed genomes of novel Dehalococcoides mccartyi strains from 1,2,3,4-tetrachlorodibenzo-p-dioxin-dechlorinating enrichment cultures reveal divergent reductive dehalogenase gene profiles. FEMS Microbiology Ecology 93:fix151. doi.org/10.1093/femsec/fix151