Donna E. Fennell

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

ABSTRACT Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.

PCB dechlorination enhancement in Anacostia River sediment microcosms

In situ treatment of PCB contaminated sediments via microbial dechlorination is a promising alternative to dredging, which may be reserved for only the most contaminated areas. Reductive dechlorination of low levels of weathered PCB mixtures typical of urban environments may occur at slow rates. Here, we report that biostimulation and bioaugmentation enhanced dechlorination of low concentration (2.1 mg PCBs/kg dry weight) historical PCBs in microcosms prepared with Anacostia River, Washington, DC, sediment. Treatments included electron donors butyrate, lactate, propionate and acetate (1 mM each); alternate halogenated electron acceptors (haloprimers) tetrachlorobenzene (TeCB, 25 microM), pentachloronitrobenzene (PCNB, 25 microM), or 2,3,4,5,6-PCB (PCB116, 2.0 microM); and/or bioaugmentation with a culture containing Dehalococcoides ethenogenes strain 195 (3 x 10(6)cells/mL). Dechlorination rates were enhanced in microcosms receiving bioaugmentation, PCNB and PCNB plus bioaugmentation, compared to other treatments. Microcosm subcultures generated after 415 days and spiked with PCB116 showed sustained capacity for dechlorination of PCB116 in PCNB, PCNB plus bioaugmentation, and TeCB treatments, relative to other treatments. Analysis of Chloroflexi 16S rRNA genes showed that TeCB and PCNB increased native Dehalococcoides spp. from the Pinellas subgroup; however this increase was correlated to enhanced dechlorination of low concentration weathered PCBs only in PCNB-amended microcosms. D. ethenogenes strain 195 was detected only in bioaugmented microcosms and decreased over 281 days. Bioaugmentation with D. ethenogenes strain 195 increased PCB dechlorination rates initially, but enhanced capacity for dechlorination of a model congener, PCB116, after 415 days occurred only in microcosms with enhanced native Dehalococcoides spp.

Evidence for widespread dechlorination of polychlorinated biphenyls in groundwater, landfills, and wastewater collection systems.

One of the few pathways for environmental transformation of polychlorinated biphenyls (PCBs) is microbial dechlorination under anaerobic conditions, which is reported to occur in contaminated sediments of rivers, lakes and harbors. The goal of this work was to determine whether PCB dechlorination occurs in built waste treatment environments. We analyzed a large database on PCB congener concentrations in effluents and some influents of facilities in the Delaware River Basin. Positive matrix factorization was used to identify the sources of PCBs and to look for evidence of dechlorination. Seven factors were resolved from the data set of 89 congeners in 645 samples. Two of the resolved factors represented dechlorination signals. One of these was dominated by PCBs 4 and 19 and represents an advanced stage of dechlorination of Aroclors to di- and trichlorinated congeners. This dechlorination signal was most prevalent in effluents from sites with contaminated groundwater and from wastewater treatment plants (WWTPs) that serve combined sewers or treat landfill leachate. The other dechlorination signal appeared to represent an intermediate stage of dechlorination, because it was dominated by two coeluting groups of tetrachlorinated congeners: PCBs 44 + 47 + 65 and 45 + 51. This partial dechlorination signal was most prevalent in the 40 WWTPs with separate (sanitary) sewer systems, where it often comprised more than 20% of the PCBs in the effluents. Both dechlorination signals were present in WWTP influents, but were not observed in stormwater runoff, suggesting that dechlorination occurs in sewers. This work represents the first convincing evidence of PCB dechlorination occurring outside of contaminated aquatic sediments or anaerobic digesters. The results suggest that PCBs are dechlorinated by anaerobic bacteria in sewers, landfills, and contaminated groundwater. These two dechlorination signals comprise about 19% of the total loads of PCBs to the Delaware River from the sampled dischargers.

Detection and characterization of a dehalogenating microorganism by terminal restriction fragment length polymorphism fingerprinting of 16S rRNA in a sulfidogenic, 2-bromophenol-utilizing enrichment

ABSTRACT Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.

Biostimulation and bioaugmentation to enhance dechlorination of polychlorinated dibenzo-p-dioxins in contaminated sediments

Dechlorination of spiked 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) was investigated in sediment microcosms from three polychlorinated dibenzo-p-dioxin and dibenzofuran (CDD/F)-contaminated sites: River Kymijoki, Finland; Gulf Island Pond, Maine; and Lake Roosevelt, Washington. Dechlorination was stimulated by addition of electron donor and halogenated priming compounds, and bioaugmentation by a mixed culture containing Dehalococcoides ethenogenes strain 195. Amendment with 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB) promoted rapid dechlorination of 1,2,3,4-TeCDD to 2-monochlorodibenzo-p-dioxin (2MCDD) in Gulf Island Pond and River Kymijoki sediments, however, only slow dechlorination to 1,4-dichlorodibenzo-p-dioxin was observed in Lake Roosevelt sediments. The dechlorination pathway in 1,2,3,4-TeCB-amended microcosms proceeded mainly via 1,3-dichlorodibenzo-p-dioxin, with less production of 2,3-dichlorodibenzo-p-dioxin in comparison with other treatments. Microbial community analyses indicated that Dehalococcoides-like bacteria were enriched with 1,2,3,4-TeCB. Quantitative real-time PCR analysis of Dehalococcoides-specific 16S rRNA genes and the D. ethenogenes strain 195 dehalogenase gene, tceA, showed at least an order of magnitude higher gene copy numbers in the bioaugmented than in the nonbioaugmented microcosms. An active-dechlorinating population is present in the River Kymijoki and biostimulation may enhance both native Dehalococcoides spp. and the bioaugmented D. ethenogenes strain 195.

Detection by PCR of reductive dehalogenase motifs in a sulfidogenic 2-bromophenol-degrading consortium enriched from estuarine sediment

Abstract Polymerase chain reaction (PCR) primers were developed based on three known reductive dehalogenase (RDH) genes (pceA from Dehalospirillum multivorans, cprA from Desulfitobacterium dehalogenans, tceA from Dehalococcoides ethenogenes) and used to amplify bands of the appropriate size from a microbial consortium inoculated with contaminated estuarine sediment and enriched under sulfidogenic conditions using 2-bromophenol (2-BP) as the sole carbon source. These PCR fragments were found to contain many of the conserved amino acids and motifs present in the known RDH genes. The three cloned PCR products (2bprdh61, 2bprdh63, 2bprdh81 - designated 2-bromophenol RDH) shared 21 of the 31 conserved amino acids present in the C-terminus of the RDHs in GenBank. (The N-terminus of the RDH protein shares very little homology for the known RDH genes.) All 2-BP PCR products and the known RDH genes were found to contain two iron-sulfur cluster binding domains as well as conserved PCR priming sites. Southern hybridization of genomic DNA revealed multiple bands, implying additional RDH-like motifs in the sulfidogenic 2-BP-degrading consortium. In order to gain information on upstream regions of the RDH-like motifs, DNA fragments containing 2bprdh61, 2bprdh63, and 2bprdh81 were cloned and sequenced via an inverse PCR approach. The results indicated the presence of transposase gene homologues upstream of 2-bprdh61A and 2-bprdh81A. Therefore, some RDH-like PCR products in our consortium are possibly pseudogenes and some RDH genetic diversity may be generated by transposition.

Anaerobic dehalogenation of organohalide contaminants in the marine environment.

Microbially mediated dehalogenation processes contribute to the global cycling of both biogenic and anthropogenic halogenated organic compounds. Detailed information on biodegradation mechanisms for a variety of organohalides and on the microorganisms mediating these processes has greatly increased our understanding of the cycling and fate of these unique and widespread compounds in our environment. The marine environment appears to be a particularly rich source of dehalogenating microorganisms. It is well established by laboratory and field studies that anaerobic dehalogenation of sediment contaminants, such as PCBs, pesticides, and dioxins, occurs intrinsically and can be enhanced via various methods. Specific dehalogenating bacterial populations can be enriched on various organohalides. Biodehalogenation processes are likely to be significantly affected by the prevailing terminal electron-accepting condition, and thus, biotransformation of organohalide contaminants in marine and estuarine environments will vary as a function of the redox conditions within the sediment profile. Fundamental knowledge of the activities and interactions of dehalogenating microorganisms is providing a strong basis for development of new bioremediation technologies for removal of harmful halogenated compounds from our environment.

Microcosms for Site-Specific Evaluation of Enhanced Biological Reductive Dehalogenation

Microcosm studies are currently one of the best tools available for the assessment of a site-specific biodehalogenation potential. Physiological and quantitative studies conducted in laboratory microcosms can be combined with molecular tools to determine which dechlorinating bacteria and supporting community members are present, and can also help to develop rate information for the design of field implementation. Until existing technologies for rapid, inexpensive detection of specific microbial populations and the active expression of important genes are improved, laboratory microcosm studies continue to be a vital tool for assessing bioremediation potential in the field and providing a design basis for implementation. Small-scale field tests or in situ microcosms are currently the only alternative to laboratory microcosm studies. Ultimately, for full-scale site remediation, reliable, flexible models are needed which can aid in design and management of these complex systems. The determination of relevant, site-specific parameters for input into the models is another important potential use for laboratory microcosm studies.

Source apportionment of polychlorinated biphenyls in the New York/New Jersey Harbor.

The New York/New Jersey Harbor (also known as the Hudson River Estuary) is heavily contaminated with polychlorinated biphenyls (PCBs) arising in part from inputs from the Upper Hudson River, which is a Superfund site containing historical PCB contamination, and also due to inputs from the New York City metropolitan area. The Contamination Assessment and Reduction Project (CARP) measured PCBs and other contaminants in ambient water samples collected throughout the Harbor region during 1998-2001. In order to investigate the sources of PCBs to the NY/NJ Harbor, this data base of PCB concentrations was analyzed using Positive Matrix Factorization (PMF). This analysis resolved seven factors that are thought to be associated with sources such as the Upper Hudson River, storm water runoff, combined sewer overflows (CSOs), and wastewater effluents. The PMF model also produced a factor that appears to be related to sites contaminated with Aroclor 1260. To the extent that the NY/NJ Harbor is typical of urbanized estuaries throughout the United States, these results suggest that storm water runoff is probably a significant source of PCBs to surface waters in urban areas.

The effect of co-substrate activation on indigenous and bioaugmented PCB dechlorinating bacterial communities in sediment microcosms

Microbial reductive dechlorination by members of the phylum Chloroflexi, including the genus Dehalococcoides, may play an important role in natural detoxification of highly chlorinated environmental pollutants, such as polychlorinated biphenyls (PCBs). Previously, we showed the increase of an indigenous bacterial population belonging to the Pinellas subgroup of Dehalococcoides spp. in Anacostia River sediment (Washington DC, USA) microcosms treated with halogenated co-substrates (“haloprimers”), tetrachlorobenzene (TeCB), or pentachloronitrobenzene (PCNB). The PCNB-amended microcosms exhibited enhanced dechlorination of weathered PCBs, while TeCB-amended microcosms did not. We therefore developed and used different phylogenetic approaches to discriminate the effect of the two different haloprimers. We also developed complementary approaches to monitor the effects of haloprimer treatments on 12 putative reductive dehalogenase (rdh) genes common to Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1. Our results indicate that 16S rRNA gene-based phylogenetic analyses have a limit in their ability to distinguish the effects of two haloprimer treatments and that two of rdh genes were present in high abundance when microcosms were amended with PCNB, but not TeCB. rdh gene-based phylogenetic analysis supports that these two rdh genes originated from the Pinellas subgroup of Dehalococcoides spp., which corresponds to the 16S rRNA gene-based phylogenetic analysis.