anaerobic dehalogenation of halogenated organic compounds

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508 Max M. Häggblom et al. dehalogenating populations are distinct under different redox conditions, e.g., sulfidogenic, iron-reducing or methanogenic (Knight et al., 1999; Häggblom et al., 2000; Rhee et al., 2003; Fennell et al., 2004a). Differences in substrate specificity, differences in apparent rate of dehalogenation, and unique community profiles indicate that distinct microbial populations are responsible for reductive dehalogenation under different dominant terminal electron accepting conditions (Knight et al., 1999; Häggblom et al., 2000, 2003). A. 181 bp 215 bp DNA: 2 -BP + Sulfate master culture B. 215 bp 224 bp RNA: Phenol + Sulfate 254 bp 298 bp C. 181 bp 212 bp RNA: 2 -BP + Hydrogen D. 181 bp DNA: Cloned SSU gene 2-BP 48 Figure 1. Comparison of T-RFLP analysis of genomic DNA from master culture 16S rRNA gene (A), reverse transcribed 16S rRNA from knock-out cultures phenol + sulfate (B), 2- bromophenol + hydrogen (C) and cloned 16S rRNA gene of 2BP-48 (from Fennell et al., 2004a). We have pursued the characterization of the structure and diversity of microbial food webs that are active during dehalogenation in anaerobic estuarine and marine sediments in order to identify the dehalogenating organisms. We have applied an assay using a starved consortium (low ribosome content culture) fed a suite of selective substrates (Fennell et al., 2004a). The newly synthesized 16S rRNA was then analyzed using terminalrestriction fragment length polymorphism (T-RFLP) analysis of reverse transcribed rRNA as a means for assigning a metabolic role to the different members of the sulfidogenic 2-bromo-phenol-dehalogenating consortium. Those organisms that became active after substrate feeding were recognized as distinct peaks in the electropherogram and could be identified based on data from clonal libraries. Direct terminal restriction fragment length polymorphism fingerprinting of ribosomes in the various subcultures (fed
Anaerobic Dehalogenation of Halogenated Organic Compounds 509 different substrate combinations) indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence reported previously; Knight et al., 1999) was responsible for the dehalogenation of 2-bromophenol (Figure 1). A stable coculture was established containing predominantly 2BP-48 and a second Desulfovibrio species capable of mineralizing 2-bromophenol coupled to sulfate reduction. Strain 2BP-48 in the co-culture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2- iodophenol with lactate or formate as an electron donor. Interestingly, this strain is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate. The use of functional genes (Rhee et al., 2003) to elucidate dehalogenating microorganisms in consortia or natural environments is an alternative to the indirect method using ribosomes. Recently, several membrane bound reductive dehalogenases (RDH) have been purified and characterized (see Holliger et al., 2003). Several sets of degenerate PCR primers to amplify RDH gene fragments were designed and putative RDH genes were cloned and identified from estuarine and marine environments (Rhee et al., 2003; Ahn et al., 2003, unpublished; Figure 2). The amino acid sequences deduced from the C-terminal region of the enzymes were compared and showed high similarity. However, several of these putative RDH genes were linked to transposon-like sequences and may, in fact, be pseudogenes (Rhee et al., 2003). Thus, additional assays must be done at the mRNA level to identify expressed RDH genes in natural samples in order to monitor dehalogenating populations. We have used 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) and 1,2,3,4-tetrachlorodibenzofuran (1,2,3,4-TeCDF) as model contaminants to examine the potential for microbial reductive dechlorination of PCDD/Fs and to develop highly enriched cultures for determination of the identities of the dechlorinating organisms (Vargas et al., 2001; Fennell et al., 2004b; Ahn et al., 2005). We have characterized the PCDD/F dechlorinating capability of native dehalogenating bacteria from using a variety of electron donor and halogenated co-amendments (Ahn et al., 2005). PCDD dechlorination was observed in different freshwater, estuarine and marine sediments, suggesting the ubiquitous nature of these bacteria. 1,2,3,4-TeCDD was sequentially dehalogenated, initially in the lateral position, leading to the formation of 2- monoCDD (Figure 3.) While reductive dechlorination of PCDDs occured in sulfate-rich environments, the rates of dechlorination were slower than those under methanogenic conditions. The pathways may also be different, possibly indicating the stimulation of phylogenetically different organisms (Ahn et al., unpublished). There have been many reports that the presence of
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anaerobic dehalogenation of halogenated organic compounds

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