Geographic and Ecological Diversity of Green Sulfur Bacteria in Hot Spring Mat Communities.

Three strains of thermophilic green sulfur bacteria (GSB) are known; all are from microbial mats in hot springs in Rotorua, New Zealand (NZ) and belong to the species Chlorobaculum tepidum. Here, we describe diverse populations of GSB inhabiting Travel Lodge Spring (TLS) (NZ) and hot springs ranging from 36.1 °C to 51.1 °C in the Republic of the Philippines (PHL) and Yellowstone National Park (YNP), Wyoming, USA. Using targeted amplification and restriction fragment length polymorphism analysis, GSB 16S rRNA sequences were detected in mats in TLS, one PHL site, and three regions of YNP. GSB enrichments from YNP and PHL mats contained small, green, nonmotile rods possessing chlorosomes, chlorobactene, and bacteriochlorophyll c. Partial 16S rRNA gene sequences from YNP, NZ, and PHL mats and enrichments from YNP and PHL samples formed distinct phylogenetic clades, suggesting geographic isolation, and were associated with samples differing in temperature and pH, suggesting adaptations to these parameters. Sequences from enrichments and corresponding mats formed clades that were sometimes distinct, increasing the diversity detected. Sequence differences, monophyly, distribution patterns, and evolutionary simulation modeling support our discovery of at least four new putative moderately thermophilic Chlorobaculum species that grew rapidly at 40 °C to 44 °C.

Genomic Characterization of Dehalococcoides mccartyi Strain JNA That Reductively Dechlorinates Tetrachloroethene and Polychlorinated Biphenyls.

Dehalococcoides mccartyi strain JNA detoxifies highly chlorinated polychlorinated biphenyl (PCB) mixtures via 85 distinct dechlorination reactions, suggesting that it has great potential for PCB bioremediation. However, its genomic and functional gene information remain unknown due to extremely slow growth of strain JNA with PCBs. In this study, we used tetracholorethene (PCE) as an alternative electron acceptor to grow sufficient biomass of strain JNA for subsequent genome sequencing and functional gene identification. Analysis of the assembled draft genome (1 462 509 bp) revealed the presence of 29 putative reductive dehalogenase (RDase) genes. Among them, JNA_RD8 and JNA_RD11 genes were highly transcribed in both PCE- and PCB-fed cultures. Furthermore, in vitro assays with crude cell lysate from PCE grown cells revealed dechlorination activity against both PCE and 2,2',3,4,4',5,5'-heptachlorobiphenyl. These data suggest that both JNA_RD8 and JNA_RD11 may be bifunctional PCE/PCB RDases. This study deepens the knowledge of organohalide respiration of PCBs and facilitates in situ PCB-bioremediation with strain JNA.

Dehalococcoides mccartyi strain JNA dechlorinates multiple chlorinated phenols including pentachlorophenol and harbors at least 19 reductive dehalogenase homologous genes.

Pentachlorophenol and other chlorinated phenols are highly toxic ubiquitous environmental pollutants. Using gas chromatographic analysis we determined that Dehalococcoides mccartyi strain JNA in pure culture dechlorinated pentachlorophenol to 3,5-dichlorophenol (DCP) via removal of the ortho and para chlorines in all of the three possible pathways. In addition, JNA dechlorinated 2,3,4,6-tetrachlorophenol via 2,4,6-trichlorophenol (TCP) and 2,4,5-TCP to 2,4-DCP and 3,4-DCP, respectively, and dechlorinated 2,3,6-TCP to 3-chlorophenol (CP) via 2,5-DCP. JNA converted 2,3,4-TCP to 3,4-DCP and 2,4-DCP by ortho and meta dechlorination, respectively. 2,3-DCP was dechlorinated to 3-CP, and, because cultures using it could be transferred with a low inoculum (0.5 to 1.5% vol/vol), it may act as an electron acceptor to support growth. Using PCR amplification with targeted and degenerate primers followed by cloning and sequencing, we determined that JNA harbors at least 19 reductive dehalogenase homologous (rdh) genes including orthologs of pcbA4 and pcbA5, pceA, and mbrA, but not tceA or vcrA. Many of these genes are shared with D. mccartyi strains CBDB1, DCMB5, GT, and CG5. Strain JNA has previously been shown to extensively dechlorinate the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1260. Collectively the data suggest that strain JNA may be well adapted to survive in sites contaminated with chlorinated aromatics and may be useful for in situ bioremediation.

Dehalococcoides mccartyi strain JNA in pure culture extensively dechlorinates Aroclor 1260 according to polychlorinated biphenyl (PCB) dechlorination Process N.

We isolated Dehalococcoides mccartyi strain JNA from the JN mixed culture which was enriched and maintained using the highly chlorinated commercial PCB mixture Aroclor 1260 for organohalide respiration. For isolation we grew the culture in minimal liquid medium with 2,2',3,3',6,6'-hexachlorobiphenyl (236-236-CB)(20 µM) as respiratory electron acceptor. We repeatedly carried out serial dilutions to extinction and recovered dechlorination activity from transfers of 10(-7) and 10(-8) dilutions. Fluorescence microscopy, DGGE and RFLP analysis of PCR amplified16S rRNA genes, and multilocus sequence typing of three housekeeping genes confirmed culture purity. No growth occurred on complex media. JNA dechlorinated most hexa- and heptachlorobiphenyls in Aroclor 1260 (50 µg/mL) leading to losses of 51% and 20%, respectively. Dechlorination was predominantly from flanked meta positions of 34-, 234-, 235-, 236-, 245-, 2345-, 2346-, and 2356-chlorophenyl rings, as indicated by the underscores. The major products were 24-24-CB, 24-26-CB, 24-25-CB, and 25-26-CB. We identified 85 distinct PCB dechlorination reactions and 56 different PCB dechlorination pathways catalyzed by JNA. Dechlorination pathways were confirmed by mass balance of substrates and products. This dechlorination pattern matches PCB Dechlorination Process N. JNA is the first pure culture demonstrated to carry out this extensive and environmentally relevant PCB dechlorination pattern.

"Dehalococcoides" sp. strain CBDB1 extensively dechlorinates the commercial polychlorinated biphenyl mixture aroclor 1260.

"Dehalococcoides" sp. strain CBDB1 in pure culture dechlorinates a wide range of PCB congeners with three to eight chlorine substituents. Congener-specific high-resolution gas chromatography revealed that CBDB1 extensively dechlorinated both Aroclor 1248 and Aroclor 1260 after four months of incubation. For example, 16 congeners comprising 67.3% of the total PCBs in Aroclor 1260 were decreased by 64%. We confirmed the dechlorination of 43 different PCB congeners. The most prominent dechlorination products were 2,3',5-chlorinated biphenyl (25-3-CB) and 24-3-CB from Aroclor 1248 and 235-25-CB, 25-25-CB, 24-25-CB, and 235-236-CB from Aroclor 1260. Strain CBDB1 removed flanked para chlorines from 3,4-, 2,4,5-, and 3,4,5-chlorophenyl rings, primarily para chlorines from 2,3,4,5-chlorophenyl rings, primarily meta chlorines from 2,3,4- and 2,3,4,6-chlorophenyl rings, and either meta or para chlorines from 2,3,4,5,6-chlorophenyl rings. The site of attack on the 2,3,4-chorophenyl ring was heavily influenced by the chlorine configuration on the opposite ring. This dechlorination pattern matches PCB Process H dechlorination, which was previously observed in situ both in the Acushnet Estuary (New Bedford, MA) and in parts of the Hudson River (New York). Accordingly, we propose that Dehalococcoides bacteria similar to CBDB1 are potential agents of Process H PCB dechlorination in the environment. This is the first time that a complex naturally occurring PCB dechlorination pattern has been reproduced in the laboratory using a single bacterial strain.

A case study for microbial biodegradation: anaerobic bacterial reductive dechlorination of polychlorinated biphenyls-from sediment to defined medium.

The history of anaerobic microbial polychlorinated biphenyl (PCB) dechlorination is traced over 20 years using a case study of PCB dechlorination in the Housatonic River (Massachusetts) as an example. The history progresses from the characterization of the PCBs in the sediment, to cultivation in sediment microcosms, to the identification of four distinct types of PCB dechlorination, to a successful field test, to the cultivation in defined medium of the organisms responsible for extensive dechlorination of Aroclor 1260, and finally to the identification of a Dehalococcoides population that links its growth to the dechlorination of Aroclor 1260. Other PCB dechlorinators have also been identified. Two bacterial strains, o-17 and DF-1, that link their growth to the dechlorination of several PCB congeners belong to a novel clade of putative dechlorinating bacteria within the phylum Chloroflexi. Dehalococcoides ethenogenes strain 195 also dechlorinates several PCB congeners when grown on chlorinated ethenes. Evidence is mounting that Dehalococcoides and other dechlorinating Chloroflexi may play a significant role in the dechlorination of commercial PCBs in situ.

The Dehalococcoides population in sediment-free mixed cultures metabolically dechlorinates the commercial polychlorinated biphenyl mixture aroclor 1260.

Microbial reductive dechlorination of commercial polychlorinated biphenyl (PCB) mixtures (e.g., Aroclors) in aquatic sediments is crucial to achieve detoxification. Despite extensive efforts over nearly two decades, the microorganisms responsible for Aroclor dechlorination remained elusive. Here we demonstrate that anaerobic bacteria of the Dehalococcoides group derived from sediment of the Housatonic River (Lenox, MA) simultaneously dechlorinate 64 PCB congeners carrying four to nine chlorines in Aroclor 1260 in the sediment-free JN cultures. Quantitative real-time PCR showed that the Dehalococcoides cell titer in JN cultures amended with acetate and hydrogen increased from 7.07 x 10(6) +/- 0.42 x 10(6) to 1.67 x 10(8) +/- 0.04 x 10(8) cells/ml, concomitant with a 64.2% decrease of the PCBs with six or more chlorines in Aroclor 1260. No Dehalococcoides growth occurred in parallel cultures without PCBs. Aroclor 1260 dechlorination supported the growth of 9.25 x 10(8) +/- 0.04 x 10(8) Dehalococcoides cells per mumol of chlorine removed. 16S rRNA gene-targeted PCR analysis of known dechlorinators (i.e., Desulfitobacterium, Dehalobacter, Desulfuromonas, Sulfurospirillum, Anaeromyxobacter, Geobacter, and o-17/DF-1-type Chloroflexi organisms) ruled out any involvement of these bacterial groups in the dechlorination. Our results suggest that the Dehalococcoides population present in the JN cultures also catalyzes in situ dechlorination of Aroclor 1260 in the Housatonic River. The identification of Dehalococcoides organisms as catalysts of extensive Aroclor 1260 dechlorination and our ability to propagate the JN cultures under defined conditions offer opportunities to study the organisms' ecophysiology, elucidate nutritional requirements, identify reductive dehalogenase genes involved in PCB dechlorination, and design molecular tools required for bioremediation applications.

Development and characterization of stable sediment-free anaerobic bacterial enrichment cultures that dechlorinate aroclor 1260.

We have developed sediment-free anaerobic enrichment cultures that dechlorinate a broad spectrum of highly chlorinated polychlorinated biphenyls (PCBs). The cultures were developed from Aroclor 1260-contaminated sediment from the Housatonic River in Lenox, MA. Sediment slurries were primed with 2,6-dibromobiphenyl to stimulate Process N dechlorination (primarily meta dechlorination), and sediment was gradually removed by successive transfers (10%) to minimal medium. The cultures grow on pyruvate, butyrate, or acetate plus H(2). Gas chromatography-electron capture detector analysis demonstrated that the cultures extensively dechlorinate 50 to 500 mug/ml of Aroclor 1260 at 22 to 24 degrees C by Dechlorination Process N. Triplicate cultures of the eighth transfer without sediment dechlorinated 76% of the hexa- through nonachlorobiphenyls in Aroclor 1260 (250 mug/ml) to tri- through pentachlorobiphenyls in 110 days. At least 64 PCB congeners, all of which are chlorinated on both rings and 47 of which have six or more chlorines, were substrates for this dechlorination. To characterize the bacterial diversity in the enrichments, we used eubacterial primers to amplify and clone 16S rRNA genes from DNA extracted from cultures grown on acetate plus H(2). Restriction fragment length polymorphism analysis of 107 clones demonstrated the presence of Thauera-like Betaproteobacteria, Geobacter-like Deltaproteobacteria, Pseudomonas species, various Clostridiales, Bacteroidetes, Dehalococcoides of the Chloroflexi group, and unclassified Eubacteria. Our development of highly enriched, robust, stable, sediment-free cultures that extensively dechlorinate a highly chlorinated commercial PCB mixture is a major and unprecedented breakthrough in the field. It will enable intensive study of the organisms and genes responsible for a major PCB dechlorination process that occurs in the environment and could also lead to effective remediation applications.

Characterization of the PCB substrate range of microbial dechlorination process LP.

We have characterized the substrate range of Process LP, a PCB dechlorination activity mediated by anaerobic bacteria, in Housatonic River sediment (Lenox, MA). Process LP has the rare ability to remove unflanked para chlorines from polychlorinated biphenyls (PCBs). We used 2,6-difluoro-4-chlorobiphenyl (DFCB) to activate Process LP in anoxic sediment microcosms and tested its ability to dechlorinate 34 potential PCB substrates, all of which are significant components of PCB mixtures found in contaminated sediments. We used gas chromatography-mass spectrometry to monitor the dechlorination of DFCB and PCBs and the appearance of products. The preferred substrates for Process LP were PCB congeners in which the target para chlorines were flanked by meta chlorines, such as those having 3,4- and 2,4,5-chlorophenyl rings. The unflanked para chlorines on PCBs with 2,4-, 2,4,6-, and sometimes 4-chlorophenyl rings, were also substrates. Furthermore, the data revealed that Process LP can also meta-dechlorinate 2,3-, 2,3,4-, and 2,3,5-chlorophenyl groups on some congeners. A single ortho chlorine on the unattacked ring generally enhanced dechlorination activity, but the presence of 3 or 4 ortho chlorines or a 4-chlorophenyl group decreased the dechlorination efficiency. PCBs with 2,4-, 2,4,6-, 2,3-, and 2,3,5-chlorophenyl rings are often terminal dechlorination products of other microbial dechlorination activities. Since these PCBs are substrates for Process LP, this dechlorination activity works especially well in tandem with other dechlorination activities and further reduces the toxicity and persistence of PCBs. The data presented here will facilitate the construction of accurate models to interpret in situ PCB dechlorination and predict PCB fate.