Effects of activated carbon on reductive dechlorination of PCBs by organohalide respiring bacteria indigenous to sediments.

Polychlorinated biphenyls (PCBs) have accumulated in aquatic sediments due to their inherent chemical stability and their presence poses a risk due to their potential toxicity in humans and animals. Granular activated carbon (GAC) has been applied to PCB contaminated sediment sites to reduce the aqueous concentration by sequestration thus reducing the PCB exposure and toxicity to both benthic and aquatic organisms. However, it is not known how the reduction of PCB bioavailability by adsorption to GAC affects bacterial transformation of PCBs by indigenous organohalide respiring bacteria. In this study, the impact of GAC on anaerobic dechlorination by putative organohalide respiring bacteria indigenous to sediment from Baltimore Harbor was examined. It was shown that the average Cl/biphenyl after dehalogenation of Aroclor 1260 was similar between treatments with and without GAC amendment. However, GAC caused a substantial shift in the congener distribution whereby a smaller fraction of highly chlorinated congeners was more extensively dechlorinated to mono- through tri-chlorinated congeners compared to the formation of tri- through penta-chlorinated congeners in unamended sediment. The results combined with comparative sequence analysis of 16S rRNA gene sequences suggest that GAC caused a community shift to putative organohalide respiring phylotypes that coincided with more extensive dechlorination of ortho and unflanked chlorines. This shift in activity by GAC shown here for the first time has the potential to promote greater degradation in situ by promoting accumulation of less chlorinated congeners that are generally more susceptible to complete mineralization by aerobic PCB degrading bacteria.

Biocorrosion and biofilm formation in a nutrient limited heating system subjected to alternating microaerophilic conditions.

Severe biofilm formation and biocorrosion have been observed in heating systems even when the water quality complied with existing standards. The coupling between water chemistry, biofilm formation, species composition, and biocorrosion in a heating system was investigated by adding low concentrations of nutrients and oxygen under continuous and alternating dosing regimes. Molecular analysis of 16S rRNA gene fragments demonstrated that the amendments did not cause changes in the overall bacterial community composition. The combined alternating dosing of nutrients and oxygen caused increased rates of pitting (bio-) corrosion. Detection of bacteria involved in sulfide production and oxidation by retrieval of the functional dsrAB and apsA genes revealed the presence of Gram-positive sulfate- and sulfite-reducers and an unknown sulfur-oxidizer. Therefore, to control biocorrosion, sources of oxygen and nutrients must be limited, since the effect of the alternating operational conditions apparently is more important than the presence of potentially corrosive biofilm bacteria.

In situ detection of bacteria involved in cathodic depolarization and stainless steel surface corrosion using microautoradiography.

AIMS: To examine the activity of bacteria involved in cathodic depolarization and surface corrosion on stainless steel in an in situ model system. METHODS AND RESULTS: The microautoradiographic technique (MAR) was used to evaluate the activity of bacterial populations on stainless steel surfaces with a single cell resolution. Anaerobic uptake and fixation of (14)C-labelled bicarbonate occurred within corrosion sites in the absence of atmospheric hydrogen or other external electron donors, whereas it was taken up and fixed by bacteria at all other stainless steel surfaces in the presence of atmospheric hydrogen. This indicates that the bacteria utilized electrons originating from the corrosion sites due to the ongoing corrosion (cathodic depolarization). CONCLUSION: Under in situ conditions, bacteria were fixating (14)C-labelled bicarbonate at corrosion sites in the absence of atmospheric hydrogen. This indicates that electrons transferred to the bacteria provided energy for bicarbonate fixation due to cathodic depolarization. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of the MAR method showed ongoing biocorrosion in the applied in situ model system and allowed in situ examination of bacterial activity on a single cell level directly on a metal surface providing information about potential corrosion mechanisms. Furthermore, application of fluorescence in situ hybridization in combination with MAR allows for identification of the active bacteria.

Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori.

The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.

Chlorophenol production by anaerobic microorganisms: transformation of a biogenic chlorinated hydroquinone metabolite.

Chlorinated hydroquinones of biological origin are fully dechlorinated to 1,4-dihydroquinone by anaerobic bacteria such as Desulfitobacterium spp. (C. E. Milliken, G. P. Meier, J. E. M. Watts, K. R. Sowers, and H. D. May, Appl. Environ. Microbiol. 70:385-392, 2004). In the present study, mixed microbial communities from Baltimore Harbor sediment and a pure culture of Desulfitobacterium sp. strain PCE1 were discovered to demethylate, reductively dehydroxylate, and dechlorinate chlorinated hydroquinones into chlorophenols. Mixed microbial cultures from a freshwater source and several other desulfitobacteria in pure culture did not perform these reactions. Desulfitobacterium sp. strain PCE1 degraded 2,3,5,6-tetrachloro-4-methoxyphenol, a metabolite of basidiomycete fungi, to 2,3,5,6-tetrachlorophenol and 2,3,5-trichlorophenol, recalcitrant compounds that are primarily synthesized anthropogenically.

Microbial anaerobic demethylation and dechlorination of chlorinated hydroquinone metabolites synthesized by basidiomycete fungi.

The synthesis and degradation of anthropogenic and natural organohalides are the basis of a global halogen cycle. Chlorinated hydroquinone metabolites (CHMs) synthesized by basidiomycete fungi and present in wetland and forest soil are constituents of that cycle. Anaerobic dehalogenating bacteria coexist with basidiomycete fungi in soils and sediments, but little is known about the fate of these halogenated fungal compounds. In sediment microcosms, the CHMs 2,3,5,6-tetrachloro-1,4-dimethoxybenzene and 2,3,5,6-tetrachloro-4-methoxyphenol (TCMP) were anaerobically demethylated to tetrachlorohydroquinone (TCHQ). Subsequently, TCHQ was converted to trichlorohydroquinone and 2,5-dichlorohydroquinone (2,5-DCHQ) in freshwater and estuarine enrichment cultures. Screening of several dehalogenating bacteria revealed that Desulfitobacterium hafniense strains DCB2 and PCP1, Desulfitobacterium chlororespirans strain Co23, and Desulfitobacterium dehalogenans JW/DU1 sequentially dechlorinate TCMP to 2,3,5-trichloro-4-methoxyphenol and 3,5-dichloro-4-methoxyphenol (3,5-DCMP). After a lag, these strains demethylate 3,5-DCMP to 2,6-DCHQ, which is then completely dechlorinated to 1,4-dihydroquinone (HQ). 2,5-DCHQ accumulated as an intermediate during the dechlorination of TCHQ to HQ by the TCMP-degrading desulfitobacteria. HQ accumulation following TCMP or TCHQ dechlorination was transient and became undetectable after 14 days, which suggests mineralization of the fungal compounds. This is the first report on the anaerobic degradation of fungal CHMs, and it establishes a fundamental role for microbial reductive degradation of natural organochlorides in the global halogen cycle.

The absence and application of stable carbon isotopic fractionation during the reductive dechlorination of polychlorinated biphenyls.

A bacterial enrichment culture (specific to doubly flanked chlorine removal) reductively dechlorinated 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) to 2,3,5-trichlorobiphenyl (2,3,5-CB) in aqueous media. Approximately 90% conversion to 2,3,5-CB occurred after 90 days, with no other products formed. The delta13C values of 2,3,4,5-CB and 2,3,5-CB were relatively constant over the course of the reaction, indicating a very small or no isotope effect. In addition, compound-specific delta13C analysis performed for every congener in three different lots of Aroclor 1268 showed an intrinsic isotopic trend of decreasing 13C abundance with increasing chlorine content, similar to observations in other commercial mixtures of polychlorinated biphenyls (PCBs). The results of this laboratory study suggest that microbial reductive dechlorination of PCBs in contaminated sediments will create congeners with more depleted delta13C values than native PCBs of similar chlorination. Such information may provide additional evidence for the occurrence of this process and aid in further understanding the biogeochemistry of these compounds.

Identification of a microorganism that links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl.

Anaerobic bacteria reductively dechlorinate polychlorinated biphenyls (PCBs) in aquatic sediments, but these microorganisms remain uncultured and, until now, unidentified. Through denaturing gradient gel electrophoresis (DGGE) of 16S rDNA from a highly enriched ortho-PCB dechlorinating culture, the growth of a single microorganism was shown to be dependent upon the presence and dechlorination of 2,3,5,6-tetrachlorobiphenyl. This is the first identification of a microorganism that catalyses the reductive dechlorination of a PCB. The organism, bacterium o-17, has high sequence similarity with the green non-sulphur bacteria and with a group that includes Dehalococcoides ethenogenes. Bacterium o-17 required acetate for dechlorination and growth. H2:CO2 (80:20 at 101 kPa) did not support dechlorination or growth of the dechlorinator. Archaeal 16S rDNA was not detected in actively dechlorinating bromoethanesulphonate-treated non-methanogenic cultures, which indicated that methanogenic Archaea were not required for dechlorination. The consistent association with dechlorinating activity combined with high similarity to other known dechlorinating microorganisms indicates that bacterium o-17 catalyses the reductive ortho-dechlorination of 2,3,5,6-tetrachlorobiphenyl.

Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screening techniques.

The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).

Synthesis of cysteinyl-tRNA(Cys) by a genome that lacks the normal cysteine-tRNA synthetase.

Synthesis of cysteinyl-tRNA(Cys) by cysteine-tRNA synthetase is required for decoding cysteine codons in all known organisms. The genome of the archaeon Methanococcus jannaschii lacks the gene for a normal cysteine-tRNA synthetase. The activity of the enzyme, however, was identified recently, and it allowed the purification of the enzyme and cloning of its gene. Sequence analysis of the gene showed that it encodes proline-tRNA synthetase and, thus, raised the possibility of dual activities in a single aminoacyl-tRNA synthetase. Assays of aminoacyl-adenylate synthesis confirmed the ability of the enzyme to activate proline and cysteine and showed that both activities were independent of tRNA. Assays of tRNA aminoacylation established the specific attachment of proline to tRNA(Pro) and cysteine to tRNA(Cys). However, in contrast to a recent report of comparable activities with cysteine and proline, results here indicate that the adenylate synthesis and aminoacylation activities with cysteine are significantly lower than the respective activity with proline. In addition, there is evidence of overlapping amino acid-binding sites and tRNA-binding sites. These considerations, among others, raised the distinct possibility that the M. jannaschii proline-tRNA synthetase may recruit additional protein or RNA factors to facilitate the synthesis of cysteinyl-tRNA(Cys).

Cellulomonas persica sp. nov. and Cellulomonas iranensis sp. nov., mesophilic cellulose-degrading bacteria isolated from forest soils.

Two newly described species of mesophilic, cellulose-degrading, aerobic bacteria were isolated from forest humus soils along the southern border of the Caspian Sea. Cellulomonas persica and Cellulomonas iranensis are proposed as new specific epithets based on comparative sequence analyses of 16S rDNA, DNA-DNA hybridization and phenotypic characteristics. Formal species descriptions are provided.

Establishment of a polychlorinated biphenyl-dechlorinating microbial consortium, specific for doubly flanked chlorines, in a defined, sediment-free medium.

Estuarine sediment from Charleston Harbor, South Carolina, was used as inoculum for the development of an anaerobic enrichment culture that specifically dechlorinates doubly flanked chlorines (i.e., chlorines bound to carbon that are flanked on both sides by other chlorine-carbon bonds) of polychlorinated biphenyls (PCBs). Dechlorination was restricted to the para chlorine in cultures enriched with 10 mM fumarate, 50 ppm (173 microM) 2,3,4, 5-tetrachlorobiphenyl, and no sediment. Initially the rate of dechlorination decreased upon the removal of sediment from the medium. However, the dechlorinating activity was sustainable, and following sequential transfer in a defined, sediment-free estuarine medium, the activity increased to levels near that observed with sediment. The culture was nonmethanogenic, and molybdate, ampicillin, chloramphenicol, neomycin, and streptomycin inhibited dechlorination activity; bromoethanesulfonate and vancomycin did not. Addition of 17 PCB congeners indicated that the culture specifically removes double flanked chlorines, preferably in the para position, and does not attack ortho chlorines. This is the first microbial consortium shown to para or meta dechlorinate a PCB congener in a defined sediment-free medium. It is the second PCB-dechlorinating enrichment culture to be sustained in the absence of sediment, but its dechlorinating capabilities are entirely different from those of the other sediment-free PCB-dechlorinating culture, an ortho-dechlorinating consortium, and do not match any previously published Aroclor-dechlorinating patterns.

An archaeal aminoacyl-tRNA synthetase missing from genomic analysis.

The complete genomic sequencing of Methanococcus jannaschii cannot identify the gene for the cysteine-specific member of aminoacyl-tRNA synthetases. However, we show here that enzyme activity is present in the cell lysate of M. jannaschii. The demonstration of this activity suggests a direct pathway for the synthesis of cysteinyl-tRNA(Cys) during protein synthesis.

Gene transfer systems for the Archaea.

The recent focus on exobiology and the potential for life in extreme environments has generated a great deal of interest in the Archaea because of their adaptation to extremes of temperature, salinity and anaerobicity. Recent advances in the development of genetic transfer systems for the Archaea provide the first glimpse of their genetic mechanisms and have the potential to serve as powerful tools for studying their unique adaptive strategies.

Characterization of a defined 2,3,5, 6-tetrachlorobiphenyl-ortho-dechlorinating microbial community by comparative sequence analysis of genes coding for 16S rRNA.

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3, 5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the delta subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the delta, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.

Microbial dechlorination of 2,3,5,6-tetrachlorobiphenyl under anaerobic conditions in the absence of soil or sediment.

Bacterial enrichment cultures developed with Baltimore Harbor (BH) sediments were found to reductively dechlorinate 2,3,5, 6-tetrachlorobiphenyl (2,3,5,6-CB) when incubated in a minimal estuarine medium containing short-chain fatty acids under anaerobic conditions with and without the addition of sediment. Primary enrichment cultures formed both meta and ortho dechlorination products from 2,3,5,6-CB. The lag time preceding dechlorination decreased from 30 to less than 20 days as the cultures were sequentially transferred into estuarine medium containing dried, sterile BH sediment. In addition, only ortho dechlorination was observed following transfer of the cultures. Sequential transfer into medium without added sediment also resulted in the development of a strict ortho-dechlorinating culture following a lag of more than 100 days. Upon further transfer into the minimal medium without sediment, the lag time decreased to less than 50 days. At this stage all cultures, regardless of the presence of sediment, would produce 2,3,5-CB and 3,5-CB from 2,3,5,6-CB. The strict ortho-dechlorinating activity in the sediment-free cultures has remained stable for more than 1 year through several transfers. These results reveal that the classical microbial enrichment technique using a minimal medium with a single polychlorinated biphenyl (PCB) congener selected for ortho dechlorination of 2,3,5,6-CB. Furthermore, this is the first report of sustained anaerobic PCB dechlorination in the complete absence of soil or sediment.

Microbial reductive dechlorination of aroclor 1260 in anaerobic slurries of estuarine sediments.

Reductive dechlorination of Aroclor 1260 was investigated in anaerobic slurries of estuarine sediments from Baltimore Harbor (Baltimore, Md.). The sediment slurries were amended with 800 ppm Aroclor 1260 with and without the addition of 350 muM 2,3,4,5-tetrachlorobiphenyl (2,3,4,5-CB) or 2,3,5,6-tetrachlorobiphenyl (2,3,5,6-CB) and incubated in triplicate at 30 degrees C under methanogenic conditions in an artificial estuarine medium. After 6 months, extensive meta dechlorination and moderate ortho dechlorination of Aroclor 1260 occurred in all incubated cultures except for sterilized controls. Overall, total chlorines per biphenyl decreased by up to 34%. meta chlorines per biphenyl decreased by 65, 55, and 45% and ortho chlorines declined by 18, 12, and 9%, respectively, when 2,3,4,5-CB, 2,3,5,6-CB, or no additional congener was supplied. This is the first confirmed report of microbial ortho dechlorination of a commercial polychlorinated biphenyl mixture. In addition, compared with incubated cultures supplied with Aroclor 1260 alone, the dechlorination of Aroclor 1260 plus 2,3,4,5-CB or 2,3,5,6-CB occurred with shorter lag times (31 to 60 days versus 90 days) and was more extensive, indicating that the addition of a single congener stimulated the dechlorination of Aroclor 1260.

Isolation of an aceticlastic strain of Methanosarcina siciliae from marine canyon sediments and emendation of the species description for Methanosarcina siciliae.

A newly described strain of the genus Methanosarcina was isolated from submarine canyon sediments and is shown by comparative sequence analyses of 16S ribosomal DNA and the gene encoding methyl coenzyme M reductase, mcrI, to be a strain of Methanosarcina siciliae. Morphological and physiological characteristics are described. In contrast to the two previously described strains that grow exclusively on methanol, methylamines, and dimethylsulfide, M. siciliae C2J is also capable of growth on and methanogenesis from acetate. We propose that the species description for M. siciliae be amended to include aceticlastic strains.

A genetic system for Archaea of the genus Methanosarcina: liposome-mediated transformation and construction of shuttle vectors.

New methods that allow, for the first time, genetic analysis in Archaea of the genus Methanosarcina are presented. First, several autonomously replicating plasmid shuttle vectors have been constructed based on the naturally occurring plasmid pC2A from Methanosarcina acetivorans. These vectors replicate in 9 of 11 Methanosarcina strains tested and in Escherichia coli. Second, a highly efficient transformation system based upon introduction of DNA by liposomes has been developed. This method allows transformation frequencies of as high as 2 x 10(8) transformants per microgram of DNA per 10(9) cells or approximately 20% of the recipient population. During the course of this work, the complete 5467-bp DNA sequence of pC2A was determined. The implications of these findings for the future of methanoarchaeal research are also discussed.

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor.

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.