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The search for microorganisms that are capable of catalyzing the reduction of an electrode within a fuel cell has primarily been focused on bacteria that operate mesobiotically. Bacteria that function optimally under extreme conditions are beginning to be examined because they may serve as more effective catalysts (higher activity, greater stability, longer life, capable of utilizing a broader range of fuels) in microbial fuel cells. An examination of marine sediment from temperate waters (Charleston, SC) proved to be a good source of thermophilic electrode-reducing bacteria. Electric current normalized to the surface area of graphite electrodes was approximately ten times greater when sediment fuel cells were incubated at 60 degrees C (209 to 254 mA/m(2)) vs 22 degrees C (10 to 22 mA/m(2)). Electricity-generating communities were selected in sediment fuel cells and then maintained without sediment or synthetic electron-carrying mediators in single-chambered fuel cells. Current was generated when cellulose or acetate was added as a substrate to the cells. The 16S ribosomal ribonucleic acid genes from the heavy biofilms that formed on the graphite anodes of acetate-fed fuel cells were cloned and sequenced. The preponderance of the clones (54 of 80) was most related to a Gram-positive thermophile, Thermincola carboxydophila (99% similarity). The remainder of clones from the community was most related to T. carboxydophila, or uncultured Firmicutes and Deferribacteres. Overall, the data indicate that temperate aquatic sediments are a good source of thermophilic electrode-reducing bacteria.

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Desulfitobacterium hafniense strain DCB2 generates electricity in microbial fuel cells (MFCs) when humic acids or the humate analog anthraquinone-2,6-disulfonate (AQDS) is added as an electron-carrying mediator. When utilizing formate as fuel, the Gram-positive, spore-forming bacterium generated up to 400 mW/m2 of cathode surface area in a single-chamber MFC with a platinum-containing air-fed cathode. Hydrogen, lactate, pyruvate, and ethanol supported electricity generation, but acetate, propionate, and butyrate did not. Scanning electron microscopy indicated that strain DCB2 colonized the surface of a current-generating anode but not of an unconnected electrode. The electricity was recovered fully within minutes after the exchange of the medium in the anode chamber and within a week after an exposure of a colonized anode to 90 degrees C for 20 min. Of the six strains of Desulfitobacteria tested, all of which would reduce AQDS, only D. hafniense strain DCB2 continued to reduce AQDS and generate electricity for more than 24 h, indicating that reduction of the humate analog alone is insufficient to sustain electrode reduction.

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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.

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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.

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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.

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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.

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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).

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The Fusarium spp. mycotoxins fusaric acid and deoxynivalenol (DON) were tested for antimicrobial activity against Ruminococcus albus and Methanobrevibacter ruminantium. The growth of both organisms was inhibited by fusaric acid as low as 15 micrograms/mL (84 microM) but not by DON, at levels as high as 100 micrograms/mL (338 microM). No synergistic inhibitory effect was observed with DON plus fusaric acid. Neither organism was able to adapt to the fusaric acid and responses of each organism to the compound were different. The optical density (OD) maximum for R. albus, but not for M. ruminantium, was diminished after 28 days incubation at concentrations of fusaric acid below 240 micrograms/mL. Inhibition of R. albus started before significant growth had occurred, while M. ruminantium doubled twice before the onset of inhibition. Responses to picolinic acid, an analog of fusaric acid, were also dramatically different between the two microorganisms with M. ruminantium exhibiting a severe lag followed by a complete recovery of growth, while R. albus was only slightly inhibited with no lag. These results suggest that the mechanism of fusaric acid inhibition is specific to each microorganism. This is the first demonstration of the common mycotoxin fusaric acid inhibiting the growth of rumen bacteria.

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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.

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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.

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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.

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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.

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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.

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An actinomycete capable of sustained aerobic growth on 1,4-dioxane was isolated from a dioxane-contaminated sludge samples. The actinomycete, CB1190, grows on 1,4-dioxane as the sole carbon and energy source with a generation time of approximately 30 h. CB1190 degrades 1,4-dioxane at a rate of 0.33 mg of dioxane min-1 mg of protein-1 and mineralizes 59.5% of the dioxane to CO2. CB1190 also grows with other cyclic and linear ethers as the sole carbon and energy sources, including 1,3-dioxane, 2-methyl-1,3-dioxolane, tetrahydrofuran, tetrahydropyran, diethyl ether, and butyl methyl ether. CB1190 is capable of aerobic autotrophic growth on H2 and CO2.

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We have employed a method of enrichment that allows us to significantly increase the rate of reductive polychlorinated biphenyl (PCB) dechlorination. This method shortens the time required to investigate the effects that culture conditions have on dechlorination and provides an estimate of the potential activity of the PCB-dechlorinating anaerobes. The periodic supplementation of sterile sediment and PCB produced an enhanced, measurable, and sustained rate of dechlorination. We observed volumetric rates of the dechlorination of 2,3,6-trichlorobiphenyl (2,3,6-CB) to 2,6-dichlorobiphenyl (2,6-CB) of more than 300 mumol liter day when the cultures were supplemented daily. A calculation of this activity that is based on an estimate of the number of dechlorinating anaerobes present indicates that 1.13 pmol of 2,3,6-CB was dechlorinated to 2,6-CB day bacterial cell. This rate is similar to that of the reductive dechlorination of 3-chlorobenzoate by Desulfomonile tiedjei. Methanogenesis declined from 585.3 to 125.9 mumol of CH(4) liter day, while dechlorination increased from 8.2 to 346.0 mumol of 2,3,6-CB dechlorinated to 2,6-CB liter day.

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An anaerobic culture capable of dechlorinating polychlorinated biphenyls was subcultured under strict anaerobic conditions on solid media containing sterilized river sediment. The dechlorination activity was transferred as a bacterial colony on a solid medium three times. After two transfers on solid medium, the culture was no longer methanogenic but still dechlorinated a mixture of tri- and tetrachlorobiphenyls. This demonstrates that anaerobic bacteria are responsible for the polychlorinated biphenyl dechlorination and can be grown without polychlorinated biphenyl on solid media.

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The regions surrounding the three strictly conserved cysteine residues (positions 70, 95 and 153) in the beta-subunit of the Azotobacter vinelandii nitrogenase MoFe protein have been proposed to provide P-cluster environments [Dean, Setterquist, Brigle, Scott, Laird & Newton (1990) Mol. Microbiol. 4, 1505-1512]. In the present study, each of these cysteine residues was individually substituted by either serine or alanine by site-directed mutagenesis of the nifK gene, which encodes the MoFe protein beta-subunit. A mutant strain for which the codon for Cys-153 is removed was also isolated. Significant structural or functional roles are indicated for the cysteine residues at positions 70 and 95, where substitution by either serine or alanine eliminates diazotrophic growth of the resulting strains and abolishes or markedly decreases both MoFe-protein acetylene-reduction activity and the intensity of the whole-cell S = 3/2 e.p.r. signal. Changes introduced at position 153 have various effects on the functional properties of the enzyme. The strains produced either by deletion of the Cys-153 residue or its substitution by serine exhibit only a moderate decrease in diazotrophic growth and MoFe-protein activity and no loss of the whole-cell e.p.r.-signal intensity. In contrast, substitution by alanine eliminates diazotrophic growth and very markedly decreases both MoFe-protein activity and e.p.r.-signal intensity. These results are interpreted in terms of a metallocluster-driven protein rearrangement. After purification of the altered MoFe protein, in which serine replaces Cys-153, an investigation of its catalytic and spectroscopic properties confirms that neither the FeMo cofactor, i.e. the substrate-reduction site, nor the component-protein interaction site has been affected. Instead, these data indicate a disruption in electron transfer within the MoFe protein, which is consistent with a role for this residue (and region) at the P clusters.

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Two components constitute Mo-dependent nitrogenase (EC 1.18.6.1)--the Fe protein (a homodimer encoded by nifH) and the MoFe protein (an alpha 2 beta 2 tetramer encoded by nifDK). The MoFe protein provides the substrate-binding site and probably contains six prosthetic groups of two types--four Fe-S centres and two Fe- and Mo-containing cofactors. To determine the distribution and catalytic function of these metalloclusters, we and others are attempting to change the catalytic and spectroscopic features of nitrogenase by substituting specific amino acids targeted as potential metallocluster ligands, particularly those to the FeMo-cofactor, which is responsible for the biologically unique electron paramagnetic resonance signal (S = 3/2) of nitrogenase, and is believed to be the N2-reducing site. Here we describe mutant strains of Azotobacter vinelandii that have single amino-acid substitutions within the MoFe protein alpha-subunit. These substitutions alter both substrate-reduction properties and the unique electron paramagnetic resonance signal, indicating that the FeMo-cofactor is associated with both the alpha-subunit and the substrate-reducing site.

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The influence of sodium molybdate and sodium tungstate on formate dehydrogenase activity was studied in H2-CO2-grown cultures of Methanobacterium formicicum. Depletion of molybdate from the growth medium resulted in a 75-fold decrease of intracellular molybdenum and a 35-fold decrease in enzyme activity; however, growth rate and cell yields were not influenced. By using an indirect enzyme-linked immunoassay, the amount of formate dehydrogenase approximated 3% of the total protein in cells grown in the presence of molybdate. Molybdenum-starved cells contained approximately 15-fold less formate dehydrogenase protein; Western blot (immunoblot) analysis revealed that both subunits of the enzyme were synthesized. Molybdenum starvation resulted in an increase in the amount of mRNA that hybridized to fdh-specific DNA. The results indicated an inverse relationship between the amount of transcript and the amount of formate dehydrogenase protein detected in response to molybdenum starvation. The addition of 1 mM tungstate to molybdate-containing media resulted in nearly complete loss of enzyme activity and decreased the intracellular concentration of molybdenum 10-fold. Cells grown in the presence of tungstate synthesized high amounts of inactive formate dehydrogenase and contained mRNA that hybridized to fdh-specific DNA in amounts similar to that in cells grown with sufficient molybdate. Inactive formate dehydrogenase, purified from cells grown in the presence of tungstate, had the same subunit composition and contained amounts of molybdopterin cofactor, albeit metal-free, comparable to those in the active enzyme.

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The genes for the two subunits of the formate dehydrogenase from Methanobacterium formicicum were cloned and their sequences determined. When expressed in Escherichia coli, two proteins were produced which had the appropriate mobility on an SDS gel for the two subunits of formate dehydrogenase and cross-reacted with antibodies raised to purified formate dehydrogenase. The genes for the two formate dehydrogenase subunits overlap by 1 base pair and are preceded by DNA sequences similar to both eubacterial and archaebacterial promoters and ribosome-binding sites. The amino acid sequences deduced from the DNA sequence were analyzed, and the arrangement of putative iron-sulfur centers is discussed.

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