Anaerobic Oxidation of n-Dodecane by an Addition Reaction in a Sulfate-Reducing Bacterial Enrichment Culture

We identified trace metabolites produced during the anaerobic biodegradation of H₂₆- and D₂₆-n-dodecane by an enrichment culture that mineralizes these compounds in a sulfate-dependent fashion. The metabolites are dodecylsuccinic acids that, in the case of the perdeuterated substrate, retain all of the deuterium atoms. The deuterium retention and the gas chromatography-mass spectrometry fragmentation patterns of the derivatized metabolites suggest that they are formed by C—H or C—D addition across the double bond of fumarate. As trimethylsilyl esters, two nearly coeluting metabolites of equal abundance with nearly identical mass spectra were detected from each of H₂₆- and D₂₆-dodecane, but as methyl esters, only a single metabolite peak was detected for each parent substrate. An authentic standard of protonated n-dodecylsuccinic acid that was synthesized and derivatized by the two methods had the same fragmentation patterns as the metabolites of H₂₆-dodecane. However, the standard gave only a single peak for each ester type and gas chromatographic retention times different from those of the derivatized metabolites. This suggests that the succinyl moiety in the dodecylsuccinic acid metabolites is attached not at the terminal methyl group of the alkane but at a subterminal position. The detection of two equally abundant trimethylsilyl-esterified metabolites in culture extracts suggests that the analysis is resolving diastereomers which have the succinyl moiety located at the same subterminal carbon in two different absolute configurations. Alternatively, there may be more than one methylene group in the alkane that undergoes the proposed fumarate addition reaction, giving at least two structural isomers in equal amounts.     

Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-¹³C]naphthalene or deuterated D₈-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [¹³C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, ¹³C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.     

Anaerobic Degradation of p-Xylene by a Sulfate-Reducing Enrichment Culture

A strictly anaerobic enrichment culture was obtained with p-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. p-Xylene was completely oxidized to CO₂. The enrichment culture depended on Fe(II) in the medium as a scavenger of the produced sulfide. 4-Methylbenzylsuccinic acid and 4-methylphenylitaconic acid were identified in supernatants of cultures indicating that degradation of p-xylene was initiated by fumarate addition to one of the methyl groups. Therefore, p-xylene degradation probably proceeds analogously to toluene degradation by Thauera aromatica or anaerobic degradation pathways for o- and m-xylene.     

Anaerobic Degradation of 2-Methylnaphthalene by a Sulfate-Reducing Enrichment Culture

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 μM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 ± 0.003 nmol min⁻¹ mg of protein⁻¹ only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.     

Polysulfides as Intermediates in the Oxidation of Sulfide to Sulfate by Beggiatoa spp.

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S⁰ under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S₈) and inorganic polysulfides (S_n²⁻). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that S_n²⁻ species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS⁻ as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S⁰ deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.     

Anaerobic sulfide oxidation with nitrate by a freshwater Beggiatoa enrichment culture

A lithotrophic freshwater Beggiatoa strain was enriched in O2-H2S gradient tubes to investigate its ability to oxidize sulfide with NO3- as an alternative electron acceptor. The gradient tubes contained different NO3- concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O2, H2S, pH, and NO3- were determined with microsensors. The more NO3- that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO3- to oxidize sulfide at depths below the depth that O2 penetrated. In the presence of NO3- Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO3- was added. These thick mats spatially separated O2 and sulfide but not NO3- and sulfide, and therefore NO3- must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O2 flux and a twofold-higher sulfide flux into the NO3- -exposed mats compared to the fluxes for controls without NO3-. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S0 with NO3- as the electron acceptor.     

Anaerobic Oxidation of Acetylene by Estuarine Sediments and Enrichment Cultures

Acetylene disappeared from the gas phase of anaerobically incubated estuarine sediment slurries, and loss was accompanied by increased levels of carbon dioxide. Acetylene loss was inhibited by chloramphenicol, air, and autoclaving. Addition of ¹⁴C₂H₂ to slurries resulted in the formation of ¹⁴CO₂ and the transient appearance of ¹⁴C-soluble intermediates, of which acetate was a major component. Acetylene oxidation stimulated sulfate reduction; however, sulfate reduction was not required for the loss of C₂H₂ to occur. Enrichment cultures were obtained which grew anaerobically at the expense of C₂H₂.     

同步厌氧生物脱氮除硫工艺性能的研究

研究了同步厌氧生物脱氮除硫工艺的性能。该工艺具有很高的硫化物和硝酸盐转化潜能,稳态运行时的容积硫化物去除率和容积硝酸盐去除率分别为3.73kg/(m3.d)和0.80kg/(m3.d);能够耐受580mg/L的硫化物浓度和110mg/L的硝酸盐浓度,适宜浓度分别为280mg/L和67.5mg/L;能够耐受较高的水力负荷,适宜的水力停留时间为0.13d,反应器运行性能会因缩短水力停留时间而突发性恶化。     

Evidence for Anaerobic CH 4 Oxidation in Freshwater Peatlands

This study involved in vitro assays of peat soil to investigate the occurrence, importance and potential mechanism(s) of anaerobic methane oxidation (AOM) in several northern peatlands ranging from ombrotrophic bog to minerotrophic fen. Although strong evidence suggests that AOM is linked to sulfate reduction in marine sediments, very little is known about AOM in freshwater systems such as northern peatlands, which have large methane (CH ₄ ) production and are a significant source of atmospheric CH ₄ . Our results showed a mean net AOM rate of 17 ± 2.6 nmol kg ^{? 1} s ^{? 1} with a maximum rate of 176 nmol kg ^{? 1} s ^{? 1} for a minerotrophic fen in central New York. AOM was demonstrated with three independent methods to verify our results: (a) additions of methanogenic inhibitors, (b) stable isotope enrichment ( ¹³ C-CH ₄ ), and (c) natural abundance stable isotope analysis ( ¹³ C-CH ₄ ). These experiments confirmed that AOM occurs simultaneously with methanogenesis, consumes a significant portion of gross CH ₄ production, and significantly fractionates C isotopes (～ ?127‰). Experiments using a variety of potential electron acceptors demonstrated that Fe(III) and SO₄ ^{2 ?} are not quantitatively important, while the role of NO ₃ ^? is uncertain and deserves more attention. The exact mechanism(s) for AOM in peat soils remains unclear; however the AOM rates reported in this study are similar to those reported for CH ₄ production and aerobic CH ₄ oxidation in northern peatlands, suggesting that AOM may be an important control on CH ₄ fluxes in northern peatland ecosystems.     

Anaerobic Degradation of Hexadecan-2-one by a Microbial Enrichment Culture under Sulfate-Reducing Conditions

A microbial enrichment culture from marine sediment was able to grow on hexadecan-2-one as the sole source of carbon and energy under sulfate-reducing conditions. Oxidation of the ketone involved carboxylation reactions and was coupled to sulfide production. This enrichment culture also grew on 6,10,14-trimethylpentadecan-2-one.     

Anaerobic Cometabolic Conversion of Benzothiophene by a Sulfate-Reducing Enrichment Culture and in a Tar-Oil-Contaminated Aquifer

Anaerobic cometabolic conversion of benzothiophene was studied with a sulfate-reducing enrichment culture growing with naphthalene as the sole source of carbon and energy. The sulfate-reducing bacteria were not able to grow with benzothiophene as the primary substrate. Metabolite analysis was performed with culture supernatants obtained by cometabolization experiments and revealed the formation of three isomeric carboxybenzothiophenes. Two isomers were identified as 2-carboxybenzothiophene and 5-carboxybenzothiophene. In some experiments, further reduced dihydrocarboxybenzothiophene was identified. No other products of benzothiophene degradation could be determined. In isotope-labeling experiments with a [¹³C]bicarbonate-buffered culture medium, carboxybenzothiophenes which were significantly enriched in the ¹³C content of the carboxyl group were formed, indicating the addition of a C₁ unit from bicarbonate to benzothiophene as the initial activation reaction. This finding was consistent with the results of earlier studies on anaerobic naphthalene degradation with the same culture, and we therefore propose that benzothiophene was cometabolically converted by the same enzyme system. Groundwater analyses of the tar-oil-contaminated aquifer from which the naphthalene-degrading enrichment culture was isolated exhibited the same carboxybenzothiophene isomers as the culture supernatants. In addition, the benzothiophene degradation products, in particular, dihydrocarboxybenzothiophene, were significantly enriched in the contaminated groundwater to concentrations almost the same as those of the parent compound, benzothiophene. The identification of identical metabolites of benzothiophene conversion in the sulfate-reducing enrichment culture and in the contaminated aquifer indicated that the same enzymatic reactions were responsible for the conversion of benzothiophene in situ.     

Anaerobic versus Aerobic Degradation of Dimethyl Sulfide and Methanethiol in Anoxic Freshwater Sediments

Degradation of dimethyl sulfide and methanethiol in slurries prepared from sediments of minerotrophic peatland ditches were studied under various conditions. Maximal aerobic dimethyl sulfide-degrading capacities (4.95 nmol per ml of sediment slurry · h⁻¹), measured in bottles shaken under an air atmosphere, were 10-fold higher than the maximal anaerobic degrading capacities determined from bottles shaken under N₂ or H₂ atmosphere (0.37 and 0.32 nmol per ml of sediment slurry · h⁻¹, respectively). Incubations under experimental conditions which mimic the in situ conditions (i.e., not shaken and with an air headspace), however, revealed that aerobic degradation of dimethyl sulfide and methanethiol in freshwater sediments is low due to oxygen limitation. Inhibition studies with bromoethanesulfonic acid and sodium tungstate demonstrated that the degradation of dimethyl sulfide and methanethiol in these incubations originated mainly from methanogenic activity. Prolonged incubation under a H₂ atmosphere resulted in lower dimethyl sulfide degradation rates. Kinetic analysis of the data resulted in apparent K_m values (6 to 8 μM) for aerobic dimethyl sulfide degradation which are comparable to those reported for Thiobacillus spp., Hyphomicrobium spp., and other methylotrophs. Apparent K_m values determined for anaerobic degradation of dimethyl sulfide (3 to 8 μM) were of the same order of magnitude. The low apparent K_m values obtained explain the low dimethyl sulfide and methanethiol concentrations in freshwater sediments that we reported previously. Our observations point to methanogenesis as the major mechanism of dimethyl sulfide and methanethiol consumption in freshwater sediments.     

Characterization Studies of an Anaerobic, Pentachlorophenol-Dechlorinating Enrichment Culture

Dechlorination studies were conducted using microbial cultures developed in a fluidized-bed reactor (FBR) that dechlorinates pentachlorophenol (PCP) to 3,4-dichlorophenol (3,4-DCP) and 4-monochlorophenol (4-MCP). Electron donor experiments demonstrated that lactate, propionate, and H₂ can serve as electron donors for chlorophenol (CP) dechlorination in mixed, anaerobic, PCP-enriched cultures. Dechlorination did not proceed in the absence of an electron donor. Acetate, which resulted in little H₂ production, was a poor electron donor. The results of inhibition studies using vancomycin and 2-bromoethanesulfonic acid implicate members of the domain bacteria in the dechlorination of CPs, whereas methanogens do not appear to be involved in dechlorination. Brief heat treatment (80°C for 90 min) of the FBR enrichment cultures implicated endospore formers in the dechlorination of CPs, primarily at the ortho position, where PCP was dechlorinated to 3,4,5-trichlorophenol (3,4,5-TCP) (the sole TCP detected) and subsequently to 3,4-DCP. Both lactate and H₂ served as electron donors in the heat-and oxygen-treated cultures. In contrast, a lactate-fed anaerobic spread-plate enrichment culture exhibited solely meta-dechlorination, where PCP dechlorinated solely to 2,4,6-TCP. The separation of ortho- and meta-specific dechlorination reactions provides evidence that PCP dechlorination in the FBR enrichment culture was catalyzed by at least the following two separate groups of CP-dechlorinating bacteria: one meta-dechlorinating group and one primarily ortho-dechlorinating group.     

Isolation of a Multiheme Protein with Features of a Hydrazine-Oxidizing Enzyme from an Anaerobic Ammonium-Oxidizing Enrichment Culture

A multiheme protein having hydrazine-oxidizing activity was purified from enriched culture from a reactor in which an anammox bacterium, strain KSU-1, was dominant. The enzyme has oxidizing activity toward hydrazine but not hydroxylamine and is a 130-kDa homodimer composed of a 62-kDa polypeptide containing eight hemes. It was therefore named hydrazine-oxidizing enzyme (HZO). With cytochrome c as an electron acceptor, the V_max and K_m for hydrazine are 6.2 ± 0.3 μmol/min · mg and 5.5 ± 0.6 μM, respectively. Hydrazine (25 μM) induced an increase in the proportion of reduced form in the spectrum, whereas hydroxylamine (500 μM) did not. Two genes coding for HZO, hzoA and hzoB, were identified within the metagenomic DNA from the culture. The genes encode the same amino acid sequence except for two residues. The sequences deduced from these genes showed low-level identities (<30%) to those of all of the hydroxylamine oxidoreductases reported but are highly homologous to two hao genes found by sequencing the genome of “Candidatus Kuenenia stuttgartiensis” (88% and 89% identities). The purified enzyme might therefore be a novel hydrazine-oxidizing enzyme having a critical role in anaerobic ammonium oxidation.     

Sulfide induces phosphate release from polyphosphate in cultures of a marine Beggiatoa strain

Sulfur bacteria such as Beggiatoa or Thiomargarita have a particularly high capacity for storage because of their large size. In addition to sulfur and nitrate, these bacteria also store phosphorus in the form of polyphosphate. Thiomargarita namibiensis has been shown to release phosphate from internally stored polyphosphate in pulses creating steep peaks of phosphate in the sediment and thereby inducing the precipitation of phosphorus-rich minerals. Large sulfur bacteria populate sediments at the sites of recent phosphorite formation and are found as fossils in ancient phosphorite deposits. Therefore, it can be assumed that this physiology contributes to the removal of bioavailable phosphorus from the marine system and thus is important for the global phosphorus cycle. We investigated under defined laboratory conditions which parameters stimulate the decomposition of polyphosphate and the release of phosphate in a marine Beggiatoa strain. Initially, we tested phosphate release in response to anoxia and high concentrations of acetate, because acetate is described as the relevant stimulus for phosphate release in activated sludge. To our surprise, the Beggiatoa strain did not release phosphate in response to this treatment. Instead, we could clearly show that increasing sulfide concentrations and anoxia resulted in a decomposition of polyphosphate. This physiological reaction is a yet unknown mode of bacterial polyphosphate usage and provides a new explanation for high phosphate concentrations in sulfidic marine sediments.     

Measurements of Photosynthetic Sulfide Oxidation by Chlorobium Using a Sulfide Ion Selective Electrode

A simple technique is described for using a sulfide sensitiveelectrode to measure the photooxidation of H₂S by a green sulfurbacterium, Chlorobium limicola forma thiosulfatophilum. Sulfidephotooxidation occurred only in the presence of bicarbonateat concentrations greater than 0.1 mM. This implies that therate-limiting carboxylating enzyme for CO₂ fixation in Chlorobiumhas a relatively low affinity for CO₂ compared to ribulose-1,5-biphosphatecarboxylase. Carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone(FCCP), an uncoupler of photophosphorylation, delays sulfideoxidation for about 15 sec after the onset of illumination at2 µM and is completely inhibitory at 10 µM. Theseeffects can be explained by the ATP requirement for CO₂ fixation.When the photooxidation of H₂S was prevented by 10 µMFCCP, a photoevolution of H₂S was observed. (Received December 24, 1981; Accepted September 10, 1982)     

Physiological and Molecular Characterisation of an Anaerobic Thermophilic Oleate-degrading Enrichment Culture

Thermophilic oleate-degrading bacteria were enriched in mineral medium in association withMethanobacterium thermoautotrophicum . The enrichment culture also produced methane from linear saturated fatty acids (two to 18 carbon atoms), and fermented crotonate to acetate and butyrate, with a stoichiometry similar to that reported forSyntrophomonaswolfei . Two thermophilic, anaerobic members of the domain Bacteria were isolated and phylogenetically characterised as Coprothermobacter proteolyticus and Anaerobaculum thermoterrenum. Amplified ribosomal DNA restriction analysis of the members of the domain Bacteria in the enrichment culture showed that there were three dominant restriction patterns, two of which corresponded to those of the isolated non-syntrophic organisms. The sequence of the third pattern clustered with a group of bacteria that degrade C4 and higher fatty acids in syntrophic association. However, it diverged considerably from the closest thermophilic relatives, Syntrophothermus lipocalidus and Thermosyntropha lipolytica, indicating the presence of a new thermophilic long-chain fatty acid-degrading bacterium in the enrichment culture.     

Anaerobic Biotransformation of High Concentrations of Chloroform by an Enrichment Culture and Two Bacterial Isolates

A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two Pantoea spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B₁₂ (approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO₂, and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation.Chloroform (CF) is a toxic organic compound that is frequently detected in groundwater. In the 2007 “CERCLA Priority List of Hazardous Substances,” CF ranks eleventh overall and is the third highest among chlorinated organics after vinyl chloride and polychlorinated biphenyls (4). When present at hazardous waste sites, CF is often a focal point for evaluating the feasibility of bioremediation, since it is toxic to many obligate anaerobic prokaryotes (44). For example, 1 mg/liter of CF completely inhibited dechlorination of tetrachloroethene (PCE) by a chlororespiring anaerobic isolate (32). Inhibition of reductive dechlorination of chloroethenes by CF is a general problem for sites cocontaminated with CF, which can only be overcome by first removing the CF (6).In spite of major recent advances in bioremediation of chlorinated organic compounds, treatment of CF, especially at high concentrations (e.g., >100 mg/liter), remains challenging. Although aerobic biotransformation of CF is possible (e.g., cometabolism by a butane-grown strain) (14), CF is more difficult to cometabolize than trichloroethene (42). Biotransformation of CF by mixed or pure cultures under methanogenic (5, 21) and sulfate-reducing (20) conditions has been reported, however, only at low-mg/liter CF concentrations.Corrinoids such as vitamin B₁₂ (i.e., cyanocobalamin) are effective catalysts for increasing the rate of halomethane biotransformation under anaerobic conditions. Addition of vitamin B₁₂ also shifts the pathway away from reductive dechlorination and toward hydrolytic and substitutive reactions, forming CO, CO₂, and organic acids as the major products (8, 23, 24). With low levels of B₁₂ added (3 to 5% molar ratios of CF), an enrichment culture grown on dichloromethane (DCM) as the sole substrate (8) and a lactate-grown sulfate-reducing enrichment culture (18) were able to biotransform up to 260 mg/liter and 270 mg/liter CF, respectively. However, use of a DCM-grown culture is not feasible for in situ bioremediation since DCM is also a regulated contaminant and the culture grows well only at 35°C. A major drawback with using a lactate-grown culture is the accumulation of DCM during CF transformation. Organohalide respiration of CF by a Dehalobacter population was recently reported (19), although DCM is the end product. Further treatment of the DCM would be necessary for bioremediation to be successful, although bioaugmentation cultures for this purpose are not yet available. It is apparent that a strategy for bioremediation of high concentrations of chloroform is still lacking.In a recent microcosm study using soil and groundwater from an industrial site, we demonstrated that bioaugmentation is a technically feasible remediation strategy for high concentrations of chloroform as well as carbon tetrachloride (CT) and trichlorofluoromethane (35). High rates of transformation were achieved with a fermentative enrichment culture that grows on corn syrup, supplemented with B₁₂ (1.3 to 1.4 mol%). The objectives of this study were to characterize the fermentative culture in terms of its maximum rate of CF transformation, its dependence on B₁₂ for transformation of CF, and its ability to grow on corn syrup in the presence of CF; to identify the transformation products from CF; to isolate and identify the microbes in the enrichment culture that are responsible for CF biotransformation; and to investigate the adaptation mechanisms used by the isolates to tolerate the toxicity of high concentrations of CF.     

Anaerobic Benzene Oxidation by Geobacter Species

The abundance of Geobacter species in contaminated aquifers in which benzene is anaerobically degraded has led to the suggestion that some Geobacter species might be capable of anaerobic benzene degradation, but this has never been documented. A strain of Geobacter, designated strain Ben, was isolated from sediments from the Fe(III)-reducing zone of a petroleum-contaminated aquifer in which there was significant capacity for anaerobic benzene oxidation. Strain Ben grew in a medium with benzene as the sole electron donor and Fe(III) oxide as the sole electron acceptor. Furthermore, additional evaluation of Geobacter metallireducens demonstrated that it could also grow in benzene-Fe(III) medium. In both strain Ben and G. metallireducens the stoichiometry of benzene metabolism and Fe(III) reduction was consistent with the oxidation of benzene to carbon dioxide with Fe(III) serving as the sole electron acceptor. With benzene as the electron donor, and Fe(III) oxide (strain Ben) or Fe(III) citrate (G. metallireducens) as the electron acceptor, the cell yields of strain Ben and G. metallireducens were 3.2 × 10⁹ and 8.4 × 10⁹ cells/mmol of Fe(III) reduced, respectively. Strain Ben also oxidized benzene with anthraquinone-2,6-disulfonate (AQDS) as the sole electron acceptor with cell yields of 5.9 × 10⁹ cells/mmol of AQDS reduced. Strain Ben serves as model organism for the study of anaerobic benzene metabolism in petroleum-contaminated aquifers, and G. metallireducens is the first anaerobic benzene-degrading organism that can be genetically manipulated.