Effect of Sulfate and Organic Carbon Supplements on Reductive Dehalogenation of Chloroanilines in Anaerobic Aquifer Slurries

When di-, tri-, and tetrachloroaniline were incubated in methanogenic groundwater slurries, they were reductively dehalogenated by the aquifer microbiota. 2,3,4-Trichloroaniline was metabolized by two pathways. Primary dehalogenation occurred at either the meta or ortho position of this substrate to form 2,4- and 3,4-dichloroaniline, respectively. The latter chemical could be stoichiometrically converted to 3-chloroaniline. 2,3,4,5-Tetrachloroaniline was degraded by the sequential removal of halogens from the para and then the ortho position to form 3,5-dichloroaniline. An additional pathway was observed with this substrate when the aquifer slurries were amended with butyrate. That is, halogens could be removed from both the meta and ortho positions of tetrachloroaniline. The amendment of sulfate to methanogenic aquifer slurries slowed the rate of 2,3,4,5-tetrachloroaniline degradation and increased the amount of substrate channeled through the additional pathway. The reported intermediates or end products are identified by their chromatographic mobility and mass-spectral profiles.     

Biodegradation of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid by dichlorophenol-adapted microorganisms from freshwater,anaerobic sediments

Reductive dechlorination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated in anaerobic sediments by non-adapted microorganisms and by microorganisms adapted to either 2,4- or 3,4-dichlorophenol (DCP). The rate of dechlorination of 2,4-D was increased by adaptation of sediment microorganisms to 2,4-DCP while dechlorination by sediment microorganisms adapted to 3,4-DCP displayed a lag phase similar to non-adapted sediment slurries. Both 2,4- and 3,4-DCP-adapted microorganisms produced 4-chlorophenoxyacetic acid by ortho-chlorine removal. Lag phases prior to dechlorination of the initial addition of 2,4,5-T by DCP-adapted sediment microorganisms were comparable to those from non-adapted sediment slurries. However, the rates of dechlorination increased upon subsequent additions of 2,4,5-T. Biodegradation of 2,4,5-T by sediment microorganisms adapted to 2,4- and/ or 3,4-DCP produced 2,5-D as the initial intermediate followed by 3-chlorophenol and phenol indicating a para > ortho > meta order of dechlorination. Dechlorination of 2,4,5-T, by either adapted or non-adapted sediment microorganisms, progressed without detection of 2,4,5-trichlorophenol as an intermediate.     

Kinetics of Microbial Dehalogenation of Haloaromatic Substrates in Methanogenic Environments

The kinetic parameters associated with the microbial dehalogenation of 3-chlorobenzoate, 3,5-dichlorobenzoate, and 4-amino-3,5-dichlorobenzoate were measured in anoxic sediment slurries and in an enriched methanogenic culture grown on 3-chlorobenzoate. The initial dehalogenation of the substrates exhibited Michaelis-Menten kinetics. The apparent K_m values for the above substrates ranged from 30 to 67 μM. The pattern of degradation, however, was unusual. The enrichment culture accumulated partially dehalogenated intermediates to 72 and 98% of that possible when incubated with either 3,5-dichloro- or 4-amino-3,5-dichlorobenzoate, respectively, but did not accumulate significant amounts of benzoate when 3-chlorobenzoate was the sole carbon and energy source. The accumulated intermediates were rapidly metabolized only after the parent substrate concentrations were nearly depleted (<5 μM). A sequential Michaelis-Menten model was developed to account for the observed pattern of biodegradation. Using this model, we found that relative differences in the K_m and V_max parameters for substrate and intermediate dehalogenations alone were insufficient to explain the transitory accumulation of intermediates. However, by inserting a competitive inhibition term, with the primary substrate as the inhibitor, the observed pattern of degradation was simulated. Apparently, the dichlorinated substrates competitively inhibit the dehalogenation of the monochlorinated substrates. Similar kinetic patterns were noted for sediments, although the rates were slower than in the enrichment culture.     

Stimulation of Reductive Dechlorination of Tetrachloroethene in Anaerobic Aquifer Microcosms by Addition of Short-Chain Organic Acids or Alcohols

The effect of the addition of common fermentation products on the dehalogenation of tetrachloroethene was studied in methanogenic slurries made with aquifer solids. Lactate, propionate, crotonate, butyrate, and ethanol stimulated dehalogenation activity, while acetate, methanol, and isopropanol did not.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

Regulation of 2,4,5-trichlorophenoxyacetic acid and chlorophenol metabolism in Pseudomonas cepacia AC1100.

The expression of the degradative genes encoding 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and pentachlorophenol (PCP) dechlorination in a 2,4,5-T-degrading strain of Pseudomonas cepacia was examined during growth on alternate carbon sources. The dechlorination mechanisms for all three compounds were expressed in 2,4,5-T- and 2,4,5-TCP-grown cells but were not expressed in cells grown on succinate, glucose, or lactate. The addition of 2,4,5-TCP or PCP to cells grown on succinate or lactate resulted in the expression of the 2,4,5-TCP dechlorination mechanism in resting cells after 1-h lag. This expression was prevented by the presence of chloramphenicol in the resting cell suspension. Succinate-plus-PCP-grown resting cells preincubated with 2,4,5-TCP fully induced the trichlorophenol dechlorination system and partially induced the PCP dechlorination system. Preincubation of succinate-plus-PCP-grown resting cells with PCP induced neither the 2,4,5-TCP nor the PCP dechlorinating system. Succinate-grown resting cells converted 2,4,5-T to 2,4,5-TCP even in the presence of chloramphenicol. Thus, the data indicate that the enzyme(s) which converts 2,4,5-T to 2,4,5-TCP is constitutively expressed, whereas those that convert 2,4,5-TCP to central intermediates are induced by 2,4,5-TCP but not by 2,4,5-T or PCP and are repressed in the presence of an alternate carbon source.     

Reductive Dechlorination of the Nitrogen Heterocyclic Herbicide Picloram

The anaerobic biodegradation of picloram (3,5,6-trichloro-4-amino-2-pyridinecarboxylic acid) in freshwater sediment was favored under methanogenic conditions but not when sulfate or nitrate was available as a terminal electron acceptor. Under the former conditions, more than 85% of the parent substrate (340 μM) was removed from nonsterile incubations in 30 days, following a 50-day acclimation period. Concomitant with substrate decay, an intermediate transiently accumulated in the sediment slurries. By liquid chromatography-mass spectrometry, the intermediate was identified as an isomer of dichloro-4-amino-2-pyridinecarboxylic acid. Proton nuclear magnetic resonance evidence suggested that a chlorine was reductively removed from the parent substrate at the position meta to the nitrogen heteroatom. Upon continued incubation, the dechlorinated product was transformed into an unidentified compound which accumulated and resisted further decay. The addition of sulfate or bromoethanesulfonic acid to sediment slurries inhibited picloram dehalogenation, but molybdate reversed the inhibitory effect of sulfate on pesticide metabolism. These findings help clarify the fate of a halogenated nitrogen heterocyclic herbicide in anaerobic environments.     

Anaerobic degradation of m-cresol in anoxic aquifer slurries: carboxylation reactions in a sulfate-reducing bacterial enrichment

The anaerobic biodegradation of m-cresol was observed in anoxic aquifer slurries kept under both sulfate-reducing and nitrate-reducing but not methanogenic conditions. More than 85% of the parent substrate (300 microM) was consumed in less than 6 days in slurries kept under the former two conditions. No appreciable loss of the compound from the corresponding autoclaved controls was measurable. A bacterial consortium was enriched from the slurries for its ability to metabolize m-cresol under sulfate-reducing conditions. Metabolism in this enrichment culture was inhibited in the presence of oxygen or molybdate (500 microM) and in the absence of sulfate but was unaffected by bromoethanesulfonic acid. The consortium consumed 3.63 mol of sulfate per mol of m-cresol degraded. This stoichiometry is about 87% of that theoretically expected and suggests that m-cresol was largely mineralized. Resting-cell experiments demonstrated that the degradation of m-cresol proceeded only in the presence of bicarbonate. 4-Hydroxy-2-methylbenzoic acid and acetate were detected as transient intermediates. Thus, the parent substrate was initially carboxylated as the primary degradative event. The sulfate-reducing consortium could also decarboxylate p- but not m-hydroxybenzoate to near stoichiometric amounts of phenol, but this reaction was not sulfate dependent. The presence of p-hydroxybenzoate in the medium temporarily inhibited m-cresol metabolism such that the former compound was metabolized prior to the latter and phenol was degraded in a sequential manner. These findings help clarify the fate of a common groundwater contaminant under sulfate-reducing conditions.     

Anaerobic degradation of m-cresol in anoxic aquifer slurries: carboxylation reactions in a sulfate-reducing bacterial enrichment.

The anaerobic biodegradation of m-cresol was observed in anoxic aquifer slurries kept under both sulfate-reducing and nitrate-reducing but not methanogenic conditions. More than 85% of the parent substrate (300 microM) was consumed in less than 6 days in slurries kept under the former two conditions. No appreciable loss of the compound from the corresponding autoclaved controls was measurable. A bacterial consortium was enriched from the slurries for its ability to metabolize m-cresol under sulfate-reducing conditions. Metabolism in this enrichment culture was inhibited in the presence of oxygen or molybdate (500 microM) and in the absence of sulfate but was unaffected by bromoethanesulfonic acid. The consortium consumed 3.63 mol of sulfate per mol of m-cresol degraded. This stoichiometry is about 87% of that theoretically expected and suggests that m-cresol was largely mineralized. Resting-cell experiments demonstrated that the degradation of m-cresol proceeded only in the presence of bicarbonate. 4-Hydroxy-2-methylbenzoic acid and acetate were detected as transient intermediates. Thus, the parent substrate was initially carboxylated as the primary degradative event. The sulfate-reducing consortium could also decarboxylate p- but not m-hydroxybenzoate to near stoichiometric amounts of phenol, but this reaction was not sulfate dependent. The presence of p-hydroxybenzoate in the medium temporarily inhibited m-cresol metabolism such that the former compound was metabolized prior to the latter and phenol was degraded in a sequential manner. These findings help clarify the fate of a common groundwater contaminant under sulfate-reducing conditions.     

Dehalogenation of dichloroethene in a contaminated soil: fatty acids and alcohols as electron donors and an apparent requirement for tetrachloroethene

Environmental soil contamination at an industrial site in Marion, Ohio (USA) with tetrachloroethene (perchloroethene, PCE) resulted in residual cis-1, 2-dichloroethene (DCE) contamination that had not declined after more than 15 years. Microcosm slurries containing 2.6% soil from this site were supplemented with different electron donors, i.e., individual fatty acids or alcohols. None of the microcosms supported complete DCE dechlorination, unless PCE was added to the microcosm at initiation. The addition of fresh PCE resulted in the dehalogenation of PCE to DCE in the microcosms supplemented with fatty acids having an even number of carbon atoms (acetate, butyrate, and caproate), but not in those with an odd number of carbon atoms (formate, propionate, and valerate), where negligible or no activity was detected. No significant further DCE degradation was observed in any of the microcosms supplied with fatty acids as electron donors. Microcosms supplemented with freshly added PCE bioconverted PCE to DCE and completely dehalogenated both the ex-novo and soil-supplied DCE within 60 days, but only if alcohols having an even number of carbon atoms (ethanol or butanol) were also added as electron donors. Odd-numbered alcohols either did not produce dehalogenation (as with methanol) or only dehalogenated PCE to DCE (as with propanol).     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

Raoultella planticola, a new strain degrading 2,4,5-trichlorophenoxyacetic acid

A new strain that degrades the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was isolated from soil, which was exposed to factors related to the petrochemical industry. According to its physiological, biochemical, cultural, and morphological traits, together with the sequence of the 16S rRNA gene, the strain was identified as Raoultella planticola 33-4ch. The strain could consume 2,4,5-T as a sole source of carbon and energy. The amount of 2,4,5-T in the culture medium decreased by 51% after five days of incubation. Raoultella planticola 33-4ch consumes 2,4,5-T to produce 4-chlorophenoxyacetic, phenoxyacetic, and 3-methyl-2,6-dioxo-4-hexenoic acids.     

Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications.

This review is a survey of bacterial dehalogenases that catalyze the cleavage of halogen substituents from haloaromatics, haloalkanes, haloalcohols, and haloalkanoic acids. Concerning the enzymatic cleavage of the carbon-halogen bond, seven mechanisms of dehalogenation are known, namely, reductive, oxygenolytic, hydrolytic, and thiolytic dehalogenation; intramolecular nucleophilic displacement; dehydrohalogenation; and hydration. Spontaneous dehalogenation reactions may occur as a result of chemical decomposition of unstable primary products of an unassociated enzyme reaction, and fortuitous dehalogenation can result from the action of broad-specificity enzymes converting halogenated analogs of their natural substrate. Reductive dehalogenation either is catalyzed by a specific dehalogenase or may be mediated by free or enzyme-bound transition metal cofactors (porphyrins, corrins). Desulfomonile tiedjei DCB-1 couples energy conservation to a reductive dechlorination reaction. The biochemistry and genetics of oxygenolytic and hydrolytic haloaromatic dehalogenases are discussed. Concerning the haloalkanes, oxygenases, glutathione S-transferases, halidohydrolases, and dehydrohalogenases are involved in the dehalogenation of different haloalkane compounds. The epoxide-forming halohydrin hydrogen halide lyases form a distinct class of dehalogenases. The dehalogenation of alpha-halosubstituted alkanoic acids is catalyzed by halidohydrolases, which, according to their substrate and inhibitor specificity and mode of product formation, are placed into distinct mechanistic groups. beta-Halosubstituted alkanoic acids are dehalogenated by halidohydrolases acting on the coenzyme A ester of the beta-haloalkanoic acid. Microbial systems offer a versatile potential for biotechnological applications. Because of their enantiomer selectivity, some dehalogenases are used as industrial biocatalysts for the synthesis of chiral compounds. The application of dehalogenases or bacterial strains in environmental protection technologies is discussed in detail.     

The metabolism of the ground water contaminant 2-hydroxybiphenyl under sulfate-reducing conditions

The metabolic fate of 2-hydroxybiphenyl under different anaerobic conditions was tested with sediment slurries and enrichment cultures obtained from a shallow anoxic aquifer. 2-Hydroxybiphenyl was depleted in aquifer slurries over the course of incubation, but substrate loss in methanogenic slurries was not significantly different from either filter-sterilized or autoclaved controls. In contrast, the rate of substrate removal was significantly higher in non-sterile, sulfate-reducing aquifer slurries relative to abiotic control incubations. A 2-hydroxybiphenyl-degrading enrichment was established that was inhibited by molybdate but not by bromoethane-sulfonic acid. For every mole of substrate consumed by the bacterial consortium, 6.1±0.2 moles of sulfate were depleted from the enrichment medium. This represents about 87% of the theoretical amount of sulfate consumed and suggests that the 2-hydroxybiphenyl was largely mineralized. Oxygen, nitrate, or carbon dioxide could not replace sulfate as a terminal electron acceptor for the enrichment. Other hydroxybiphenyl isomers were not metabolized by these cultures. This study shows that aromatic substrates with multiple ring systems can undergo biotransformation by anaerobic microorganisms under some ecological conditions.     

Reductive Dehalogenation of a Nitrogen Heterocyclic Herbicide in Anoxic Aquifer Slurries

We studied the metabolic fate of bromacil in anaerobic aquifer slurries held under denitrifying, sulfate-reducing, or methanogenic conditions. Liquid chromatograhy-mass spectrometry of the slurries confirmed that bromacil was debrominated under methanogenic conditions but was not degraded under the other incubation conditions. This finding extends the range of aryl reductive dehalogenation reactions to include nitrogen heterocyclic compounds.     

Aromatic and Volatile Acid Intermediates Observed during Anaerobic Metabolism of Lignin-Derived Oligomers

Anaerobic enrichment cultures acclimated for 2 years to use a ¹⁴C-labeled, lignin-derived substrate with a molecular weight of 600 as a sole source of carbon were characterized by capillary and packed column gas chromatography. After acclimation, several of the active methanogenic consortia were inhibited with 2-bromoethanesulfonic acid, which suppressed methane formation and enhanced accumulation of a series of metabolic intermediates. Volatile fatty acids levels in 2-bromoethanesulfonic acid-amended cultures were 10 times greater than those in the uninhibited, methane-forming consortia with acetate as the predominant component. Furthermore, in the 2-bromoethanesulfonic acid-amended consortia, almost half of the original substrate carbon was metabolized to 10 monoaromatic compounds, with the most appreciable quantities accumulated as cinnamic, benzoic, caffeic, vanillic, and ferulic acids. 2-Bromoethanesulfonic acid seemed to effectively block CH₄ formation in the anaerobic food chain, resulting in the observed buildup of volatile fatty acids and monoaromatic intermediates. Neither fatty acids nor aromatic compounds were detected in the oligolignol substrate before its metabolism, suggesting that these anaerobic consortia have the ability to mediate the cleavage of the β-aryl-ether bond, the most common intermonomeric linkage in lignin, with the subsequent release of the observed constituent aromatic monomers.     

Natural Biological Attenuation of Phenoxy Herbicides in Groundwater: Dow AgroSciences Paritutu Site, New Zealand

Groundwater beneath a manufacturing site previously used for herbicide production has been shown to contain low levels of chlorinated phenols and phenoxy herbicides. The importance of biological processes in the natural attenuation of the groundwater contaminants was examined as part of an ongoing investigation. Analysis of the groundwater chemistry indicated that the aquifer is essentially aerobic in the area of interest. Laboratory microcosm experiments demonstrated that the naturally occurring microorganisms rapidly degraded a mixture of the predominant organic contaminants under conditions that simulate those in the aquifer. The time required for 50% degradation ranged from 7 to 27 days for 2,4-dichlorophenoxyacetic acid (2,4-D) and 9 to 49 days for 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The rapid biodegradation rates were consistent with the results of microbiological analyses, which demonstrated that a substantial proportion of the culturable bacteria were capable of growth on 2,4-D as a sole carbon source. Results of gene probe assays suggested the numbers of bacteria with the potential to degrade 2,4-D were one to two orders of magnitude higher than were detected using plate counts. Computer model simulations illustrated that biodegradation would be expected to significantly contribute to the attenuation of 2,4-D and 2,4,5-T in the aquifer. On the basis of the various lines of evidence and the distances the groundwater must travel, the groundwater contaminants would be expected to naturally biodegrade to below levels of concern before the plume reaches potential environmental receptors.     

Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by Pseudomonas cepacia.

The strain of Pseudomonas cepacia, AC1100, capable of utilizing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy can degrade 2,4,5-T in contaminated soil, removing more than 99% of 2,4,5-T present at 1 mg/g of soil within 1 week. Repeated application of AC1100 even allowed more than 90% removal of 2,4,5-T within 6 weeks from heavily contaminated soil containing as much as 20,000 ppm 2,4,5,-T (20 mg/g of soil). Microbial removal of 2,4,5-T allowed the soil to support growth of plants sensitive to low concentrations of 2,4,5-T. After 2,4,5-T removal, the titer of AC1100 in the soil rapidly fell to undetectable levels within a few weeks.     

Detoxification of 2,4,5-trichlorophenoxyacetic acid from contaminated soil by Pseudomonas cepacia

The strain of Pseudomonas cepacia, AC1100, capable of utilizing 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy can degrade 2,4,5-T in contaminated soil, removing more than 99% of 2,4,5-T present at 1 mg/g of soil within 1 week. Repeated application of AC1100 even allowed more than 90% removal of 2,4,5-T within 6 weeks from heavily contaminated soil containing as much as 20,000 ppm 2,4,5,-T (20 mg/g of soil). Microbial removal of 2,4,5-T allowed the soil to support growth of plants sensitive to low concentrations of 2,4,5-T. After 2,4,5-T removal, the titer of AC1100 in the soil rapidly fell to undetectable levels within a few weeks.     

Microbial Assimilation of Hydrocarbons II. Fatty Acids Derived from 1-Alkenes

The utilization of 1-alkenes by Micrococcus cerificans was investigated with respect to characteristic fatty acid profiles resulting from growth at the expense of these substrates. Saturated fatty acids containing even numbers of carbon atoms were produced from 1-dodecene and 1-tetradecene. Unsaturated fatty acids related to the parent alkene were not detected. The fatty acid profile from 1-pentadecene utilization resulted in the identification of 14-pentadecenoic acid, indicating preferential methyl-group attack. Studies with 1-hexadecene and 1-octadecene indicated simultaneous methyl-group and double-bond attack. Omega-Unsaturated fatty acids related to carbon number of parent alkene and odd-carbon fatty acids one carbon less than the substrate molecule were identified. A mechanism involving double bond epoxidation and oxidative cleavage was supported by measuring the release of formaldehyde. It appears that a dichotomous mechanism is functional in the assimilation of higher carbon number alkenes.