Transformations of 1- and 2-carbon halogenated aliphatic organic compounds under methanogenic conditions

Several 1- and 2-carbon halogenated aliphatic organic compounds present at low concentrations (less than 100 micrograms/liter) were degraded under methanogenic conditions in batch bacterial cultures and in a continuous-flow methanogenic fixed-film laboratory-scale column. Greater than 90% degradation was observed within a 2-day detention time under continuous-flow methanogenic conditions with acetate as a primary substrate. Carbon-14 measurements indicated that chloroform, carbon tetrachloride, and 1,2-dichloroethane were almost completely oxidized to carbon dioxide, confirming removal by biooxidation. The initial step in the transformations of tetrachloroethylene and 1,1,2,2-tetrachloroethane to nonchlorinated end products appeared to be reductive dechlorination to trichloroethylene and 1,1,2-trichloroethane, respectively. Transformations of the brominated aliphatic compounds appear to be the result of both biological and chemical processes. The data suggest that transformations of halogenated aliphatic compounds can occur under methanogenic conditions in the environment.     

Transformations of halogenated organic compounds under denitrification conditions.

Trihalomethanes, carbon tetrachloride, 1,1,1-trichloroethane, 1,2-dibromoethane, chlorinated benzenes, ethylbenzene, and naphthalene at concentrations commonly found in surface and groundwater were incubated under anoxic conditions to study their transformability in the presence of denitrifying bacteria. None of the aromatic compounds showed significant utilization relative to sterile controls at initial concentrations from 41 to 114 micrograms/liter after 11 weeks of incubation. Of the halogenated aliphatic compounds studied, transformations of carbon tetrachloride and brominated trihalomethanes were observed after 8 weeks in batch denitrification cultures. Carbon from the decomposition of carbon tetrachloride was both assimilated into cell material and mineralized to carbon dioxide. How this was possible remains unexplained, since carbon tetrachloride is transformed to CO2 by hydrolysis and not by oxidation-reduction. Chloroform was detected in bacterial cultures with carbon tetrachloride initially present, indicating that reductive dechlorination had occurred in addition to hydrolysis. The data suggest that transformations of certain halogenated aliphatic compounds are likely to occur under denitrification conditions in the environment.     

Transformations of halogenated organic compounds under denitrification conditions

Trihalomethanes, carbon tetrachloride, 1,1,1-trichloroethane, 1,2-dibromoethane, chlorinated benzenes, ethylbenzene, and naphthalene at concentrations commonly found in surface and groundwater were incubated under anoxic conditions to study their transformability in the presence of denitrifying bacteria. None of the aromatic compounds showed significant utilization relative to sterile controls at initial concentrations from 41 to 114 micrograms/liter after 11 weeks of incubation. Of the halogenated aliphatic compounds studied, transformations of carbon tetrachloride and brominated trihalomethanes were observed after 8 weeks in batch denitrification cultures. Carbon from the decomposition of carbon tetrachloride was both assimilated into cell material and mineralized to carbon dioxide. How this was possible remains unexplained, since carbon tetrachloride is transformed to CO2 by hydrolysis and not by oxidation-reduction. Chloroform was detected in bacterial cultures with carbon tetrachloride initially present, indicating that reductive dechlorination had occurred in addition to hydrolysis. The data suggest that transformations of certain halogenated aliphatic compounds are likely to occur under denitrification conditions in the environment.     

Effects of 2-Bromoethanesulfonic Acid and 2- Chloroethanesulfonic Acid on Acetate Utilization in a Continuous-Flow Methanogenic Fixed-Film Column

2-Bromoethanesulfonic acid (BESA) and 2-chloroethanesulfonic acid (CESA) have been reported to be potent inhibitors of methane formation during methanogenic decomposition in batch cultures. However, in a laboratory-scale continuous-flow methanogenic fixed-film column containing a predominance of acetate-decarboxylating methanogens, BESA at 6 × 10⁻⁴ M produced only a 41% inhibition of acetate utilization, and CESA at 5.4 × 10⁻⁴ M produced a 37% inhibition of acetate utilization. BESA and CESA concentrations were not monitored in the effluent, so their fate is unknown. The organisms in the column were capable of degrading trace halogenated aliphatic compounds (~30 μg/liter) with acetate (100 mg/liter) as the primary substrate. Previous exposure of the cells to halogenated organic compounds may have conferred resistance to BESA and CESA. Degradation of the inhibitor compounds is another possible explanation for the observed effects.     

Role of sulfate in microbial transformations of environmental contaminants: Chlorinated aromatic compounds

Despite recent progress made in describing microbial transformations that occur under anaerobic conditions, our understanding of the role sulfate‐reducing bacteria may play in the remediation of environmental contaminants is still very limited. The objective of this mini‐review is to summarize what is currently known of the metabolism of chlorinated aromatic compounds in the presence of sulfate. Sulfidogenic processes are discussed with respect to the thermodynamics of haloaromatic oxidation and to their potential use in the in situ bioremediation of hazardous organic wastes. A comprehensive listing is made of anaerobic transformations that involve both halogenated and nonhalogenated monoaromatic substrates by denitrifiers, dissimilatory iron‐reducing bacteria, and methanogenic consortia. In contrast to other anaerobic processes, studies involving sulfate‐mediated metabolism of hazardous organic compounds have been neglected; however, the recent success in defining methanogenic transformations, in particular, has enhanced expectations of defining an analogous role for sulfate‐reducing microbial communities in low redox environments that have become contaminated with hazardous substances.     

Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the delta subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.     

Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

Methanogenic transformation of aromatic hydrocarbons and phenols in groundwater aquifers

Fermentative and methanogenic bacteria have been found repeatedly as important members of microbial flora in anoxic zones of the subsurface—in pristine as well as in contaminated groundwater aquifers. These bacteria, which together with obligate proton reducers form complex methanogenic communities, are significant as decomposers of organic matter under conditions of exogenous electron acceptor depletion. Their metabolic activity has been demonstrated in laboratory microcosms derived from aquifer material, and also in the subsurface in situ. Methanogenic communities have been shown to transform numerous organic pollutants, or even to completely degrade these compounds with the production of carbon dioxide and methane. Depending on the chemical structure of the pollutant, such a compound can be used as an electron donor and a carbon/energy source for fermentative microorganisms (which is typically the case with highly reduced compounds); alternatively, a highly oxidized pollutant can be used as a potential electron acceptor or electron sink. This review addresses fermentative/methanogenic degradation of chlorinated and nonchlorinated aromatic hydrocarbons and phenols by subsurface microorganisms; for comparison, it briefly relates also other types of anaerobic transformations (under sulfate‐reducing, iron‐reducing, and denitrifying conditions). Furthermore, it outlines transformation pathways, those that are proposed as well as those that are already partially proved, for aromatic hydrocarbons and phenols under fermentative/methanogenic conditions; finally, it discusses the relevance of these processes to bioremediation of contaminated groundwater aquifers.     

Ethylene dibromide transformation under methanogenic conditions.

Ethylene dibromide present at a low concentration (less than 100 micrograms/liter) was transformed by reductive dehalogenation under methanogenic conditions in batch bacterial cultures and in a continuous-flow, methanogenic, fixed-film, laboratory-scale column.     

Ethylene dibromide transformation under methanogenic conditions

Ethylene dibromide present at a low concentration (less than 100 micrograms/liter) was transformed by reductive dehalogenation under methanogenic conditions in batch bacterial cultures and in a continuous-flow, methanogenic, fixed-film, laboratory-scale column.     

Utilization rates of trace halogenated organic compounds in acetate-grown biofilms

Trace concentrations of chlorinated benzenes and chlorinated aliphatics were biotransformed by acetate-supported biofilms, the former under aerobic conditions and the latter under methanogenic conditions. The rates of transformation of the halogenated organic compounds (secondary substrates) differed from that of acetate, the primary substrate; some were higher, some were lower, and some were similar. Factors affecting the relative rates of utilization in multisubstrate systems are not known.     

Versatility of soil column experiments to study biodegradation of halogenated compounds under environmental conditions

Soil column experiments were performed to obtain insight in the different biological and physico-chemical processes affecting biodegradation of halogenated compounds under natural conditions in a water infiltration site. Lower chlorinated aromatic compounds could be degraded under aerobic conditions, whereas highly chlorinated compounds and chlorinated aliphatic compounds were mainly transformed under anaerobic conditions. Microorganisms which derive energy from reductive dechlorination were enriched and characterized. It was found that microbes could adapt to using chlorinated benzenes by evolution of new enzyme specificities and by exchange of genetic material. For halogenated pollutants, which are generally hydrophobic, sorption processes control the concentration available for biodegradation. The effects of very low concentrations of halogenated compounds on their biodegradability are described. The use of isolated bacterial strains to enhance biodegradation was evaluated with respect to their temperature-related activity and to their adhesion properties.Abbreviations 3-CB 3-chlorobenzoate - DCB dichlorobenzene - HCH hexachlorocyclohexane - IS insertion sequence - PER tetrachloroethylene - S_min minimal substrate concentration for growth - TCB trichlorobenzene - TRI trichloroethylene - filtration coefficient     

Production of Ethane, Ethylene, and Acetylene from Halogenated Hydrocarbons by Methanogenic Bacteria

Several methanogenic bacteria were shown to produce ethane, ethylene, and acetylene when exposed to the halogenated hydrocarbons bromoethane, dibromo- or dichloroethane, and 1,2-dibromoethylene, respectively. They also produced ethylene when exposed to the coenzyme M analog and specific methanogenic inhibitor bromoethanesulfonic acid. The production of these gases from halogenated hydrocarbons has a variety of implications concerning microbial ecology, agriculture, and toxic waste treatment. All halogenated aliphatic compounds tested were inhibitory to methanogens. Methanococcus thermolithotrophicus, Methanococcus deltae, and Methanobacterium thermoautotrophicum ΔH and Marburg were completely inhibited by 7 μM 1,2-dibromoethane and, to various degrees, by 51 to 1,084 μM 1,2-dichloroethane, 1,2-dibromoethylene, 1,2-dichloroethylene, and trichloroethylene. In general, the brominated compounds were more inhibitory. The two Methanococcus species were fully inhibited by 1 μM bromoethanesulfonic acid, whereas both Methanobacterium strains were only partly inhibited by 2,124 μM. Coenzyme M protected cells from bromoethanesulfonic acid but not from any of the other inhibitors.     

Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers

An enzyme that is capable of hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols was purified from a 1,6-dichlorohexane-degrading bacterium. The dehalogenase was found to be a monomeric protein of relative molecular mass 28,000. The affinity for its substrates was relatively low with Km values for short-chain haloalkanes in the range 0.1-0.9 mM. The aliphatic dehalogenase showed a much broader substrate range than has been reported for halidohydrolases so far. Novel classes of substrates include dihalomethanes, C5-C9 1-halo-n-alkanes, secondary alkylhalides, halogenated alcohols and chlorinated ethers. Several of these compounds are important environmental pollutants, e.g. methylbromide, dibromomethane, 1,2-dibromoethane, 1,3-dichloropropene, and bis(2-chloroethyl)ether. The degradation of chiral 2-bromoalkanes appeared to proceed without stereochemical preference. Optically active 2-bromobutane was converted with inversion of configuration at the chiral carbon atom, suggesting that the dehalogenase reaction proceeds by a nucleophilic substitution involving a carboxyl group or base catalysis.     

Anaerobic production and transformation of aromatic hydrocarbons and substituted phenols by ferulic acid-degrading BESA-inhibited methanogenic consortia

Abstract Sewage sludge-derived methanogenic enrichments degrading ferulic acid as sole carbon and energy source were partially inhibited with 2-bromoethanesulfonic acid. The various intermediates and products formed under inhibition of methanogenesis were studied using gas chromatography/mass spectrometry (GC/MS). In addition to aromatic, alicyclic, and aliphatic acids previously shown to be intermediates of ferulate degradation to CO₂ and CH₄, the following compounds were detected: toluene, ethylbenzene, phenol, p -cresol, 2-ethylphenol, catechol, and 3-hydroxy-4-ethylphenol. The character and the sequence of appearance of the compounds indicate that fermentative bacteria which initiate the anaerobic transformation of ferulic acid, in case of disruption of interspecies hydrogen transfer, dispose of electrons by converting part of the substrate to reduced derivatives. Aromatic hydrocarbons are further partially oxidized through hydroxylation of the ring (and, to a lesser extent, the side-chain), and partially reduced to saturated alicyclic rings. Some of these compounds seem to be gradually degraded to branched or straight- chain aliphatic acids. Some compounds, like catechol and ethylphenol, accumulate transiently or persistently in high concentrations (up to 16 mM carbon out of the initial concentration of 30 mM substrate carbon), indicating that hydroxylation of the aromatic ring might be an important metabolic reaction in these systems.     

Transformations of TNT and related aminotoluenes in groundwater aquifer slurries under different electron-accepting conditions

The transport and fate of pollutants is often governed by both their tendency to sorb as well as their susceptibility to biodegradation. We have evaluated these parameters for 2,4,6-trinitrotoluene (TNT) and several biodegradation products. Slurries of aquifer sediment and groundwater depleted TNT at rates of 27, 7.7 and 5.9 μM day⁻¹ under methanogenic, sulfate-reducing and nitrate-reducing conditions, respectively. Abiotic losses of TNT were determined in autoclaved controls. Abiotic TNT loss and subsequent transformation of the products was also observed. These transformations were especially important during the first step in the reduction of TNT. Subsequent abiotic reactions could account for all of the transformations observed in bottles which were initially nitrate-reducing. Other controls removed TNT reduction products at much slower rates than slurries containing live organisms. 2-Amino-4,6-dinitrotoluene was produced in all slurries but disappeared in methanogenic and in sulfate-reducing slurries within several weeks. This compound was converted to 2,4-diamino-6-nitrotoluene in all slurries with subsequent removal of the latter from methanogenic and sulfate-reducing slurries, while it persisted in autoclaved controls and in the nitrate-reducing slurries. Aquifer slurries incubated with either 2,4- or 2,6-diaminotoluene showed losses of these compounds relative to autoclaved controls under nitrate-reducing conditions but not under sulfate-reducing or methanogenic conditions. These latter compounds are important as reduced intermediates in the biodegradation of dinitrotoluenes and as industrial chemicals. In experiments to examine sorption, exposure to landfill sediment resulted in losses of approximately 15% of diaminotoluene isomers and 25% of aminodinitrotoluene isomers from initial solution concentrations within 24 h. Isotherms confirmed that the diaminotoluenes were least strongly sorbed and the amino-dinitrotoluenes most strongly sorbed to this sediment, while TNT sorption capacity was intermediate. In our studies, 2,4,6-triaminotoluene sorption capacity was indeterminate due to its chemical instability. Coupled with biodegradation information, isotherms help describe the likelihood of contaminant removal, persistence, and movement at impacted sites. Received 11 March 1996/ Accepted in revised form 24 July 1996     

Comparative mutagenicity of aliphatic epoxides in Salmonella

37 aliphatic epoxides comprising 6 subclasses (unsubstituted aliphatic epoxides, halogenated aliphatic epoxides, glycidyl esters, glycidates, glycidyl ethers and diglycidyl ethers) were tested, under code, for mutagenicity in Salmonella strains TA98, TA100, TA1535 and TA1537 and/or TA97 with and without metabolic activation using a standardized protocol. The 4 halogenated aliphatic epoxides and the 4 diglycidyl ethers were all mutagenic. The 2 glycidates were negative in all strain/activation systems used while all 5 glycidyl esters were mutagenic. 3 of the 8 unsubstituted aliphatic epoxides and 11 of the 12 glycidyl ethers were mutagenic. Glycidol also was mutagenic whereas 9,10-epoxyoctadecanoic acid, 2-ethylhexyl ester was not mutagenic. Of the 28 mutagenic compounds, all but neodecanoic acid, 2,3-epoxypropyl ester and 2-ethylhexyl glycidyl ether were detected in TA100 without activation. The latter two were detected only with activation in TA100 and TA1535. The majority of the other 26 chemicals were also mutagenic in TA1535 without activation. Good intra- and interlaboratory reproducibility was seen in the results of each of the 4 chemicals tested in more than one set of experiments. The current results confirm and extend the observations of other investigators regarding structural effects on the mutagenicity of members of the aliphatic epoxide class of chemicals.     

Dechlorination of trichlorofluoromethane (CFC-11) by sulfate-reducing bacteria from an aquifer contaminated with halogenated aliphatic compounds.

Groundwater samples were obtained from a deep aquifer contaminated with halogenated aliphatic compounds. One-milliliter samples contained 9.2 x 10(5) total bacteria (by acridine orange microscopic counts) and 2.5 x 10(3) sulfate-reducing bacteria (by most probable number analysis). Samples were incubated anaerobically in a basal salts medium with acetate as the electron donor and nitrate and sulfate as the electron acceptors. Residual levels of trichlorofluoromethane (CFC-11) in samples were biotically degraded, while trichloroethylene was not. When successively higher levels of CFC-11 were added, increasingly rapid degradation rates were observed. Concomitant with CFC-11 degradation was the near stoichiometric production of fluorodichloromethane (HCFC-21); the production of HCFC-21 was verified by mass spectrometry. CFC-11 degradation was dependent on the presence of acetate (or butyrate) and sulfate but was independent of nitrate. Other carbon sources such as lactate and isopropanol did not support the degradation. The addition of 1 mM sodium sulfide completely inhibited CFC-11 degradation; however, degradation occurred in the presence of 2 mM 2-bromoethanesulfonic acid. These results indicate that the anaerobic dechlorination of CFC-11 is carried out by sulfate-reducing bacteria and not by denitrifying or methanogenic bacteria.     

Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under methanogenic conditions

Tetrachloroethylene (PCE) and trichloroethylene (TCE), common industrial solvents, are among the most frequent contaminants found in groundwater supplies. Due to the potential toxicity and carcinogenicity of chlorinated ethylenes, knowledge about their transformation potential is important in evaluating their environmental fate. The results of this study confirm that PCE can be transformed by reductive dehalogenation to TCE, dichloroethylene, and vinyl chloride (VC) under anaerobic conditions. In addition, [14C]PCE was at least partially mineralized to CO2. Mineralization of 24% of the PCE occurred in a continuous-flow fixed-film methanogenic column with a liquid detention time of 4 days. TCE was the major intermediate formed, but traces of dichloroethylene isomers and VC were also found. In other column studies under a different set of methanogenic conditions, nearly quantitative conversion of PCE to VC was found. These studies clearly demonstrate that TCE and VC are major intermediates in PCE biotransformation under anaerobic conditions and suggest that potential exists for the complete mineralization of PCE to CO2 in soil and aquifer systems and in biological treatment processes.