Sequential anaerobic/aerobic biodegradation of chloroethenes--aspects of field application

Because of a range of different industrial activities, sites contaminated with chloroethenes are a world-wide problem. Chloroethenes can be biodegraded by reductive dechlorination under anaerobic conditions as well as by oxidation under aerobic conditions. The tendency of chloroethenes to undergo reductive dechlorination decreases with a decreasing number of chlorine substituents, whereas with less chlorine substituents chloroethenes more easily undergo oxidative degradation. There is currently a growing interest in aerobic metabolic degradation of chloroethenes, which demonstrates advantages compared to cometabolic degradation pathways. Sequential anaerobic/aerobic biodegradation can overcome the disadvantages of reductive dechlorination and leads to complete mineralization of the chlorinated pollutants. This approach shows promise for site remediation in natural settings and in engineered systems.     

Enhanced dechlorination of chloroethenes by granular sludge under microaerophilic conditions

This study investigates an innovative dechlorination process using anaerobic granular sludge that was partially exposed to oxygen. The exposure supported a synchronously anaerobic and aerobic bioconversion process that combined reductive dechlorination with aerobic co-oxidation in a sludge granule. Experimental results showed that the highest dechlorination rates of tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride were 6.44, 2.98, 1.70 and 0.97 nmol/gVS day, at initial O₂ concentrations of 10, 100, 5 and 0%, respectively. Strictly anaerobic conditions favored the dechlorination of vinyl chloride while absolutely aerobic conditions were preferred for trichloroethene dechlorination. Microaerophilic conditions are suggested to ensure the overall biodegradation of the chlorinated ethenes present in groundwater as a mixture.     

Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

Transition Metal Catalyst-Assisted Reductive Dechlorination of Perchloroethylene by Anaerobic Aquifer Enrichments

Bioremediation of groundwater contaminated with chlorinated solvents, such as perchloroethylene (PCE) or carbon tetrachloride, can be accomplished by adding nutrients to stimulate a microbial community capable of reductive dechlorination. However, biotransformation of these solvents, especially PCE, typically occurs very slowly or not at all. Experiments were conducted to evaluate whether the addition of transition metal tetrapyrrole catalysts would increase the reductive transformation of PCE to trichloroethylene (TCE) by sulfate-reducing enrichment cultures. Batch assays were used to test vitamin B₁₂ and two synthetic sulfonatophenyl porphine catalysts for the stimulation of reductive dechlorination of PCE by sulfate-reducing bacteria (SRB) enriched from aquifer sediments from two locations at Dover Air Force Base. Cells from the enrichments were concentrated and added to batch assay vials. Vials containing SRB cells amended with vitamin B12 exhibited enhanced transformation of PCE to TCE compared with reactors amended with either synthetic catalysts or reactors containing cells alone. Methane production was observed in reactors that exhibited maximum levels of dechlorination. Storage of aquifer sediments between enrichments led to decreased levels of PCE dechlorination in subsequent assays.     

Regiospecificity of chlorophenol reductive dechlorination by vitamin b(12s)

Vitamin B(12), reduced by titanium (III) citrate to vitamin B(12s), catalyzes the reductive dechlorination of chlorophenols. Reductive dechlorination of pentachlorophenol and of all tetrachlorophenol and trichlorophenol isomers was observed. Reaction of various chlorophenols with vitamin B(12) favored reductive dechlorination at positions adjacent to another chlorinated carbon, but chlorines ortho to the hydroxyl group of a phenol were particularly resistant to reductive dechlorination, even if they were also ortho to a chlorine. This resulted in a reductive dechlorination pattern favoring removal of para and meta chlorines, which differs substantially from the pattern exhibited by anaerobic microbial consortia.     

Growth of Dehalobacter and Dehalococcoides spp. during degradation of chlorinated ethanes

Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.     

Microbial reductive dechlorination of PCBs

Reductive dechlorination is an advantageous process to microorganisms under anaerobic conditions because it is an electron sink, thereby allowing reoxidation of metabolic intermediates. In some organisms this has been demonstrated to support growth. Many chlorinated compounds have now been shown to be reductively dechlorinated under anaerobic conditions, including many of the congeners in commercial PCB mixtures. Anaerobic microbial communities in sediments dechlorinate Aroclor at rates of 3 µg Cl/g sediment × week. PCB dechlorination occurs at 12° C, a temperature relevant for remediation at temperate sites, and at concentrations of 100 to 1000 ppm. The positions dechlorinated are usually meta > para > ortho. The biphenyl rings, and the mono-ortho- and diorthochlorobiphenyls were not degraded after a one year incubation. Hence subsequent aerobic treatment may be necessary to meet regulatory standards. Reductive dechlorination of Arochlors does reduce their dioxin-like toxicity as measured by bioassay and by analysis of the co-planar congeners. The most important limitation to using PCB dechlorination as a remediation technology is the slower than desired dechlorination rates and no means yet discovered to substantially enhance these rates. Long term enrichments using PCBs as the only electron acceptor resulted in an initial enhancement in dechlorination rate. This rate was sustained but did not increase in serial transfers. Bioremediation of soil contaminated with Aroclor 1254 from a transformer spill was dechlorinated by greater than 50% following mixing of the soil with dechlorinating organisms and river sediment. It is now reasonable to field test reductive dechlorination of PCBs in cases where the PCB concentration is in the range where regulatory standards may be directly achieved by dechlorination, where a subsequent aerobic treatment is feasible, where any co-contaminants do not pose an inhibitory problem, and where anaerobic conditions can be established.This paper was presented at the Pacific Basin Conference on Hazardous Waste, April, 1992, Bangkok, Thailand. Published by permission of the Pacific Basin Consortium for Hazardous Waste Research, East-West Center, Honolulu, HI     

Regiospecificity of Chlorophenol Reductive Dechlorination by Vitamin B12s

Vitamin B₁₂, reduced by titanium (III) citrate to vitamin B_12s, catalyzes the reductive dechlorination of chlorophenols. Reductive dechlorination of pentachlorophenol and of all tetrachlorophenol and trichlorophenol isomers was observed. Reaction of various chlorophenols with vitamin B₁₂ favored reductive dechlorination at positions adjacent to another chlorinated carbon, but chlorines ortho to the hydroxyl group of a phenol were particularly resistant to reductive dechlorination, even if they were also ortho to a chlorine. This resulted in a reductive dechlorination pattern favoring removal of para and meta chlorines, which differs substantially from the pattern exhibited by anaerobic microbial consortia.     

Complete biological reductive transformation of tetrachloroethene to ethane.

Reductive dechlorination of tetrachloroethene (perchloroethylene; PCE) was observed at 20 degrees C in a fixed-bed column, filled with a mixture (3:1) of anaerobic sediment from the Rhine river and anaerobic granular sludge. In the presence of lactate (1 mM) as an electron donor, 9 microM PCE was dechlorinated to ethene. Ethene was further reduced to ethane. Mass balances demonstrated an almost complete conversion (95 to 98%), with no chlorinated compounds remaining (less than 0.5 micrograms/liter). When the temperature was lowered to 10 degrees C, an adaptation of 2 weeks was necessary to obtain the same performance as at 20 degrees C. Dechlorination by column material to ethene, followed by a slow ethane production, could also be achieved in batch cultures. Ethane was not formed in the presence of bromoethanesulfonic acid, an inhibitor of methanogenesis. The high dechlorination rate (3.7 mumol.l-1.h-1), even at low temperatures and considerable PCE concentrations, together with the absence of chlorinated end products, makes reductive dechlorination an attractive method for removal of PCE in bioremediation processes.     

Complete biological reductive transformation of tetrachloroethene to ethane.

Reductive dechlorination of tetrachloroethene (perchloroethylene; PCE) was observed at 20 degrees C in a fixed-bed column, filled with a mixture (3:1) of anaerobic sediment from the Rhine river and anaerobic granular sludge. In the presence of lactate (1 mM) as an electron donor, 9 microM PCE was dechlorinated to ethene. Ethene was further reduced to ethane. Mass balances demonstrated an almost complete conversion (95 to 98%), with no chlorinated compounds remaining (less than 0.5 micrograms/liter). When the temperature was lowered to 10 degrees C, an adaptation of 2 weeks was necessary to obtain the same performance as at 20 degrees C. Dechlorination by column material to ethene, followed by a slow ethane production, could also be achieved in batch cultures. Ethane was not formed in the presence of bromoethanesulfonic acid, an inhibitor of methanogenesis. The high dechlorination rate (3.7 mumol.l-1.h-1), even at low temperatures and considerable PCE concentrations, together with the absence of chlorinated end products, makes reductive dechlorination an attractive method for removal of PCE in bioremediation processes.     

Sequential application of electron donors and humic acids for the anaerobic bioremediation of chlorinated aliphatic hydrocarbons

In situ anaerobic bioremediation of chlorinated solvents such as perchloroethene (PCE) frequently faces the problem of accumulating toxic, lower chlorinated compounds such as dichloroethene (cis-DCE) and vinyl chloride (VC). In the present study, the efficacy of the sequential application of electron donors, supporting reductive dechlorination, and of humic acids, acting as extracellular electron shuttles facilitating the anaerobic oxidation of recalcitrant intermediates, was explored in microcosm studies. Upon one initial dose of lactose, supplied in a 1000-fold superstoichiometric electron equivalent ratio, PCE was completely converted into cis-DCE within 35 days. Repeated electron donor additions did not entail exhaustive cis-DCE degradation over incubation time (120 days). Although the electron donor was quickly converted into fatty acids, about 30% of added reducing equivalents were recovered as acetate after four months of operation, indicating the inhibition of acetoclastic methanogenesis. In the next step, the substoichiometric addition of anthraquinone-2,6-disulfonate, a humic acid model compound, effected the complete removal of the accumulated cis-DCE within 15 days, probably as a result of the participation of the quinone in the biotic or abiotic anaerobic oxidation of cis-DCE. Cis-DCE degradation was not connected to the accumulation of VC, rendering the proposed two-step treatment an efficient and environmentally compliant remedy for anaerobic groundwater bodies contaminated with chlorinated solvents.     

Reductive dechlorination of Tri- and tetrachloroethylenes depends on transition from aerobic to anaerobic conditions.

Aerobic enrichment cultures from contaminated groundwaters dechlorinated trichloroethylene (TCE) (14.6 mg/liter; 111 mumol/liter) and tetrachloroethylene (PCE) (16.2 mg/liter; 98 mumol/liter) reductively within 4 days after the transition from aerobic to anaerobic conditions. The transformation products were equimolar amounts of cis-1,2-dichloroethylene and traces of 1,1-dichloroethylene. No other chlorinated product and no methane were detected. The change was accompanied by the release of sulfide, which caused a decrease in the redox potential from 0 to -150 mV. In sterile control experiments, sulfide led to the abiotic formation of traces of 1,1-dichloroethylene without cis-1,2-dichloroethylene production. The reductive dechlorination of PCE via TCE depended on these specific transition conditions after consumption of the electron acceptor oxygen or nitrate. Repeated feeding of TCE or PCE to cultures after the change to anaerobic conditions yielded no further dechlorination. Only aerobic subcultures with an air/liquid ratio of 1:4 maintained dechlorination activities; anaerobic subcultures showed no transformation. Bacteria from noncontaminated sites showed no reduction under the same conditions.     

Reductive dechlorination of Tri- and tetrachloroethylenes depends on transition from aerobic to anaerobic conditions

Aerobic enrichment cultures from contaminated groundwaters dechlorinated trichloroethylene (TCE) (14.6 mg/liter; 111 mumol/liter) and tetrachloroethylene (PCE) (16.2 mg/liter; 98 mumol/liter) reductively within 4 days after the transition from aerobic to anaerobic conditions. The transformation products were equimolar amounts of cis-1,2-dichloroethylene and traces of 1,1-dichloroethylene. No other chlorinated product and no methane were detected. The change was accompanied by the release of sulfide, which caused a decrease in the redox potential from 0 to -150 mV. In sterile control experiments, sulfide led to the abiotic formation of traces of 1,1-dichloroethylene without cis-1,2-dichloroethylene production. The reductive dechlorination of PCE via TCE depended on these specific transition conditions after consumption of the electron acceptor oxygen or nitrate. Repeated feeding of TCE or PCE to cultures after the change to anaerobic conditions yielded no further dechlorination. Only aerobic subcultures with an air/liquid ratio of 1:4 maintained dechlorination activities; anaerobic subcultures showed no transformation. Bacteria from noncontaminated sites showed no reduction under the same conditions.     

Bioremediation of Hexachlorocyclohexane Isomers,Chlorinated Benzenes,and Chlorinated Ethenes in Soil and Fractured Dolomite

Groundwater at an industrial site is contaminated with α hexachlorocyclohexane (HCH) and γ -HCH (i.e., lindane) (0.3 to 0.5 ppm). Other contaminants in the 1 to 15 ppm range include 1,2,4-trichlorobenezene (TCB), 1,2-dichlorobenzene (DCB), 1,3-DCB, 1,4-DCB, chlorobenzene (CB), benzene, trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). The aquifer consists of a shallow layer of soil over fractured dolomite, where most of the contaminant mass resides. The objective of this study was to compare (1) anaerobic reductive dechlorination of the polychlorinated contaminants, followed by aerobic biodegradation of the daughter products (mainly DCBs, CB, and benzene); and (2) aerobic biodegradation of α - and γ -HCH, TCB, DCBs, CB, and benzene, followed by anaerobic reduction of TCE and cDCE to ethene. Conventional wisdom suggests that sequential anaerobic and aerobic conditions are desirable for bioremediating sites contaminated by mixtures of polychlorinated organics. The results of this microcosm study suggest that a sequential aerobic and anaerobic approach may be more successful, although implementing this in the field presents some major challenges. In the dolomite microcosms incubated under aerobic conditions first (59 days), α - and γ -HCH were biodegraded close to the maximum contaminant level for lindane; all of the aromatic compounds were consumed; and there was partial removal of TCE and cDCE (presumptively via cometabolism). The subsequent switch to anaerobic conditions (day 101) yielded reductive dechlorination of the remaining TCE; a significant level of ethene was produced, although some cDCE and VC persisted. In contrast, sequential anaerobic (393 days) and aerobic treatment (498 days) for the dolomite microcosms was ineffective in completely removing the aromatic compounds, α -HCH, cDCE, and VC. For the soil microcosms, both treatment sequences were effective, most likely reflecting a greater abundance of the necessary microbes and electron donor in this part of the site.     

Successive rapid reductive dehalogenation and mineralization of pentachlorophenol by the indigenous microflora of farmyard manure compost

AIMS: To determine whether composting with animal manure can be used to effectively remediate soil from a pentachlorophenol (PCP)-contaminated site, and to establish the fate of the degraded xenobiotic. METHODS AND RESULTS: Contaminated soil from a sawmill site was mixed with farm animal manure and composted in a 0.5 m3 silo under fully aerobic conditions. The disappearance and fate of PCP was monitored by gas chromatography (GC-ECD) and extensive mineralization confirmed in experiments with 14C-radiolabelled PCP. The disappearance of PCP was rapid and virtually complete within 6 days, prior to the onset of thermophilic conditions. Dechlorination of the PCP was found to be both reductive and sequential. CONCLUSIONS: PCP removal from contaminated soil by aerobic composting with animal manure is efficient and proceeds via reductive dechlorination to virtually complete mineralization. This contrasts with other chlorophenol composting regimes in which mineralization is achieved but dechlorination intermediates do not accumulate to detectable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate that anaerobic reductive dechlorination can proceed in an aerobic composting environment and contribute to efficient pentachlorophenol removal. Farmyard manure composts may represent a rapid, low-cost, low-technology option for treatment of chlorophenol-contaminated soils.     

Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes

Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.     

氯酚类化合物的微生物降解研究进展

综述了近年在具有降解氯酚类化合物能力的微生物的筛选、氯酚类化合物的好氧和厌氧降解机制以及现代生物技术的开发利用研究．阐述了氯酚类化合物在不同条件下的降解路径．在好氧条件下,单氯酚和二氯酚在氧化酶的攻击下形成氯代邻二酚,邻二酚开环生成相应的氯代粘康酸或半醛,粘康酸内酯化过程中释放氯离子;高度氯代的化合物则是在氢氧化酶作用下生成氯代醌,并逐步脱去所有的氯原子生成苯酚后才开环．在厌氧或缺氧条件下,氯酚进行还原脱氯,在得到电子的同时去掉一个氯取代基．     

Formulation of the CBC-Model for Modelling the Contaminants and Footprints in Natural Attenuation of BTEX

The Refuse Hideaway Landfill (23-acre) received municipal, commercial, and industrial waste between 1974 and 1988. It was designed as a "natural attenuation" landfill and no provision was made to collect and treat contaminated water. Natural biological degradation through sequential reductive dechlorination had been an important mechanism for natural attenuation at the site. We used the concentration of hydrogen to forecast whether reductive dechlorination would continue over time at particular locations in the plume. Based on published literature, reductive dechlorination and natural attenuation of PCE, TCE, and cis-DCE can be expected in the aquifer if the concentration of molecular hydrogen in monitoring wells are adequate (> 1 nanomolar). Reductive dechlorination can be expected to continue as the ground water moves down gradient. Natural attenuation through reductive dechlorination is not expected in flow paths that originate at down gradient monitoring wells with low concentrations of molecular hydrogen (< 1 nanomolar). In three monitoring wells at the margin of the landfill and in five monitoring wells down gradient of the landfill, ground water maintained a molecular hydrogen concentration, ranging from 1.30 to 9.17 nanomolar, that is adequate for reductive dechlorination. In three of the monitoring wells far down gradient of the landfill, the concentration of molecular hydrogen (0.33 to 0.83 nanomolar) was not adequate to support reductive dechlorination. In wells with adequate concentrations of hydrogen, the concentrations of chlorinated volatile organic compounds were attenuated over time, or concentrations of chlorinated volatile organics were below the detection limit. In wells with inadequate concentrations of hydrogen, the concentrations of chlorinated organic compounds attenuated at a slower rate over time. In wells with adequate hydrogen the first order rate of attenuation of PCE, TCE, cis-DCE and total chlorinated volatile organic compounds varies from 0.38 to 0.18 per year. In wells without adequate hydrogen the rate varies from 0.015 to 0.006 per year.     

Modeling Natural Attenuation of Chlorinated Ethenes Under Spatially Varying Redox Conditions

A three-dimensional model for the transport and reductive dechlorination of chlorinated ethenes in ground-water systems with variable redox conditions is demonstrated and applied to a pilot test for accelerated natural attenuation of trichloroethene (TCE). The rate and extent of biotransformation of TCE and chlorinated progeny is controlled by the dominant terminal electron accepting process (TEAP) that is simulated over space and time. The solute transport code, Sequential Electron Acceptor Model, 3D-transport, (SEAM3D) which simulates aerobic and sequential anaerobic biodegradation of organic carbon, is modified to implement the equations. Results of a generic model for TCE transport in ground-water systems with different redox conditions demonstrate that the degree of chlorinated ethene attenuation is influenced by background concentrations of aqueous- and solid-phase electron acceptors, but that model results are sensitive to other input parameters (inhibition coefficients, maximum rate of reductive dechlorination, biomass concentrations, and ground-water velocity). Simulation results of enhanced in situ bioremediation using dissolved organic carbon as a reducing agent show that spatial and temporal changes in the dominant TEAP and the subsequent rate of reductive dechlorination are adequately represented with the model. Initial concentrations of Fe(III) and the dechlorinating microbial population influence the simulated time lag observed during the pilot test.