Isolation of a Xanthobacter sp. degrading dichloromethane and characterization of the gene involved in the degradation

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with μ_max of 0.094 h⁻¹ and S _m of 1,435 mg l⁻¹. Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM.     

Continuous dechlorination of tetrachloroethene in an upflow anaerobic sludge blanket reactor

Influences of hydraulic retention time (HRT) on dechlorination of tetrachloroethene (PCE) were investigated in an upflow anaerobic sludge blanket (UASB) reactor inoculated with anaerobic granular sludge non-pre-exposed to chlorinated compounds. PCE was introduced into the reactor at a loading rate of 3 mg/l d. PCE removal increased from 51 +/- 5% to 87 +/- 3% when HRT increased from 1 to 4 d, corresponding to an increase in the PCE biotransformation rate from 10.5 +/- 2.3 to 21.3 +/- 3.7 mumol/d. A higher ethene production rate, 0.9 +/- 0.2 mumol/d, was attained without accumulation of dichloroethenes at the HRT of 4 d. Dehalococcoides-like species were detected in sludge granules by fluorescence in situ hybridization, with signal strength in proportion to the extent of PCE dechlorination.     

Dechlorination of Pesticides by a Specific Bacterial Glutathione S‐transferase,BphKLB400: Potential for Bioremediation

The bphK gene located in the bph operon of Burkholderia xenovorans LB400 encodes a protein, BphK^LB400, with significant sequence similarity to glutathione‐S‐transferases (GSTs). GSTs are a superfamily of enzymes involved in the detoxification of many endobiotic and xenobiotic substances. Recently, BphK^LB400 was shown to catalyze the dechlorination of a number of toxic chlorinated organic compounds. Comparison of the amino acid sequence of BphK^LB400 with GSTs from other bacteria that degrade polychlorinated biphenyls identified a number of highly conserved amino acids in the C‐terminal region of the protein thought to be associated with substrate specificity. Mutating the conserved amino acid at position 180 of BphK^LB400 from an alanine to a proline residue resulted in an increase in GST activity of bacterial cell extracts towards a number of chlorinated organic substrates tested including commonly used pesticides. Laboratory scale plant protection experiments suggested that E. coli expressing BphK^LB400 [wildtype and mutant (Ala180Pro)] could protect pea plants from the effects of chloromequat chloride. Therefore, BphK^LB400, identified as having dechlorination activity towards toxic chlorinated organic compounds used in the environment, could have potential in bioremediation.     

Microcosm evaluation of bioaugmentation after field-scale thermal treatment of a TCE-contaminated aquifer

This paper investigates effects of combining thermal and biological remediation, based on laboratory studies of trichloroethene (TCE) degradation. Aquifer material was collected 6 months after terminating a full-scale Electrical Resistance Heating (ERH), when the site had cooled from approximately 100°C to 40°C. The aquifer material was used to construct bioaugmented microcosms amended with the mixed anaerobic dechlorinating culture, KB-1^TM, and an electron donor (5 mM lactate). Microcosms were bioaugmented during cooling at 40, 30, 20, and 10°C, as temperatures continually decreased during laboratory incubation. Redox conditions were generally methanogenic, and electron donors were present to support dechlorination. For microcosms bioaugmented at 10°C and 20°C, dechlorination stalled at cis-dichloroethene (cDCE) and vinyl chloride (VC) 150 days after bioaugmentation. However, within 300 days of incubation ethene was produced in the majority of these microcosms. In contrast, dechlorination was rapid and complete in microcosms bioaugmented at 30°C. Microcosms bioaugmented at 40°C also showed rapid dechlorination, but stalled at cDCE with partial VC and ethene production, even after 150 days of incubation when the temperature had decreased to 10°C. These results suggest that sequential bioremediation of TCE is possible in field-scale thermal treatments after donor addition and bioaugmentation and that the optimal bioaugmentation temperature is approximately 30°C. When biological and thermal remediations are to be applied at the same location, three bioremediation approaches could be considered: (a) treating TCE in perimeter areas outside the source zone at temperatures of approximately 30°C; (b) polishing TCE concentrations in the original source zone during cooling from approximately 30°C to ambient groundwater temperatures; and (c) using bioremediation in downgradient areas taking advantages of the higher temperature and potential release of organic matter.     

Isolation and characterization of novel Serratia marcescens (AY927692) for pentachlorophenol degradation from pulp and paper mill waste

Seven aerobic bacterial strains were isolated from pulp paper mill waste and screened for pentachlorophenol (PCP) tolerance on PCP containing mineral salt agar medium (MSM). The organism was characterized by 16S rDNA sequencing which showed 99.7% sequence similarity with Serratia marcescens. PCP degradation was routinely monitored with spectrophotometric analysis and further confirmed by HPLC analysis. Among seven strains, ITRC S₇ was found to degrade up to 90.33% of 1.127 mM (300 mg/l) of PCP and simultaneous release of chloride ion (2.435 mM) emphasized the bacterial dechlorination in the medium in presence of glucose as an additional carbon and energy source under optimized condition within 168 h incubation. In absence of glucose bacterium was unable to utilize PCP indicating the phenomenon of co-metabolism. Bacterium was identified as S. marcescens (AY927692), was a novel and potential aerobic bacterial strain capable of degrading PCP in axenic condition. Further, this strain may be used for bioremediation of PCP containing pulp paper mill waste in the environment.     

Cometabolic degradation of mono-chloro benzoic acids by <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. R04 grown on organic carbon sources

The aerobic cometabolism of chlorobenzoic acids (CBAs) by Rhodococcus sp. R04 was accomplished by augmenting the medium with organic carbon sources. In mineral medium supplemented with glucose (MMG), 0.5 mM 2-CBA was incompletely metabolized after the 5-day incubation, while the near-complete disappearance of 0.5 mM 4-CBA was monitored. Over the 5-day incubation period, the concentration of chloride increased to 0.17 mM in bottles containing 4-CBA, glucose and strain R04; whereas in cultivation with 2-CBA the chloride content was about 0.1 mM. After 5-day incubation, 28.5% 4-CBA was remained in mineral medium supplemented with ethanol (MME), and the relatively low values of chloride were released. To our knowledge, it is first report that the feasibility of using ethanol as an added substrate for cometabolic degradation of CBA by aerobic polychlorinated biphenyl (PCB)-degrading bacteria. The specific activities of (chloro)benzoate 1,2-dioxygenase and (chloro)catechol 1,2-dioxygenase activities were detected in cell-free extracts (CFEs) of strain R04. These results suggest that the initial degradation of CBAs occurred most likely prior to chloride release.     

Microbial degradation of chlorinated phenols

Chlorophenols have been introduced into the environment through their use as biocides and as by-products of chlorine bleaching in the pulp and paper industry. Chlorophenols are subject to both anaerobic and aerobic metabolism. Under anaerobic conditions, chlorinated phenols can undergo reductive dechlorination when suitable electron-donating substrates are available. Halorespiring bacteria are known which can use both low and highly chlorinated congeners of chlorophenol as electron acceptors to support growth. Many strains of halorespiring bacteria have the capacity to eliminate ortho-chlorines; however only bacteria from the species Desulfitobacterium hafniense (formerly frappieri) can eliminate para- and meta-chlorines in addition to ortho-chlorines. Once dechlorinated, the phenolic carbon skeletons are completely converted to methane and carbon dioxide by other anaerobic microorganisms in the environment. Under aerobic conditions, both lower and higher chlorinated phenols can serve as sole electron and carbon sources supporting growth. The best studied strains utilizing pentachlorophenol belong to the genera Mycobacterium and Sphingomonas. Two main strategies are used by aerobic bacteria for the degradation of chlorophenols. Lower chlorinated phenols for the most part are initially attacked by monooxygenases yielding chlorocatechols as the first intermediates. On the other hand, polychlorinated phenols are converted to chlorohydroquinones as the initial intermediates. Fungi and some bacteria are additionally known that cometabolize chlorinated phenols.     

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.     

The aerobic dechlorination activities of two bacterial species isolated from a refuse dumpsite in Nigeria

Two bacterial species isolated using enrichment culture techniques from the topsoil of a main refuse dumpsite in Nigeria were assessed for their dehalogenation potentials. The bacterial isolates were identified as belonging to the Bacillus and Pseudomonas genera. Axenic cultures of the isolates utilized monochloroacetic acid (MCA), trichloroacetic acid (TCA), trichloromethane (CHCl₃) and tetrachloromethane (CCl₄) as the sole source of carbon for growth up to a final substrate concentration of 0.1% (w/v). The mean generation times of the isolates in all the growth media ranged significantly (P<0.05) from 2.41 to 10.04 h and were generally higher than that observed in glucose medium (1.46–1.51 h). The numbers of the chloride atoms in the different organochlorides were negatively correlated with the ability of the organisms to degrade the compounds. Dehalogenase specific activities of the cell-mediated cultures ranged from 0.1 to 0.96 μg ml^–1 chloride release (mg protein)^–1 h^–1 and were significantly (P <0.05) higher than that of the cell-free extract [0.09–0.8 μg ml^–1 chloride release (mg protein)^–1 h^–1]. The optimal pH of the dehalogenase activity was found to be 8.0, and the optimal temperature was between 30 and 35 °C. Electronic Publication     

Microbial degradation of chlorinated benzenes

Chlorinated benzenes are important industrial intermediates and solvents. Their widespread use has resulted in broad distribution of these compounds in the environment. Chlorobenzenes (CBs) are subject to both aerobic and anaerobic metabolism. Under aerobic conditions, CBs with four or less chlorine groups are susceptible to oxidation by aerobic bacteria, including bacteria (Burkholderia, Pseudomonas, etc.) that grow on such compounds as the sole source of carbon and energy. Sound evidence for the mineralization of CBs has been provided based on stoichiometric release of chloride or mineralization of (14)C-labeled CBs to (14)CO(2). The degradative attack of CBs by these strains is initiated with dioxygenases eventually yielding chlorocatechols as intermediates in a pathway leading to CO(2) and chloride. Higher CBs are readily reductively dehalogenated to lower chlorinated benzenes in anaerobic environments. Halorespiring bacteria from the genus Dehalococcoides are implicated in this conversion. Lower chlorinated benzenes are less readily converted, and mono-chlorinated benzene is recalcitrant to biotransformation under anaerobic conditions.