Enrichment and Properties of a 1,2,4-Trichlorobenzene-Dechlorinating Methanogenic Microbial Consortium

A methanogenic microbial consortium capable of reductively dechlorinating 1,2,4-trichlorobenzene (1,2,4-TCB) was enriched from a mixture of polluted sediments. 1,2,4-TCB was dechlorinated via 1,4-dichlorobenzene (1,4-DCB) to chlorobenzene (CB). Lactate, which was used as an electron donor during the enrichment, was converted via propionate and acetate to methane. Glucose, ethanol, methanol, propionate, acetate, and hydrogen were also suitable electron donors for dechlorination, whereas formate was not. The addition of 5% (wt/vol) sterile Rhine River sand was necessary to maintain the dechlorinating activity of the consortium. The addition of 2-bromoethanesulfonic acid (BrES) inhibited methanogenesis completely but had no effect on the dechlorination of 1,2,4-TCB. The consortium was also able to dechlorinate other chlorinated benzenes via various simultaneous pathways to 1,3,5-TCB, 1,2-DCB, 1,3-DCB, or CB as an end product. The addition of BrES inhibited several of the simultaneously occurring dechlorination pathways of 1,2,3,4- and 1,2,3,5-tetrachlorobenzene and of pentachlorobenzene, which resulted in the formation of CB as the only final product. Hexachlorobenzene and polychlorinated biphenyls (PCBs) were dechlorinated after a lag phase of ca. 15 days, showing a dechlorination pattern that is different from those observed for lower chlorinated benzenes: only chlorines with two adjacent chlorines were removed. The results show that the consortium possesses at least three distinct dechlorination activities toward chlorinated benzenes and PCBs.     

Dehalorespiration with hexachlorobenzene and pentachlorobenzene by<Emphasis Type="Italic"> Dehalococcoides</Emphasis> sp. strain CBDB1

The chlororespiring anaerobe Dehalococcoides sp. strain CBDB1 used hexachlorobenzene and pentachlorobenzene as electron acceptors in an energy-conserving process with hydrogen as electron donor. Previous attempts to grow Dehalococcoides sp. strain CBDB1 with hexachlorobenzene or pentachlorobenzene as electron acceptors failed if these compounds were provided as solutions in hexadecane. However, Dehalococcoides sp. strain CBDB1 was able to grow with hexachlorobenzene or pentachlorobenzene when added in crystalline form directly to cultures. Growth of Dehalococcoides sp. strain CBDB1 by dehalorespiration resulted in a growth yield (Y) of 2.1±0.24 g protein/mol Cl^– released with hexachlorobenzene as electron acceptor; with pentachlorobenzene, the growth yield was 2.9±0.15 g/mol Cl^–. Hexachlorobenzene was reductively dechlorinated to pentachlorobenzene, which was converted to a mixture of 1,2,3,5- and 1,2,4,5-tetrachlorobenzene. Formation of 1,2,3,4-tetrachlorobenzene was not detected. The final end-products of hexachlorobenzene and pentachlorobenzene dechlorination were 1,3,5-trichlorobenzene, 1,3- and 1,4-dichlorobenzene, which were formed in a ratio of about 3:2:5. As reported previously, Dehalococcoides sp. strain CBDB1 converted 1,2,3,5-tetrachlorobenzene exclusively to 1,3,5-trichlorobenzene, and 1,2,4,5-tetrachlorobenzene exclusively to 1,2,4-trichlorobenzene. The organism therefore catalyzes two different pathways to dechlorinate highly chlorinated benzenes. In the route leading to 1,3,5-trichlorobenzene, only doubly flanked chlorine substituents were removed, while in the route leading to 1,3-and 1,4-dichlorobenzene via 1,2,4-trichlorobenzene singly flanked chlorine substituents were also removed. Reductive dehalogenase activity measurements using whole cells pregrown with different chlorobenzene congeners as electron acceptors indicated that different reductive dehalogenases might be induced by the different electron acceptors. To our knowledge, this is the first report describing reductive dechlorination of hexachlorobenzene and pentachlorobenzene via dehalorespiration by a pure bacterial culture.     

Reductive dechlorination of hexachlorobenzene to tri- and dichlorobenzenes in anaerobic sewage sludge.

Hexachlorobenzene was dechlorinated to tri- and dichlorobenzenes in anaerobic sewage sludge. The complete biotransformation of 190 microM hexachlorobenzene (approximately 50 ppm) occurred within 3 weeks. The calculated rate of hexachlorobenzene dechlorination was 13.6 mumol liter-1 day-1. Hexachlorobenzene was dechlorinated via two routes, both involving the sequential removal of chlorine from the aromatic ring. The major route was hexachlorobenzene----pentachlorobenzene----1,2,3,5-tetrachlorobenzene--- -1,3,5- trichlorobenzene. Greater than 90% of the added hexachlorobenzene was recovered as 1,3,5-trichlorobenzene, and there was no evidence for further dechlorination of 1,3,5-trichlorobenzene. The minor route was hexachlorobenzene----pentachlorobenzene----1,2,4,5-tetrachlorobenzene--- -1,2,4- trichlorobenzene----dichlorobenzenes. These results extend reductive dechlorination to poorly water soluble aromatic hydrocarbons which could potentially include other important environmental pollutants like polychlorinated biphenyls.     

Biodegradation of 1,2,3- and 1,2,4-Trichlorobenzene in Soil and in Liquid Enrichment Culture

The biodegradation of radiochemically pure (99%) 1,2,3- and 1,2,4-trichlorobenzene (TCB) in soil was investigated. Experimental difficulties posed by the high volatility and slow biodegradation rate of the TCBs were partially overcome by using a specially designed incubation and trapping apparatus. Evolution of ¹⁴CO₂ from active versus poisoned soil dosed with 50 μg of the individual TCBs per g gave conclusive proof that both isomers are biodegradable. At 20°C, 1,2,4-TCB was mineralized at an approximate rate of 1 nmol/day per 20 g of soil sample, and 1,2,3-TCB was mineralized at one-half to one-third that rate. Mineral fertilizers or cosubstrates failed to increase TCB mineralization rates in soil. Anaerobic conditions had a negative effect on mineralization, and increased temperatures had a positive effect. With increasing 1,2,4-TCB concentrations, ¹⁴CO₂ evolution exhibited saturation kinetics with an apparent K_m of 55.5 nmol per g of soil. Recovery of total radioactivity was good from soil containing high organic matter concentrations. From low-organic-matter soil, some of the radioactivity was recovered only on combustion, and overall recovery was lower. In soil-inoculated liquid culture, the cosubstrates glucose and benzene caused a slight stimulation of 1,2,4-TCB mineralization. Cochromatography of known standards with the extracts of soil pretreated with [¹⁴C]TCBs indicated that 3,4,5-trichlorophenol, 2,6-dichlorophenol and, to a lesser degree, 2,3-dichlorophenol were present in soils incubated with 1,2,3-TCB. 2,4-, 2,5-, and 3,4-dichlorophenol were present in soils incubated with 1,2,4-TCB.     

Biodegradation of 1,2,3- and 1,2,4-trichlorobenzene in soil and in liquid enrichment culture.

The biodegradation of radiochemically pure (99%) 1,2,3- and 1,2,4-trichlorobenzene (TCB) in soil was investigated. Experimental difficulties posed by the high volatility and slow biodegradation rate of the TCBs were partially overcome by using a specially designed incubation and trapping apparatus. Evolution of (14)CO(2) from active versus poisoned soil dosed with 50 mug of the individual TCBs per g gave conclusive proof that both isomers are biodegradable. At 20 degrees C, 1,2,4-TCB was mineralized at an approximate rate of 1 nmol/day per 20 g of soil sample, and 1,2,3-TCB was mineralized at one-half to one-third that rate. Mineral fertilizers or cosubstrates failed to increase TCB mineralization rates in soil. Anaerobic conditions had a negative effect on mineralization, and increased temperatures had a positive effect. With increasing 1,2,4-TCB concentrations, (14)CO(2) evolution exhibited saturation kinetics with an apparent K(m) of 55.5 nmol per g of soil. Recovery of total radioactivity was good from soil containing high organic matter concentrations. From low-organic-matter soil, some of the radioactivity was recovered only on combustion, and overall recovery was lower. In soil-inoculated liquid culture, the cosubstrates glucose and benzene caused a slight stimulation of 1,2,4-TCB mineralization. Cochromatography of known standards with the extracts of soil pretreated with [(14)C]TCBs indicated that 3,4,5-trichlorophenol, 2,6-dichlorophenol and, to a lesser degree, 2,3-dichlorophenol were present in soils incubated with 1,2,3-TCB. 2,4-, 2,5-, and 3,4-dichlorophenol were present in soils incubated with 1,2,4-TCB.     

Effects of Polychlorinated Biphenyl Congener Concentration and Sediment Supplementation on Rates of Methanogenesis and 2,3,6-Trichlorobiphenyl Dechlorination in an Anaerobic Enrichment

We have employed a method of enrichment that allows us to significantly increase the rate of reductive polychlorinated biphenyl (PCB) dechlorination. This method shortens the time required to investigate the effects that culture conditions have on dechlorination and provides an estimate of the potential activity of the PCB-dechlorinating anaerobes. The periodic supplementation of sterile sediment and PCB produced an enhanced, measurable, and sustained rate of dechlorination. We observed volumetric rates of the dechlorination of 2,3,6-trichlorobiphenyl (2,3,6-CB) to 2,6-dichlorobiphenyl (2,6-CB) of more than 300 μmol liter^-1 day^-1 when the cultures were supplemented daily. A calculation of this activity that is based on an estimate of the number of dechlorinating anaerobes present indicates that 1.13 pmol of 2,3,6-CB was dechlorinated to 2,6-CB day^-1 bacterial cell^-1. This rate is similar to that of the reductive dechlorination of 3-chlorobenzoate by Desulfomonile tiedjei. Methanogenesis declined from 585.3 to 125.9 μmol of CH₄ liter^-1 day^-1, while dechlorination increased from 8.2 to 346.0 μmol of 2,3,6-CB dechlorinated to 2,6-CB liter^-1 day^-1.     

Diversity of dechlorination pathways and organohalide respiring bacteria in chlorobenzene dechlorinating enrichment cultures originating from river sludge

Anaerobic reductive dechlorination of hexachlorobenzene (HCB) and three isomers of tetrachlorobenzene (TeCB) (1,2,3,4-, 1,2,3,5- and 1,2,4,5-TeCB) was investigated in microcosms containing chloroaromatic contaminated river sediment. All chlorobenzenes were dechlorinated to dichlorobenzene (DCB) or monochlorobenzene. From the sediment, a methanogenic sediment-free culture was obtained which dechlorinated HCB, pentachlorobenzene, three TeCB isomers, three trichlorobenzene (TCB) isomers (1,2,3-, 1,2,4- and 1,3,5-TCB) and 1,2-DCB. Dechlorination involved multiple pathways including the removal of doubly flanked, singly flanked and isolated chlorine substituents. 454-pyrosequencing of partial bacterial 16S rRNA genes amplified from selected chlorobenzene dechlorinating sediment-free enrichment cultures revealed the presence of a variety of bacterial species, including Dehalobacter and Dehalococcoides mccartyi, that were previously documented as organohalide respiring bacteria. A genus with apparent close relationship to Desulfitobacterium that also has been associated with organohalide respiration, composed the major fraction of the operational taxonomic units (OTUs). Another major OTU was linked with Sedimentibacter sp., a genus that was previously identified in strict co-cultures of consortia reductively dehalogenating chlorinated compounds. Our data point towards the existence of multiple interactions within highly chlorinated benzene dechlorinating communities.     

Degradation of 1,2,4-Trichloro- and 1,2,4,5-Tetrachlorobenzene by Pseudomonas Strains

Two Pseudomonas sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon and energy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grew on monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). Strain PS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds both strains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and 1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. The intermediary cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCB was rearomatized by an NAD⁺-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from the initially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent ortho cleavage was catalyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which was channeled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present case included 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitro group was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for the oxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on these haloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.     

Mineralization of mono- and dichlorobenzenes and simultaneous degradation of chloro- and methyl-substituted benzenes by the white rot fungus Phanerochaete chrysosporium.

Phanerochaete chrysosporium extensively degraded and mineralized chlorobenzene and o-, m-, and p-dichlorobenzenes. The rate of degradation was in the following order: monochlorobenzene > m-dichlorobenzene > o-dichlorobenzene > p-dichlorobenzene. Net level of degradation was generally higher than mineralization. Maximal degradation and mineralization of chlorobenzenes were observed in malt extract cultures in which the lignin peroxidases and manganese peroxidases are not known to be produced. The fungus degraded both chlorobenzene and toluene when presented as a mixture, indicating its ability to simultaneously degrade chloro-substituted and methyl-substituted benzenes.     

Anaerobic ortho Dechlorination of Polychlorinated Biphenyls by Estuarine Sediments from Baltimore Harbor

Reductive dechlorination of the ortho moiety of polychlorinated biphenyls (PCBs) as well as of meta and para moieties is shown to occur in anaerobic enrichments of Baltimore Harbor sediments. These estuarine sediments ortho dechlorinated 2,3,5,6-chlorinated biphenyl (CB), 2,3,5-CB, and 2,3,6-CB in freshwater or estuarine media within a relatively short period of 25 to 44 days. ortho dechlorination developed within 77 days in marine medium. High levels of ortho dechlorination (>90%) occurred when harbor sediments were supplied with only 2,3,5-CB. Incubation with 2,3,4,5,6-CB or 2,3,4,5-CB resulted in the formation of the ortho dechlorination product 3,5-CB; however, para dechlorination of these congeners always preceded ortho chlorine removal. ortho dechlorination of PCBs is an exceedingly rare event that has not been reported previously for marine or estuarine conditions. The activity was reproducible and could be sustained through sequential transfers. In contrast, freshwater sediments incubated under the same conditions exhibited only meta and para dechlorinations. The results indicate that unique anaerobic dechlorinating activity is catalyzed by microorganisms in the estuarine sediments from Baltimore Harbor.     

Induction of hydroxyl radical production in Trametes versicolor to degrade recalcitrant chlorinated hydrocarbons

Extracellular hydroxyl radical (OH) production via quinone redox cycling in Trametes versicolor, grown in a chemically defined medium, was investigated to degrade trichloroethylene (TCE), perchloroethylene (PCE), 1,2,4- and 1,3,5-trichlorobenzene (TCB). The activity of the enzymes catalyzing the quinone redox cycle, quinone reductase and laccase, as well as the rate of OH production, estimated as the formation of thiobarbituric acid reactive substances (TBARS) from 2-deoxyribose, increased rapidly during the first 2–3 days and then remained at relatively constant levels. Under quinone redox cycling conditions, TCE degradation was concomitant to TBARS production and chloride release, reaching a plateau after 6 h of incubation. Similar results were obtained in PCE, 1,2,4- and 1,3,5-TCB time course degradation experiments. The mole balance of chloride release and 1,2,4-TCB and TCE degraded suggests that these chemicals were almost completely dechlorinated. Experiments using [¹³C]-TCE confirmed unequivocal transformation of TCE to ¹³CO₂. These results are of particular interest because PCE and 1,3,5-TCB degradation in aerobic conditions has been rarely reported to date in bacterial or fungal systems.     

Reducing effect of aerobic selector on the toxicity of synthetic organic compounds in activated sludge process

The effects of phenol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP) and 1,2,4-trichlorobenzene (1,2,4-TCB) on the biodegradation kinetics of the conventional activated sludge system (CASS) and the selector activated sludge system (SASS) were investigated. Experiments were carried out using a respirometric method on unacclimated biomass from two lab-scale systems that were operated with the sludge age of 8 days. Toxicity of the test compounds for both reactors were arranged according to EC₅₀ (effective concentration) values in order as: 1,2,4-TCB > 2,4-DCP > 2-CP > phenol. All selected test compounds induced higher inhibition effect in the CASS. The SASS appeared to reduce inhibition effect in comparison to the CASS, by 21.36%, 66.95%, 64.37% and 33.33% for phenol, 2-CP, 2,4-DCP and 1,2,4-TCB, respectively. Consequently, the SASS may be recommended as a promising configuration alternative for the waste streams containing toxic compounds.     

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.     

Aerobic Mineralization of Hexachlorobenzene by Newly Isolated Pentachloronitrobenzene-Degrading Nocardioides sp. Strain PD653

A novel aerobic pentachloronitrobenzene-degrading bacterium, Nocardioides sp. strain PD653, was isolated from an enrichment culture in a soil-charcoal perfusion system. The bacterium also degraded hexachlorobenzene, a highly recalcitrant environmental pollutant, accompanying the generation of chloride ions. Liberation of ¹⁴CO₂ from [U-ring-¹⁴C]hexachlorobenzene was detected in a culture of the bacterium and indicates that strain PD653 is able to mineralize hexachlorobenzene under aerobic conditions. The metabolic pathway of hexachlorobenzene is initiated by oxidative dechlorination to produce pentachlorophenol. As further intermediate metabolites, tetrachlorohydroquinone and 2,6-dichlorohydroquinone have been detected. Strain PD653 is the first naturally occurring aerobic bacteria capable of mineralizing hexachlorobenzene.Hexachlorobenzene (C₆Cl₆; HCB) is one of the most persistent environmental pollutants. Its average half-life in soil is approximately 9 years (2). When HCB is liberated in environment, it is bioaccumulated in plants, zooplankton, and shellfish. Finally, HCB is accumulated in the human body via the food chain, whereupon its possible toxicity adversely affects human health as a result of long-term exposure and accumulation. Therefore, HCB was listed as one of the 12 persistent organic pollutants in the Stockholm Convention.A number of studies have been attempted to develop cleanup technology for environmental pollutants. Microbial degradation is a promising effective way to remediate environmental pollutants, including persistent organic pollutants. However, heavily chlorinated benzenes, especially HCB, are resistant to microbial degradation. Several studies have been reported on the reductive dechlorination of HCB. Reductive dechlorination of HCB to pentachlorobenzene by cytochrome P-450 was found in rat hepatic microsomes (22). Microbial transformation of HCB to trichlorobenzene and dichlorobenzene by reductive dechlorination was observed in anaerobic sewage sludge and a mixed culture (5, 7). Yeh and Pavlostathis maintained such an HCB-dechlorinating mixed culture for more than 1 year by adding surfactants as carbon sources (30). One of the microorganisms that reductively dechlorinates HCB is “Dehalococcoides” sp. strain CBDB1 (12). Dehalococcoides sp. strain CBDB1 dechlorinated HCB and pentachlorobenzene via dehalorespiration and gave a final end product mixture comprised of 1,3,5-trichlorobenzene, 1,3-dichlorobenzene, and 1,4-dichlorobenzene. These reductive dechlorinating processes take a longer time and leave less-chlorinated compounds such as trichlorobenzene and dichlorobenzene as end products.Strictly aerobic, naturally occurring microorganisms that degrade and completely mineralize HCB have not been found. On the other hand, a microorganism capable of mineralizing pentachlorophenol (PCP), Sphingobium chlorophenolicum strain ATCC 39723, was isolated, and its gene organization involved in PCP metabolism was shown (4). Conversion of HCB to PCP was reported by using the genetically engineered mutant of cytochrome P-450_cam (CYP101) (13). Wild-type CYP101 from Pseudomonas putida had low degrading activity for dichlorobenzene and trichlorobenzene but did not decompose more highly chlorinated benzenes. The F87W/Y96F/V247L mutant showed improved di- and trichlorobenzene-degrading activity, but activity toward highly chlorinated benzenes including HCB was still low. The activity upon highly chlorinated benzenes was further improved in the mutant CYP101, F87W/Y96F/L244A/V247L (6). The rate of HCB degradation was increased 200-fold in the mutant. Yan et al. introduced the mutant CYP101 gene into S. chlorophenolicum strain ATCC 39723 by homologous recombination, to produce a complete HCB degrader (28). This genetically engineered bacterium degraded HCB almost completely within 12 h, together with formation of PCP as an intermediate. However, the application of genetically engineered microorganisms in natural areas is strictly restricted in many countries. HCB-degrading aerobes derived from natural sources are still required for remediation of HCB-contaminated areas.We describe here isolation and identification of a novel aerobic soil bacterial species capable of aerobically mineralizing HCB. The characterization of metabolites caused by oxidative removal of the chlorine groups from HCB is also described.     

Substrate Interactions during the Biodegradation of Benzene, Toluene, Ethylbenzene, and Xylene (BTEX) Hydrocarbons by the Fungus Cladophialophora sp. Strain T1

The soil fungus Cladophialophora sp. strain T1 (= ATCC MYA-2335) was capable of growth on a model water-soluble fraction of gasoline that contained all six BTEX components (benzene, toluene, ethylbenzene, and the xylene isomers). Benzene was not metabolized, but the alkylated benzenes (toluene, ethylbenzene, and xylenes) were degraded by a combination of assimilation and cometabolism. Toluene and ethylbenzene were used as sources of carbon and energy, whereas the xylenes were cometabolized to different extents. o-Xylene and m-xylene were converted to phthalates as end metabolites; p-xylene was not degraded in complex BTEX mixtures but, in combination with toluene, appeared to be mineralized. The metabolic profiles and the inhibitory nature of the substrate interactions indicated that toluene, ethylbenzene, and xylene were degraded at the side chain by the same monooxygenase enzyme. Our findings suggest that soil fungi could contribute significantly to bioremediation of BTEX pollution.     

Chlorophenol dechlorination and subsequent degradation in denitrifying microcosms fed low concentrations of nitrate

The potential of using nitrate as a terminal electron acceptor to stimulate anaerobic degradation of mixtures of monochlorophenols (MCPs) or dichlorophenols (DCPs) was evaluated. Contaminated and non-contaminated soils were added to water saturated anaerobic microcosms supplemented with 1 mM or 5 mM nitrate. Denitrification and dechlorination activity were present in three diverse soil types and were maintained upon refeeding both nitrate and the appropriate chlorophenol. However, dechlorination activity could only be serially transferred in enrichments with an added electron donor such as acetate. Dehalogenation activity in enrichments from four of the primary microcosms showed at least five different dechlorination reactions, each mediated by different microbial communities. Three of these are distinct ortho-dechlorinating paths; two are meta-dechlorinating and one is the para-dechlorination of 3,4-DCP. Simultaneous dechlorination and denitrification was observed and both activities could be maintained in microcosms but only in the presence of low nitrate concentrations. Dechlorination and denitrification were mediated by two separate microbial communities; one that dechlorinates without use of nitrate and one that denitrifies while oxidizing the dechlorinated aromatic ring. There was no evidence that dechlorination is mediated by the denitrifying community, however the maintenance of a denitrification potential using low (< 1 mM) nitrate concentrations may be useful for completing the food chain by stimulating the mineralization of phenol and benzoate.     

Bacterial cometabolic degradation of chlorinated paraffins

Summary Cometabolic dechlorination of chlorinated paraffins was demonstrated in the presence of n-hexadecane by bacterial strains (HK-3, HK-6, HK-8, and HK-10) isolated from soil samples.Eleven per cent of chlorine of chlorinated paraffin-150 (CP-150) was released by strain HK-3. The mixed culture of strain HK-3, catalyzing the dechlorination of terminal chlorine of chloroalkane, and strain H15-4, capable of releasing the chlorine from 2-chlorinated fatty acids, dechlorinated CP-150 up to 13%. The mixed culture of the four strains (HK-3, HK-6, HK-8, and HK-10) performed the dechlorination of CP-150 by cometabolism in a jar fermentor pH at 7.0. The amount of chloride released from the chlorinated paraffins tested was in the range of 15–57%.The activated sludge acclimatized to n-hexadecane for 60 days showed a little dechlorination activity to CP-150.     

Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT

The cooperation of Bacillus subtilis strain DKT and Comamonas testosteroni KT5 was investigated for biofilm development and toluenes and chlorobenzenes degradation. Bacillus subtilis strain DKT and C. testosteroni KT5 were co-cultured in liquid media with toluenes and chlorobenzenes to determine the degradation of these substrates and formation of dual-species biofilm used for the degradation process. Bacillus subtilis strain DKT utilized benzene, mono- and dichlorinated benzenes as carbon and energy sources. The catabolism of chlorobenzenes was via hydroxylation, in which chlorine atoms were replaced by hydroxyl groups to form catechol, followed by ring fission via the ortho-cleavage pathway. The investigation of the dual-species biofilm composed of B. subtilis DKT and C. testosteroni KT5 (a toluene and chlorotoluene-degrading isolate with low biofilm formation) showed that B. subtilis DKT synergistically promoted C. testosteroni KT5 to develop biofilm. The bacterial growth in dual-species biofilm overcame the inhibitory effects caused by monochlorobenzene and 2-chlorotoluene. Moreover, the dual-species biofilm showed effective degradability toward the mixture of these substrates. This study provides knowledge about the commensal relationships in a dual-culture biofilm for designing multispecies biofilms applied for the biodegradation of toxic organic substrates that cannot be metabolized by single-organism biofilms.

     

Degradation of polychlorinated phenols by Streptomyces rochei 303

The strain Streptomyces rochei 303 (VKM Ac-1284D) is capable of utilizing 2-chloro-,2,4-,2,6-dichloro- and 2,4,6-trichlorophenols as the sole source of carbon. Its resting cells completely dechlorinated and degraded 2-, 3-chloro-; 2,4-, 2,6-, 2,3-, 2,5-, 3,4-, 3,5-dichloro-; 2,4-, 2,6-dibromo-; 2,4,6-, 2,4,5-, 2,3,4-, 2,3,5-, 2,3,6-trichlorophenols; 2,3,5,6-tetrachloro- and pentachlorophenol. During chlorophenol degradation, a stoichiometric amount of chloride ions was released and chlorohydroquinols were formed as intermediates. In cell-free extracts of S. rochei, the activity of hydroxyquinol 1,2-dioxygenase was found. The enzyme was induced with chlorophenols. Of all so far described strains degrading polychlorophenols, S. rochei 303 utilized a wider range of chlorinated phenols as the sole sourse of carbon and energy.Abbreviations CP chlorophenol - DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - DBrP dibromophenol - CHQ chlorohydroquinol - DCHQ dichlorohydroquinol - HHQ hydroxyhydroquinol - CHHQ chlorohydroxyhydroquinol - CC chlorocatechol - TLC thin layer chromatography - GC/MC chromato-mass-spectrometry - HPLC high-performance liquid chromatography     

Toxicity and kinetic parameters of the aerobic biodegradation of the phenol and alkylphenols by a mixed culture

A mixed culture aerobically metabolized phenol, cresol isomers (o-,m-,p-), 2-ethylphenol and xylenol isomers (2,5-DMP and 3,4-DMP) as the sole carbon and energy source. This culture had a high tolerance towards phenol with values of maximum degradation rate (V_\max) of 47 M phenol mg^–1 protein h^–1 and inhibition substrate constant (Ki) of 10 mM. These kinetic parameters were considerably diminished and the toxicity increased with the alkylphenols. For example with 2,5-xylenol, V_\max and Ki values of 0.8 M 2,5-xylenol mg^–1 protein h^–1 and 1.3 mM, respectively, were obtained. The cresols were 5-fold more toxic than phenol, whereas 2-ethylphenol and 3,4-xylenol were 11-fold more toxic, and 2,5-xylenol was 34-fold more toxic than phenol.