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PubMed1988

Sensitivity of ruminal microorganisms to pentachlorophenol.

Pentachlorophenol (PCP) is used extensively as a biocidal agent, and there is considerable concern about the adverse effects of this compound in biological ecosystems. The effects of PCP on the growth and fermentative activity of cultures of mixed ruminal microorganisms and the sensitivity of 14 ruminal bacterial species to PCP in pure culture were examined in this study. Increasing concentrations of PCP (9.4 to 375.4 microM) depressed growth and propionate concentrations in cultures of mixed ruminal microorganisms. Wide differences in the sensitivities of ruminal bacterial strains to various concentrations of PCP were observed. Cellulolytic strains were highly sensitive to PCP, while amylolytic, sugar-utilizing, and intermediate acid-utilizing strains were more resistant. Growth of major succinate-producing strains was depressed by PCP. Strains which depend on substrate level phosphorylation appeared to be more resistant. The data suggest that the adverse effects of PCP on ruminal microorganisms may be the result of its role as both an uncoupler of electron transport and a protonophore.

Animals↗
PubMed1988

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).

Biodegradation, Environmental↗
PubMed1989

Degradation of pentachlorophenol by polyurethane-immobilized Flavobacterium cells.

Polyurethane-immobilized Flavobacterium cells (ATCC 39723) degraded pentachlorophenol (PCP) at initial concentrations as high as 300 mg liter-1. The reversible binding of PCP to the polyurethane was shown to be important in the protection of the cells from inhibition of PCP degradation. The degradation activity of the bacteria was monitored for 150 days in semicontinuous batch reactors. The degradation rate dropped by about 0.6% per day. PCP was degraded in a continuous-culture bioreactor at a rate of 3.5 to 4 mg g of foam-1 day-1 for 25 days. Electron micrographs of the polyurethane suggested that the cells were entrapped within 50- to 500-microns-diameter pockets in the foam.

Biodegradation, Environmental↗
PubMed1990

Degradation of pentachlorophenol by a Flavobacterium species grown in continuous culture under various nutrient limitations.

A Flavobacterium sp. was grown in continuous culture limited for growth with ammonium, phosphate, sulfate, glucose, glucose + pentachlorophenol (PCP) (0.065 h -1), or PCP. Cells ere harvested, washed, and suspended to 3 x 10(7) cells ml (-1) in shake flasks containing a complete mineral salts medium without added carbon or supplemented with 50 mg of PCP ml(-1) or 50 mg of PCP ml(-1) + 100 mg of glucose ml(-1). The PCP concentration and the viable cell density were determined periodically. Cells that were grown under phosphate, glucose, or glucose + PCP limitation were more sensitive to PCP and took longer to degrade 50 mg of PCP ml(-1) than did cells that very were grown under ammonium, sulfate, or PCP limitation. Glucose stimulated viability and PCP degradation in all cases except when the cells were grown under carbon limitation with glucose and PCP added together as the carbon source. These results indicate that there is a relationship between nutrient limitation, phenotypic variation, and the sensitivity to and degradation of PCP by this organism.

Biodegradation, Environmental↗
PubMed1991

Biodegradation of creosote and pentachlorophenol in contaminated groundwater: chemical and biological assessment.

Shake flask studies examined the rate and extent of biodegradation of pentachlorophenol (PCP) and 42 components of coal-tar creosote present in contaminated groundwater recovered from the American Creosote Works Superfund site, Pensacola, Fla. The ability of indigenous soil microorganisms to remove these contaminants from aqueous solutions was determined by gas chromatographic analysis of organic extracts of biotreated groundwater. Changes in potential environmental and human health hazards associated with the biodegradation of this material were determined at intervals by Microtox assays and fish toxicity and teratogenicity tests. After 14 days of incubation at 30 degrees C, indigenous microorganisms effectively removed 100, 99, 94, 88, and 87% of measured phenolic and lower-molecular-weight polycyclic aromatic hydrocarbons (PAHs) and S-heterocyclic, N-heterocyclic, and O-heterocyclic constituents of creosote, respectively. However, only 53% of the higher-molecular-weight PAHs were degraded; PCP was not removed. Despite the removal of a majority of the organic contaminants through biotreatment, only a slight decrease in the toxicity and teratogenicity of biotreated groundwater was observed. Data suggest that toxicity and teratogenicity are associated with compounds difficult to treat biologically and that one may not necessarily rely on indigenous microorganisms to effectively remove these compounds in a reasonable time span; to this end, alternative or supplemental approaches may be necessary. Similar measures of the toxicity and teratogenicity of treated material may offer a simple, yet important, guide to bioremediation effectiveness.

Animals↗
PubMed1991

Potential for thermophilic (50 degrees C) anaerobic dechlorination of pentachlorophenol in different ecosystems.

Thermophilic (50 degrees C) anaerobic biodegradation of pentachlorophenol (PCP) was investigated by using different inocula from natural ecosystems and anaerobic digesters. The inocula tested were three freshwater sediments, four anaerobic sewage sludge samples from digesters treating sludge from wastewater plants with various industrial inputs, and digested manure from an anaerobic reactor. Only one digested-sludge sample and the manure sample were from thermophilic environments. The initial PCP concentration was 7.5 or 37.5 microM. After 8 months, PCP had disappeared from the sediment samples and various, less chlorinated intermediates were present. Additions of extra PCP were degraded within 4 weeks, and a maximal observed dechlorination rate of 1.61 mumol/liter/day in the vials with addition of 7.5 microM PCP and 7.50 mumol/liter/day in the vials with addition of 37.5 microM PCP were measured for a freshwater sediment. In contrast, only 2.8 to 17.5% of the initial PCP added had disappeared from the sludge samples after 8 months of incubation. The complex pattern of intermediates formed indicated that the dechlorination of PCP proceeded via different pathways, involving at least two different populations in the dechlorination processes.

Anaerobiosis↗
PubMed1991

Regiospecific dechlorination of pentachlorophenol by dichlorophenol-adapted microorganisms in freshwater, anaerobic sediment slurries.

The reductive dechlorination of pentachlorophenol (PCP) was investigated in anaerobic sediments that contained nonadapted or 2,4- or 3,4-dichlorophenol (DCP)-adapted microbial communities. Adaptation of sediment communities increased the rate of conversion of 2,4- or 3,4-DCP to monochlorophenols (CPs) and eliminated the lag phase before dechlorination was observed. Both 2,4- and 3,4-DCP-adapted sediment communities dechlorinated the six DCP isomers to CPs. The specificity of chlorine removal from the DCP isomers indicated a preference for ortho-chlorine removal by 2,4-DCP-adapted sediment communities and for para-chlorine removal by 3,4-DCP-adapted sediment communities. Sediment slurries containing nonadapted microbial communities either did not dechlorinate PCP or did so following a lag phase of at least 40 days. Sediment communities adapted to dechlorinate 2,4- or 3,4-DCP dechlorinated PCP without an initial lag phase. The 2,4-DCP-adapted communities initially removed the ortho-chlorine from PCP, whereas the 3,4-DCP-adapted communities initially removed the para-chlorine from PCP. A 1:1 mixture of the adapted sediment communities also dechlorinated PCP without a lag phase. Dechlorination by the mixture was regiospecific, following a para greater than ortho greater than meta order of chlorine removal. Intermediate products of degradation, 2,3,5,6-tetrachlorophenol, 2,3,5-trichlorophenol, 3,5-DCP, 3-CP, and phenol, were identified by a combination of cochromatography (high-pressure liquid chromatography) with standards and gas chromatography-mass spectrometry.

Anaerobiosis↗
PubMed1992

Influence of a supplemental carbon source on anaerobic dechlorination of pentachlorophenol in granular sludge.

Anaerobic dechlorination of pentachlorophenol (PCP) was studied in two upflow anaerobic sludge blanket reactors. One reactor received glucose (0.9 g liter-1) as an additional carbon source; the other one served as a control. The concentration of PCP in the medium was 4.5 and 3.0 mg liter-1 in the experimental and control reactors, respectively. The reactors were inoculated with granular sludge previously grown on sugar-containing wastewater. After 10 months of continuous operation, the removal of PCP was 99% in the glucose-amended reactor, whereas the removal in the control reactor varied between 32 and 77%. Furthermore, 94% of the PCP was completely dechlorinated in the glucose reactor compared with a maximum of 20% in the control reactor. In the same period, the amount of biomass in the glucose reactor had increased by approximately 150% compared with that in the control reactor, where no growth of the sludge bed occurred. Batch culture activity tests showed that the addition of glucose had a stimulatory effect on the dechlorination rate of PCP per gram of volatile solids. This indicated that the better performance of the glucose-amended reactor was due to a higher concentration of biomass and a direct stimulatory effect of glucose on the dechlorination rate. The pattern of dechlorination of PCP showed that an initial para cleavage was followed by two ortho cleavages.

Anaerobiosis↗
PubMed1992

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)

Biodegradation, Environmental↗
PubMed1993

Growth and enrichment of pentachlorophenol-degrading microorganisms in the nutristat, a substrate concentration-controlled continuous culture.

The nutristat, a substrate concentration-controlled continuous culture, was used to grow pentachlorophenol (PCP)-degrading microorganisms. The PCP concentration control system consisted of on-line measurement of the PCP concentration in the culture vessel with a tangential filter and a flowthrough spectrophotometer. With PCP concentrations between 45 and 77 microM, a stable situation was established in the nutristat, with an average dilution rate of 0.035 +/- 0.003 h-1. Compared with those of fed-batch cultures and chemostat cultures, the growth rates of microorganisms in the PCP nutristat were significantly higher, leading to considerable time savings in the enrichment procedure. In addition, PCP accumulation to severe inhibitory levels in the culture is prevented because the set point determines the (maximum) PCP concentration in the culture. The use of the nutristat as a tool for the growth of bacteria that degrade toxic compounds is discussed.

Biodegradation, Environmental↗
PubMed1993

Performance of anaerobic granules for degradation of pentachlorophenol.

Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28 degrees C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99% was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14C]PCP to 14CH4 and 14CO2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP.

Acetates↗
PubMed1997

Mutational analysis of pcpA and its role in pentachlorophenol degradation by Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723.

Sphingomonas (Flavobacterium) chlorophenolica ATCC 39723 degrades pentachlorophenol (PCP) through a catabolic pathway encoded by multiple genes. One gene required for PCP degradation is pcpA, which encodes information for a 30-kDa polypeptide, PcpA, found in the periplasm of the bacterium. The biological role of PcpA has remained unknown. We disrupted pcpA by replacing it with a defective copy through homologous recombination. The pcpA recombinant, mutant strains accumulated 2,6-dichlorohydroquinone (2,6-DiCH) as a metabolite of PCP. This work confirms that pcpA is essential for degradation of PCP by S. chlorophenolica ATCC 39723 and suggests that it encodes a protein involved in hydrolytic dehalogenation of 2,6-DiCH, an already established primary metabolite of the PCP catabolic pathway.

Bacterial Proteins↗
PubMed1997

In vivo levels of chlorinated hydroquinones in a pentachlorophenol-degrading bacterium.

Sphingomonas chlorophenolica RA-2 is a soil microorganism that can grow on pentachlorophenol (PCP) as a sole carbon source. In this microorganism, PCP is converted to tetrachlorohydroquinone (TCHQ), trichlorohydroquinone, and 2,6-dichlorohydroquinone. The remainder of the pathway has not yet been defined. The ability to grow on PCP as a sole carbon source is remarkable because of the toxicity of PCP and its chlorinated hydroquinone metabolites. Experiments in which the levels of PCP and chlorinated hydroquinones were measured in cells metabolizing [U-14C]PCP revealed that the levels of chlorinated hydroquinones in the cytoplasm are in the low micromolar range. The toxicity of chlorinated hydroquinones was evaluated by exposure of Escherichia coli cells that had been treated with EDTA (to remove the outer membrane) to TCHQ. Significant toxicity due to TCHQ was not apparent until concentrations of 500 microM and higher. Thus, an important part of the explanation for why S. chlorophenolica RA-2 is able to grow on PCP as a sole carbon source is undoubtedly that it can process sufficient carbon for growth without accumulating high levels of toxic intermediates.

Cell Extracts↗
PubMed1999

Biodegradation of pentachlorophenol in a continuous anaerobic reactor augmented with Desulfitobacterium frappieri PCP-1.

In this work, a strain of anaerobic pentachlorophenol (PCP) degrader, Desulfitobacterium frappieri PCP-1, was used to augment a mixed bacterial community of an anaerobic upflow sludge bed reactor degrading PCP. To estimate the efficiency of augmentation, the population of PCP-1 in the reactor was enumerated by a competitive PCR technique. The PCP-1 strain appeared to compete well with other microorganisms of the mixed bacterial community, with its population increasing from 10(6) to 10(10) cells/g of volatile suspended solids within a period of 70 days. Proliferation of strain PCP-1 allowed for a substantial increase of the volumetric PCP load from 5 to 80 mg/liter of reaction volume/day. A PCP removal efficiency of 99% and a dechlorination efficiency of not less than 90.5% were observed throughout the experiment, with 3-Cl-phenol and phenol being observable dechlorination intermediates.

Bacteria, Anaerobic↗
PubMed2001

Phosphorus-31 nuclear magnetic resonance study of the effect of pentachlorophenol (PCP) on the physiologies of PCP-degrading microorganisms.

Free and agarose-encapsulated pentachlorophenol (PCP)-degrading Sphingomonas sp. isolates UG25 and UG30 were compared to Sphingomonas chlorophenolica ATCC 39723 with respect to the ability to degrade PCP. Pretreatment of the UG25 and UG30 strains with 50 microg of PCP per ml enabled the cells to subsequently degrade higher levels of this environmental pollutant. Similar treatment of ATCC 39723 cells had no effect on the level of PCP degraded by this strain. Phosphorus-31 nuclear magnetic resonance spectra of agarose-immobilized strains UG25 and UG30 grown in the absence of PCP showed that there was marked deenergization of the cells upon exposure to a nonlethal concentration of PCP (120 microg/ml). For example, no transmembrane pH gradient was observed, and the ATP levels were lower than the levels obtained in the absence of PCP. The transmembrane pH gradient and ATP levels were restored once the immobilized cells had almost completely degraded the PCP in the perfusion medium. PCP-pretreated cells, on the other hand, maintained their transmembrane pH gradient and ATP levels even in the presence of high levels of PCP. The ability of PCP-pretreated strain UG25 and UG30 cells to remain energized in the presence of PCP was shown to correlate with an altered membrane phospholipid profile; these cells had a higher concentration of cardiolipin than cells cultured in the absence of PCP. Strain ATCC 39723, which did not degrade higher levels of PCP after PCP pretreatment, did not show this response.

Biodegradation, Environmental↗
PubMed2004

Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723.

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.

Base Sequence↗
PubMed2005

Desulfitobacterium hafniense is present in a high proportion within the biofilms of a high-performance pentachlorophenol-degrading, methanogenic fixed-film reactor.

We developed a pentachlorophenol (PCP)-degrading, methanogenic fixed-film reactor by using broken granular sludge from an upflow anaerobic sludge blanket reactor. This methanogenic consortium was acclimated with increasing concentrations of PCP. After 225 days of acclimation, the reactor was performing at a high level, with a PCP removal rate of 1,173 muM day(-1), a PCP removal efficiency of up to 99%, a degradation efficiency of approximately 60%, and 3-chlorophenol as the main chlorophenol residual intermediate. Analyses by PCR-denaturing gradient gel electrophoresis (DGGE) showed that Bacteria and Archaea in the reactor stabilized in the biofilms after 56 days of operation. Important modifications in the profiles of Bacteria between the original granular sludge and the reactor occurred, as less than one-third of the sludge DGGE bands were still present in the reactor. Fluorescence in situ hybridization experiments with probes for Archaea or Bacteria revealed that the biofilms were composed mostly of Bacteria, which accounted for 70% of the cells. With PCR species-specific primers, the presence of the halorespiring bacterium Desulfitobacterium hafniense in the biofilm was detected very early during the reactor acclimation period. D. hafniense cells were scattered in the biofilm and accounted for 19% of the community. These results suggest that the presence of PCP-dehalogenating D. hafniense in the biofilm was crucial for the performance of the reactor.

Anaerobiosis↗