Degradation of chlorinated phenols by a pentachlorophenol-degrading bacterium

A pentachlorophenol (PCP)-degrading Flavobacterium sp. was tested for its ability to dechlorinate other chlorinated phenols by using resting cells that had been grown in the presence or absence of PCP. Phenols with chlorine atoms at positions 2 and 6 of the phenol ring were dechlorinated completely by PCP-induced cells. Other chlorinated phenols were not significantly mineralized. When PCP was added to a culture growing on L-glutamate, there was a lag period before the start of PCP degradation. When similar cells were treated with chloramphenicol prior to the addition of PCP, they did not degrade added PCP, even after prolonged incubations. Thus, the enzymes necessary for PCP degradation appeared to be inducible. Suspensions of cells grown in the presence of 2,4,6-trichlorophenol or 2,3,5,6-tetrachlorophenol did not show a lag period for mineralization of PCP, 2,4,6-trichlorophenol, or 2,3,5,6-tetrachlorophenol, indicating that one enzyme system probably was induced for the biodegradation of all three compounds. Nondegradable chlorophenols were toxic toward the Flavobacterium sp., probably acting as uncouplers of oxidative phosphorylation.     

Degradation of chlorinated phenols by a pentachlorophenol-degrading bacterium.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. was tested for its ability to dechlorinate other chlorinated phenols by using resting cells that had been grown in the presence or absence of PCP. Phenols with chlorine atoms at positions 2 and 6 of the phenol ring were dechlorinated completely by PCP-induced cells. Other chlorinated phenols were not significantly mineralized. When PCP was added to a culture growing on L-glutamate, there was a lag period before the start of PCP degradation. When similar cells were treated with chloramphenicol prior to the addition of PCP, they did not degrade added PCP, even after prolonged incubations. Thus, the enzymes necessary for PCP degradation appeared to be inducible. Suspensions of cells grown in the presence of 2,4,6-trichlorophenol or 2,3,5,6-tetrachlorophenol did not show a lag period for mineralization of PCP, 2,4,6-trichlorophenol, or 2,3,5,6-tetrachlorophenol, indicating that one enzyme system probably was induced for the biodegradation of all three compounds. Nondegradable chlorophenols were toxic toward the Flavobacterium sp., probably acting as uncouplers of oxidative phosphorylation.     

Investigation of the biodegradation of pentachlorophenol by the predominant bacterial strains in a mixed culture

A pentachlorophenol (PCP) degrading mixed culture contained three predominant strains identified as Flavobacterium gleum, Agrobacterium radiobacter and Pseudomonas sp. The relative abilities of the three strains to degrade PCP were tested individually and in combination. Rates of PCP degradation by individual isolates were lower than that observed for the three isolates combined. Of the individual strains, Flavobacterium gleum manifested highest PCP degradation ability. A biodegradation medium inoculated with a combination of the three isolates exhibited PCP degradation patterns similar to the original mixed culture. Varying low amounts of tetrachlorophenol were found in degradation medium inoculated with individual isolates, but this intermediate was absent from media inoculated with the mixed culture.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

Purification and properties of pentachlorophenol hydroxylase, a flavoprotein from Flavobacterium sp. strain ATCC 39723.

A pentachlorophenol (PCP) hydroxylase which catalyzed the conversion of PCP to 2,3,5,6-tetrachlorohydroquinone and released iodide from triiodophenol in the presence of NADPH and oxygen was identified. The enzyme was purified by protamine sulfate precipitation, ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography, gel filtration chromatography, and crystallization. The enzyme was a monomer with a molecular weight of 63,000. Under certain conditions, dimer and multimer conformations were also observed. The pI of the enzyme was pH 4.3. The optimal conditions for activity were a pH of 7.5 to 8.5 and a temperature of 40 degrees C. Each enzyme molecule contained one flavin adenine dinucleotide molecule. The Km for PCP was 30 microM and the Vmax was 16 mumol/min/mg of protein. The enzymatic reaction required 2 mol of NADPH per mol of halogenated substrate. On the basis of the data we present, it is likely that PCP hydroxylase is a flavoprotein monooxygenase. The addition of flavins to the reaction mixture did not stimulate the enzymatic reaction; however, we identified the photodegradation of triiodophenol and tribromophenol, but not PCP, by flavin mononucleotide or riboflavin and light.     

Confirmation of oxidative dehalogenation of pentachlorophenol by a Flavobacterium pentachlorophenol hydroxylase.

Pentachlorophenol (PCP) hydroxylase purified from Flavobacterium sp. strain ATCC 39723 converted PCP or 2,3,5,6-tetrachlorophenol to tetrachloro-p-hydroquinone (TeCH) with the co-consumption of O2 and NADPH. The purified enzyme incorporated 18O from 18O2 but not from H218O into the reaction end product TeCH. The results clearly demonstrate that PCP is oxidatively converted to TeCH by a monooxygenase-type enzyme from Flavobacterium sp. strain ATCC 39723.     

PCP degradation is mediated by closely related strains of the genus Sphingomonas

There have been numerous reports in the literature of diverse bacteria capable of degrading pentachlorophenol (PCP). In order to gain further insight into the phylogenetic relationships of PCP-degrading bacteria, we examined four strains: Arthrobacter sp. strain ATCC 33790, Flavobacterium sp. strain ATCC 39723, Pseudomonas sp. strain SR3, and Sphingomonas sp. strain RA2. These organisms were isolated from different geographical locations and all of them degrade high concentrations (100–200 mg/L) of PCP. Southern blot analyses determined that these bacteria all harbour DNA that encodes similar, if not identical, genes involved in PCP degradation. Comparison of the 16S rRNA nucleotide sequences revealed that these organisms were very closely related and, in fact, represent a monophyletic group. The 16S rRNA analyses together with fatty acid and sphingolipid analyses strongly suggest that the four strains are members of the genus Sphingomonas . The close relationship of the four organisms is supported by nucleotide sequence analysis data of the pcpB locus encoding PCP-4-monooxygenase, the first enzyme in the PCP degradative pathway.     

Catabolism of pentachlorophenol by a Flavobacterium sp

The pathway employed for pentachlorophenol (PCP) degradation by an aerobic, chlorophenol-utilizing Flavobacterium sp. was initiated by conversion of PCP to tetrachloro-p-hydroquinone (TCH). 18O labelling experiments demonstrated that the first dechlorination, where a hydroxyl replaced the chlorine at PCP ring position number 4, involved a hydrolytic reaction. Then two reductive dechlorinations of TCH followed to yield firstly trichlorohydroquinone (TrCH) and then 2,6-dichlorohydroquinone (DCH). Thus, the initial steps in catabolism of PCP by the Flavobacterium were: PCP----TCH----TrCH.     

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.     

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.     

Pentachlorophenol biodegradation by Pseudomonas spp. UG25 and UG30

Eighty-nine bacterial isolates obtained by enrichment from pentachlorophenol (PCP)-contaminated soil samples were tested for PCP dechlorination activity and hybridization to pcpB (encoding PCP-4-monooxygenase) and pcpC (encoding tetrachlorohydroquinone reductive dehalogenase) gene probes synthesized by polymerase chain reaction from Flavobacterium sp. ATCC 39723 genomic DNA. Seven isolates were able to dechlorinate PCP, hybridize to both pcpB and pcpC DNA probes, and mineralize sodium pentachlorophenate (NaPCP) at an initial concentration of 100g/ml. Although the seven PCP-mineralizing isolates possessed DNA sequences homologous to the Flavobacterium pcpB and pcpC genes, restriction analysis revealed sequence differences between the isolates and the Flavobacterium PCP dechlorination genes. Two isolates, designated UG25 and UG30, with the fastest onset and highest extent of PCP mineralization were selected for further study. Both isolates were tentatively identified as Pseudomonas spp. and exhibited stoichiometric release of Cl– ions as PCP was degraded. The release of Cl– began concomitantly with PCP disappearance from the medium. Both UG25 and UG30 degraded NaPCP at a concentration of 250 g/ml in a minimal salt medium. Supplementation of the medium with glutamate increased the NaPCP degradation threshold of UG25 to a concentration of 300 g/ml but did not affect that of UG30. 31P-NMR spectra of UG25 and UG30 cell suspensions exposed to PCP showed lower intracellular ATP levels and a more acidic cytoplasmic pH relative to untreated cells. This de-energization may explain the lack of cell growth in the presence of high PCP concentrations.     

Enrichment, isolation and characterization of pentachlorophenol degrading bacterium Acinetobacter sp. ISTPCP-3 from effluent discharge site

Three pentachlorophenol (PCP) degrading bacterial strains were isolated from sediment core of pulp and paper mill effluent discharge site. The strains were continuously enriched in mineral salts medium supplemented with PCP as sole source of carbon and energy. One of the acclimated strains with relatively high PCP degradation capability was selected and characterized in this study. Based on morphology, biochemical tests, 16S rDNA sequence analysis and phylogenetic characteristics, the strains showed greatest similarity with Acinetobacter spp. The strain was identified as Acinetobacter sp. ISTPCP-3. The physiological characteristics and optimum growth conditions of the bacterial strain were investigated. The results of optimum growth temperature revealed that it was a mesophile. The optimum growth temperature for the strain was 30°C. The preferential initial pH for the strain was ranging at 6.5–7.5, the optimum pH was 7. The bacterium was able to tolerate and degrade PCP up to a concentration of 200 mg/l. Increase in PCP concentration had a negative effect on biodegradation rate and PCP concentration above 250 mg/l was inhibitory to its growth. Acinetobacter sp. ISTPCP-3 was able to utilize PCP through an oxidative route with ortho ring-cleavage with the formation of 2,3,5,6-tetrachlorohydroquinone and 2-chloro-1,4-benzenediol, identified using gas chromatograph–mass spectrometric (GC–MS) analysis. The degradation pathway followed by isolated bacterium is different from previously characterized pathway.     

Degradation of pentachlorophenol by <Emphasis Type="Italic">Kocuria</Emphasis> sp. CL2 isolated from secondary sludge of pulp and paper mill

A pentachlorophenol (PCP) degrading bacterium was isolated and characterized from sludge of pulp and paper mill. This isolate used PCP as its sole source of carbon and energy and was capable of degrading this compound, as indicated by stoichiometric release of chloride and biomass formation. Based on morphology, biochemical tests, and 16S rRNA gene sequence analysis this strain was identified as Kocuria sp. CL2. High Performance Liquid Chromatography (HPLC) analysis revealed that this strain was able to degrade PCP up to a concentration of 600 mg/l. This is first time we are reporting the degradation of PCP by the Kocuria species. This isolate was also able to remove 58.64% of PCP from the sludge within two weeks. This study showed that the removal efficiency of PCP by CL2 was found to be very effective and can be used in degradation of PCP containing pulp paper mill waste in the environment.     

Chlorophenol and nitrophenol metabolism by Sphingomonas sp UG30

Sphingomonas sp UG30 is a pentachlorophenol (PCP)-degrading bacterial strain capable of degrading several nitrophenolic compounds, including p-nitrophenol (PNP), 2,4-dinitrophenol (2,4-DNP), p-nitrocatechol and 4,6-dinitro-o-cresol (DNOC). The ability to degrade both chlorophenolic and nitrophenolic compounds is probably not restricted to UG30, but may also be possessed by other pentachlorophenol-degrading Sphingomonas spp. The interesting question arises as to whether there is any point of convergence between the initial pathways of PCP and nitrophenol degradation in these microorganisms. There is some experimental evidence that PCP-4-monooxygenase is involved in metabolism of both p-nitrophenol and 2,4-dinitrophenol. Further studies are needed to confirm this and to examine the role(s) of other PCP-degrading enzymes in nitrophenol metabolism by this microorganism. In this paper, we review some of the taxonomic, biochemical, physiological and ecological properties of Sphingomonas sp UG30 with respect to biodegradation of PCP and nitrophenolic compounds. Received 19 April 1999/ Accepted in revised form 21 August 1999     

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of readily metabolizable carbon on pentachlorophenol metabolism by a pentachlorophenol-degrading Flavobacterium sp.

The influence of high concentrations of pentachlorophenol (PCP) and readily metabolizable carbon on the activity and viability of a PCP-degrading Flavobacterium sp. was examined in a mineral salts medium. Lags preceding PCP removal by glutamate-grown Flavobacterium cells were greatly attenuated by the addition of glutamate, aspartate, succinate, acetate, glucose, or cellobiose. The effect of these supplementary carbon sources on the apparent lag was not mediated entirely through the stimulation of growth since PCP metabolism accompanied the onset of growth. The specific activity of PCP-degrading cells in the absence of supplementary carbon was 1.51 x 10(-13) +/- 0.08 x 10(-13) g of PCP per cell per h and in the presence of supplementary carbon was 0.92 x 10(-13) +/- 0.09 x 10(-13) g of PCP per cell per h. Glutamate in combination with glucose or cellobiose partially repressed PCP metabolism. PCP removal by PCP-induced, glutamate-grown cells suspended in the presence of 4 g of sodium glutamate per liter was sensitive to shock loads of PCP, with a Ki of about 86.8 micrograms/ml. Subsequent removal rates, however, were more resistant to PCP. Optimal stimulation of PCP removal by sodium glutamate required 3.0 g/liter, about the same concentration as that which saturated growth in the absence of PCP. PCP removal rates decayed within minutes following the transfer of PCP-induced, glutamate-grown cells to media containing PCP without supplementary carbon, and increasing PCP concentrations accelerated the decay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of ethylamine degrading bacteria to atrazine degradation in soils

Bacterial communities that cooperatively degrade atrazine commonly consist of diverse species in which the genes for atrazine dechlorination and dealkylation are variously distributed among different species. Normally, the first step in degradation of atrazine involves dechlorination mediated by atzA, followed by stepwise dealkylation to yield either N-ethylammelide or N-isopropylammelide. As the liberated alkylamine moieties are constituents of many organic molecules other than atrazine, it is possible that a large number of alkylamine-degrading bacteria other than those previously described might contribute to this key step in atrazine degradation. To examine this hypothesis, we isolated 82 bacterial strains from soil by plating soil water extracts on agar media with ethylamine as a sole carbon source. Among the relatively large number of isolates, only 3 were able to degrade N-ethylammelide, and in each case were shown to carry the atzB gene and atzC genes. The isolates, identified as Rhizobium leguminosarum, Flavobacterium sp., and Arthrobacter sp., were all readily substituted into an atrazine-degrading consortium to carry out N-ethylammelide degradation. The distribution of these genes among many different species in the soil microbial population suggests that these genes are highly mobile and over time may lead to generation of various atrazine-degrading consortia.     

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 μg 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 μg/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.     

Effects of p-cresol and chlorophenols on pentachlorophenol biodegradation

The effects of three aromatic compounds, p-cresol, 2,4-dichlorophenol (DCP), and 2,4,6-trichlorophenol (TCP), on cell growth and pentachlorophenol (PCP) degradation bya Flavobacterium species were investigated. While p-cresol was not degraded by this bacterium, DCP and TCP were simultaneously degraded with PCP. Both DCP and TCP lowered cell growth and PCP degradation rate. Cell growth was modeled by cell death, because p-cresol, DCP, and TCP were toxic to the organism. A new model was used to predict cell death rate in a mixture of two toxic compounds from the cell death kinetics for each individual compound. PCP degradation rates were modeled by conventional inhibition models, but only over a small concentration range for the secondary toxic compound. However, a new empirical model described PCP degradation over a wider concentration range of the secondary toxic compound. (c) 1995 John Wiley & Sons Inc.