The survival of the pentachlorophenol-degrading Rhodococcus chlorophenolicus PCP-1 and Flavobacterium sp. in natural soil

The survival of two different pentachlorophenol (PCP)-degrading bacteria were studied in natural soil. The PCP-degraders Rhodococcus chlorophenolicus and Flavobacterium sp., both able to mineralize PCP into CO₂ and chloride in axenic culture, were tested for the capacity to survive and degrade PCP in natural soil. These bacteria were immobilized on polyurethane (PUR) foam and introduced into natural peaty soil to give about 10⁹ cells g^-1 of soil (dry weight). R. chlorophenolicus induced PCP-degrading activity in soil remained detectable for 200 days whether or not a carbon source was added (distillery waste or wood chips). Electron microscopic investigation performed almost a year after inoculation, revealed the presence of R. chlorophenolicus-like cells in the PUR foam particles. PCP-degrading activity of Flavobacterium sp. declined within 60 days of burial in the soil without enhancing the PCP removal. R. chlorophenolicus degraded PCP in soil at a mean rate of 3.7 mg of PCP day^-1 kg^-1 of soil, which corresponds to ca. 5×10^-3 pg of PCP degraded per inoculated R. chlorophenolicus cell day^-1. The solvent extractable organic chlorine contents of the soil decreased stoichiometrically (>95%) with that of PCP indicating that PCP was essentially mineralized.Abbreviations ATCC American type culture collection - DSM Deutsche Sammlung für Mikroorganismen - DW distillery waste - EM electron microscopy - EOX extractable organic halogen - GC/ECD gas chromatograph/electron capture detector - GC/MS gas chromatograph/mass spectrometer - PCP pentachlorophenol - WC wood chips - d.wt. dry weight - w.wt. wet weight - d.s. dry soil - d.H₂O distilled water - PCA polychlorinated aromatics     

Cell division across the volume of colonies of flavobacterium sp. 22

Six-day-old colonies ofFlavobacterium sp. 22 were studied by electron microscopy. Direct evidence was obtained of bacterial cell division across the entire colony volume, indicating that the colony growth ofFlavobacterium sp. 22 is not purely peripheral. It is argued that the colony shape is determined not only by peripheral growth but also by physical forces acting upon a droplet of liquid on the surface. For bacterial colonies developing on solid nutrient media, the intercellular matrix plays the role of such a liquid.     

Detecting Surfactant-producing Microorganisms by the Drop-collapse Test

Summary A rapid method, ’drop-collapse’, was used for screening biosurfactant production by Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Phanerochaete chrysosporium liquid cultures. Before measuring the total biosurfactant, the drop-collapse method was used in order to detect rhamnolipid presence in the culture broths. The method was performed in a microwell plate; the polystyrene platform with small wells. If the culture broth contained biosurfactant, the droplets of the broth in the oil-coated wells collapsed. If not, there was no change in the shape of the droplets. Pseudomonas aeruginosa and Bacillus subtilis culture supernatants showed spreading movement, meaning that they produced biosurfactants. However, Candida albicans and Phanerochaete chrysosporium supernatants remained beaded, meaning they did not produce any type of microbial surfactant.     

Remediation of pentachlorophenol-contaminated soil by composting with immobilized Phanerochaete chrysosporium

Summary To reduce and eliminate the hazards of pentachlorophenol (PCP) to the soil, the method of inoculating free and immobilized white rot fungi, Phanerochaete chrysosporium to PCP-polluted soils was investigated. Three parallel beakers A, B, C are adopted with the same components of soil, yard waste, straw and bran for aerated composting to degrade the PCP in soil. A was with no inoculants as control, B was added with the inoculants of immobilized P.␣chrysosporium, C was inoculated with non-immobilized P. chrysosporium, and additionally D contained only PCP-contaminated soils also as control. By contrastive analyses, the feasibility of applying composting to the bioremediation of the PCP-polluted soil was discussed. From the experimental results, it could be seen that the degradation rate of PCP by the immobilized fungi exceeded 50% at day 9, while that of the non-immobilized fungi achieved the same rate at day 16. However, the final degradation rates of PCP for both of them were beyond 90% at day 60 and that the rate of A was much lower than the others. The above data have shown that the degradation effect of inoculating P. chrysosporium was better than that of no inoculation, and that of the immobilized fungi was better than that of non-immobilized ones. Meanwhile, shown by all the indicators the composts of A, B and C were mature and stabilized at the end of the experiment. Therefore, the method of composting with immobilized P.␣chrysosporium is effective for the bioremediation of PCP-contaminated soil.     

Degradation of major compounds of creosotes (PAH and phenols) byPhanerochaete chrysosporium

Summary A mixture of naphtalene, phenanthrene, anthracene,p-cresol, pentachlorophenol (PCP) and phenol, representing the major components of creosotes, was degraded byPhanerochaete chrysosporium. Intermediate products such as quinones did accumulate. A slight difference in biodegradation rates was observed between single, individual compounds and the same compounds in the mixture.     

Biological potential of fungal inocula for bioaugmentation of contaminated soils

The suitability of the fluorescein diacetate hydrolyzing activity (FDA) assay for determining the biological potential (ie fungal biomass produced per unit of substrate) of solid pelleted fungal inoculum intended for use in the bioaugmentation of contaminated soils with white-rot fungi, was evaluated. FDA activity of the white-rot fungusPhanerochaete chrysosporium grown on pelleted substrates and on agar was found to be proportional to quantities of fungal ergesterol and fungal dry matter, respectively. Inoculum biological potential was found to be greatly influenced by substrate formulation and structure, and temperature. Biological potential and the type of carrier influenced the ability ofP. chrysosporium to tolerate pentachlorophenol (PCP).Phanerochaete chrysosporium andTrametes versicolor introduced into PCP-contaminated soil on pellets with higher biological potential and higher nitrogen content (C:N ratio of 501), did not remove PCP more efficiently than when the fungi were introduced on pellets with a lower biological potential (C:N ratio of 3091). However, under the latter conditions most of the PCP was transformed to pentachloroanisole (PCA). In soil inoculated withT. versicolor on pellets with high biological potential, higher manganese peroxidase activity was detected compared to soil inoculated with pellets with a lower biological potential.     

Use of lignin-degrading fungi in the disposal of pentachlorophenol-treated wood

Summary The lignin-degrading fungiPhanerochaete chrysosporium, P. sordida, Trametes hirsuta, andCeriporiopsis subvermispora were evaluated for their ability to decrease the concentration of pentachlorophenol (PCP) and to cause dry weight loss in PCP-treated wood. Hardwood and softwood materials from PCP-treated ammunition boxes that were chipped to pass a 3.8-cm screen were used. All four fungi caused significant weight losses and decreases in the PCP concentration. The largest PCP decrease (84% in 4 weeks) was caused byT. hirsuta, and the smallest decrease was caused byC. subvermispora (37% in 4 weeks). After 4 weeks, the fate of spiked¹⁴C[PCP] in softwood chips inoculated withT. hirsuta was as follows: 27% was mineralized, 42.5% was non-extractable and bound to the chips, 23.5% was associated with fungal hyphae, and 6% was organic-extractable. Decreases of PCP byP. chrysosporium andP. sordida averaged 59% and 57%, respectively. PCP decreases caused byPhanerochaete spp. were not significantly affected by wood type or sterilization and were primarily due to methylation of PCP that resulted in accumulation of pentachloroanisole. Softwood weight losses caused byT. hirsuta, P. chrysosporium andC. subvermispora were respectively, 24, 6.5, and 17%, after 4 weeks. These weight losses are comparable to reported weight losses by these organisms in non-treated softwood. Nutrient supplementation significantly increased weight loss but not percentage decrease of PCP. The results of this research demonstrate the potential for using lignin-degrading fungi to destroy PCP-treated wood.     

Degradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium grown in ammonium lignosulphonate media

Removal and degradation of pentachlorophenol (PCP) by Phanerochaete chrysosporium in static flask cultures was studied using ammonium lignosulphonates (LS), a waste product of the papermill industry, as a carbon and nitrogen source. After 3 days, cultures of P. chrysosporium grown in either a 2% LS (nitrogen-sufficient) medium or a 0.23% LS and 2% glucose (nitrogen-deficient) medium removed 72 to 75% of PCP, slightly less than the 95% removal seen using nitrogen-deficient glucose and ammonia medium. PCP dehalogenation occurred despite the fact that extracellular enzyme (LiP) activity, measured by a veratryl alcohol oxidation assay and by PCP disappearance in cell-free extracts, was inhibited by LS. This inactivation of LiP likely contributed to the lower percent of PCP dehalogenation observed using the LS media. In order to better understand the relationship between PCP disappearance and dehalogenation, we measured the fate of the chlorine in PCP. After 13 days, only 1.8% of the initial PCP added was recoverable as PCP. The remainder of the PCP was either mineralized or transformed to breakdown intermediates collectively identified as organic halides. The largest fraction of the original chlorine (58%) was recovered as organic (non-PCP) halide, most of which (73%) was associated with the cell mass. Of the remaining chlorine, 40% was released as chloride ion, indicating a level of dehalogenation in agreement with previously reported values.     

Molecular analysis of pentachlorophenol degradation

A limited number of microorganisms have been described for their ability to partially degrade pentachlorophenol (PCP), or to completely mineralize it. Several years ago we chose one of these microorganisms,Flavobacterium sp. strain ATCC 39723, for use in a detailed molecular analysis of the catabolism of PCP. This strain was chosen because it had previously been studied in great detail for its growth characteristics in relation to degradation of PCP. In this paper we provide an overview of the degradation pathway of PCP to 2,6-dichloro-p-hydroquinone byFlavobacterium. The specific biochemical reactions and the genes encoding the enzymes are reviewed. The successful transformation and site specific mutagenesis ofFlavobacterium, as well as the discovery of two newpcp alleles is also presented.     

Establishment of the white rot fungus Phanerochaete chrysosporium on unsterile straw in solid substrate fermentation systems intended for degradation of pesticides

The effects of different inoculum-loading rates and pre-treatment of wheat straw with formic acid and hot water (50 °C) on the establishment of Phanerochaete chrysosporium on unsterile straw were studied in laboratory scale and in a 1.5-m³ bioreactor. The establishment of P. chrysosporium on unsterile straw was satisfactory. Phanerochaete chrysosporium and other fungi, which developed simultaneously, were able to produce the activity necessary to degrade two herbicides, bentazon and MCPA (4-chloro-2-methylphenoxyacetic acid) in 20 days (65 and 75%, respectively). The decrease of both herbicides coincided with the presence of the activity of the lignin-degrading enzymes lignin peroxidase and manganese peroxidase/laccase. Extensive growth of P. chrysosporium or other lignin-degrading fungi on unsterile straw would be excellent for inexpensive solid substrate systems intended for degradation of pesticides.     

Evaluation of pentachlorophenol-degrading potentiality of Pseudomonas sp. in a soil microcosm

Pseudomonas sp. strain IST103 obtained from a stable consortium was capable of degrading pentachlorophenol (PCP) as sole carbon and energy source. The PCP-degrading potentiality of the strain was determined by growth of bacteria in culture medium, utilization of PCP by high performance liquid chromatography (HPLC), chloride release and ring cleavage. The strain was applied in two set of soil microcosms containing 20 and 40% moisture, each having different concentrations, 0, 10, 100, 500, and 1000 mg/l, of PCP. The result showed significant utilization of PCP (77% in 45 days) and higher growth of bacterial strain when PCP was applied in 100 mg/l concentration at 40% moisture. Inhibitory effects on the growth of bacterial strain were seen in 500 and 1000 mg/l concentration.     

Enrichment and characterization of a microbial community from tannery effluent for degradation of pentachlorophenol

A bacterial community obtained by continuous enrichment from the microbial population of tannery effluent using pentachlorophenol (PCP) as sole source of carbon and energy, contained four different bacterial species including Serratia marcescens (three isolates, TE₁, TE₂ and TE₄) and Pseudomonas fluorescens (one isolate, TE₃). The members of the community grew separately on various chlorinated compounds, carbon and nitrogen sources and exhibited a remarkable ability to utilize PCP. Biodegradation studies revealed a time-dependent disappearance of PCP and its intermediary metabolites, tetrachloro-p-hydroquinone and chlorohydroquinone, and indicated the individual role of members of the community in the degradation of PCP.     

Physiological properties and substrate specificity of a pentachlorophenol-degradingPseudomonas species

A bacterial strain capable of utilizing pentachlorophenol (PCP) as sole source of carbon and energy for growth was isolated from enrichment cultures containing 100 mg/l PCP in a mineral salts medium inoculated with contaminated soil from a lumber treatment waste site. The isolate, designated strain SR3, was identified as a species ofPseudomonas by virtue of its physiological and biochemical characteristics. Mineralization of PCP byPseudomonas sp. strain SR3 was demonstrated by loss of detectable PCP from growth medium, stoichiometry of chloride release (5 equivalents of chloride per mole of PCP), and formation of biomass consistent with the concentration of PCP mineralized. PCP-induced cells of strain SR3 showed elevated rates of oxygen consumption in the presence of PCP, and with different chlorinated phenols, with complete degradation of 2,3,5,6-, 2,3,6-, 2,4,6-, 2,4-, and 2,6-chloro-substituted phenols. Concentrations of PCP up to 175 mg/liter supported growth of this organism, but maximal rates of PCP removal were observed at a PCP concentration of 100 mg/liter. Based on its degradative properties,Pseudomonas sp. strain SR3 appears to have utility in bioremediation of soil and water contaminated with PCP.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol Contribution No. 750 from the United States Environmental Protection Agency Environmental Research Laboratory, Gulf Breeze, FL32561, USA. A preliminary report of this work has appeared in abstract form (Resnick & Chapman 1990; Abstr. Annu Meet Amer Soc Microbiol Q-70, p. 300).     

Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.     

Enhanced ligninolytic enzyme production and degrading capability of Phanerochaete chrysosporium and Trametes versicolor

Ligninolytic enzyme production by the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor precultivated with different insoluble lignocellulosic materials (grape seeds, barley bran and wood shavings) was investigated. Cultures of Phanerochaete chrysosporium precultivated with grape seeds and barley bran showed maximum lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP) activities (1000 and 1232 U/l, respectively). Trametes versicolor precultivated with the same lignocellulosic residues showed the maximum laccase activity (around 250 U/l). For both fungi, the ligninolytic activities were about two-fold higher than those attained in the control cultures. In vitro decolorization of the polymeric dye Poly R-478 by the extracellular liquid obtained in the above-mentioned cultures was monitored in order to determine the respective capabilities of laccase, LiP and MnP. It is noteworthy that the degrading capability of LiP when P. chrysosporium was precultivated with barley bran gave a percentage of Poly R-478 decolorization of about 80% in 100 s, whereas control cultures showed a lower percentage, around 20%, after 2 min of the decolorization reaction.     

Metabolism of mono- and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporium cytochromes P450

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min⁻¹) and 2-MCDD (13 min⁻¹) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichloro-dibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzo-p-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.     

The recent evolution of pentachlorophenol (PCP)-4-monooxygenase (PcpB) and associated pathways for bacterial degradation of PCP

Man-made polychlorinated phenols such as pentachlorophenol (PCP) have been used extensively since the 1920s as preservatives to prevent fungal attack on wood. During this time, they have become serious environmental contaminants. Despite the recent introduction of PCP in the environment on an evolutionary time scale, PCP-degrading bacteria are present in soils worldwide. The initial enzyme in the PCP catabolic pathway of numerous sphingomonads, PCP-4-monooxygenase (PcpB), catalyzes the para-hydroxylation of PCP to tetrachlorohydroquinone and is encoded by the pcpB gene. This review examines the literature concerning pcpB and supports the suggestion that pcpB/PcpB should be considered a model system for the study of recent evolution of catabolic pathways among bacteria that degrade xenobiotic molecules introduced into the environment during the recent past.     

Degradation of the insecticide Hydramethylnon byPhanerochaete chrysosporium

The decomposition of the amidinohydrazone-type insecticide Hydramethylnon (HMN) by soil fungi has been investigated. A simple spectrophotometric method was developed for the estimation of HMN in soil and fungal culture media. HMN was found to be degraded in soil with a half life of 14 to 25 days.Degradation of HMN by the lignolytic fungus,Phanerochaete chrysosporium yielded two major breakdown products;p-(trifluoromethyl)-cinnamic acid (TFCA) andp-(trifluoromethyl)-benzoic acid (TFBA). TFCA was converted to TFBA which was subsequently metabolised via themeta-fission pathway. Fluoride release from HMN could not be detected.Abbreviations BzDAc benzene, dioxane, acetic acid (60: 36: 4) - DCM dichloroethane - DNPH 2,4-dinitro-phenylhydrazine - HMN Hydramethylnon - TDAc toluene, dioxane, acetic acid (90: 30: 1) - TFCA p-(trifluoromethyl)-cinnamic acid - TFBA p-(trifluoromethyl)-benzoic acid - TFP 1,5-bis(trifluoro-p-tolyl)-1,4-pentadien-3-one - VA veratryl alcohol     

Phylogeny of Sphingomonas species that degrade pentachlorophenol

Four pentachlorophenol (PCP)-degrading bacteria isolated from geographically diverse areas have been examined in detail as regards their physiology and phylogeny. According to traditional biochemical methods, these strains had been classified as members of the genera Arthrobacter, Flavobacterium, Pseudomonas, and Sphingomonas. The PCP degradation pathway has been studied extensively in Sphingomonas (Flavobacterium) sp strain ATCC 39723 and the first three degradation steps catalyzed by a PCP-4-monooxygenase (PcpB) and a reductive dehalogenase (PcpC) that functions twice are well established. A fourth step appears to involve ring-fission of the aromatic nucleus (PcpA). Molecular analyses revealed that the PCP degradation pathway in these four strains was rather conserved, leading to a phylogenetic analysis using 16S rDNA. The results revealed a much closer phylogenetic relationship between these organisms than traditional classification indicated, placing them into the more recently established genus Sphingomonas where they may even represent a single species. With 16S rDNA analysis, many bacterial isolates involved in degradation of xenobiotic compounds that were previously classified into diverse genera have been reclassified into the genus Sphingomonas. Received 14 April 1999/ Accepted in revised form 20 July 1999     

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.