Bioremediation of a Chilean Andisol contaminated with pentachlorophenol (PCP) by solid substrate cultures of white-rot fungi

This study provides a first attempt investigation of a serie of studies on the ability of Anthracophyllum discolor, a recently isolated white-rot fungus from forest of southern Chile, for the treatment of soil contaminated with pentachlorophenol (PCP) to future research on potential applications in bioremediation process. Bioremediation of soil contaminated with PCP (250 and 350 mg kg⁻¹ soil) was investigated with A. discolor and compared with the reference strain Phanerochaete chrysosporium. Both strains were incorporated as free and immobilized in wheat grains, a lignocellulosic material previously selected among wheat straw, wheat grains and wood chips through the growth and colonization of A. discolor. Wheat grains showed a higher growth and colonization of A. discolor, increasing the production of manganese peroxidase (MnP) activity. Moreover, the application of white-rot fungi immobilized in wheat grains to the contaminated soil favored the fungus spread. In turn, with both fungal strains and at the two PCP concentrations a high PCP removal (70–85%) occurred as respect to that measured with the fungus as free mycelium (30–45%). Additionally, the use of wheat grains in soil allowed the proliferation of microorganisms PCP decomposers, showing a synergistic effect with A. discolor and P. chrysosporium and increasing the PCP removal in the soil.     

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.     

Influence of environmental parameters on pentachlorophenol biotransformation in soil by Lentinula edodes and Phanerochaete chrysosporium

 The influences of temperature, soil moisture potential and initial pH on the biotransformation of pentachlorophenol (PCP) by the lignicolous fungi Lentinula edodes and Phanerochaete chrysosporium were examined. At 10^°C, L. edodes was more effective in degrading PCP (P<0.05) than P. chrysosporium. At 15^°C similar results were obtained for the two fungi. The highest levels of degradation occurred for both fungi at 25^°C. With P. chrysosporium, the extent of PCP elimination was directly related to soil moisture content and optimal at approximately 47%. With L. edodes, in contrast, the process was inversely related to moisture content and maximal at 26%. The initial soil pH also had a marked influence, and pH 4.0 was optimal for both fungi. Received: 7 August 1995/Accepted: 22 August 1995     

Biostimulation and Bioaugmentation of Anaerobic Pentachlorophenol Degradation in Contaminated Soils

The effects of bioaugmentation with a pentachlorophenol (PCP)-adapted consortium and biostimulation with glucose as a carbon source on anaerobic bioremediation of PCP-contaminated soil were investigated in terms of the initial PCP removal rate and the extent of PCP dechlorination and mineralization. Samples from two PCP-contaminated sites were prepared, put into a series of Hungate tubes, inoculated, and fed under different conditions. Chlorophenols in the tubes were monitored over a 4-month period to measure PCP transformation in the soil. In less contaminated soil (10 mg PCP/kg soil), it was found that biostimulation with glucose at 1 g/kg soil or bioaugmentation at 0.14 g volatile suspended solids (VSS)/kg soil could greatly improve PCP degradation. The best PCP degradation was obtained when both bioaugmentation and biostimulation were applied, but higher levels of glucose (2 g/kg soil) or inoculum (0.56 g VSS/kg soil) had little additional effect. The highest initial PCP-removal rate reached 8.1 μmol/kg soil-d, which is almost 20 times greater than in the unamended controls. PCP was dechlorinated to lesser chlorinated phenols with 0.6 chlorine remaining on average, and the extent of mineralization approached 70% in 4 months. In highly PCP-contaminated soil (90 mg PCP/kg soil), PCP degradation was partially inhibited, but the relative effects of augmentation, stimulation, and combined treatments were the same as in the less contaminated soil.     

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.     

The Co-application of Willow and Earthworms/Horseradish for Removal of Pentachlorophenol from Contaminated Soils

Pentachlorophenol (PCP) was once commonly used as a pesticide worldwide, and is now a toxic and recalcitrant environmental pollutant. To explore a practical approach in the remediation of PCP-contaminated soils in China, we evaluated the efficacy of a local willow (Salix × aureo-pendula CL “J1011”) on removing PCP and assessed the potential of a native earthworm (Metaphire guillelmi) and horseradish (Armoracia rusticana Gaerth), alone and in combination with willow, to enhance the efficiency of removing PCP from spiked field soils. Willow, horseradish, and earthworms alone significantly increased PCP removal from soil. After 45 days, only 47.2% of the PCP remained in the presence of willow alone; 68.4, 51.4, and 46.3% of the PCP remained with 50, 100, and 200 g horseradish m^?2, respectively; and 41.1% of the PCP remained in the presence of earthworms. The removal of PCP from soils significantly increased with co-application of willow and horseradish (23.1% remaining) and especially with the co-application of willow and earthworms (2.2% remaining). Our results indicate that in terms of remediation efficiency, economic value, ecological benefits, and ease of use, a system consisting of this autochthonous willow and indigenous earthworms has great potential for the remediation of PCP-polluted soil in China.     

Biodegradation of textile azo-dyes byPhanerochaete chrysosporium

Of 18 commercially used textile dyes, eight were degraded by the white rot fungus,Phanerochaete chrysosporium, by 40 to 73% based on decrease of colour. Both the lignin-degrading enzyme system ofP. chrysosporium and adsorption to its cell mass were involved in the degradation of the diazo dye, Reactofix Gold Yellow. Degradation was best achieved by adding the dye to the medium and then inoculating with pre-grown mycelium; inoculation with spores resulted mainly in dye adsorption.     

五氯酚（PCP）污染土壤厌氧生物修复技术的初步研究

研究土壤泥浆反应器在投加厌氧颗粒污泥条件下修复PCP污染土壤的性能.结果表明,对PCP浓度30mg     

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.     

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.     

Screening pentachlorophenol degradation ability by environmental fungal strains belonging to the phyla Ascomycota and Zygomycota

Pentachlorophenol (PCP) bioremediation by the fungal strains amongst the cork-colonising community has not yet been analysed. In this paper, the co- and direct metabolism of PCP by each of the 17 fungal species selected from this community were studied. Using hierarchical data analysis, the isolates were ranked by their PCP bioremediation potential. Fifteen isolates were able to degrade PCP under co-metabolic conditions, and surprisingly Chrysonilia sitophila, Trichoderma longibrachiatum, Mucor plumbeus, Penicillium janczewskii and P. glandicola were able to directly metabolise PCP, leading to its complete depletion from media. PCP degradation intermediates are preliminarily discussed. Data emphasise the significance of these fungi to have an interesting potential to be used in PCP bioremediation processes.     

Sensitivity to and Degradation of Pentachlorophenol by Phanerochaete spp

This research measured mycelial extension rates of selected strains of Phanerochaete chrysorhiza, Phanerochaete laevis, Phanerochaete sanguinea, Phanerochaete filamentosa, Phanerochaete sordida, Inonotus circinatus, and Phanerochaete chrysosporium and the ability of these organisms to tolerate and degrade the wood preservative pentachlorophenol (PCP) in an aqueous medium and in soil. Most of the tested species had mycelial extension rates in the range of ≤0.5 to 1.5 cm day⁻¹, but there were large interspecific differences. A notable exception, P. sordida, grew very rapidly, with an average mycelial extension rate of 2.68 cm day⁻¹ at 28°C. Rank of species by growth rate was as follows: P. chrysosporium > P. sordida > P. laevis > P. chrysorhiza = P. sanguinea > I. circinatus = P. filamentosa. There were also significant intraspecific differences in mycelial extension rates. For example, mycelial extension rates among strains of P. sordida ranged from 1.78 to 4.81 cm day⁻¹. Phanerochaete spp. were very sensitive to PCP. Growth of several species was prevented by the presence of 5 ppm (5 μg/g) PCP. However, P. chrysosporium and P. sordida grew at 25 ppm PCP, albeit at greatly decreased mycelial extension rates. In an aqueous medium, mineralization of PCP by P. sordida 13 (ca. 12% after 30 days) was significantly greater than that by all other tested P. sordida strains and P. chrysosporium. After 64 days, the level of PCP had decreased by 96 and 82% in soil inoculated with P. chrysosporium and P. sordida, respectively. Depletion of PCP by these fungi occurred in a two-stage process. The first stage was characterized by a rapid depletion of PCP that coincided with an accumulation of pentachloroanisole (PCA). At the end of the first stage, ca. 64 and 71% of the PCP was converted to PCA in P. chrysosporium and P. sordida cultures, respectively. In the second stage, levels of PCP and PCA were reduced by 9.6 and 18%, respectively, in soil inoculated with P. chrysosporium and by 3 and 23%, respectively, in soil inoculated with P. sordida. PCA was mineralized by both P. chrysosporium and P. sordida in an aqueous medium.     

Isolation of a novel pentachlorophenol-degrading bacterium, Pseudomonas sp. Bu34

A pentachlorophenol (PCP)-degrading bacterium was isolated from possible PCP-contaminated soil from Pusan, Korea and identified as a member of the genus Pseudomonas. It used PCP as its sole source of carbon and energy. This micro-organism was capable of degrading PCP more effectively, certified by the increase in cell density and the decrease in PCP substrate. Pseudomonas sp. Bu34 was able to degrade a much higher concentration of PCP (4000 mg l⁻¹) than any previously reported PCP-degrading bacteria and fungi and to grow in mineral salts solution containing one of a variety of chlorophenols. In non-acclimated strain Bu34, the cell number decreased from 87 to 99·9% in 75–4000 mg l⁻¹ PCP at 24 h. In the acclimated strain the PCP toxic effect did not appear with 75 mg l⁻¹ PCP treatment, but 1000–4000 mg l⁻¹ PCP decreased the cell number of strain Bu34 by 25% to 24 h and then the cell number slightly increased at 48 h. Therefore, it suggested that the maximum resistance of acclimated strain Bu34 to PCP was 4000 mg l⁻¹ PCP. We suggest that strain Bu34 could be used as a micro-organism for the bioremediation of highly PCP-contaminated soils, water or wood products.     

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.     

Phytoremediation of Pentachlorophenol in the Crested Wheatgrass (Agropyron cristatum × desertorum) Rhizosphere

Pentachlorophenol (PCP) is a widespread, highly toxic contaminant of soil and water that is generally recalcitrant to microbial breakdown and thus may be considered a good candidate for phytoremediation. PCP toxicity and rates of mineralization were compared in crested wheatgrass seedlings that were either sterile or root-inoculated with microbial consortia derived from soil at a PCP-contaminated site. Inoculated seedlings were more tolerant to PCP and mineralized threefold more ¹⁴C-PCP than sterile seedlings. Only 10% of the recovered radioactivity from sterile seedlings represented mineralized PCP, indicating that rhizosphere microorganisms are primarily responsible for PCP mineralization. The levels of PCP degradation exhibited by several microbial consortia and isolates in liquid culture were not correlated with their ability to protect crested wheatgrass seedlings from PCP toxicity. Most probable number estimates showed that the presence of crested wheatgrass root exudates enhanced the number of PCP-degrading microorganisms by 100-fold in liquid culture, indicating that exudate components provide some nutritive benefit, possibly as PCP co-metabolites. A close association of plants and rhizosphere microorganisms appears to be necessary for crested wheatgrass survival in PCP-contaminated soil, although understanding the molecular details of this association requires further research.     

Phytoremediation Efficiency of a PCP-Contaminated Soil using Four Plant Species as Mono- and Mixed Cultures

Bioremediation of soil polluted by pentachlorophenol (PCP) is of great importance due to the persistence and carcinogenic properties of PCP. Phytoremediation has long been recognized as a promising approach for removal of PCP from soil. The present study was conducted to investigate the capability of four plant species; white clover, ryegrass, alfalfa, and rapeseed grown alone and in combination to remediate pentachlorophenol contaminated soil. After 60 days cultivation, white clover, raygrass, alfalfa, and rapeseed all significantly enhanced the degradation of PCP in soils. Alfalfa showed highest efficiency for the removal of PCP in single cropping flowed by rapeseed and ryegrass. Mixed cropping significantly enhanced the remediation efficiencies as compared to single cropping; about 89.84% of PCP was removed by mixed cropping of rapeseed and alfalfa, and 72.01% of PCP by mixed cropping of rape and white clover. Mixed cropping of rapeseed with alfalfa was however far better for the remediation of soil PCP than single cropping. An evaluation of soil biological activities as a monitoring mechanism for the bioremediation process of a PCP-contaminated soil was made using measurements of microbial counts and dehydrogenase activity.     

Bioremediation of Crude Oil–Contaminated Soil By Immobilized Bacteria on an Agroindustrial Waste—Sunflower Seed Husks

This study reports the immobilization and performance of a hydrocarbon-degrading Rhodococcus sp. strain (designated as QBTo) on sunflower seed husks (SH) for the bioremediation of soils polluted with crude oil. The SH performance as inoculants carrier was compared with peat, which is a vegetal material traditionally used in carrier-based inoculants production. The stability of the immobilized culture under storage conditions was assessed by viability at different times when stored at 25°C and 10°C. The catabolic activity of immobilized and free QTBo cells introduced into sandy loam soil, freshly contaminated with crude oil, was studied in microcosms. A higher number of viable QTBo cells were recovered from the inoculants formulated with SH (QTBo-SH) after prolonged storage at 10°C and 25°C. The microcosms amended with QTBo-SH inoculants showed a removal of about 66% of total petroleum hydrocarbons (TPH), whereas in those inoculated with QTBo-peat inoculants, the decrease was of about 47%. In the control microcosms (noninoculated) and liquid culture–amended soils, the TPH removal was about 28%. SH is a waste of edible oil industry, nontoxic, and biodegradable and has demonstrated to confer to the immobilized cultures greater potential to survive not only during storage but also in the soil environment, improving bioremediation process.     

Screening of New Zealand Native White-Rot Isolates for PCP Degradation

From a pool of 367 white-rot fungi native to New Zealand (over 77 genera), isolates were screened for their bioremediation potential of pentachlorophenol (PCP). Fungi were tested for their ligninolytic activity (Poly R-478, 367 isolates; wood decay, 235 isolates), tolerance to temperature (261 isolates), resistance to PCP (253 isolates), and PCP degradation potential plus laccase expression (20 isolates). Of the isolates tested, 26% showed a discolouration in the polymeric dye assay, but all caused wood decay (5 to 169 mm) on willow cuttings. In the temperature tolerance tests, all isolates survived incubation from 0 to 30°C, however, 18% and 40% did not survive incubation at 35 and 40°C, respectively. In the PCP resistance tests, 23 isolates (9%) were able to grow on 200 mg/L PCP amended agar, of which 20 isolates were further studied for laccase expression and PCP degradation in vitro. All 20 isolates reduced (P < 0.05) PCP in the liquid fraction in the absence or presence of laccase and five of the isolates produced no detectable levels of PCP. None of the screening tests were predictive for PCP degradation in vitro. The requirements to build a database to select a superior white-rot fungal isolates for bioremediation is discussed.     

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics

The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals. Received: 25 April 2000 / Received revision: 05 June 2000 / Accepted: 04 July 2000     

Comparison of ligninolytic activities of selected white-rot fungi

Summary Six fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.Dedicated to Professor Dr. Hans-Jürgen Rehm on the occasion of his 60th birthday