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The ability of tropical Brazilian basidiomycetes to degrade pentachlorophenol (PCP) in soils from areas contaminated with organochlorine industrial residues ...
 Springer 2005

World Journal of Microbiology & Biotechnology (2005) 21: 297–301 DOI 10.1007/s11274-004-3693-z

Biodegradation of pentachorophenol by tropical basidiomycetes in soils contaminated with industrial residues Ka´tia Maria Gomes Machado1,*, Da´cio Roberto Matheus2, Regina Teresa Rosim Monteiro3 and Vera Lu´cia Ramos Bononi2 1 Universidade Cato´lica de Santos, Santos, and Fundac¸a˜o Centro Tecnolo´gico de Minas Gerais, Belo Horizonte, Brazil 2 Instituto de Botaˆnica, Secretaria do Meio Ambiente do Estado de Sa˜o Paulo, Brazil 3 Centro de Energia Nuclear na Agricultura, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil *Author for correspondence: UniSantos, Av. Conselheiro Ne´bias 300, Santos, Sa˜o Paulo, CEP11015-002, Brazil. Tel.: +55-13-32055555, Fax: +55-13-32844146, E-mail: [email protected]

Keywords: Non-lignicolous basidiomycetes, organochlorine compounds, pentachloroanisole, pentachlorophenolmineralization, soil bioremediation, soil pollution, tropical fungi

Summary The ability of tropical Brazilian basidiomycetes to degrade pentachlorophenol (PCP) in soils from areas contaminated with organochlorine industrial residues was studied. Thirty-six basidiomycetes isolated from different tropical ecosystems were tested for tolerance to high PCP concentrations in soil. Peniophora cinerea and Psilocybe castanella, two strains of Trametes villosa, Agrocybe perfecta, Trichaptum bisogenum and Lentinus villosus were able to colonize soil columns containing up to 4600 mg pentachlorophenol kg)1 soil. The first four species were inoculated into soil containing 1278 mg pentachlorophenol kg)1 soil supplemented with gypsum and sugar cane bagasse. P. cinerea, P. castanella, T. villosa CCB176 and CCB213 and Agrocybe perfecta reduced the PCP present in the contaminated soil by 78, 64, 58, 36 and 43%, respectively, after 90 days of incubation. All fungi mineralized [14C] pentachlorophenol, mainly P. cinerea and T. villosa with the production of 7.11 and 8.15% 14CO2, respectively, during 120 days of incubation. All fungi produced chloride ions during growth on soil containing PCP, indicating dehalogenation of the molecule. Conversion of PCP to pentachloroanisole was observed only after 90 days of incubation in soils inoculated with A. perfecta, P. cinerea and one of T. villosa strain. The present study shows the potential of Brazilian fungi for the biodegradation of toxic and persistent pollutants and it is the first to report fungal growth and PCP depletion in soils with high pentachlorophenol concentrations.

Introduction Pentachlorophenol (PCP), synthesized for the first time in 1872, has been used since the late 30s as a wood preservative together with its salt, sodium pentachlorophenate, due to its broad spectrum and low cost. Its esters have also been used as biocides. This generalized use has led to the contamination of many ecosystems, with PCP currently considered to be a product of priority for decontamination studies according to the European Community and the American Environmental Protection Agency. In Brazil, PCP has been widely used as a herbicide to clean pasture fields and in deforestation, for the construction of hydroelectric plants and dams for water reservoirs. In the State of Sa˜o Paulo, the most industrialized region of Brazil, there is widespread concern over locating, monitoring and treating soil areas that were contaminated with organic

pollutants, particularly in the sixties and seventies, among them residuals containing organochlorine compounds such as hexachlorobenzene and PCP (Matheus et al. 2000). The capacity of basidiomycetes to degrade PCP, as well the possibility of using these organisms in processes of reclamation of PCP-contaminated soils, have been the subject of much previous research (Lamar & Dietrich 1992; Liang & McFarland 1994; Reddy & Gold 2000; Leontievsky et al. 2002). An enormous diversity of basidiomycetes exists, with 29,914 species known worldwide (Kirk et al. 2001). Thus it is clear that a selection must be made of species with greater potential to degrade xenobiotics, and that at the same time are able to grow in contaminated environments or in materials that must be treated. Moreover, in many countries, including Brazil, there are restrictions about the use of certain allochthonous microorganisms in

298 treatment systems open to the environment. Thus it is desirable to screen indigenous species. In the present study, we assessed the potential use of basidiomycetes from Brazilian ecosystems in the bioremediation of soils contaminated with organochlorine compounds in terms of (i) tolerance to organochlorine compounds in the soil, and (ii) capacity to biodegrade PCP.

Materials and methods

K.M.G. Machado et al. Biodegradation of pentachlorophenol A 25 g (dry weight) mixture of soil–CaSO4–sugar cane bagasse (95:2.5:2.5, dry weight) was placed in bottles of 800 ml. The moisture content was adjusted to 75% of the mixture’s water retention capacity and corrected weekly. A 2.5 g amount of inoculum was used. In the control treatment, the fungi were replaced with sterilized wheat grains. All treatments were carried out in quadruplicate. Free chlorides were determined in aqueous extract using a silver electrode.

Fungi Chromatographic analyses The 36 fungal strains studied were previously selected for Remazol brilliant blue R decolorization and a growth rate exceeding 1.0 cm day)1 in a solid medium (Faleiro et al. 1996 ; Okino et al. 2000). All cultures were from Brazilian ecosystems and were maintained in 2% (w/v) malt extract agar (MEA) at 4 C. They are deposited in the culture collection of basidiomycetes (CCB) of the Instituto de Botaˆnica, Sa˜o Paulo, SP, Brazil.

The compounds were extracted from the total content of each bottle with a soxhlet apparatus and the concentrations of organochlorines were determined by high-resolution gas chromatography (Matheus et al. 2000). The analyses were standardized for: PCP, pentachloroanisole (PCA), hexachlorethane, hexachlorobutadiene, tetrachlorobenzene, pentachlorobenzene and hexachlorobenzene. The factor of recovery ranged from 98.9 to 100.7%. The detection limit was 10 mg kg)1 of soil.

Soil Mineralization of The soil was collected from an area situated in Sa˜o Vicente, SP, in a region where organochlorine industrial residues with high concentrations of PCP had been inadequately disposed. The soil contained 98.0% (w/w) sand, pH 3.65, had a cation exchange capacity of 5.5 mEq 100 g)1 soil, and contained 2.3% (w/w) organic matter, 0.06 g nitrogen per 100 g)1 soil, 1.0 lg phosphorus per g)1 soil, and 0.01 mEq potassium per 100 ml)1. No bacteria or fungi were isolated from this soil. Before use, the soil was air dried, sieved through a 2 mm mesh, diluted in non-contaminated soil and autoclaved for 1 h at 100 C for three consecutive days. Fungal tolerance This test was performed as described by Matheus et al. (2000). Fungi were inoculated into non-inclined test tubes containing MEA and incubated at 30 C for about one week. The soil, with 46,300 mg of PCP kg)1 of soil, was diluted with non-contaminated soil. When fungal growth had started, a column of 15 g sterilized soil was placed over each colony, in quadruplicate, for 10, 50 and 100% (w/w) of PCP-soil. Non-contaminated soil was used as control. The fungi that could colonize more than 25% of the soil column were considered to be tolerant to organochlorine compounds. Fungal inoculum For the experiments of PCP degradation and mineralization, the inoculum of each fungus was prepared using wheat grains (Matheus et al. 2000).

14

C-PCP

An amount of 3.0 · 106 d.p.m. of [U-14C] pentachlorophenol (Sigma, St. Louis, MO) per 100 g of soil was placed in 300 ml bottles with 100 g of the mixture of soil–gypsum–bagasse. The culture conditions and the treatment control were the same as described to the PCP biodegradation. Incubation was carried out in a dark chamber at 23 ± 2 C. The initial radioactivity applied was determined by combustion and the 14CO2 produced was periodically captured in a trap of soda lime and extracted as described by Matheus et al. (2000). Thin layer chromatography (TLC) The extracts were evaporated in a rotary evaporator and resuspended in 2 ml of acetonitrile. PCP and PCA were identified by coelution of the 14C-labelled products with added standards on TLC plates (60F254 Merck). Mass balance analysis Soil samples were extracted for the activity remaining in the flasks. Five ml of acetonitrile was added to 10 g of sample in a stainless steel container, shaken in a Vortex apparatus (5 min) and later centrifuged (14,000 rpm, 20 min). The supernatant was collected and the extraction operation repeated. The activity was measured in 1 ml from combined supernatants. The residual soil was air dried at room temperature and then homogenized using a ceramic pulverizer. The bound residues was determined by combustion of 1 g of these material in a biological oxidizer (R.J. Harvey, model OX-500) and the 14CO2 produced was determined. The total radioactivity recovered was calculated by summing the

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Pentachlorophenol biodegradation by basidiomycetes radioactivity in the acetonitrile extract, in the trapping solution and bound in the soil matrix.

14

CO2

Table 1. Colonization of PCP contaminated soil by basidiomycetes. Basidiomycetes

PCP concentration in column of soil (mg PCP kg)1 soil)

Agrocybe perfecta CCB1611 Antrodiella semidupina CCB446 Antrodiella sp. CCB426 Coprinus jamaicensis CCB318 Gymnopillus chrysopellus CCB381 Gymnopillus eartei CCB375 Hydnopolyporus fimbriatus CCB289 Irpex lacteusCCB196 Lentinus crinitus CCB217 Lentinus crinitus CCB356 Lentinus strigellus CCB380 Lentinus velutinus CCB268 Lentinus villosus CCB271 Lentinus zeyheri CCB274 Oudemansiella canarii CCB179 Oudemansiella canarii CCB241 Peniophora cinerea CCB204 Peniophora cremea CCB286 Phanerochaete chrysosporium CCB478 Phelinus lividus CCB305 Psathyrella tenuis CCB376 Psilocybe castanella CCB444 Pycnoporus sanguineus CCB175 Pycnoporus sanguineus CCB277 Pycnoporus sanguineus CCB458 Ripartitella brasiliensis CCB467 Stereum ostrea CCB267 Trametes versicolor CCB202 Trametes versicolor CCB372 Trametes villosa CCB165 Trametes villosa CCB213 Trametes villosa CCB291 Trichaptum byssogenum CCB203 Trogia buccinalis CCB390 Tyromyces pseudolacteus CCB193

4600.0 ng2 0.0 ng 0.0 0.0 ng ng 0.0 ng 0.0 ng 4600.0 0.0 ng ng 0.0 ng 0.0 0.0 ng 4600.0 0.0 ng 0.0 0.0 0.0 0.0 ng 0.0 4600.0 ng 4600.0 ng ng

Statistical analysis

Results and discussion Tolerance to organochlorine compounds in the soil The soil used contained 46,300 mg PCP kg)1 soil, the concentrations in the diluted soils being proportional to the executed dilutions, corresponding to 4600 and 23,000 mg of PCP kg)1 soil, respectively. Among the 36 fungi, Agrocybe perfecta (Rick) Sing. CCB161, Trametes villosa (Fr.) Kreisel CCB176 and CCB213, Trichaptum bisogenum (Jungh.) Riv. CCB203, Lentinus villosus Klotzsch CCB271 and Psilocybe castanella Peck CCB444 were capable of colonizing the columns of soil with 4600 mg PCP kg)1 soil (Table 1). Although the majority of the isolates studied are lignicolous species (Okino et al. 2000), the results showed that many of them developed well in the soil column. Due to the toxic effect of PCP as an inhibitor of oxidative phosphorylation, the growth of these fungi at the higher PCP concentration was unexpected. P. castanella, a saprotrophic species, was isolated from sandy organochlorine-contaminated soil in an area where industrial dumping had occurred. A. perfecta, a non-lignicolous species, was isolated from a sugar cane bagasse stack in Sa˜o Paulo city. T. villosa CCB176 and CCB213 are lignicolous fungi and both strains were isolated from forests distant from the contaminated areas. In another study, Peniophora cinerea (Fr.) Cooke CCB204 a lignicolous fungus isolated from the Atlantic forest, Sa˜o Vicente, SP, grew in soil with 1300 mg PCP kg)1 soil (results not shown) and was also evaluated. L. villosus and T. bissogenum are lignicolous species isolated from ‘restinga’ forests near the contaminated areas, but were not screened for the experiments of PCP biodegradation, because they did not show good growth in the incubation system. PCP depletion PCP depletion was evaluated on the basis of the residual concentrations of the compound in relation to the initial concentration of 1278 mg PCP kg)1 of soil. P. cinerea caused the greatest decrease of PCP (77.97%), followed by P. castanella (64.46%) and T. villosa CCB176 (58.07%), after 90 days of incubation. These results

1 2

Depletion of PCP in soil (%)

This was done according to a fully randomized experimental design. Analyses of variance (ANOVA) were conducted (a ¼ 0.05) using the ANOVA program of Excel 5.0 for Windows. Whenever a significant effect of treatments was observed, differences among treatment means were determined by the Tukey test (P £ 0.05).

CCB = code in the culture collection of basidiomycetes. ng = no growth.

65 days of incubation 90 days of incubation

100 90 80 70 60 50 40 30 20 10 0 CCB161

CCB204

CCB444

CCB176

CCB213

Control

Figure 1. Depletion of PCP by basidiomycete in organochlorinecontaminated soil (the bars indicate the standard deviation of the mean). CCB161 ¼ Agrocybe perfecta, CCB213 and CCB176 ¼ Trametes villosa, CCB444 ¼ Psilocybe castanella, CCB204 ¼ Peniophora cinerea, control ¼ without fungus.

(Figure 1) were significantly greater (P < 0.05) than those observed in control treatment (27.74%). The other fungi did not differ statistically from the control treatment. The biotic loss of 996.73, 824.07 and

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K.M.G. Machado et al.

742.4 mg PCP kg)1 of soil incubated with the strains P. cinerea, P. castanella and T. villosa CCB 176, respectively, are close to those reported in other studies (44–89%) using Phanerochaete spp. and Lentinula edodes, in soils with initial concentration between 200 and 700 mg kg)1 of PCP (Lamar & Evans 1993; Okeke et al. 1993). Several factors can influence organochlorine depletion in soils (soil type, availability of nutrients, oxygenation, moisture content, pH and compound concentration). The conditions employed in the present experiment were compatible with the good colonization of the fungus in the soil and the optimization of manageable parameters was not evaluated. At this study, the soil had high concentrations of organochlorines, since this is a frequently encountered condition in the region. Chloride ion production The initial concentration of PCP could produce 851 mg Cl) kg)1 soil. The largest Cl) concentration was encountered in the soil incubated with P. cinerea and A. perfecta (440 and 418 mg Cl) kg)1 soil) corresponding to 51.7 and 51.3% of the initial concentration, respectively (Figure 2). The other treatments did not differ from each other, although all of them were significantly different (P £ 0.05) from the control. The results indicate transformation of PCP and molecule dehalogenation. In soil inoculated with A. perfecta and T. villosa CCB213, the levels of chloride ions did not show a direct relation to the loss of PCP. One explanation is that degradation by these fungi resulted in a transformation product that had more chlorine atoms than those produced by the other fungi. PCA production In soil treated with T. villosa CCB213, P. cinerea and A. perfecta, 16, 25.5 and 35 mg PCA kg)1 soil were detected, respectively, only at 90 days of incubation. The rates of PCP conversion to PCA (Figure 3) were lower than those presented by Phanerochaete chrysosporium,

Control 65 days of incubation

CCB213

90 days of incubation CCB176 CCB444 CCB204 CCB161 0

0,5

1

1,5

2

2,5

3

PCP to PCA conversion (%)

Figure 3. Transformation of PCP to PCA in organochlorinecontaminated soil after incubation with basidiomycetes. CCB161= Agrocybe perfecta, CCB213 and CCB176 ¼ Trametes villosa, CCB444 ¼ Psilocybe castanella, CCB204 ¼ Peniophora cinerea, control ¼ without fungus.

P. sordida and L. edodes (Lamar & Dietrich 1990; Okeke et al. 1993). PCA was not detected in the control treatment nor in soil with P. castanella and T. villosa CCB176 as described for other fungi as Trametes hirsuta and Ceriporiopsis subvermispora (Lamar & Dietrich 1990). Mineralization and mass balance of

14

C-PCP

The initial concentration of PCP in this test was 1180 mg PCP kg)1 soil. The extent of PCP mineralization was 3.07, 3.17, 4.95, 7.11 and 8.15% when the soil was incubated for 120 days with T. villosa CCB213, A. perfecta, P. castanella, P. cinerea and T. villosa CCB176, respectively (Figure 4). These rates are comparable to those described in the literature (Lamar et al. 1990; Liang & Mcfarland 1994). Indeed, the effect of the C/N ratio on these organisms could also have produced differences in the amounts of PCP degraded, mineralized or converted to PCA by the several fungi, as observed by Lamar & White (2001). Table 2 presents the 14C balance of masses. The low recovery of 14C from treatments with P. castanella,

9,00

400 350 300 250

7,00 CO2 (%)

65 days of incubation 90 days of incubation

450

14

-1

mg of inorganic chloride kg of soil

8,00 500

6,00 5,00 4,00

200

3,00

150

2,00

100

1,00

50

0,00

0 CCB161

CCB204

CCB444

CCB176

CCB213

Control

Figure 2. Inorganic chloride in organochlorine-contaminated soil after incubation with basidiomycetes. Initial concentration of 851 mg Cl) kg)1 of soil (the bars indicate the standard deviation of the mean). CCB161 ¼ Agrocybe perfecta, CCB213 and CCB176 ¼ Trametes villosa, CCB444 ¼ Psilocybe castanella, CCB204 =Peniophora cinerea, control =without fungus.

0

20

40 60 80 Time of incubation (days)

100

120

Figure 4. Mineralization of 14C-PCP by basidiomycetes in organochlorine-contaminated soil. (r) Agrocybe perfecta CCB161, (·) Trametes villosa CCB176, (m) T. villosa CCB213, (n) Psilocybe castanella CCB444, (d) Peniophora cinerea CCB204; (s) control= without fungus.

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Table 2. Mass balance of radioactivity from [14C] pentachlorophenol in soil bioaugmented with basidiomycetes during 120 days of incubation. Basidiomycetes

Agrocybe perfecta CCB1611 Peniophora cinerea CCB204 Psilocybe castanella CCB444 Trametes villosa CCB176 Trametes villosa CCB213 Control

Radioactivity recovery (%) Mineralization

Acetonitrile extract

Soil combustion

Total balance

3.17 7.11 4.95 8.15 3.07 0.53

47.84 nd2 28.21 16.16 37.93 67.30

31.41 nd 38.08 37.37 24.41 17.64

82.42 nd 71.24 61.68 65.41 85.47

1

CCB = code in the culture collection of basidiomycetes. nd = not determined.

2

T. villosa CCB213 and CCB176 may be justified by the lack of determination of volatilized compounds and adsorption to the bottles, which can correspond to as much as 10% (Liang & Mcfarland 1994). The main fate of PCP in soils inoculated with basidiomycetes is the transformation to extractable metabolites (as PCA) and bound residues, depending on the kind of soil. These residues can be formed by oxidative reactions mediated by a variety of biotic and non-biotic catalysts, producing polymers that become incorporated into humus matter (Bollag & Dec 1995; Ullah et al. 2000). In the soil bioremediation process, the most desirable outcome is the conversion from PCP to CO2 and bound residues, reducing the bioavailability and consequently the toxicity of this pollutant. Among the fungi evaluated, P. castanella and T. villosa CCB176 showed 43.03 and 45.52% of the recovered radioactivity as 14CO2 from PCP mineralization and soil combustion. This is the first study in which initial PCP levels of more than 1200 mg kg)1 soil were used. The performance of P. castanella, also emphasizes the possibility of use non-lignicolous basidiomycetes in bioremediation.

Acknowledgements This project was the result of an agreement between Rhodia Brazil Ltda and Universidade Estadual Paulista, together with Instituto de Botaˆnica of Sa˜o Paulo, Brazil. We are grateful to FUNDUNESP, CNPq and FAPEMIG for financial support.

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