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ENVOLVIMENTO DAS ENZIMAS LIGNINOLÍTICAS DE Ganoderma lucidum NA REMOÇÃO DOS HERBICIDAS DIURON E BENTAZON JAQUELINE DA SILVA COELHO Maringá 2009 UNIVERSIDADE ESTADUAL DE MARINGÁ CENTRO DE CIÊNCIAS BIOLÓGICAS Programa de Pós-Graduação em Ciências Biológicas

ENVOLVIMENTO DAS ENZIMAS LIGNINOLÍTICAS DE Ganoderma

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Page 1: ENVOLVIMENTO DAS ENZIMAS LIGNINOLÍTICAS DE Ganoderma

ENVOLVIMENTO DAS ENZIMAS LIGNINOLÍTICAS

DE Ganoderma lucidum NA REMOÇÃO DOS

HERBICIDAS DIURON E BENTAZON

JAQUELINE DA SILVA COELHO

Maringá

2009

UNIVERSIDADE ESTADUAL DE MARINGÁ CENTRO DE CIÊNCIAS BIOLÓGICAS

Programa de Pós-Graduação em Ciências Biológicas

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JAQUELINE DA SILVA COELHO

ENVOLVIMENTO DAS ENZIMAS LIGNINOLÍTICAS

DE Ganoderma lucidum NA REMOÇÃO DOS

HERBICIDAS DIURON E BENTAZON

Dissertação apresentada ao programa de Pós Graduação em Ciências Biológicas da Universidade Estadual de Maringá, como parte dos requisitos para obtenção do título de mestre em Ciências (Área: Biologia Celular e Molecular).

Maringá

2009

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Dados Internacionais de Catalogação-na-Publicação (CIP) Biblioteca Central da UEM. Maringá-PR

B672p Coelho, Jaqueline da Silva Envolvimento das enzimas ligninolíticas de Ganoderma lucidum na remoção dos

herbicidas diuron e bentazon - Maringá: UEM, 2007. 51 p

Orientadora: Dra. Cristina Giatti Marques de Souza Co-orientadora: Dra. Rosane Marina Peralta.

Dissertação (mestrado em Ciências Biológicas) Departamento de Bioquímica.

Universidade Estadual de Maringá, 2009 Tese apresentada na forma de artigos científicos (2)

1.Ganoderma lucidum. 2. enzimas. 3. biodegradação. 4. herbicidas. 5. lacases 6. Biotecnologia. 7. Degradação de resíduos. I. Universidade Estadual de Maringá. Departamento de Bioquímica

CDD 21. Ed. 572.76 CIP-NBR 12899-AACR2

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Orientadora

Profa. Dra. Cristina Giatti Marques de Souza

Co-Orientadora

Profa. Dra. Rosane Marina Peralta

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BIOGRAFIA

Jaqueline da Silva Coelho nasceu em São Jorge do Patrocínio/PR em 12/12/1983. Possui graduação em Ciências Biológicas pela Universidade Estadual de Maringá (2006) Tem experiência nas áreas de Biologia Celular e Bioquímica, com ênfase em bioquímica de microrganismos, atuando principalmente nos seguintes temas: biotecnologia aplicada a processos de biorremediação ambiental, fisiologia e bioquímica de microrganismos, produção de enzimas ligninolíticas por fungos basidiomicetos.

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Dedico

A Deus, mestre maior e presença constante em minha

vida.

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AGRADECIMENTOS

Às professoras Drª Rosane Marina Peralta e Drª Cristina Giatti Marques de Souza, pela oportunidade, ensinamentos, paciência e ajuda. Ao professor Dr Sérgio Paulo Severo de Souza Diniz pelos incentivos, indicações e amizade. Aos meus amigos, em especial: Débora Bastos, Venessa Algarte, Gabrielle Jacklin Eller, pela compreensão nos momentos de ausência, sincera amizade, torcida e pelos doces momentos que passamos juntas. Aos meus pais pela educação base para minha vida e apoio nos meus estudos. Aos colegas e funcionários do laboratório de Bioquímica e Fisiologia de Microrganismos da UEM, em especial à Maria Aparecida (Pingo) pela ajuda, apoio e amizade. À CAPES pelo fornecimento da bolsa de estudos que garantiu o sustento financeiro necessário à realização desta dissertação de mestrado.

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APRESENTAÇÃO

Esta dissertação de mestrado está apresentada na forma de dois artigos

científicos

1 COELHO JS, OLIVEIRA AL, BRACHT A, SOUZA, CGM, PERALTA RM.

Effect of the herbicides bentazon and diuron on the production of

ligninolytic enzymes by Ganoderma lucidum a ser submetido ao

periódico científico Applied Microbiology and Biotechnology.

2 COELHO JS, ZILLY A, OLIVEIRA AL, BRACHT A, PERALTA RM, SOUZA,

CGM. Removal of bentazon by Ganoderma lucidum in liquid and solid

state cultures a ser submetido ao periódico científico Chemosphere.

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GENERAL ABSTRACT

INTRODUCTION. Throughout the past century farming and agricultural activities have released many chemical pesticides into the environment including insecticides, fungicides, nematicides, rodenticides and herbicides. The intensive use of pesticides results in environmental and human contamination due to their slow degradation and high toxicity. The herbicides diuron (N-3,4-dichlorophenyl-N’,N’-dimethylurea) and bentazon (3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide), both of them classified as dangerous for the environment, are largely used in Brazil mainly for sugar-cane and rice cultivation, respectively. Bioremediation using various microbial organisms is one safe and cost-effective way which has been shown to remove several persistent chemicals from the environment. Among the microorganisms used in this technique, the white rot fungi have generated considerable research in the last years. These fungi are the only microorganisms known to be able to degrade the highly recalcitrant natural polymer lignin due to the existence of a powerful enzymatic system formed mainly by peroxidases and laccases. These enzymes are secreted into the extracellular environment and involve the formation of highly reactive free radical intermediate which attacks the substrates and are responsible for the non-specificity of the system. This provides some basis for the attack of a wide range of recalcitrant compounds including several herbicides, structurally related to lignin. Phanerochaete chrysosporium is the most frequently used white-rot fungus (WRF) for bioremediation processes. Its secreted enzymes have demonstrated to possess high potential for detoxification of several xenobiotics, including the herbicides bentazon and diuron. Ganoderma lucidum, a medicinal WRF widely distributed in the world, is associated with the degradation of a wide variety of woods. It shows a great ability to produce ligninolytic enzymes, mainly laccases. Until now, G. lucidum is known to be a great degrader of dyes but with regard to its capacity to degrade other xenobiotics studies are still scarce. Thus, the application of G. lucidum and its enzymes to bioremediation processes is still an open field demanding exploration. AIMS. The objectives of this work were: 1) to evaluate the effect of the herbicides bentazon and diuron on growth and production of ligninolytic enzymes by G. lucidum cultured in glucose liquid media and to verify the capability of the fungus to remove these two compounds from the liquid media; 2) to compare the removal of bentazon by liquid and solid state cultures of G. lucidum cultured under conditions adequate to obtain elevated levels of ligninolytic enzymes.

MATERIAL AND METHODS. G. lucidum was cultivated in liquid stationary and solid state conditions at 28 oC in the dark. The liquid cultures were supplemented with glucose (1%) as carbon source or corn cob (1%). Several amounts of bentazon (0-20 mM) and diuron (0-80 µM) were added to the liquid medium and after 7 days the cultures were filtered to determine the dry biomass and to obtain the culture filtrates used as source of laccases and manganese peroxidases. Alternatively, the fungus inoculated in liquid medium was allowed to grow for 3 days before the addition of herbicides. The cultures were filtered at periodic intervals and the culture filtrates were used to determine the enzymatic activities and the residual herbicides. The cultivation under solid state conditions was performed using corn cob milled with moisture content adjusted to 75%. Several different amounts of bentazon (0-50 mM) were added to this medium. At periodic intervals the cultures were stopped by the addition of cold water and the mixtures were shaken for 1 h at 4 oC. The mixtures were filtered to retain

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insoluble materials, and the aqueous extracts were used as source of enzymes. To extract the bentazon sorbed on the fungal mycelia and residual corn cob, 20 ml of methanol were added to the insoluble materials obtained after aqueous extraction and the mixtures were shaken for 2 h. The mixtures were then filtered to retain the mycelia and corn cob e the residual bentazon was determined. All the analysis of residual herbicides was performed by HPLC (High performance liquid chromatography). RESULTS AND DISCUSSION. Strong improvement of the laccase activity associated with growth reduction was observed during the first 48 h after the application of both herbicides to the G. lucidum culture in glucose liquid media. No growth was observed upon the addition of 25 mM bentazon and 100 µM diuron. In the absence of the herbicides, the activity of laccase on 7-days culture-filtrate was 20 U/g. In the presence of bentazon and diuron, laccase activities improved up to 170 and 207 U/g, respectively. The Mn peroxidase activity was induced only by diuron and the presence of bentazon caused a slight improvement in the Mn peroxidase activity. Native PAGE analysis of the G.

lucidum laccases revealed two bands with laccase activities. The herbicides bentazon and diuron strongly improved only one isoform. Analysis of the residual herbicides showed that both herbicides were removed from the culture filtrates, but bentazon was more efficiently removed than diuron. Laccase was equally produced in liquid and solid state cultures of G. lucidum using corn cob as substrate, but activities of Mn peroxidase in solid state cultures were 10 times superior to those found in liquid cultures. Despite of the apparent growth inhibition caused by bentazon, the herbicide enhanced the production of laccase to a maximal value of 1,800 U/L, using concentrations of 2.5 mM and 30 mM in liquid and solid state cultures, respectively. The Mn peroxidase activity was not significantly affected by bentazon. After 10 days of cultivation, the amounts of residual bentazon present in the combined extracts were 47% and 12% of the initial amounts added to liquid and solid state cultures, respectively. These data show that the best degradation was obtained in the solid state conditions, where laccase and Mn peroxidase were produced at high levels. The results suggest the possibility of both enzymes to have a role in bentazon degradation.

CONCLUSIONS. The results obtained in this research show that Ganoderma lucidum and its enzymes can be useful in the control of environmental pollution caused by the herbicides diuron and bentazon. Further studies, using purified enzymes from Ganoderma lucidum are necessary to elucidate the types and toxicity of reaction products produced under these conditions. Such studies are important for developing effective bioremediation programs based on the ligninolytic system of G. lucidum. Key words: bioremediation, Ganoderma lucidum, laccase, ligninolytic enzymes, pesticides, white rot fungus.

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RESUMO GERAL

INTRODUÇÃO. Durante o século passado as atividades agrícolas foram responsáveis pela liberação de muitos pesticidas (inseticidas, fungicidas e herbicidas) no ambiente. O uso intensivo de tais compostos resultou em contaminações ambientais e humanas devido ao seu lento processo de degradação e alta toxicidade. Os herbicidas diuron (N'-3,4-diclorofenil-N,N-dimetiluréia) e bentazon (3-isopropil-1H-2,1,3-benzothiadizin-4-(3H)-ona 2,2-dióxido), ambos classificados como perigosos ao meio ambiente, são amplamente utilizados no Brasil principalmente em lavouras de cana-de-açúcar e arroz, respectivamente. A biorremediação utilizando vários microorganismos é uma alternativa segura e de baixo custo para remover diversos químicos persistentes do ambiente. Entre os microorganismos usados nesta técnica, os fungos da podridão branca da madeira têm sido objetos de várias pesquisas nos últimos anos. Estes basidiomicetos são os únicos organismos conhecidos capazes de degradar o polímero recalcitrante natural da madeira, a lignina. Isto é possível devido à existência de um sistema enzimático formado principalmente por lacases e peroxidases. Estas enzimas agem extracelularmente e, frequentemente, produzem radicais livres altamente reativos que atacam os substratos e são responsáveis pela baixa especificidade do sistema. Tais características possibilitam o ataque de uma ampla variedade de compostos químicos recalcitrantes estruturalmente relacionados à lignina. Phanerochaete chrysosporium é o basidiomiceto mais utilizado em processos de biorremediação. Suas exo-enzimas têm demonstrado possuir um grande potencial para detoxificação de vários xenobióticos, incluindo os herbicidas diuron e bentazon. Ganoderma lucidum, um fungo medicinal amplamente encontrado na natureza está associado à degradação de uma ampla variedade de madeiras. Alguns estudos revelaram seu grande potencial para a produção das enzimas modificadoras de lignina, principalmente lacases e manganês peroxidases. Até o presente, G. lucidum é conhecido ser um bom degradador de corantes sintéticos. A despeito de sua capacidade de degradar outros xenobióticos, os estudos de biorremediação utilizando G. lucidum ainda são escassos. Assim, a aplicação deste fungo e suas enzimas a processos de biorremediação é ainda um campo aberto que demanda exploração. OBJETIVOS. Os objetivos deste trabalho foram: 1) avaliar o efeito dos herbicidas diuron e bentazon sobre o crescimento e produção de enzimas ligninolíticas por G. lucidum cultivado em meio líquido contendo glicose e verificar a capacidade do fungo em remover ambos os compostos do meio; 2) comparar a remoção de bentazon em culturas líquidas e em estado sólido por G. lucidum cultivado em condições adequadas para elevada produção de enzimas ligninolíticas. MATERIAL E METODOS. G. lucidum foi cultivado em meio líquido estacionário e em estado sólido a 28 oC no escuro. As culturas líquidas foram suplementadas com glicose (1%) como fonte de carbono ou sabugo de milho (1%). Nestas culturas várias concentrações dos herbicidas bentazon (0-20 mM) e diuron (0-80 µM) foram adicionadas e, após 7 dias de cultivo, as culturas foram filtradas para determinar a biomassa seca e obter os filtrados fontes das enzimas lacase e manganês peroxidase. Alternativamente, o fungo foi cultivado nos meios líquidos por 3 dias antes da adição dos herbicidas. As culturas foram periodicamente filtradas para determinação de biomassa seca e atividades enzimáticas, além da determinação do conteúdo residual de herbicidas. O cultivo em estado sólido foi

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realizado utilizando sabugo de milho triturado com umidade inicial de 75%. Várias quantidades de bentazon (0-50 mM) foram adicionadas ao meio e, periodicamente, as culturas foram interrompidas pela adição de água destilada fria e agitadas por 1 h a 4 oC. As misturas foram filtradas para reter os materiais insolúveis e os filtrados aquosos foram usados como fontes das enzimas. Para extrair o bentazon adsorvido ao micélio ou ao resíduo de sabugo de milho, adicionou-se 25 ml de metanol ao material insolúvel obtido após a extração aquosa, sendo as misturas agitadas por 2 h. Os extratos metanólicos obtidos por filtração foram analisados quanto ao conteúdo residual de bentazon. Todas as análises da quantidade de herbicida residual foram realizadas por HPLC (Cromatografia Líquida de Alta Performace). RESULTADOS E DISCUSSÃO. Elevada atividade de lacase associada a uma redução de crescimento foi observado durante as primeiras 48 h após a aplicação de ambos os herbicidas às culturas de G. lucidum crescidas em meios líquidos com glicose. Nenhum crescimento foi observado com a adição de 25 mM de bentazon e 100 µM de diuron. Na ausência dos herbicidas, a atividade da lacase nos filtrados das culturas de sete dias foi 20 U/g. Na presença de bentazon e diuron, a atividade de lacase atingiu 170 e 207 U/g, respectivamente. A atividade de Mn peroxidase foi induzida apenas por diuron e a presença de bentazon causou apenas um ligeiro aumento na atividade desta enzima. Análise por eletroforese em gel de poliacrilamida das proteínas dos filtrados das culturas de G. lucidum revelou duas bandas com atividade lacase, sendo que a adição dos herbicidas bentazon e diuron induziu apenas uma isoforma. As análises de herbicidas residuais mostraram que ambos foram removidos dos filtrados das culturas, porém o bentazon foi mais eficientemente removido que o diuron. Lacase foi igualmente produzida nas culturas de G. lucidum em meio líquido e em estado sólido usando sabugo de milho como substrato, porém as atividades de Mn peroxidase nos cultivos em estado sólido foram 10 vezes superiores às encontradas em culturas líquidas. Apesar da aparente inibição do crescimento causado por bentazon, o herbicida elevou a produção de lacase para um valor máximo de 1800 U/L usando concentrações de 2,5 mM e 30 mM em culturas líquidas e em estado sólido, respectivamente. A atividade de Mn peroxidase não foi significantemente alterada pela adição do bentazon. Após 10 dias de cultivo, as quantidades de bentazon residual presentes nos extratos foram 47% e 12% das quantidades iniciais adicionadas aos meios líquidos e em estado sólido, respectivamente. Estes dados mostram que os cultivos em estado sólido foram mais eficientes na degradação do herbicida, onde as enzimas lacase e Mn peroxidase foram produzidas em altos níveis. Os resultados sugerem a possibilidade de ambas as enzimas estarem envolvidas na degradação do herbicida bentazon.

CONCLUSÕES. Os resultados obtidos nesta pesquisa mostram que Ganoderma lucidum e suas enzimas podem ser utilizadas no controle da poluição ambiental causada por bentazon. O entendimento do papel das enzimas ligninolíticas na degradação de compostos químicos é importante para o desenvolvimento efetivo de programas de biorremediação. Contudo, mais estudos necessitam ser realizados utilizando enzimas purificadas de G. lucidum para elucidar os tipos e toxicidade dos produtos de reação gerados em tais condições. Palavras chaves: biorremediação, enzimas ligninolíticas, fungos da podridão branca, Ganoderma lucidum, lacase, pesticidas.

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ARTICLE 1

Effect of the herbicides bentazon and diuron on

the production of ligninolytic enzymes

by Ganoderma lucidum

Jaqueline da Silva Coelho, Andrea Luiza de Oliveira, Cristina Giatti

Marques de Souza, Adelar Bracht and Rosane M. Peralta

Abstract

The effect of the herbicides bentazon and diuron on growth and production

of ligninolytic enzymes by the white rot fungus Ganoderma lucidum

cultured in glucose liquid media was evaluated in this work. Strong

improvement of the laccase activity associated with growth reduction was

observed during the first 48 h after the application of both herbicides.

Native PAGE analysis of the G. lucidum laccases revealed that the

improved activity in response to the herbicides was not due to the

expression of a new laccase, but that it was due to the over production of

an already existing isoform in the non-induced cultures. Analysis of the

residual herbicides showed that G. lucidum was able to remove both

bentazon and diuron and, as other white rot fungus, can be considered to

be potentially useful in the development of methods aiming to reduce the

contamination with herbicides.

Key words: herbicides, bentazon, bioremediation, diuron, Ganoderma

lucidum, white rot fungus

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Introduction

Throughout the past century farming and agricultural activities have

released many persistent and toxic chemical pesticides into the

environment including insecticides, fungicides, nematicides, rodenticides

and herbicides. The use of pesticides coincides with the chemical age,

which has transformed society since the 1950s. In areas where intensive

monoculture is practiced, pesticide use has been the standard method for

pest control. Unfortunately, the use of pesticides can also result in

environmental problems, such as disruption of predator-prey relationships

and loss of biodiversity. Additionally, the slow degradation of pesticides in

the environment can lead to environmental contamination of water, soil,

air, several types of crops and indirectly of humans (Navalon et al. 2002).

The consumption of herbicides in Brazil has increased strongly in

the last 10 years, mainly due to the improvement of the use of the no-

tillage technique. The herbicides diuron (N-3,4-dichlorophenyl-N’,N’-

dimethylurea) and bentazon (3-isopropyl-1H-2,1,3-benzothiadiazin-4(3H)-

one 2,2-dioxide) are largely used in Brazil mainly for sugar-cane and rice

cultivation, respectively. Both of them are classified as toxicity class III,

slightly toxic, and dangerous for the environment (Anvisa 2009).

Herbicides are persistent in the environment, are highly mobile and

can accumulate in the animal tissues producing a variety of ill effects.

Removing these pollutants from the environment in an ecologically

responsible, safe, and cost-effective way is a top concern for land

management agencies. Bioremediation using various microbial organisms

is one way to do this (Watanabe 2001). In the last years, the capability of

white rot fungi (WRF) to biodegrade several xenobiotics and recalcitrant

pollutants has generated a considerable research interest in this area of

industrial/environmental microbiology.

WRF are the only microorganisms known to be able to degrade the

highly recalcitrant natural polymer lignin (a heterogeneous polyphenolic

polymer) due to the existence of a powerful enzymatic system formed

mainly by peroxidases (lignin peroxidase, EC 1.11.1.12 and Mn

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peroxidase, EC 1.11.1.13) and laccases (EC 1.10.3.2) (Boerjan et al.

2003; Asgher et al. 2008; Novotný et al. 2004). Since these enzymes are

non-specific, they can also attack a wide range of recalcitrant compounds

including several herbicides, structurally related to lignin, accumulated in

soil and water due to an unsatisfactory management of chemicals at

farms, industries and society in general (Pointing 2001).

Fungal lignin-degrading systems have demonstrated capability to

transform these herbicides and can be an alternative to reduce the

ecological problems caused by the accumulation of these products in

nature. The most frequently used white-rot fungus for these applications

is Phanerochaete chrysosporium, whose secreted enzymes have

demonstrated to possess high potential for detoxification of several

xenobiotics (Bennet et al. 2002; Asgher et al. 2008). Other WRF largely

studied in detoxification processes include the genera Pleurotus, Trametes

and Coriolus (Pointing 2001; Alleman et al. 1992; Lamar and Dietrich

1990; Machado et al. 2005; Mileski et al. 1988, Tortella et al. 2005,

Asgher et al. 2008; Bennet al. 2002).

Ganoderma lucidum is one of the most important and widely

distributed WRF in the world and is associated with the degradation of a

wide variety of woods (D’Souza et al. 1999). Most studies with G. lucidum

are related to its medicinal and pharmacological properties (Boh et al.

2007; Sliva 2004; Zhong et al. 2004). However, the fungus has

demonstrated to possess a great ability to produce ligninolytic enzymes,

mainly laccases (D’Souza et al. 1999; Ko et al. 2001) and some studies

have explored its capability to degrade xenobiotics, including dyes

(Muregesan et al. 2007; 2009) and organic compounds (Jeon et al. 2008).

The potential of G. lucidum and its enzymes in bioremediation processes is

still far from being fully explored. Within this context, the objectives of

this work were to evaluate the effects of the herbicides diuron and

bentazon on the production of ligninolytic enzymes by G. lucidum and to

verify the capability of the fungus to remove these two compounds in

liquid media.

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Material and methods

Microorganism and inoculum

Ganoderma lucidum was obtained from the Culture Collection of the São

Paulo Botany Institute and cultured on potato dextrose agar Petri dishes

(PDA) for up to 2 weeks at 28 °C. When the Petri dish was fully covered

with mycelia, mycelial plugs measuring 10 mm in diameter were made

and used as inoculum for liquid cultures.

Cultivation of G. lucidum under liquid conditions in the presence of diuron

and bentazon

G. lucidum was cultivated in liquid stationary conditions at 28 °C in the

dark. Three disks from the growing edge of the mycelium on PDA plates

(approximately 1 cm of diameter) were transferred to 125 ml Erlenmeyer

flasks containing 25 ml of mineral solution (Vogel 1956) supplemented

with glucose at 1% as carbon source. The medium was previously

sterilized by autoclaving at 121 °C for 15 min. Several amounts of

bentazon (0-20 mM) and diuron (0-80 µM) were added to the liquid

medium. After 7 days, the cultures were filtered through Whatman no 1

filter paper to retain the mycelia. The culture filtrates were used to

determine the enzymatic activities. Alternatively, the fungus was

inoculated in glucose medium and allowed to grow for 3 days before the

addition of herbicides. In these cases, the cultures were periodically

interrupted by filtration, and the culture filtrates used to determine the

enzymatic activities and the residual herbicides. Abiotic controls which did

not receive the inoculum were incubated under the same conditions.

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Determination of dry biomass and amounts of diuron and bentazon

sorbed on the fungal mycelia

The mycelia were washed twice with distilled water and dried to constant

weight at 50 °C. The dry biomasses were determined gravimetrically. To

extract herbicides possibly adsorbed to the cells, 5 ml of methanol were

added to each dry mycelium and maintained shaken at 110 rpm for 3 h.

The extracts were obtained by centrifugation at 5000g for 15 min and

analyzed for herbicides by HPLC.

Enzyme Assay

Laccase activity was determined with 2,2′-azino-di-(3-ethylbenzothialozin-

6-sulfonic acid) (ABTS) as the substrate. The reaction mixture contained

0.5 mM ABTS, 20 mM sodium acetate buffer (pH 4.5) and the culture

filtrate. Oxidation of ABTS was monitored by an absorbance increase at

420 nm (ε420=36,000 M−1 cm−1) at 30 °C (Hou et al. 2004). The Mn

peroxidase activity was assayed spectrophotometrically by following the

oxidation of 1 mM MnSO4 in 0.05 M sodium malonate, pH 4.5, in the

presence of 0.1 mM H2O2. Manganic ions, Mn3+, form a complex with

malonate, which absorbs at 270 nm (ε270=11.59 mM-1 cm-1) (Wariishi et

al. 1992). One unit (U) of enzymatic activity was defined as the amount of

enzyme required to produce 1 µmol product per min and was expressed

as U/g dry mycelial biomass (U/g).

Analysis of residual herbicides

A HPLC system (Shimadzu, Tokyo) with a LC-20AT Shimadzu system

controller, Shimadzu SPD-20 A UV-VIS detector, equipped with a reversed

Shimpack C18 column (4.6 x 250 mm), maintained at 30 °C was used for

determining the residual amounts of bentazon and diuron in the culture

filtrates. All samples in duplicate were filtered through a 0.22 µm filter

unit (Millex®-GV, Molsheim, France) before injection and the solvents

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were filtered through a 0.45 µm filter (Whatman, Maidstone,

England). For the bentazon analyses, the mobile phase used was

methanol: acetic acid 0.1 M (50:50) and for the diuron analyses, the

mobile phase was methanol:water (70:30). For both solvents the flow

rate was 1 ml/min. Detection was done spectrophotometrically at 245 and

254 nm for diuron and bentazon, respectively. The herbicides

concentrations were determined using calibration curves with peak areas

of authentic standards.

Native polyacrilamide gel electrophoresis (SDS-PAGE)

Native SDS-PAGE was carried out on 12% polyacrilamide gel (Laemmli

1970). The laccase activity was visualized in the gel with ABTS as the

substrate (Yaver et al. 1996).

Chemicals

The herbicides Basagran® 600 (commercial formulation of bentazon) and

diuron (Sigma Chemical Corp., St. Louis, Mo) were used in this work.

Stock solutions of bentazon (600 mg/ml in water) and diuron (10 mg/ml

in DMSO) were prepared, filtered through a Millipore membrane (0.45 µm)

and stored at 4 ºC. The enzymatic substrates were obtained from Sigma

Chemical Corp. (St Louis, MO). PDA was obtained from Difco Laboratories

(Detroit, MI). The solvents used in the HPLC analysis were of

chromatographic grade and all other reagents were of analytical grade.

Statistical analyses

The data from the different treatments were compared using paired t-test

with a significance level of p<0.05. The experiments were conducted in

triplicate. The data are presented as mean± standard error. The analyses

were conducted using the GraphPad Prism® statistical program pack

(Graph Pad Software, San Diego, USA).

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19

Results

The effect of diuron and bentazon on the growth and enzyme

production by liquid cultures of G. lucidum is shown in Fig. 1. In these

experiments, increasing amounts of herbicides were added at the

beginning of the cultivation. Both herbicides had a negative effect in the

mycelial growth and no growth was observed upon the addition of 25 mM

bentazon and 100 µM diuron. However, the herbicides acted as strong

inducers of laccase. In the absence of the herbicides, the activity of

laccase on 7-days culture-filtrate was 20 U/g. In the presence of bentazon

and diuron, laccase activities improved up to 170 and 207 U/g,

respectively (Fig. 1A-B). The Mn peroxidase activity was induced only by

diuron (from 0.7 U/g in control media to 8.6 U/g) (Fig. 1B). The presence

of bentazon caused a slight improvement in the Mn peroxidase activity

(Fig. 1A).

Fig. 2 shows the effects of two different concentrations of diuron and

bentazon on the production of mycelial biomass when the herbicides were

added to the actively growing fungus. In these experiments the fungus

was allowed to grow for 3 days in glucose basal medium before the

addition of the herbicides. At the lowest concentrations (5 mM bentazon

and 30 µM diuron), the herbicides reduced only slightly the mycelial

biomass obtained after 10 days of cultivation. At the highest

concentrations (20 mM bentazon and 80 µM diuron), the growth was

drastically reduced and less than 60% of the mycelial biomasses were

obtained after 10 days of cultivation. Time courses of enzyme production

under both conditions are shown in Fig. 3. In control media, the maximal

laccase activity was obtained in the 3-days culture filtrate (around 25 U/g)

and the Mn peroxidase activity was less than 0.5 U/g. The addition of 5

and 20 mM bentazon drastically improved the laccase in the first 2 days of

cultivation and maximal activities of 90 and 140 U/g respectively, were

obtained after 4 days following the herbicide addition (Fig. 3A). Similar

results were obtained by the addition of diuron, although the inductive

effect was less accentuated (Fig. 3B). In relation to Mn peroxidase, the

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20

enzyme was induced by diuron (at low and high concentrations) and

by bentazon (at low concentration) (Fig.3C-D). The addition of 20 mM

bentazon had a negative effect in the Mn peroxidase activity (Fig. 3C).

Non-denaturing SDS-PAGE analysis was carried out using 10 days culture

filtrates (7 days of addition of herbicides) with and without herbicides (Fig.

4). The zymogram revealed two bands with laccase activities (lac1, with

minor electrophoretic mobility, and lac2, with major electrophoretic

mobility) in glucose culture filtrate (line 1). The addition of the herbicides

bentazon and diuron strongly improved only the isoform lac2 (lines 2 and

3, respectively)

Table 1 shows the residual amounts of herbicides after 7 and 10

days of cultivation (4 and 7 days after the addition of herbicides). Both

herbicides were removed from the culture filtrates, but bentazon was

more efficiently removed than diuron. To both herbicides, less than 8%

was sorbed to the fungal mycelium (data not shown).

Discussion

The data obtained in this work confirm the previous report (D’Souza

et al. 1999) that laccase is the most important ligninolytic enzyme of G.

lucidum considering that Mn peroxidase was marginally produced in

glucose basal media. The laccases of G. lucidum were strongly induced by

the herbicides diuron and bentazon. Native PAGE analysis of G. lucidum

laccases revealed that the improvement in the laccase activity in response

to the herbicides was not due to the expression of a new laccase, but that

it was due to the over production of an already existing isoform in the

non-induced cultures. Similar results were obtained with Trametes

versicolor and Abortiporus biennis (Jaszek et al. 2006) and Rhizoctonia

solani (Crowe and Olsson 2001), where their constitutive laccases were

overproduced in the presence of paraquat. It is well known that a

constitutive laccase is usually produced in small amounts by WRF and its

production can be significantly enhanced by a wide variety of substances,

including phenolic compounds such as 2,5-xylidine (Jang et al. 2006),

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21

ferulic acid and vanillin (Souza et al. 2004) or by the addition of metal

ions such as copper (Galhaup and Haltrich 2001; Tychanowicz et al. 2006)

or cadmium (Jarosz-Wilkołazka et al. 2002). On the basis of our results,

bentazon and diuron seem to be a novel way to achieve laccase activity

stimulation in a chemically defined medium. Mn peroxidase is not the

main ligninolytic enzyme of G. lucidum, its activity being several times

lower than that of laccase (Songulashvili et al. 2007). It is important to

note, however, that diuron also enhanced the Mn peroxidase activity.

Due the fact that several xenobiotics act as inducers of ligninolytic

enzymes, it has been frequently suggested that these enzymes are

important in the removal or transformation of those molecules. In fact, in

some white-rot fungi such as Pleurotus and Coriolus the biodegradation of

xenobiotics appear to be conducted mostly by laccases (Levin et al. 2003;

Ricotta et al. 1996; Sedarati et al. 2003; Ullah et al. 2000a-b, Gorbatova

et al. 2006). On the other hand, the oxidation of xenobiotics by other

white-rot fungi such as Nematoloma frowardii (Hofrichter et al. 1998;

Sack et al. 1997), P. chrysosporium (Moen and Hammel 1994), Irpex

lacteus (Baboravá et al. 2006) and Bjerkandera sp (Eibes et al. 2007;

Longoria et al. 2008; Rubilar et al. 2007) is mainly due to the action of Mn

peroxidases. In recent studies, purified ligninolytic enzymes from different

WRF were used to degrade in vitro pesticides such as chorophenols

(Zhang et al. 2008), hydroxyphenylureas (Jolivalt et al. 2006) and

glyphosate (Pizzul et al. 2009). These results show that these types of

enzymes have, at least in part, an important role in the degradation of

pollutants under in vitro conditions. However, some researchers have found

no correlation between ligninolytic enzymes and xenobiotics oxidation.

These researchers have suggested that, besides extracellular enzymes,

intracellular factors may also be involved in the capability of white rot

fungi to degrade xenobiotics. For example, cytochrome P450 (P450)-

mediated oxygenation reactions apparently do play an important role

during fungal metabolism of recalcitrant xenobiotic compounds in

Pleurotus ostreatus (Bezalel et al. 1996) and Coriolus versicolor (Hiratsuka

et al. 2001). Also, the degradation of the pesticide lindane by P.

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22

chrysosporium has been found to occur via detoxification by a

cytocrome P450 monooxygenase system, independent of the production

of ligninolytic peroxidase enzymes (Mougin et al. 1996; Matsuzaki and

Wariishi 2004). Moreover, the ligninolytic enzymes seem not to be

essential for the biodegradation of pentachlorophenol by P. chrysosporium

(Ryu et al. 2000).

In addition to stimulation by phenolic compounds chemically related

to lignin, the laccase activity in WRF can be regulated by some

environmental stress conditions such as high nitrogen levels (Gianfrieda

et al. 1999), nitrogen limitation (Pointing et al. 2000), or changes in

temperature (Fink-Boots et al. 1999). More recently, a role for laccase as

an element of the general stress response in several white rot fungi has

been suggested by many reports (Mayer and Staples 2002; Jaroz-

Wilkolazka et al. 2002; Galhaup and Haltrich 2001, Jaszek et al. 2006).

For example, the addition of the herbicide paraquat to the cultures of

Trametes versicolor and Abortiporus biennis significantly stimulated the

lacccase activity in association with an evident improvement of both

superoxide dismutase and catalase activities, well-known stress oxidative

markers (Jaszek et al. 2006). The strong growth reduction suggests that

the herbicides could be causing oxidative stress in G. lucidum. However,

additional efforts are needed to correlate the overproduction of laccase as

one more element of the general stress response.

Environmental contamination with herbicides and other aromatic

pollutants are a serious concern world-wide. Many studies have shown

that these persistent compounds can be degraded by the ligninolytic

system of white rot fungi. In this study, it was discovered that bentazon

and diuron strongly improved the production of laccase by Ganoderma

lucidum and the fungus was able to efficiently remove the herbicides from

the media. This suggests that Ganoderma lucidum and its laccase, as

other white rot fungus, could be useful as a biomarker to assess

environmental contamination and as an agent for xenobiotics removal.

Further studies are necessary to elucidate the types and toxicity of the

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reaction products generated during the degradation of bentazon,

diuron and possibly other herbicides.

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0 5 10 15 20 25

0

40

80

120

160

200

240

0

10

20

30

40

50

bentazon (mM)

lacc

ase

( �, U

/g),

dry

bio

mas

s (o

, mg)

Mn peroxidase (

�, U

/g)

0 20 40 60 80 100

0

40

80

120

160

200

240

0

10

20

30

40

50

diuron (µM)

lacc

ase

(�

, U/g

), d

ry b

iom

ass

(O, m

g)

Mn peroxidase (

�, U

/g)

A

B

Figure 1. Effects of bentazon and diuron on growth and production of

ligninolytic enzymes by G. lucidum. The herbicides were added at time

zero. The cultures were developed under static conditions at 28 °C for 7

days.

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30

0 2 4 6 8 10 12

0

50

100

150

200

culture time (d)

dry

biom

ass

(mg)

0 2 4 6 8 10 12

0

50

100

150

200

culture time (d)

dry

biom

ass

(mg)

A B

Figure 2. Effects of bentazon and diuron on growth of G. lucidum. The

fungus was cultured in glucose basal medium under static conditions for 3

days before the addition of bentazon (A) and diuron (B). The cultures

were maintained for more 7 days under the same conditions. Control:�; 5

mM bentazon and 30 µM diuron:�; 20 mM bentazon and 80 µM diuron:

�.

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0 2 4 6 8 10 12

0

30

60

90

120

150

culture time (d)

lacc

ase

(U/g

)

0 2 4 6 8 10 12

0.0

0.5

1.0

1.5

2.0

culture time (d)

Mn

pero

xida

se (

U/g

)

0 2 4 6 8 10 12

0

30

60

90

120

150

culture time (d)la

ccas

e (U

/g)

0 2 4 6 8 10 12

0.0

0.5

1.0

1.5

2.0

culture time (d)

Mn

pero

xida

se (

U/g

)

A B

C D

Figure 3. Time course of laccase and Mn peroxidase production by G.

lucidum. The fungus was cultured in glucose basal medium under static

condition for 3 days before the addition of bentazon (A end C) and diuron

(B end D). The cultures were maintained for more 7 days under the same

conditions. Control:�; 5 mM bentazon and 30 µM diuron:�; 20 mM

bentazon and 80 µM diuron: �.

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Figure 4. Native SDS-PAGE electrophoresis of extracellular laccase from G.

lucidum after 10 days of cultivation (7 days after the addition of the

herbicides). A volume of 25 µl of each culture filtrate was loaded in each

lane. Lane 1: sample of control culture; lane 2: sample of 20 mM

bentazon culture; Lane 3: sample of 30 µM diuron culture.

lac 1

lac 2

1 2 3

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33

Table 1. Residual bentazon and diuron in glucose cultures of

Ganoderma lucidum

Herbicide Residual herbicide (%)

Abiotic control 7 day-culture filtrate

10 day-culture filtrate

Diuron 30 µM 95±7.2A 68±4.8B 45±7.0C

Diuron 80 µM 93±7.0A 90±7.0A 74±5.1B

Bentazon 5 mM 94±6.0A 55±4.1B 12±2.0C

Bentazon 20 mM 96±8.0A 78±6.0B 61±6.0C

Means, within a row, followed by different letters differ statistically for

p<0.05

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ARTICLE 2

Removal of bentazon by Ganoderma lucidum in

liquid and solid state cultures

Jaqueline da Silva Coelho, Adriana Zilly, Andréia Luisa de Oliveira, Adelar

Bracht, Rosane Marina Peralta and Cristina Giatti Marques de Souza

Abstract

Bentazon removal by Ganoderma lucidum in liquid and solid state corn

cob cultures was studied in this work. In solid state cultures, the fungus

produced both ligninolytic enzymes, laccase and Mn peroxidase. In liquid

cultures, the main ligninolytic enzyme produced was laccase. In both

types of cultures bentazon improved the production of laccase without

significant alteration in the production of Mn peroxidase. In solid state

cultures, the fungus was more resistant to the presence of the herbicide

and more efficient in removing bentazon. The data obtained suggest that

laccase and Mn peroxidase may have an important role in the degradation

of bentazon by G. lucidum.

Key words: Ganoderma lucidum, herbicides, bentazon, white rot fungus,

ligninolytic enzymes, laccase.

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Introduction

The herbicide bentazon (3-isopropyl-1H-2,1,3-benzothiadiazin-

4(3H)-one 2,2-dioxide), CAS registry number 25057-89-0, is commonly

used as a post-emergence herbicide in cereal crops. Its application is

regulated in many countries such as Australia, New Zealand, India,

Philippines, South Africa, South America and Canada. In Brazil, bentazon

is mainly used on peanuts, rice, beans, corn, soy-beans and wheat. In

other countries it is used on these and several others, such as grassy,

leguminous and also leafy cultures (Pinto and Jardim 1999). Bentazon is

slightly toxic by ingestion and by dermal absorption. Human ingestion of

high doses of this herbicide causes vomiting, diarrhea, trembling,

weakness, and irregular or difficult breathing. It is moderately irritating to

the skin, eyes, and respiratory tract.

Bentazon is degraded at a moderate rate by microorganisms in the

soil environment. Lysimeter experiments with bentazon showed that its

half-life ranged from 24 to 65 days (Kordel et al. 1991). Huber and Otto

(1994) reported half-lives of bentazon from 4 to 21 days at five different

field sites in Germany and from 3 to 19 days at six different sites in the

United States. As consequence, after pesticide application, residues may

remain in the crops, soil and natural water and constitute a health risk

because of their toxicity. Removing bentazon from the environment in an

ecologically responsible, safe, and cost-effective way is a top concern for

land management agencies. Bioremediation using various microbial

organisms is one way of doing it (Watanabe 2001). In the last years, the

capability of white rot fungi (WRF) to biodegrade several xenobiotics and

recalcitrant pollutants has generated a considerable research interest in

the area of industrial/environmental microbiology (Boerjan et al., 2003,

Asgher et al. 2008; Novotný et al. 2004). WRF are the only

microorganisms known to be able to degrade the highly recalcitrant

natural polymer lignin (a heterogeneous polyphenolic polymer) because

they possess a powerful enzymatic system formed mainly by peroxidases

(lignin peroxidase, EC 1.11.1.12 and Mn peroxidase, EC 1.11.1.13) and

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36

laccases (EC 1.10.3.2). Since these enzymes are non-specific, they

can also attack a wide range of recalcitrant compounds, structurally

related to lignin, accumulated in soil and water.

Fungal lignin-degrading systems have demonstrated capability to

transform several herbicides and can be an alternative to reduce the

ecological problems caused by the accumulation of these products in

nature (Asgher et al. 2008). In relation to the capability to degrade

bentazon, Phanerochaete chrysosporium is the only well studied WRF

(Castillo et al. 2000a,b; Knauber et al. 2000). In principle at least, other

WRF could be useful in the degradation of bentazon. Ganoderma lucidum

is one of the most important and widely distributed WRF in the world and

it is associated with the degradation of a wide variety of woods (D’Souza

et al. 1999). Most studies with G. lucidum are related to its medicinal and

pharmacological properties (Boh et al. 2007; Sliva 2004; Zhong et al.

2004). However, the fungus has demonstrated to possess a great ability

to produce ligninolytic enzymes, mainly laccases (D’Souza et al. 1999; Ko

et al. 2001) and some studies have explored its capability to degrade

xenobiotics, including dyes (Muregesan et al. 2007; 2009) and organic

compounds (Jeon et al. 2008). We recently reported that two herbicides,

diuron and bentazon, effectively stimulated the production of laccase by

G. lucidum in glucose liquid cultures (Coelho et al. submitted). However,

the potential of G. lucidum and its enzymes in bioremediation processes is

still far from being fully explored. Within this context, the objective of this

work was to compare the removal of bentazon by liquid and solid state

cultures of G. lucidum. The cultures were done under conditions suitable

for obtaining elevated levels of ligninolytic enzymes.

Material and methods

Microorganism and inoculum

Ganoderma lucidum was obtained from the Culture Collection of the São

Paulo Botany Institute and cultured on potato dextrose agar Petri dishes

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37

(PDA) for up to 2 weeks at 28 °C. When the Petri dish was fully

covered with mycelia, mycelial plugs measuring 10 mm in diameter were

made and used as inoculum for liquid cultures.

Cultivation of G. lucidum under liquid conditions in the presence and

absence of bentazon

G. lucidum was cultivated under liquid stationary conditions at 28 °C in

the dark. Three disks from the growing edge of the mycelium on PDA

plates (approximately 1 cm of diameter) were transferred to 125 ml

Erlenmeyer flasks containing 25 ml of mineral solution (Vogel 1956)

supplemented with corn cob powder at 1% as substrate. The medium was

previously sterilized by autoclaving at 121 °C for 15 min. Several different

amounts of bentazon (0-20 mM) were added to the liquid medium. After 7

days, the cultures were filtered through Whatman no 1 filter paper to

retain the mycelia. The culture filtrates were used to determine the

enzymatic activities. Alternatively, the fungus was inoculated in the corn

cob liquid medium and allowed to grow for 3 days before the addition of

2.5 mM bentazon. In these cases, the cultures were periodically

interrupted by filtration, and the culture filtrates used to determine the

enzymatic activities and the residual herbicide.

Cultivation of G. lucidum under solid state conditions in the presence and

absence of bentazon.

Three mycelial plugs were transferred to 125 ml Erlenmeyer flasks

containing 5 g of corn cob powder. Mineral solution (Vogel 1956) was used

to adjust the moisture content to 75%. Dry weight of the substrate and

moisture content were determined gravimetrically, after drying samples at

60 oC. Incubation was carried out at 28 °C. The medium was previously

sterilized by autoclaving at 121 °C for 15 min. Several different amounts

of bentazon (0-50 mM) were added to the medium. At periodic intervals

25 ml of cold water were added to the cultures and the mixtures were

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38

shaken for 1 h at 4 °C. The mixtures were filtered, and the filtrates

were used as source of enzymes.

Extraction of bentazon sorbed on the fungal mycelia and residual corn

cob.

For extracting the bentazon possibly sorbed on the fungal mycelia and

corn cob, 25 ml of methanol were added to the insoluble materials

obtained after aqueous extraction and the mixtures were shaken at 120

rpm in an orbital shaker for 2 h. The mixtures were then filtered through

Whatman no 1 filter paper to retain the mycelia and corn cob.

Analysis of residual herbicides

To evaluate the residual bentazon in the cultures, the combined aqueous

and methanolic extracts were concentrated just to dryness by using a

rotary evaporator. Each residue was reconstituted in 10 ml of a mixture

of methanol:acetic acid 0.1 M (50:50). A HPLC system (Shimadzu, Tokyo)

with a LC-20AT Shimadzu system controller, Shimadzu SPD-20 A UV-VIS

detector, equipped with a reversed Shimpack C18 column (4.6 x 250

mm), maintained at 30 °C, was used for determining the residual amounts

of bentazon. All samples in duplicate were filtered through a 0.22 µm filter

unit (Millex®-GV, Molsheim, France) before injection and the solvents

were filtered through a 0.45 µm filter (Whatman, Maidstone, England).

For the bentazon analyses, the mobile phase was methanol: acetic acid

0.1 M (50:50) and the flow rate was 1 ml/min. Detection was done

spectrophotometrically at 254 nm. The herbicide concentrations were

determined using a calibration curve constructed with peak areas of

authentic standards. Identification of bentazon in the samples was based

on retention time (6.38 min) and fortification of the samples with

standards.

Enzyme assays

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39

Laccase activity was determined with 2,2′-azino-di-(3-ethylbenzothialozin-

6-sulfonic acid) (ABTS) as the substrate. The reaction mixture contained

0.5 mM ABTS, 20 mM sodium acetate buffer (pH 4.5) and the culture

filtrate. Oxidation of ABTS was monitored as absorbance increase at

420 nm (ε420=36,000 M−1 cm−1) at 30 °C (Hou et al. 2004). The Mn

peroxidase activity was assayed spectrophotometrically by following the

oxidation of 1 mM MnSO4 in 0.05 M sodium malonate, pH 4.5, in the

presence of 0.1 mM H2O2. Manganic ions, Mn3+, form a complex with

malonate, which absorbs at 270 nm (ε270=11.59 mM-1 cm-1) (Wariishi et

al. 1992). One unit (U) of enzymatic activity was defined as the amount of

enzyme required to produce 1 µmol product per min and was expressed

as U/L.

Chemicals

The herbicide Basagran® 600 (commercial formulation of bentazon) was

used in this work. Stock solution of bentazon (600 mg/ml in water) was

prepared, filtered through a Millipore membrane (0.45 µm) and stored at

4 °C. The enzymatic substrates were obtained from Sigma Chemical Corp.

(St Louis, MO). PDA was obtained from Difco Laboratories (Detroit, MI).

The solvents used in the HPLC analysis were of chromatographic grade

and all other reagents were of analytical grade.

Statistical analyses

The data from the different treatments were compared using paired t-test

with a significance level of p<0.05. The experiments were conducted in

triplicate. The data are presented as mean± standard error. The analyses

were conducted using the GraphPad Prism® statistical program pack

(Graph Pad Software, San Diego, USA).

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Results

G. lucidum was able to grow in liquid and solid state cultures using

corn cob as substrate. In control cultures, identical maximal laccase

activities were 1,000 U/L in both types of cultivation. In relation to Mn

peroxidase, solid state conditions allowed the obtainment of high Mn

peroxidase activity (230 U/L), in comparison to that one obtained in liquid

cultures (15.3 U/L).

The effects of bentazon on the production of laccase and Mn

peroxidase by G. lucidum are shown in Fig. 1 and 2, respectively. In these

experiments, increasing amounts of herbicides were added at the

beginning of the cultivation. Bentazon had a negative effect on the

mycelial growth (visual analysis) in both types of cultures. No growth was

observed upon the addition of 25 and 60 mM bentazon in liquid and solid

state cultures, respectively. In despite of the apparent growth inhibition,

the herbicide enhanced the production of laccase to a maximal value of

1,800 U/L, using 2.5 mM and 30 mM bentazon in liquid and solid state

cultures, respectively (Fig. 1). The Mn peroxidase activity was only slightly

improved by bentazon: using 10 mM of bentazon, the production of Mn

peroxidase was 21 and 262 U/L in liquid and solid state cultures,

respectively (Fig. 2). Time courses of enzyme production under both

conditions are shown in Fig. 3. These curves confirm that bentazon

significantly improved the production of laccase (p<0.05) while the

production of Mn peroxidase was not significantly affected (p>0.05).

Typical HPLC profiles of combined residual bentazon (aqueous and

methanolic extracts) from solid state cultures at three times of cultivation

(0, 5 and 10 days) are shown in Fig. 4. Similar results were obtained

analyzing the combined extracts from liquid cultures (data not shown).

Phenolic compounds from substrate (corn cob) were eluted between 2 and

3 min and bentazon eluted at 6.38 min. The amounts of residual bentazon

were calculated from the areas under the chromatographic profiles based

on appropriate standard curves (Fig. 5). After 5 days of cultivation, in

spite of a decreasing tendency, the amount of residual bentazon was not

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41

statistically different from the initial bentazon amount (p>0.05).

However, after 10 days of cultivation, the residual bentazon present in the

combined extracts was 47 and 12% of the initially added to liquid and

solid state cultures, respectively.

Discussion

Ganoderma lucidum belongs to a highly specialized group of

microorganisms able to degrade the recalcitrant polymer lignin because

they possess oxidative lignin-modifying enzymes (peroxidases and

laccase). These enzymes present a wide range of biotechnological

applications including bioremediation of xenobiotic compounds. Laccase is

described as the main ligninolytic enzyme produced by G. lucidum

(D’Souza et al. 1999; Ko et al. 2001), although in a recent work, Mn

peroxidase has been described as the only ligninolytic enzyme produced

by this fungus (Bibi et al. 2009). The results obtained in this work showed

that laccase was produced in both liquid and solid state cultures using

corn cob as substrate, but activities of Mn peroxidase in solid state

cultures were 10 times superior than those found in liquid cultures.

Lignocellulosic agricultural crop residues are frequently used for the

cultivation of WRF. The choise of corn cob was due to the facility of

obtainment, the low amounts of natural colored pigments found in this

material and by the capability of G. lucidum to grow in corn cob based

medium without the necessity of supplementation with additional carbon

sources. Corn cob has been used by several researchers as substrate in

liquid and solid state cultures for enhanced enzyme production (Boer et al.

2004; Oliveira et al. 2006; Kadowaki et al. 1997; Tychanowicz et al.

2006).

Due to the low specificity of oxidative lignin-modifying enzymes, and

the chemical structure of bentazon, it is reasonable to suppose that this

herbicide can be degraded by WRF. However, until now, only

Phanerochaete chrysosporium was more properly evaluated as a bentazon

decomposer. A culture of P. chrysosporium and its purified laccase

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42

converted 8-hydroybentazon, one of the metabolite products of

bentazon, to a dimeric derivative (Knauber et al. 2000). A relationship

between bentazon degradation and the production of ligninolytic enzymes

has already been reported in solid state cultures of P. chrysosporium.

Moreover, it was also shown that a purified lignin peroxidase from P.

chrysosporium efficiently oxidizes bentazon (Castillo et al. 2000).

In the present work G. lucidum showed a considerable tolerance to

bentazon when cultured on solid state conditions. The data suggest that

under both types of cultivation, the fungus was able to degrade bentazon.

Degradation, however, was more efficient under solid state conditions,

where high levels of both laccase and Mn peroxidase activitites were

found. These observations suggest that both enzymes may have a role in

bentazon degradation. The extracellular ligninolytic system of WRF is

mainly based on free radical oxidative reactions, which represent an

unspecific and efficient way to reach recalcitrant compounds (Davila-

Vazquez et al. 2005).

Studies of bentazon degradation by soil microorganisms have

identified 6 and 8 hydroxybentazon as the main metabolic products of

bentazon (Otto et al. 1979). However, the identification of these

metabolites is frequently difficult because within 24 h, hydroxylated

bentazons are incorporated as insoluble, bound residues on humic and

fulvic acids (Wagner et al. 1996). In Ganoderma lucidum cultures,

bentazon was undoubtly transformed, but no metabolite product could be

found in the combined aqueous and methanolic extracts. Possibly

insoluble molecules were produced. If this occurred such compounds were

discarded in the various filtration procedures. In support to this idea it

should be mentioned that both laccase and Mn peroxidase are able to

polymerize phenolic compounds generating high molecular weight

products. For example, Mn peroxidase from Bjerkandera adusta was able

to polymerize guaiacol, o-cresol, 2,6 dimethoxyphenol and other phenolic

compounds and aromatic amines generating molecules with elevated

molecular weight, some of them insoluble in methanol (Iwahara et al.

2000). Additionally, a purified versatile peroxidase of B. adusta produced

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43

several polymers from the pesticide bromoxynil (Davila-Vazquez et al.

2005). Concerning laccase, a purified enzyme from Trametes versicolor,

immobilized onto a hydrophilic PVDF microfiltration membrane,

transformed the herbicide phenylurea via an oxidative reaction resulting

into an insoluble polymerized product (Jolivalt et al. 2000).

Increasing amounts of agrochemical and industrial effluents enter

soil and water environments each day, and it is essential to develop a

thorough understanding of the role of ligninolytic enzymes in the

degradation of these chemicals. The results obtained in this research show

that Ganoderma lucidum and its enzymes can be useful in the control of

environmental pollution caused by bentazon. Further studies, using

purified enzymes from Ganoderma lucidum are necessary to elucidate the

types and toxicity of reaction products produced under these conditions.

Such studies are important for developing effective bioremediation

programs based on the ligninolytic system of G. lucidum.

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0 10 20 30 40 50 60

0

500

1000

1500

2000

2500

bentazon concentration (mM)

lacc

ase

(U/L

)

Figure 1. Effect of bentazon on the production of laccase by G. lucidum in

liquid (�) and solid state corn cob cultures (�). The herbicide was added

at time zero. The cultures were developed under static conditions at 28 °C

for 7 days.

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45

0 10 20 30 40 50 60

0

100

200

300

0

10

20

30

bentazon concentration (mM)

Mn

pero

xida

se

(�

,U/L

) Mn peroxidase ( �

, U/L)

Figure 2. Effect of bentazon on the production of Mn peroxidase by G.

lucidum in liquid (�) and solid state (�) corn cob cultures. The herbicide

was added at time zero. The cultures were developed under static

conditions at 28 °C for 7 days.

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46

0 2 4 6 8 10

0

500

1000

1500

2000

culture time (d)

Lacc

ase

(U/L

)

0 2 4 6 8 10

0

100

200

300

culture time (d)

Mn

pero

xida

se (

U/L

)

0 2 4 6 8 10

0

500

1000

1500

2000

culture time (d)

Lacc

ase

(U/L

)

0 2 4 6 8 10

0

10

20

30

culture time (d)

Mn

pero

xida

se (

U/L

)

A B

CD

Figure 3. Time course of laccase and Mn peroxidase production by G.

lucidum. The fungus was cultured in solid state end liquid corn cob

cultures. In solid state cultures (A and C), the bentazon ((�:10mM; �:

30mM) was added at time zero. In liquid cultures (B and D), the fungus

was allowed to grow for 3 days before the addition of bentazon (2.5mM).

Without bentazon (�).

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47

Figure 4. Typical HPLC profiles of combined residual bentazon (aqueous

and methanolic extracts) from Ganoderma lucidum solid state cultures at

three times of cultivation (0, 5 and 10 days). Standard= bentazon

standard; control cultures= G. lucidum solid state culture developed

without bentazon.

10 days

0 days

0 1 2 3 4 5 6

12 7 8 9 10

Elution time (minutes)

1800

1500

1200

900

0

mV

Control culture, 5 days

Standard

5 days

600

300

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48

0 5 100

20

40

60

80

100

120

culture time (d)

Ext

ract

ed b

enta

zon

(%)

0 5 100

20

40

60

80

100

120

time course (d)E

xtra

cted

ben

tazo

n (%

)

A B

Figure 5. Residual bentazon in liquid and solid state cultures of

Ganoderma lucidum. A: Bentazon was added to the solid state cultures to

a final concentration of 30 mM. The residual bentazon extracted

(combined aqueous and methanolic extractions) from the 5 and 10 days-

cultures was determined by HPLC. B: Bentazon was added to the liquid

cultures in the third day of cultivation to a final concentration of 2.5 mM.

The residual bentazon extracted (combined aqueous and methanolic

extractions) from the 5 and 10 days-cultures was determined by HPLC. In

both types of cultivation, abiotic controls (hatched columns) containing

the same amounts of bentazon were submitted at the same extraction

process.

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49

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