UNIVERSIDADE ESTADUAL DE MARINGÁ
CENTRO DE CIÊNCIAS BIOLÓGICAS
Programa de Pós-Graduação em Ciências Biológicas
ENVOLVIMENTO DAS ENZIMAS LIGNINOLÍTICAS
DE Ganoderma lucidum NA REMOÇÃO DOS
HERBICIDAS DIURON E BENTAZON
JAQUELINE DA SILVA COELHO
Maringá
2009
2
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
3
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
4
Orientadora
Profa. Dra. Cristina Giatti Marques de Souza
Co-Orientadora
Profa. Dra. Rosane Marina Peralta
5
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.
6
Dedico
A Deus, mestre maior e
presença constante em minha
vida.
7
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.
8
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.
9
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-dichlorophenylN’,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
10
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.
11
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,Ndimetiluréia) e bentazon (3-isopropil-1H-2,1,3-benzothiadizin-4-(3H)-ona 2,2dióxido), 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
12
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.
13
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
14
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 notillage
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
15
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.
16
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.
17
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-ethylbenzothialozin6-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
18
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).
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
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),
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.
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; JarozWilkolazka 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
23
reaction products generated during the degradation of bentazon,
diuron and possibly other herbicides.
References
Alleman BC, Logan BE, Gilbertson RL (1992) Toxicity of pentachlorophenol
to six species of white rot fungi as a function of chemical dose.
Applied and Environmental Microbiology 58: 4048-4050.
Anvisa. Brasilian National Health Vigilance Agency: Information system
about
agrotoxics
(2009)
Available
in :
http://www4.anvisa.gov.br/AGROSIA/asp/frm_dados_agrotoxico.asp.
Acessed on June 17
th
2009.
Asgher M, Bhatti HN, Ashraf M, Legge RL (2008) Recent developments in
biodegradation of industrial pollutants by white rot fungi and their
enzyme system. Biodegradation 19:771-783.
Baboravá P, Moder M, Baldrian P, Cajthamlová K, Cajthaml T
(2006)
Purification of a new manganese peroxidase of the white-rot fungus
Irpex lacteus and degradation of polycyclic aromatic hydrocarbons by
the enzyme. Chemosphere 43:167-182.
Bennet J, Wunch K, Faison B (2002) Use of Fungi in Biodegradation. In:
Hurst CJ (ed) Manual of Environmental Microbiology, 2nd edn. ASM
Press, Washington DC, pp 960–971.
Bezalel L, Hadar Y, Cerniglia CE (1996) Mineralization of polycyclic
aromatic hydrocarbons by the white-rot fungus Pleurotus ostreatus.
Appl Envirom Microbiol 62:292-295.
Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant
Biol 54:519–546.
Boh B, Berovic M, Zhang J, Zhi-Bin L (2007) Ganoderma lucidum and its
pharmaceutically active compounds. Biotechnol Ann Rev 13:265-301.
Crowe JD, Olsson S (2001) Induction of laccase activity in Rhizoctonia
solani by antagonistic Pseudomonas fluorescens strains and a range
of chemical treatments. Appl Environ Microbiol 67:2088-2094.
24
D’Souza TM, Merrit CS, Reddy CA (1999) Lignin-modifying enzymes of
the
white-rot basidiomycete
Ganoderma
lucidum.
Appl Environ
Microbiol 65:5307-5313.
Eibes G, Moreira MT, Feijoo G, Daugulis AJ, Lema JM (2007) Operation of
a two-phase partitioning bioreactor for the oxidation of anthracene by
the enzyme manganese peroxidase. Chemosphere 66:1744-1751.
Fink- Boots M, Malarczyk E, Leonowicz A (1999) Increased enzymatic
activities and levels of superoxide anion and phenolic compounds in
cultures of basidiomycetes. Acta Biotechnol. 19: 319–330.
Galhaup C, Haltrich D (2001) Enhanced formation of laccase activity by
the white-rot fungus Trametes pubescens in the present of copper.
Appl Microbiol Biotechnol 56:225-232.
Gianfrieda L, Xu F, Bollag JM (1999) Laccases: a useful group of
oxidoreductive enzymes. Bioremediation J. 3: 1–25.
Gorbatova ON, Koroleva OV, Landesman EO, Stepanova EV, Zherdev AV
(2006) Increase of the detoxification potential of basidiomycetes by
induction
of
laccase
biosynthesis.
Applied
Biochemistry
and
Microbiology 42:1608-3024.
Hiratsuka N, Wariishi H, Tanaka H (2001) Degradation of diphenyl ether
herbicides by the lignin-degrading basiomycete Coriolus versicolor.
Appl Microbiol Biotechnol 57:563-57.
Hofrichler M, Scheibner K, Schneegab K, Fritsche W (1998) Enzymatic
combustion of aromatic and aliphatic compounds by manganese
peroxidase
from
Nematoloma
frowardii.
Appl
Environ
Microbiol
64:399-404.
Hou H, Zhou J, Wang J, Du C, Yan B (2004) Enhancement of laccase
production by Pleurotus ostreatus and its use for the decolorization of
anthraquinone dye. Proc Biochem 30:1415-1419.
Jang MY, Ryu WY, Cho MH (2006) Enhanced production of laccase from
Trametes sp. by combination of various inducers. Biotechnol Biopro
Eng 11:96–99.
Jarosz-Wilkolazka A, Malarczyk E, Pirszel J, Skowronski T, Leonowicz A
(2002) Uptake of cadmium ions in white-rot fungus Trametes
25
versicolor: effect of Cd(II) ions on the activity of laccase. Cell Boil
Intern 26:605-613.
Jaszek M, Grzywnowicz K, Malarczyk E, Leonowicz A (2006) Enhanced
extracellular activity as a part of the response system of white rot
fungi: Trametes versicolor and Abortiporus biennis to paraquatcaused oxidative stress conditions. Pestic Biochem Phys 85:147-154.
Jeon J-R, Murugesan K, Kim Y-M, Kim E-J, Chang Y-S (2008) Synergistic
effect
of
laccase
mediators
on
pentachlorophenol
removal
by
Ganoderma lucidum laccase. Appl Microbiol Biotechnol 81:783-790.
Jolivalt C, Neuville L, Boyer FD, Kerhoas L, Mougin C (2006) Identification
and formation pathway of laccase-mediated oxidation products
formed from hydroxyphenylureas. J Agric Food Chem 54:5046-5054.
Ko E-M, Leem Y-E, Choi HT (2001) Purification and characterization of
laccase isoenzymes from the white-rot basidimycete Ganoderma
lucidum. Appl Microbiol Biotechnol 57:98-102.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
Lamar RT, Dietrich DM (1990) In situ depletion of pentachlorophenol from
contaminated soil by Phanerochaete spp. Appl Environ Microbiol 56:
3093-3100.
Levin L, Viale A, Forchiassin A (2003) Degradation of organic pollutants by
the white rot basidiomycete Trametes trogii. Int Biodet Biodeg 52:15.
Longoria A, Tinoco R, Vázquez-Duhalt R (2008) Chloroperoxidasemediated
transformation
of
highly
halogenated
monoaromatic
compounds. Chemosphere 72: 485–490.
Machado KMG, Matheus DR, Bononi VLR (2005) Ligninolytic enzymes
production and Remazol Brilliant blue R decolorization by tropical
Brazilian Basidiomycetes fungi. Brazilian Journal of Microbiology
36:246-252.
Matsuzaki F, Wariishi H (2004) Functional diversity of cytochrome P450 s
of the white-rot fungus Phanerochaete chrysosporium. Biochem
Biophys Res Commun 324:387–393.
26
Mayer AM, Staples RC (2002) Laccase: new function for an old
enzyme. Phytochemistry 60:551-565.
Mileski GJ, Bumpus JA, Jurek MA, Aust SD (1988) Biodegradation of
pentachlorophenol
by
the
white-rot
fungus
Phanerochaete
chrysosporium. Appl Environ Microbiol 54:2885-2889.
Moen MA, Hammel KE (1994) Lipid peroxidation by the manganese
peroxidase
by
Phanerochaete
chrysosporium
is
the
basis
for
phenanthrene oxidation by the intact fungus. Appl Environ Microbiol
60:1956-1961.
Mougin
C,
Pericaud
C,
Biotransformation
of
Malosse
the
C,
Laugero
insectide
C,
lindane
Asther
by
the
M
(1996)
white
rtol
basidiomycete Phanerochaete chrysosporium. Pestic Sci 47:51-59.
Muregesan K, Yang I-H, Kim Y-M, Jeon J-R, Chang Y-S (2009) Enhanced
transformation of malachite green by laccase of Ganoderma lucidum
in the presence of natural phenolic compounds. Appl Microbiol
Biotechnol 82:341-350.
Murugesan K, Nam IH, Kim YM, Chang YS (2007) Decolorization of
reactive dyes by a thermostable laccase produced by Ganoderma
lucidum in solid state culture. Enz Microbial Technol 40:1662–1672.
Navalon A, Prielo A, Araujo L, Vilchez JL (2002) Determination of
oxadiazon residues by headspace solid-phase micro-extraction and
gas chromatography-mass spectrometry. J Chromatogr A 946:239245.
Novotný Č, Svobodová K, Erbanová P, Cajthaml T, Kasinath A, Lang E,
Šašek V (2004) Ligninolytic fungi in bioremediation: extracellular
enzyme production and degradation rate. Soil Biol Biochem 36:1545–
1551.
Pizzul L, Castillo MP, Stenström J (2009) Degradation of glyphosate and
other pesticides by ligninolytic enzymes. Biodegradation in press
Pointing SB (2001) Feasibility of bioremediation by white rot fungi. Appl
Microbiol Biotechnol 57:20-33.
27
Pointing SB, Jones EBG, Vrijmoed LLP (2000) Optimization of laccase
production by Pycnoporus sanquineus in submerged liquid culture.
Mycologia 92:139–144.
Ricotta A, Unz RF, Bollag JM (1996) Role of laccase in the degradation of
pentachlorophenol. Bull Environ Contam Toxicol 57:560-567.
Rubilar O, Feijoo G, Diez MC, Lu-Chau TA, Moreira MT, Lema JM (2007)
Biodegradation
of
pentachlorophenol
in
soil
slurry
cultures
by
Bjerkandera adusta and Anthracophyllum discolor. Ind Eng Chem Res
46:6744–6751.
Ryu WR, Shim SH, Jang MY, Jeon YJ, Oh KK, Cho MH (2000)
Biodegradation of pentachlorophenol by white rot fungus under
ligninolytic and nonligninolytic conditions. Biotechnol Bioprocess Eng
5:211-214.
Sack U, Heinze TM, Deck J, Cerniglia CE, Martens R, Zadrazil F, Fritsche W
(1997) Comparison of phenanthrene and pyrene degradation by
different wood-decaying fungi. Appl Environ Microbiol 63:3919-3925.
Sedarati
MR,
Keshavarz
T,
Leontievsky
AA,
Evans
CS
(2003)
Transformation of high concentrations of chlorophenols by the whiterot basidiomycete Trametes versicolor immobilized on nylon mesh.
Electron J Biotechnol 6:104–114.
Sliva D (2004) Cellular and physiological effects of Ganoderma lucidum
(Reishi). Mini Rev Med Chem 4: 873-879.
Songulashvili G, Elisashvili V, Wasser SP, Nevo E, Hadar Y (2007)
Basidiomycetes
laccase
and
manganese
peroxidase
activity
in
submerged fermentation of food industrial wastes. Enz Microb Technol
41:57-61.
Souza CGM, Tychanowica GK, Souza DF, Peralta RM (2004) Production of
laccase isoforms by Pleurotus pulmonarius in response to presence of
phenolic and aromatic compounds. J Bas Microbiol 2:129-136.
Tortella GR Diez MC (2005) Fungal diversity and use in decomposition of
environmental pollutants. Crit Rev Microbiol 34:189-206.
Tychanowicz GK, Souza DF, Souza CGM, Kadowaki MK, Peralta RM (2006)
Copper improves the production of laccase by the white-rot fungus
28
Pleurotus pulmonarius in solid state fermentation. Braz Arch
Biol
Technol 49:699-704.
Ullah MA, Bedford CT, Evans CS (2000)a Reactions of pentachlorophenol
with laccase from Coriolus versicolor. Appl Microbiol Biotechnol
53:230-234.
Ullah MA, Kadhim H, Rastall RA, Evans CS (2000)b Evaluation of solid
substrates for enzyme production by Coriolus versicolor, for use in
bioremediation of chlorophenols in aqueous effluents. Appl Microbiol
Biotechnol 54:832-837.
Vogel HA (1956) A convenient growth medium for Neurospora crassa.
Microb Genet Bull 13:42-43.
Wariishi H, Valli
manganese
K, Gold MH (1992) Manganese(II)
peroxidase
from
the
Basidiomycete
oxidation
by
Phenerochaete
chrysosporium. J. Biol. Chem. 267:23688–23695.
Watanabe K (2001) Microorganisms relevant to bioremediation. Curr Opin
Biotechnol 12:237–241.
Yaver DS, Xu F, Golightly DJ, Brown KM, Brown SH, Rey MW, Scheider P,
Halkier T, Mondorf K, Dalboge H (1996) Purification, characterization,
molecular cloning and expression of two laccase genes from the
white-rot basidiomycete Trametes villosa. Appl Environ Microbiol
62:834–841.
Zhang J, Liu X, Xu Z, Chen H, Yang
Y (2008) Degradation of
chlorophenols catalyzed by laccase. Int Biodeter Biodegr 61:351-356
Zhong JJ, Tang YJ (2004) Submerged cultivation of medicinal mushrooms
for production of valuable bioactive metabolites. Adv Biochem Eng
Biotechnol 87:25-59.
240
50
A
200
40
160
30
120
20
80
Mn peroxidase ( , U/g)
laccase ( , U/g), dry biomass (o, mg)
29
10
40
0
0
0
5
10
15
20
25
240
50
B
200
40
160
30
120
20
80
10
40
0
Mn peroxidase (, U/g)
laccase ( , U/g), dry biomass (O, mg)
bentazon (mM)
0
0
20
40
60
80
100
diuron (µM)
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.
30
200
B
A
150
dry biomass (mg)
dry biomass (mg)
200
100
150
100
50
50
0
0
0
2
4
6
8
10 12
culture time (d)
0
2
4
6
8
10 12
culture time (d)
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:
.
31
150
150
B
A
120
laccase (U/g)
laccase (U/g)
120
90
60
90
60
30
30
0
0
0
2
4
6
8
10 12
0
2
2.0
8
10
12
2.0
D
Mn peroxidase (U/g)
C
Mn peroxidase (U/g)
6
culture time (d)
culture time (d)
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
0
2
4
6
8
10
12
culture time (d)
Figure 3.
4
0
2
4
6
8
10
12
culture time (d)
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
bentazon and 80 µM diuron: .
and 30 µM diuron:; 20 mM
32
lac 1
lac 2
1
2
3
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.
33
Table 1. Residual bentazon and diuron in glucose cultures of
Ganoderma lucidum
Herbicide
Residual herbicide (%)
Abiotic control
Diuron 30 µM
95±7.2A
7 day-culture
filtrate
68±4.8B
10 day-culture
filtrate
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
34
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.
35
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
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
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
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
39
Laccase activity was determined with 2,2′-azino-di-(3-ethylbenzothialozin6-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).
40
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
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
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 (DavilaVazquez 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
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.
44
2500
laccase (U/L)
2000
1500
1000
500
0
0
10
20
30
40
50
60
bentazon concentration (mM)
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.
300
30
200
20
100
10
0
Mn peroxidase (, U/L)
Mn peroxidase ( ,U/L)
45
0
0
10
20
30
40
50
60
bentazon concentration (mM)
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.
46
2000
A
B
1500
Laccase (U/L)
Laccase (U/L)
2000
1000
1500
1000
500
500
0
0
0
2
4
6
8
10
0
2
culture time (d)
30
C
Mn peroxidase (U/L)
Mn peroxidase (U/L)
6
8
10
culture time (d)
300
200
100
0
D
20
10
0
0
2
4
6
8
culture time (d)
Figure 3.
4
10
0
2
4
6
8
10
culture time (d)
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 ().
47
1800
1500
mV
1200
10 days
5 days
900
0 days
600
Standard
300
0
Control culture, 5 days
0
1
2
3
4
5
6
7
12
Elution time (minutes)
8
9
10
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.
48
120
120
B
Extracted bentazon (%)
Extracted bentazon (%)
A
100
80
60
40
20
80
60
40
20
0
0
0
5
10
culture time (d)
Figure 5.
100
0
5
10
time course (d)
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 dayscultures 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.
49
References
Asgher M, Bhatti HN, Ashraf M, Legge RL (2008) Recent developments in
biodegradation of industrial pollutants by white rot fungi and their
enzyme system. Biodegradation 19:771-783.
Bibi I, Bhatti HN, Asgher M (2009) Decolourisation of direct dyes with
manganese peroxidase from white rot basidiomycete Ganoderma
lucidum-IBL-5.
The
Canadian
Journal
of
Chemical
Engineering
87:435-440.
Boer CG, Obici L, Souza CGM, Peralta RM (2004) Decolorization of
synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes
producing manganese peroxidase as the main ligninolytic enzyme.
Bioresour Technol 94:107-112.
Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant
Biol 54:519–546.
Boh B, Berovic M, Zhang J, Zhi-Bin L (2007) Ganoderma lucidum and its
pharmaceutically active compounds. Biotechnol Ann Rev 13:265-301
Castillo MP, Andersson A, Ander P, Stenstróm J, Torstensson L (2000a)
Establisment of the white rot fungus Phanerochaete chrysosporium on
unsterile straw in solid substrate fermentation systems intended for
degradation of pesticides. World J Microbiol Biotechnol 17:627-633.
Castillo MP, Ander P, Stenstróm J, Torstensson L (2000b) Degradation of
the
herbicide
bentazon
as
related
to
enzyme
production
by
Phanerochaete chrysosporium in two solid substrate fermentation
systems. World J Microbiol Biotechnol 16:289-295.
Coelho JS, Oliveira AL, Bracht A, Souza, CGM, Peralta RM (2009) Effect of
the herbicides bentazon and diuron on the production of ligninolytic
enzymes
by
Ganoderma
lucidum.
Appl
Microbiol
Biotechnol,
submitted.
Davila-Vazquez G, Tinoco R, Pickard MA, Vazques-Duhalt R (2005)
Transformation of halogenated pesticides by versatile peroxidase from
Bjerkandera adusta. Enz Microb Technol 36:223-231.
50
D’Souza TM, Merrit CS, Reddy CA (1999) Lignin-modifying enzymes of
the
white-rot basidiomycete
Ganoderma
lucidum.
Appl Environ
Microbiol 65:5307-5313.
Hou H, Zhou J, Wang J, Du C, Yan B (2004) Enhancement of laccase
production by Pleurotus ostreatus and its use for the decolorization of
anthraquinone dye. Proc Biochem 30:1415-1419.
Huber R, Otto S (1994) Environmental behavior of bentazon herbicide.
Rev Environ Contam Toxicol 137: 111-134.
Iwahara K, Honda Y, Watanabe T, Kuwahara M (2000) Polymerization of
guaiacol by lignin-degrading manganese peroxidase from Bjerkandera
adusta in aqueous organic solvents. Appl Microbiol Biotechnol 54: 104111.
Jeon J-R, Muregesan K, Kim Y-M, Kim E-J, Chang Y-S 92008) Synergistic
effect
of
laccase
mediators
on
pentachlorophenol
removal
by
Ganoderma lucidum laccase. Appl Microbiol Biotechnol 81:783-790.
Jolivalt
C,
Brenon
S,
Caminade
E,
Mougin
C,
Ponti
M
(2000)
Immobilization of laccase from Trametes versicolor on a modified PVDF
microfiltration membrane: characterization of the grafted support and
application in removing a phenylurea pesticide in wastewater. J. Membr
Sci 180:103–113.
Kadowaki, MK, Souza CGM, Simão RCG, Peralta RM (1997) Xylanase
production by Aspergillus tamari. Appl Biochem Biotechnol 66:97-106.
Knauber WR, Krotzky AJ, Schink B (2000) Microbial metabolism and
further fate of bentazon in soil. Environ. Sci. Technol 34:598–603.
Ko E-M, Leem Y-E, Choi HT (2001) Purification and characterization of
laccase isoenzymes from the white-rot basidimycete Ganoderma
lucidum. Appl Microbiol Biotechnol 57:98-102.
Kordel W, Herrchen M, Hamm RT (1991) Lysimeter experiments on
bentazon. Chemosphere 23:83-97.
Muregesan K, Yang I-H, Kim Y-M, Jeon J-R, Chang Y-S (2009) Enhanced
transformation of malachite green by laccase of Ganoderma lucidum
in the presence of natural phenolic compounds. Appl Microbiol
Biotechnol 82:341-350.
51
Murugesan K, Nam IH, Kim YM, Chang YS (2007) Decolorization of
reactive dyes by a thermostable laccase produced by Ganoderma
lucidum in solid state culture. Enz Microbial Technol 40:1662–1672.
Novotný Č, Svobodová K, Erbanová P, Cajthaml T, Kasinath A, Lang E,
Šašek V (2004) Ligninolytic fungi in bioremediation: extracellular
enzyme production and degradation rate. Soil Biol Biochem 36:1545–
1551.
Oliveira LA, Porto ALF, Tambourgi EB (2006) Production of xylanase and
protease by Penicillium janthinellum CRC 87M-115 from different
agricultural wastes. Bioresour Technol 97:862–867.
Otto S, Beutel P, Drescher N, Huber R (1979) Investigations into the
degradation of bentazon in plant and soil. IUPAC Advances in
Pesticide ScienceGeisbuhler, H., Ed.; Pergamon Press: Oxford 551556.
Pinto GM, Jardim ICSF (1999) Determination of bentazon residues in
water by high-performance liquid chromatography: validation of the
method. J Chromatog A 846:369-374.
Sliva D (2004) Cellular and physiological effects of Ganoderma lucidum
(Reishi). Mini Rev Med Chem 4: 873-879.
Tychanowicz GK, Souza DF, Souza CGM, Kadowaki MK, Peralta RM (2006)
Copper improves the production of laccase by the white-rot fungus
Pleurotus pulmonarius in solid state fermentation. Braz Arch Biol
Technol 49:699-704.
Vogel HA (1956) A convenient growth medium for Neurospora crassa.
Microb Genet Bull 13:42-43.
Wagner SC, Zablotowicz RM, Gaston LA, Locke MA, Kinsella J (1996)
Bentazon degradation in soil: influence of tillage and history of
bentazon application. J Agric Food Chem 44:1593-1598.
Wariishi H, Valli
manganese
K, Gold MH (1992) Manganese(II)
peroxidase
from
the
Basidiomycete
oxidation
by
Phenerochaete
chrysosporium. J. Biol. Chem. 267:23688–23695.
Watanabe K (2001) Microorganisms relevant to bioremediation. Curr Opin
Biotechnol 12:237–241.
52
Zhong JJ, Tang YJ (2004) Submerged cultivation of medicinal
mushrooms for production of valuable bioactive metabolites. Adv
Biochem Eng Biotechnol 87:25-59.