Degradation of Diuron by Phanerochaete chrysosporium: Role of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 251354 9 pageshttpdxdoiorg1011552013251354

Research ArticleDegradation of Diuron by Phanerochaete chrysosporiumRole of Ligninolytic Enzymes and Cytochrome P450

Jaqueline da Silva Coelho-Moreira Adelar Bracht Aline Cristine da Silva de SouzaRoselene Ferreira Oliveira Anacharis Babeto de Saacute-NakanishiCristina Giatti Marques de Souza and Rosane Marina Peralta

Department of Biochemistry State University of Maringa 87020-900 Maringa PR Brazil

Correspondence should be addressed to Rosane Marina Peralta rosanemperaltagmailcom

Received 9 October 2013 Revised 26 November 2013 Accepted 27 November 2013

Academic Editor Kannan Pakshirajan

Copyright copy 2013 Jaqueline da Silva Coelho-Moreira et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquidstationary cultures The presence of diuron increased the production of lignin peroxidase in relation to control cultures but onlybarely affected the production of manganese peroxidaseThe herbicide at the concentration of 7120583gmL did not cause any reductionin the biomass production and it was almost completely removed after 10 days Concomitantly with the removal of diuron twometabolites DCPMU [1-(34-dichlorophenyl)-3-methylurea] and DCPU [(34-dichlorophenyl)urea] were detected in the culturemedium at the concentrations of 074 120583gmL and 006 120583gmL respectively Crude extracellular ligninolytic enzymes were notefficient in the in vitro degradation of diuron In addition 1-aminobenzotriazole (ABT) a cytochrome P450 inhibitor significantlyinhibited both diuron degradation andmetabolites production Significant reduction in the toxicity evaluated by the Lactuca sativaL bioassay was observed in the cultures after 10 days of cultivation In conclusion P chrysosporium can efficientlymetabolize diuronwithout the accumulation of toxic products

1 Introduction

Agricultural practices are among the main activities respon-sible for the release of hazardous chemicals into the environ-ment [1] Among these chemicals the pesticides (fungicidesherbicides and insecticides) have been used for decades with-out any control resulting in a strong contamination of waterair and foods as well as in the development of pesticide resis-tant organisms This problem became more serious duringthe last years resulting in high risks to human health

Herbicides are the main class of pesticides used exten-sively in home gardens and farms all over the world [2]Diuron is a phenylurea herbicide applied to a wide variety ofcrops especially sugar-cane cultures The compound acts inphotosynthetic organisms by blocking electron transport inphotosystem II thus inhibiting photosynthesis In the envi-ronment diuron can be transformed abiotically via hydrolysisand photodegradation reactions but under natural condi-tions these reactions occur at very low rates [3] Due to

this diuron is known as a potential water contaminantbeing frequently detected at concentrations ranging from27 120583gmL to 2849 120583gmL in surface water and 034 to 537120583gmL in groundwater [4] The dissipation of diuron fromthe environment is thus mainly due to biotic processes theaerobic microbial metabolism being the major form ofdiuron transformation [3 5] The main reactions involvedare N-demethylation and hydrolysis of the amide bond Inmost cases mono- and didemethylated compounds and 34-dichloroaniline appear as the main products of microbialmetabolism [3 6] These metabolites accumulate in the envi-ronment and some of them are more toxic than diuron [7]

The aerobic biodegradation pathway for diuron is wellestablished (Figure 1) It proceeds by successive demethy-lation steps to form DCPMU [1-(34-dichlorophenyl)-3-methylurea] DCPU [1-(34-dichlorophenyl)urea] and 34DCA (34-dichloroaniline) Several studies describe the capa-bility of soil fungi to degrade diuron with accumulation of

2 BioMed Research International

NH

NH

NH

H

H

H

CH3

CH3

CH3

N

N

N

O

O

O

C

C

C

Diuron

DCPMU

DCA

DCPU

NH2

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Figure 1 Chemical structure of diuron and its main metabolitesDCPMU DCPU and DCA

these N-demethylated metabolites [7ndash9] Among white-rotfungi the degradation of diuron is generally attributed tothe action of extracellular enzymes the lignin-modifyingenzymes typically produced by these fungi [10ndash13] Howeverthe products generated in the degradation have not yet beencharacterized and no efforts have been done to evaluate theirtoxicity

The model of white-rot fungus for many bioremediationstudies is Phanerochaete chrysosporium [14] Its ability todegrade pollutants appears to be related especially to theproduction of lignin peroxidase and manganese peroxidasetwo lignin-modifying enzymes generally expressed undernitrogen-limited culture conditions [15] as well as to theintracellular cytochrome P450 system [16] The transforma-tion of diuron by P chrysosporium in liquid cultures hasalready been documented in both stationary and shakenconditions [11 12] Stationary cultures are advantageousover shaken cultures because they work without mechanicalenergy requirements thus increasing the feasibility of thetechnique for application in large scale treatment of wastew-ater The metabolic processing of diuron by P chrysosporiumis still not completely clarified specially with respect to themetabolites that are produced and the role of cytochromeP450 in the degradation Taking this into considerationthe objectives of this work were to study the removal ofdiuron from liquid cultures of P chrysosporium with special

interest in the role of cytochrome P450 and identificationof demethylated metabolites Attempts were also done tocompare the toxicity of diuron metabolites with the parentmolecule

2 Materials and Methods

21 Chemicals The enzymatic substrates diuron (ge98)DCPMU DCPU 34-DCA (34-dichloroaniline) and ABT(1-aminobenzotriazole) were obtained from Sigma ChemicalCorp (St Louis MO) Stock solutions of diuron DCPMUDCPU 34-DCA and ABT were prepared by dissolvingstandards in dimethyl sulfoxide (DMSO) filtering through amillipore membrane (045mm) and storing at 4∘C PDAwasobtained fromDifco Laboratories (Detroit MI)The solventsused in the HPLC analyses were of chromatographic gradeand all other reagents were of analytical grade

22 Microorganism and Inoculum Phanerochaete chrysospo-rium was obtained from the Andre Tosello Foundation(ATCC 24725) and cultured on potato dextrose agar (PDA)for 7 days at 28∘C Mycelial plugs measuring 15mm indiameter weremade and used as inoculum for liquid cultures

23 Culture Conditions The experiments were performed inliquid medium under stationary conditions at 28∘C in thedark P chrysosporium was cultivated in 125mL Erlenmeyerflasks using three mycelial disks on PDA plates (approxi-mately 15mm in diameter) for up to 12 days Each flaskcontained 25mL of a medium prepared with a mineralsolution without nitrogen source [17] containing 12mmolLammonium tartrate in order to obtain a nitrogen-limitedmedium that is favorable to ligninolytic enzyme productionAdditionally to induce the ligninolytic enzymes a corncob extract rich in phenolic compounds was used Forpreparation of the extract an aqueous suspension containing3 corn cob powder (wv) was boiled for 5 minutes andfiltered throughWhatman filter paper number 1 to retain theresidues and to avoid diuron adsorption on the insoluble corncob Afterwards the mineral solution was prepared using thecorn cob extract enriched with 1 glucose as carbon sourceThemediumwas previously sterilized by autoclaving at 121∘Cfor 15min

24 Effect of Diuron in the Biomass Production of P chrysospo-rium Increasing amounts of diuron dissolved in DMSO(30 to 100 120583molL) were added to the liquid medium atthe beginning of the cultivation The DMSO volume addedwas not superior to 30 120583L in order to not affect the fungalmetabolism After 10 days the cultures were interrupted byfiltration and the mycelia were washed three times withdistilled water and dried to constant weight at 50∘C

25 Time Course of Enzyme Production and Diuron Degrada-tion To evaluate the capability ofP chrysosporium liquid cul-tures to degrade diuron each culture flask received a volumeof a diuron stock solution inDMSO (10mgmL) to give a finalconcentration of 30120583molL (7 120583gmL) The flasks were then

BioMed Research International 3

agitated tomix the herbicide solution and inoculatedwith thefungus Two types of controls were conducted in parallel Inthe first the fungus was inoculated in liquidmediumwithoutherbicide The second control consisted of sterile mediumcontaining the same amount of diuron (abiotic control) Allflasks were incubated for up to 12 days At periodic intervalsthe cultures were interrupted by filtration and the cultureextracts were used to evaluate the lignin-modifying enzymeslignin peroxidase and manganese peroxidase as well as theresidual diuron and its metabolites by high-performanceliquid chromatography (HPLC)

26 Lignin-Modifying Enzyme Assays Manganese peroxi-dase (Mn peroxidase) activity was assayed by the oxidation of1mmolL MnSO

4in 50mmolL sodium malonate buffer pH

45 in the presence of 01mmolL H2O2 Manganic ions

(Mn3+) form a complex with malonate which absorbs at270 nm (120576

270= 11590Mminus1 cmminus1) [18] Lignin peroxidase

activitywas determined by spectrophotometricmeasurementat 310 nm of the H

2O2-dependent veratraldehyde formation

from 04mmolL veratryl alcohol in 01molL tartrate bufferof pH 30 [19] Laccase activity was assayed by measuringthe oxidation of 271015840-azinobis [3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) [20] The enzymaticactivities were expressed as International Units (U) definedas the amount of enzyme required to produce 1 120583mol productper min at 40∘C

27 Analysis of Residual Diuron and Identification of DiuronMetabolites For extraction of diuron and its metabolitesfrom the mycelia a volume of 6mL of acetonitrile was addedto the cells and the mixture was maintained for 2 h underagitation at 130 rpmThemycelial extracts were obtained aftercentrifugation at 5000 rpm for 15min at 4∘CThe concentra-tions of diuron and itsmetabolites in the culturemedia and inthemycelial extracts were determined using anHPLC system(Shimadzu Tokyo) with an LC-20AT Shimadzu system con-troller Shimadzu SPD-20 A UV-VIS detector equipped witha reversed ShimpackC18 column (46times 250mm)maintainedat 40∘C The mobile phase consisted of water (solvent A)and acetonitrile (solvent B) at a flow rate of 08mLmin Thegradient program started with 25 acetonitrile increasingto 85 in 15 minutes The column was equilibrated for 10minutes before the next injection The UV detection wasmade at 245 nm and the injection volume was 20120583L Allsamples in triplicate were filtered through a membrane filter(045120583m) before injectionThe concentrations of diuron andits metabolites were determined using calibration curvesconstructed with peak areas of authentic standards (diuronDCPMU DCPU and DCA) The identification of the com-pounds was based on their respective retention times andon the fortification of the samples with standards Under theconditions employed diuron was eluted at 116min DCPMUat 106min DCPU at 95min and 34-DCA at 121min

28 Cytochrome P450 Inhibition Studies The cytochromeP450 inhibitor 1-aminobenzotriazole (ABT) was added to Pchrysosporium cultures at the start of the cultivation to obtain

a final concentration of 1mmolL A diuron stock solutionwas added to a final concentration of 30120583molL (7 120583gmL)Control cultures without cytochrome P450 inhibitor wereincubated in parallel The cultures were inoculated andincubated as described above The diuron degradation in thepresence and absence of ABT was measured after 5 and 10days of cultivation

29 In Vitro Degradation of Diuron by Extracellular P chry-sosporium Crude Enzymes The capability of extracellular Pchrysosporium crude enzymes to degrade diuron was eval-uated using 7-day culture filtrates according to the proto-col described previously [21] The reaction mixture (1mL)contained (final concentration) 7120583gmL diuron 40ULcrude lignin peroxidase 50UL manganese peroxidase and01mmolL H

2O2in tartrate buffer 01molL pH 30 The

effects of adding 05mmolLMnSO4and 05mmolL veratryl

alcohol were tested separately or in combination All reac-tions were conducted in sterile tubes at room temperatureAfter 24 h the reaction mixtures were filtrated with a mem-brane filter (045120583m) before the HPLC analyses

210 Toxicological Test The toxicity assessment of crudeculture filtrates and abiotic control samples was conductedusing lettuce seeds (Lactuca sativa) The crude filtrates wereobtained from 10-day cultures using an initial concentrationof diuron of 7 120583gmL The bioassay was conducted with fivedilutions of each sample in water (vv) (100 80 50 20 and10) Twenty seeds were placed in 90mm diameter Petridishes containing filter paper saturated with 3mL of differentdilutions of samples or water (control) [22] After 5 days thenumber of germinated seeds was counted and the lengths ofthe radicles and hypocotyls were measured The data wererepresented as absolute germination percentage and relativegrowth compared to the control as follows

Absolute germination () = GSTStimes 100

Relative growth of the radiclehypocotyl () = LSLCtimes 100

(1)

where GS = number of germinated seeds TS = number oftotal seeds LS = average length of the sample and LC =average length of the control

To calculate the dose that produced 50 inhibition ofgermination or growth (LD

50) the coefficient of inhibition

119868 () for each parameter was calculated as follows

119868 ()germination =GSC minus GSS

GSCtimes 100

119868 ()radiclehypocotyl growth =LC minus LS

LCtimes 100

(2)

where GSC = number of germinated seeds in control andGSS = number of germinated seeds in sample


0 3 6 9 12 150

50

100

150

Culture time (d)

Dry

bio

mas

s (m

g)

(a)

0 3 6 9 12 15

0

25

50

75

100

Culture time (d)

Lign

in p

erox

idas

e (U

L)

(b)

0 3 6 9 12 15

0

10

20

30

40

Culture time (d)

Mn

pero

xida

se (U

L)

(c)

Figure 2 Effect of diuron in the biomass production (a) production of lignin peroxidase (b) and production of manganese peroxidase (c)Absence of diuron (Control cultures) (e) with 7120583gmL diuron (I)

3 Results

31 Effects of Diuron on Biomass Production and Produc-tion of Ligninolytic Enzymes Concentrations of diuron upto 80120583molL (186 120583gmL) only barely affected the fungalbiomass produced after 10 days of cultivationwhen comparedwith the fungal biomass obtained in the absence of herbicideAn increase to 100 120583molL (232 120583gmL) in the concentrationof diuron resulted in high inhibition of the biomass produc-tion At 7 120583gmL of diuron the growth curve was very similarto the growth curve in the absence of herbicide (Figure 2(a))The addition of diuron increased the LiP activities from

47UL (at day 7) to 88UL (at day 10) (Figure 2(b)) Themaximal Mn peroxidase activity was barely affected by thepresence of diuron After 5 days of cultivation the levels ofMn peroxidase were 224 and 200UL in the presence andabsence of diuron (Figure 2(c)) After 10 days of cultivationthe levels of Mn peroxidase found in the culture filtratesin the presence and absence of diuron were 294 and 154respectively Laccase activity was not detected under anycondition with or without diuron

32 Diuron Degradation Experiments P chrysosporiumshowed high rates of diuron removal in the corn cob liquid


Table 1 Effects of 1-aminobenzotriazole on diuron transformation and metabolites production by P chrysosporium

P chrysosporium cultureRecupered diuron and produced metabolites (120583gmL)

5 days 10 daysDiuron DCPMU DCPU Diuron DCPMU DCPU

Inhibitor free-control 172 plusmn 041 054 plusmn 020 006 plusmn 001 026 plusmn 003 016 plusmn 006 006 plusmn 001

ABT (1mmolL) 525 plusmn 050 022 plusmn 001 004 plusmn 001 407 plusmn 012 032 plusmn 001 004 plusmn 002

medium under the nitrogen-limited condition used in thiswork (Figure 3) and this fact seems to be associated with themycelium growing phase After 5 days of cultivation thefungus was able to remove approximately 52 of diuron andat the end of the experiment the removal reached 94 Con-comitantly with the removal of diuron the formation ofdiuron metabolites was detected namely DCPMU andDCPU Identification of the metabolites was carried out bycomparison of their retention times with those obtained byinjecting standards under the same conditions as well asby spiking the samples with stock standard solutions Theconcentration of DCPMU reached 074120583gmL at day 5 anddecreased to 008 120583gmL at the end of the experiment Onlytraces of the metabolite DCPU were observed (less than006120583gmL) during the whole cultivation An unidentifiedpeak (retention time of 152min) was detected with amaximal area at day 7 and decreased until the end of theexperiment No 34-DCA production was detected in theP chrysosporium cultures All these compounds were notobserved in cultures without diuron

33 Effect of CytochromeP450 Inhibitor on FungalMetabolismThe involvement of the cytochrome P450 in the diurondegradation was examined by adding the cytochrome P450inhibitor (ABT) to the cultures containing the herbicide atthe beginning of the cultivationThe diuron degradation wasclearly inhibited by the addition of 1mmolL ABT As shownin Figure 4 in cultures where ABT was added higher con-centrations of diuron were observed whereas in inhibitor-free cultures diuron was almost completely removed whencompared to the uninoculated controls The addition of ABTdid not affect the fungal biomass production (not shown)

The production of N-demethylated metabolites wasclearly affected by the presence of the cytochrome P450inhibitor The formation of the metabolite DCPMU was themost strongly inhibited As shown in Table 1 in control cul-tures the levels of DCPMU decreased until the end of theexperiments suggesting that this compoundwasmetabolizedby the fungus In contrast in the presence of ABT this metab-olite accumulated in the medium as can be observed by theslight increase of its concentration at day 10 which indicatesthat the metabolization of DCPMU was also inhibited byABT The effect of ABT on the formation of the metaboliteDCPU was less pronounced causing a slight decrease of itslevel when compared with the control

The amounts of diuron and its metabolites in the culturefiltrate and mycelial extract after 5 days of cultivation areshown in Table 2 It is noteworthy to mention that infractional terms the amounts of DCPMU and DCPU in

0 3 6 9 12 15

00

15

30

45

60

75

0

20000

40000

60000

Culture time (d)

Peak

area

(mV

)

Com

poun

d (120583

gm

L)

Figure 3 Time course of diuron degradation (e) and formationof DCPMU (998771) DCPU (I) and an unknown product (◻) elutedat 152min Recovery of diuron (◼) from the uninoculated controlculture is also shown

the mycelial extract exceeded those of the parent compounddiuron by factors of 173 and 199 respectively Furthermorethe total amount of diuron and its metabolites inside and out-side of the cells corresponds to 616 of the amount of diuronadded to the culturesThis indicates that a substantial fractionwas removed Unfortunately chromatographic analyses of 10d-mycelial extracts could not be evaluated considering thepresence of interfering molecules also present in the cellextracts obtained in the absence of diuron

34 Degradation of Diuron by Crude Enzymatic Extract Thecapability of crude enzymatic extracts to degrade diuron invitro was tested under different conditions (Table 3) Nosignificant differences (119875 lt 005) were observed betweenthe control and the treatments containing enzymes andmediators

35 Toxicity Tests Thesamples showedmoderate toxic effectswithout dilution (100) or when the samples were dilutedby a factor of 125 (80) (Table 4) Toxic effects of the sam-ples were observed on the radicles and hypocotyl such as


5 100

2

4

6

8

Culture time (d)

Recu

pere

d di

uron

(120583g

mL)

Figure 4 Effect of the cytochrome P450 inhibitor (ABT) on diurondegradation Treatments abiotic controls (striped bars) inhibitor-free control (grey bars) and cultures with ABT (black bars)

Table 2 Distribution of residual diuron and itsmetabolites betweenculture filtrates andmycelial extracts of P chrysosporium after 5 daysof cultivation

Compound Residual diuron and metabolites after 5 days (120583g)Culture filtrate Mycelial extract Total

Diuron 713 plusmn 33 110 plusmn 08 823DCPMU 185 plusmn 05 56 plusmn 01 241DCPU 11 plusmn 01 037 plusmn 01 15An amount of 175120583g of diuron was added at zero time in each culture

reduction in size necrosis and fragility In relation to theradicle growth no reduction in the toxicity between treatedand nontreated samples (abiotic control) was observed Inrelation to the hypocotyl development it was possible toobserve a reduction in the toxicity after transformation ofdiuron by P chrysosporium while nontreated samples pro-moted an effective inhibition of the hypocotyl growth treatedsamples allowed a better development of this structurewhat demonstrates a detoxification of the medium by thefungal treatment In these analyses the inhibition coefficientallowed to calculate the LD

50 In this case the LD

50indicates

the acute toxicity of the samples and its value represents thesample dilution (vv) that produced 50 inhibition of germi-nation or growth The lower the LD

50value the more toxic

the sample Therefore it is possible to compare the toxicitiesof different samples For radicle growth the LD

50values

were 154 and 587 for nontreated and treated samples

respectively For hypocotyl growth the LD50

was of 516for nontreated and of 951 for treated samples The highestLD50

values were achieved with treated samples indicatingthat there is a smaller toxicity for 10-day-treated samplesTherefore a detoxification process seems to have occurred inpresence of the fungus

4 Discussion

It is well known that P chrysosporium possesses a great abilityto degrade herbicides for example isoproturon [23] atrazine[24] propanil [11] bentazon [25] and also diuron [10] Moststudies have obtained the greatest degradation levels in solidstate cultures whereas in liquidmedium the degradation effi-ciency seems to be smaller In this study the stationary liquidculture was used because it is specially suitable for waste-water treatment due to its simplicity and low cost providingconditions for high removal of diuron and identification ofthe main metabolites

The expression of ligninolytic enzymes by P chrysospo-rium in the liquid system used in this work was favored bythe limitation of nitrogen and by the presence of soluble corncob phenolics Different studies where the capability of Pchrysosporium to degrade herbicides was evaluated have sug-gested the participation of ligninolytic enzymes in the processbased on a temporal coincidence between herbicide degrada-tion andmaximal enzyme activities [10 23]Theparticipationof extracellular enzymes in the transformation of several her-bicides by some white-rot fungi including P chrysosporiumwas conclusively demonstrated by studies performed withpurified enzymes and compiled in a recent review [2] Inthe present work a temporal correlation between productionof ligninolytic enzymes and initial diuron degradation wasevidenced However no transformation of the herbicide wasobserved when the crude culture filtrates were incubated invitro with diuron These results indicate that the first steps ofdiuron transformation seem to be more associated with themycelial components than with the extracellular enzymes Inrelation to the increase in the activity of lignin peroxidasethis phenomenon is not necessarily related to the herbi-cide transformation since this effect can be due to severalalterations in cell physiology or in plasmatic membranestructure which reflect the adaptation of the fungus to thepolluted environment [26] These considerations support thehypothesis that diuron degradation involves an intracellularenzymatic system and weaken the hypothesis of extracellularLiP andMnP participations in the initial reactions Two otherobservations of the present work reinforce the hypothesisthat the degradation of diuron started intracellularly theconsiderable amounts of diuron DCPMU and DCPU foundin fresh mycelia and the inhibition of degradation caused byABT a cytochrome P450 inhibitor

The participation of an intracellular enzymatic mecha-nism represented mainly by cytochrome P450 in the degra-dation of different xenobiotics has been extensively consid-ered in the last years Purification of fungal cytochrome P450in order to obtain conclusive data has been accomplishedin only a few studies due to the difficulties in keeping


Table 3 Degradation of diuron by enzymatic crude filtrate from P chrysosporium cultures after 24 h

Treatment Veratryl alcohol(05 mM)

H2O2(01mM)

MnSO4(05mM) Crude filtratelowast Recupered diuron

(120583gmL)Control minus minus minus minus 647 plusmn 043

1 + + + + 740 plusmn 046

2 minus + minus + 634 plusmn 072

3 + + minus + 598 plusmn 035

4 minus + + + 627 plusmn 031

5 + minus minus + 566 plusmn 048

6 + + + + 739 plusmn 039

lowast400UL of lignin peroxidase activity and 50UL ofmanganese peroxidase activity All the treatment contained diuron (7120583gmL) and sodiummalonate buffer50mM pH 45 Control was run only with diuron and buffer Treatment 6 was performed using boiled crude enzyme Degradation values are means plusmn SD(119899 = 3)

Table 4 Parameters measured for L sativa bioassay

Absolute germination () Relative growth averages ()Sample dilution (vv) Radicle Hypocotyl

Abiotic control 10-day treatment Abiotic control 10-day treatment Abiotic control 10-day treatment10 841 plusmn 20 950 plusmn 31 422 plusmn 37 770 plusmn 41

lowast

1131 plusmn 70 1162 plusmn 42

20 803 plusmn 36 883 plusmn 28lowast

363 plusmn 86 1026 plusmn 85lowast











The percentage of the absolute germination and the growth averages for lettuce seed bioassays were calculated at five dilutions of nontreated (abiotic control)and 10-day-treated samples in triplicates lowastSignificant differences between samples for the same parameter analyzed (119875 lt 005) by 119905-test

the activation of the enzymes duringmicrosome preparationHence most conclusions were drawn from the results of indi-rect experiments consisting in the addition of specific cyto-chrome P450 inhibitors to the culturemedium such as piper-onyl butoxide and 1-aminobenzotriazole the same strategyused in the present work Two recent studies reinforced theimportance of this system in theP chrysosporiumdegradationof pentachlorophenol [27] and phenanthrene [28] In thelatter study strong evidence has been presented for the partic-ipation of cytochrome P450 monooxygenases in anthracenemetabolism by P chrysosporium

The chromatographic analysis demonstrated that diuronwas effectively transformed by the fungus DCPMU wasthe major N-demethylated metabolite identified and did notaccumulate in the medium suggesting that this compoundwas further degraded by the fungus Another metaboliteappeared in the medium but unfortunately it could not beidentified by the methods employed in the present work

Besides P chrysosporium [10ndash12] other white-rot speciessuch as Bjerkandera adusta and Trametes versicolor [12 13]and Pleurotus ostreatus [11 12] have also been reported todegrade diuron in liquid cultures In these studies no effortswere made to identify the transformation products Ourresults show that diuron transformation by P chrysosporiumappears to be similar to that described for some soil fungi thatis by successive N-demethylation but with no formation andaccumulation of 34-DCA considered to be the most toxicand persistent metabolite of diuron [3]

Although diuron has been efficiently degraded in fun-gal cultures conclusions drawn from this kind of experi-ments should not be extrapolated straightforwardly to naturebecause the biological degradation is frequently incompleteand may produce metabolites that are more toxic than theinitial compound The toxicological tests using lettuce seedsas bioindicators represent an effort to compare the toxicity oftreated and nontreated diuron samples and thus to assess theeffectiveness of P chrysosporium in reducing environmentalcontamination [22] Previous studies showed that diuron istransformed by fungal cultures producing N-demethylatedmetabolites with a higher toxicity than diuron [7 29] In suchworks the toxicity was evaluated according to the Microtoxtest after isolation and concentration of the metabolites Thepresentworkwas conducted to evaluate the general toxicity ofthe culture extracts after just a single centrifugation step usedin order to clean the samplesThe results showed a significantdecrease in the toxicity of culture extracts at the end of thefungal treatment which coincided with the decrease of themedium concentrations of both diuron and its metabolitesdue to the degradation processes and also to the uptake ofthese compounds into the cells

5 Conclusion

The present study emphasizes the capability of P chrysospo-rium to degrade the herbicide diuron The fungus was ableto remove 94 of the herbicide after 10 days of cultivation


with no apparent accumulation of toxic products This workcomplements the results obtained by other authors whichdemonstrate that demethylation at the terminal nitrogen ofthe diuron molecule is the initial degradation reaction infungal metabolism To our knowledge this is the first studythat demonstrates the involvement of cytochromeP450 in thetransformation of diuron and its metabolites by P chrysospo-rium

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by the Conselho Nacional dePesquisa e Desenvolvimento (CNPq) and Coordenacao deAperfeicoamento de Pessoal de Nivel Superior (CAPES)Rosane Marina Peralta and Adelar Bracht are Research Fel-lows of CNPq

References

[1] H Harms D Schlosser and L Y Wick ldquoUntapped potentialexploiting fungi in bioremediation of hazardous chemicalsrdquoNature Reviews Microbiology vol 9 no 3 pp 177ndash192 2011

[2] J S Coelho-Moreira G M Maciel R Castoldi et al ldquoInvolve-ment of lignin-modifying enzymes in the degradation of herbi-cidesrdquo in HerbicidesmdashAdvances in Research pp 165ndash187 2013

[3] S Giacomazzi andN Cochet ldquoEnvironmental impact of diurontransformation a reviewrdquoChemosphere vol 56 no 11 pp 1021ndash1032 2004

[4] United States Environmental Protection Agency ldquoEPArdquo 2013httpwwwepagovespplitstatuseffectsdiuron efed chapterpdf

[5] J A Field R L Reed T E Sawyer S M Griffith and P JWigington Jr ldquoDiuron occurrence and distribution in soil andsurface and ground water associated with grass seed produc-tionrdquo Journal of EnvironmentalQuality vol 32 no 1 pp 171ndash1792003

[6] J E Cullington andAWalker ldquoRapid biodegradation of diuronand other phenylurea herbicides by a soil bacteriumrdquo SoilBiology and Biochemistry vol 31 no 5 pp 677ndash686 1999

[7] C TixierM Sancelme F Bonnemoy A Cuer andH Vescham-bre ldquoDegradation products of a phenylurea herbicide diuronsynthesis ecotoxicity and biotransformationrdquo EnvironmentalToxicology and Chemistry vol 20 no 7 pp 1381ndash1389 2001

[8] N Badawi S Roslashnhede S Olsson et al ldquoMetabolites of thephenylurea herbicides chlorotoluron diuron isoproturon andlinuron produced by the soil fungus Mortierella sprdquo Environ-mental Pollution vol 157 no 10 pp 2806ndash2812 2009

[9] L Ellegaard-Jensen J Aamand B B Kragelund A H Johsenand S Rosendahl ldquoStrains of the soil fungus Mortierella showdifferent degradation potentials for the phenylurea herbicidediuronrdquo Biodegradation vol 24 no 6 pp 765ndash774 2013

[10] L E Fratila-Apachitei J A Hirst M A Siebel and H J GijzenldquoDiuron degradation by Phanerochaete chrysosporium BKM-F-1767 in synthetic and natural mediardquo Biotechnology Letters vol21 no 2 pp 147ndash154 1999

[11] C Torres-Duarte R Roman R Tinoco and R Vazquez-Duhalt ldquoHalogenated pesticide transformation by a laccase-mediator systemrdquo Chemosphere vol 77 no 5 pp 687ndash6922009

[12] A Khadrani F Seigle-Murandi R Steiman and T VroumsialdquoDegradation of three phenylurea herbicides (chlortoluron iso-proturon and diuron) by micromycetes isolated from soilrdquoChemosphere vol 38 no 13 pp 3041ndash3050 1999

[13] G D Bending M Friloux and A Walker ldquoDegradation ofcontrasting pesticides by white rot fungi and its relationshipwith ligninolytic potentialrdquo FEMSMicrobiology Letters vol 212no 1 pp 59ndash63 2002

[14] C O Adenipekun and R Lawal ldquoUses of mushrooms in biore-mediation a reviewrdquo Biotechnology Molecular Biology Reviewvol 7 no 3 pp 62ndash68 2012

[15] S W Kullman and F Matsumura ldquoMetabolic pathways uti-lized by Phanerochaete chrysosporium for degradation of thecyclodiene pesticide endosulfanrdquo Applied and EnvironmentalMicrobiology vol 62 no 2 pp 593ndash600 1996

[16] D Ning H Wang and Y Zhuang ldquoInduction of functionalcytochrome P450 and its involvement in degradation of benzoicacid by Phanerochaete chrysosporiumrdquo Biodegradation vol 21no 2 pp 297ndash308 2010

[17] H A Vogel ldquoA convenient growth medium for NeurosporacrassardquoMicrobial Genetic Bulletin vol 13 pp 42ndash43 1956

[18] H Wariishi K Valli and M H Gold ldquoManganese(II) oxida-tion by manganese peroxidase from the basidiomycete Phane-rochaete chrysosporium Kinetic mechanism and role of chela-torsrdquo Journal of Biological Chemistry vol 267 no 33 pp 23688ndash23695 1992

[19] M Tien and T K Kirk ldquoLignin-degrading enzyme from Phane-rochaete chrysosporium purification characterization and cat-alytic properties of a unique H

2

O2

-requiring oxygenaserdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 81 pp 2280ndash2284 1984

[20] H Hou J Zhou J Wang C Du and B Yan ldquoEnhancement oflaccase production by Pleurotus ostreatus and its use for thedecolorization of anthraquinone dyerdquo Process Biochemistry vol39 no 11 pp 1415ndash1419 2004

[21] N Hiratsuka M Oyadomari H Shinohara H Tanaka and HWariishi ldquoMetabolic mechanisms involved in hydroxylationreactions of diphenyl compounds by the lignin-degradingbasidiomycete Phanerochaete chrysosporiumrdquo BiochemicalEngineering Journal vol 23 no 3 pp 241ndash246 2005

[22] M C Sobrero and A Ronco ldquoEnsayo de toxicidad aguda con-semillas de lechuga (Lactuca sativa L)rdquo in Ensayos Toxicologicosy Metodos de Evaluacion de Calidad de Aguas pp 71ndash79 2004

[23] M Del Pilar Castillo S Von Wiren-Lehr I Scheunert andL Torstensson ldquoDegradation of isoproturon by the white rotfungus Phanerochaete chrysosporiumrdquo Biology and Fertility ofSoils vol 33 no 6 pp 521ndash528 2001

[24] C Mougin C Laugero M Asther and V Chaplain ldquoBio-transformation of s-triazine herbicides and related degradationproducts in liquid culture by thewhite rot fungusPhanerochaetechrysosporiumrdquo Pesticide Science vol 49 pp 169ndash177 1997

[25] M D P Castillo P Ander J Stenstrom and L TorstenssonldquoDegradation of the herbicide bentazon as related to enzymeproduction by Phanerochaete chrysosporium in two solid sub-strate fermentation systemsrdquoWorld Journal of Microbiology andBiotechnology vol 16 no 3 pp 289ndash295 2000

[26] G M Zeng A W Chen G Q Chen et al ldquoResponses ofPhanerochaete chrysosporium to toxic pollutants physiological


flux oxidative stress and detoxificationrdquo Environmental Scienceand Technology vol 46 pp 7818ndash7825 2012

[27] D Ning and H Wang ldquoInvolvement of cytochrome P450 inpentachlorophenol transformation in a white rot fungusrdquoPhanerochaete Chrysosporium vol 7 no 9 Article ID e458872012

[28] N L Chigu S Hirosue C Nakamura H Teramoto HIchinose andHWariishi ldquoCytochrome P450monooxygenasesinvolved in anthracene metabolism by the white-rot basid-iomycete Phanerochaete chrysosporiumrdquo Applied Microbiologyand Biotechnology vol 87 no 5 pp 1907ndash1916 2010

[29] C Tixier P Bogaerts M Sancelme et al ldquoFungal biodegrada-tion of a phenylurea herbicide diuron structure and toxicityof metabolitesrdquo Pesticide Management Science vol 56 pp 455ndash462 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


NH

NH

NH

H

H

H

CH3

CH3

CH3

N

N

N

O

O

O

C

C

C

Diuron

DCPMU

DCA

DCPU

NH2

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Figure 1 Chemical structure of diuron and its main metabolitesDCPMU DCPU and DCA

these N-demethylated metabolites [7ndash9] Among white-rotfungi the degradation of diuron is generally attributed tothe action of extracellular enzymes the lignin-modifyingenzymes typically produced by these fungi [10ndash13] Howeverthe products generated in the degradation have not yet beencharacterized and no efforts have been done to evaluate theirtoxicity

The model of white-rot fungus for many bioremediationstudies is Phanerochaete chrysosporium [14] Its ability todegrade pollutants appears to be related especially to theproduction of lignin peroxidase and manganese peroxidasetwo lignin-modifying enzymes generally expressed undernitrogen-limited culture conditions [15] as well as to theintracellular cytochrome P450 system [16] The transforma-tion of diuron by P chrysosporium in liquid cultures hasalready been documented in both stationary and shakenconditions [11 12] Stationary cultures are advantageousover shaken cultures because they work without mechanicalenergy requirements thus increasing the feasibility of thetechnique for application in large scale treatment of wastew-ater The metabolic processing of diuron by P chrysosporiumis still not completely clarified specially with respect to themetabolites that are produced and the role of cytochromeP450 in the degradation Taking this into considerationthe objectives of this work were to study the removal ofdiuron from liquid cultures of P chrysosporium with special

interest in the role of cytochrome P450 and identificationof demethylated metabolites Attempts were also done tocompare the toxicity of diuron metabolites with the parentmolecule

2 Materials and Methods

21 Chemicals The enzymatic substrates diuron (ge98)DCPMU DCPU 34-DCA (34-dichloroaniline) and ABT(1-aminobenzotriazole) were obtained from Sigma ChemicalCorp (St Louis MO) Stock solutions of diuron DCPMUDCPU 34-DCA and ABT were prepared by dissolvingstandards in dimethyl sulfoxide (DMSO) filtering through amillipore membrane (045mm) and storing at 4∘C PDAwasobtained fromDifco Laboratories (Detroit MI)The solventsused in the HPLC analyses were of chromatographic gradeand all other reagents were of analytical grade

22 Microorganism and Inoculum Phanerochaete chrysospo-rium was obtained from the Andre Tosello Foundation(ATCC 24725) and cultured on potato dextrose agar (PDA)for 7 days at 28∘C Mycelial plugs measuring 15mm indiameter weremade and used as inoculum for liquid cultures

23 Culture Conditions The experiments were performed inliquid medium under stationary conditions at 28∘C in thedark P chrysosporium was cultivated in 125mL Erlenmeyerflasks using three mycelial disks on PDA plates (approxi-mately 15mm in diameter) for up to 12 days Each flaskcontained 25mL of a medium prepared with a mineralsolution without nitrogen source [17] containing 12mmolLammonium tartrate in order to obtain a nitrogen-limitedmedium that is favorable to ligninolytic enzyme productionAdditionally to induce the ligninolytic enzymes a corncob extract rich in phenolic compounds was used Forpreparation of the extract an aqueous suspension containing3 corn cob powder (wv) was boiled for 5 minutes andfiltered throughWhatman filter paper number 1 to retain theresidues and to avoid diuron adsorption on the insoluble corncob Afterwards the mineral solution was prepared using thecorn cob extract enriched with 1 glucose as carbon sourceThemediumwas previously sterilized by autoclaving at 121∘Cfor 15min

24 Effect of Diuron in the Biomass Production of P chrysospo-rium Increasing amounts of diuron dissolved in DMSO(30 to 100 120583molL) were added to the liquid medium atthe beginning of the cultivation The DMSO volume addedwas not superior to 30 120583L in order to not affect the fungalmetabolism After 10 days the cultures were interrupted byfiltration and the mycelia were washed three times withdistilled water and dried to constant weight at 50∘C

25 Time Course of Enzyme Production and Diuron Degrada-tion To evaluate the capability ofP chrysosporium liquid cul-tures to degrade diuron each culture flask received a volumeof a diuron stock solution inDMSO (10mgmL) to give a finalconcentration of 30120583molL (7 120583gmL) The flasks were then





















(1)






GSCtimes 100


LCtimes 100

(2)



0 3 6 9 12 150

50

100

150

Culture time (d)

Dry

bio

mas

s (m

g)

(a)

0 3 6 9 12 15

0

25

50

75

100

Culture time (d)

Lign

in p

erox

idas

e (U

L)

(b)

0 3 6 9 12 15

0

10

20

30

40

Culture time (d)

Mn

pero

xida

se (U

L)

(c)


3 Results














0 3 6 9 12 15

00

15

30

45

60

75

0

20000

40000

60000

Culture time (d)

Peak

area

(mV

)

Com

poun

d (120583

gm

L)






5 100

2

4

6

8

Culture time (d)

Recu

pere

d di

uron

(120583g

mL)







50indicates




50values





4 Discussion







H2O2(01mM)



1 + + + + 740 plusmn 046


3 + + minus + 598 plusmn 035

4 minus + + + 627 plusmn 031


6 + + + + 739 plusmn 039





lowast



















5 Conclusion






Acknowledgments


References




















2

O2





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















(1)






GSCtimes 100


LCtimes 100

(2)



0 3 6 9 12 150

50

100

150

Culture time (d)

Dry

bio

mas

s (m

g)

(a)

0 3 6 9 12 15

0

25

50

75

100

Culture time (d)

Lign

in p

erox

idas

e (U

L)

(b)

0 3 6 9 12 15

0

10

20

30

40

Culture time (d)

Mn

pero

xida

se (U

L)

(c)


3 Results














0 3 6 9 12 15

00

15

30

45

60

75

0

20000

40000

60000

Culture time (d)

Peak

area

(mV

)

Com

poun

d (120583

gm

L)






5 100

2

4

6

8

Culture time (d)

Recu

pere

d di

uron

(120583g

mL)







50indicates




50values





4 Discussion







H2O2(01mM)



1 + + + + 740 plusmn 046


3 + + minus + 598 plusmn 035

4 minus + + + 627 plusmn 031


6 + + + + 739 plusmn 039





lowast



















5 Conclusion






Acknowledgments


References




















2

O2





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0 3 6 9 12 150

50

100

150

Culture time (d)

Dry

bio

mas

s (m

g)

(a)

0 3 6 9 12 15

0

25

50

75

100

Culture time (d)

Lign

in p

erox

idas

e (U

L)

(b)

0 3 6 9 12 15

0

10

20

30

40

Culture time (d)

Mn

pero

xida

se (U

L)

(c)


3 Results














0 3 6 9 12 15

00

15

30

45

60

75

0

20000

40000

60000

Culture time (d)

Peak

area

(mV

)

Com

poun

d (120583

gm

L)






5 100

2

4

6

8

Culture time (d)

Recu

pere

d di

uron

(120583g

mL)







50indicates




50values





4 Discussion







H2O2(01mM)



1 + + + + 740 plusmn 046


3 + + minus + 598 plusmn 035

4 minus + + + 627 plusmn 031


6 + + + + 739 plusmn 039





lowast



















5 Conclusion






Acknowledgments


References




















2

O2





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











0 3 6 9 12 15

00

15

30

45

60

75

0

20000

40000

60000

Culture time (d)

Peak

area

(mV

)

Com

poun

d (120583

gm

L)






5 100

2

4

6

8

Culture time (d)

Recu

pere

d di

uron

(120583g

mL)







50indicates




50values





4 Discussion







H2O2(01mM)



1 + + + + 740 plusmn 046


3 + + minus + 598 plusmn 035

4 minus + + + 627 plusmn 031


6 + + + + 739 plusmn 039





lowast



















5 Conclusion






Acknowledgments


References




















2

O2





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


5 100

2

4

6

8

Culture time (d)

Recu

pere

d di

uron

(120583g

mL)







50indicates




50values





4 Discussion







H2O2(01mM)



1 + + + + 740 plusmn 046


3 + + minus + 598 plusmn 035

4 minus + + + 627 plusmn 031


6 + + + + 739 plusmn 039





lowast



















5 Conclusion






Acknowledgments


References




















2

O2





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




H2O2(01mM)



1 + + + + 740 plusmn 046


3 + + minus + 598 plusmn 035

4 minus + + + 627 plusmn 031


6 + + + + 739 plusmn 039





lowast



















5 Conclusion






Acknowledgments


References




















2

O2





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References




















2

O2





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Degradation of Diuron by Phanerochaete chrysosporium: Role of