Biodegradation of a Model Azo Disperse Dye by the White Rot Fungus

8/20/2019 Biodegradation of a Model Azo Disperse Dye by the White Rot Fungus

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International Biodeterioration & Biodegradation 57 (2006) 1–6

Biodegradation of a model azo disperse dye by the white rot fungus

Pleurotus ostreatus

Xueheng Zhaoa,, Ian R. Hardinb, Huey-Min Hwanga

aDepartment of Biology, Jackson State University, Jackson, MS 39217, USAbDepartment of Textiles, Merchandising, and Interiors, University of Georgia Athens, GA 30602, USA

Received 2 June 2005; accepted 17 October 2005

Available online 5 December 2005

Abstract

Disperse Orange 3, 4-(4-nitrophenylazo)aniline, was chosen as a model to study biodegradation of azo dyes by the white-rot fungus

Pleurotus ostreatus (strain Florida) which was grown in submerged culture under controlled conditions. Degradation was investigated

using a commercial preparation of Disperse Orange 3 that contained 20% dye plus dispersing agents, and an high-performance liquid

chromatography purified preparation of the dye. The metabolites generated by Pleurotus ostreatus were identified as 4-nitroaniline, 4-

nitrobenzene, 4-nitrophenol, and 4-nitroanisole. Veratryl alcohol, a redox mediator for lignin peroxidase of white-rot fungi, and its

oxidant veratraldehyde were also detected in cultures grown in the presence of Disperse Orange 3. 4-Nitroanisole was the major

metabolite when 4-nitrophenol was incubated with Pleurotus ostreatus. Kinetic profiles of these degradation products were determined

and a partial degradation pathway is proposed.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Azo dye; Biodegradation; Products; White rot fungi

1. Introduction

Between 10% and 15% of the total dye consumed in

dyeing processes of textile industries pollutes massive

quantities of the wastewater generated (Jarosz-Wilkolazka

et al., 2002). Dyes in textile wastewater present aesthetic

problems and can pose threats to public health (Chung,

1983; Achwal, 1997), although most liquid and solid

effluents from textile industries are treated before being

discharged into the environment. In wastewater the textile

dyes can be physically or chemically removed by floccula-

tion, adsorption, filtration and oxidation. Most of thephysical methods, however, simply accumulate and con-

centrate dyes and create solid waste, and so the problem of

disposal still exists. Chemical oxidation with either

peroxide or ozone can destroy dyestuffs but this approach

is costly (Robinson et al., 2001a). The possibility of using

white-rot fungi to decolorize wastewater containing dyes

has received much attention because their ligninolytic

enzymes have the ability to degrade many recalcitrant

pollutants, including synthetic dyes. Biocatalytic processes

based on white-rot fungi provide alternative methods to

decolorize textile effluents (Swamy and Ramsay, 1999;

Selvam et al., 2003).

The basidiomycete Phanerochaete chrysosporium is one

of the ligninolytic fungi that was extensively used to

degrade dyes (Cripps et al., 1990; Goszczynski et al., 1994;

Spadaro et al., 1992; Wesenberg et al., 2003). This fungus

produces several extracellular ligninolytic enzymes that

have been associated with the degradation of dyes (Jarosz-

Wilkolazka et al., 2002; Wesenberg et al., 2003). Otherspecies of ligninolytic fungi, such as Bjerkandera adusta,

Pleurotus ostreatus and Irpex lacteus also showed potential

for decolorization of various dyes including azo dyes

(Novotny et al., 2001; Robinson et al., 2001b; Zhao, 2004),

however, the biochemical pathways involved in azo dye

degradation and decolorization by white rot fungi are still

unclear (Wesenberg et al., 2003).

Disperse azo dyes, account for about 70% of the total

commercial disperse dyes used in the textile industry

(Zollinger, 1991). Therefore understanding the pathway

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Corresponding author. Tel.: +1 601979 1226; fax: +1 601979 2778.

E-mail address: [email protected] (X. Zhao).

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by which these dyes are metabolized by white-rot fungi, is

of importance for the application of fungi in the treatment

of textile effluents. In this study, Disperse Orange 3, 4-(4-

nitrophenylazo)aniline, a monoazo dye, was chosen as a

model compound to investigate the pathways involved in

the metabolism of an azo dye by Pleurotus ostreatus. It has

a simple structure (Fig. 1) that is typical of many disperse

azo dyes.

2. Materials and methods

2.1. Chemicals

Disperse Orange 3 (Color Index No. 11005, 20% colorant content) and

Disperse Orange 3 (90% colorant content) used in this work were

purchased from Aldrich Chemical Co. (Milwaukee, Wis.). The commercial

form of this dye contains dispersing agents and surfactants. Compounds

used as standards: 4-nitroaniline, 4-nitrophenol, 4-nitroanisole, nitroben-

zene, veratryl alcohol, veratraldehyde, 1,4-phenylenediamine, 1,4-dinitro-

benzene, 4-nitrocatechol, 2-amino-5-nitrophenol, aniline, 2-nitroaniline,

and 3-nitroaniline were analytical grade reagents and were obtained from

Aldrich. Acetonitrile (Aldrich) and methanol (EMD Chemicals, Gibbs-

town, NJ) used in high-performance liquid chromatography (HPLC)

analysis and sample preparation were of HPLC grade. Phosphoric acid

(85%), potassium dihydrogen phosphate and sodium hydroxide pellets

were analytical grade (J.T.Baker, Phillipsburg NJ). All other chemicalsused throughout this study were reagent-grade chemicals. Purified water

was obtained from an ion exchange and membrane filtration system from

US Filter (Warrendale, PA).

2.2. Culture conditions and biodegradation assays

Pleurotus ostreatus (strain Florida) was provided by K.-E. Eriksson

(University of Georgia, USA) and was maintained on malt agar medium

and subcultured every month as described previously (Zhao and Hardin,

2002). A 10-mm agar plug taken from a fungal colony growing on a malt

agar plate was used to inoculate 250-ml Erlenmeyer flask containing

125ml Kirk’s medium (Kirk et al., 1978) at pH 5.0. The cultures were

incubated for 3 days at 30 1C, and shaken at 200rpm.

Commercial disperse dyes always contain dispersing agents andsurfactants, so, to eliminate interference from such impurities, technical

grade Disperse Orange 3 (90% content) was further purified by

recrystallization with acetonitrile. The purified product was free of

impurities when examined by HPLC. For the degradation assays, the

purified colorant was finely ground and added to 3-day fungal cultures to

a final concentration of 80 mg l1 (w/v). The shaking rate of the cultures

was reduced to 150 rpm. Controls without dyes and/or inoculum were run

under identical conditions. Duplicate cultures were sampled daily for 5

days. At the time of sampling, the entire contents of each culture (120 ml)

was gravity filtered (Fisher P8 filter paper) and the filtrate from each flask

extracted three times with methylene chloride (10ml). The combined

organic layers, approximately 30 ml, were concentrated to about 1 ml

using a rotary vacuum evaporator (Buchi Analytical, New Castle, DE)

after drying with sodium sulphate. The methylene chloride extract was

analyzed by GC–MS.

A set of identical cultures grown under the same conditions was used to

investigate the degradation of the non-purified commercial preparation of

Disperse Orange 3 (20% colorant content) at a concentration of

200mgl1 in the cultures. Samples (3 ml) were taken from the culture daily

for 9 days to monitor substrate depletion and metabolite production. The

same amount of liquid medium containing 200 mgl1 of commercial

disperse dye was added after each sampling to keep a constant volume in

the culture flask. Four replicate flasks with the same dye concentrationwere used for the study and results are reported as the mean. An equal

volume of methanol was mixed with the culture samples to ensure

complete solubilization prior to measurement (Zhao and Hardin, 2002).

The concentration of Disperse Orange 3 in the growth medium was

monitored spectrophotometrically at its maximum wavelength of absorp-

tion (lmax ¼ 415nm). The initial concentration of Disperse Orange 3

corresponded to 100% of the dye. During the fungal degradation of

Disperse Orange 3 metabolites were monitored by HPLC analysis. No

significant variation (o5%) in concentration was induced by photo-

degradation, and no degradation products were detected in control

samples.

2.3. GC–MS analyses

GC–MS was performed using a QP5000 mass spectrometer (Shimadzu)fitted with a GC17A gas chromatograph (Shimadzu). The ionization

voltage was 70 eV. Gas chromatography was conducted in the tempera-

ture-programming mode with a Restek column (0.25 mm30 m; XTI-5).

The initial column temperature was 40 1C for 4 min, then increased linearly

at 10 1Cmin1, to 270 1C, and held for 4 min. The temperature of the

injection port was 275 1C and the GC/MS interface was maintained at

300 1C. The helium carrier gas flow rate was 1.0ml min1. Degradation

products were identified by comparison of retention time and fragmenta-

tion pattern with known reference compounds, as well as with mass

spectra in the NIST spectral library stored in the computer software

(version 1.10 beta, Shimadzu) of the GC–MS.

2.4. High-performance liquid chromatography (HPLC)

A Hewlett-Packard 1100 series HPLC system (Hewlett-PackardGmbH, Germany), consisting of a model G1311A quaternary pump, a

model G1322A degasser and a model G1315A diode array detector, was

used for liquid chromatography. HP ChemStation software (version 3.1)

was used for data processing and reporting. A stainless steel ODS column

5mm packing (Phenomenex, Ultracarbs, 150mm4.6mm I.D.) and a

RP-C18 guard column was used in the analysis. The volume of each

injection was 100ml delivered by a model HP1313A automatic injector.

The separation temperature was fixed at 25 1C. The mobile phase was a

gradient 0.025M phosphate buffer (pH ¼ 3.0) (A); acetonitrile (B): from 0

to 20 min, B increased linearly from 5% to 25%, then increased to 40% in

the next 10min, B was kept at 40% for 10 min and then decreased to 5%

in 5 min. The flow rate was 1ml min1 and the sample was filtered through

a 0.45mm membrane filter prior to HPLC analysis.

The identity of products was confirmed by comparison of both

retention time and spectrum with those of standard compounds and wasconsistent with the GC–MS analysis. For quantification, standards were

prepared by dilution of stock solutions that were prepared in methanol or

water (1mg ml1) and were stored at 4 1C, to give concentrations of 0.05,

0.10, 0.25, 0.50, and 1.25 mg ml1. Calibration curves were constructed by

regressing peak area against concentrations of the standard solutions.

Concentration of products in the samples was calculated using linear

regression equations from the calibration curves. Peak areas based on the

lmax of each compound were used for determination. All calibration

curves were linear (r240:999) over the concentration range investigated.

3. Results and discussion

Total ion chromatograms (TIC, Fig. 2) obtained from

GC–MS analysis of the degradation products of purified

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NNO2

NH2

N

Fig. 1. Structure of Disperse Orange 3 (C.I. 11005).

X. Zhao et al. / International Biodeterioration & Biodegradation 57 (2006) 1–6 2



Disperse Orange 3 (I) colorant revealed the presence of

several peaks other than the dye. Mass spectral analysis of

the metabolites produced from the culture of Pleurotus

ostreatus incubated with Disperse Orange 3 for 3 days

allowed the identification of 4-nitroaniline (II), nitroben-

zene (III), 4-nitrophenol (IV) and 4-nitroanisole (V) which

are probable metabolites of the dye. Both retention timesand mass spectra of these products matched those of

authentic standards. None of these compounds was

detected in the control samples. 4-Nitroaniline (II) was

detected in a previous investigation when Pleurotus

ostreatus was grown under similar conditions (Zhao and

Hardin, 2002). Veratryl alcohol (VI) and its oxidation

product, veratraldehyde (VII), were also detected on the

GC–MS chromatogram, (Fig. 2). The other peaks in the

TIC shown in Fig. 2 were also present in the control

cultures. In order to verify the presence of cell-bound

metabolites, the fungal mycelium was extracted with

methylene chloride. When the supernatant was extracted

with methlyene chloride under acid (pH 2) and basic (pH

13) conditions, no metabolites other than those obtained

when the extraction was carried out under neutral

conditions, were detected.

Owing to the unsatisfactory recovery rate of degradation

products from Disperse Orange 3 by liquid–liquid extrac-

tion (with methylene chloride), GC–MS was not used to

quantify these compounds. Instead, HPLC analysis was

employed to quantify the metabolites because of its

accuracy and simplicity.

HPLC analysis verified that commercial Disperse Or-

ange 3 and purified dye produced identical products during

degradation (data not shown). Therefore, commercialDisperse Orange 3 (20% colorant content) was used in

the remaining studies.

The kinetic profiles of the dye and its four degradation

products during a period of 9 days are shown in Fig. 3.

More than half the added dye was seen to be removed from

the medium during this period by monitoring the

maximum wavelength at 415 nm. Adsorption of dye by

the fungal mycelium contributed in part to this removal

and accounted for about 10–15% of total decolorization.

One of the products, 4-nitroaniline (II), reached a

maximum on day 2 and then decreased, indicating that 4-

nitroaniline was not the final product in the degradation.

From day 2 onwards, nitrobenzene concentration increased

markedly and linearly for 9 days. Another product, 4-

nitrophenol, was detected after 4 days along with itsmethylated product, 4-nitroanisole. Taking into considera-

tion the absorption of the substrate by the mycelium and

the loss of products during sample transfer, it was

estimated that these four metabolites contributed to

approx. 15–20% of the Disperse Orange 3 that was

removed after incubation for 9 days. Other possible

products were sought by extraction of fungal mycelium

and using acid or basic extraction conditions. However, no

other products could be found by these methods.

Oligomeric coupling of azo dye by peroxidases was

reported by Spadaro and Renganathan (1994). A similar

reaction might produce some dimers or oligomers of

Disperse Orange 3 in the fungal transformation, which

could not be identified in this work.

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V

VII

IV

VI

I

II

III

4.00E + 06

3.00E + 06

2.00E + 06

1.00E + 06

0.00E + 06

3.00 5.00 7.00 9.00 11.00 13.00 15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00 31.00

Time (min)

TIC

C

C

C

Fig. 2. Total ion chromatogram of the metabolites generated from purified Disperse Orange 3. Peak I, Disperse Orange 3; Peak II, 4-nitroaniline; Peak

III, nitrobenzene; Peak IV, 4-nitrophenol; Peak V, 4-nitroanisole; Peak VI, veratryl alcohol; Peak VII, veratraldehyde; Peaks C, peaks also detected in

control samples. Sample was taken after degradation for 3 days by Pleurotus ostreatus, supernatant extracted by methylene chloride.

5.00

4.00

3.00

2.00

1.00

0.00

0 2 4 6 8 10

Time (days)

100

80

60

40

20

0

% r

e m a i n o f d y e

C o

n c e n t r a t i o n o f p r o d u c t s ( µ m )

4 - Nitroaniline 4 - Nitrobenzene 4 - Nitrophenol

DO34 - Nitroanisole

Fig. 3. Partial kinetic profiles of some degradation products of Disperse

Orange 3 by HPLC.




To examine a possible pathway responsible for 4-

nitroanisole production, a separate experiment was con-

ducted, in which 4-nitrophenol was added to the cultures

and its degradation was examined by GC–MS. The only

metabolite identified was 4-nitroanisole (Fig. 4). This

shows that methylation occurs in the Pleurotus ostreatus

culture. Methylation of phenolic compounds by Phaner-

ochaete chrysosporium has also been reported by other

researchers (Valli and Gold, 1991; Valli et al., 1992).

Compared with Phanerochaete chrysosporium, Pleurotusostreatus produces laccases instead of LiP as the major

enzyme responsible for the biodegradation of lignin and

textile dyes (Robinson et al., 2001b; Rodriguez et al., 1999).

However, in Kirk’s medium, Pleurotus ostreatus produced

all three types of essential extracellular enzyme, LiP, MnP,

and laccases (Robinson et al., 2001b). The activity of

laccases was also detected in this study (data not shown

here). Veratryl alcohol (VI), a natural product synthesized

by fungi, is the mediator in the action of LiP. In a previous

investigation (Paszczynski and Crawford, 1991), LiP

compound I oxidized azo dyes and was converted to

compound II, which was then reduced by veratryl alcohol

to complete the catalytic cycle of the enzyme. The pathway

to re-oxidize veratryl alcohol involves two successive one-

electron oxidations to form veratraldehyde (Tien, 1987;

Zapanta and Tien, 1997). Furthermore, Paszczynski and

Crawford (1991) found that lignin peroxidase was only

capable of catalyzing the oxidization of recalcitrant azo

dyes effectively when veratryl alcohol was present.

Although the data are not shown, in this study, it was

found that veratryl alcohol (VI) was detectable in the

culture before nitrobenzene (III), 4-nitrophenol (IV),

and 4-nitroanisole (V). These observations suggest that

veratryl alcohol was involved in the degradation of this

azo dye although more biochemical investigations will

be required to demonstrate its role in the conversion of

azo dye.

A partial degradation pathway is proposed in Fig. 5,

showing the results expected from both reduction and

oxidation of Disperse Orange 3 (I) by Pleurotus ostreatus.

It is presumed that Pleurotus ostreatus peroxidases and

laccases were also involved in the degradation of Disperse

Orange 3 (I) and the methylation of 4-nitrophenol (IV)

observed in this study. Spadaro and Renganathan (1994)

proposed a degradation pathway for another disperse azodye, Disperse Yellow 3, by lignin peroxidase suggesting an

oxidation mechanism involving carbonium ion and phenyl

radicals. These results agree with their pathway for

nitrobenzene (III) production. Hydroxylation of aromatic

ring concomitant with cleavage of azo bond (NQN) is

presumed to be responsible for 4-nitrophenol (IV) produc-

tion.

The 4-nitroaniline (II) detected on the first day of

degradation does not seem to be generated by an

extracellular lignolytic enzyme. A reduction procedure

rather than an oxidation might be responsible for its

generation through a symmetric breakage of the azo bond.

Rodriguez et al. (1999) suggested that enzymes other than

those of the ligninolytic system were engaged in the dye

decolorization by Pleurotus ostreatus. The formation of 4-

nitroaniline may also be related to the reduction ability of

this fungus. However, this needs confirmation since no 1,4-

phenylenediamine, another likely reduction product, was

found in our investigation. This pathway may result from

the presence, in the azo dye, of an amino functional group

which is a weaker electron donor than a hydroxyl group,

and is thus more difficult to oxidize.

Data showed that white-rot fungi degrade the azo dye

Disperse Orange 3 via an oxidative mechanism, which is

different from that in anaerobic bacteria, and aromatic

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1.00E + 07

8.00E + 06

6.00E + 06

4.00E + 06

2.00E + 06

0.00E + 00

1.00E + 07

8.00E + 06

6.00E + 06

4.00E + 06

2.00E + 06

0.00E + 00

TIC

TIC

0 Day

5 Days

IV

IVV

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

3 5 7 9 11 13 15 17 19 21 23 25 27 29 31

Time (min)

Fig. 4. Identification of 4-nitroanisole as the major transformation product of 4-nitrophenol by GC–MS.




amines are not exclusive products. Exactly the same

degradation products were released from Disperse Red 1,

which is structurally similar to Disperse Orange 3, by

cultures of Pleurotus ostreatus (Lu and Hardin, 2005). This

indicates that the degradation pathway of Disperse Orange

3 can be used to assess degradation and predict the

degradation pathway of other azo dyes with similarstructure. Further study of the biodegradation of these

products is continuing in order to understand the complete

degradation pathway of azo dyes by white-rot fungi.

Acknowledgment

Authors appreciate the discussion of results and help

from Professor George L. Baughman at the University of

Georgia.

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O2N

O2N

O2N

O2N

O2N

I

NH2

NH2

OH

OCH3

III IV

V

LiP

Veratryl alcohol

II

LiP/Laccases

Veratraldehyde

Symmetric azo bond cleavage

N N

Fig. 5. Proposed degradation pathway of Disperse Orange 3 by Pleurotus ostreatus.




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Biodegradation of a Model Azo Disperse Dye by the White Rot Fungus