Accepted Manuscript

Title: Efficient decolorization and detoxification of the

sulfonated azo dye Reactive Orange 16 and simulated textile
wastewater containing Reactive Orange 16 by the white-rot
fungus Ganoderma sp. En3 isolated from the forest of
Tzu-chin Mountain in China

Author: Li Ma Rui Zhuo Huahua Liu Dong Yu Mulan Jiang

Xiaoyu Zhang Yang Yang

PII: S1369-703X(13)00296-9
DOI: http://dx.doi.org/doi:10.1016/j.bej.2013.10.015
Reference: BEJ 5823

To appear in: Biochemical Engineering Journal

Received date: 21-5-2013

Revised date: 20-9-2013
Accepted date: 17-10-2013

Please cite this article as: L. Ma, R. Zhuo, H. Liu, D. Yu, M. Jiang, X. Zhang, Y. Yang,
Efficient decolorization and detoxification of the sulfonated azo dye Reactive Orange
16 and simulated textile wastewater containing Reactive Orange 16 by the white-rot
fungus Ganoderma sp. En3 isolated from the forest of Tzu-chin Mountain in China,
Biochemical Engineering Journal (2013), http://dx.doi.org/10.1016/j.bej.2013.10.015

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Efficient decolorization and detoxification of the sulfonated azo dye Reactive Orange

16 and simulated textile wastewater containing Reactive Orange 16 by the white-rot

fungus Ganoderma sp. En3 isolated from the forest of Tzu-chin Mountain in China

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Li Maa, Rui Zhuoa, Huahua Liua, Dong Yua, Mulan Jiangb, Xiaoyu Zhanga, Yang

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Yanga*

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. College of Life Science and Technology, Huazhong University of Science and

Technology, Wuhan, 430074, China

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b
. Key Laboratory of Oil Crops Biology of Ministry of Agriculture in China, Oil

Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan,

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430064, China
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* Corresponding author:
Yang Yang, College of Life Science and Technology, Huazhong University of Science
p

and Technology, Wuhan, 430074, China.

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Tel: +86-27-87792108, E-mail: yangyang@mail.hust.edu.cn

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Page 1 of 40
Abstract

The sulfonated azo dye Reactive Orange 16 is the commonly used representative

of reactive dyes, but is hard to be degraded by some conventional treatment methods.

In order to develop more efficient and more cost-effective treatment methods for

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ip
degrading this recalcitrant dye, the capability of the white-rot fungus Ganoderma

cr
sp.En3 isolated by our laboratory to decolorize and detoxify Reactive Orange 16 was

investigated in this study. Ganoderma sp.En3 had a strong ability to decolorize high

us
concentrations of Reactive Orange 16 and simulated textile wastewater containing

an
Reactive Orange 16 in submerged cultures. Decolorization of Reactive Orange 16 and

its simulated dye effluents by this fungus resulted in the decrease of phytotoxicity.
M
Ganoderma sp.En3 had strong adaptability and tolerance to high concentrations of

Reactive Orange 16. Compared with some previous research, Ganoderma sp.En3 was
d

superior to some other fungal strains reported previously in the rate and extent of
te

decolorizing Reactive Orange 16. It was also found that the real textile wastewater
p

could be efficiently decolorized by Ganoderma sp.En3 in submerged cultures. The

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crude enzyme produced by Ganoderma sp.En3 could also efficiently decolorize

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Reactive Orange 16 and simulated textile wastewater under in vitro conditions.

Keywords: Decolorization; Detoxification; Biodegradation; Enzymes; Filamentous

Fungi; Waste-Water Treatment

Page 2 of 40
1. Introduction

Synthetic dyes have been widely used in many fields of industry, such as

textiles, dyeing, print, and leather industry. Although various synthetic dyes showing

considerable structural diversity have great value in the industrial applications, the

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extensive use of synthetic dyes has also led to a serious environmental problem [1,2].

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It has been reported that about 10-15 percent of dyes is released into wastewater and

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industrial effluents during textile processing [1]. Because many dyes discharged from

the textile industry have been shown to be toxic, carcinogenic and mutagenic, release

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of synthetic dyes into the environment may bring on the severe environmental

pollution and threaten human health [3,4]. Thus it is very necessary and important to
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remove these dyes from the effluents before discharged to the environment.

In recent years, white-rot fungi and their non-specific extracellular ligninolytic

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enzymes have been shown to possess strong ability to degrade a wide range of

synthetic dyes with different structure [5-7]. White-rot fungus is one kind of main
p

wood decay fungi. The most unique feature of white-rot fungus is its powerful ability
ce

of extensively degrading lignin. The extracellular and non-specific ligninolytic

Ac

enzymes that mainly comprise of lignin peroxidase (LiP), mangnese dependent

peroxidase (MnP) and laccase have been shown to be involved in the degradation of

lignin and various recalcitrant xenobiotics by white-rot fungus [5-7]. Previous

research has demonstrated that structurally different dyes including anthraquinone,

azo, indigoid, triphenylmethane and heterocyclic dyes can be efficiently decolorized

by various species of white-rot fungi such as Phanerochaete chrysosporium, Trametes

Page 3 of 40
versicolor, Pleurotus ostreatus, Pycnoporus sanguineus, Irpex flavus, Phellinus gilvus

etc [6-8]. Decolorization of different synthetic dyes by white-rot fungi is more

efficient, cost-effective and environmentally friendly compared with other techniques.

Thus, biodegradation of dyes in textile waste effluents by white-rot fungi is a very

t
ip
promising alternative to the traditional methods for removal of recalcitrant dyes [9].

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Although white-rot fungus has great potential for the application in the

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biodegradation of recalcitrant dyes, there are still some problems which restrict the

more effective decolorization of dyes by white-rot fungus. Firstly, little work has been

an
done to evaluate the ability of white-rot fungi to decolorize high concentration of

industrial dyes. Secondly, previous research has found that high concentration of dye
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may be toxic to fungal growth. The inhibition of dyes on fungal growth may impede

the efficient decolorization of high concentration of dyes by white-rot fungi [10,11].

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Therefore, searching for new white-rot fungi strains with stronger ability to tolerate

and decolorize high concentrations of dyes is needed for the application of white-rot
p

fungi in the biodegradation of recalcitrant dyes. Thirdly, although there have been
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many reports about the fungal decolorization of structurally different dyes, few
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studies have been conducted on determining the toxicity of dye effluent after

decolorization by white-rot fungi [12].

In our previous work [13], a new white-rot fungi strain Ganoderma sp. En3,

which was isolated from the forest of Tzu-chin Mountain in China, has been shown to

have a strong ability to decolorize and detoxify four synthetic dyes-methyl orange,

malachite green, bromophenol blue, crystal violet and the textile dye effluents.

Page 4 of 40
Laccase played an important role in the efficient decolorization of different dyes by

this fungus [13]. Based on our previous research, in order to make better use of this

fungus in the area of environmental biotechnology and evaluate the feasibility and

effectiveness of using this fungus to decolorize other types of dyes more recalcitrant to

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degradation, this work focuses on studying the capability of Ganoderma sp. En3 to

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decolorize and detoxify a very important sulfonated azo dye-Reactive Orange 16 that is

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difficult to be degraded. Reactive Orange 16 (C.I. 17757) is a reactive dye bearing an

azo group as chromophore and a sulphatoethylsulfone as the reactive group. Because

an
this dye has excellent properties for dyeing silk and cotton, Reactive Orange 16 has

been widely used in the textile industries. However, Reactive Orange 16 is hard to be
M
degraded by some conventional treatment methods [14-16]. In this study, we found

that not only the pure Reactive Orange 16 dye but also the simulated textile
d
te

wastewater containing Reactive Orange 16 could be efficiently decolorized and

detoxified by Ganoderma sp. En3. Ganoderma sp. En3 had a strong ability to
p

decolorize and tolerate high concentrations of Reactive Orange 16 and its simulated
ce

dye effluents. Compared with some previous research, Ganoderma sp. En3 was
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superior to other fungal strains reported previously in the rate and extent of

decolorizing Reactive Orange 16. The crude enzyme produced by Ganoderma sp. En3

could also efficiently decolorize Reactive Orange 16 and simulated textile wastewater

under in vitro conditions. In addition, it was found that the real textile wastewater

could be efficiently decolorized by Ganoderma sp. En3.

Page 5 of 40
2. Material and methods

2.1 Strains and media

Ganoderma sp. En3 was isolated from the forest of Tzu-chin Mountain in China

in our previous work [13] and preserved in Institute of Environment and Resource

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Microbiology, Huazhong University of Science and Technology, Wuhan, China.

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Ganoderma sp. En3 was maintained on potato-dextrose agar (PDA) medium. The

basal liquid medium contained (g l-1 of distilled water): Glucose 20g, Yeast extract

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2.5g, KH2PO4 1g, Na2HPO4 0.05g, MgSO4 ·7H2O 0.5g, CaCl2 0.01g, FeSO4 · 7H2O

an
0.01g, MnSO4 · 4H2O 0.001g, ZnSO4 · 7H2O 0.001g, CuSO4 · 5H2O 0.002g. The pH

of the medium was adjusted to 5.5 [17,18].

M
2.2 Decolorization of Reactive Orange 16 in submerged cultures of Ganoderma sp.

En3
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250 ml Erlenmeyer flasks containing 100 ml basal liquid medium were each

inoculated with the actively growing mycelium pellets of Ganoderma sp. En3. Then
p

Ganoderma sp. En3 was incubated at 28 ºC in a shaking incubator (150 rpm) for 5
ce

days. The sulfonated azo dye Reactive Orange 16 (C.I. 17757, purchased from
Ac

Sigma–Aldrich, the chemical structure of Reactive Orange 16 was shown in Fig.1)

was added into the actively growing 5-day-old cultures of Ganoderma sp. En3 at the

final concentrations: 100 and 1000 mg/l. After adding the dye, the fungal cultures

were then incubated at 28 ◦C with shaking at 150 rpm continuously. Samples from

triplicate flasks were withdrawn at different time intervals, centrifuged at 8000g for

20 min, and the clear supernatant was used for determination of decolorization

Page 6 of 40
efficiency spectrophotometrically. Decolorization was monitored by measuring the

absorbance of the culture supernatant at 492 nm for Reactive Orange 16. The

decolorization of dye, expressed as dye decolorization (%), was calculated as the

following formula: decolorization (%)=[(Ai-At)/Ai]*100, where, Ai: initial absorbance

t
ip
of the dye, At: absorbance of the dye along the time. Effect of different carbon sources

cr
on dye decolorization was studied by replacing glucose with glycerol, fructose,

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sucrose and starch in the basal liquid medium. Effect of varying nitrogen sources on

dye decolorization was studied by replacing yeast extract with L-asparagine,

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ammonium sulphate, ammonium tartrate, peptone and urea.

2.3 Decolorization of simulated textile wastewater containing Reactive Orange 16

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in submerged cultures of Ganoderma sp. En3

Simulated textile wastewater containing Reactive Orange 16 designated as

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STWRO16-I (Simulated Textile Wastewater containing Reactive Orange 16-I) was

prepared as described in reference [19,20]. The composition of STWRO16-I was

p

based on instructions of the manufacturer Bezema AG (Montlingen, Switzerland) for

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reactive dyes [19,20]. It consisted of 0.5 g/l Reactive Orange 16, 30 g/l NaCl, 5 g/l
Ac

Na2CO3 and 1.5 ml/l of 32.5% (w/v) NaOH in deionized water. The pH was adjusted

to 4.5 with HCl. The effluent sample was scanned using UV–Vis Spectrophotometry,

which showed the maximum absorbance at 490 nm.

Flasks containing basal liquid medium were each inoculated with the actively

growing mycelium pellets of Ganoderma sp. En3 and incubated at 28 ºC in a shaking

incubator (150 rpm) for 5 days. Then STWRO16-I was added into the 5-day-old

Page 7 of 40
cultures at different final concentrations, respectively. After adding the simulated

textile wastewater containing Reactive Orange 16, the fungal cultures were then

incubated at 28 ◦C with shaking at 150 rpm continuously. Samples from triplicate

flasks were withdrawn at different time intervals, centrifuged at 8000g for 20 min, and

t
ip
the clear supernatant was used for determination of decolorization efficiency

cr
spectrophotometrically. Decolorization was monitored by measuring the absorbance

us
of the culture supernatant at 490 nm. The decolorization of dye effluent, expressed as

dye decolorization (%), was calculated as the following formula: decolorization

an
(%)=[(Ai-At)/Ai]*100, where, Ai: initial absorbance of the dye effluent, At:

absorbance of the dye effluent along the time.

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2.4 Decolorization of real textile wastewater in submerged cultures of Ganoderma

sp. En3
d
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The real textile wastewater used in this work was the indigo jean dyeing

wastewater obtained from a textile factory in China. This wastewater mainly

p

contained the indigoid and sulphur dyestuff as well as some other dyeing auxiliaries.
ce

The physicochemical property of this wastewater was as follows: COD=20393.2

Ac

mg/L, pH=9.8, the maximum absorbance of this wastewater scanned by UV–Vis

spectrophotometry was 710 nm.

Flasks containing 100 ml textile wastewater were inoculated with the actively

growing mycelium pellets of Ganoderma sp. En3 and incubated at 28 ºC in a shaking

incubator (150 rpm). Samples from triplicate flasks were taken every day, centrifuged

at 8000g for 20 min, and the clear supernatant was used for determination of

Page 8 of 40
decolorization efficiency and measuring the laccase activity. Decolorization was

detected by measuring the absorbance of the culture supernatant at 710 nm. The

decolorization of wastewater, expressed as dye decolorization (%), was calculated as

the following formula: decolorization (%)=[(Ai-At)/Ai]*100, where, Ai: initial

t
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absorbance of the wastewater, At: absorbance of the wastewater along the time.

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2.5 Detection of the tolerance of Ganoderma sp. En3 to Reactive Orange 16 and

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simulated textile wastewater

The tolerance of Ganoderma sp. En3 to different concentrations of Reactive

an
Orange 16 and simulated textile wastewater was detected by measuring the mycelial

dry weight of fungal cultures (100 ml) supplemented with different concentrations of
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dye and dye effluent. Determination of mycelial dry weight was performed according

to the method described by Manubens et al. [21]. Triplicate flasks were harvested and
d
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filtered through Whatman No. 1 paper that had previously been dried at 100 ºC to a

constant weight. The mycelium retained on the filter paper was dried at 100 ºC to a
p

constant weight and the mycelial weight was determined [21].

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2.6 Phytotoxicity study of Reactive Orange 16 and simulated textile wastewater

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containing Reactive Orange 16 before and after fungal treatment

The phytotoxicity of Reactive Orange 16 and simulated textile wastewater

containing Reactive Orange 16 before and after fungal treatment were detected as

follows. The relative sensitivities towards the original dyes and their degradation

products in relation to Triticum aestivum and Oryza sativa seeds were evaluated

according to the method described by reference [22]. The phytotoxicity study was

Page 9 of 40
performed (at room temp) in relation to Triticum aestivum and Oryza sativa (10 seeds

of each) by watering separately 10 ml sample of original dyes and their decolorization

products per day. Seeds were also treated with distilled water at the same time as the

control. The length of shoot and root of Triticum aestivum treated under different

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conditions was measured after 4 days. The length of shoot and root of Oryza sativa

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treated under different conditions was measured after 7 days. Values are mean of

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germinated seeds of three experiments.

2.7 Enzyme activity assay

an
The activity of extracellular ligninolytic enzymes, such as laccase, manganese

peroxidase, lignin peroxidase before and after decolorization were measured as

M
following. Laccase activity was determined with 2,2′-azino-bis

(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as the substrate [23]. Assay mixtures

d

contained 0.5 mM ABTS, 0.1 M sodium acetate (pH=5.0) and 100 μl culture fluid.
te

Oxidation of ABTS was monitored by determining the increase in A420 (Ɛ=36,000 M−1
p

cm−1). One unit of laccase activity was defined as the amount of enzyme required to
ce

oxidize 1 μmol of ABTS per min [23]. Manganese peroxidase activity was measured
Ac

as described by reference [24]. Lignin peroxidase activity was measured as described

by reference [25].

2.8 In vitro decolorization of Reactive Orange 16 and simulated textile

wastewater by crude enzyme produced by Ganoderma sp. En3

The culture supernatants prepared from Ganoderma sp. En3 at the peak of

laccase activity were used to decolorize Reactive Orange 16 and simulated textile

10

Page 10 of 40
wastewater under in vitro conditions. The decolorization of Reactive Orange 16 by the

crude enzyme was carried out as follows. The reaction mixture in a total volume of 2

ml contained (final concentration): acetate buffer (50 mM, pH: 5.0), 500 µl crude

enzyme (laccase activity was 280 U/l) and Reactive Orange 16 (50, 100, 200 and 500

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mg/l). During incubation at 30 ºC, the time course of decolorization was detected

cr
at designated time intervals by measuring the absorbance of the reaction mixture at

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492 nm for Reactive Orange 16 in a UV–Vis Spectrophotometer. The reaction

mixture was also scanned using UV–Vis Spectrophotometry. The decolorization of

an
dye, expressed as dye decolorization (%), was calculated as the formula:

decolorization (%)=[(Ai-At)/Ai]*100, where, Ai: initial absorbance of the dye, At:

M
absorbance of the dye along the time. In order to test the effect of redox mediator on

the dye decolorization, the decolorization of higher concentrations of Reactive Orange

d
te

16 (500 and 2000 mg/l) by the crude enzyme was also performed with addition of a

natural redox mediator-syringaldehyde (1 mM).

p

The decolorization of simulated textile wastewater containing Reactive Orange

ce

16 (STWRO16-I) by the crude enzyme was performed as follows. 2 ml of reaction

Ac

mixture contained acetate buffer (50 mM, pH: 5.0), appropriate amount of crude

enzyme and STWRO16-I. During incubation at 30 ºC, the time course of

decolorization was detected at designated time intervals by measuring the absorbance

at 490 nm for STWRO16-I. STWRO16-I was also decolorized by crude enzyme with

addition of 1 mM syringaldehyde to the reaction mixture.

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Page 11 of 40
3. Results

3.1 Decolorization and detoxification of Reactive Orange 16 in submerged

cultures of Ganoderma sp. En3

The ability of Ganoderma sp. En3 to decolorize the sulfonated azo dye Reactive

t
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Orange 16 was evaluated in submerged cultures according to the method described in

cr
Materials and methods. Our research suggested that Ganoderma sp. En3 could

us
decolorize this azo dye efficiently. As shown in Fig.2A, after Reactive Orange 16 (100

mg/l) was added into the actively growing 5-day-old cultures of Ganoderma sp. En3,

an
100 mg/l Reactive Orange 16 was decolorized up to 95.1% by Ganoderma sp. En3

within 96 hours. However, the decolorization efficiency decreased as the

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concentration of Reactive Orange 16 added into the basal liquid medium increased.

When the concentration of Reactive Orange 16 increased from 100 to 1000 mg/l, the
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te

efficiency of decolorization after 96 hours decreased from 95.1% to 49.1% (Fig.2A).

In order to substantially improve the capability of decolorizing higher

p

concentration of Reactive Orange 16, the operational conditions such as carbon and
ce

nitrogen sources in the media were optimized. Firstly, the effect of different carbon
Ac

sources on dye decolorization was investigated. As shown in Fig.2B, among all of the

carbon sources tested, glycerol was the best carbon source for achieving the highest

decolorization efficiency. When glycerol was used as the carbon source, the maximum

decolorization (%) of Reactive Orange 16 (1000 mg/l) could reach 92.3% within only

24 hours. After 96 hours of incubation, 1000 mg/l Reactive Orange 16 was

decolorized up to 96.6% by Ganoderma sp.En3 (Fig.2B). Thus, glycerol was shown

12

Page 12 of 40
to be the best carbon source for the decolorization of Reactive Orange 16 by

Ganoderma sp.En3. Secondly, the effect of different nitrogen sources on dye

decolorization was also determined. As shown in Fig.2C, among the different nitrogen

sources, peptone was the best with 95.5% decolorization within 96 hours. Hence,

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peptone was selected as the best nitrogen source for the decolorization of Reactive

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Orange 16 by Ganoderma sp.En3.

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After finding the best carbon (glycerol) and nitrogen source (peptone) and

optimizing the medium components for the decolorization of Reactive Orange 16,

an
higher concentrations of Reactive Orange 16 were decolorized in submerged cultures

simultaneously using glycerol and peptone. As shown in Fig.2D, the capability of

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Ganoderma sp.En3 to decolorize higher concentration of Reactive Orange 16 was

significantly improved by using the optimized carbon and nitrogen source

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simultaneously. After 96 hours of incubation, the highest decolorization efficiency for

Reactive Orange 16 (1000, 2000, 4000 and 8000 mg/l ) reached 98.2%, 95.1%, 93.7%
p

and 74.6%, respectively (Fig.2D). The decolorization rate was also greatly increased.
ce

1000 mg/l Reactive Orange 16 could be decolorized up to 91.4% within only 12 hours
Ac

(Fig.2D).

The change of the activity of ligninolytic enzymes before and after

decolorization of Reactive Orange 16 was investigated (Table S1). The activities of

laccase, manganese peroxidase and lignin peroxidase were assayed in the supernatant

medium before and after decolorization. Extracellular laccase activities of Ganoderma

sp.En3 significantly increased after decolorization of Reactive Orange 16 (2000 mg/l ).

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Page 13 of 40
Neither manganese peroxidase nor lignin peroxidase was detected during the

decolorization process (Table S1). Significant induction in the activity of laccase

during the decolorization process suggested that laccase may be involved in the

decolorization of Reactive Orange 16 by Ganoderma sp. En3. The change of COD

t
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(chemical oxygen demand) before and after decolorization was also studied.

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Significant reductions in COD were observed after decolorization of Reactive Orange

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16 by Ganoderma sp. En3 (Table S1).

In order to evaluate the tolerance of Ganoderma sp. En3 to Reactive Orange 16

an
and determine the toxicity of Reactive Orange 16 to the growth of Ganoderma sp.

En3, the effect of different concentrations of Reactive Orange 16 on the fungal growth
M
was detected by measuring the mycelial dry weight of cultures supplemented with

different concentrations of Reactive Orange 16 (Fig.S1). It was found that the growth
d
te

of Ganoderma sp. En3 in the presence of high concentrations of Reactive Orange 16

were very similar to that of control without adding any dye (Fig.S1). This result
p

suggested that Ganoderma sp. En3 had strong adaptability and tolerance to high
ce

concentrations of Reactive Orange 16.

Ac

The phytotoxicity of Reactive Orange 16 before and after fungal treatment was

further evaluated. The relative sensitivities towards 1000 mg/l Reactive Orange 16

and its decolorization product in relation to Triticum aestivum and Oryza sativa seeds

were shown in Fig.S2. When the seeds were treated with the original Reactive Orange

16 (1000 mg/l), the growth of shoot and root of Triticum aestivum and Oryza sativa

seeds were significantly inhibited. However, the decolorization product after fungal

14

Page 14 of 40
treatment exhibited lower phytotoxicity than the untreated dye with respect to

Triticum aestivum and Oryza sativa (Fig.S2). This result demonstrated that

decolorization of Reactive Orange 16 by Ganoderma sp. En3 resulted in the decrease

of phytotoxicity.

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3.2 Decolorization and detoxification of simulated textile wastewater containing

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Reactive Orange 16 in submerged cultures of Ganoderma sp. En3

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Although above research has demonstrated that Ganoderma sp. En3 had a strong

ability to decolorize the pure sulfonated azo dye-Reactive Orange 16, more study has

an
to be done to evaluate the ability of Ganoderma sp. En3 to decolorize the dye

effluents with high concentration of salts and high ionic strength. Therefore,
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Ganoderma sp. En3 was further used to decolourise the simulated textile wastewater

containing Reactive Orange 16 (STWRO16-I), which was prepared as described in

d
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reference [19,20].

Higher concentrations of STWRO16-I were decolorized in submerged cultures

p

simultaneously using sucrose and peptone. As shown in Fig.3, STWRO16-I (final

ce

concentration: 30% and 60%) could be decolorized up to 89.5% and 73.2% by

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Ganoderma sp. En3 within 10 days (Fig.3).

The change of the activity of ligninolytic enzymes before and after

decolorization of STWRO16-I was also investigated ( Table S1). It was found that

extracellular laccase activities of Ganoderma sp. En3 significantly increased after

decolorization of STWRO16-I. Induction of the extracellular laccase activity during

the decolorization process suggested its involvement in the decolorization of

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Page 15 of 40
STWRO16-I by Ganoderma sp. En3. After fungal treatment of STWRO16-I (30%)

for 10 days, 61.6 % reduction of COD were observed respectively (Table S1).

Our results also suggested that Ganoderma sp. En3 could tolerate different

concentrations of STWRO16-I. The mycelial dry weights of 10-day-old cultures

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supplemented with different concentrations of STWRO16-I (30% and 60%) were very

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similar to that of control without adding STWRO16-I (Fig.S3).

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The phytotoxicity of STWRO16-I before and after fungal treatment were also

evaluated. Our results suggested that decolorization of STWRO16-I by Ganoderma

an
sp. En3 could also result in the decrease of phytotoxicity. Decolorization of

STWRO16-I was accompanied by reduction in phytotoxicity (data not shown).

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3.3 Decolorization of real textile wastewater in submerged cultures of Ganoderma

sp. En3
d
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The ability of Ganoderma sp. En3 to decolorize the real textile wastewater was

also evaluated as described in Material and methods. As shown in Fig.4A,

p

Ganoderma sp. En3 showed an effective decolorization of this textile wastewater. The
ce

textile wastewater could be decolorized up to 85.1% by this fungus within 8 days

Ac

(Fig.4A). It was also found that the extracellular laccase activity of Ganoderma sp.

En3 significantly enhanced along with the decolorization of this textile wastewater

(Fig.4B). In addition, the COD value of this textile wastewater decreased along with

the decolorization process (Fig.4C). It was also found that Ganoderma sp. En3 could

tolerate this real textile wastewater.

3.4 In vitro decolorization of Reactive Orange 16 by crude enzyme produced by

16

Page 16 of 40
Ganoderma sp. En3

The crude enzyme was prepared from the cultures of Ganoderma sp. En3 grown

in liquid medium and then used to decolorize Reactive Orange 16 under in vitro

conditions. As shown in Fig.5A, during 30 h incubation, maximum of 66.7% and

t
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60.4% decolorization of Reactive Orange 16 (50 and 100 mg/l) were observed in the

cr
absence of any redox mediator, respectively. However, the decolorization efficiency

us
decreased as the concentration of Reactive Orange 16 increased. 200 and 500 mg/l

Reactive Orange 16 could only be decolorized up to 42.1% and 18.2% by the crude

an
enzyme within 30 hours (Fig.5A).

Nevertheless, the crude enzyme exhibited more efficient decolorization of higher

M
concentrations of Reactive Orange 16 when syringaldehyde was added into the

reaction mixture as the natural redox mediator. As shown in Fig.5B, addition of 1 mM

d
te

syringaldehyde to the reaction mixture significantly enhanced the decolorization of

Reactive Orange 16. In the presence of 1 mM syringaldehyde, 500 and 2000 mg/l
p

Reactive Orange 16 could be decolorized up to 88.1% and 71.6% by the crude

ce

enzyme within 24 hours (Fig.5B). We also noticed that addition of the natural
Ac

phenolic compound syringaldehyde could greatly increase the rate of decolorization.

For example, in the presence of 1 mM syringaldehyde, 500 mg/l Reactive Orange 16

could be decolorized up to 71.1% within only 1 hour (Fig.5B). Fig.5C showed the

UV–visible absorbance spectrum for 2000 mg/l Reactive Orange 16 treated by crude

enzyme in the presence of 1 mM syringaldehyde. The absorbance peak gradually

decreased with time after treated by the crude enzyme of Ganoderma sp. En3, which

17

Page 17 of 40
was associated with decolorization. This result further confirmed that high

concentrations of Reactive Orange 16 could be efficiently decolorized by the crude

enzyme of Ganoderma sp. En3 in the presence of a natural redox

mediator-syringaldehyde. According to the method described in reference [26],

t
ip
Native PAGE of the crude enzyme was performed to test the involvement of laccase

cr
in decolorization of Reactive Orange 16. As shown in Fig.5D, the crude enzyme

us
showed a single laccase activity against ABTS (lane 1). The gel was also stained with

Reactive Orange 16 (50 mg/l) and Reactive Orange 16 (100 mg/l) in sodium acetate

an
buffer (0.1 mM, pH: 5.0) for 2 hours at 30 ◦C with addition of 0.5 mM syringaldehyde.

After incubation for 2 hours, a clear white band of decolorized zone was observed in
M
both the gels stained with 50 and 100 mg/l Reactive Orange 16 respectively (lane 2

and 3).
d
te

3.5 In vitro decolorization of simulated textile wastewater containing Reactive

Orange 16 by crude enzyme produced by Ganoderma sp. En3

p

The same crude enzyme prepared from the cultures of Ganoderma sp. En3 was
ce

also used to decolorize simulated textile wastewater containing Reactive Orange 16

Ac

(STWRO16-I) under in vitro conditions. As shown in Fig.6A, in the absence of any

redox mediator, STWRO16-I (final concentration: 40%) could only be decolorized up

to 18.5% by the crude enzyme within 30 hours (Fig.6A). It was found that the

efficiency of crude enzyme to decolorize STWRO16-I could be significantly

enhanced by the natural redox mediator-syringaldehyde. As shown in Fig.6A, 40% of

STWRO16-I could be decolorized up to 80.3% by the crude enzyme within 30 hours

18

Page 18 of 40
in the presence of 1 mM syringaldehyde. Fig.6B showed the UV–visible absorbance

spectrum for STWRO16-I (20%) after 30 hours of incubation in the presence of 1 mM

syringaldehyde. The absorbance peak gradually decreased with time after treated by

the crude enzyme of Ganoderma sp. En3. This result further proved that STWRO16-I

t
ip
could be efficiently decolorized by the crude enzyme of Ganoderma sp. En3.

cr
4. Discussion

us
In this study, the white-rot fungus Ganoderma sp. En3 isolated by our

laboratory was used to decolorize Reactive Orange 16 by different methods. After we

an
test the fungus whole strain and crude enzyme, we think that the fungus whole strain

is more feasible to be used in the practical wastewater treatment. The reason is

M
described below. Our study suggested that high concentrations of Reactive Orange

16 could be effectively decolorized and detoxified by fungal whole cultures.

d
te

Ganoderma sp.En3 also had strong adaptability and tolerance to high concentrations

of Reactive Orange 16. However, the high concentrations of Reactive Orange 16

p

could not be efficiently decolorized by the crude enzyme unless the redox mediator
ce

was added. Thus, from the point view of practical application, the use of fungus
Ac

whole strain for dye decolorization is more economical and cost effective.

Although there have been some reports about the decolorization of Reactive

Orange 16 by white-rot fungi [14,16,28], bacteria [29] and plant consortium [30],

Ganoderma sp. En3 is superior to some other fungal strains reported previously in the

respect of decolorizing Reactive Orange 16. The detailed comparison between our

work and previous studies is described as follows. Ganoderma sp. En3 had the more

19

Page 19 of 40
powerful capability for decolorizing Reactive Orange 16 compared with some other

fungal strains reported previously [14,16,28]. Baldrian et al. have reported that some

litter-decomposing fungi such as Collybia dryophila, Mycena inclinata, Stropharia

rugosoannulata, and white-rot fungi Trametes versicolor, Pleurotus ostreatus could

t
ip
decolorize Reactive Orange 16 in whole fungal cultures. But the decolorization

cr
efficiency of these fungal strains was relatively low. The decolorization of 100 mg/l

us
Reactive Orange 16 after 28 days of incubation ranged only 45–82% [16]. Novotny et

al. have studied the capability of another white-rot fungus Irpex lacteus to decolorize

an
Reactive Orange 16. In submerged culture, 150 mg/l Reactive Orange 16 was

decolorized up to only about 16% by Irpex lacteus within 7 days. Addition of Tween
M
80 to the submerged culture increased the decolorization efficiency to about 87% after

7 days of incubation [14]. Previous research also showed that the decolorization of
d
te

Reactive Orange 16 by the white-rot fungus Phanerochaete chrysosporium was rather

limited. The maximum decolorization (%) of Reactive Orange 16 (150 mg/l) by

p

agitated cultures of Phanerochaete chrysosporium could only reach 30.3% within 24

ce

hours [28]. In our research, we found that Ganoderma sp. En3 showed 88.2 %
Ac

decolorization of Reactive Orange 16 (100 mg/l) within 48 hours (Fig.2A). After

optimizing the carbon and nitrogen source in the medium, higher concentration of

Reactive Orange 16 (1000 mg/l) could be decolorized up to 91.1% and 94.3% by

Ganoderma sp. En3 within 12 and 24 hours, respectively (Fig.2D). Compared with

previous research [16], the time required for complete decolorization of Reactive

Orange 16 by Ganoderma sp. En3 was much less than some other fungi such as

20

Page 20 of 40
Trametes versicolor, Pleurotus ostreatus. The decolorization efficiency of Reactive

Orange 16 by Ganoderma sp. En3 was also higher than other fungal strains reported

previously [14,16,28]. The strong tolerance of Ganoderma sp. En3 to Reactive

Orange 16 may be one of the reasons why Ganoderma sp. En3 had a strong ability to

t
ip
decolorize high concentrations of Reactive Orange 16.

cr
In our previous research [13], we have cloned the laccase gene lac-En3-1

us
encoding laccase and its corresponding full-length cDNA from Ganoderma sp.En3

(GenBank accession no. HM569745). The functionality of lac-En3-1 gene encoding

an
active laccase was verified by expressing this gene in the yeast Pichia pastoris

successfully [13]. The sequence of laccase gene lac-En3-1 from Ganoderma sp.En3
M
has been compared with other laccase genes from other white-rot fungi. No significant

differences have been found between the laccase gene lac-En3-1 from Ganoderma
d
te

sp.En3 and other laccase genes. LAC-En3-1 protein encoded by lac-En3-1 gene

contains four copper-binding conserved domains of typical laccase: CuI (HWHGFFQ),

p

CuII (HSHLSTQ), CuIII (HPFHLHG) and CuIV (HCHIDFHL) [13]. We have

ce

purified and studied the characteristics of laccase protein from Ganoderma sp.En3. It
Ac

is found that some enzymology properties of purified laccase from Ganoderma

sp.En3 are superior to some other laccases from other fungi. Compared with some

other laccases reported previously, laccase from Ganoderma sp.En3 shows higher

thermal stability and better alkali-resistance. Laccase from Ganoderma sp.En3 also

exhibits strong tolerance to different organic solvents such as acetonitrile, methanol

and ethanol (data not published). The excellent enzymology property of laccase from

21

Page 21 of 40
Ganoderma sp.En3 may be one of the reasons accounting for the efficient

decolorization and detoxicity abilities possessed by Ganoderma sp.En3.

In order to better use the white-rot fungus Ganoderma sp.En3 in the future

wastewater treatment, we will try the following methods to enhance the feasibility and

t
ip
efficacy of using this fungus to treat the dye wastewater in future studies. Firstly,

cr
previous research has suggested that the treatment systems composed of mixed

us
microbial cultures (microbial consortium) result in a higher degree of biodegradation

due to the synergistic metabolic activities and have considerable advantages over the

an
use of pure cultures in the biodegradation of dye wastewater [31-34]. Microbial

consortium may be consisted of bacteria, fungi, or a mixture of both [35,36]. The

M
synergetic reaction of single strains in the microbial consortium can significantly

improve the decolorization efficiency and decolorization rate [37]. Fungal-bacterial

d
te

co-cultures have been used to take advantage of the diversity of oxidoreductive

enzymes produced by these two types of microorganisms [36,38]. Thus, in the further
p

study, we plan to construct the microbial consortium consisted of the white-rot fungus
ce

Ganoderma sp.En3 and bacteria and try to use this consortium to decolorize the real
Ac

wastewater more effectively under the optimal conditions. We will attempt to enhance

the decolorization efficiency and rate by establishing a fungi-bacteria co-cultures

system in which the two groups of microorganisms work synergistically. Secondly,

the various molecular biology techniques can be used to construct the genetically

engineered fungus with stronger ability of treating the dye effluents and higher

biodegradation capacities [36]. Gene manipulation can enhance the capacity of

22

Page 22 of 40
microorganisms to decolorize dyes [39]. Our study has demonstrated that laccase

played a very important role in the decolorization of synthetic dye and dye effluent by

Ganoderma sp. En3. The laccase gene lac-En3-1 has been cloned from Ganoderma

sp.En3 in our previous work [13]. In order to further improve the capacity of this

t
ip
fungus to decolorize the real textile wastewater and produce more active enzyme to

cr
degrade dyes faster, we will construct the genetically modified strain with

us
over-expression of laccase gene in Ganoderma sp.En3 in our future work. In addition,

the modified laccase with higher biodegradation capacity may be obtained through the

an
molecular biology techniques such as the enzyme directed evolution, random

mutagenesis, site-directed mutagenesis and gene recombination technique [36].

M
Thirdly, the different operation modes may be applied to enhance the efficacy of dye

wastewater treatment by fungus. Biodegradation of dyes by Ganoderma sp.En3 in

d
te

bioreactor mode may be developed for more efficient decolorization of recalcitrant

dyes and practical treatment of textile wastewater at the industrial scale. In addition,
p

decolorization processes using immobilised fungus may be more promising and more
ce

efficient than those with free cells because the immobilisation allows using the
Ac

microbial cells repeatedly and continuously [40]. Thus we will try to use the

immobilised fungus or immobilized microbial consortium to increase the

decolorization efficacy in the future study.

5. Conclusions

The white-rot fungus Ganoderma sp. En3 isolated from the forest of Tzu-chin

Mountain in China had a strong ability to decolorize and detoxify the sulfonated azo

23

Page 23 of 40
dye Reactive Orange 16 and simulated textile wastewater containing Reactive Orange

16. The white-rot fungus Ganoderma sp. En3 isolated by our laboratory had great

potential for the practical application in the field of environmental biotechnology such

as biodegradation of different industrial dyes and textile dye effluents.

t
ip
Acknowledgments

cr
This work was supported by the National Natural Sciences Foundation of China

us
(No. 31070069, 31370122 and 31170104), National High Technology Research and

Development Program of China (No. 2012AA022301D), The Fundamental Research

an
Funds for the Central Universities (HUST 2013TS082), Open Fund of State Key

Laboratory of Agricultural Microbiology.

M
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p
ce
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28

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Figure legends:

Fig.1 Chemical structure of the sulfonated azo dye Reactive Orange 16 studied in

this work.

t
ip
Fig.2 Decolorization of Reactive Orange 16 by Ganoderma sp. En3 in submerged

cr
cultures. (A). Decolorization of Reactive Orange 16 by whole fungal cultures in the

us
basal liquid medium containing glucose as the carbon source, yeast extract as the

nitrogen source. (B). Effect of different carbon sources on the decolorization of

an
Reactive Orange 16 (1000 mg/l). (C). Effect of different nitrogen sources on the

decolorization of Reactive Orange 16 (1000 mg/l). (D). Decolorization of higher

M
concentrations of Reactive Orange 16 (1000, 2000, 4000, 8000 mg/l) by using the

optimized carbon (glycerol) and nitrogen source (peptone) simultaneously.

d
te

Fig.3 Decolorization of simulated textile wastewater containing Reactive Orange

p

16 (STWRO16-I) by Ganoderma sp. En3 in submerged cultures.

ce
Ac

Fig.4 Decolorization of real textile wastewater in submerged cultures of

Ganoderma sp. En3. (A). Time course of the decolorization efficiency. (B). Time

course of the extracellular laccase activity. (C). Time course of the COD (chemical

oxygen demand) of wastewater.

29

Page 29 of 40
Fig.5 In vitro decolorization of Reactive Orange 16 by crude enzyme produced by

Ganoderma sp. En3. (A). Decolorization of Reactive Orange 16 by the crude enzyme

prepared from the cultures of Ganoderma sp. En3 without addition of any redox

mediator. (B). Decolorization of higher concentrations of Reactive Orange 16 (500

t
ip
and 2000 mg/l) by crude enzyme prepared from the cultures of Ganoderma sp. En3

cr
with addition of a natural redox mediator-syringaldehyde (1 mM). (C). UV–visible

us
absorbance spectrum for 2000 mg/l Reactive Orange 16 after 30 hours of incubation

in the presence of 1 mM syringaldehyde. (D). Detection of laccase activity and

an
decolorization activity of crude enzyme from Ganoderma sp. En3 on Native PAGE.

Lane 1: gel stained with 1mM ABTS. Lane 2: gel incubated with Reactive Orange 16
M
(50 mg/l) and 0.5 mM syringaldehyde for decolorization showing single band of

decolorized zone. Lane 3: gel incubated with Reactive Orange 16 (100 mg/l) and 0.5
d
te

mM syringaldehyde for decolorization showing single band of decolorized zone.

p

Fig.6 In vitro decolorization of simulated textile wastewater containing Reactive

ce

Orange 16 (STWRO16-I) by crude enzyme produced by Ganoderma sp. En3. (A).

Ac

Decolorization of STWRO16-I (final concentration: 40%) by crude enzyme prepared

from the cultures of Ganoderma sp. En3. (B). UV–visible absorbance spectrum for

20% STWRO16-I after 30 hours of incubation in the presence of 1 mM

syringaldehyde.

30

Page 30 of 40
Highlights
Ganoderma sp.En3 had a strong ability to decolorize Reactive Orange 16 and its simulated textile
wastewater
Decolorization of Reactive Orange 16 and its simulated dye effluent resulted in the decrease of
phytotoxicity.
Ganoderma sp.En3 could tolerate and decolorize high concentration of Reactive Orange 16.
Laccase played an important role in the decolorization of Reactive Orange 16 by Ganoderma

t
sp.En3.

ip
cr
us
an
M
d
p te
ce
Ac

32

Page 31 of 40
Figure(s)

Fig.1

t
ip
cr
us
an
M
ed
pt
ce
Ac

Page 32 of 40
Figure(s)

Fig.2

100 Reactive orange 16

90 (100mg/l)
80
Decolorization (%)

Reactive orange 16
70 (1000mg/l)

t
60

ip
50
40

cr
30
20
10

us
0
0 6 24 48 96

an
Time (hour)

B
M
4 hour
100
8 hour
ed

90
12 hour
80 24 hour
Decolorization (%)

70 48 hour
pt

60 96 hour
50
ce

40
30
20
Ac

10
0
Glucose Fructose Sucrose Starch Glycerol

Page 33 of 40
C

4 hour
100
90 8 hour
Decolorization (%)

80 12 hour
70 24 hour
60 48 hour
50 96 hour
40

t
30

ip
20
10

cr
0

e
t

te

a
e

e
c

at

re
in

on
tra

ha

us
rtr
ag

pt
lp
ex

ta
ar

Pe
su
t

sp

m
as

iu
A
Ye

iu

on
L-

on

an
m

m
m

A
A

M
D

Reactive orange 16
100
ed

(1000mg/l)
90 Reactive orange 16
80 (2000mg/l)
Decolorization (%)

pt

70 Reactive orange 16
(4000mg/l)
60
Reactive orange 16
ce

50 (8000mg/l)
40
30
Ac

20
10
0
0 4 8 12 24 48 96
Time (hour)

Page 34 of 40
Figure(s)

Fig.3

100 STWRO16-I（ 30% ）

90
80 STWRO16-I（ 60% ）
Decolorization (%)

70
60

t
50

ip
40
30

cr
20
10

us
0
0 2 4 6 8 10
Time (day) an
M
ed
pt
ce
Ac

Page 35 of 40
Figure(s)

Fig.4

100
90
Decolorization (%)

80
70

t
60

ip
50
40

cr
30
20
10

us
0
0 1 2 3 4 5 6 7 8
an
Time (day)

B
M
50
45
ed
Laccase activity (U/L)

40
35
pt

30
25
20
ce

15
10
Ac

5
0
0 1 2 3 4 5 6 7 8
Time (day)

Page 36 of 40
C

25000

20000
COD (mg/L)

15000

t
10000

ip
5000

cr
0
0 1 2 3 4 5 6 7 8

us
Time (day)

an
M
ed
pt
ce
Ac

Page 37 of 40
Figure(s)

Fig.5

Reactive orange 16
70 (50mg/l)
60 Reactive orange 16
Decolorization (%)

(100mg/l)
50 Reactive orange 16

t
(200mg/l)

ip
40 Reactive orange 16
(500mg/l)
30

cr
20

us
10
0
0 6 24 30
Time (hour)
an
M
B
ed

100 Reactive orange 16

(500mg/l)+1 mM
90 syringaldehyde
80
pt

Reactive orange 16
Decolorization (%)

70 (2000mg/l)+1 mM
syringaldehyde
ce

60
50
40
Ac

30
20
10
0
0 0.5 1 2 6 24 30
Time (hour)

Page 38 of 40
C

2.5 0 hour

0.5 hour
2
1 hour
Absorbance

1.5
24 hour

t
30 hour

ip
1

cr
0.5

us
350 370 390 410 430 450 470 490 510 530 550 570 590
Wavelength

an
M
D
ed
pt
ce
Ac

Page 39 of 40
Figure(s)

Fig.6

90 STWRO16-I（ 40% ）

80
STWRO16-I（ 40% )+1 mM
70 syringaldehyde
Decolorization (%)

t
60

ip
50

40

cr
30

us
20

10

0
0 6 24
an 30
Time (hour)
M
B
ed

0 hour
3
3 hour
2.5
6 hour
pt

24 hour
Absorbance

2
ce

30 hour
1.5

1
Ac

0.5

0
410 430 450 470 490 510 530 550 570 590
Weavelength (nm)

Page 40 of 40