Identiﬁcation of naphthalene metabolism by white rot...

ORIGINAL PAPER

Identification of naphthalene metabolism by whiterot fungus Pleurotus eryngii

Tony Hadibarata • Zee Chuang Teh • Rubiyatno •

Meor Mohd Fikri Ahmad Zubir • Ameer Badr Khudhair •

Abdull Rahim Mohd Yusoff • Mohd Razman Salim •

Topik Hidayat

Received: 24 October 2012 / Accepted: 5 January 2013 / Published online: 19 January 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract The use of biomaterials or microorganisms in

PAHs degradation had presented an eye-catching perfor-

mance. Pleurotus eryngii is a white rot fungus, which is

easily isolated from the decayed woods in the tropical rain

forest, used to determine the capability to utilize naphtha-

lene, a two-ring polycyclic aromatic hydrocarbon as source

of carbon and energy. In the meantime, biotransformation

of naphthalene to intermediates and other by-products

during degradation was investigated in this study. Pleurotus

eryngii had been incubated in liquid medium formulated

with naphthalene for 14 days. The presence of metabolites

of naphthalene suggests that Pleurotus eryngii begin the

ring cleavage by dioxygenation on C1 and C4 position to

give 1,4-naphthaquinone. 1,4-Naphthaquinone was further

degraded to benzoic acid, where the proposed terepthalic

acid is absent in the cultured extract. Further degradation of

benzoic acid by Pleurotus eryngii shows the existence of

catechol as a result of the combination of decarboxylation

and hydroxylation process. Unfortunately, phthalic acid

was not detected in this study. Several enzymes, including

manganese peroxidase, lignin peroxidase, laccase, 1,2-

dioxygenase and 2,3-dioxygenase are enzymes responsible

for naphthalene degradation. Reduction of naphthalene and

the presence of metabolites in liquid medium showed the

ability of Pleurotus eryngii to utilize naphthalene as carbon

source instead of a limited glucose amount.

Keywords Pleurotus eryngii � Naphthalene �1,4-Naphthaquinone � Laccase � Lignin peroxidase

Introduction

Environmental pollutions caused by aromatic xenobiotic,

such as polyaromatic hydrocarbons (PAHs) and synthetic

dyes, have become a serious global issue and threat to

human, wildlife, and marine life [1]. PAHs are formed as a

result of different activities, including incomplete com-

bustion of organic matter, automobile exhaust, domestic

matter and other activities [2]. Persistent organic pollutants

are distributed worldwide which can easily be seen or used

in our daily activities. PAHs are toxic chemicals possessing

high-bioaccumulation ability [3] in the food chain and food

web, and are persistent and resistant to environmental

degradation through chemical, biological and photolytic

processes [4]. They are also prone to long range transport

and may adversely affect the human health [5, 6]. The

Unites States Environmental Protection Agency (USEPA)

has recognized 16 PAHs as priority pollutants including

naphthalene [7]. Numerous approaches have been devel-

oped to eliminate and remove PAHs from environment,

including volatilization, photo-oxidation, chemical oxida-

tion, absorption and biodegradation [8, 9].

T. Hadibarata � Z. C. Teh (&) � Rubiyatno �M. M. F. A. Zubir � A. B. Khudhair �A. R. M. Yusoff � M. R. Salim

Faculty of Civil Engineering, Institute of Environmental

and Water Resource Management, Universiti Teknologi

Malaysia, 81310 Skudai, Johor, Malaysia

e-mail: [email protected]

T. Hadibarata

e-mail: [email protected]

A. R. M. Yusoff

Department of Chemistry, Faculty of Science, Universiti

Teknologi Malaysia, 81310 Johor Bahru, Johor, Malaysia

T. Hidayat

Department of Biology Education, Faculty of Mathematic

and Natural Science, University of Education (UPI),

Jalan Dr. Setiabudhi No. 229, 40154 Bandung, Indonesia

123

Bioprocess Biosyst Eng (2013) 36:1455–1461

DOI 10.1007/s00449-013-0884-8

Naphthalene is a bicyclic aromatic hydrocarbon that

possesses all PAH properties. Naphthalene has low water

solubility [10]. Naphthalene is found naturally in crude oil.

It is a major component of the coal and tar-based industries

and it has been detected in soil, oil contaminated sediments

and in both industrial and urban waste water [11]. Since, it

is known as highly toxic xenobiotic compound, various

studies have been established to remove it from soil or water

environment, including using of microbes or bacteria, either

aerobic or anaerobic biodegradation pathway [12–14].

Pleurotus eryngii belong to genus Pleurotus and are the

largest edible species in oyster mushroom genus. It is an

interesting white rot fungus species which was originally

isolated from our laboratory due to its dye decolorization

ability. Dyes tested on Pleurotus eryngii include azo,

anthraquinone and triphenylmethanes, where Pleurotus

eryngii are able to degrade and utilize dyes as carbon and

energy source for growth [15]. Generally, carbon and nitrogen

are highly needed by fungi for growth and reproduction

[16, 17]. When simple carbon or nitrogen sources are not

available, fungi will secrete an enzyme system which is able to

degrade a polymer to simpler molecules [18]. White-rot fungi

have been reported to be able to secrete ligninolytic enzymes,

which are lignin peroxidase (LiP), manganese peroxidase

(MnP) and laccase that are associated with ligninolyric

activities [19]. These enzymes possess ability in pollutants

degradation, including PAHs [20]. In order to isolate Pleurotus

eryngii, it was grown in a temperature range of 25–30 �C,

where 25 �C was optimum temperature for mycelial growth

for Pleurotus strains [21]. The current study was to charac-

terize the naphthalene metabolites produced by Pleurotus

eryngii under thermophilic condition had been carried out by

using UV–Vis spectrophotometer, model DR 5000 and gas

chromatography mass spectrometry (GC–MS) analysis.

In this study, white rot fungus Pleurotus eryngii had been

used for naphthalene degradation purpose. Due to high-

operational cost of traditional physical chemistry treatment

methods, such as composting and biosparging, application of

fungus for PAHs degradation would seem as an economic

solution. The objective of the study was to identify the

metabolites of naphthalene produced during naphthalene

degradation by Pleurotus eryngii treatment. With the aid of

analytical instruments such as GS–MS, metabolites or by-

products produced from naphthalene degradation are able to

be detected.

Materials and methods

Microorganism and growth conditions

Pleurotus eryngii isolated from the tropical rain forest in

Indonesia was used for naphthalene degradation in this

study. The macroscopic and morphological characteriza-

tions were performed on petri dishes with PDA and malt

extract agar (MEA), respectively, as described in the pre-

vious study [15]. Pleurotus eryngii culture was maintained

by using 2 % (w/v) malt extract, 2 % (w/v) glucose, 0.1 %

(w/v) polypeptone and 1.5 % (w/v) agar in a plastic petri

dish at 4 �C prior to use. Pleurotus eryngii had been

selected based on its ability to grow on 20 mL MEA

containing naphthalene which dissolved in dim-

ethylformamyde (DMF), 1 % Tween 80 and 300 mg/L

benomill. Benomill was added to inhibit bacterial growth.

After inoculation had been done, Pleurotus eryngii had

been incubated at room temperature during a period of

2 weeks. After 2 weeks, naphthalene degrading fungus had

been selected based on its ability to grow on naphthalene

containing media. A single colony will be transferred to

naphthalene-containing mineral liquid media that con-

tained 20 mL of mineral salt broth (MSB), containing (in

g/L distilled water): glucose (10), KH3PO4 (2),

MgSO4.7H2O (0.5), CaCI2.2H2O (0.1), ammonium tartrate

(0.2) and trace element (10 mL) [22]. Growing naphtha-

lene-degrading fungus on MEA at 25 �C for 1 week and

subsequently inoculated it into MSB medium. The cultures

were shaken at 120 rpm at 35 �C for 14 days. At the

meantime, control experiments have been carried out using

autoclaved Pleurotus eryngii and all analysis will be done

as same as sample.

Morphologic and molecular characterization

In order to observe microscopic and macroscopic

behavior of Pleurotus eryngii, PDA and MEA were

prepared in petri dishes, respectively. Molecular identi-

fication of Pleurotus eryngii was carried out via 18S

rRNA. Expected DNA genome was amplified through

polymerase chain reaction (PCR) process and initiated

by universal primers, NS1 and NS8. Important compo-

nents used in PCR include MgCI2, buffer PCR, primers,

heat stable Taq polymerase, dNTPs mix and DNA

template. PCR had been carried out through a series of

thermal cycling, consisting of cycles of heating and

cooling. The process began with 1 cycle at 94 �C for

3 min, followed by 25 cycles at 94 �C for 30 s, 50 �C

for 30 s and 72 �C for 2 min. This amplification process

ended with 1 cycle at 72 �C for 10 min. After the gene

had been amplified, the products were cloned into

pGEM-T Easy (Promega) before sending them to 1st

BASE Laboratory Sdn. Bhd. Malaysia for sequencing

purpose. The resulting DNA sequence was read and

edited by BIOEDIT. After the gene had been edited and

sequenced, it was further compared with known gene

sequences from NCBI GenBank database. Phylogenetic

analysis was conducted.

1456 Bioprocess Biosyst Eng (2013) 36:1455–1461

123

Chemical

Organic solvents used in the experiment were purchased

from New Zealand manufacturer, Qrec. While, the main

compound for this study, naphthalene and reference com-

pounds ([98 % pure) for metabolite identification purpose

were obtained from Sigma-Aldrich, supplier from Detroit,

USA. Malt extract and polypeptone were supplied by Difco

(Detroit, USA). Merck from Darmstadt, Germany is the

main supplier for thin layer chromatography (TLC) alu-

minium sheets (Silica gel 60 F264, 20 cm 9 20 cm).

Extraction and analysis of metabolites

Once fungus screening process is done, flasks with

obvious fungi growth have been selected for further

extraction and analysis process. The flasks which con-

tained liquid medium and fungus bodies were mixed and

blended with ethyl acetate and acidified with 1.0 M HCI

[7]. The residue (fungal bodies) was separated from fil-

trate (liquid medium) by filtration. Extraction will be

carried out by mixing of three equal volumes of ethyl

acetate with supernatant. Next, the residual extracts were

dried under reduced pressure at room temperature using

anhydrous sodium sulfate and after that metabolites of

naphthalene’s degradation in culture extracts were deter-

mined using TLC on silica gel (thickness 0.25 mm).

Separation by using TLC technique will be carried out in

two phases, short phase and long phase, only long phase

had been done in this study. Short-term analysis utilize

solvent system with a combination of hexane: chloroform

in the ratio of 30:10 (v/v) and dichloromethane: ethyl

acetate with ratio of 10:30 (v/v) will be used as solvent

system for long-term analysis. The location of compounds

or metabolites on TLC plates were detected using UV

light, while the Rf value of each compound will be

compared with known suspected compounds or metabo-

lites. Extracts from supernatant portion will be determined

using DR 5000 UV–Vis spectrophotometer. Once TLC

and UV–Vis analyses are obtained, the metabolites will

be re-confirmed again with GC–MS.

Naphthalene, carboxylic acid derivatives and aromatic

hydroxyl groups were analyzed and determined using

GC–MS. Sample preparation was done by mixing small

portion of the extract with solvent system of N,O-bis-tri-

methylsilyl acetaminde (40 %), pyridine (40 %) and tri-

methylchlorosilane (20 %) at 80 �C for 10 min. Small drop

of sample will be injected into a TC-1 capillary column

(30 m 9 0.25 mm) ID 0.25 lm using a temperature gra-

dient of 60 �C for 2 min, raised gradually to 300 �C at

25 �C/min, and maintained at 300 �C for 6 min [7]. Injector

and interface temperatures were 260 �C. GC–MS used to

determine naphthalene degradation pathway is Shimadzu

QP-5050, where the conditions include detector at 1.3 eV,

scan intervals of 1 s, and a mass range of 50–500. Suspected

compound will be compared with authentic known standard

in term of retention time and identified using Wiley 275L

MS data base.

Results and discussion

Isolation and identification of fungi

Pleurotus eryngii, largest edible oyster mushroom species,

isolated from most hardwoods. This strain produces white

spores, possesses white thick stem, with grey–brown cap.

Physically, fresh Pleurotus eryngii had a diameter of

2–4.5 cm, length of 6–15 cm. Pleurotus eryngii is becom-

ing popular due to its high fruitbodies quality, strong tex-

ture, delicious aroma and good preservation. Based on these

macroscopic, morphological and phylogenetic position of

Tetrapisispora sp FJ153120.1

Armillaria spp AF454743.1

Sample F032

Pleurotus eryngii FJ379286.1

Pichia spartinae FJ153139.1

Candida parapsilosis AY055856.

Saccharomyces spp FM178249.1

100

48

97

100

Fig. 1 Phylogenetic tree of

Pleurotus species

Bioprocess Biosyst Eng (2013) 36:1455–1461 1457

123

this sample, eryngii is classified and belongs to the genus of

Pleurotus (Fig. 1).

UV, TLC and GC–MS analyses for naphthalene’s

metabolites

UV–Vis spectra, TLC and GC–MS have been used for

naphthalene degradation pathway analyses. Those instru-

ments had been used for metabolites determination which

results from biodegradation by Pleurotus eryngii. GC–MS

identification had figured out three metabolites formed

from degradation of naphthalene, stated in Table 1, which

are 1,4-naphthaquinone, benzo acid-TMS derivative and

catechol-TMS derivative that had been confirmed with a

standard based on retention time. The authentic standards

were chosen according to our proposed naphthalene

degradation pathway. Analysis of TLC using ethyl ace-

tates showed the presence of three metabolites. Metabolite

I, having Rf value 0.65, showed UV spectrum with kmax

of 437, 463 and 482 (Fig. 3a), similar to that of 1,4-

naphthaquinone. Spectrum of compound I (m/z 208, M?)

which GC retention time recorded at 8.6 min is shown in

Fig. 4a. The GC retention time at 8.6 min, having MS

properties of the M? at m/z 158, with significant fragment

ions at m/z 102 and 130 (M?28), formed due to the

sequential losses of –CO, were identical to authentic

1,4-naphthaquinone.

Naphthalene degradation by white rot fungus resembles

the degradation pathway which had been discussed earlier

[7] via 1,4-naphthaquinone to phthalic acid. However,

terepthalic acid and phthalic acid were unable to be iden-

tified in the culture extract. GC chromatograph in Fig. 2

showed that three metabolites exist after naphthalene had

been degraded by Pleurotus eryngii for 14 days. The

fragmentation pattern and retention time of compound II

and III had been compared with known compounds and

proved matching with authentic benzoic acid-TMS deriv-

ative and catechol-TMS derivative, respectively. However,

naphthalene peak still exists after 14 incubation days.

Naphthalene was not fully degraded may be due to insuf-

ficient incubation.

TLC analysis of ethyl acetate-extractable metabolites of

naphthalene showed degradation and reduction of naph-

thalene, with the presence of metabolite. Metabolite II

(benzoic acid-TMS derivative) having Rf value of 0.28.

Analysis using UV spectrophotometer showed that

metabolite II had the same UV characteristics (kmax 220

and 273 nm) with authentic benzoic acid standard

(Fig. 3b). Identification of compound II using mass spec-

trum found that TMS compound exists with GC retention

time at 5.8 min (Fig. 4b). Mass spectrum analysis for

benzoic acid-TMS derivative showed that metabolite II had

a molecular ion (M?) at m/z 194 for TMS-derivatives and

base peak (significant fragment ions) at m/z 179, due to lose

Table 1 Gas chromatography retention times and mass spectral data of naphthalene metabolites formed by Pleurotus eryngii

No Metabolites Retention

time (min)

Prominent fragment ion (m/z) (% relative intensity)

I 1,4-Naphthaquinone

(confirmed with standard)

8.6 158 (100, M?), 64 (31), 65 (34), 74 (34), 75 (46), 76 (84), 86 (33), 101 (24), 102 (95),

103 (83), 104 (86), 130 (51, M? -28), 131 (48), 157 (48), 159 (57)

II Benzoic acid-TMS derivatives


5.8 194 (25, M?), 60 (38), 61 (27), 73 (26), 76 (47), 77 (69), 78 (59), 105 (95), 120 (34),

135 (51), 145 (28), 160 (27), 178 (62), 179 (100, M? -15), 180 (51)

III Catechol-TMS derivative


6.6 254 (9, M?), 59 (20), 73 (100), 74 (21), 91 (19), 105 (22), 115 (23), 136 (36), 151 (38),

166 (41), 239 (39, M? -15), 240 (18), 254 (6), 255 (5), 256 (3)

Fig. 2 GC chromatogram of

naphthalene metabolites


123

of methyl group (–CH3) to form the most stable fragment

ion. Other expected fragment ions were recorded at 135,

105 and 73 for metabolite II.

Metabolite III, catechol-TMS derivative (catechol also

known as pyrocatechol or 1,2-dihydroxybenzene) was

found in crude extract, gave an UV spectrum with kmax at

278 nm [13], which is compatible with kmax of authentic

catechol standard (Fig. 3c). TLC analysis determines that

metabolite III possesses Rf value similar to catechol stan-

dard, giving a value of 0.39. Mass spectrum of compound

III eluted at retention time of 6.6 (Fig. 4c), recorded

molecular ion at m/z 254, producing significant ions at

m/z 73 and 239, which are the most stable fragment ions

produced from metabolite III in mass spectrum.

Degradation of naphthalene by Pleurotus eryngii was a

complicated process which involved enzyme systems.

During naphthalene degradation, numerous catabolic

pathways occurred and intermediate products were pro-

duced and consumed. Pleurotus eryngii was cultured in a

liquid medium with naphthalene as source of carbon and

energy. Ability of Pleurotus eryngii to utilize the naph-

thalene as nutrient source depends on the ability of their

enzyme system to cleave the persistent naphthalene struc-

ture to smaller compounds. Based on the identification of

various metabolites produced during aromatic ring oxida-

tion and cleavage, naphthalene degradation pathway by

Pleurotus eryngii was explored.

Naphthalene degradation gave 1,4-naphthaquinone

suggesting the lignolytic activities in Pleurotus eryngii.

This pathway differs from a typical naphthalene degrada-

tion showed by bacteria, which will cleave naphthalene at

carbon number 1 and 2 (C1 and C2) to produce 1,2-dihy-

dro-1,2-dihydroxynaphthalene and 1,2-dihydroxynaphtha-

lene [13, 23]. Typical naphthalene degradation pathway

followed the sequences in Fig. 5, where dioxygenation of

naphthalene yield 1,4-naphthaquinone. Further degradation

by Pleurotus eryngii produced benzoic acid and followed

by catechol, which produced as combination of decarbox-

ylation and hydroxylation process. Unfortunately, phthalic

acid and hexa-2,4-dienedioic acid were not detected as

shown in our proposed degradation pathway.

Previous research had discussed naphthalene degrada-

tion and its metabolites by another fungus species, Armil-

laria sp. F022 in 2012. The present study showed simpler

degradation pathway and less metabolites produced com-

pared to Armillaria sp. F022. Naphthalene degradation

produced 1,4-naphthaquinone, benzoic acid and catechol

showed simpler process. This may be due to the enzyme

system present in fungus secretion in liquid medium.

Conclusions

Pleurotus eryngii is a white rot fungus that can utilize

polycyclic aromatic hydrocarbon, a nutrient source by

secreting enzyme system which is able to cleave compli-

cated aromatic compounds into smaller molecules for its

carbon and energy source. 1,4-Naphthaquinone, benzoic

Fig. 3 UV spectra of a 1,4-naphthaquinone, b benzoic acid and

c catechol. Solid lines standard, dashed lines for metabolite


123

acid and catechol were detected and identified as metabo-

lites produced as a result of naphthalene degradation by

Pleurotus eryngii. Reduction in naphthalene concentration

and the presence of metabolites showed that the ability of

Pleurotus eryngii to utilize naphthalene as carbon source

instead of limited glucose in liquid medium. Naphthalene

intermediate products were toxic and harmful to micro

flora and might degrade itself when accumulated to a cer-

tain level. Hence, choosing suitable PAHs, degraders

should be taken into serious consideration, since incom-

plete degradation or by-products from degradation would

be even more toxic than the parent compounds. Efficiency

of Pleurotus eryngii in naphthalene degradation saw it as a

suitable candidate in oil contaminated soils.

Acknowledgments A part of this project was financially supported

by Universiti Teknologi Malaysia (RUG Vote QJ1.3000.2522.02H65)

and Ministry of High Education, Malaysia (ERGS Vote R.J130000.

7822.4L053).

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Fig. 4 Mass spectral profiles

of naphthalene metabolites:

a 1,4-naphthaquinone;

b benzoic acid-TMS derivatives

and c catechol-TMS derivatives

Fig. 5 Proposed pathway for

the degradation of naphthalene

by Pleurotus eryngii. The

metabolites in brackets had not

been identified in our culture

extract


123

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