Degradation of Polycyclic Aromatic Hydrocarbons by ... - Springer Link

Curr Microbiol (2010) 60:336–342 DOI 10.1007/s00284-009-9546-0

Degradation of Polycyclic Aromatic Hydrocarbons by Crude Extracts from Spent Mushroom Substrate and its Possible Mechanisms Xuanzhen Li • Xiangui Lin • Jing Zhang Yucheng Wu • Rui Yin • Youzhi Feng • Yong Wang

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Received: 5 August 2009 / Accepted: 5 November 2009 / Published online: 19 November 2009 Ó Springer Science+Business Media, LLC 2009

Abstract Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by pure laccase has been reported, but the high cost limited its application in environmental bioremediation. Here, we reported a study about PAHs degradation by crude extracts (CEs) containing laccase, which were obtained by extracting four spent mushroom (Agaricus bisporus, Pleurotus eryngii, Pleurotus ostreatus, and Coprinus comatus) substrates. The results showed that anthracene, benzo[a]pyrene, and benzo[a]anthracene were top three degradable PAHs by CEs while naphthalene was most recalcitrant. The PAHs oxidation was enhanced in the presence of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Laccase included in CE might play a major role in PAHs degradation. The maximum degradation rate of anthracene and benzo[a]pyrene was observed by using crude extracts from P. eryngii while the highest laccase activities were found in crude extracts from A. bisporus, moreover, crude extracts from P. eryngii, which contained less laccase activities, degraded more anthracene and benzo[a]pyrene than pure laccase with higher laccase activities. The lack of correlation between laccase activity and PAHs degradation rate indicated that other factors might also influence the PAHs degradation.

X. Li X. Lin (&) J. Zhang Y. Wu R. Yin Y. Feng Y. Wang State Key Laboratory of Soil and Sustainable Agriculture, CAS Key Laboratory of Soil Environment and Pollution Remediation, Joint Open Laboratory of Soil and the Environment, Hongkong Baptist University & Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China e-mail: [email protected] X. Li Graduate University of Chinese Academy of Sciences, Beijing 100049, China

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Boiled CEs were added to determine the effect on PAHs degradation by laccase. The results showed that all four boiled CEs had improved the PAHs oxidation. The maximum improvement was observed by adding CEs from P. eryngii. It suggested that some mediators indeed existed in CEs and CEs from P. eryngii contained most. As a result, CEs from P. eryngii has the most application potential in PAHs bioremediation.

Introduction Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds consisting of two or more fused benzene rings that arranged in various structures. For their high toxicity and recalcitrance, 16 PAHs were recognized as priority pollutant by the US Environmental Protection Agency (EPA) [1]. Numerous studies indicated that low molecular weight PAHs(LMW PAHs) with one, two, or three rings are acutely toxic and high molecular weight (HMW PAHs) with more than three rings are genotoxic [2]. Fungi have advantage in biodegrading HMW PAHs compared with bacteria [3], and the mechanism can be concluded to two ways, namely, path of cytochrome P450 monoxygenase enzyme system and ligninolytic enzyme system [4]. Laccase is extracellular copper-containing polyphenol oxidases, and it is one of most important members in ligninolytic enzyme system [5, 6]. Laccase was first discovered in the Japanese lacquer tree Rhus vernicifera, and latter, it has been also found in most higher fungi, including cultivated edible fungi [7]. Unlike other oxidized enzymes which need additives to catalyze (e.g., hydrogen peroxide for peroxidase), dissolved oxygen is the only requirement for laccase-catalyzed reaction [8]. It can catalyze the reduction

X. Li et al.: Degradation of Polycyclic Aromatic Hydrocarbons

of one dioxygen molecule to two molecules of water, simultaneously oxidizing aromatic substrates [9]; therefore, laccase was considered to be environmental friendly and was nominated ‘‘blue enzymes for green chemistry’’ [10]. Laccase can degrade many kinds of xenobiotic organic compounds, such as phenols [11, 12], chlorophenols [13], PAHs [5, 14–16], and so on. With mediator, which acted as ‘‘electron shuttle’’ between enzyme and substrates [17], such as 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT), enzymatic oxidation efficiency could be improved greatly [16, 17]. Besides, some natural mediator, such as 4-hydroxybenzoic acid and 4-hydroxybenzyl alcohol, which was secreted extracellular by fungi, also play a positive role in fungi biodegradation of substrates [17]. Partly because the natural mediators present, no strong correlation was found between the extracellular enzymes activity and the biodegradation of aromatic xenobiotic compounds by laccase-producing fungi [5, 17]. Patrick had further proved that there indeed existed some compounds B10 kDa in size, secreted by Trametes versicolor, mediated the anthracene oxidation [5]. The mechanism of laccase-mediator system in xenobiotic degradation was expatiated elaborately by Morozova [7]. More and more attention was attracted to potential of laccase in pollutants biodegradation [8, 16, 18, 19]. However, the cost of the production and purification of laccase was so high that it was impossible to apply laccase to large scale remediation. Approach to produce cheap laccase must be established. Extracting laccase from spent mushroom substrate (SMS) would be a good solution. SMS is a byproduct of mushroom industry and millions tons of SMS were discarded each year in China. Large amount of laccase were produced during growth of mycelium on substrate. After harvest, a considerable amount of laccase was left in the SMS. SMS would be a potential source of ligninolytic enzymes such as laccase [20]. Some reports had introduced the recovery methods of laccase from SMS [21, 22]. SMS were already proved to be effective in organic pollutants bioremediation in some studies [20, 23]. In this study, the crude extracts (CEs) from SMS were extracted and their laccase activities were measured. The aims of this study were to determine the potential of CE in PAHs biodegradation, and further more, the effect of artificial and natural mediators on the PAHs oxidation by CE were explained.

Materials and Methods Chemicals and Materials The 15 PAHs were purchased from Supelco. Pure laccase (from Trametes versicolor) and ABTS were purchased from Sigma-Aldrich (Shanghai, China). Four SMS (A.

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bisporus, P. eryngii, P. ostreatus, and C. comatus) were obtained from Academy of Agriculture Science, Henan province. The mushroom was grown on corncob mixed with different amendments. Preparation of Crude Extracts The preparation of CE was produced according to D’Annibale’s methods [24]. 10 g of SMS was extracted with 50 ml Buffer A, which contained 0.1 M sodium acetate, 5 mM CaCl2, 0.05% Tween80, and 1% polyvinylpolypyrrolidone, on rotary shaker (180 rpm, 25°C) for 1 h. The aqueous suspensions were centrifuged (11,000g, 30 min) and the supernatants were used directly for the laccase activity assay. Assay of Enzymes Activity Laccase activity was determined by the oxidation of ABTS at 30°C. The 2 ml reaction mixture included 1.8 ml B&R buffer (0.1 M boracic acid, 0.1 M phosphoric acid, and 0.1 M acetic acid, pH adjusted to 5.0 with NaOH), 0.1 ml ABTS (20 mM), and 0.1 ml CE. The increase in absorbance at 420 nm was monitored with a spectrophotometer (model752, CANY, China) to calculate the laccase activity (e420 = 36,000 M-1 cm-1). The laccase activity was calculated by formula of DA*20*106/36000. DA was increment of absorbance per min when it was stable. One unit of laccase activity was defined as the amount of enzyme able to oxidize 1 lmol ABTS min-1. Lignin peroxidase (LiP) and manganese peroxidase (MnP) activities were determined by methods described by Ryu [25]. Oxidation of PAHs by Crude Extracts and Pure Laccase To determine the profile of degradable PAHs by CEs, the experiments were performed in 15 ml tubes with 4.5 ml CE and 0.5 ml acetonitrile containing 15 PAHs, and their final concentration was listed in Table 1. The effect of mediator on PAHs oxidation was also tested by adding ABTS to A. bisporus’ CE treatments, providing a concentration of 1 mM. Reaction tubes were closed tightly with screw cups and shaken violently by hand, and then incubated in the dark for 24 h (25°C). Another 5 ml acetonitrile was added to terminate the reaction. The screw caps were closed tightly, and the tubes were shaken again. After 1 h incubated, reaction mixture was centrifuged at 13,000g for 10 min, and 20 ll supernatant was analyzed by Ultra Fast Liquid Chromatograph system. Control samples were prepared by adding different deactivated CE (by boiling 30 min) instead of fresh CE. All treatments, including controls, were in triplicates.

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To compare the catalysis efficiency of CE and pure laccase, 15 PAHs were replaced by anthracene or benzo[a]pyrene with a final concentration of 1 mg l-1 in all treatments, 4 ml of different CE or buffer containing pure laccase (45 U) were added in treatments, respectively. The follow procedures were performed as described below. To determine the effect of artificial and natural mediators possibly present in CE on PAHs oxidation, a series of experiments were performed as described in Table 2. CE from different SMS, boiled for 30 min to inactivate all the enzymes in the extracts, were used as possible natural mediators. The final activity of reaction mixture was 2 U ml-1 and the concentration of anthracene or benzo[a]pyrene was 1 mg l-1.

Removal of Toxic Equivalency of Total Fifteen PAHs by Crude Extracts

Ultra Fast Liquid Chromatograph (UFLC) Determinations PAHs samples were analysis by Shimadzu UFLC-20 system. A reversed phase column C18 (3 mm 9 150 mm, particle size 2.2 lm), using a mobile phase with acetonitrile/water (gradient elution 20 min, at a constant flow rate 0.8 ml/min, 50°C), was used to separate PAHs. Statistical Analysis SPSS for Windows software was used for statistical analysis and one-Way ANOVA to determine difference between treatments at a significant level of 0.01 or 0.05.

Results and Discussion Laccase activities of Crude Extracts

Toxic equivalency factors (TEF) was introduced to evaluate the detoxification of PAHs by CE. The TE values of 15 PAHs were listed in Table 1 according to Nesbit [26]. The TEF removal rate of total 15 PAHs was calculated by the formula: Removal of TE ¼ P concentration Degradation rate TE value 15PAHs Final P 15PAHs Final concentration TE value Final concentration meant that PAHs concentration in the reaction mixture at the beginning of reaction.

Table 1 The initial concentration of PAHs in reaction mixture and their toxic equivalency (TE) values PAHs

Abbreviation Rings Concentration (lg l-1)

TE value

Naphthalene

Nap

2

1000

0.001

Acenaphthylene

Ace

3

2000

0.001

Fluorene

Flu

3

200

0.001

Phenanthrene

Phe

3

100

0.001

Anthracene

AnT

3

100

0.01

Fluoranthrene

FluA

4

200

0.001

Pyrene

Pyr

4

100

0.001

Benzo[a]anthracene

BaA

4

100

0.1

Chrysene

Chry

4

100

0.01

Benzo[b]fluoranthene

BbF

5

200

0.1

Benzo[k]fluoranthene Benzo[a]pyrene

BkF BaP

5 5

100 100

0.1 1

Dibenzo[a,h]anthrecene DBA

5

200

5

Benzo[g,h,i]perylene

6

200

0.01

6

100

0.1

BghiP

Indeno[1,2,3-cd]pyrene IP

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The extracellular enzymes produced by fungi, including LiP, MnP, and laccase have been reported to detoxification of xenobiotic compounds [27, 28]. The activities of three important ligninolytic enzymes in crude extracts from four mushroom SMS were determined, but only laccase activity was found. The activities of laccase in extracts of four SMS were determined and the highest laccase activity was found in extracts of A. bisporus, followed by P. eryngii, C. comatus, and P. ostreatus (Fig. 1). The great variation indicated that the extractable laccase of different SMS differed so much, and it might owe to the different laccaseproducing ability of edible mushroom. A. bisporus has been considered as a potent laccase-producing organism [29], and the SMS of A. bisporus has been used for laccase recovery [20, 21]. P. ostreatus was also a well-known laccase-producing mushroom and the SMS has been already reported to apply in bioremediation [30, 31], but, unfortunately, no considerable amount of laccase was found in P. ostreatus SMS in this study. Less was known about laccase of P. eryngii [32]and C. comatus; however, more laccase activity was observed in extract from those two mushroom than P. ostreatus (P \ 0.05). Generally, SMS of A. bisporus could be treated as one of the most potential source of laccase due to high laccase activity, moreover, CE from A. bisporus SMS was expected to be most potent in PAHs bioremediation. Potential of Crude Extracts and Pure Laccase in Oxidizing PAHs The degradation of 15 PAHs by CE was determined. All four CE could oxidize the some kinds of 15 PAHs (Table 3). The maximum total degradation rate of 15 PAHs


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Table 2 The experimental design to test the effect of potential mediators in CEs on enzymatic oxidationa Treatments

Boiled CE (ml)

Buffer A (ml)

Pure laccase (U)

ABTS (g)

Acetonitrile (ml)

PAH (lg)

Control

–

4.5

–

–

0.5

5

1

–

4.5

10

–

0.5

5

2

–

4.5

10

0.0055

0.5

5

3

A. bisporus, 4.5

–

10

–

0.5

5

4

P. eryngii, 4.5

–

10

–

0.5

5

5

P. ostreatus, 4.5

–

10

–

0.5

5

6

C. comatus, 4.5

–

10

–

0.5

5

a

Symbols: ‘‘–’’ indicated that it was not added

were produced by CE from P. eryngii SMS, followed by A. bisporus, P. ostreatus and C. comatus. In all treatments, anthracene and benzo[a]pyrene were more readily degradable than other 13 PAHs. Almost all the anthracene and benzo[a]pyrene were removed completely by CE from A. bisporus, P. eryngii and P. ostreatus SMS. Benzo[a]anthracene was the third transformable PAHs by four CE. Naphthalene, which was more hydrosoluble and could be metabolized by many microbes, was recalcitrant to the attack by CE. The degradable profile of PAH by four CE seem to be similar to those by pure laccase [13], and it suggested that laccase presence in CE might play an important role in PAHs degradation. CE from A. bisporus SMS was selected to test the effect of ABTS on PAHs enzymatic oxidation (Table 3). Removal of other 13 PAHs, except anthracene and benzo[a]pyrene, were all enhanced significantly (P \ 0.05) by the addition of ABTS. When compared with the treatment without ABTS, the biodegradation rate of Indeno[1,2,3cd]pyrene increased by 69.8% (P \ 0.05), which was enhanced most. For the naphthalene, which almost could not be degraded by CE individually, 14.3% of increment was observed (P \ 0.05). It was reported widely that

Laccase activity (Uml-1)

10

a

8 6 4 b

2 d

c

P. ostreatus

C. comatus

0 A. bisporus

P. eryngiu

Fig. 1 Laccase activities of CE from different SMSs. CE was obtained by extracting 10 g SMS by 50 ml Buffer A solution for 1 h (180 rpm, 25°C). Values were means of triplicates, and error bars stood for standard deviations. Means with the same letter are not significantly different (P [ 0.05)

benzo[a]pyrene and anthracene could be oxidized to a larger extent in the presence of ABTS [5, 7, 16]; however, in this study, benzo[a]pyrene and anthracene with final concentration of 100 lg l-1 were removed completely by CE from A. bisporus individually, the possible promotion to enzymatic catalysis by adding ABTS probably was not exhibited. The degradation rate increment might correlate to the number of rings of PAHs. The biodegradation rate increment of six rings PAHs were maximum (53.2–69.8%), followed by 29.6–42.0% for 5 rings, 2.8–22.7% for 4 rings, 3.6–15.4% for 3 rings and 14.3% for 2 rings. The data indicated that ABTS had positive effect on oxidation of 15 PAHs by CE, and the effect was greater for HMW PAHs than for LMW. ABTS is a well-known mediator in laccase catalysis [7]. The observed enhancement of PAHs degradation by CE in the presence of ABTS likely contributed to the mediation of ABTS to laccase catalysis. It suggested that laccase might be the major factor in PAHs degradation by CE. Bogan and Lamar expounded the mechanism of laccase catalysis by ionization potentials (IPs) which refer to the energy required to remove an electron and to form a cation radical, and the disappearance of PAHs was considered to have a strong correlation with the IPs [33]. The compounds with high IP were resistant to oxidized [14]. According to Majcherczyk, the IP values is 8.13 eV for naphthalene, 7.12 eV for benzo[a]pyrene, and 7.43 eV for anthracene, 7.44 eV for benzo[a]anthracene and laccase can oxidize PAHs which IP is below 7.45 eV [14]. The theory could be used to explain the results of this study, in which CE from SMS containing laccase could oxidize benzo[a]pyrene, anthracene, and benzo[a]anthracene but not naphthalene. Toxic equivalency factors (TEF) was introduced to evaluate the detoxification of PAHs by CE (Fig. 2). The treatment of CE from A. bisporus had removed 92.3 ± 5.1 and 54.6 ± 2.8% of TE with and without ABTS, which were more than other treatment extremely significantly (P \ 0.01). CE from P. ostreatus, P. eryngii and C. comatus had diminished the TE by 33.0 ± 1.5, 34.8 ± 1.4 and 34.4 ± 3.1%, respectively. The data indicated that all four

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Table 3 Oxidation 15 PAHs by CE from different SMSs PAHs

Oxidation of 15 PAHs by CE (% of control) A. bisporus

A. bisporusa

P. ostreatus

P. eryngii

C. comatus

Nap

3.5 ± 1.7b

17.8 ± 12.8a

0

0

0

Ace

43.6 ± 1.3c

53.8 ± 8.2b

40.2 ± 3.8c

68.5 ± 0.6a

24.3 ± 3.4d

Flu

31.3 ± 1.0cd

35.0 ± 12.5cd

53.5 ± 0.4b

78.5 ± 0.6a

25.4 ± 2.5d

Phe AnT

4.4 ± 0.3b 100.0 ± 0a

19.9 ± 16.4a 100.0 ± 0a

5.2 ± 5.6b

4.7 ± 1.5b

6.4 ± 0.4b

100 ± 0a

99.9 ± 0a

73.5 ± 0.4b

FluA

8.0 ± 6.4a

11.6 ± 12.9a

6.3 ± 3.9a

6.7 ± 0.3a

9.9 ± 3.1a

Pyr BaA

30.5 ± 0.8c 66.7 ± 0.2b

48.2 ± 10.7a 69.5 ± 6.6b

37.6 ± 2.7ab 79.2 ± 2.2a

33.4 ± 1.4b 76.6 ± 0.8ab

13.7 ± 2.7d 35.6 ± 4.2d

Chry

9.1 ± 0.9c

31.8 ± 14.1a

3.5 ± 1.6d

4.2 ± 1.4d

18.2 ± 3.9b

BbF

10.2 ± 1.4b

40.5 ± 12.8a

0

7.5 ± 0.1c

33.7 ± 2.5a

BkF BaP DBA

19.1 ± 1.2b 100.0 ± 0a 51.7 ± 8.4b

48.7 ± 10.8a 100.0 ± 0a 93.6 ± 6.2a

16.8 ± 2.3b

19.3 ± 1.1b

34.4 ± 5.6a

97.0 ± 1.4b

100.0 ± 0a

80.1 ± 3.7c

27.0 ± 2.1c

28.6 ± 1.4c

29.9 ± 5.1c

BghiP

24.8 ± 13.0b

77.9 ± 12.7a

23.0 ± 0.7c

22.8 ± 3.0c

21.3 ± 5.0c

IP

16.7 ± 26.5d

86.5 ± 7.4a

25.8 ± 4.3c

26.4 ± 0.4c

32.1 ± 2.9b

Total

31.4 ± 0.8c

47.4 ± 8.5a

28.9 ± 2.7c

42.1 ± 0.7b

21.3 ± 3.2d

The fianl fifteen PAHs concentration was present in Table 1. The reaction system was incubated in the dark for 24 h (25°C). Data are presented as mean ± S.D. Means in same row with the same letter are not significantly different (P [ 0.05) a

Indicated that 1 mM ABTS was added

CE could decrease the TE of PAHs remarkably. CE from A. bisporus was most excellent in detoxification and with the assistance of ABTS, the efficiency of detoxification was greatly enhanced. TEF was more and more used in assessment of hazards and risks of toxic chemicals, such as PAHs in environment [34–37]. However, it was reported that laccase only could cleavage the benzene rings and couldn’t induce the mineralization of PAHs completely [4], and the toxicity of intermediate metabolite of PAHs might be more than parent compound [2]. So, more information about toxicity of metabolites of enzymatic catalysis of PAHs must be known by other test. A higher concentration (1 mg l-1) was applied to investigate the potential of CE to remove anthracene and benzo[a]pyrene (Fig. 3). CE from P. eryngii removed the most of anthracene and benzo[a]pyrene (99.9 ± 0 and 87.5 ± 0.8%, respectively). However, CE from A. bisporus, which showed the strongest laccase activity, had only degraded anthracene and benzo[a]pyrene by 89.8 ± 0.6 and 48.6 ± 1.4%, which was less than P. eryngii (P \ 0.05). The oxidation rates of anthracene and benzo[a]pyrene by CE from P. ostreatus or C. comatus were 38.0 ± 0.1 and 31.0 ± 0.5 or 9.8 ± 0.2 and 9.3 ± 2.4%, respectively. The pure laccase, with a final laccase activity of 9 U ml-1, which was stronger than that of any CE, only degraded anthracene and benzo[a]pyrene by 74.7 ± 3.9 and 38.4 ± 3.5%. The results indicated that laccase activities in CE had less relation with PAHs biodegradation

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rates than expected, and it suggested that there existed other factors, besides laccase, participating in PAHs removal. Oxidative enzymes might be one reason. So far, three extracellular enzymes (LiP, MnP and laccase) were reported to have ability in PAH-biodegradation [8, 38]. On account of activity of LiP and MnP had not being found, the possibility that there existed some enzymes unknown, with the ability of PAHs biodegradation, was valid. Besides, some low molecular compounds, which were observed to be secreted by fungi and called ‘‘natural mediators’’, might also exist in the CE and play a role in laccase catalysis [5, 17]. Anyhow, CE from P. eryngii, instead of A. bisporus, was more efficient in PAHs biodegradation.

Fig. 2 Removal of toxic equivalency of total fifteen PAHs by CEs. ‘‘A. bisporus ? ABTS’’ indicated that ABTS was added to the reaction mixture. Values were means of triplicates, and error bars stood for standard deviations. Means with the same letter are not significantly different (P [ 0.05)


Fig. 3 The oxidation of PAHs by pure laccase and CE from different SMSs. The laccase activity in pure laccase treatment was 9 U ml-1. Both benzo[a]pyrene and anthracene concentration was 1 mg l-1. The reaction system was incubated in the dark for 24 h (25°C). Values were means of triplicates, and error bars stood for standard deviations. Means with the same letter are not significantly different (P [ 0.05)

Oxidation of Anthracene and Benzo[a]pyrene by Laccase with ABTS and Possible Natural Mediators To determine if some natural mediators existed in CE and were responsible for the abnormity of PAHs degradation, CE were boiled to destroy all the enzymes and used as possible natural mediator. ABTS and possible natural mediators were added to reaction mixtures, respectively, to investigate their effects on oxidation of anthracene and benzo[a]pyrene (Fig. 4). 2 U ml-1 of pure laccase could only degrade anthracene and benzo[a]pyrene by 2.1 ± 0.9 and 1.2 ± 0.6%, and in the presence of ABTS, the degradation rate increased to 7.1 ± 0.5 and 27.1 ± 1.8%. Besides, all four boiled CE improved the PAHs oxidation rate significantly (P \ 0.05). Addition of boiled CE from P. eryngii was most effective, and 90.5 ± 8.9% of anthracene and 59.8 ± 27.9% of benzo[a]pyrene were removed. The effect

341

of addition of boiled CE from A. bisporus was least and only 4.6 ± 0.1% of anthracene and 5.3 ± 1.9% of benzo[a]pyrene were transformed. When boiled CE from P. ostreatus or C. comatus were in present, the degradation rate of anthracene and benzo[a]pyrene were 39.4 ± 1.1 and 27.4 ± 3.7 or 33.6 ± 0.2 and 23.1 ± 0.4%, respectively. The data indicated that some chemicals, indeed existed in the CE and influenced the PAHs oxidation, on count of CE had being inactivated. The equivalence amount of natural mediator varied from one CE to another and CE from P. eryngii contained the most. These might partially explain why CE from P. eryngii removed more PAHs than A. bisporus and why laccase activity of CE individual was not parallel with the ability to degrade PAHs. The interaction of natural mediators and laccase activities, probably could account for the degradation of PAHs better. As a result, for no natural mediators contained, pure laccase might be less effective in PAHs removal than CE. However, if the natural mediators were secreted by mycelium of mushroom or from decomposition of lignin substrate were remain undetermined.

Conclusions The activity of three important ligninolytic enzymes in crude extracts from four mushroom SMS was determined, but only laccase activity was found. Each CE had the ability of PAHs degradation and the degradation could be improved in the presence of ABTS. Anthracene, benzo[a]pyrene, and benzo[a]anthracene were three more degradable PAHs while naphthalene was most undegradable by CE. The degradation profile was similar to that of pure laccase; therefore, it was suggested that laccase catalysis might be the major factor of PAHs degradation. However, partly for natural mediators presenting in CE, there was no correlation between the laccase activity and PAHs degradation rate, moreover, CE were more efficient in PAHs-biodegradation than pure laccase with equal laccase activity. The study could not predicate if there existed other oxidized enzymes unknown to oxidize PAHs. Any how, for high efficiency and low cost, it might be very meaningful to use CE in environmental remediation and CE from P. eryngii was most potential. Acknowledgment This study was supported by the Ministry of Science and Technology of the People’s Republic of China (2007AA061101) and National Natural Science Foundation of China (40801091); WU Y-C was partly supported by a IFS grant (C/4471-1).

Fig. 4 Effect of ABTS and possible natural mediators on PAHs oxidation by pure laccase. The treatments were listed in Table 2. The laccase activity was 2 U ml-1. Both anthracene and benzo[a]pyrene concentration was 1 mg l-1. The reaction system was incubated in the dark for 24 h (25°C). Values were means of triplicates, and error bars stood for standard deviations. Means with the same letter are not significantly different (P [ 0.05)

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