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Edible fungus degrade bisphenol A with no harmful effect on its fatty acid composition
Ecotoxicology and Environmental Safety 118 (2015) 126–132
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Edible fungus degrade bisphenol A with no harmful effect on its fatty acid composition Chengdong Zhang a,n, Mingzhu Li a, Xiaoyan Chen a, Mingchun Li b,nn a Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Tianjin Key Laboratory of Environmental Remediation and Pollution Control and College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China b Department of Microbiology, College of Life Science, Nankai University, Tianjin 300071, China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 February 2015 Received in revised form 16 April 2015 Accepted 17 April 2015 Available online 1 May 2015
Bisphenol A (BPA) is an endocrine-disrupting chemical that is ubiquitous in the environment because of its broad industrial use. The authors report that the most widely cultivated mushroom in the world (i.e., white-rot fungus, Pleurotus ostreatus) efficiently degraded 10 mg/L of BPA in 7 days. Extracellular laccase was identified as the enzyme responsible for this activity. LC–MS analysis of the metabolites revealed the presence of both low- and high-molecular-weight products obtained via oxidative cleavage and coupling reactions, respectively. In particular, an analysis of the fatty acid composition and chemical structure of the fungal mycelium demonstrated that exposure to BPA resulted in no harmful effects on this edible fungus. The results provide a better understanding of the environmental fate of BPA and its potential impact on food crops. & 2015 Elsevier Inc. All rights reserved.
Keywords: BPA Biodegradation Edible fungus Fatty acid composition
1. Introduction Bisphenol A (BPA) is widely used for the production of flame retardants, polycarbonate plastics, food-drink packaging coatings, epoxy resins, and other specialty chemicals (Michałowicz, 2014). More than one million tons of BPA are produced annually worldwide; in the United States and China alone, 7.6 108 and 5.6 107 kg of BPA are produced per year, respectively (Kusvuran and Yildirim, 2013). BPA can be released into the environment during its production, use and subsequent disposal. It has been detected in various environmental media, including soil, sediment, sewage sludge and waste water (Huang et al., 2012b). For example, BPA is present ubiquitously in aquatic environments, with concentrations in the range of 0.01 to 21 μg/L in surface waters (Flint et al., 2012) and 1.3 to 17,200 μg/L in land leachate (Yamamoto et al., 2001). Recently, considerable attention has focused on the endocrine-disrupting ability of BPA, which has been shown to alter the function of the endocrine systems in both wildlife and humans (Michałowicz, 2014). The prevalence of BPA and its potential exposure to humans (Zhang et al., 2011) warrants the need to understand the fate of BPA in the environment and its possible toxic effects. n
Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Zhang), [email protected] (M. Li). nn
http://dx.doi.org/10.1016/j.ecoenv.2015.04.020 0147-6513/& 2015 Elsevier Inc. All rights reserved.
BPA can be biodegraded by microorganisms, including bacteria, fungi and algae (Kang et al., 2006). BPA biodegradation by fungi is primarily caused by lignin-degrading fungi or white-rot fungi, which produce extracellular enzymes (i.e., peroxidase, laccase) with low substrate specificity and are consequently suitable for degrading various aromatic compounds (Cajthaml et al., 2009; Čvančarová et al., 2013). Among the white-rot fungi, those of genus Pleurotus are of particular interest because they are among the most cultivated mushrooms in the world. China is the largest producer of this genus of mushroom, and its production of mushrooms has increased continuously (Reis et al., 2012). Because of the ubiquitous presence of BPA in water systems and the potential use of reclaimed water for irrigation to mediate water shortages, interest in determining whether BPA accumulates in food crops has been increasing (Liao and Kannan, 2013; Mezcua et al., 2012). The concentrations of BPA in lettuce and collard tissue have been reported to reach 23 ng/kg and 331 ng/kg, respectively (Dodgen et al., 2013). However, limited information is available on the degradation of BPA by these edible fungi. Moreover, the need to determine whether the nutritional value, in terms of fatty acid composition, is affected by the degradation of BPA by these fungi is urgent. For these reasons, the objectives of the present study were: (i) to investigate the potential of six edible white-rot fungi to degrade BPA; (ii) to identify the major degradation products and determine which enzymes were responsible for the degradation; and (iii) to evaluate the change of the fatty acid composition of the fungi following their incubation with BPA.
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Fig. 2. Effects of (A) glucose and (B) BPA concentrations on BPA degradation by fungus f2. The concentration of BPA was 10 mg/L for Fig. 2A, and the concentration of glucose was 10 mg/L for Fig. 2B. The error bars represent standard deviations of 3 replicate samples. C0 and C referred to the initial BPA concentration and the BPA concentration at the respective time.
2. Materials and methods 2.1. Chemicals BPA (92%), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS), veratryl alcohol, 2,6-dimethoxyphenol and fatty acid methyl ester reference standards were purchased from Sigma-Aldrich Chemical Co., USA. All other chemicals were of analytical grade or better. 2.2. Fungi and cultivation Six types of edible white-rot fungi, Pleurotus ostreatus (f1–f3), Pleurotus abalones (f4), Pleurotus nebrodensis (f5) and Ganoderma lucidum (f6) were provided by Professor Mingchun Li from the Department of Microbiology at Nankai University. All fungi were pre-cultured in potato dextrose liquid medium at 30 °C for 3 d and collected at the late logarithmic growth phase by centrifugation at 3000g for 15 min. The fungi were then washed twice with liquid culture medium. The culture medium (per liter of distilled water) comprised 10 g of glucose, 2 g of NH4NO3, 0.8 g of KH2PO4, 0.4 g of Na2HPO4, 0.5 g of MgSO4·7H2O, and 2 g of yeast extract. All following BPA degradation experiments were performed by using this culture medium except that indicated glucose concentration was used when required. Fig. 1. (A) Growth curves of six fungi. (B) Degradation of BPA by six fungi. (C) Degradation efficiencies of various treatments. The concentration of BPA was 10 mg/L in all cases. The error bars represent standard deviations of 3 replicate samples. In Fig. 1B, C0 and C referred to the initial BPA concentration and the BPA concentration at the respective time.
2.3. Biodegradation of BPA in fungal cultures The inoculum was prepared by growing the fungi on a rotary shaker at 160 rpm and 30 °C in 250 mL flasks containing 100 mL of the culture medium with 10 mg/L of BPA. Three replicates were
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prepared for each fungus. After each indicated interval (i.e., 0, 1, 2, 3, 5, 7 d), the sample was mixed well and 2 mL of the samples were withdrawn and extracted with ethyl acetate (1:1, v-v) for 3 times. BPA adsorbed onto the fungal biomass can also be extracted by ethyl acetate. The organic phase was evaporated and redissolved in a methanol:water mixture (1:1, v-v) for analysis by high-performance liquid chromatography (HPLC). The recovery efficiency was determined by adding 10 mg/L of BPA to heat inactivated fungi medium and extracting following the same procedure described above. The recovery efficiency was over 99%. For the analysis of the metabolites by liquid chromatography mass spectroscopy (LC–MS), the sample dissolved in the methanol/water mixture was further filtered through a 0.22 μm membrane (syringe filter with hydrophilic polyethersulfone membrane, Keyilong Scientific Instrument Co., China). When required, 0 or 5 g/L of glucose or 20 or 30 mg/L of BPA were used instead. A culture medium containing only BPA was used as the control. 2.4. Identification of the degradation enzyme in f2 culture Aliquots of the extracellular fluid were collected at 1, 2, 3, 4, 6, and 9 d by centrifugation at 9000g for 2 min. Laccase activity was determined by monitoring the oxidation of ABTS to its cation radical at 420 nm (Cabana et al., 2007). The relative activity was expresses as laccase activity at the respective time in various treatments versus laccase activity in control (without BPA) at the beginning. The activity of lignin peroxidase was determined by the oxidation of veratryl alcohol at 310 nm (Walter et al., 2004). The activity of manganese peroxidase was assayed by the degradation of 2,6-dimethoxyphenol at 450 nm (Walter et al., 2004). The role of the cytochrome P450 system on BPA degradation was examined by adding the cytochrome P450 inhibitor (Prieto et al., 2011) 1-aminobenzotriazole at a final concentration of 5 mM to the f2 culture at the beginning of the incubation period. 2.5. Analysis of BPA and its metabolites The concentration of BPA was measured by HPLC (Alliance 2695, Waters, USA) on an instrument equipped with a symmetry reversed phase C18 column (4.6 150 mm) and a Waters 2489 UV/visible detector. The sample (50 μL) was injected and then eluted from the column at a flow rate of 0.8 mL/min using an acetonitrile:water mixture (11: 9, v-v). The column temperature was 35 °C. BPA was detected at 224 nm. The detection limit was determined to be 0.02 mg/L. The analysis of the metabolites was performed on a Waters Ultra Performance liquid chromatograph equipped with an Acquity UPLC BEH C18 column (1.7 μm, 2.1 50 mm) and connected to a Xevo TQ S mass spectrometer. The mobile phase began with 1% methanol and 99% water, was increased to 50% methanol and 50% water in a linear fashion within 2 min, and was then increased to 100% methanol in a linear fashion within 12 min. Finally, the eluent was changed back to 1% methanol and 99% water in a linear fashion within 13 min and was maintained at this ratio for 2 min. The signals were monitored at 280 nm, and the flow rate was 0.45 mL/min. The mass spectrometer was operated in the negative-ion mode at a mass range of 50–500m/z. The electrospray unit was operated with the source temperature set at 150 °C, and the voltages for the capillary entrance and exit were 2700 and 40 V, respectively.
Fig. 3. Changes in laccase activities as a function of time in the presence of (A) different concentrations of glucose and 10 mg/L of BPA and (B) different concentrations of BPA and 10 mg/L of glucose. (C) BPA (10 mg/L) degradation as a function of time with the addition of a cytochrome P450 inhibitor. The error bars represent standard deviations of 3 replicate samples. In Fig. 3C, C0 and C referred to the initial BPA concentration and the BPA concentration at the respective time.
2.6. Characterization of the fungal mycelium structure and the fatty acid composition Fugal mycelia were collected after incubation with 10 mg/L of BPA for 7 days. The mycelia were then washed and dried in a freeze-drier (Ly-8-FM-ULE, Snijders, The Netherlands). Pellets
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Table 1 Identifications of metabolites and tentative structure assignments. Metabolites
Retention time (min)
Molecular weight
C9H12O2
0.353
152
C7H6O2
0.411
122
C8H8O2
0.411
136
C8H8O2
0.411
136
C15H16O4
0.454
260
C15H16O3
0.525
244
C15H16O2
0.591
228
C18H20O2
4.274
268
were prepared by mixing 5 mg of lyophilized mycelia with 50 mg of KBr and compressing the mixture under vacuum. The spectra were recorded between 4000 and 400 cm 1 using a Bio-Rad FTS 6000 Fourier-transform infrared (FTIR) spectrometer (Bio-Rad, USA) with 4 cm 1 resolution. Fatty acid composition analysis was performed according to Huang's method (Huang et al., 2012a). Briefly, 5 mL of 0.5 M sodium hydroxide (in methanol) was added to 50 mg of freeze-dried fungi mycelium. Samples were heated in 80 °C water baths for
Structure assignment
10 min to induce saponification. After the samples were cooled, 5 mL of 2 M sulfuric acid (in methanol) was added to act as a catalyst for transesterification. These reactions were performed for 15 min at 80 °C. Saturated saline (2 mL) was added to prevent emulsification. Then, 1 mL of n-hexane was added, and the transesterified fatty acid methyl esters were extracted to the n-hexane layer and subsequently analyzed by gas chromatography (GC). GC (7890A, Agilent Technologies Co., USA) analysis involved the use of HP-5 columns (30 m 0.25 mm 0.25 μm, Agilent
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Technologies Co., USA). The oven temperature was initially set at 100 °C for 1 min and was increased at a rate of 10 °C/min to 185 °C and maintained at this temperature for 1 min. Then, the temperature was increased at 0.3 °C/min to 190 °C and was maintained at this temperature for 2 min. The temperature was then increased at a rate of 10 °C/min to 250 °C and maintained at this
temperature for 3 min. The injector and flame ionization detector temperatures were set at 260 °C and 270 °C, respectively. The nitrogen flow rate was 1 mL/min, the hydrogen flow rate was 25 mL/ min, and the air flow rate was 250 mL/min. Sample injection aliquots were 1 μL with a split ratio of 1:10. Fatty acid methyl ester reference were used to allow a comparison of the fatty acid methyl
Fig. 4. Proposed BPA degradation pathways by f2. (A) BPA is degraded to low-molecular-weight metabolites. (B) The formation of high-molecular-weight metabolites via coupling reactions.
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Table 2 Fatty acid composition of the studied edible fungus f2 with or without exposure to 10 mg/L of BPA. Fatty acid methyl ester
Fatty acid content (%) In the absenceof In the presenceof BPA BPA
C14:0 C16:0 C18:2
0 18.357 1.32 55.577 3.23
0 20.03 7 1.57 50.22 7 1.83
21.39 7 2.08
23.22 7 1.94
0 4.687 0.55 0 0 23.03 76.96
0 6.53 7 0.63 0 0 26.56 73.44
Methyl myristate Methyl palmitate Methyl linoleateLinolelaidic acid methyl ester C18:1 Trans-9-elaidic methyl ester Cis9-oleic methyl ester C18:3 Methyl linolenate C18:0 Methyl stearate C20:0 Methyl arachidate C22:0 Methyl behenate Total saturation Total unsaturation
ester peaks in the chromatograms of the samples with those in the chromatograms of the standards, and the concentrations of single fatty acid methyl esters in the samples were determined. 2.7. Statistical analysis Each experiment was performed in triplicate, and the results are expressed as the mean values and standard deviations. The results were evaluated by t-tests using Statistical Packages for the Social Sciences (SPSS) version 19.0.
3. Results and discussion 3.1. BPA biodegradation and the effects of glucose and BPA The strains of six different edible ligninolytic fungi were exposed to BPA at an initial concentration of 10 mg/L. As shown in Fig. 1A, all the fungi grew well, and the optical densities reached 1.5–2.0 after 48 h of incubation. No significant differences were observed among the six fungi. BPA was removed rapidly by these fungi over a seven-day incubation period (Fig. 1B). The biodegradation efficiency was defined as the amount of BPA degraded versus the amount of initial BPA. The overall biodegradation efficiency was greater than 78%, with a maximum of 99% obtained for f2 (Fig. 1C) after 7 days. Sample f2, also known as gray oyster mushroom, is one of the most common edible mushrooms on the market; thus, f2 was chosen to examine the degradation pathway and nutritional value change in the presence of BPA. The effects of glucose on the degradation of BPA by f2 were examined (Fig. 2A). The results show that the degradation efficiency increased from 25% to 99% when the concentration of glucose was increased from 0 to 10 g/L. This result indicates that proportionality exists between the fungal biomass and the extent of BPA degradation. Previous investigations of eight ligninolytic fungi on the biodegradation of BPA at an initial concentration of 10 mg/L indicated a comparable time frame (14 days) for the complete removal of BPA. They also observed that P. ostreatus was the most efficient fungus to degrade BPA (Cajthaml et al., 2009). However, the degradation products were not identified. Fig. 2B shows that a high concentration of BPA significantly affected microbial activity. The degradation efficiencies were 99%, 76% and 70% for BPA concentrations of 10, 20 and 30 mg/L, respectively. Notably, in general, the BPA concentrations in surface water and reclaimed water were approximately in the ng/L and μg/L ranges, respectively (Huang et al., 2012b). Sample f2 was
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inferred to effectively degrade BPA at environmentally relevant concentrations without any inhibitory effect to the fungus. 3.2. Identification of the main degradation enzyme, metabolites and a proposed pathway White-rot fungi degrade a broad spectrum of xenobiotics through the action of a nonspecific enzymatic system that includes extracellular lignin-degrading enzymes (especially laccase, lignin peroxidase and manganese peroxidase) and intracellular enzymes, such as those in the cytochrome P450 family (Prieto et al., 2011). Sample f2 constitutively produced laccase, and the activity correlated well with the degradation efficiency (Fig. 3). Laccase activity increased with increasing incubation time, and a comparison of the activity of day 7 with that of day 1 revealed that the activities increased approximately 13- and 24-fold in the presence of 5 and 10 g/L of glucose, respectively (Fig. 3A). Nevertheless, laccase activity was inhibited when f2 was incubated with a high concentration of BPA (Fig. 3B). The activities of lignin peroxidase and manganese peroxidase were not considered because they were present at negligible concentrations in the medium (data not shown). Although the cytochrome P450 system in white-rot fungi has been shown to be important in catalyzing the detoxification of several organic pollutants (Doddapaneni and Yadav, 2004), our findings indicate that BPA degradation was not inhibited in the presence of a cytochrome P450 inhibitor (Fig. 3C), which implies that cytochrome P450 enzymes were not involved in the degradation process. Overall, our results suggest that laccase from f2 plays an important role in BPA degradation. To elucidate the BPA biodegradation pathway and its corresponding metabolic mechanism, HPLC-MS was used to identify the degradation intermediates. Fig. S1 displays the HPLC chromatograph and MS spectra of the metabolites observed after a 7 day incubation period. The principal mass peaks in the spectra correspond to compounds with molecular masses of 122, 134, 136, 152, 244, 258 and 260; tentative structures of these compounds are proposed in Table 1. With the exception of the product with a mass of 260, all of the identified intermediates exhibited retention times shorter than that of BPA, which indicates that they are more polar than BPA. The intermediates agree well with those identified in the enzymatic transformation of BPA by white-rot fungi (Hou et al., 2014; Huang and Weber, 2005; Yang et al., 2013). Highmolecular-weight products (i.e., MW 4300) formed during the coupling reactions were not detected, which is probably because metabolites generated from such reactions precipitated from the medium and could not be extracted by ethyl acetate. Nevertheless, the laccase-catalyzed polymerizations of BPA have been reported in other studies (Mita et al., 2003; Uchida et al., 2001). A possible degradation pathway was proposed on the basis of the identified intermediate products (Fig. 4). The mechanism is described as follows: 1) BPA is oxidized to form hydroquinone and the isopropylphenol carbocation (Kolvenbach et al., 2007); 2) 4-isopropenylphenol (MW ¼134) and 4-isopropylphenol (MW¼136) are formed from isopropylphenol carbocations by the loss of H þ or through the addition of H ; isopropenylphenol is further transformed to hydroacetophenone (MW ¼ 136) and phydroxybenzaldehyde (MW ¼ 122) (Li et al., 2012); 3) the isopropylphenol carbocation can also react with water to form 4-(2hydroxylpropan-2-yl)phenol (MW ¼ 152) (Huang and Weber, 2005); 4) products with masses of 244, 260 and 268 may arise from radical coupling reactions involving the isopropylphenol carbocation and other intermediates (Kabiersch et al., 2011). Notably, Suzuki et al. (2004) reported a significantly reduced estrogenic activity because of metabolite formation (e.g., hydroxyacetophenone and p-hydroxybenzaldehyde) during BPA biodegradation. The polymerization and coupling processes catalyzed
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by laccase also efficiently removed BPA estrogenic activity (Fukuda et al., 2001; Suzuki et al., 2004; Uchida et al., 2001). 3.3. Impact on the fatty acid compositions and mycelium structure of the mushrooms Unsaturated fatty acids are essential in the human diet; in particular, the high percentage of linoleic acid in mushrooms is an important factor when regarding mushrooms as a health food (Reis et al., 2012). The growth conditions of cultivated mushrooms are known to influence their fatty acid composition (Öztürk et al., 2011). The fatty acid composition of f2 in the absence and presence of BPA are given in Table 2. The elution time and the corresponding gas chromatographs of the samples and standards are provided in Table S1 and Fig. S2 of the Supporting information. The dominant fatty acids were observed to be linoleic acid (C18:2), oleic acid (C18:1) and palmitic acid (C16:0). Steric acid (C18:0) was detected in a low concentration. Other fatty acids, such as C14:0, C20:0 and C22:0, were not detected in f2. However, no significant difference was observed between BPA-treated fungus and the BPA-free control. The percentage of unsaturated fatty acids shifted slightly from 77% in the BPAfree sample to 73% in the BPA-treated fungus. Furthermore, FTIR analysis (Fig. S3) revealed the mycelia exhibited the same pattern with or without exposure to BPA for 7 days, indicating that no remarkable qualitative changes in the composition of the fungal biomass. Herein, no obvious harmful effects were observed for the edible fungus f2. At the same time, the results show that f2 exhibits a high capability to degrade BPA.
4. Conclusion The present study demonstrated that BPA can be degraded efficiently by edible white-rot fungi at environmentally relevant concentrations. Notably, no obvious harmful effects that would affect the nutritional value of the fungi were observed. The results are expected to provide additional information about the effect of estrogen disruptor on food crops. However, reclaimed water and surface water are sources for many organic pollutants, including pharmaceutical and personal care products; whether the co-existence of contaminants could influence the biodegradation of estrogen disruptor in food crops requires additional investigation.
Acknowledgments This work was supported by the Tianjin Municipal Science and Technology Commission (Grant 13JCZDJC35900).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.04.020.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Edible fungus degrade bisphenol A with no harmful effect on its fatty acid composition Chengdong Zhang a,n, Mingzhu Li a, Xiaoyan Chen a, Mingchun Li b,nn a Ministry of Education Key Laboratory of Pollution Processes and Environmental Criteria, Tianjin Key Laboratory of Environmental Remediation and Pollution Control and College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China b Department of Microbiology, College of Life Science, Nankai University, Tianjin 300071, China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 February 2015 Received in revised form 16 April 2015 Accepted 17 April 2015 Available online 1 May 2015
Bisphenol A (BPA) is an endocrine-disrupting chemical that is ubiquitous in the environment because of its broad industrial use. The authors report that the most widely cultivated mushroom in the world (i.e., white-rot fungus, Pleurotus ostreatus) efficiently degraded 10 mg/L of BPA in 7 days. Extracellular laccase was identified as the enzyme responsible for this activity. LC–MS analysis of the metabolites revealed the presence of both low- and high-molecular-weight products obtained via oxidative cleavage and coupling reactions, respectively. In particular, an analysis of the fatty acid composition and chemical structure of the fungal mycelium demonstrated that exposure to BPA resulted in no harmful effects on this edible fungus. The results provide a better understanding of the environmental fate of BPA and its potential impact on food crops. & 2015 Elsevier Inc. All rights reserved.
Keywords: BPA Biodegradation Edible fungus Fatty acid composition
1. Introduction Bisphenol A (BPA) is widely used for the production of flame retardants, polycarbonate plastics, food-drink packaging coatings, epoxy resins, and other specialty chemicals (Michałowicz, 2014). More than one million tons of BPA are produced annually worldwide; in the United States and China alone, 7.6 108 and 5.6 107 kg of BPA are produced per year, respectively (Kusvuran and Yildirim, 2013). BPA can be released into the environment during its production, use and subsequent disposal. It has been detected in various environmental media, including soil, sediment, sewage sludge and waste water (Huang et al., 2012b). For example, BPA is present ubiquitously in aquatic environments, with concentrations in the range of 0.01 to 21 μg/L in surface waters (Flint et al., 2012) and 1.3 to 17,200 μg/L in land leachate (Yamamoto et al., 2001). Recently, considerable attention has focused on the endocrine-disrupting ability of BPA, which has been shown to alter the function of the endocrine systems in both wildlife and humans (Michałowicz, 2014). The prevalence of BPA and its potential exposure to humans (Zhang et al., 2011) warrants the need to understand the fate of BPA in the environment and its possible toxic effects. n
Corresponding author. Corresponding author. E-mail addresses: [email protected] (C. Zhang), [email protected] (M. Li). nn
http://dx.doi.org/10.1016/j.ecoenv.2015.04.020 0147-6513/& 2015 Elsevier Inc. All rights reserved.
BPA can be biodegraded by microorganisms, including bacteria, fungi and algae (Kang et al., 2006). BPA biodegradation by fungi is primarily caused by lignin-degrading fungi or white-rot fungi, which produce extracellular enzymes (i.e., peroxidase, laccase) with low substrate specificity and are consequently suitable for degrading various aromatic compounds (Cajthaml et al., 2009; Čvančarová et al., 2013). Among the white-rot fungi, those of genus Pleurotus are of particular interest because they are among the most cultivated mushrooms in the world. China is the largest producer of this genus of mushroom, and its production of mushrooms has increased continuously (Reis et al., 2012). Because of the ubiquitous presence of BPA in water systems and the potential use of reclaimed water for irrigation to mediate water shortages, interest in determining whether BPA accumulates in food crops has been increasing (Liao and Kannan, 2013; Mezcua et al., 2012). The concentrations of BPA in lettuce and collard tissue have been reported to reach 23 ng/kg and 331 ng/kg, respectively (Dodgen et al., 2013). However, limited information is available on the degradation of BPA by these edible fungi. Moreover, the need to determine whether the nutritional value, in terms of fatty acid composition, is affected by the degradation of BPA by these fungi is urgent. For these reasons, the objectives of the present study were: (i) to investigate the potential of six edible white-rot fungi to degrade BPA; (ii) to identify the major degradation products and determine which enzymes were responsible for the degradation; and (iii) to evaluate the change of the fatty acid composition of the fungi following their incubation with BPA.
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Fig. 2. Effects of (A) glucose and (B) BPA concentrations on BPA degradation by fungus f2. The concentration of BPA was 10 mg/L for Fig. 2A, and the concentration of glucose was 10 mg/L for Fig. 2B. The error bars represent standard deviations of 3 replicate samples. C0 and C referred to the initial BPA concentration and the BPA concentration at the respective time.
2. Materials and methods 2.1. Chemicals BPA (92%), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS), veratryl alcohol, 2,6-dimethoxyphenol and fatty acid methyl ester reference standards were purchased from Sigma-Aldrich Chemical Co., USA. All other chemicals were of analytical grade or better. 2.2. Fungi and cultivation Six types of edible white-rot fungi, Pleurotus ostreatus (f1–f3), Pleurotus abalones (f4), Pleurotus nebrodensis (f5) and Ganoderma lucidum (f6) were provided by Professor Mingchun Li from the Department of Microbiology at Nankai University. All fungi were pre-cultured in potato dextrose liquid medium at 30 °C for 3 d and collected at the late logarithmic growth phase by centrifugation at 3000g for 15 min. The fungi were then washed twice with liquid culture medium. The culture medium (per liter of distilled water) comprised 10 g of glucose, 2 g of NH4NO3, 0.8 g of KH2PO4, 0.4 g of Na2HPO4, 0.5 g of MgSO4·7H2O, and 2 g of yeast extract. All following BPA degradation experiments were performed by using this culture medium except that indicated glucose concentration was used when required. Fig. 1. (A) Growth curves of six fungi. (B) Degradation of BPA by six fungi. (C) Degradation efficiencies of various treatments. The concentration of BPA was 10 mg/L in all cases. The error bars represent standard deviations of 3 replicate samples. In Fig. 1B, C0 and C referred to the initial BPA concentration and the BPA concentration at the respective time.
2.3. Biodegradation of BPA in fungal cultures The inoculum was prepared by growing the fungi on a rotary shaker at 160 rpm and 30 °C in 250 mL flasks containing 100 mL of the culture medium with 10 mg/L of BPA. Three replicates were
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prepared for each fungus. After each indicated interval (i.e., 0, 1, 2, 3, 5, 7 d), the sample was mixed well and 2 mL of the samples were withdrawn and extracted with ethyl acetate (1:1, v-v) for 3 times. BPA adsorbed onto the fungal biomass can also be extracted by ethyl acetate. The organic phase was evaporated and redissolved in a methanol:water mixture (1:1, v-v) for analysis by high-performance liquid chromatography (HPLC). The recovery efficiency was determined by adding 10 mg/L of BPA to heat inactivated fungi medium and extracting following the same procedure described above. The recovery efficiency was over 99%. For the analysis of the metabolites by liquid chromatography mass spectroscopy (LC–MS), the sample dissolved in the methanol/water mixture was further filtered through a 0.22 μm membrane (syringe filter with hydrophilic polyethersulfone membrane, Keyilong Scientific Instrument Co., China). When required, 0 or 5 g/L of glucose or 20 or 30 mg/L of BPA were used instead. A culture medium containing only BPA was used as the control. 2.4. Identification of the degradation enzyme in f2 culture Aliquots of the extracellular fluid were collected at 1, 2, 3, 4, 6, and 9 d by centrifugation at 9000g for 2 min. Laccase activity was determined by monitoring the oxidation of ABTS to its cation radical at 420 nm (Cabana et al., 2007). The relative activity was expresses as laccase activity at the respective time in various treatments versus laccase activity in control (without BPA) at the beginning. The activity of lignin peroxidase was determined by the oxidation of veratryl alcohol at 310 nm (Walter et al., 2004). The activity of manganese peroxidase was assayed by the degradation of 2,6-dimethoxyphenol at 450 nm (Walter et al., 2004). The role of the cytochrome P450 system on BPA degradation was examined by adding the cytochrome P450 inhibitor (Prieto et al., 2011) 1-aminobenzotriazole at a final concentration of 5 mM to the f2 culture at the beginning of the incubation period. 2.5. Analysis of BPA and its metabolites The concentration of BPA was measured by HPLC (Alliance 2695, Waters, USA) on an instrument equipped with a symmetry reversed phase C18 column (4.6 150 mm) and a Waters 2489 UV/visible detector. The sample (50 μL) was injected and then eluted from the column at a flow rate of 0.8 mL/min using an acetonitrile:water mixture (11: 9, v-v). The column temperature was 35 °C. BPA was detected at 224 nm. The detection limit was determined to be 0.02 mg/L. The analysis of the metabolites was performed on a Waters Ultra Performance liquid chromatograph equipped with an Acquity UPLC BEH C18 column (1.7 μm, 2.1 50 mm) and connected to a Xevo TQ S mass spectrometer. The mobile phase began with 1% methanol and 99% water, was increased to 50% methanol and 50% water in a linear fashion within 2 min, and was then increased to 100% methanol in a linear fashion within 12 min. Finally, the eluent was changed back to 1% methanol and 99% water in a linear fashion within 13 min and was maintained at this ratio for 2 min. The signals were monitored at 280 nm, and the flow rate was 0.45 mL/min. The mass spectrometer was operated in the negative-ion mode at a mass range of 50–500m/z. The electrospray unit was operated with the source temperature set at 150 °C, and the voltages for the capillary entrance and exit were 2700 and 40 V, respectively.
Fig. 3. Changes in laccase activities as a function of time in the presence of (A) different concentrations of glucose and 10 mg/L of BPA and (B) different concentrations of BPA and 10 mg/L of glucose. (C) BPA (10 mg/L) degradation as a function of time with the addition of a cytochrome P450 inhibitor. The error bars represent standard deviations of 3 replicate samples. In Fig. 3C, C0 and C referred to the initial BPA concentration and the BPA concentration at the respective time.
2.6. Characterization of the fungal mycelium structure and the fatty acid composition Fugal mycelia were collected after incubation with 10 mg/L of BPA for 7 days. The mycelia were then washed and dried in a freeze-drier (Ly-8-FM-ULE, Snijders, The Netherlands). Pellets
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Table 1 Identifications of metabolites and tentative structure assignments. Metabolites
Retention time (min)
Molecular weight
C9H12O2
0.353
152
C7H6O2
0.411
122
C8H8O2
0.411
136
C8H8O2
0.411
136
C15H16O4
0.454
260
C15H16O3
0.525
244
C15H16O2
0.591
228
C18H20O2
4.274
268
were prepared by mixing 5 mg of lyophilized mycelia with 50 mg of KBr and compressing the mixture under vacuum. The spectra were recorded between 4000 and 400 cm 1 using a Bio-Rad FTS 6000 Fourier-transform infrared (FTIR) spectrometer (Bio-Rad, USA) with 4 cm 1 resolution. Fatty acid composition analysis was performed according to Huang's method (Huang et al., 2012a). Briefly, 5 mL of 0.5 M sodium hydroxide (in methanol) was added to 50 mg of freeze-dried fungi mycelium. Samples were heated in 80 °C water baths for
Structure assignment
10 min to induce saponification. After the samples were cooled, 5 mL of 2 M sulfuric acid (in methanol) was added to act as a catalyst for transesterification. These reactions were performed for 15 min at 80 °C. Saturated saline (2 mL) was added to prevent emulsification. Then, 1 mL of n-hexane was added, and the transesterified fatty acid methyl esters were extracted to the n-hexane layer and subsequently analyzed by gas chromatography (GC). GC (7890A, Agilent Technologies Co., USA) analysis involved the use of HP-5 columns (30 m 0.25 mm 0.25 μm, Agilent
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Technologies Co., USA). The oven temperature was initially set at 100 °C for 1 min and was increased at a rate of 10 °C/min to 185 °C and maintained at this temperature for 1 min. Then, the temperature was increased at 0.3 °C/min to 190 °C and was maintained at this temperature for 2 min. The temperature was then increased at a rate of 10 °C/min to 250 °C and maintained at this
temperature for 3 min. The injector and flame ionization detector temperatures were set at 260 °C and 270 °C, respectively. The nitrogen flow rate was 1 mL/min, the hydrogen flow rate was 25 mL/ min, and the air flow rate was 250 mL/min. Sample injection aliquots were 1 μL with a split ratio of 1:10. Fatty acid methyl ester reference were used to allow a comparison of the fatty acid methyl
Fig. 4. Proposed BPA degradation pathways by f2. (A) BPA is degraded to low-molecular-weight metabolites. (B) The formation of high-molecular-weight metabolites via coupling reactions.
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Table 2 Fatty acid composition of the studied edible fungus f2 with or without exposure to 10 mg/L of BPA. Fatty acid methyl ester
Fatty acid content (%) In the absenceof In the presenceof BPA BPA
C14:0 C16:0 C18:2
0 18.357 1.32 55.577 3.23
0 20.03 7 1.57 50.22 7 1.83
21.39 7 2.08
23.22 7 1.94
0 4.687 0.55 0 0 23.03 76.96
0 6.53 7 0.63 0 0 26.56 73.44
Methyl myristate Methyl palmitate Methyl linoleateLinolelaidic acid methyl ester C18:1 Trans-9-elaidic methyl ester Cis9-oleic methyl ester C18:3 Methyl linolenate C18:0 Methyl stearate C20:0 Methyl arachidate C22:0 Methyl behenate Total saturation Total unsaturation
ester peaks in the chromatograms of the samples with those in the chromatograms of the standards, and the concentrations of single fatty acid methyl esters in the samples were determined. 2.7. Statistical analysis Each experiment was performed in triplicate, and the results are expressed as the mean values and standard deviations. The results were evaluated by t-tests using Statistical Packages for the Social Sciences (SPSS) version 19.0.
3. Results and discussion 3.1. BPA biodegradation and the effects of glucose and BPA The strains of six different edible ligninolytic fungi were exposed to BPA at an initial concentration of 10 mg/L. As shown in Fig. 1A, all the fungi grew well, and the optical densities reached 1.5–2.0 after 48 h of incubation. No significant differences were observed among the six fungi. BPA was removed rapidly by these fungi over a seven-day incubation period (Fig. 1B). The biodegradation efficiency was defined as the amount of BPA degraded versus the amount of initial BPA. The overall biodegradation efficiency was greater than 78%, with a maximum of 99% obtained for f2 (Fig. 1C) after 7 days. Sample f2, also known as gray oyster mushroom, is one of the most common edible mushrooms on the market; thus, f2 was chosen to examine the degradation pathway and nutritional value change in the presence of BPA. The effects of glucose on the degradation of BPA by f2 were examined (Fig. 2A). The results show that the degradation efficiency increased from 25% to 99% when the concentration of glucose was increased from 0 to 10 g/L. This result indicates that proportionality exists between the fungal biomass and the extent of BPA degradation. Previous investigations of eight ligninolytic fungi on the biodegradation of BPA at an initial concentration of 10 mg/L indicated a comparable time frame (14 days) for the complete removal of BPA. They also observed that P. ostreatus was the most efficient fungus to degrade BPA (Cajthaml et al., 2009). However, the degradation products were not identified. Fig. 2B shows that a high concentration of BPA significantly affected microbial activity. The degradation efficiencies were 99%, 76% and 70% for BPA concentrations of 10, 20 and 30 mg/L, respectively. Notably, in general, the BPA concentrations in surface water and reclaimed water were approximately in the ng/L and μg/L ranges, respectively (Huang et al., 2012b). Sample f2 was
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inferred to effectively degrade BPA at environmentally relevant concentrations without any inhibitory effect to the fungus. 3.2. Identification of the main degradation enzyme, metabolites and a proposed pathway White-rot fungi degrade a broad spectrum of xenobiotics through the action of a nonspecific enzymatic system that includes extracellular lignin-degrading enzymes (especially laccase, lignin peroxidase and manganese peroxidase) and intracellular enzymes, such as those in the cytochrome P450 family (Prieto et al., 2011). Sample f2 constitutively produced laccase, and the activity correlated well with the degradation efficiency (Fig. 3). Laccase activity increased with increasing incubation time, and a comparison of the activity of day 7 with that of day 1 revealed that the activities increased approximately 13- and 24-fold in the presence of 5 and 10 g/L of glucose, respectively (Fig. 3A). Nevertheless, laccase activity was inhibited when f2 was incubated with a high concentration of BPA (Fig. 3B). The activities of lignin peroxidase and manganese peroxidase were not considered because they were present at negligible concentrations in the medium (data not shown). Although the cytochrome P450 system in white-rot fungi has been shown to be important in catalyzing the detoxification of several organic pollutants (Doddapaneni and Yadav, 2004), our findings indicate that BPA degradation was not inhibited in the presence of a cytochrome P450 inhibitor (Fig. 3C), which implies that cytochrome P450 enzymes were not involved in the degradation process. Overall, our results suggest that laccase from f2 plays an important role in BPA degradation. To elucidate the BPA biodegradation pathway and its corresponding metabolic mechanism, HPLC-MS was used to identify the degradation intermediates. Fig. S1 displays the HPLC chromatograph and MS spectra of the metabolites observed after a 7 day incubation period. The principal mass peaks in the spectra correspond to compounds with molecular masses of 122, 134, 136, 152, 244, 258 and 260; tentative structures of these compounds are proposed in Table 1. With the exception of the product with a mass of 260, all of the identified intermediates exhibited retention times shorter than that of BPA, which indicates that they are more polar than BPA. The intermediates agree well with those identified in the enzymatic transformation of BPA by white-rot fungi (Hou et al., 2014; Huang and Weber, 2005; Yang et al., 2013). Highmolecular-weight products (i.e., MW 4300) formed during the coupling reactions were not detected, which is probably because metabolites generated from such reactions precipitated from the medium and could not be extracted by ethyl acetate. Nevertheless, the laccase-catalyzed polymerizations of BPA have been reported in other studies (Mita et al., 2003; Uchida et al., 2001). A possible degradation pathway was proposed on the basis of the identified intermediate products (Fig. 4). The mechanism is described as follows: 1) BPA is oxidized to form hydroquinone and the isopropylphenol carbocation (Kolvenbach et al., 2007); 2) 4-isopropenylphenol (MW ¼134) and 4-isopropylphenol (MW¼136) are formed from isopropylphenol carbocations by the loss of H þ or through the addition of H ; isopropenylphenol is further transformed to hydroacetophenone (MW ¼ 136) and phydroxybenzaldehyde (MW ¼ 122) (Li et al., 2012); 3) the isopropylphenol carbocation can also react with water to form 4-(2hydroxylpropan-2-yl)phenol (MW ¼ 152) (Huang and Weber, 2005); 4) products with masses of 244, 260 and 268 may arise from radical coupling reactions involving the isopropylphenol carbocation and other intermediates (Kabiersch et al., 2011). Notably, Suzuki et al. (2004) reported a significantly reduced estrogenic activity because of metabolite formation (e.g., hydroxyacetophenone and p-hydroxybenzaldehyde) during BPA biodegradation. The polymerization and coupling processes catalyzed
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by laccase also efficiently removed BPA estrogenic activity (Fukuda et al., 2001; Suzuki et al., 2004; Uchida et al., 2001). 3.3. Impact on the fatty acid compositions and mycelium structure of the mushrooms Unsaturated fatty acids are essential in the human diet; in particular, the high percentage of linoleic acid in mushrooms is an important factor when regarding mushrooms as a health food (Reis et al., 2012). The growth conditions of cultivated mushrooms are known to influence their fatty acid composition (Öztürk et al., 2011). The fatty acid composition of f2 in the absence and presence of BPA are given in Table 2. The elution time and the corresponding gas chromatographs of the samples and standards are provided in Table S1 and Fig. S2 of the Supporting information. The dominant fatty acids were observed to be linoleic acid (C18:2), oleic acid (C18:1) and palmitic acid (C16:0). Steric acid (C18:0) was detected in a low concentration. Other fatty acids, such as C14:0, C20:0 and C22:0, were not detected in f2. However, no significant difference was observed between BPA-treated fungus and the BPA-free control. The percentage of unsaturated fatty acids shifted slightly from 77% in the BPAfree sample to 73% in the BPA-treated fungus. Furthermore, FTIR analysis (Fig. S3) revealed the mycelia exhibited the same pattern with or without exposure to BPA for 7 days, indicating that no remarkable qualitative changes in the composition of the fungal biomass. Herein, no obvious harmful effects were observed for the edible fungus f2. At the same time, the results show that f2 exhibits a high capability to degrade BPA.
4. Conclusion The present study demonstrated that BPA can be degraded efficiently by edible white-rot fungi at environmentally relevant concentrations. Notably, no obvious harmful effects that would affect the nutritional value of the fungi were observed. The results are expected to provide additional information about the effect of estrogen disruptor on food crops. However, reclaimed water and surface water are sources for many organic pollutants, including pharmaceutical and personal care products; whether the co-existence of contaminants could influence the biodegradation of estrogen disruptor in food crops requires additional investigation.
Acknowledgments This work was supported by the Tianjin Municipal Science and Technology Commission (Grant 13JCZDJC35900).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.04.020.
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