Biodegradation and metabolite transformation of pyrene by ...

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Aug 15, 2012 - by basidiomycetes fungal isolate Armillaria sp. F022. Tony Hadibarata • Risky Ayu Kristanti. Received: 26 June 2012 / Accepted: 30 July 2012 ...

Bioprocess Biosyst Eng (2013) 36:461–468 DOI 10.1007/s00449-012-0803-4


Biodegradation and metabolite transformation of pyrene by basidiomycetes fungal isolate Armillaria sp. F022 Tony Hadibarata • Risky Ayu Kristanti

Received: 26 June 2012 / Accepted: 30 July 2012 / Published online: 15 August 2012 Ó Springer-Verlag 2012

Abstract Armillaria sp. F022 is a white-rot fungus isolated from a tropical rain forest in Indonesia that is capable of utilizing pyrene as a source of carbon and energy. Enzymes production during the degradation process by Armillaria sp. F022 was certainly related to the increase in biomass. In the first week after incubation, the growth rate rapidly increased, but enzyme production decreased. After 7 days of incubation, rapid growth was observed, whereas, the enzymes were produced only after a good amount of biomass was generated. About 63 % of pyrene underwent biodegradation when incubated with this fungus in a liquid medium on a rotary shaker (120 rpm, 25 °C) for 30 days; during this period, pyrene was transformed to five stable metabolic products. These metabolites were extracted in ethyl acetate, isolated by column chromatography, and then identified using thin layer chromatography (TLC) and gas chromatography–mass spectrometry (GC–MS). 1-Hydroxypyrene was directly identified by GC–MS, while 4-phenanthroic acid, 1-hydroxy-2-naphthoic acid, phthalic acid, and protocatechuic acid were identified to be present in their derivatized forms (methylated forms and silylated forms). Protocatechuic acid was the end product of pyrene degradation by Armillaria sp. F022. Dynamic profiles of two key enzymes, namely laccase and 1,2-dioxygenase,

T. Hadibarata (&) Institute of Environmental and Water Resources Management, Faculty of Civil Engineering, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor Bahru, Malaysia e-mail: [email protected] R. A. Kristanti Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 4-3-11, Takeda, Kofu, Yamanashi 400-8511, Japan

were revealed during the degradation process, and the results indicated the presence of a complicated mechanism in the regulation of pyrene-degrading enzymes. In conclusion, Armillaria sp. F022 is a white-rot fungus with potential for application in the degradation of polycyclic aromatic hydrocarbons such as pyrene in the environment. Keywords Armillaria sp. F022  Biotransformation of pyrene  Phenoloxidase and dioxygenase  Effect of biomass and glucose  Phthalic acid route

Introduction As products of incomplete combustion of organic materials such as fossil fuels and other industrial processes, polycyclic aromatic hydrocarbons (PAHs) are worldwide environmental pollutants that contribute to the accumulation of toxic chemicals in the food chains of living organisms. Pyrene, a symmetrical compound consisting of four fused aromatic rings, is one of the most abundant PAHs in the environment [1, 2]. Although pyrene is not genotoxic in nature, it has been used as an indicator for assessing PAH-containing pollutants, since its molecular structures is known to be potentially carcinogenic [3]. Microbial degradation of PAHs is considered an important natural process for the decomposition of these contaminants, and it represents an alternative solution for the problems related to environmental pollution. White-rot fungi have been extensively exploited for their ability to transform PAHs in the environment. These fungi produces extracellular enzymes such as phenoloxidases (laccase and tyrosinase), H2O2-producing enzymes, and dioxygenases with very low substrate specificity, making them suitable for the degradation of a variety of organic pollutants [4–6].



It is known that ligninolytic enzymes perform one-electron oxidation, producing cation radicals of pollutants, and leading to the formation of quinines [7]. Manganese peroxidase (MnP), one of the most common ligninolytic enzymes produced by white-rot fungi, is a heme containing glycoprotein that needs hydrogen peroxide as an oxidant while lignin peroxidase (LiP) is containing one high-spin ferric heme group per molecule of enzyme [8–10]. Laccase is a multicopper oxidase, plays an important role in the global carbon cycle and could help in degrading a wide range of xenobiotic compounds such as synthetic dyes [11]. While biodegradation of low molecular weight PAHs has been extensively studied, the biotransformation of PAHs containing four or more rings is less explored, and therefore, perhaps less well understood [12]. The genus Armillaria, a white-rot fungus, has been extensively investigated for its application in decolorization of some synthetic dyes and degradation of naphthalene [13, 14]. Its capacity to degrade and transform high molecular weight PAHs is not well known, thus deeming the characterization of microorganism as well as studies on pyrene utilization quite interesting. Since the objectives of this study were to evaluate the ability of Armillaria species to degrade pyrene in liquid culture, we quantified biomass production, glucose consumption, and extracellular enzymes production. The metabolites involved during degradation of pyrene were also identified and characterized.

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production with pyrene utilization, sufficient pyrene (5 mg/L) was dissolved in dimethylformamide (DMF), tween 80, and benomyl (300 mg/L) for inhibiting bacterial growth, and then this solution was added to the liquid medium with continuous stirring in order to disperse the insoluble pyrene. Modified mineral salt broth medium (MSB) was used according to the details reported in previous study [4]. Experiments were performed using 100-mL Erlenmeyer flasks containing 20 ml of fungal cultures and 5 mg/L of pyrene. For each experiment, 20 mL of pre-grown Armillaria sp. F022 cultures were autoclaved and used as positive controls. All the cultures were incubated at 25 °C on a rotary shaker in the dark for 7, 15, 22, and 30 days. All experiments were performed in triplicate. For analyzing the pyrene-derived metabolites in the liquid medium, the fungi were incubated in MSB supplemented with 10 mg/L pyrene for 0–30 days. Extraction and analyses of the pyrene metabolites were performed as described in our previous study [4]. Biomass production was determined by weighing the cell mass produced, which was pelleted by the centrifugation of the culture broth at 1,000 rpm for 45 min, followed by removal, washing, and filtering the pellet through a pre-dried and pre-weighed filter paper. The filter paper was dried until a fixed weight was achieved. The biomass of fungi was defined in mg/L. Enzyme assays

Malt extract and polypeptone were purchased from Difco (Detroit, USA). TLC aluminum sheets (Silica gel 60 F254, 20 9 20 cm) were obtained from Merck (Darmstadt, Germany). Pyrene and all other chemicals with highest analytical standard were purchased from Sigma-Aldrich (Milwaukee, USA).

Cells grown in the presence of pyrene were harvested at different time intervals and filtered through glass wools to eliminate residual pyrene. The cultures were centrifuged (10,000 rpm, 15 min) at 25 °C and rapidly washed thrice with 50 mM phosphate buffer (pH 7) after which the cells were disintegrated with a probe-type sonic oscillator for 10 min. The extract was centrifuged at 15,000 rpm at 4 °C for 20 min to remove whole cells and large amounts of debris. An enzymatic analysis of phenoloxidases and dioxygenases was undertaken in accordance with a previous study [4].

Microorganism and growth conditions

Extraction and analysis of metabolites

Armillaria sp. F022 used in this study was isolated from a fruiting body found in decayed wood in Samarinda, Indonesia [13]. It was isolated by cultivating a small part of the internal tissue removed from the fruiting body on a malt extract agar (MEA) plate at room temperature. The MEA contained 20 g/L of malt extract, 20 g/L of glucose, 1 g/L of polypeptone, and 15 g/L of agar. The white-rot fungus Armillaria sp. F022 was selected for this investigation because of the extracellular enzymes activities demonstrated during the degradation of low molecular weight PAHs such as naphthalene [14]. To correlate biomass

To determine the amount of residual pyrene in solution, aliquots were withdrawn at regular interval, the aqueous solution and fungal mycelia were separated by filtration, and the solution was extracted with thrice with equal volumes of ethyl acetate (EtAc, 40 mL). Recovery of pyrene under the extraction conditions was 98 %. The extracts were then dried (anhydrous Na2SO4) and the volumes were reduced to 1 mL (N2). The degradation products of residual pyrene in the solution were detected by TLC using hexane– dichloromethane (40:10 v/v) as the running solvent system for short- and medium-term incubations, and

Materials and methods Chemicals


Bioprocess Biosyst Eng (2013) 36:461–468

dichloromethane-ethyl acetate (20:30 v/v), for long-term incubation. The spots on the TLC were visualized under UV light, and individual compounds, namely pyrene, and the metabolic products were identified by comparison of their Rf values and the color of spot fluorescence with those of standard compounds in a specific solvent system. The GC–MS analyses were performed on Agilent 5975E FID GC–MS and Shimadzu QP2010 equipped with DB-1 capillary column (30 m length, 0.25 mm diameter, and 0.25-lm film thickness). The temperature program for GC was set as follows: 80 °C (2-min isothermal), 80–150 °C (at 15 °C/min), 150–320 °C (at 30 °C/min) and 320 °C (5-min isothermal). Flow rate of the helium carrier gas was 1 mL/min. The following conditions were applied for mass analysis: injector and interface temperatures of 260 °C; ionization mode, detector at 1.3 eV, 1-s scan intervals, and mass range m/z of 50–500. The mass spectra from individual total ion peaks were identified by comparison with corresponding authentic standards and the mass spectra database of Wiley 275L [15]. Derivatization Derivatization is routinely carried out to transform the chemical structure and this increases both volatility and/or thermal stability of the compounds to make them more readable by GC. The frequently used derivatization methods are silylation and methylation, which can be used to convert compounds containing active hydrogen groups, thus changing polar-reactive compounds into nonpolarinert ones. In our research, silylation was achieved by introducing trimethylsilyl group using N,O-bis-trimethylsilyl acetamide (40 lL), pyridine (40 lL), and trimethylchlorosilane (20 lL). The reaction was effected at 80 °C for 10 min without intervention of moisture. Methylation was accomplished by diazomethanylation using phenoxyacetamidopenicillanic acid 1-oxide dissolved in methylene dichloride and ethanol. The end-point was indicated by the change in the color of the solution to yellow.


Fig. 1 Utilization of pyrene and glucose by Armillaria sp. F022 during degradation

time under these conditions was 30 days and the rate of pyrene utilization was 0.195 mg/L/day. It was also evident that pyrene degradation by Armillaria sp. F022 was correlated with the rate of glucose consumption in the culture. As anticipated, pyrene utilization was slightly slow in the first 2 weeks after incubation when glucose was rapidly consumed by the fungus for its growth, followed by breakdown of pyrene as a source of carbon and energy, leading to an increase in the biomass. Previous studies have reflected on inability of the white-rot fungi to utilize the organic pollutant as a sole source of carbon and energy for mycelial growth and enzyme production. A carbon source, such as glucose, had to be added to the culture medium as a co-metabolic substrate. Glucose could play a major role in converting the short chain fatty acids in the enhanced biological phosphorus removal system. Microorganisms, such as bacteria and fungi, have been found to grow more rapidly when the polluted liquid culture was supplemented with glucose. The organic compound was more rapidly degraded when glucose was used as a co-substrate for growth [16–19]. On the contrary, in most basidiomycetes,

Results and discussion Utilization of pyrene and glucose by Armillaria sp. F022 during degradation To correlate pyrene utilization with glucose consumption, Armillaria sp. F022 was inoculated into MSB containing 5 mg/L pyrene (Fig. 1). Residual pyrene was B19 % after 30 days of incubation, while the pyrene in the autoclaved control showed stagnan elimination (96 % remaining). The most rapid utilization of pyrene (0.61 mg/L/day) was observed in the third week after incubation, the generation

Fig. 2 Biomass production and enzyme activity by Armillaria sp. F022 during pyrene degradation



Bioprocess Biosyst Eng (2013) 36:461–468

Fig. 3 GC chromatogram of pyrene metabolisms after short-term incubation (a), medium-term incubation (b), and long-term incubation (c)

glucose or other carbon sources supplied the important substrates for enzyme production and cell growth. While the production of ligninolytic enzymes occurs during the secondary metabolism, and is affected by nutrient limitation, the process of degradation occurs even after enzyme production [20]. Another study showed that degradation was repressed by glucose, because glucose can inhibit the utilization of the target compound [21]. It is slightly different in case of Armillaria species, compared to most basidiomycetes, in that degradation occurred throughout the growth phase of fungal cells, and mostly depended on the availability of sufficient nutrients. Degradation by basidiomycetes thus suggested a different role for glucose, and therefore, an unknown mechanism of degradation.


Biomass production and enzyme activity by Armillaria sp. F022 during degradation The process of enzyme activity against biomass production was detected as shown in Fig. 2. The cell weight of Armillaria sp. F022 was C1,800 mg/L after 30 days of incubation. The average growth of the fungus was 61.5 mg/L/day, while the fastest growth was shown in 2 weeks of incubation (113.2 mg/L/day). Enzymes production during the degradation process by Armillaria sp. F022 was certainly related to the increase in biomass. In the first week after incubation, the growth rate rapidly increased, but enzyme production decreased. After 7 days of incubation, rapid growth was observed, whereas the

Bioprocess Biosyst Eng (2013) 36:461–468


Fig. 4 Mass spectra of metabolite I identified as 1-hydroxypyrene (a) and methylated derivative of metabolite II identified as 4-phenanthroic acid (b)

enzymes were produced only after a good amount of biomass was generated. Several enzymes such as MnP, LiP, laccase, 1,2-dioxygenase, and 2,3-dioxygenase were detected in the culture. The highest activity were displayed by laccase (117.3 U/L) after 30 days of incubation, followed by 1,2-dioxygenase (62.3 U/L). Minor activities were shown by MnP, LiP, and 2,3-dioxigenase towards the end of the incubation period. The ligninolytic and dioxygenase enzymes are important for the conversion of various organic pollutants, such as PAHs, halogenated compounds, and synthetic dyes. Dioxygenase is commonly considered as the bacterial enzyme responsible for degradation of PAHs [22, 23]. The enzyme activity proceeded in a manner similar to that of pyrene degradation by Armillaria sp. F022. Laccase and 1,2-dioxygenase activities rapidly increased and were expended within 2 weeks of incubation, which was in line with the rapid rate of pyrene degradation on 15 days of incubation. Identification of metabolites Examination of the acidic extract revealed the presence of some substantial metabolites, which, on partial purification by silica gel column chromatography, indicated that the compounds were derived from pyrene. The presence of different intermediates in the degradative pathway was detected using UV–Vis spectrophotometry by combining

the short- (1–7 days), medium- (8–16 days) and long-term (17–30 days) incubation extracts. GC–MS analyses of the extracts from the three sets revealed the presence of 5 metabolites (Fig. 3). Finding from TLC analyses which obtained using the ethyl acetate-extractable metabolites of short-term incubation of pyrene formed by Armillaria sp. F022, showed the presence of 1 metabolite. This metabolite (I) had an Rf value of 0.65, similar to that of 1-hydroxypyrene. The spectrum of compound I (m/z 218, M?) which had a retention time (tR) of 9.4 min and the significant fragment ions at m/z 94, 136, 148, 189, and 190 (M?-17) corresponding to the respective sequential losses of –OH was identical to that of the standard 1-hydroxypyrene. Armillaria sp. F022 degraded pyrene by converting 1-hydroxypyrene to pyrenequinone within the period of short-term incubation. Unfortunately, the presence of pyrenequinone was not confirmed in our culture extract. However, when a medium-term incubation extract was analyzed in subsequent samples, other peaks from the short-term incubation extract were decreased, and new peaks appeared. The retention time and fragmentation patterns of compound II matched with those of authentic 4-phenanthroic acid, respectively. Pyrene peaks were still apparent on the chromatogram, probably because pyrene was not completely degraded after medium-term incubation. Metabolite II had a Rf value of 0.47, and the retention



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Fig. 5 Mass spectra of silylation derivative of metabolite III identified as 1-hydroxy-2-naphthoic acid (a), metabolite IV identified as phthalic acid (b), and metabolite V identified as protocatechuic acid (c)

time peak at 8.3 min refers to authentic 4-phenanthroic acid, with a molecular ion (M?) at m/z 236, corresponding to the molecular mass of monomethylated 4-phenanthroic acid and fragmentation ions at m/z 131, 151, 177 (M?-59), 205 (M?-31, CH3O loss) and m/z 221 (M?-15, CH3 loss), which are characteristic of a methylated carboxylic acid. This fragmentation pattern as well as a search of the mass spectral library suggested that this metabolite was 4-phenanthroic acid (Fig. 4). TLC of the ethyl acetate-extractable metabolites from long-term incubation with pyrene showed 3 metabolites. Compound III showed a Rf value of 0.57, similar to that of standard 1-hydroxy-2-naphthoic acid. The retention time peak at 8.6 min refers to authentic 1-hydroxy-2-naphthoic acid. MS analysis of the 1-hydroxy-2-naphthoic acid


produced from pyrene-1,6-quinone gave an apparent molecular ion [M?] at m/z 332 for TMS-derivatives and apparent losses of M?-15 at m/z 317 corresponding to the respective sequential losses of methyl (–CH3), as well as some expected fragment ions at m/z 114, 147, and 73 which are characteristic of a silylation of 1-hydroxy-2-naphthoic acid. Based on data obtained above, the compound was proposed to be 1-hydroxy-2-naphthoic acid. Compound IV was possibly phthalic acid as reflected by a major peak at 9.1 min and its mass spectrum. MS analysis of the phthalic acid produced from 1-hydroxy-2-naphthoic acid gave an apparent molecular ion (M?) at m/z 310 for TMS-derivatives and apparent losses of M?-15 at m/z 295 corresponding to the respective sequential losses of methyl (–CH3), and other fragment ions at m/z 73, 147, 221, and

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Fig. 6 Proposed pathway for the degradation of pyrene by Armillaria sp. F022. Compounds between brackets were not identified in our culture extract

265. Based on the data above, the compound should be phthalic acid. Compound V had a GC retention time of 11.3 min. MS revealed this metabolite to have a molecular ion (M?) at m/z 370, fragmentation ion at m/z at 355 (M?15), representing a probable loss of CH3 as well as the expected fragment ions at m/z 73, 193, 281, and 311 which are characteristic of a silylation of protocatechuic acid. The mass spectral fragmentation pattern suggested that this metabolite was protocatechuic acid. This indicates that 1-hydroxy-2-naphthoic acid was converted into protocatechuic acid via phthalic acid (Fig. 5). The metabolism of pyrene by white-rot fungi is a complex process involving some enzymes and different catabolic pathways, as well as the production and accumulation of several metabolic products. The present research confirmed the essential features of the major pathway of pyrene as illustrated in Fig. 6. In case of Armillaria sp. F022 grown on pyrene as the source of carbon and energy, the initial attack occurs at the 4-position, by monooxygenase or dioxygenases, to form a hydrodiol, and it is further


converted to hydroxypyrene. This is followed by ring cleavage to form phenanthrene-4,5-diol, which on decarboxylation provides 4-phenanthroic acid. During the degradation process, Armillaria sp. F022 did not accumulate large concentration of aromatic metabolites, and this may be correlated to the physical interactions between the fungal mycelia and hydrophobic compounds [24]. 1-Hydroxypyrene can be further degraded into a major product 1-hydroxy-2-naphthoic acid. 1-Hydroxy-2-naphthoic acid could then be subjected to meta-cleavage to yield phthalic acid. Since, phthalic acid was isolated, it implied that one of the two routes for the degradation of 1-hydroxy-2-naphthoic acid degradation was operating. The presence of other salicylic acid-producing pathways identified by Hadibarata et al. was not observed in Armillaria sp. F022 cultures, indicating that either this pathway may not be operating in this fungus or the metabolites formed were not in concentrations adequate for detection. Phthalic acid can also be further converted to benzoic acid, which could undergo to form catechol intermediates [25, 26]. Although our finding is similar to pyrene metabolism in bacteria reported by Walter et al. in that the recyclization of the initial ring cleavage product was performed by Rhodococcus sp. UW1 in pyrene degradation, the site of the first attack could not be established as the 1, 2 or 4, 5 positions [27]. In addition, previous studies have shown that the metabolites of pyrene were discovered through the identification of 1-hydroxypyrene, 4-hydroxyperinaphthenone, 4-phenanthroic, acid, cinnamic acid, and phthalic acid. Dioxygenase played an important role in the formation of 4,5-phenanthrenedioic acid, which, on the loss of a carboxyl group, formed 4-phenanthroate. Further metabolism confirms that 4-phenanthroic acid was converted to give 1-hydroxy-2-naphthoic acid, phthalic acid and cinnamic acid, suggesting that two metabolic pathways are involved in pyrene degradation [28, 29]. However, cinnamic acid was not detected in our culture extract. In this study, protocatechuic acid was identified to be present in the culture extract, and growth was observed in the medium containing protocatechuic acid.

Conclusions In view of the results obtained in this study, it can be concluded that a white-rot fungus, Armillaria sp. F022, has great capability in the degradation of high molecular weight PAHs. The activities of two key enzymes, i.e., laccase and 1,2-dioxygenase, played an important role in the complicated mechanism of pyrene degradation by Armillaria sp. F022. Currently, more studies in degradation of high molecular weight PAHs, such as benzo[a]pyrene and perylene, are underway in our laboratory.


468 Acknowledgments A part of this research was financially supported by a Research University Grant of Universiti Teknologi Malaysia (Vote QJ1.3000.2522.02H65), which is gratefully acknowledged.

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