Journal 
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants produced from incompletely burned organic materials, oil, petroleum gas, coal, wood (Arun et al. 2008). PAH contamination has been received public and scientific attention owing to their recalcitrant properties and mutagenic or carcinogenic effects (Lei et al. 2007). Human bodies also can be damaged by PAHs via skin contact by direct inhaling and ingesting. Exposures to PAHs have been linked to skin, lung, liver, intestine, and pancreas cancers (Ting et al. 2011). For these reasons, many attempts have been tried to remove hazardous PAH compounds from contaminated environments. Among many process to degrade and cleavage PAHs by a variety of microbes, fungal degradation has been received great attention. One of the top abilities of white rot fungi (WRF) is to degrade lignin, a biopolymer from wood chemicals, and also a variety of hazardous chemical pollutants, such as PAHs (Suhara et al. 2003). WRF produce extracellular ligninolytic enzymes, laccases, manganese dependent peroxidases (MnP), and lignin peroxidases (LiP), because their very low specificity for substrate and they are able to break down the irregular structure of lignin (Casas et al. 2009). These enzymes are directly involved in the biodegradation of a wide range of xenobiotic pollutants. The ligninolytic enzymes can be induced by aromatic or phenolic compounds, such as ABTS, 2,5-xylidine, ferulic acid, or veratryl alcohol (Niladevi and Prema 2008). In addition, the use of stimulators to enhance the production of ligninolytic enzymes has been extensively approached in WRF where enzyme stimulation by aromatic compounds is well recognized (Terron et al. 2004; Lee et al. 2015).
Phlebia brevispora, a white rot fungus, has been investigated to degrade endocrine disruptors, such as polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDD/PCDFs) and 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) (Kamei et al. 2005; Xiao et al. 2011). It was also reported that chloronaphthalene, PCBs (Polychlorinated biphenyls) and PAHs were significantly mineralized by Phlebia species (Mori et al. 2003; Kamei et al. 2005). However, Phlebia brevispora is not well known to degrade recalcitrant organic pollutant.
Phenanthrene, anthracene, fluoranthene, and pyrene are listed on the standard sixteen PAHs by the U.S. EPA as priority pollutants (ATSDR 1995). And these four PAHs display representative structures of 3- and 4-fused benzene rings, respectively (Lee et al. 2014). PAHs are existed with mixture of PAHs and other pollutants, such as dye, heavy metal in soil, sediment environment (ATSDR 1995). Owing to its vivid biotechnological applications, ligninolytic enzyme induced by RBBR can be capable of simultaneous degradation of PAHs and decolorization of dye waste water. In this study we evaluated the degrading ability of P. brevispora KUC9045 against four PAHs and its ligninolytic enzymes were also quantified.
Materials and Methods
1.Chemicals
Four PAHs used in this study, phenanthrene (PHE), anthracene (ANT), and fluoranthrene (FLT), and pyrene (PYR) with 99.0% analytical standards were purchased from the Aldrich Chemical Co., St. Louis, USA, Steinheim, Germany and China, respectively. Acetone used a solvent was purchased from Samchun Chemical Co. Ltd., Pyeongtaek-si, Korea. Remazol Brilliant Blue R (RBBR) with ~50% of dye content was purchased from Aldrich Chemical Co., Milwaukee, WI, USA. Individual PAH was initially dissolved in acetone at a concentration of 50,000 mg L-1. PAHs mixture was prepared by mixing four PAHs into one stock; thus, the concentration of each PAH in this mixture was 50,000 mg L-1.
2.Organisms and medium
Phlebia brevispora KUC9045 was obtained from Picea abies logs in Icheon, Korea and isolated on 2% malt extract agar (MEA, malt extract 20 g, agar 15 g, distilled water 1 L) amended with benomyl (4 mg) to support only the growth of basidiomycetes (Wang and Zabel 1990). To identify white rot fungi specifically, fungal DNA extraction and PCR were performed following the experimental protocols by Lim et al. (2005). To amplify partial large subunit (LSU) ribosomal DNA region, the PCR was conducted using the fungal universal primers, LR0R/LR3, which were used to conduct a BLAST search of the GenBank database (Altschul et al. 1994). DNA sequencing of PCR products were performed using the aforementioned primers and an ABI 3730XL (Macrogen, Korea). The sequences obtained in this study were deposited under the GenBank accession no. AB084614. The fungus was subcultivated every five days, and all isolates were deposited. All isolates were deposited in sterile water at 4℃ in the Korea University Culture Collection (KUC), Korea University, Korea.
3.Experimental design
Experiments were performed using Erlenmeyer flasks (250 mL) containing 100 mL of 2% ME liquid medium. And they were aseptically inoculated with ten agar plugs (5 mm diameter) of fungal pre-culture. The cultures were incubated on a rotary shaker at 150 rpm (27℃) after the addition of PAHs stocks. The concentration of individual PAH was determined by 50 mg L-1. Uninoculated flasks were treated similarly as an abiotic control, and P. chrysosporium KCTC 6293 was used as a positive control, which is well known as an effective degrader of PAHs (Bamforth and Sigleton 2005). For enhancement of degradation of PAHs, RBBR was supplemented into liquid fungal culture at concentration of 100 mg L-1 and 200 mg L-1, respectively. With the supplement of RBBR, four PAHs stock was aseptically spiked with the concentration of 50 mg L-1, respectively. And the cultures were incubated in agitation for two weeks since fungi were inoculated in ME liquid media. All samples were carried out in triplicates.
4.Analytical methods
The entire liquid and solid part of fungal culture of P. brevispora KUC9045 was extracted every other day for 14 days. And the abiotic and positive control cultures were extracted on 14th day after incubation. Each sample was extracted tree times of 100 mL of dichloromethane (Duksan, Korea) to finally obtain 300 mL of each extracts. Each extracted sample was concentrated to 10 mL using a vacuum rotary evaporator (Eyela N-1000 series, Japan). The concentrations of PAHs in the extracts were measured by injecting 1 μL of the extract into a gas chromatograph-mass spectrometer (GC-MS, Agilent technologies, Model 7890, Santa Clara, CA, USA). Separation was achieved using a DB-5MS (0.25 mm diameter by 30 m length, film thickness 0.25 μm). The GC-MS analysis was conducted according to the method described by Lee et al. (2010).
5.Enzyme assays
For three ligninolytic enzyme activity, laccase, manganese- dependent peroxidase (MnP), Lignin peroxidase (LiP), were measured using UV-VIS spectrophotometer after removal of mycelium using a syringe filter (0.45 μm). 2% ME liquid media were used for comparing enzyme activity. The ligninolytic enzyme activity was processed according to the method described by Kirk et al. (1986). The LiP was assayed by measuring the oxidation of veratryl alcohol to veratraldehyde based on the increase of absorbance at 310 nm in pH 2.5 sodium tartrate buffer solution (Tien and Kirk 1988). The MnP was determined by the oxidation of 2,6-dimethoxylphenol as indicated by the increase of absorbance at 469 nm in pH 4.5 sodium malonated buffer (Wang et al. 2008). The laccase was measured with 2,2-azinobis-3-ethylbenzothiazoline- 6-sulfonic acid as the substrate at 420 nm in pH 4.5 sodium acetate buffer (Novotný et al. 2004). One unit of enzyme activity procuced 1 μmol of reaction product per minute under the assay conditions at room temperature and the activity was expressed in U mL-1 (Hadibarata et al. 2009).
6.Data analysis
Data in terms of degradation were analyzed using the Statistical Analysis Systems (SAS 9.1, SAS Institute, Inc., Cary NC, USA). One-way analysis of variance (ANOVA) was used to assay significance of degradation among species in the tukey test. The mean values were compared by using the least significant difference index at P<0.05.
Results and Discussion
1.Comparison of P. brevispora with controls on PAHs degradation
The PAH concentrations in the cultures were determined at the end of the 14 days incubation. The percentage of degradation of phenanthrene, anthracene, fluoranthene, and pyrene in abiotic control showed 22.4%, 17.8%, 25.1%, and 14.5%, respectively (Table 1). And as a positive control, P. chrysosporium KCTC6293 degraded PHE (35.7%), ANT (43.1%), FLT (28.6%), and PYR (32.6%), respectively. By compared with the controls, P. brevispora KUC9045 demonstrated significantly higher rates of degradation of all PAHs examined in this study with PHE (66.3%), ANT (67.4%), FLT (61.6%), and PYR (63.3%), respectively (P< 0.05). Thus, it was concluded that the PAHs were degraded in all of the following experiments by the fungal action. In the previous study, P. brevispora KUC9045 showed high tolerance against the four PAHs over 90%. It means the percentage of mycelial growth inhibition (MGI) of the species on MEA solid media amended with the four PAHs were expressed as 0≤%MGI<10 (Lee et al. 2014). With the high resistance, P. brevispora KUC9045 were grown in the presence of four PAHs and the species took carbon source as a nutrient from the PAHs, mineralizing their recalcitrant structure.
2.Time course of PAHs degradation by P. brevispora
As time goes by, degradation rates of four PAHs by P. brevispora KUC9045 was increased within 14 days (Fig. 1). Among four PAHs, degradation of ANT was demonstrated with the lowest efficiency after two days of incubation. Meanwhile, at the end of 14 days incubation, P. brevispora KUC9045 degraded ANT with the highest rate (67.4%). It is considered that three PAHs except for ANT, were initially used as a carbon source. However, it cannot be speculated that the chemical structure of three PAHs were easy to be digested. Consequently, the species degraded ANT with the highest rate and PHE, FLT, PYR were followed by that. It indicates that probability to access three PAHs is relatively high due to more carbon presented than ANT. With the results, P. brevispora KUC9045 shows a valuable fungal resource for the degradation of ANT. In fact, Peniophora incarnata, which is known as one of the greatest degrader of PAHs, were enhanced its draw back in the degradation of ANT (Lee et al. 2015). Meanwhile, P. brevispora KUC9045 degraded ANT more than other PAHs. It indicates that there may be a major target such as ANT for the species in spite of the high tolerance against PAHs.
In addition, molar mass of three-benzen ring PAHs, which is ANT and PHE is C14H10 and four-benzene ring, which is FLT and PYR is C16H10 (Bamforth and Sigleton 2005). Because of Carbon number and molar mass might related with accessibility of fungal mycelium. In addition, the reason that densities of ANT (1.25 g m-3) and PYR (1.27 g m-3) were known higher than PHE (1.18 g m-3) and FLT (1.25 g m-3), it demonstrated slightly differ in degradation rates on 14 days.
3.Ligninolytic enzyme during degradation of PAHs
Three main ligninolytic enzymes, LiP, laccase, and MnP were measured every day during degradation of PAHs for 14 days. Among three enzymes, LiP was not produced from P. brevispora KUC9045. Meanwhile, laccase and MnP were produced with high efficiency during degradation of PAHs (Fig. 2).
It is considered that laccase and MnP are mainly secreted and functioned during the degradation of PAHs by the species among many known enzymes. During the degradation of PAHs, there is a main enzyme for the individual fungi (Janusz et al. 2013). Promoter region to secrete enzyme as a protein is differentially expressed in the fungi during the mineralization of the target compound (Jang et al. 2012).
In time course of ligninolytic enzymes produced, trends of production of two enzymes were similarly demonstrated. On ninth day of incubation since four PAHs were spiked, laccase and MnP were produced with high efficiency and then these enzymes were temporarily decreased and eventually increased higher than on ninth day. The activity of these enzymes might be related to selectively degrade PAHs. As degradable trends of ANT were changed from 10 day of incubation, the species produced ligninolytic enzymes to focus on the target PAH. And ANT and PHE were target PAHs by P. brevispora KUC9045 in this study.
4.E nhanced degradation of PAHs with decolorization of RBBR
To brief the results, RBBR was supplemented into liquid media to enhance the degradation of PAHs (Fig. 3). After two weeks of the incubation, RBBR was fully decolorized (data not shown). It was photometrically monitored using 1 mL of dye content from the culture at 593 nm of UV-VIS spectrophotometer, which is the maximum wavelength for visible absorbance of this dye (Casieri et al., 2010). As increased decolorization rate of RBBR, four PAHs simultaneously degraded according to the different concentration of RBBR. Entirely, degradation rates of four PAHs were increased after two weeks more than one week. It indicates that PAHs can be degraded in the presence of RBBR as time goes by, and the simultaneous degradation of PAHs and dye was achieved. Interestingly, three PAHs, PHE, FLT, and PYR, were degraded in 200 ppm of RBBR more than in the media with lower concentration of RBBR. It means that the stronger and more hazardous environment force P. brevispora KUC9045 to degrade PAHs with higher rate. The degradation of PAHs is correlated with the production of ligninolytic enzymes. On the basic knowledge of the study, laccase and MnP were considered to play important roles in the degradation of recalcitrant organic pollutants by their induction. Laccase and MnP can be induced with supplement, and the removal of PAHs can be enhanced by the induction of ligninolytic enzymes (Lee et al. 2015). RBBR was used as a supplement in the study, and the degradation of the three PAHs were enhanced with decolorization of the highest concentration of RBBR. Nevertheless ANT was removed with the highest degradation rate (Fig. 1), ANT was not degraded more in the presence of the RBBR by the species. However, P. brevispora KUC9045 was recognized as a great degrader of ANT with the statistical degradation rate among four PAHs.
Thus, we suggest P. brevispora KUC9045 is valuable fungal resource for remediation of mixed contaminated envirionments with recalcitrant organic pollutants and dye. And it might be applicable for dedgradation of PAHs and decolorizaiton of dye waste water simultaneously.
Conclusion
Korean indigenous fugal isolates, P. brevispora KUC9045 demonstrated effective degradation of four PAHs, which is anthracne, phenanthrene, fluorancene, and pyrene, coincidentally. And the ligniniolytic enzymes could be helpful to degrade PAHs. As RBBR was supplemented, laccase and MnP were induced to produce and the degradation was enhanced by ligninolytic enzyme inducer. In addition, dye contents of RBBR were completely decolorized with the mineralization of PAHs into liquid media. In the results of our study, degradation of four PAHs could be coincidentally operated by decolorized waste water due to large amounts of ligniolytic enzymes induced by RBBR dye. Therefore simultaneous biodegradation of recalcitrant chemical pollutants and decolorization of dye waste water can be operated by indigenous fungal isolates, P. brevispora KUC9045. And the species is capable of biotechnological application for degradation of a various xenobiotics.
