Degradative pathways of polycyclic aromatic hydrocarbons (PAHs) by Phanerochaete chrysosporium under optimum conditions

ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) constitute one group of priority environmental contaminants which must be disposed. Phanerochaete chrysosporium is one of the white rot fungi (WRF) used in the present study to investigate different conditions affecting its ability to utilize PAHs as a sole carbon and energy sources. P. chrysosporium was grown under optimum conditions, and its degradation percentage and degradative pathways have been determined by HPLC and GC/MS. The results showed that the optimum condition for the growth of P. chrysosporium was five discs inoculum size at 25°C for 7 days incubation period on 100 mg/L of each PAHs in BSM supplemented with 2000 µM MnSO4 with shaking. Four groups (I–IV) of optimum conditions were used to determine degradation percentage. The results cleared that the best PAHs in degradation was pyrene (Pyr.). P. chrysosporium degraded (100%) Pyr. under the four groups. P. chrysosporium degraded the six PAHs (Acen.; Anth.; Flu.; Naph.; Phen.; and Pyr.) efficiently. The intermediates resulted from degradation indicated that P. chrysosporium first oxidized the middle ring or hetero ring followed by ring fission. P. chrysosporium followed the phthalate route in its degradative pathway. The intermediates finally interred TCA cycle and give CO2 and H2O or short chain aliphatic polymerized to give long chains.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are fused ring aromatic compounds of two or more fused rings. Science PAHs are carcinogenic, mutagenic, and teratogenic, and they represent a great environmental concern (Torres-Farrada et al., 2019;Abo-State & El-kelani, 2020).
The United States Environmental Protection Agency (USEPA) classified 16 PAHs as the most priority pollutants that must be disposed (White, 1986;Eggen & Majcherczyk, 1998). As the number of rings in PAHs increased, resistance to degradation and their risk increased (Garon et al., 2000;Bishnoi et al., 2005;Bishnoi et al., 2008;Agrawal et al., 2019;Lee et al., 2020). With increasing molecular weight, PAHs tend to have increased in hydrophobicity (low aqueous solubility) and accumulate in the environment (soil and/or water) (Haritash & Kaushik, 2009).
White rot fungi (WRF) can represent as a good candidate to remove such PAH compounds efficiently and ecofriendly (Biotechnological approach) for both low molecular weight (LMW) and high molecular weight (HMW) -PAHs. And can transform these compounds to non-harmful, simpler nontoxic or even CO 2 and H 2 O, which means complete mineralization (Berekaa, 2013;Kuppusamy et al., 2017;Adnan et al., 2018;Agrawal et al., 2019;Lee et al., 2020).
Optimization of PAHs degradation depends on efficient screening and selection of good candidates WRF and changing degradation conditions. Different microorganisms have optimum conditions for growth and also have different degrading efficiencies due to toxic compounds (Bao et al., 2013;El-Borai et al., 2016). For improving biodegradation, WRF must remove these toxic compounds. To enhance degradation capacities, a wide range of factors were included (molecular structure of PAHs, culture media composition, and fungal species). However, temperature, pH values, inoculums size, moisture, and toxic compound concentrations are known to be conditions that affect the WRF degradation rate (Hofrichter et al., 1998;Chang et al., 2012;Yang et al., 2013).
Biodegradation can be done even at extremely cold temperatures (5°C), so biodegradation will be more efficient and can occur over a wide range of temperatures (Eriksson et al., 2001;Leys et al., 2004). But PAHs solubility increased with an increase in temperature (Margesin & Schinner, 2001), which increased PAHs bioavailability. Also, oxygen solubility decreases with increasing temperature, which may reduce the activity of aerobic microorganisms agitation that may have a bad effect on the ligninolytic enzyme secretion, which contributes in PAHs degradation. All these various factors were connected with biodegradation efficiency (Bishnoi et al., 2008;Abo-State et al., 2011a, Abo-State et al., 2011bHadibarata & Chuang, 2014;El-Borai et al., 2016;Lee et al., 2020).
P. chrysosporium secretes MnP and LiP, which may be the primary enzymes responsible for degradation of PAHs (Wang et al., 2009). The peroxidases are heme -containing enzymes having known catalytic cycles. One molecule of H 2 O 2 oxidizes the resting enzyme generating two electrons. The peroxidase is reduced back in two steps of one electron oxidation (Kadri et al., 2017). For better understanding of degradation pathways by WRF, the degradation process includes the presence of ligninolytic enzymes and cytochrome P450 monooxygenase (P450s) (Z. Li et al., 2018).
The aim of the present study was to study the factors affecting the degradation process by the white rot fungus (P. chrysosporium) and its pathway in the degradation of six PAHs (Acenaphthene, Anthracene, Fluoranthene, Naphthalene, Phenanthrene, and Pyrene) especially a little have been mentioned on WRF-biodegradation pathways comparing by bacterial degradation pathways.

Microorganism
Phanerochaete chrysosporium ATCC 32629 is one of the white rot fungi (WRF). P. chrysosporium used in the present study was purchased from Microbiological Resources Center (MIRCEN), Faculty of Agriculture, Ain-Shams University, Cairo, Egypt.

Culturing of WRF
Phanerochaete chrysosporium ATCC 32629 was cultured on 2% (w/v) Malt Extract Agar (ME) (Oxoid, 1982) plates and slants. The slants were kept at 4°C until needed. Phanerochaete chrysosporium can be activated by subculturing on Potato dextrose agar (PDA) plates (Oxoid, 1982) or on ME plates and incubated at 28°C for 14 days.

Culture medium for PAHs degradation
Basal Salt Medium (BSM) (Wen et al., 2011) was a liquid medium with modification (Abo-state et al., 2013a; Abo-State et al., 2013b, 2014 and 2018) was used for biodegradation of PAHs by P. chrysosporium. This medium was used for PAHs degradation by P. chrysosporium. BSM was amended by 100 mg/L of each PAH compound.

P. chrysosporium inoculum preparation
From the margin of actively growing (14 days) fungal culture (P. chrysosporium) on PDA agar plates, agar discs (6 mm diameter) were cut out. These discs were used to inoculate BSM for biodegradation of PAHs.

Optimizing biodegradation of PAHs by P. chrysosporium under certain conditions
In 500 ml conical flasks containing 150 ml BSM and sterilized by autoclaving, then amended by 100 mg L −1 concentration of each one of the six examined PAHs, the biodegradation experiment was performed.
Three replicates were used for each PAH compound, for each parameter. After incubation period, all BSM were filtrate, extracted and analyzed to know the fungal growth, fungal activity and degradation rate of six PAHs tested.

P. chrysosporium growth and protein determination
The P. chrysosporium mycelia at the end of incubation period were removed from BSM cultures by filtration via Whatman filter paper and dried at 60°C for constant weight. Biomass (fungal mycelia) was weighed for the quantitative determination of P. chrysosporium growth (Dry weight).
The fungal filtrates of cultures were utilized for the determination of the efficient degradation rate of PAHs. The fungal activity was determined by measuring the extracellular protein secreted according to Lowry et al. (1951) periodically for each incubation period by using a Spectrophotometer (LW-V-200 RS UV/VIS, Germany) at National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), Naser City, Cairo, Egypt.

High performance liquid chromatography (HPLC) analysis
Quantitative analysis of residual PAHs in BSM was performed by Liquid/Liquid (1:1 v/v) extraction. P. chrysosporium (BSM): Chloroform (1: 1 v/v) was used. The chloroform layer (extracted samples) was dried by evaporation to a fixed volume, then analyzed by HPLC at Micro Analytical Center, Fac. Sci, Cairo University, Giza, Egypt. HPLC system (YL 9100), (South Korea) with pump No. YL 9110, occupied by UV/V detector No. 9120 and Column compartment No. 9131 with 150 mm reversed phase column (hypersil) ODS-C 18 , 5 µm. Acetonitrite and deionized water (85:15 v/v) represented the mobile phase. Manual injection with flow rate (1 ml/min) at 40°C and 254 nm UV was used.
All the experiments and measurements were done in duplicates, and arithmetic averages were used throughout the data analysis and calculations. Efficient degradation percentages were analyzed and calculated for 100 mg L −1 concentrations of each PAHs examined for each parameter.

Determination of biodegradation intermediates by GC/MS analysis
The optimum conditions of group IV were used to determine the degradative pathways of the six PAHs (Ace., Anth., Flu., Naph., Phen., and Pyr.) according to Abo-State et al. (2018b).
Abo-State et al. (2018a) illustrated the qualitative and quantitative determination of various compounds performed using Gas Chromatography/Mass Spectrometry (GC/MS) in The Regional Center for Food and Feed (R.C.F.F.), Giza, Egypt. The analysis was carried out using a GC (Agilent Technologies 7890A) interfaced with a mass-selective detector (MSD, Agilent 7000 Triple, Quad) equipped with Agilent HP5ms (5%-phenyl methyl polysiloxane) capillary column (30 m × 0.25 mm i. d. and 0.25 μm film thickness) Santa Clara, California, USA. The carrier gas was helium with the linear velocity of 1 ml/min. The injector and detector temperatures were 200°C and 250°C, respectively. The volume injected 1 μl of the sample. The MS operating parameters were as follows: ionization potential 70 eV, interface temperature 250°C, and acquisition mass range 50-600 (Oberoi et al., 2015). The identification of components was based on a comparison of their mass spectra and retention time with those of the authentic compounds and by computer matching with NIST and WILEY library as well as by comparison of the fragmentation pattern of the mass spectral data with those reported in the literature.

Growth and protein secretion by P. chrysosporium under different conditions
Biodegradation of PAH compounds (100 mg/L) after different incubation periods using different inoculum sizes, MnSO 4 concentrations, incubation temperatures under shaking or stagnant states have been determined.
The growth of P. chrysosporium on PAHs was indicated in Figures 1-6. The results revealed that maximum growth of P. chrysosporium was recorded at 5 and 6 discs of inoculum after 7 days of incubation period except Naph. need more time (14 d.). Also, protein secretion followed the same trend of growth (i.e. five out of the six PAHs secreted maximum protein with 5 discs after 7 days incubation except Naph. need more time (14 days)).
Growth of P. chrysosporium on BSM supplemented by different concentrations of MnSO 4 as inducer for MnP enzyme were shown in Figures 7-12. Results cleared that the highest concentrations of MnSO 4 (2000, 3000 µM) gave the highest growth but it may need more incubation period (14 d.). P. chrysosporium secreted maximum protein at concentration (1500-3000 µM) after 14 days incubation.    Maximum growth of P. chrysosporium has been recorded at 25°C incubation period for all PAHs tested in the present study after 7 days incubation (Figures 13-18). However, the maximum protein secretion for Flu., Naph., and Phen. was recorded at 40°C as shown in Figures 13-18. Figure 19 shows the maximum growth has been recorded for Phen. with shaking after 7 d. incubation. Generally, all PAHs supporting the growth of P. chrysosporium under shaking conditions than the stagnant state. Also, the maximum protein secretion is shown in Figure 20.     Table 1 shows that pyrene was the best compound degraded by P. chrysosporium (100%) under four groups (I-IV) followed by Ace. (I, III, IV), and Phen. (III, IV). However, the worst PAHs in degradation was Anth. (III, IV) by degradation percentage 66.99%. The previous results were confirmed by the results of other investigators as follows:

Degradation rate under optimum conditions
Initial biodegradation indicated 75.2% and 54.3% phenanthrene and pyrene degraded by C. sakazakii MMO45 (KT933253) within 24 h. After CCD optimization,    100% degradation was achieved for each of phenanthrene and pyrene, resulting in the formation of intermediate metabolites (Umar et al., 2017).

Degradation intermediates as determined by GC/MS
P. chrysosporium grown in BSM amended by 100 mg/L of each PAHs under optimum condition degraded these compounds by oxidation followed by ring fission.
Anthracene (Anth.) degradation by P. chrysosporium was first oxidized to 9, 10 Anthracenedione with further oxidation followed by ring fission gave 1,2-    benzene dicarboxylic acid, bis -(1-methyl ethyl) ester, which inter TCA cycle to gave finally CO 2 and H 2 O as shown in Figure 22.
Fluoranthene (Flu.) was degraded by P. chrysosporium via oxidation and hetero ring fission to give benzene methanol, 3-phenoxy with more oxidation it gave Di -n-octyl phthalate (i.e. followed the phthalic pathway) as cleared in Figure 23.     However, Naphthalene (Naph.) first gives 1,2 -Naphthalene dihydro-, followed by oxidation and ring fission to give benzoic acid, which finally converted to short aliphatic chains. It may be via polymerization converted to long chain of aliphatic compound as indicated in Figure 24.
Phenanthrene (Phen.) degradation showed that Phen. may be degraded first from the middle ring to give Cis -Stibene or from peripheral ring to give Naph. via oxidation and ring fission as shown in Figure 25.
Pyrene (Pyr.) was first oxidized to give 3, 4-dihydrophenantherene followed by ring fission to convert to phenanthrene as shown in Figure 26.
In spite of a large number of research being conducted on PAHs degradation by different bacteria and their pathways nearly screened by Nzila (2019) and Abo-State and El-kelani (2020), little has been known about PAHs pathways by fungi. White rot fungi (WRF) take great attention in biodegradation of PAHs, because of their ability to secrete a battery of ligninolytic enzymes.
It was clear that P. chrysosporium undergo oxidation followed by ring fission in the proposed six pathways for the degradation of different PAHs used in the present study. Also, from the previous proposed pathways, it was obvious that P. chrysosporium followed the phthalic pathway then inter TCA cycle, and convert finally to CO 2 and H 2 O (Abo-State & El-kelani, 2020;Lyu et al., 2014;Peng et al., 2008;Sawulski et al., 2014).  The previous findings of the present study were confirmed by other investigates as shown in Figure  26. Biodegradation pathways encompass the breakdown of PAHs, being ring fission by intracellular oxidation and hydroxylation, which represent the typical initial steps (Abo-State et al., 2018a). The microorganisms cleave the benzene ring in different ways. Ortho-or meta-cleavage path leading to the formation of central intermediates which further converted to tricarboxylic acid (TCA) cycle intermediate (Abbasian et al., 2015). It was also observed the formation of long linear chains of aliphatic polymers (Tetradecane 2, 6, 10 trimethyl-; Octacosane; Hexacosane) and branched chains of polymer (Triacontane,11,. These chains may be used by the microorganisms to build up their cell wall; it was observed the same phenomena by other investigators. Octanoic acid may inter tricarboxylic acid cycle (TCC) or with reduction and polymerization converted to nanodecane or heneicosane. These aliphatic intermediates degraded to CO 2 and H 2 O or may serve in the formation of the cell wall of the microorganisms as Bacillus spp. and Rhodococcus sp. (Fritsche & Hofrichter, 2008;Saleh et al., 2013).  Initial oxidative attach followed by ring cleavage of the benzene ring is the key step in degradation of PAHs. The oxidation results in the formation of a diol with further cleavage forming dicarboxylic acids (Hendrickx et al., 2006;Abo-State et al., 2014). The first step is oxidation catalyzed by monooxygenase and dioxygenase (Kanaly & Harayama, 2000;Zhang et al., 2011).
Degradation of Naphthalene starts through Naphthalene dioxygenase, which converts Naph. to Cisdihydrodiol, which transformed to 1, 2 dihydroxynaphthalene followed by metabolites of ring fission leading the formation of phthalic or salicylic pathway. Both the two pathways enter the Krebs cycle (TCA) (Haritash & Kaushik, 2009;Se et al., 2009).

Conclusion
Phanerochaete chrysosporium was able to utilize each one of PAHs (Acen., Anth., Flu., Naph., Phen., and Pyr.) as a sole carbon and energy source and reached to the maximum growth and extracellular protein secretion under four certain conditions [5 agar plugs inoculum size (6 mm diameter), 2000 µM/L MnSO 4 concentration, 25°C temperature degree with shaking stagnant].
Pyrene was the best compound degraded by P. chrysosporium (100%) under four groups (I-IV) of optimum conditions followed by Ace. (I, III, IV) and Phen. (III, IV). P. chrysosporium followed the phthalate route in its degradative pathway for PAHs, and finally, its intermediates interred TCA cycle and gave CO 2 and H 2 O or short chain aliphatic polymerized to give long chains.

Disclosure statement
No potential conflict of interest was reported by the author(s).