Biodegradation and detoxification of phenanthrene in in vitro and in vivo conditions by a newly isolated ligninolytic fungus Coriolopsis byrsina strain APC5 and characterization of their metabolites for environmental safety

Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant organic pollutants generated from agricultural, industrial, and municipal sources, and their strong carcinogenic and teratogenic properties pose a harmful threat to human beings. The present study deals with the bioremediation of phenanthrene by a ligninolytic fungus, Coriolopsis byrsina (Mont.) Ryvarden strain APC5 (GenBank; KY418163.1), isolated from the fruiting body of decayed wood surface. During the experiment, Coriolopsis byrsina strain APC5 was found as a promising organism for the degradation and detoxification of phenanthrene (PHE) in in vitro and in vivo conditions. Further, HPLC analysis showed that the C. byrsina strain degraded 99.90% of 20 mg/L PHE in in vitro condition, whereas 77.48% degradation of 50 mg/L PHE was reported in in vivo condition. The maximum degradation of PHE was noted 25 °C temperature under shaking flask conditions at pH 6.0. Further, GC-MS analysis of fungal treated samples showed detection of 9,10-Dihydroxy phenanthrene, 2,2-Diphenic acid, phthalic acid, 4-heptyloxy phenol, benzene octyl, and acetic acid anhydride as the metabolic products of degraded PHE. Furthermore, the phytotoxicity evaluation of degraded PHE was observed through the seed germination method using Vigna radiata and Cicer arietinum seeds. The phytotoxicity results showed that the seed germination index and vegetative growth parameters of tested plants were increased in the degraded PHE soil. As results, C. byrsina strain APC5 was found to be a potential and promising organism to degrade and detoxify PHE without showing any adverse effect of their metabolites.

Introduction studies because it contains K-region and bay-region in their structure, which are especially found in the structure of higher molecular weight PAHs (Garcia-uitz et al. 2016;Luo et al. 2020). Due to the presence of K-region and bay-region in their structure, PHE is structurally stable and recalcitrant in nature. PHE showed toxic effects on the human respiratory system and caused photosensitization of the skin (Budavari et al. 1989;Xu et al. 2006); therefore, globally, it is necessary to remediate PHE from the contaminated environment.
A wide array of various physical, chemical, and biological methods has been applied for the degradation and remediation of PAHs from contaminated environment. The physicochemical methods such as incineration, photodegradation (Nguyen et al. 2020), nano-filtration (Li et al. 2019), electrokinetic remediation (Pourfadakari et al. 2021), thermal desorption (Falciglia et al. 2020;Li et al. 2020), soil washing (Gitipour et al. 2018), radiocolloid treatment, electron beam irradiation (Xu et al. 2020), and chemical immobilization have been found efficient for degradation of PAHs. However, these methods of remediation are expensive; in many cases, they only transfer the toxic compounds from one phase to another phase and cause a secondary pollution and unable to degrade high molecular weight (HMW) PAHs (Antosova et al. 2020;Li et al. 2014). Thus, cost-effective, efficient, and sustainable biological methods involving microorganisms for the degradation of PAHs have been developed (Zhao et al. 2009). Bioremediation has gained much attention in the recent past for the remediation of hazardous and toxic environmental pollutants from contaminated sites (Asgher et al. 2008;Haritash and Kaushik 2009;Romantschuk et al. 2002). Various microbes such as bacterial species, Pseudomonas aeruginosa PFL-P1 (Mahto and Das 2020), Paracoccus aminovorans HPD-2 (Teng et al. 2010), Burkholderia cepacia (Chen et al. 2013), Roseobacter clade , and Sphingomonas sp. GY2B (Liu et al. 2017), and fungal species, Trametes hirsuta D7 (Hidayat and Yanto 2018), Ganoderma lucidum (Agrawal et al. 2018), Pleurotus ostreatus (Pozdnyakova et al. 2010), Leucoagaricus gongylophorus (Ike et al. 2019), and Armillaria sp. FO22 (Hadibarata and Kristanti 2012), have been reported for the remediation of PAHs from contaminated soil; however, as compared with bacteria, fungi is a promising alternative for the remediation of PAHs. Bacteria secrete inter-and extracellular narrow substrate-specific enzymes (Madhavi and Lele 2009;Singh 2006); fungi secreted non-specific, non-selective, ligninolytic enzymes viz., lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (LAC) that are associated with their ligninolytic activities; other than this, fungi degrade more than four ring containing HMW PAHs. Fungi secrete extracellular broad substrate-specific ligninolytic enzymes that penetrate the polluted soil, sludge, or matrix and then remove the pollutants; they involve less energy, time, and chemicals and grow on a broad range of substrates (Balaji and Ebenezer 2008;Bankole et al. 2021;Czaplicki et al. 2018). However, white rot fungi are considered the most favorable fungi for the investigation of PAHs degradation and their efficiency depends on the microbial strain and the structure of organic pollutants (Agrawal and Shahi 2017;Jia et al. 2019;Llorens-Blanch et al. 2018). White rot LAC belongs to the multicopper containing phenol oxidase (EC 1.10.3.2: benzenediol, oxygen oxidoreductase, or p-diphenol oxidase), which catalyzes the oxidation of aromatic-ring containing phenols, polyphenols, and non-aromatic substrates through the radical catalyzed mechanism (Moon et al. 2018;Shafiei et al. 2019;Wulandari et al. 2021); LiP (EC 1.11.1.14, 1, 2-bis (3, 4-dimethoxyphenyl) propane-1, 3-diol: hydrogenperoxide oxidoreductase) catalyzes the hydrogen peroxidasedependent cleavage of C α -C β and aryl C α bond, and oxidizes phenolic and non-phenolic lignin model compounds (Biko et al. 2020;Naghdi et al. 2018). MnP (EC 1.11.1.13; Mn (II) hydrogen-peroxide oxidoreductase) catalyzes the manganese (Mn)-dependent oxidation of phenolic compounds in presence of hydrogen peroxide (Emami et al. 2020;Nowak et al. 2020;Wong 2009). The rate of the PAHs degradation depends on the culture conditions like pH, temperature, oxygen, the concentration of PAHs, accessibility of nutrients, and agitated culture (Kadri et al. 2017;Ting et al. 2011). Several authors have been reported the degradation of PHE by white rot fungi such as Polyporus sp. S133 (Hadibarata and Tachibana 2010), Pleurotus eryngii (Wu et al. 2016), and P. ostreatus D1 (Pozdnyakova et al. 2018). However, few reports are available on the in vitro and in vivo degradation and detoxification of PHE by white rot fungi.
In the present study, PHE degradation by Coriolopsis byrsina (Mont.) Ryvarden strain APC5 was assessed in the in vitro and in vivo conditions. Further, degradation of PHE was evaluated by HPLC, FT-IR, and GC-MS techniques. Furthermore, ligninolytic enzyme activity and PHE degradation were optimized at concentration of PHE in various environmental conditions like pH, temperature, and salinity. Afterward, the formulation of C. byrsina fungus was prepared in the different substrate to check the PHE degradation efficiency of fungus in vivo condition. Furthermore, the phytotoxicity of PHE metabolites on the growth of Vigna radiata and Cicer arietinum was also investigated.

Chemicals and reagents
All the chemicals, solvents, and reagents used in the present investigation were of analytical grade and used without further purification. PHE (gt;98.0% analytical standard), Sabouraud dextrose agar (SDA), azure B, guaiacol, 2,6-dimethoxy phenol, and ethyl acetate (HPLC grade) were purchased from Merck Life Science, Mumbai, India. Bovine serum albumin (BSA) used as a protein standard was supplied from Sigma-Aldrich Chemical Co. Ltd. (St. Louis, USA). All the media and working solutions were prepared by using ultrapure water generated by the Milli-Q system (ELIX, Merck Millipore, India) Fungi culture and medium for PHE degradation C. byrsina (Mont.) Ryvarden strain APC5 (KY418163.1), isolated from the fruiting body of decayed wood surface in our laboratory, was used for the investigation of PHE degradation (Agrawal and Shahi 2017). For the degradation and enzyme study, SDA medium containing 7 days old culture of strain APC5 (one disc of 8 mm diameter) was used as the inoculum. The mineral salt broth (MSB) medium used for PHE degradation and ligninolytic enzyme activity investigation contains (g/L) glucose, 10; KH 2 PO 4 , 2.0; MgSO 4 .7H 2 O, 0.5; CaCl 2 .2H 2 O, 0.1; ammonium tartrate, 0.2; and trace element solution, 10 (mL) (Arora and Gill 2001). MSB medium was supplemented with an antibacterial compound (to inhibit the bacterial growth) and 0.45-μm membrane filter was sterilized with 20 mg/L concentration of PHE. The pH of the MSB medium was adjusted to 7.0 with the help of 0.1 N NaOH or 0.1N HCl.

Analysis of PHE degradation by plate assay
Preliminary Bushnell Haas Agar (BHA) medium (composition, g/L: MgSO 4 (0.2), CaCl 2 (0.02), KH 2 PO 4 (1), K 2 HPO 4 (1.0), NH 4 NO 3 (1.0), FeCl 3 (0.05), and agar (20)) supplemented with 20 mg/L PHE (dissolved in acetonitrile) was ( Fig. 1a) used for the qualitative analysis of PHE degradation. In this methods, one BHA medium plate was used as control, and in another plate, one mycelial disc (8.0 mm in diameter) of strain APC5 was transferred, and then incubated at 27°C for 10 days. After incubation, clearing zone formation was observed around the inoculated mycelia of C. byrsina strain APC5 after the degradation of PHE from the medium.

Measurement of PHE degradation efficiency
The PHE degradation experiment was performed in Erlenmeyer flasks (250 mL) containing 20 mL of previously prepared sterile MSB medium supplemented with PHE. Triplicate flasks were inoculated with culture mycelium of strain APC5 and incubated at 27°C under rotary shaker (120 rpm) for 15 days. The sample was collected at regular 2 days of the time interval during the incubation period, and residual PHE in the MSB medium and the ligninolytic enzyme production were investigated. At the same time, the control (without fungus culture) was incubated. The PHE degradation efficiency was measured as percent (%) of degradation.
where C i is the initial concentration of PHE (mg/L), and C t is the remaining concentration of PHE (mg/L) (Bishnoi et al. 2008). After the extraction, filtrate culture mycelium of strain APC5 was kept in a hot air oven at 50°C, and dried weight of mycelium (biomass) was observed.

Extraction of PHE
PHE and other existing organic compounds present in treated and untreated samples of PHE were extracted by liquid-liquid extraction procedure with ethyl acetate as described previously by Agrawal and Shahi (2017). An equal volume of PHE degraded medium and ethyl acetate were taken for the extraction of metabolites. The obtained organic solvent phase of metabolites was dried under vacuum using a rotary evaporator. Then obtained residues were dissolved in HPLC grade 2.0 mL acetonitrile and used for HPLC, FTIR, and GC-MS analysis.

HPLC analysis
The residual concentration of PHE was assessed with the help of HPLC chromatogram peak area. HPLC analysis was performed on UFLC model equipped with a PDA detector system ((Shimadzu prominence, Japan), C 18 , 100 A°column (Luna5u, 250 × 4.6 nm) and pump (LC-20AT), detector (PDA) was used at 254 nm). A gradient of solvent A and solvent B acetonitrile and water (70:30) was applied at1.0 mL/min flow rate for 20 min. The detection was observed at a wavelength of 254 nm to measure the degradation of PHE. The remaining concentration of PHE was measured by the following formula; Concentration of PHE in sample mg=L ð Þ where A SAM is the peak area of chromatogram of a sample, A STD is the peak area of standard PHE compound, and C STD is the concentration of standard PHE compound (mg/L)

Identification of PHE degraded metabolites by FTIR spectroscopy and GC-MS analysis
To access the bond modification of metabolites after degradation, FTIR spectroscopy was performed in the range of 375-4000/cm using spectrophotometer (IR affinity-1, Shimadzu, Japan). The separated extract of metabolites was mixed with spectroscopic grade potassium bromide to prepare the pellet for FTIR analysis. At the same time, IR spectra of PHE (control) were examined. Further, the metabolites of degraded PHE were identified by GC-MS (Thermo Fisher Scientific, USA, GC model-Trace GC ultra, MS model-Polaris Q) with DB-5 MS capillary column (30 m length, 0.25 mm internal diameter, and 0.25 μm film thickness). The injection volume of sample was 1 mL. The ion source of GC-MS was kept at 230°C and transfer line temperature along with inlet temperature was maintained at 280°C. The temperature program kept for 2 min hold at 70°C, an increase to 200°C at 10°C per min, kept for 1 min in 200°C, then again increase temperature to 325°C at 5°C per min and hold for 15 min at 325°C . Helium gas (1 mL per min) was used as the carrier gas. The mass spectrometer was operated in an electron ionization mode of 70 eV energy Ion trap detector. The detected metabolites were identified by comparing the mass spectrum of detected compounds with NIST (National institute of standard technology, USA) database attached with the GC-MS instrument (Ghosal et al. 2010).

Ligninolytic enzyme activities
To assess the ligninolytic enzyme activities during PHE degradation, the culture supernatant was taken as per the protocol described earlier ("Measurement of PHE degradation efficiency"). LAC enzyme assay of collected, treated supernatant was performed according to the method described by Sandhu and Arora (1985); take 3 mL of solution containing 1.5 mL sodium acetate buffer (10 mM, pH 5.0), 0.5 mL of the enzyme extract, and 1.0 mL guaiacol (2 mM, Ɛ 450 = 12100/M / cm), and then incubate for 2 h and absorbance read at 450 nm using double beam UV-Visible spectrophotometer (ELICO SL 218, Andhra Pradesh, India). For the LiP assay, total 3 mL of reaction mixture containing 1.0 mL of 125 mM sodium tartrate buffer (pH 3.0), 1.0 mL of 0.16 mM azure B (Ɛ 651 = 48800/M/ cm), and 0.5 mL of the culture filtrate and then add 0.5 mL of 2 mM hydrogen peroxide. Then absorbance was recorded at 651 nm (Archibald 1992). MnP activity was analyzed by the method reported by de Jong et al. (1992). Take 3 mL of reaction mixture containing 1 mL of sodium tartrate buffer (50 mM, pH 4.0), 0.5 mL culture filtrate, and 1 mL of 2 mM 2,6-DMP (Ɛ 468 = 49600/M/cm). The reaction was started with the addition of 0.5 mL hydrogen peroxide (0.4 mM) by the oxidation of 2,6-DMP. The enzyme activity was expressed in terms of international units per liter of enzyme extract (U/L).

Effect of environmental parameters
Ligninolytic enzyme production and PHE degradation by strain APC5 were optimized in different environmental parameter conditions like pH, temperature, and salinity. During optimization experiment, MSB (20 mL) supplemented with 20 mg/L PHE was inoculated by 8 mm diameter mycelial disc of strain APC5. Afterwards, inoculated flasks were incubated at varying pH (3.0-8.0), temperature (15-55°C), and salinity (10 and 32 g/L) in the rotary shaker incubator (120 rpm) for 10 days. The PHE degradation and ligninolytic enzyme activity were determined as described earlier, and protein concentration was analyzed according to the standard protocol of Lowry et al. (1951).

Effect of different concentration of PHE
To evaluate the effect of different concentration of PHE on the degradation by strain APC5, Erlenmeyer flasks containing different concentrations of PHE (10-100 mg/L) in MSB were inoculated by mycelial disc (8 mm diameter) of strain APC5, and flasks were incubated at 27°C in shaking flask condition (120 rpm) for 10 days. After incubation, the degradation of PHE was evaluated by HPLC analysis.

Degradation of PHE in soil: in vivo study
PHE degradation by C. byrsina strain APC5 was also investigated in the soil (in vivo condition). Briefly, in this experiment, the soil was collected from village Sendri, Bilaspur (Chhattisgarh, India) as prescribed earlier in Agrawal and Shahi (2017). The soil type was classified as loam soil, with 0.225 carbon (5%), 275 nitrogen (kg/ ha), 11.25 sulfur (kg/ha), 168 potassium (kg/ha), and 5.8 pH. Electrical conductivity (0.14) was analyzed by soil testing laboratory at the Department of Agriculture, Bilaspur, Chhattisgarh. Sterilized soil was treated with 50 mg/kg of PHE (dissolved in acetonitrile) (Agrawal and Shahi 2017). After that, PHE incorporated soil was inoculated by wheat bran and talc powder substrate-based inoculum separately. Wheat bran inoculum (100 g) was prepared in the sterilized spawn bags with 14000 CFU (colony forming unit)/g. To prepare talc powder inoculum, homogenized culture broths (100 mL, 13300 CFU/ mL) were transferred and mixed with sterilized talc powder (1.0 kg) containing carboxymethyl cellulose (20 g, as a binder) and then dried. Subsequently, after drying, inoculum was ready for application. Wheat bran inoculumtreated soil (100 g/kg) and control sets (soil containing PHE and without any fungal inoculum) were kept in sterilized polythene bags and incubated at 27°C for 30 days and talc powder inoculum-treated soil and control sets (soil containing PHE and without any fungal inoculum) were transferred in nursery bags. Nursery bags were incubated for 30 days in the outer environmental condition.

PHE degradation analysis by HPLC
PHE degraded metabolites were extracted from soil according to the method described by Zebulum et al. (2011). Briefly, control and treated soil samples were taken separately in Erlenmeyer flasks and mixed with 20 mL of acetonitrile. Further, the reaction mixture was shake for 10 min. To extract the large number of metabolites from treated sample, the extraction procedure was repeated thrice. The collected extracts were concentrated by vacuum evaporator. Further, the concentrated extract was investigated with the help of HPLC to calculate the PHE degradation percentage.

Phytotoxicity assessment of PHE degraded metabolites
The phytotoxicity assessment of control (untreated) and bioremediated soil was studied in the plants Vigna radiata and Cicer arietinum. V. radiata (seed variety: LeelA seeds, Ahamdabad) and C. arietinum (seed variety: Research Bengal Gram Daftari 21) which seeds were collected from Krishi Raksha Kendra, Bilaspur, Chhattisgarh. Prior to experiment, the surface of collected seeds was sterilized with 70% (v/v) ethanol and 2% (v/v) solution of sodium hypochlorite. Furthermore, repeated washing of seeds were performed with distilled water to remove the remaining sodium hypochlorite. Sterilized seeds of V. radiata and C. arietinum were sowed in control and treatment sets (In control sets, 50 mg/L of PHE was incorporated in 1 kg soil. In treatment sets, 50 mg/L of PHE incorporated in 1 kg soil was treated by fungal inoculum, and after 30 days of incubation, bioremediated soil was used as treatment sets.). V. radiata seeds were sowed in wheat bran inoculated soil and C. arietinum seeds were sowed in talc powder inoculated soil. The control and treatment sets were kept in outer environmental condition for 30 days. After that, plants were harvested from the soil. The length and weight (both dry and fresh) of the root and shoot, germination percentage, root elongation, and germination index (GI) of tested seeds were examined with the help of formulae as described earlier by Agrawal and Shahi (2017).

Statistical analysis
To avoid any type of experimental errors, each test of experiment was conducted in triplicate. The standard deviation was investigated with the help of Microsoft Excel (ver. 2019, Microsoft, UK) and the results of the experiments were presented in mean ± SD value. The mean value of various tests of control and experiment samples was investigated by one-way ANOVA (analysis of variance) test by applying Dunnett's multiple comparison through Graph Pad Prism 8.0, (USA), statistical software.

Results
Analysis of PHE degradation by plate assay C. byrsina culture was grown in the BHA media containing petri plates (one plate of BHA media containing PHE was used as a control (Fig. 1a); after the incubation period, 7 mm clearing zone formation was observed around the mycelium because of the degradation of PHE (Fig. 1b)).

Measurement of PHE degradation efficiency
PHE degradation by C. byrsina strain APC5 was examined after 2 days of regular intervals by HPLC chromatogram. The HPLC chromatogram of control sample showed (Fig. 1c) a single major peak of PHE, whereas in fungi treated sample, chromatogram (Fig. 1d) showed the small peak of PHE and many extra peaks of other newly formed metabolites that indicated the PHE degradation efficiency of strain APC5 by their ligninolytic enzyme activity. It was observed that as the incubation period increased, PHE degradation and biomass (Fig. 1e) of C. byrsina also increased, after the 8th day, 83.40% PHE (20 mg/L) degradation was examined at pH 6.0 and 27°C, while at the 14th day of incubation, 99.90% PHE degradation was observed (Fig. 1e).

Identification of metabolites after PHE degradation
FTIR analysis of C. byrsina treated PHE sample supported the degradation of PHE as compared with control due to the change in the functional groups of compounds (Fig. 2). The vibrational band frequencies of PHE were found in FTIR spectroscopy between 2800 and 3200 cm −1 (3057 cm −1 ) (Wu et al. 2010) (Fig. 2a, b). Infrared spectra (Fig. 2a, b) of compounds demonstrated different skeleton and stretching vibration of functional groups. A peak at 1716-1740 cm −1 was observed because of the existence of stretching vibration of CO bond in carboxyl, carboxylic acids, and ketones. The absorption band at 1450-1625 cm −1 might be due to the result of the skeleton vibration of the benzene ring. At 1463 cm −1 , 610-700 cm −1 , skeleton vibration of the aromatic ring, -C ≡ C-H: C-H bend in alkynes, was investigated, because of ring cleavage reaction. In this experiment, the GC-MS analysis of ethyl extracted untreated PHE (control), the peak of PHE was detected at the retention time (RT) 13.28 min (m/z 178) (Fig. 3a). However, after the 4th day treated sample, at the RT value 17.58 min (m/z 210), 9,10-dihydroxyphenanthrene was investigated (Fig. 3b). At RT value of 16.23 min (m/z 166), phthalic acid was identified, after the 8th day of incubation (Fig. 4a). Acetic acid anhydride (RT 4.05 min, m/z 102), benzene octyl (RT 11.03 min, m/z 190), 4-heptyloxy phenol (RT 15.73 min, m/z 208), 2,2-diphenic acid (RT 18.36 min, m/z 242), and 9,10dihydroxyphenanthrene (RT 21.10 min, m/z 210) were observed (Fig. 4b) after the 12th day of incubation. These metabolites were degraded products of PHE after fungi treatment.
On the basis of above-identified metabolites, the PHE degradation pathway was postulated as shown in Fig. 5. PHE was first oxidized in K-region and transformed into 9,10dihydroxyphenanthrene by the action of LAC, LiP, and MnP enzymes. Further 2,2-diphenic acid and phthalic acid were produced due to the oxidation and ortho-ring cleavage reaction caused by LAC, LiP, and MnP enzymes. Due to side group removal, phthalic acid was converted into 4-heptyloxy phenol, benzene octyl, then ring cleavage reaction occurred by the ligninolytic enzyme, finally the conversion of acetic acid anhydride was observed after degradation.

Ligninolytic enzyme activities
LAC, LiP, and MnP activity of C. byrsina was investigated at regular 2 days time intervals during PHE degradation. In the present study, MnP activity was found maximum at the initial stage of PHE degradation, whereas LAC activity was noted maximum 2629.00 U/L after the 8th day incubated sample. LiP and MnP activity was found to be noted maximum 1727.00 U/L and 20147.00 U/L after 6 and 8 days of incubation, respectively (Table 1). It was observed that the degradation of PHE was significantly increased in the 6th day treated sample.

Effect of environmental parameters
The effect of abiotic environmental parameters like pH, temperature, and saline condition on the degradation of PHE showed the maximum ligninolytic activity (1213.00 U/L LAC, 1028.00 U/L LiP, 7300.00 U/L MnP), and PHE degradation (85.60%) was observed at pH 6.0 (Fig. 6a). It showed that when the production of enzyme increased, the degradation of PHE was simultaneously increased. The highest biomass (25.00 mg) and protein concentration (261.50 mg/L) was observed at pH 7.0 and 8.0, respectively. Furthermore, an increase in pH inhibited the growth of fungi, enzyme production, and PHE degradation. Increasing the incubation temperature up to 35°C enhanced the PHE degradation and increased enzyme activities. The maximum activity of ligninolytic enzymes (LAC 1640.00 U/L, LiP 957.00 U/L, MnP 20800.00 U/L, and degradation of PHE (87.50%) and growth (248.50 mg biomass, 110.50 mg/L protein) was observed at optimum temperature of 25°C (Fig. 6b). Further, an increase in the temperature up to 55°C adversely affected the degradation efficiency, enzyme activity, and growth of fungi. Similarly, at low salt concentration of 10 g/L, more enzyme activity (290.90 U/L LAC, 6799.00 U/L LiP, 2113.00 U/L MnP), degradation of PHE, (34.50%), and growth (150.00 mg biomass, 85.30 mg/L protein) of fungi were observed as compared to high salt concentration (32 g/L: 10.30 U/L LAC, 2193.90 U/L LiP, 604.80 U/L MnP, 20.50% PHE degradation, 100 mg biomass, 15.70 mg/L protein) (Fig. 6c).

Effect of different concentration of PHE
When the concentration of PHE increased in the medium, it was observed that after 10 days of incubation, the ligninolytic enzyme activity of organisms significantly increased (p<0.0001) ( Table 2) and the degradation % of PHE decreased (Fig. 6d). At the 10 and 20 mg/L concentration of PHE, maximum degradation up to 90.50% and 85.60% was observed. At 100 mg/L concentration of PHE, maximum Lac (3729.00 U/L), LiP (12911.00 U/L), and MnP (30247.00 U/L) activity was reported.

Degradation of PHE in soil: in vivo study
In the present experiment, the degradation of PHE was investigated in soil through the analysis of HPLC chromatogram peak area. When wheat bran prepared inoculum of strain APC5 fungus was applied, 77.48% of PHE degradation was observed, whereas 68.74% of PHE degradation was investigated with the talc powder prepared inoculum of strain APC5 fungus.

Phytotoxicity assessment of PHE degraded metabolites
The phytotoxicity of control (PHE contaminated) as well as treated soil (bioremediated soil) sample was investigated by observing GI index and germination percentage, and measuring the length and weight of root and shoot of the both  Table 3. In the present study, 100% seed germination was observed in control and treated soil sample plants of V. radiata and C. arietinum. It was observed that control sample was highly toxic; only 51.89% and 38.89% GI was found in the control plants of V. radiata and C. arietinum, respectively; however, 218.56% and 101.83% GI were found in the treated sample plants of V. radiata and C. arietinum, respectively, which defines no toxicity of PHE metabolites towards the tested plants. Values represent of three independent replicate (n=3) with mean ± standard deviation. According to one-way ANOVA, values represent in column are significant different, ns-non significant p>0.05, ****p<0.0001, ***p<0.001, ** p<0.01 and *p<0.05 As compared to control the root, shoot length of treated V. radiata plants were significantly increased (p<0.05, p<0.001). The root length of V. radiata plants in bioremediated soil was observed, 4.2 and 2.2 times longer than the seed sowed in the soil of PHE contaminated and native soil, respectively. In the case of shoot length of V. radiata plants, 4.27 and 1.73 times reduction were found in PHE contaminated and bioremediated soil, as compared to native plant. A total of 51.89 germination index was found in the PHE contaminated soil, whereas 218.56 GI was observed in the bioremediated soil that represents the nontoxic condition of soil after the degradation of PHE by C. byrsina fungus.
In the case of C. arietinum plants, as compared to control the root, shoot length of bioremmediated soil plants was increased p<0.01, p<0.001 significantly. The root length of C. arietinum plant was observed in bioremediated soil, 2.62 times longer than the seed shown in the soil of PHE contaminated. The 2.44 times reduction in shoot length of C. arietinum was found in PHE contaminated soil as compared to native plant. GI (38.89) was found in the PHE contaminated soil, whereas GI (101.83) was observed in the bioremediated soil which represents the nontoxic condition of soil after the degradation of PHE by C. byrsina fungus. On the basis of above data, it was investigated that after the degradation of PHE from soil, any types of toxic metabolites were not formed; GI index and the vegetative growth parameters of V. radiata and C. arietinum plants were increased significantly.   Values represent of three independent replicates (n=3) with mean ± standard deviation. According to one-way ANOVA analysis, the values represent in column are significant different, ns-non significant p>0.05, ****p<0.0001, ***p<0.001, ** p<0.01 and *p<0.05 Fig. 7 The effect of PHE metabolites on the growth of Vigna radiata (a) and Cicer arietinum (b) growth cultivated in PHE degraded soil

Discussion
PAHs transformation by wood decaying ligninolytic white rot fungi might be a universal phenomenon (Han et al. 2004;Mao and Guan 2016;Park et al. 2019;Sutherland et al. 1991 (Hadibarata and Yuniarto 2020) as shown in Table 4. Therefore, we can say that as compared to previous study, fungus C. byrsina degraded PHE in less incubation time with minimal carbon source. As we know, PHE is degraded by different white rot fungi like P. ostreatus, Ganoderma lucidum, Phanerochaete chrysosporium, and Trametes versicolor (Bumpus 1989;Dhawale et al. 1992;Han et al. 2004;Sutherland et al. 1991;Ting et al. 2011), but in this study, white rot fungus C. byrsina strain APC5 is first time reported for the degradation of PHE. The metabolites of PHE degradation identified by GC-MS analysis were 9,10-dihydroxyphenanthrene, 4-heptyloxy phenol, benzene octyl, and acetic acid anhydride. During degradation, toxic metabolites like quinones and phenanthrene-9,10-dione were not detected, which are formed after the degradation of PHE by many other fungi (McConkey et al. 1997;Singh 2006 ). PHE was oxidized at the K-region by the l i g n i n o l y t i c e n z y m e a n d f o r m e d 9 , 1 0 -Dihydroxyphenanthrene. Wulandari et al. (2021) also suggested the formation of 9,10-dihydroxyphenanthrene after the PHE degradation by the action of laccase enzyme of Trametes polyzona PBURU 12 fungus. Bohmer et al. (1998) and Pozdnyakova et al. (2018) also supported that ligninolytic enzymes are responsible for the initial oxidation of PHE ring. Afterwards, the formation of 2,2-diphenic acid was investigated by the oxidation and ring cleavage reaction occurred by the action of ligninolytic enzymes. 2,2-Diphenic acid produced in P. chrysosporium by the action of MnP and LiP enzyme was investigated by Hammel et al. (1992) and Hammel (1995). Further, generated phthalic acid entered into basal metabolism (also suggested by Pozdnyakova et al. (2018)). Our data support the degradation pathway of PHE as described by Bezalel et al. (1997), Hadibarata and Tachibana (2010), and Torres-Farrada et al. (2019). On the basis of the above results, the detected metabolites of PHE were transformed and mineralized by C. byrsina strain APC5 through their ligninolytic activities.
Ligninolytic enzyme of white rot fungi was described as the key for degradation of PAHs (Cajthaml et al. 2008;Ghosal et al. 2016;Kunjadia et al. 2016;Qian and Chen 2012). Our data represented that C. byrsina fungi produced significant amounts (p<0.0001) of ligninolytic enzyme; therefore, PHE was degraded more efficiently. C. byrsina strain APC5 produced LAC (2629 U/L), LiP (1727 U/L), and MnP (20147 U/L) in the PHE containing media, while previously maximum 2000 U/L LAC activity was observed in C. byrsina SXS16 in the wheat bran substrate; however, the activity of LiP and MnP was very low as compared to LAC reported by Gomes et al. (2009); other than this, PHE degrading Coriolopsis caperata BM-172 showed 880 U/L highest LAC activity between 15 and 20 days of incubation in MSB  (Hadibarata and Tachibana 2010). PHE is considered a model compound for the PAHs degradation study, because of their presence in high concentration in the PAHs polluted sites and they contain K-region and bayregion which are present in the structure of higher molecular weight PAHs (Bezalel et al. 1996;Sack et al. 1997). The efficiency of PAHs degradation depends on the production of ligninolytic enzyme Kim et al. 1998). Thus, to check the PAHs removal efficiency by strain APC5 in the contaminated area, different physical parameters and the concentration of PHE were optimized for the production of ligninolytic enzymes and PHE degradation. In this study, Coriolopsis fungi showed ligninolytic enzyme activity in the temperature range of 15-55°C at 3.0 to 8.0 pH, therefore able to degrade PHE in temperature and pH stress condition, while most fungi have shown maximum growth and ligninolytic activity at pH 3.0 to 6.0 (Yamanaka et al. 2008). Han et al. (2004) investigated the PHE degradation by Trametes versicolor was found highest at pH 6.0 and 30°C temperature. Ting et al. (2011) reported that the maximum LAC production and PHE degradation take place by the fungus Ganoderma lucidum BCRC36021 at pH 4.0 and 30°C temperature. In the present investigation, the best temperature for the ligninolytic enzymes production, PHE degradation, and optimum growth of C. byrsina was 25°C at pH 6.0. Salinity stress has also been affecting the PHE degradation, fungal growth, and enzyme production. Dixon et al. (1993) reported that every single fungus has revealed their different salt tolerance capacity due to their changed cell characteristics. In the presence of 10 g/L of salt, Coriolopsis fungi showed high growth, PHE degradation, and enzymatic activities. As the salt concentration increased fungal growth, PHE degradation and enzyme production decreased. Kamei et al. (2008) also reported that the growth of P. chrysosporium and T. versicolor was suppressed in 32 g/L sea salt concentration. But at the salt concentration of 32 g/L, Coriolopsis fungus showed their growth, enzyme production, and PHE degradation.
The concentration of carbon and nitrogen in the growth medium influences the production of ligninolytic enzymes by fungi (D ' Souza et al. 1999;Hamman et al. 1997). It was investigated that as the concentration of PHE increased in the medium, the production of ligninolytic enzyme was increased significantly (p<0.0001), which assists in the degradation of PHE at higher concentration by Coriolopsis fungus.
Formulation of C. byrsina effectively degraded PHE from soil. Degradation of PAHs in soil can not be considered complete bioremediation of soil due to the formation of toxic metabolites after degradation or incomplete degradation of PAHs enhanced the soil toxicity (Singh 2006). PHE is also toxic to plant growth; Alkio et al. (2005) reported that the greater concentration (>50 μM) of PHE adversely affects the root and shoot growth, late flowering, deformed trichomes in the Arabidopsis thaliana plant in in vitro condition. Thus, phytotoxicity of PHE and their degraded metabolites were determined in the present study. It was observed that after the degradation of PHE by C. byrsina fungus, vegetative growth parameters of V. radiata and C. arietinum plants increased significantly. After that, we can say that C. byrsina strain APC5 is a potent PHE degrader from the environment and also promotes the growth of plants.

Conclusions
This study shows that C. byrsina strain APC5 is capable of degrading recalcitrant compound, PHE (PAHs), in in vitro and in vivo conditions. C. byrsina APC5 produced significant amounts (p<0.0001) of ligninolytic enzymes at different concentrations of PHE. C. byrsina APC5 also degraded PHE and produced ligninolytic enzyme in worst environmental condition like pH (3.0 to 8.0), temperature (15 to 55°C), and salinity (10 and 32 g/L). After the degradation of PHE by C. byrsina, any types of toxic metabolites like quinone was not detected and toxicity of soil reduces; therefore, germination index and plant growth increased significantly. This work concluded that C. byrsina can be used further in the process of phytoremediation as well as plant growth improvement. Furthermore, genetic engineering plays a significant role in the enhancement of phytoremediation; by combining the knowledge of system biology with gene editing and gene manipulation technique, PHE phytoremediation efficiency of the plant can be increased.