APEB Journal

Introduction

A group of several hundred of organic compound that are chemically related and environmentally persistent and known as Polycyclic Aromatic Hydrocarbons (PAHs) [1-4].PAHs pass varied toxicity, genotoxic, mutagenic and/or carcinogenic properties[5,6]. PAHs enter the environment via varied ways, which include natural, human activity sources and found as mixture involving two or more of these chemical compounds. The disposal of petroleum products and forest fires represent the natural sources. While thermal decomposition and industrial processing represent the human activity sources [7]. The thermal decomposition occurs via pyrolysis and pyrosynthesis of organic molecules. Otherwise, industrial processing caused by incineration or combustion such as wood combustion, fuel combustion (gas, oil, cool) and waste incineration resulted in PAHs release [8-12].

PAHs found in polluted atmosphere transferred to human and plant. However, it can be filtered from soil into water. Accidental spills and industrial effluent pollute the water (Sihage et al., 2014) [13] PAHs can be found in meats, fishes, vegetables and fruits [14]. Exposure to high levels of PAHs cause diarrhea, nausea, vomiting and eye irritation [15]. Chronic Human health effects include kidney and liver damage, lung function abnormalities, decrease immune function, asthma, skin inflammation, cataracts and breathing problems could be from long exposure to PAHs [16,17].

Ability of microorganisms to transform, degrade or detoxify organic compounds by converting harmful compounds to non - toxic compound is called biodegrading. Degradation of Permanent Organic Pollutants (POPs) like PAHs, PCBs, PPCBs and Pesticides is an important research activity [18-22]. Biodegradation, biotransformation, biostimulation and bio augmentation process have been carried out via various microorganisms by breaking down the complex compounds via potential enzymes [23,24].

Fungi can degrade PAHs[25-28].White -Rot Fungi (WRF) can degrade Low Molecular Weight (LMW) as well as High Molecular Weight (HMW) PAHs with four or more fused benzene rings. This issue represent a great interest for the research groups. WRF are the most important fungi that can eliminate lignin, cellulose and hemicellulose at nearly equal rates [29-33].

WRF produce Laccase Enzyme (Lac) (Benzene diol: oxygen oxidoreductase), a multi-copper; Lignin-Peroxidase (Lip) and Manganese Peroxidase (MnP) which are lignin degrading enzymes. The ionization potential is the main aspect for removal of PAHs by those low substrate specify extracellular enzymes [5,30,34-39].

Phanerochaete chrysosporium; Ganoderma strains; Pleurotus ostratus; Pleurotus sajor-caju; Pleurotus plumonarius, Pseudotrametes gibbosa; Bjerkamdera adusta and Pycnoporus sp. are the most commonly species used for degradation of PAHs as a sole carbon and energy source [33,40-44].

The aim of the present study is to determine the ability of Phanerochaete chrysosporium as a White Rot Fungus (WRF) to degrade six different PAHs with low and High Molecular Weight (LMW-PAHs and HMW-PAHs) as a soul carbon and energy source. Also studying the role of gamma radiation in inducing mutants with hyper-activities.

Materials and Methods

Microorganism

The White-Rot Fungus (WRF) used in the present study was Phanerocheate chrysosporium ATCC 32629 purchased from Microbiological Resources Center (MIRCEN), Faculty of Agriculture, Ain-Shams University, Cairo, Egypt.

Maintenance of WRF

Phanerocheate chrysosporium ATCC 32629 was maintained on 2% (w/v) malt agar (Oxoid, 1982) [45] slants and kept at 4°C until need. P. Chrysosporium can be activated by sub- culturing on Malt Extract (ME) agar plates and Potato Dextrose Agar (PDA) plates (Oxoid, 1982) [45] for 14 day at 28±2°C.

Chemicals used

Six Polycyclic Aromatic Hydrocarbons (PAHs) with a low and high molecular weight were used in the present study. Naphthene (99٪ purity), was purchased from El- Gomhoria Company, Cairo, Egypt. While Pyrene, phenantherene, fluoranthene, Anthracene and acenaphthene (all 99٪ purity), purchased from Sigma-Aldrich, USA. All PAHs were dissolved in chloroform (Stock solution).

Degradation Culture media

The liquid Basal Salt Medium (BSM) [41] with modification [18-21,46] was used for biodegradation of PAHs by Phanerocheate chrysosporium. The BSM was amended by five concentrations (5, 10, 50, 100, 150 mgL-1) of each PAHs as sole carbon and energy source.

Preparation of inoculum

Five agar plugs (6 mm diameter) were cut out from the margin of a 14 days actively growing fungal culture of Phanerocheate chrysosporium ATCC 32629 on Malt Extract (ME) agar plates. These plugs were used as inoculum.

Biodegradation of PAHs by Phanerocheate Chrysosporium

The biodegradation experiment was performed in 500ml conical flasks containing 150ml of BSM amended by five concentrations (5, 10, 50, 100, 150 mgL-1) of six examined PAHs (naphthene, anthracene, phenantherene, Pyrene, fluoranthene and acenaphthen). Every flask was inoculated by 5 agar plugs (6mm) of the actively growing fungal strain as inoculum. Three replicates were used for each concentration of each PAH compound. The inoculated BSM as mention above were incubated at 28 ±2°C with continuous shaking at 150 rpm for zero, 3, 7, 14, 21 and 28 days.

Growth and protein determination

After each incubation period, mycelia of Phanerocheate chrysosporiumwere removed by filtration from the cultures through Whitman filter paper No.1. The fungal mycelia (biomass) were used for the quantitative determination of the fungal growth. The fungal filtrates were used for protein determination according to Lowery et al., (1951) [47] as well as for determination of degradation rates by HPLC.

HPLC Analysis

Liquid / Liquid (1:1 v/v) extraction; of P. Chrysosporium BSM / Chloroform were used for quantitative analysis of residual PAHs. The extracted sample (Chloroform layer) was analyzed by High Performance Liquid Chromatography (HPLC) at Micro Analytical Center, Faculty of Science, Cairo University, Giza, Egypt. PAHs were quantified by YL 9100 HPLC system made in South Korea (Quaternary pump No. YL9110, UV/Vis detector No. YL 9120 and Column compartment No. YL 9131) with the 150 mm reversed phase column hypersil ODS-C18, 5µm. The mobile phase consisted of acetonitrite and deionized water. The acetonitrite / deionized water ratio is 85:15 by manual injection with 1 ml/min flow rate at 254nm UV and 40°C.

All the experiments and measurements were done in duplicates and arithmetic averages were used throughout the data analysis and calculations. Degradation percentages were analysis and calculated for two concentrations (5, 150 mgL^-1) of each PAHs examined.

Gamma radiation

Preparation of spore suspension

Five Erlenmeyer flasks (250 ml) containing 100 ml [48,49] of Malt Extract (ME) agar medium were sterilized by autoclaving. The flasks after sterilization were left to cool on a horizontal surface. After cooling and solidification of ME agar medium, the flasks were inoculated by P. Chrysosporium disc (6 mm) on the center of ME agar medium. After that, the inoculated flasks were incubated stagnant for 7 days at 28°C.

Well-grown P. Chrysosporium spores were harvested by sterile saline (30 ml) containing tween-80 (0.1 % v/v). Flasks were shacked gently and spore suspension were collected from each flask into new sterile one to form a pool. This pool represent stock spores suspension to be used for inoculation. Five ml spore suspension (~ 7 × 107 CFU/ml) was distributed in sterile screw-capped tubes. These tubes were exposed to different doses (0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 KGy) of gamma radiation from Indian Chamber of Cobalet-60 (60CO) at National Center for Radiation Research And Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), and have been conducted at dose rate 1 KGy / 12.5 min. at ambient temperature at the time of experiment [50].

Determination of dose-response curve

Surface plate technique have been followed by inoculating serially diluted control (non-irradiated and irradiated suspension on the surface of ME agar plates (0.1 ml). After that, inoculated plates were incubated for 7 days at 28°C to determine their count (dose-response curve). The count of P. Chrysosporium (CFU/ml) was determined and log number of survivors have been plotted against absorbed gamma radiation doses [50,51].

Induction of mutants

From the edge of 14 days actively growing P. Chrysosporium exposed to different doses of gamma radiation and having a morphological changes, five plugs (6 mm) were cut out from each colony of gamma irradiated and non-irradiated (parent strain). These P. Chrysosporium discs were used to inoculate 150 ml of BSM supplemented with 100 mg/L of the six examined PAHs. The inoculated BSM was incubated at 28 ±2°C with continuous shaking at 150 rpm for zero and 14d. The growth (dry weight) and enzyme activity (extracellular protein secretion) have been determined.

Results and Discussion

Growth and extracellular protein of Phanerochete Chrysosporium on different concentrations of different PAHs

The growth of P. Chrysosporium on the five concentrations of fluoranthene reached the maximum growth at day 21 for the first four concentrations except for the higher concentration 150 mg/L which reached its maximum growth after 7 days incubation period as indicated in Figure 1. An extracellular protein secreted reached their maximum productivity at 3rd day of incubation for the first four concentrations of fluoranthene except for the highest concentration 150 mg/L which reached the highest protein at day 7 as indicated in Figure 2.

The maximum growth of 100 mg/L of phenanthrene has been reached after 21 days while the higher concentration of 150 mg/L reached its maximum growth at 14 days. The lowest concentrations (5 and 10 mg/L) need more time to reach their maximum productivity (28 days) as shown in Figure 3.

However, protein secretion has been recorded after 3rd day for the lowest concentrations (5 and 50 mg/L) phenanthrene as indicated in Figure 4.

The maximum growth of P. Chrysosporium on different concentrations of acenaphthene was recorded after 21 days of incubation and also the maximum protein secretion as shown in Figure 5 and Figure 6respectively.

Results of the growth of P. Chrysosporium on naphthene revealed that the maximum growth was recorded at the end of the incubation period (28 days) as indicated in Figure 7. While the maximum protein secretion reached maximum production after 14 days Figure 8.

The results revealed that growth of P. Chrysosporium on the first four concentrations on anthracene reached their maximum growth at the end of incubation (28 days) except for the higher concentration (150 mg/L) which reached its maximum growth earlier (14 days) as shown in Figure 9. Secreted protein by P. Chrysosporium reached their maximum productivity at 7th day while at the highest concentration of anthracene (150 mg/L) the maximum productivity has been recorded after 21 days Figure 10.

The maximum growth of P. Chrysosporium on pyrene has been reached at the end of the incubation period (28 days) for all the five concentrations Figure 11. However, the maximum protein secretion was recorded at 3rd and 7th days except for concentration (10 mg/L) which reached maximum protein secretion at 21 days as indicated in Figure 12.

The results of the present study, showed that growth and extracellular protein secreted by P. Chrysosporium reached their maximum activities at concentrations (5, 50, 100 and 150 mgL^-1) of the six examined PAH compounds i.e at low and high concentration of PAHs. While most studies are based on low concentrations only [26, 67].

Degradation of different PAHs

Degradation of six different PAHs by Phanerochete Chrysosporium has been determined after 28 days of the incubation period (the end of incubation). The results as indicated in Table 1 revealed that Phanerochete Chrysosporium degraded 87.77 % and 86.38 %, 61,27 % , 57.17 % , 51.90% and 29.23% of 150 mg/L of fluoranthene (Flu.), anthracene (Anth.), phenantherene (Phen.), naphthene (Naph.), pyrene (Pyr.) and acenaphthen (Acen.) respectively. The results also of the higher concentration of Flu.,Anth., Phen. , Naph. And Pyr. were degraded by P. Chrysosporium more than the lower concentration. However Phanerochete Chrysosporium degraded the lower concentration (5 mg/L) of Acen. more than the higher concentrations (150 mg/L). The degradation of Acen. Was 64.80 %. The results of Bishnoi et al., (2008) and Wang et al., (2009) [52,53] proved that Phanerochete Chrysosporium having the ability to best degradation of PAHs than other WRF such as Trichoderma (Yao, 2015)[54], Pycropous (Li et al., 2018) [33]or other fungi such as Aspergillus sp.[26] (Lahkar and Deka, 2016).

The results of the present study were confirmed by the results of other investigators. Oxidoreductase enzyme-based bio-catalytic processes for the biodegradation and biotransformation of a wide array of harmful environmental contaminants have been studied [55]. Immobilized white-rot fungus P. Chrysosporium removed greater than 90 % after 60 h. of phenantherene, pyrene, and benzo[a] pyrene in Rotating Biological Contactor (RBC) reactor [56]. The same authors (Zheng and obbard, 2002 b),[57] found that the key factors affecting the oxidation of PAH P. Chrysosporium inclusive Mn2+ concentrations on the extracellular enzyme production.

P. Chrysosporiumis known to produce the MnP enzyme[35]. P. Chrysosporium act synergistically with soil indigenous microorganisms in the oxidation of low molecular weight PAHs (LMW-PAHs) e.g Acenaphthene, fluorine, Phenantherene, Fluoranthene, and Pyrene, where oxidation was enhanced by up to 43 % in the presence of fungus. [57]. Meanwhile, the whiterot fungus Trametestrogii degraded more than 90 % of 250 - 500 ppm of nitrobenzene and anthracene within 12 - 24 days [58]. P. chrysosporium was able to degrade the highly persistent irgarol compound also. Via MnP enzyme [59].

Immobilized MnP enzyme was able to degrade Pyrene (> 86 %), anthracene (>65%) alone or in a mixture, and to less extent fluoranthene (< 15.2 %) and phenantherene (< 8.6 %) by white-rot fungi, Anthracophyllum discolor [60,61].

A defined mixture of fluoranthene, phenantherene, anthracene, pyrene, and benzo [a] anthracene have been degraded by + 60 % fluoaranthene + 40 % phenantherene, and benzo [a] anthracene was 15.2 % and 15.8 % being achieved with Phanerochete Chrysosporium [62].

MnP enzyme was able to degrade the three PAHs anthracene, dibenzothiophene, and pyrene[63]. In sterile soil, degradation in 42 days incubation of five PAHs by P. Chrysosporium was as follows: Phen. 98.96 %; anth. 92.6 %; Pyrene 92.2 %; acenaphthene 83.8 % and fluoranthene 79.8 % [52].While, P. Chrysosporium degraded phenantherene; pyrene and benzo [a] pyrene with rang 72.77 % to 25.50 % (Wang et al., 2009)[53].When a consortium of WRF (CW-1) was used for PAHs degradation, the removal of Phen. (70 - 80 %) and Pyr. (90 %) by live fungi [64].

The oxidation of Anth.,Pyr. andFluo. Detected within 24 h. were 59.7 % , 50.7 % and 49.8 % respectively, whereas only 25.3 % of benzo [a] pyrene and 32.4 % of Phen. were oxidized by white rot fungus Pycnoporus coccineus enzyme actively [65].Another WRF, Pseudotrametes gibbosa was proved to be able to utilize pyrene as the sole carbon and energy source [41].

The results of metabolic peculiarities of Anth. andPyr. by WRF Pycnoporus sanguineus H1 showed that both extracellular laccase and intracellular catabolic enzymes may perform a significant function in the initial oxidation of Anth. Whereas Pyr. could be only oxidized by catabolic enzymes [66].Another WRF, Armillaria sp. F022 is capable to degrade a wide range of four-ring aromatic hydrocarbon like Fluoranthene. Armillaria sp. was able to degrade 10 mg/L of Fluo. by 100 % within 30 days [67].

Effect of Gamma radiation on the viable count of Phanerochete Chrysosporium (dose - response Curve)

Results of exposing of P. Chrysosporium to different doses of gamma radiation revealed that as the dose of gamma radiation increased, the viable count of spore suspension decreased gradually. The results also showed that 7.0 KGy reduced the viability of P. chrysosporium completely as indicated in Table 2 and Figure 13. Dose 6.0 KGy reduced the viable count by 7.70 log cycles.

Effect of Gamma radiation on growth and extracellular protein non-irradiated (control) and irradiated Phanerochete Chrysosporium

Table 3 indicated the effect of gamma radiation on growth and extracellular protein secreted by P. Chrysosporium on different PAHs. The low dose of gamma radiation - induced the growth of P. Chrysosporium (i.e 0.5 and 1.0 KGy ) and utilized PAHs as a sole carbon and energy source. The growth of P. Chrysosporium was more than its dry weight at the beginning by 6.0, 6.6, 6.6, 3.8, 4.3 and 6 times on 100 mg/L of Flu.,Phen., Acen., Naph. , Anth. andPyr. expose to 0.5 KGy respectively. Also, exposing to 1.0 KGy induced the growth of P. Chrysosporium than their beginning by 7.1, 5.0, 5.0, 3.8, 5.5, and 3.3 times for Flu.,Phen., Acen., Naph., Anth., and Pyr. respectively.

Exposure of P. Chrysosporium to 0.5 and 1.0 kGy gamma radiation induced the growth on Flu. , Phen., Acen.,Naph., Anth. And Pyr. More than parent strain ( non- irradiated control ) by 2.4,2.8; 2.6,2.0 ; 3.8,2.9 ; 1.5,1.5 ; 2.8,3.6 and 2.2,1.2 times respectively. The highest growth rate, in general, was recorded at 0.5 and 1.0 KGy doses of gamma - irradiated P. Chrysosporium. Also, in general, the highest growth rate has been recorded by P. chrysosporium exposed to 1.0 KGy on 100 mg/L Flu. After 14 days incubation period by 7.1 times.

The results of protein secreted by P. Chrysosporium exposed to 1.5 KGy revealed that protein was more than protein determined at their beginning on different PAHs by 3.7, 3.7, 2.3, 3.1, 1.7, and 2.4 times for Flu., Phen., Acen., Naph., Anth., and Pyr. Respectively.

Generally, gamma radiation at low doses induced growth of P. Chrysosporium than its parent strain (non-irradiated) P. chrysosporium and 1.5 KGy induced protein secretion more than non-irradiated P. Chrysosporium showed their maximum at 14 days of incubation with 100 mgL-1concentrations of six examined PAHs. Gradually decrease (dose - response curve) of the present studytill the lethal dose may be explained on the basis that gamma radiation causes a variety of damage to DNA in cells [68]. Survival curves of Phanerocheate chrysosporium spores showed a rise in viability due to radiation - induced germination before a logarithmic decline [69].

Several studies show that gamma radiation can change the genomic structure and induce mutants [70-73]. Also, Abo-State (2003), [48] indicated that Fusarium neoceras mutant number 1 and number 6 were exposed to 1 min. UV- radiation recorded the highest CM Case activity while the CM Case activity from Fusarium oxysporum was mutant number 4 which was exposed to 4 min. UV - radiation. El-Batal and Abo-State (2006) [74], found that enhanced production in Avicelase CM Case, Pictinase, FPase, and xylanase and proteinase by gamma irradiation at dose 1.0 KGy with increased percentage 10 %; 8 %; 31 %; 20 %; 4 % and 34 % respectively. Rajoka (2006) [75] reported enhanced production (1.6 fold) of extracellular endoglucanase enzyme activity over the parent strain.

Abo-State et al., (2010) [49], proved that gamma radiation reduced the viability of Aspergillus MAM-F23 and MAM-F35 gradually. Mutant number 4, which exposed to 0.5 KGy produced higher cellulases than the parent strain.

A gradual decrease in the growth of P. sajor-caju was observed as a gamma - ray dose increased. However, irradiated by gamma - ray, enhanced MnP production at low doses by 4.5 times that of the parent strain [36].

The dose - response curve of two fungal isolates MAM-F15 and MAM-F48 isolated from antibiotics revealed that 6.0 KGy and 5 KGy reduced the viability completely. These doses reduced the viable count of MAM-15 by 4.48 log cycles [76].

In another study, Abo-State et al., (2019)[77]. Found that three fungi isolated from third and fourth - generation of cephalosporins when exposed to gamma radiation doses the viable count reduced gradually as the doses increased. Doses 6.0 and 7.0 KGy reduced the viable count completely.

Conclusion

Phanerochete Chrysosporiumwas able to utilize PAHs as a sole carbon and energy source.

P. Chrysosporium was able to grow on the six tested PAHs (Flu.,Phen., Anth., Naph., Acen., and Pyr.).

P. Chrysosporium degraded 87.77 %; 86.38 %; 61.27 %, 29.23%, 51.90% and 57.17 %, of 150 mg/L Flu., Anth. , Phen., Acen. , Pyr. And Naph.respectively.

As the dose of gamma radiation increased, the viable count of P. Chrysosporium decreased gradually.

Dose 6.0 KGy reduced the viable count by 7.7 KGy, while 7.0 KGy reduced the viability of P. Chrysosporium completely.

Low doses of gamma radiation (0.5 and 1.0 KGy) induced the growth of Phanerochete Chrysosporium than the non-irradiated ones (parent strain).

Dose (1.5 KGy) induced protein secretion by P. Chrysosporium than the non -irradiated ones.

P. Chrysosporium can be used as a good candidate for the biodegradation of PAHs (biotechnological treatment).

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