Isolation and screening of the various fungal isolates for the remediation of fuel oil derived polycyclic aromatic hydrocarbons (PAHS)

Introduction

Polycyclic Aromatic Hydrocarbons (PAHs) are compounds which are made up of two or more fused benzene rings. They are both hazardous to the environment as well as to the organism by being carcinogenic (Bamforth & Singleton., 2005) [7] . As there is increase in the molecular weight of PAHs the water solubility and vapour pressure decreases while hydrophobicity increases. Long term accumulation of PAHs in the nature can lead to the negative impact on natural environmental receptors (Haritash & Kaushik., 2009) [17] . Fossil fuel, coal tar, lubricating oil, crude oil and municipal solid waste are the common anthropogenic source of PAHs, while natural sources include forest fire, oil seepage and volcanic eruptions (Kadri et al., 2017) [19] . There are various techniques to clean up the contaminants which includes physical, chemical, thermal and biological remediation. Bioremediation is widely regarded as less damaging, less energy intensive and cost-effective method to clean up the contaminants from the environment as compared to the conventional methods (Alegbeleye et al., 2017) [2] . The ability of various fungi and bacteria have been reported to degrade PAHs (Mao & Guan., 2016) [20] Previous research has stated the advantage of fungal bioremediation over bacterial remediation in the following ways: (i) release of enzymes such as laccase, lignin peroxidase and manganese peroxidase having poor substrate specificity; (ii) many fungal species are osmo-and xerotolerant which allow them to live in harsh environment. (iii) the mycelia which is hydrophobic in nature, helps to improve the access of hydrocarbon contaminants (Medaura et al., 2021) [22] . These properties are especially necessary when it comes to the less water-soluble heavy PAHs, which are strongly adsorbed with the organic compounds and are less accessible for microbial degradation (Ghosal et al., 2016) [14] . According to earlier studies, the degradation of PAHs due to native fungal strains are studied which showed various fungal strains of genera like Cladosporium, Alternaria, Fusarium, Trichoderma, Aspergillus, Penicillium, Paecilomyces, Coriolus, Pycnoporus, Pleurotus, Cephalosporium, Mucor, Fomitopsis, and Daedalea (Marchand et al., 2017) [21] . Due to the success rate, researchers are currently working to identify suitable fungal strains that can degrade the PAHs. Hence, majority of studies now focuses on the use of native fungal isolates and its ability to break down PAHs in the environment (Benguenab & Chibani., 2021) [8] . The objective of this study is to find out capability of 22 fungal strains to degrade fuel oil and find the best suitable fungal isolate by a step-by-step method to find the best strain that can help to degrade these environmental contaminants.

Materials and Methods

Collection and Isolation of Fungal Strains

A variety of fungal isolates were collected from the various regions of The Maharaja Sayajirao University of Baroda and some strains were also procured from The Seed Anatomy Lab of The Maharaja Sayajirao University of Baroda. The collected fungal strains were isolated on Potato Dextrose Agar (PDA) by the method given by Sharma and Pandey (2010) [26] and the fruiting body was isolated using the method described by Cheng et al. (2004) [9] .

Screening of Suitable Fungal Isolates

Screening of potential fungal strain for the remediation of PAHs were done using qualitative and quantitative methods. Qualitative screening Each fungal isolate was subcultured for 5 days on PDA media prior to the inoculation on the experimented media. The screening was done using the method described by Hamad et al. (2021) [16] . The subcultured fungal strain was inoculated on the Czapek Dox Agar (CZA) media [potassium chloride, 1; sodium nitrate, 2; dipotassium hydrogen phosphate, 1; ferrous sulfate heptahydrate, 0.01; magnesium sulfate hepta-hydrate, 0.5; and agar, 20 g/L.] devoid of sucrose and supplemented with 0.3ml fuel oil and incubated for 15 days. The plates were observed at 7 th and 15 th day, zone of clearance was taken into consideration for the screening.

Quantitative Screening

Quantitative screening was conducted by using Three Different Methods a. Tolerance Test Tolerance test was conducted to evaluate the ability of fungal strains to grow on the contaminated media. Tolerance test was carried out using the method described by Asemoloye et al. (2020) [6] . Fungal isolates were grown on Bushnell-Haas (BH) agar [magnesium sulphate, 0.20g; calcium chloride, 0.20g; monopotassium phosphate, 1.0g; dipotassium phosphate, 1.0 g; ammonium nitrate, 1.0g; ferric chloride, 0.05g in 1000ml] devoid of any carbon source and supplemented with 5% fuel oil while the control plates were supplemented with 10g/L dextrose. The experiment was performed in triplicate and the readings were taken on 7 th and 15 th day. The formula to calculate tolerance of each fungus is described below

b. Paired Interaction Test

This method was used to find the compatibility of different fungal strains able to coexist with each other. An 8mm agar plug was taken from 7 days old sub cultured plate of different fungi and inoculated at the opposite corner of PDA media and allowed to grow. The plates were observed for 15 days. Monocultures of each strain were taken as control.

Fungal interaction was classified based on the classification given by Stahl and Christensen (1992) [27] namely (i) Neutral intermingling: one or both mycelia grows adjacent to one another without intermingling and causing adverse effect (ii) Replacement: one mycelium grows on other and replaces it.

(iii) Deadlock: mycelium of both the strains does not enter each other's territory.

c. Colorimetric Assay

Fungal strains exhibiting more than 70% tolerance and a positive zone of clearance were selected for the assessment of degradation rate using 2,6-dichlorophenolindophenol (DCPIP) indicator. The selected strains were subcultured for 5 days before the inoculation. The strains were then inoculated on media [MgSO4.7H2O (0.2g), CaCl2 (0.02g), KH2PO4 (1g), K2HPO4 (1g), FeCl3 (0.05g), NH4NO3 (1g) and distilled water (1L).] containing 0.005%w/v DCPIP, 0.1% Tween 80 and 1% fuel oil and was continuously shaken at 120rpm according to the method by Dhar K., et al.

(2013) till 15 days. The readings were taken at 7 th and 15 th day by using spectrometer at 600nm. The degradation rate was calculated using the method given below.

𝑫𝒆𝒈𝒓𝒂𝒅𝒂𝒕𝒊𝒐𝒏 𝒓𝒂𝒕𝒆 = (𝟏 -𝑶. 𝑫. 𝒐𝒇 𝒆𝒙𝒑𝒆𝒓𝒊𝒎𝒆𝒏𝒕𝒂𝒍 𝒔𝒂𝒎𝒑𝒍𝒆 𝑶. 𝑫. 𝒐𝒇 𝒄𝒐𝒏𝒕𝒓𝒐𝒍 𝒔𝒂𝒎𝒑𝒍𝒆 ) × 𝟏𝟎𝟎

d. GC-MS Analysis

Gas Chromatography-Mass Spectrometry (GC-MS) was carried out to find the best fungal isolate to degrade the PAHs. Five days old culture was inoculated on the BH broth supplemented with 1% fuel oil according to the method described by Asemoloye et al. (2020) [6] . The cultures were kept for 15 days and continuously shaken at 120rpm. The tube without fungal isolate was kept as control. For GC-MS analysis the aliquots were mixed in dichloromethane in 1:1 ratio and mixed vigorously and vortexed for a minute and allowed to settle for 5 minutes for the phase separation. The organic layer was carefully extracted and centrifuged for 3000rpm for 3 minutes. The GC-MS was carried out at Gujarat Biotechnology Research Centre (GBRC), Gandhinagar. The instrument condition was set to split mode, with injection temperature of 260 ℃, initial oven temperature was set at 50 ℃ for 5 minutes and then increased at the rate of 20℃/minute to 260 ℃ where it was held for 14.5 minutes.

Result and Discussion Collection and Isolation of Fungal Strain

A total of 22 fungal strains were collected and isolated from the above-mentioned places and labelled as SA 1 and SA 22. The isolates were cultured on PDA media in triplicate and tentative identification was carried out using the morphological and microscopic characteristics. Based on the characters observed five distinct species of Aspergillus, three species of Penicillium, and three species of Trichoderma were identified. Additionally, single species of Phanerochaete, Flavodon, Agaricus, Fusarium, Mucor and Rhizopus were also identified.

Screening of Suitable Fungal Isolates Qualitative screening

The collected fungal strains were cultured for the qualitative screening, zone of clearance around the fungal biomass was taken into consideration for finding the potential strains for PAHs degradation. Out of 22 fungal isolates only 8 strains showed positive result for zone of clearance, while 12 strains showed no results. Additionally, two strains i.e., SA 19 and SA 20 failed to grow on the contaminated media.

The detail finding and the images is given in table 1 and figure 1 respectively. Among the fungal isolate that exhibited the zone of clearance, SA 4 demonstrated the earliest result, followed by SA 8, SA 5 and SA 15. Although SA 2, SA 14 and SA 17 exhibited positive result, the intensity was comparatively lower.

Positive result for the zone of clearance was observed in Penicillium sp. which was aligning with the results of Hamad et al. (2021) [16] and Adigwe et al. (2022) [1] who found zone of clearance for Penicillium chrysogenum. However, according to Daâssi and Almaghribi (2021) [11] smaller zone of clearance was observed for Penicillium sp., contradicting to the present results. Aspergillus sp. i.e., SA 5 and SA 15 also showed significant clearance zone while SA 2 and SA 14 showed minimal results. This contrasts the previous results of Ogbonna et al. (2013) [23] and Adigwe et al. (2022) [1] where Aspergillus flavus and Aspergillus niger exhibited higher degradation. Trichoderma sp. showed no zone of clearance, contradicting to the earlier reports of Trichoderma harzianum exhibiting degradation potential (Osazee et al., 2019; Ameko et al., 2013) [24,4] . Similarly, Fusarium solani showed positive result which was consistent with the study by Al-Otibi et al.

(2023) [3] . No zone of clearance was observed in Mucor sp. and Rhizopus sp., which was differing from the previous studies (Osazee et al., 2019; Ferdinand et al., 2020) [24,13] .

There was absence of zone of clearance for Flavodon sp. and Agaricus sp. with the lack of the prior reports for both.

Quantitative screening a. Tolerance test

The ability of fungal isolate to tolerate the contaminant was compared by monitoring their growth in 1% fuel oil contaminated media. Out of 22 fungal isolates tested eight strains i.e., SA 1 to SA 6, SA 11 and SA 15 showed tolerance value above 70%. Highest tolerance was recorded in SA 1 and SA 11, SA 2 and SA 3 also showed significant tolerance. These results suggest that certain strains can survive and grow in the contaminated area. Table 2 provides details of the tolerance test of various isolates while figure 2 shows rate of tolerance at different days.

Several strains in this study showed low tolerance.

Trichoderma species reported less tolerance like the study done by Tiso et al. (2015) [28] , who found no tolerance in [5,25,30] . In contrast, high tolerance was recorded in Aspergillus flavus, A. fumigatus and A. oryzae which was consistent with previous studies. However, A. oryzae and A. niger showed moderate tolerance in contrast to Jawhari (2014) [18] where A. niger showed high tolerance. Penicillium oxalicum exhibited low tolerance rate while average tolerance was observed in Penicillium chrysogenum and Penicillium spp., which aligned with the research done by Zafra et al. (2014) [30] but contradicted with the results of Velez et al. (2019) [29] . Two studies conducted by Shakam and Gaber (2024) [25] and Cruz-Izquierdo et al. (2021) [10] reported low tolerance for Fusarium solani which was average for this study. Similar result was seen in SA 4 and SA 17, where SA 17 overgrows on SA 4. SA 5 and SA 15 showed compatibility through neutral intermingling. Table 3 describes the detail of co-culture test. 2019) [15] high colour change was noted for the Penicillium sp. which was similar to the results of the present study. According to the previous studies Fusarium solani showed the least colour change which was consistent with the present result. However, no reports on DCPIP test for Phanerochaete chrysosporium was found which was shown to be positive in this study. indicated peaks for the heavy PAHs at the RT between 11-18 while not much peak at the earlier RTs were seen for the lighter compounds as compared to the degraded samples. In contrast all the other degraded samples showed the peaks at the RT between 3-10 indicating the possibility of the formation of the lower weight compounds after degradation. SA 15 showed higher number of peaks at early RTs showing the possibility of higher number of lighter compounds formed after degradation of heavy weight PAHs. There was decrease in the peak of the heavy compounds which was seen in the control samples proving the ability of SA 15 to degrade PAHs. Similar results were observed in SA 4 and SA 5 where there was disappearance of peaks from 10-18 RTs and increase in the size of peaks from 4-9 RTs depicting the degradation of heavy compounds to the lightweight compounds. SA 2 and SA 8 showed almost similar degradation profile where there was disappearance of the higher weight compounds while most of the peaks were seen at low and mid RT range which shows the probability of partial degradation of the heavy compounds. From the above interpretation SA 4, SA 5 and SA 15 showed the highest potential for the degradation of PAHs as compared to the other samples.

Conclusion

Overall, this study investigated the capacity of 22 fungal strains to bioremediate fuel oil contaminated media by breaking down hydrocarbons. The potential fungal isolates were selected based on the methodological process which included qualitative and quantitative analysis using tolerance test, colorimetric assay, compatibility tests, DCPIP assay, and GC-MS analysis. As indicated by the method three fungal isolates namely SA 4, SA 5 and SA 15 from Aspergillus and Penicillium sp. showed the potential to degrade the hydrocarbons. The degradation of the hydrocarbon was validated by the change in the coloration during the DCPIP assay as well as the shift in the retention time of the GC-MS analysis. As observed, the potential fungal isolates can be further evaluated for the field-based application. Moreover, further research is needed to find out the enzymes responsible for the degradation of PAHs and its quantification. Furthermore, the potential of the strains can be increased by the optimization of the various parameters which can help in faster degradation of the contaminants.

Acknowledgement

AcknowledgementThe authors would like to acknowledge Gujarat Biotechnology Research Centre, Department of Science and Technology, Government of Gujarat for providing with GC-MS required for the analytical part of this study. The authors would also like to acknowledge SHODH-ScHeme of Developing High quality research for providing research fellowship that supported this work.

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