Introduction

Rapid industrial development and the dramatic increase in heavy metal addition revealed soil heavy metal contamination as a major issue (1). Metalliferous mining and mining, metal smelting, metallurgical industry activities, metal corrosion, and petroleum exploration are the primary methods of introducing heavy metals into the environment.

Pressure on the ecosystem due to adding heavy metals like cadmium (Cd), arsenic andAsbestos (As) and lead (Pb) are toxic to plants, animals, and humans (2-3).Naturally, interactions between metal concentration and availability, as well as medium nature, metal type, and microflora composition, influenced microbial response to heavy metals (4).

Bioaccumulation and biosorption are considered the main strata for the removal of heavy metals by different microorganisms  through uptaking heavy metal ions passively (biosorption) or actively (bioaccumulation). Furthermore, microorganisms introduce varied processes that are dedicated to cleaning up or removing metal from the environment, like permeability barrier blockage, sequestration by intracellular and extracellular, detoxification by enzyme activities, and cellular targets reduction sensitivity to metal ions (5-6).

Resistant microbial populations cause continuous exposure to heavy metals which belong to the genus Corynebacterium, Bacillus, and Ralstonia (7-9). Also, heavy metal leaching by fungi is considered a promising biological strategy based on the induction of weak organic acids by fungi, which could remove heavy metals by forming water soluble complexes (10- 11). Cd and Cr biosorption activities were demonstrated by Aspergillus sp. and Rhizopus sp.Thus, filamentous fungi are considered the most commonly applied fungi for heavy metal leaching (12).

This investigation was carried out to isolate and identify the most nominated resistant fungal strains against heavy metals from of  Ghawar oil field in Al-Ahsa Governorate. These fungal isolates may develop heavy metal resistance mechanisms, making them suitable for use as bioremediation tools.

Materials and methods

For fungal culturing and colony morphological studies, potato dextrose agar (PDA) media, Czapek yeast extract agar (CYA) and Sabouraud dextrose agar (SDA) media with 0.05% chloramphenicol were used (13-14).

Sampling

Heavy metal polluted soils were sampled from subsoil (020 cm) of Ghawar oil field in Al-Ahsa Governorate, Eastern Province, Saudi Arabia., 25.43oN, 49.62oE), Saudi Arabia for various locations from May 1 to 20, 2022 (Fig. 1).Sterile bags were used for keeping samples in sterile, which were directly transported to the laboratory and stored at 4 °C until further use.  After air drying for two weeks, a 1.5 mm polyethylene sieve was employed for soil sieving and dried in an oven at 75 °C. Different physicochemical parameters such as pH, EC and heavy metal contents were evaluated and analysed in triplicate. The Polarized Zeeman Atomic Absorption Spectrophotometer ZA3000 Series (HITACHI, Japan) was used to evaluate the total metal contents of soil samples.

Figure 1: Ghawar oil field in Al-Ahsa Governorate, Eastern Province, Saudi Arabia. Click here to view figure

(https://www.researchgate.net/figure/Map-showing-the-location-of-the-Ghawar-oil-field-and-Zagros-tectonic-belt-in-the-Arabian_fig6_222517325)

Fig (1). Ghawar oil field in Al-Ahsa Governorate, Eastern Province, Saudi Arabia. 

Fungi that can withstand heavy metalsIsolation

The serial dilution method [15] was applied to isolate nominated heavy metal resistant fungal isolates. To achieve this goal, 1000-ppm stock solutions of Cr (NO3)3 and Pb(NO3) were used to prepare 50, 75, 150, 300, 500, and 750 ppm concentrations. Then, sterilised 50, 75, 150, 300, 500, and 750 ppm concentrations were added to potato dextrose agar (PDA) sterilised media petri dishes. As follows, the spread plate protocol was used for isolating resistant fungal heavy metals. After serial dilution with saline solution, polluted soil samples were poured onto potato dextrose agar (PDA) media plates supplemented with 50, 75, 150, 300, 500, and 750 ppm of Cr(NO3) and Pb(NO3).For comparing the growth rate of fungi on different concentrations of heavy metals , normal growth was performed via streaking of fungal isolates on normal SDA media and used as a control after 96 h of incubation.

Evaluation of Cr and Pb were recorded through one-way ANOVA followed by post-hoc multiple comparisons by Duncan’s method with P 0.05 as the significant level.

Microscopic examination 

CYA, SDA and PDA plates supplemented with 0.5 gL-1 chloramphenicol were used for incubating growing fungal hyphae (2 days old) at 30°C for 7 days and observed for colony morphology under these conditions.

Fluorescence Microscope (MSC-F201, Bioevopeak) In the end, varied colonies were purified and preserved on the PDA plates and stored at -80°C.

Molecular Characterization

Fungi isolates were identified using 18S rRNA. To achieve this goal, total fungal DNA was extracted and purified using the E.Z.N.A.® Fungal DNA Mini Kit (D3390-01, Omega BIO-TEK, USA) according to manufacturer protocol. DreamTaq Green PCR Master Mix (2X)( K1081, Thermo Fisher, USA) was used for specific gene amplification according to manufacturer protocol through a Creacon (Holland, Inc) Polymerase Chain Reaction (PCR) system cycler. The reaction consists of 1 min of denaturation for 1 min at 95ºC, annealing for 45 sec at 55ºC, and extension for 1 min 30 sec at 72oC. A final extension at 72ºC was performed for 5 min (16). 1.0 % agarose was applied for visulations. Specific DNA fragments were eluted from the agarose gel. The E.Z.N.A.® Gel Extraction Kit (D2500-01, Omega BIO-TEK, USA) was used to purify the PCR products. Sequence analysis was employed using the ABI PRISM® 3100 Genetic Analyzer (Micron-Corp.Korea). Gel documentation system (Geldoc-it, UVP, England) was applied for data analysis using Totallab analysis software, ww.totallab.com, (Ver.1.0.1).

Phyllogenetic tree construction

The obtained sequences were aligned on the NCBI website (http://www.ncbi.nlm.nih.gov/webcite) using BLAST to confirm their identity. Genetic distances and MultiAlignments were computed by the Pairwise Distance method using ClusteralW software analysis (www.ClusteralW.com). The nucleotide sequences were also compared to highly similar isolate sequences from the GenBank database.

Results

Soil characterization from two studied sites

As shown by Table (1), heavy metal concentration, electrical conductivity (EC), and pH values reflected significant variation between the two sites. The first site was superior for Cr and Pb concentrations and EC values compared with the second sites. pH values showed acidic and alkaline prosperities for the first and second sites, respectively.

Table 1: metals concentration, Electrical conductivity (EC) and pH form different to sites.

Morphological analysis of fungi

Growing fungal isolates on PDA, CYA, MEA, and Sabouraud dextrose agar (SDA) media were selected for morphological studies. Seventeen heavy metal resistant fungi were identified in heavy metal soil samples. After incubation for 24 h at 30°C, fungal colonial morphology, colour, texture, shape, and diameter were used for initial characterization. Moreover, septation in mycelium, distinguishable reproductive structures, conidia shape and structure were applied for macroscopic examination. Based on previous morphological features, nine and eight fungal isolates from the first and second sites respectively showed resistant patterns against Cr and led and were identified as Aspergillus, Rhizomucor, Neosartorya, Penicilliumand Rhizomucor , Aspergillus , Penicillium , Cunninghamella and Fusarium for the second site.

Resistance to heavy metals in fungal strains

Among seventeen heavy metal-resistant fungal isolates

 As shown by (tables 2 and 3), identified microorganisms from two sites reflected varied resistant patterns against Cr and lead. Among nine microorganisms isolated from the first site which reflected resistant against Cr, Aspergillus flavus reflected highly resistant against all Cr concentrations. Also, Aspergillus Ochraceus and Penicillium oxalicum were resistant to all Cr concentrations except the highest (750 ppm). Furthermore, Penicillium aurantiogriseum, Rhizomucor pusillus and A. tubingensis showed resistant patterns only against Cr at 50, 75, 150, and 300 ppm. Aspergillus terreus, Penicillium aurantiogriseum and Aspergillus wentii which were isolated from the second site, reflected noticeable resistance to all Cr concentrations.

Only Aspergillus flavus, which was isolated from the first site, reflected distinguishable resistance to all Pb concentrations. Also, A. terreus showed resistant patterns against Cr at 50, 75, 150, and 300 ppm of Pb.

Table 2: characterized microorganisms from two sites with different resistant level against Cr.

Data are expressed with grams as fresh weight, ± standard deviation of triplicates.

Table 3: characterized microorganisms from two sites with different resistant level against Lead.

Data are expressed with grams as fresh weight, ± standard deviation of triplicates.

Molecular Identification for Heavy Metal Resistance of Fungi

Using 18S rDNA as a molecular identification marker, four selected fungi which reflected resistance against all concentrations of Cr and Pb were identified and assigned to Genebank as follows: Aspergillus flavus (OP113794), Aspergillus terreus(OP113795), Penicillium aurantiogriseum (OP113796), and Aspergillus wentii (OP113797). As shown by (Table 4), the phyllogenetic tree represented genetic similarity among nominated isolates and high similarity.

Table 4: Identified microorganisms from two sites.

Discussion

Heavy metal pollution is the main concern for researchers due to its spreading influence in developing countries [17].

Variation of pH values among two sites refers to ions H+ adsorbed replacement of soil absorbing complexes by soil solution, which shows the direct influence of accumulated heavy metals on general soil features. According to findings by Smith (1996) who indicated that heavy metal bioavailability was affected by pH, accumulated heavy metals in our experiment could be explained in the light of decreasing heavy metal mobility as a direct influence of high pH values due to hydroxides, carbonate precipitation, or insoluble organic complex formation.

In accordance of many studies, potato dextrose agar (PDA) and Czapk yeast extract agar (CYA) were used as selective media for morphological fungal identification (18).

Different fungal genera were frequently found in heavily metal-contaminated environments (19). The identified specific fungal genus that exhibited high heavy metal resistance could be explained by the following findings.They indicated that R. pusillus is considered the most common species which isolated from contaminated sites and products (20).

In agreements with (21) findings. Morphological characterization was used successfully to identify fungal strains, particularly for the Aspergillus genus, which is commonly considered fungus in the environment due to its high spore spreading.

Many studies have shown that microorganisms can remove heavy metals with high performance and at a lower cost than traditional methods (22).Our obtained results for detecting biosorption activity against heavy metals for different fungal isolates were in agreement with many studies which indicated similar activity for the same fungal isolates (23 and 24). Moreover, chrysogenum, Aspergillus nidulans, Aspergillus flavus, Rhizopus arrhizus, and Trichoderma viride isolates were characterised by distinguishable Pb and Cr resistant activity (25).

The use of fungal biomass for such purposes has been hindered due to problems such as small particle size, poor mechanical strength, low density, and rigidity. More support was added to our findings for using Aspergillus as a heavy metal removal agent (26, 27 and 28). They indicated that Aspergillus niger could be used successfully for removing lead and cadmium ions, especially from wastewater.   In accordance with our results for molecular deification of heavy metal removal fungi, A. terreus and A. tubingensis were identified respectively with unique heavy metal resistant activity (29, 30 and 31).

Many advantageous were detected for applying fungi with heavy metal resistance due to penetrate contaminated soil by hyphae to remove heavy metals. Furthermore, more attention was needed for heavy metals bioremediation and biodegradation processes. Finally, conducted investigations for fungi tolerance heavy metals indicated huge potentials for applied fungi as heavy metals remover.

Conclusion

In this investigation, fungi with significant lead and chromium resistant patterns were isolated from highly heavy metal contaminated soils. For the second site, seventeen identified heavy metal fungal strains that reflected resistance to Cr and led were identified as Aspergillus, Rhizomucor, Neosartorya, Penicillium, Cunninghamella, and Fusarium based on morphological features.Based on the 18S rDNA molecular identification marker, Aspergillus flavus which was isolated from first and second sites, was highly resistant to all Cr and Pb concentrations. From the second site, Aspergillus terreus, Penicillium aurantiogriseum and Aspergillus wentii showed resistant patterns against all Led concentrations. Our obtained results could be reflected by the application of fungi as heavy metal removal for highly contaminated soils.  

Reference

1. Ajaz HM, Arasuc RT, Narayananb VKR, Zahir HMI. Bioremediation of heavy metal contaminated soil by the Exigobacterium and accumulation of Cd, Ni, Zn and Cu from soil environment. Int J Biol Tech nol 2010; 1:94–101.).
2. (Renge,V.C., Khedkar, S. V. and Pandey Shraddha, V. (2012): Removal of heavy metals from wastewater using low cost adsorbents: are view. Scientific Reviews and Chemical Communications 2(4), 580–584.
3. Ayangbenro,S.A. and Babalola, O. O. (2017): A New Strategy for Heavy Metal Polluted Environments: A Review of Microbial Biosorbents Int. J. Environ. PublicHealth 2017, 14, 94; doi: 10.3390/ijerph14010094. www.mdpi.com/journal/ijerph.
4. HERRERA-ESTRELLA L.R., GUEVARA-GARCIA A.A., LOPEZ-BUCIO J. Heavy metal adaptation. Nature. Encyclopedia of Live Science, pp. 1-6, 2001.)
5. Poole RK, Gadd GM. Metals: microbe interactions. Oxford: IRL Press; 1989. 1–37.
6. Ezaka E, Anyanwa CU. Chromium (VI) tolerance of bacterial strains isolated from sewage oxidation ditch. Int J Environ Sci 2011; 1:1725– 1734.
7. Ajaz HM, Arasuc RT, Narayananb VKR, Zahir HMI. Bioremediation of heavymetal contaminated soil by the Exigobacterium and accumulation of Cd, Ni,Zn and Cu from soil environment. Int J Biol Tech nol 2010; 1:94–101.
8. Kozdro JJ, Van Elsas JD. Structural diversity of microbial communities in arable soils of a heavily industrialized area determined by PCRDGGE fi ngerprinting and FAME profi ling. Appl Soil Ec ol 2001; 17:31–42.
9. Ellis RJ, Morgan P, Weightman AJ, Fry JC. Cultivation-dependen and -independent approaches for determining bacterial diversit in heavy-metal-contaminated soil. Appl Environ Microbiol 2003; 69:3223–3230.
10. Yu, K.C., Tsai, L.J., Chen, S.H. and Ho, S.T., 2001. Correlation analyses on binding behavior of heavy metals with sediment matrices. Water Res., 35: 2417-2428.
11. Bosecker, K., 1997. Bioleaching: Metal solubilization bymicroorganisms. FEMS Microbiol. Rev., 20: 591-604.).
12. Ezzouhri, L., Castro, E., Moya, M., Espinola, F. and Lairini, K. 2009. Heavy metal tolerance of filamentous fungi isolate from polluted sites in Tangier, Morocco. 2009. J. Microbiol. Res., 3: 035.
13. Khan, I., Awais, M., Shah , M., Ali, T., Ahmad, S., Junaid, M. and Ahmad, S., 2014. Bacteriological analysis of Siran river system for fecal contamination and metallo-betalactamase blaNDM-1 gene. Pakistan J. Zool., 46: 707 716.
14. Nonaka, K., Masuma, R., Iwatsuki, M., Shiomi, K., Otoguro, K. and Omura, S., 2011. Penicillium viticola, a new species isolated from a grape in Japan. Mycoscience, 52:338–343.
15. WAKSMAN S.A. A method of counting the number of fungi in soil. J. Bacteriol. 7, (3), 339, 1922.
16. Demirel, Yigit & Uzun, Dogancan & Zhang, Yansheng & Fang, Ho-Chun & Day, Alexander & Turan, Osman. (2017). Effect of barnacle fouling on ship resistance and powering. Biofouling. 33. 1-16. 10.1080/08927014.2017.1373279.
17. Ong MC, Kamarruzzaman BY. An assessment of metals (Pb and Cu) contamination in bottom sediment from South China sea coastal waters, Malaysia. Am J Appl Sci 2009; 6:1418–1423
18. Mossel, D.A.A., Kleynen-Semmeling, A.M.C., Vincentie,H.M., Beerens, H. and Catsaras, M., 1970.Oxytetracycline-glucose-yeast extract agar for selectiveenumeration of moulds and yeasts in foods orclinicalmaterials. appl. Bact., 33: 454-457
19. Ren, W., Peijun, L.I., Geng, Y. and Xiaojun, L.I., 2009. Biological leaching of heavy metal from a contaminated soil by Aspergillus niger. Hazard. Mater., 167: 164−169.
20. Kiran, B., Jagdish, C., Uma, H., Rajpal, S.P., Rishi, R. andSalil, A., 2014. Isolation and identification of Rhizomucorpusillus from rhinofacial mucormycosis in a diabeticpatient. JMM Case Reports.DOI 10.1099/jmmcr.0.002931.
21. Henry, E., Iheanacho. Patrick, B., Njobeh, F.M., Dutton, P.A.,Steenkamp, L.S., Julian, Q.M., Barnabas, H.D. andAnthony, H.M., 2014. Morphological and molecularidentification of filamentous Aspergillus flavus andAspergillus parasiticus isolated from compound feeds inSouth Africa. Microbiol., 44:180-184.
22. Congeevaram S, Dhanara ni S, Park J, Dexilin M, Thamaraiselvi K.Biosorption of chromium and nickel by heavy metal resistant fungal andbacterial isolates. J Hazard Mater 2007; 146:270–277.
23. Pardo, R., Herguedas, M., Barrado, E. and Vega, M. (2003): ―Biosorption of cadmium, copper,lead and zinc by inactive biomass of Pseudomonasputida,‖ Analytical and BioanalyticalChemistry 376, 26-32.
24. Ayangbenro,S.A. and Babalola, O. O. (2017): A New Strategy for Heavy Metal Polluted Environments: A Review of Microbial Biosorbents Int. J. Environ. Res. PublicHealth 2017, 14, 94; doi: 10.3390/ijerph14010094. mdpi.com/journal/ijerph.
25. Kumar,R., Singh,P.,Dhir,B.,Sharma,A.K. and Mehta,D. (2014): Potential of some fungal and bacterial species in bioremediation of heavy metals. J.Nuclear Physics, Material Sciences, Radiation and Application.1 (2): 213-223.
26. Kapoor, A., Viraraghavan, T. and Roy Cullimore,D. (1999): Removal of heavy metals using the fungus Aspergillusniger. Bioresour. Technol.70:95-104.
27. Huang,C. and Huang.C.P. (1996): Application of Aspergillus oryzae and Rhizopusoryzae for Cu (II) removal. Water Res.30.1985-1990.
28. Huang, C.P., Westman, D., Huang, C. and Morehart,A.L. (1988): The removal of cadmium (II) from dilute aqueous solutions by fungal biosorpent. Water Sci.Technol. 20:369-376.
29. Lass-Florl, C., 2012, Aspergillus terreus: How inoculum sizeand host characteristics affect its virulence. Infect. Dis.,205: 1192–1194.
30. Houbraken, J. and Samson, R.A., 2011. Phylogeny ofPenicillium and the segregation of Trichocomaceae intothree families. Mycol., 70: 1–51.
31. Samson, R., Hong, A., Peterson, S.S.W., Frisvad, J.C. andVarga, J., 2007. Polyphasic taxonomy of Aspergillussection Fumigati and its teleomorph Neosartorya. Mycol., 59:147–20