Abstract

Haloalkalitolerant funguslar, tuz konsantrasyonunun ve pH değerinin yüksek olduğu ortamlarda yaşayabilirler. Stresli koşullar altında üretilen biyoaktif bileşiklerin potansiyel biyoteknolojik uygulamaları vardır. Bu çalışmada, poliekstrem koşullara sahip Acıgöl/Türkiye'den izole edilen 52 mikrofungus, bazı biyoteknolojik özellikler açısından taranmıştır. Antimikrobiyal ve antioksidan aktiviteler sırasıyla agar difüzyon ve 2,2-difenil-1-pikrilhidrazil (DPPH) radikal süpürücü etki yöntemi kullanılarak belirlenmiştir. Enzim aktiviteleri, agar difüzyon tekniğinin kullanıldığı çeşitli yöntemlerle belirlenmiştir. Gümüş nanopartikül (AgNP) sentezi, hücre içermeyen mikrofungus filtratı kullanılarak gerçekleştirilmiştir. İzolatların %40'ı test mikroorganizmalarından en az birine karşı antimikrobiyal aktivite göstermiştir. Penicillium dipodomyicola, E. coli ve S. aureus’a karşı en yüksek antibakteriyel aktiviteyi gösterirken, P. brevicompactum Candida albicans'a karşı en yüksek aktiviteyi göstermiştir. Penicillium dipodomyicola ve P. bilaiae'nin pozitif kontrol ile rekabet edebilecek düzeyde (%90 ve üzeri) serbest radikal süpürücü aktiviteye sahip olduğu belirlenmiştir. Amilaz aktivitesine sahip izolatların tamamı Aspergillus ve Penicillium cinslerine ait olup, A. ochraceous, A. flavus ve P. brevicompactum en yüksek aktiviteye sahip türler olarak belirlenmiştir. İzolatların %55'i proteolitik aktivite gösterirken, A. alliaceus en yüksek aktiviteye sahip izolat olmuştur. Hemen hemen tüm izolatlar (%92) lipolitik aktivite göstermiştir. Özellikle A. amstelodami, P. sizovae ve P. solitum izolatlarının önemli düzeyde lipolitik aktiviteye sahip olduğu belirlenmiştir. İzolatların %35'i selülolitik aktivite göstermiş ve Cladosporium pseudocladosporioides, P. dipodomyicola ve P. bilaiae izolatlarında yüksek aktivite gözlenmiştir. İzolatların sekizi 24 saat içinde AgNP sentezini gerçekleştirmiştir. Tüm sonuçlar değerlendirildiğinde Aspergillus amstelodami, A. ochraceus, Penicillium dipodomyicola ve P. brevicompactum gibi izolatların farklı endüstriyel alanlarda hizmet verme potansiyeline sahip olduğu belirlenmiştir.

SCREENING OF MICROFUNGI ISOLATED FROM ACIGÖL, TÜRKİYE FOR HYDROLYTIC ENZYMES, BIOACTIVE METABOLITES AND SILVER NANOPARTICLE PRODUCTION

Abstract

Haloalkalitolerant fungi can survive in environments with high salt concentrations and pH values. The bioactive compounds produced under stressful conditions have potential biotechnological applications. In this study, 52 microfungi isolated from Acıgöl Lake in Türkiye, offering polyextreme conditions, were screened for some biotechnological properties. The antimicrobial and antioxidant activities of the isolates were determined using the agar diffusion and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging methods, respectively. Enzyme activities were determined by various methods using the agar diffusion technique. Synthesis of silver nanoparticles (AgNPs) was carried out using cell-free filtrate of microfungi. 40% of the isolates showed antimicrobial activity against at least one of Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213 and Candida albicans ATCC 90028 used as the test microorganisms. Penicillium dipodomyicola showed the highest antibacterial activity against E. coli and S. aureus, while P. brevicompactum showed the highest activity against C. albicans. Penicillium dipodomyicola and P. bilaiae were found to have free radical scavenging activity of a level (90% and above) that can compete with positive control. All of the isolates with amylase activity belonged to Aspergillus and Penicillium and the most prominent three of them were A. ochraceous, A. flavus and P. brevicompactum. 55% of the isolates showed proteolytic activity, among which A. alliaceus had the highest activity. Almost all isolates (92%) showed lipolytic activity. Aspergillus amstelodami, P. sizovae and P. solitum had a significant level of lipolytic activity. 35% of the isolates showed cellulolytic activity with highest values Cladosporium pseudocladosporioides, P. dipodomyicola and P. bilaiae. Eight of the isolates carried out AgNP synthesis within 24 h. When all the results were evaluated, Aspergillus amstelodami, A. ochraceus, Penicillium dipodomyicola, and P. brevicompactum appeared to have the potential to serve in different industrial areas.

Keywords

• polyextremophiles
• alkaline lake
• biological activity
• microfungi

References

1. 1. Ahmed, A.-A., Hamzah, H. & Maaroof, M. 2018. Analyzing formation of silver nanoparticles from the filamentous fungus Fusarium oxysporum and their antimicrobial activity. Turkish Journal of Biology, 42(1): 54-62.
2. 2. Al-Fakih, A.A. & Almaqtri, W.Q.A. 2019. Overview on antibacterial metabolites from terrestrial Aspergillus spp. Mycology, 10(4): 191-209.
3. 3. Ali, I., Akbar, A., Anwar, M., Prasongsuk, S., Lotrakul, P. & Punnapayak, H. 2015. Purification and characterization of a polyextremophilic α-Amylase from an obligate halophilic Aspergillus penicillioides isolate and its potential for souse with detergents. Biomed Research International, 1-8. https://doi.org/10.1155/2015/245649
4. 4. Attia, E.Z., Farouk, H.M., Abdelmohsen, U.R. & Mo'men, H. 2020. Antimicrobial and extracellular oxidative enzyme activities of endophytic fungi isolated from alfalfa (Medicago sativa) assisted by metabolic profiling. South African Journal of Botany, 134: 156-162.
5. 5. Ayva, F., Demirel, R., İlhan, S., Usakbek Kyzy, L., Çiğdem, U., Zorluer, N., İrdem, E., Özbiçen, E., Ocak, E. & Tunca, G. 2021. Haloalkalitolerant and Haloalkaliphilic Fungal Diversity of Acıgöl/Turkey. Mantar Dergisi, 12(1): 33-41.
6. 6. Bodade, R. & Lonkar, K. 2022. Extremophilic Fungal Amylases: Screening, Purification, Assay, and Applications, pp. 465-487. In: Sahay, S. (ed.). Extremophilic Fungi, Springer, Singapore, 748 pp.
7. 7. Bunbamrung, N., Intaraudom, C., Dramae, A., Komwijit, S., Laorob, T., Khamsaeng, S. & Pittayakhajonwut, P. 2020. Antimicrobial, antimalarial and anticholinesterase substances from the marine-derived fungus Aspergillus terreus BCC51799. Tetrahedron, 76(41): 131496. https://doi.org/10.1016/j.tet.2020.131496
8. 8. Cantürk, Z., Kocabıyık, E., Öztürk, N. & İlhan, S. 2017. Evaluation of antioxidant and antiproliferative metabolites of Penicillium flavigenum isolated from hypersaline environment: Tuz (Salt) Lake by Xcelligence technology. Microbiology, 86(3): 346-354. https://doi.org/10.1134/S0026261717030055

9. 9. Chandra, H., Kumari, P., Prasad, R., Gupta, S.C. & Yadav, S. 2021. Antioxidant and antimicrobial activity displayed by a fungal endophyte Alternaria alternata isolated from Picrorhiza kurroa from Garhwal Himalayas, India. Biocatalysis and Agricultural Biotechnology, 33: 101955.
10. 10. Chandra, P. & Arora, D.S. 2017. Antioxidant potential of two strains of Aspergillus wentii isolated from soil of different areas of Punjab, India and its optimization using different statistical approaches. Journal of Microbiology and Biotechnology Research, 4(4): 31-41.
11. 11. Chandrasekaran, S., Kumaresan, S.S.P. & Manavalan, M. 2015. Production and optimization of protease by filamentous fungus isolated from paddy soil in Thiruvarur District Tamilnadu. Journal of Applied Biology and Biotechnology, 3(6): 066-069.
12. 12. CLSI. 2004. Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts CLSI document M44-A. Approved Guideline: Clinical and Laboratory Standards Institute.
13. 13. CLSI. 2015. Performance Standards for Antimicrobial Disk Susceptibility Tests CLSI document M02-A12. Approved Standard-Twelfh Edition: Wayne, PA: Clinical and Laboratory Standards Institute.
14. 14. de Carvalho, M.L.d.A., Carvalho, D.F., de Barros Gomes, E., Maeda, R.N., Santa Anna, L.M.M., de Castro, A.M. & Pereira Jr, N. 2014. Optimisation of cellulase production by Penicillium funiculosum in a stirred tank bioreactor using multivariate response surface analysis. Enzyme Research, 2014. https://doi.org/10.1155/2014/703291
15. 15. Demirel, R., Yılmaz Sarıözlü, N. & İlhan, S. 2008. Production of fungal amylase enzyme from Aspergillus species in solid-state culture, Journal of Biotechnology, 136: S31.
16. 16. El-Nahrawy, S., Metwally, M., El-Kodoos, A., Rizk, Y., Belal, E.-S.B., Shabana, S.A. & El-Refai, I.M. 2017. Optimization of culture conditions for production of cellulase by Aspergillus tubingensis KY615746 using rice straw waste. Environment, Biodiversity and Soil Security, 1(2017): 177-189.
17. 17. Farha, A.K. & Hatha, A.M. 2019. Bioprospecting potential and secondary metabolite profile of a novel sediment-derived fungus Penicillium sp. ArCSPf from continental slope of Eastern Arabian Sea. Mycology, 10(2): 109-117. https://doi.org/10.1080/21501203.2019.1572034
18. 18. Fossi, B.T., Tavea, F., Jiwoua, C. & Ndjouenkeu, R. 2009. Screening and phenotypic characterization of thermostable amylases producing yeasts and bacteria strains from some Cameroonian soils. African Journal of Microbiology Research, 3(9): 504-514.
19. 19. Gopinath, S.C., Anbu, P., Arshad, M.M., Lakshmipriya, T., Voon, C.H., Hashim, U. & Chinni, S.V. 2017. Biotechnological processes in microbial amylase production. Biomed Research International, 2017, 1272193. https://doi.org/10.1155/2017/1272193
20. 20. Gouda, M. & Elbahloul, Y. 2008. Statistical optimization and partial characterization of amylases produced by halotolerant Penicillium sp. World Journal of Agricultural Sciences, 4(3): 359-368.
21. 21. Griebeler, N., Polloni, A.E., Remonatto, D., Arbter, F., Vardanega, R., Cechet, J.L., Di Luccio, M., de Oliveira, D., Treichel, H. & Cansian, R.L. 2011. Isolation and screening of lipase-producing fungi with hydrolytic activity. Food and Bioprocess Technology, 4(4): 578-586.
22. 22. Grossart, H.-P., Van den Wyngaert, S., Kagami, M., Wurzbacher, C., Cunliffe, M. & Rojas-Jimenez, K. 2019. Fungi in aquatic ecosystems. Nature Reviews Microbiology, 17(6): 339-354.
23. 23. Gunde-Cimerman, N., Zalar, P., de Hoog, S. & Plemenitaš, A. 2000. Hypersaline waters in salterns–natural ecological niches for halophilic black yeasts. FEMS Microbiology Ecology, 32(3): 235-240. https://doi.org/10.1111/j.1574-6941.2000.tb00716.x
24. 24. Gunde-Cimerman, N., Zalar, P., Petrovič, U., Turk, M., Kogej, T., de Hoog, G.S. & Plemenitaš, A. 2004. Fungi in salterns, pp. 103-113. In A. Ventosa (ed.), Halophilic Microorganisms, Springer Berlin Heidelberg, Berlin, Heidelberg, 350 pp.
25. 25. Hozzein, W.N., Ali, M.I.A. & Ahmed, M.S. 2013. Antimicrobial activities of some alkaliphilic and alkaline-resistant microorganisms isolated from Wadi Araba, the eastern desert of Egypt. Life Science Journal, 10(4): 1823-1828.
26. 26. Kathiresan, K. & Manivannan, S. 2006. Amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. African Journal of Biotechnology, 5(10): 829-832.
27. 27. Kis-Papo, T., Grishkan, I., Oren, A., Wasser, S. & Nevo, E. 2001. Spatiotemporal diversity of filamentous fungi in the hypersaline Dead Sea. Mycological Research, 105(06): 749-756.
28. 28. Kluepfel, D. 1988. Screening of prokaryotes for cellulose-and hemicellulose-degrading enzymes, Methods in Enzymology, 160: 180-186.
29. 29. Koul, M. & Singh, S. 2017. Penicillium spp.: prolific producer for harnessing cytotoxic secondary metabolites. Anticancer Drugs, 28(1): 11-30. https://doi.org/10.1097/CAD.0000000000000423
30. 30. Lee, Y.M., Kim, M.J., Li, H., Zhang, P., Bao, B., Lee, K.J. & Jung, J.H. 2013. Marine-derived Aspergillus species as a source of bioactive secondary metabolites. Marine Biotechnology, 15(5): 499-519.
31. 31. Malpure, P.P., Shah, A.S. & Juvekar, A.R. 2006. Antioxidant and anti-inflammatory activity of extract obtained from Aspergillus candidus MTCC 2202 broth filtrate. Indian Journal of Experimental Biology, 44(6): 468.
32. 32. Mehta, A., Bodh, U. & Gupta, R. 2018. Isolation of a novel lipase producing fungal isolate Aspergillus fumigatus and production optimization of enzyme. Biocatalysis and Biotransformation, 36(6): 450-457.
33. 33. Niknejad, F., Moshfegh, M., Najafzadeh, M.J., Houbraken, J., Rezaei, S., Zarrini, G., Faramarzi, M.A. & Nafissi-Varcheh, N. 2013. Halotolerant ability and α-amylase activity of some saltwater fungal isolates. Iranian Journal of Pharmaceutical Research: IJPR, 12: 113.
34. 34. Oren, A. 2002. Halophilic microorganisms and their environments: Kluwer Academic Publishers, XXI, 575 pp.
35. 35. Orwa, P., Mugambi, G., Wekesa, V. & Mwirichia, R. 2020. Isolation of haloalkaliphilic fungi from Lake Magadi in Kenya. Heliyon, 6(1): e02823. https://doi.org/10.1016/j.heliyon.2019.e02823
36. 36. Ottoni, C.A., Simoes, M.F., Fernandes, S., Dos Santos, J.G., da Silva, E.S., de Souza, R.F.B. & Maiorano, A.E. 2017. Screening of filamentous fungi for antimicrobial silver nanoparticles synthesis. Amb Express, 7(1): 31. https://doi.org/10.1186/s13568-017-0332-2
37. 37. Özgök, Ö. & İlhan, S. 2020. Diversity and distribution of Dematiaceous fungi in Çamaltı Saltern in İzmir province, Turkey. Mantar Dergisi, 11(1): 29-39. doi: 10.30708/mantar.645828
38. 38. Pachauri, P., Aranganathan, V., More, S., Sullia, S. & Deshmukh, S. 2017. Purification and characterization of cellulase from a novel isolate of Trichoderma longibrachiatum. Biofuels, 11(1): 85-91. https://doi.org/10.1080/17597269.2017.1345357
39. 39. Patel, G. & Shah, K. 2020. Isolation, screening and identification of Lipase producing fungi from cotton seed soapstock. Indian Journal of Science and Technology, 13: 3762-3771.
40. 40. Pavlukova, E., Belozersky, M. & Dunaevsky, Y. 1998. Extracellular proteolytic enzymes of filamentous fungi. Biochemistry. Biokhimiia, 63(8): 899-928.
41. 41. Pérez, M.M., Gonçalves, E.C.S., Vici, A.C., Salgado, J.C.S. & de Moraes Polizeli, M.d.L.T. 2019. Fungal lipases: versatile tools for white biotechnology, pp. 361-404. In: Yadav, A.N., Mishra, S., Singh, S. & Gupta, A. (eds). Recent Advancement in White Biotechnology Through Fungi, Springer, 528 pp.
42. 42. Qader, M.M., Hamed, A.A., Soldatou, S., Abdelraof, M., Elawady, M.E., Hassane, A.S., Belbahri, L., Ebel, R. & Rateb, M.E. 2021. Antimicrobial and antibiofilm activities of the fungal metabolites isolated from the marine endophytes Epicoccum nigrum M13 and Alternaria alternata 13A. Marine Drugs, 19(4): 232. https://doi.org/10.3390/md19040232
43. 43. Raghav, D., Jyoti, A., Siddiqui, A.J. & Saxena, J. 2022. Plant‐associated endophytic fungi as potential bio‐factories for extracellular enzymes: Progress, Challenges and Strain improvement with precision approaches. Journal of Applied Microbiology, 133(2): 287-310. https://doi.org/10.1111/jam.15574
44. 44. Romano, I., Vitiello, G., Gallucci, N., Di Girolamo, R., Cattaneo, A., Poli, A. & Di Donato, P. 2022. Extremophilic Microorganisms for the Green Synthesis of Antibacterial Nanoparticles. Microorganisms, 10(10): 1885.
45. 45. Sabotič, J., Trček, T., Popovič, T. & Brzin, J. 2007. Basidiomycetes harbour a hidden treasure of proteolytic diversity. Journal of Biotechnology, 128(2): 297-307. https://doi.org/10.1016/j.jbiotec.2006.10.006
46. 46. Salwan, R. & Sharma, V. 2020. Molecular and biotechnological aspects of secondary metabolites in actinobacteria. Microbiological Research, 231: 126374.
47. 47. Sánchez‐Moreno, C., Larrauri, J.A. & Saura‐Calixto, F. 1998. A procedure to measure the antiradical efficiency of polyphenols. Journal of the Science of Food Agriculture Ecosystems & Environment, 76(2): 270-276.
48. 48. Sikandar, A., Zhang, M., Wang, Y., Zhu, X., Liu, X., Fan, H., Xuan, Y., Chen, L. & Duan, Y. 2020. Mycochemical screening and analysis, antioxidant activity, and biochemical composition of fermentation strain Snef1216 (Penicillium chrysogenum). Journal of Analytical Methods in Chemistry, 1-8. https://doi.org/10.1155/2020/3073906
49. 49. Sohail, M., Ahmad, A. & Khan, S.A. 2016. Production of cellulase from Aspergillus terreus MS105 on crude and commercially purified substrates. 3 Biotech, 6(1): 1-8.
50. 50. Souza, M.T.S., Marinho, B.M., Pasin, T.M., Nelson, D.L. & Benassi, V.M. 2020. Prospection of Filamentous Fungi and the Production of Amylase by Aspergillus sp. M1. 7.2. The Journal of Engineering and Exact Sciences, 6(3): 0365-0376.
51. 51. Srikar, S.K., Giri, D.D., Pal, D.B., Mishra, P.K. & Upadhyay, S.N. 2016. Green synthesis of silver nanoparticles: a review. Green Sustainable Chemistry, 6(1): 34-56. https://doi.org/10.4236/gsc.2016.61004
52. 52. Sun, Y., Qian, Y., Zhang, J., Wang, Y., Li, X., Zhang, W., Wang, L., Liu, H. & Zhong, Y. 2021. Extracellular protease production regulated by nitrogen and carbon sources in Trichoderma reesei. Journal of Basic Microbiology, 61(2): 122-132.
53. 53. Topal, Ş., Pembeci, C., Borcaklı, M., Batum, M. & Çeltik, Ö. 2000. Türkiye’nin tarımsal mikoflorasının endüstriyel öneme sahip bazı enzimatik aktivitelerinin incelenmesi-I: Amilaz, proteaz, lipaz. Turkish Journal of Biology, 24: 79-93.
54. 54. Vansteelandt, M., Kerzaon, I., Blanchet, E., Tankoua, O.F., Du Pont, T.R., Joubert, Y., Monteau, F., Le Bizec, B., Frisvad, J.C., Pouchus, Y.F. & Grovel, O. 2012. Patulin and secondary metabolite production by marine-derived Penicillium strains. Fungal Biology, 116(9): 954-961. https://doi.org/10.1016/j.funbio.2012.06.005
55. 55. Vázquez-Montoya, E.L., Castro-Ochoa, L.D., Maldonado-Mendoza, I.E., Luna-Suárez, S. & Castro-Martínez, C. 2020. Moringa straw as cellulase production inducer and cellulolytic fungi source. Revista Argentina De Microbiologia, 52(1): 4-12.
56. 56. Ventosa, A. 2004. Halophilic microorganisms. Berlin, Germany: Springer, 350 pp.
57. 57. Verma, V.C., Kharwar, R.N. & Gange, A.C. 2010. Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus. Nanomedicine, 5(1): 33-40. https://doi.org/10.2217/nnm.09.77
58. 58. Wadia, T. & Jain, S.K. 2017. Isolation, screening and identification of lipase producing fungi from oil contaminated soil of Shani Mandir Ujjain. International Journal of Current Microbiology and Applied Sciences, 6(7): 1872-1878.
59. 59. Wei, Y. & Zhang, S.-H. 2018. Abiostress resistance and cellulose degradation abilities of haloalkaliphilic fungi: applications for saline–alkaline remediation. Extremophiles, 22: 155-164.
60. 60. Wei, Y. & Zhang, S.-H. 2019. Haloalkaliphilic Fungi and Their Roles in the Treatment of Saline-Alkali Soil, pp. 535-557. In Tiquia-Arashiro, S. & Grube, M. (eds.). Fungi in Extreme Environments: Ecological Role and Biotechnological Significance, Springer, XVIII, 626 pp.
61. 61. Yike, I. 2011. Fungal proteases and their pathophysiological effects. Mycopathologia, 171(5): 299-323.
62. 62. Zhang, X., Li, S.-J., Li, J.-J., Liang, Z.-Z. & Zhao, C.-Q. 2018. Novel natural products from extremophilic fungi. Marine Drugs, 16(6): 194. https://doi.org/10.3390/md16060194