Marine-derived Penicillium oxalicum M6A as a halotolerant biocatalyst for enzymatic biodegradation and detoxification of the azo dye Direct Red 75

Year 2025, Volume: 26 Issue: 2, 143 - 155, 15.10.2025

Ugochukwu Ozojiofor , Mohammed Abdulsalami Nkechi Egbe Ahmed Haroun

Abstract

The structural complexity and synthetic origin of azo dyes such as Direct Red 75 (DR75) make them environmentally persistent and challenging to remove from industrial effluents. In this study, Penicillium oxalicum M6A, a halotolerant marine-derived fungus isolated from the Nigerian coastline, was evaluated for its capacity to biodegrade and detoxify DR75. The influence of pH, temperature, salt, and dye concentration on degradation efficiency was assessed, along with enzymatic activity. Fourier-transform infrared (FTIR) spectroscopy and gas chromatography–mass spectrometry (GC– MS) analyses were employed to identify degradation products and predict metabolic pathways. Toxicity was determined using three bacterial strains and two crop plant seeds. Optimal degradation was achieved at a pH of 5 (90.37%), a temperature of 35°C (66.82%), a dye concentration of 50 mg/L (91.53%), and a NaCl concentration of 4% (67.08%). Enzymatic assays revealed significant upregulation of laccase (24.17 U/mL), azoreductase (13.14 U/mL), and lignin peroxidase (12.54 U/mL), indicating their involvement in dye breakdown. FTIR analysis confirmed the disappearance of characteristic azo and sulfonic peaks, while GC–MS identified key metabolites such as 2,3-dihydrobenzofuran, m-hydroquinone, and 6-ethoxy-6-methyl-2-cyclohexenone. Microtoxicity and phytotoxicity assessments revealed that the degradation products of DR75 by P. oxalicum M6A did not inhibit the bacterial strains and exhibited low toxicity to the seeds of the crop plants used. These findings establish that P. oxalicum M6A can effectively degrade and detoxify DR75, converting it into less toxic metabolites. These observations highlight the biocatalytic potential of P. oxalicum M6A for treating dyecontaminated saline wastewater and encourage further scale-up for environmental applications.

Keywords

Penicillium oxalicum M6A , Laccase , recalcitrant pollutants , peroxidase , synthetic dyes , ecotoxicity assessment

Ethical Statement

Since the article does not contain any studies with human or animal subject, its approval to the ethics committee was not required.

Thanks

The authors are grateful to the Department of Biotechnology, Faculty of Science, Nigerian Defence Academy, and the National Research Institute for Chemical Technology, Zaria, for carrying out the research and analysis in their laboratories. Also, the authors extend their appreciation to the Tertiary Education Trust Fund-Institutional Based Research for providing the grant for the study.

References

• Akdogan, H. A., Topuz, M. C., & Urhan, A. A. (2014). Studies on decolorization of reactive blue 19 textile dye by Coprinus plicatilis. Journal of Environmental Health Science and Engineering, 12, 49–56.
• Ali, N., Hameed, A., Ahmed, S., & Haque, A. (2009). Decolorization and degradation of textile azo dye by Bacillus subtilis. Environmental Technology, 30(10), 1103–1110.
• Al-Tohamy, R., Ali, S. S., Xie, R., Schagerl, M., Khalil, M. A., & Sun, J. (2023). Decolorization of reactive azo dye using novel halotolerant yeast consortium HYC and proposed degradation pathway. Ecotoxicology and Environmental Safety, 263, 115436.
• Ameenudeen, S., Unnikrishnan, S., & Ramalingam, K. (2021). Statistical optimization for the efficacious degradation of reactive azo dye using Acinetobacter baumannii JC359. Journal of Environmental Management, 279, 111512.
• Amend, A., Burgaud, G., Cunliffe, M., Edgcomb, V. P., Ettinger, C. L., & Gutiérrez, M. H. (2019). Fungi in the marine environment: Open questions and unsolved problems. mBio, 10(3), e01189-18. https://doi.org/10.1128/ mBio.01189-18
• Asses, N., Ayed, L., Hkiri, N., & Hamdi, M. (2018). Congo red decolorization and detoxification by Aspergillus niger: Removal mechanisms and dye degradation pathway. BioMed Research International, 2018, 7148537. https:// doi.org/10.1155/2018/7148537
• Bagewadi, Z. K., Sikandar, I. M., & Harichandra, Z. N. (2017). Purification and immobilization of laccase from Trichoderma harzianum strain HZN10 and its application in dye decolorization. Journal of Genetic Engineering and Biotechnology, 15, 139–150.
• Basu, A., & Kumar, G. S. (2014). Targeting proteins with toxic azo dyes: A microcalorimetric characterization of the interaction of the food colorant amaranth with serum proteins. Journal of Agricultural and Food Chemistry, 62, 7955–7962.
• Ben Mbarek, W., Issa, M., Salvadó, V., Escoda, L., Khitouni, M., & Suñol, J. J. (2023). Degradation of azo dye solutions by a nanocrystalline Fe-based alloy and the adsorption of their by-products by cork. Materials, 16(18), 7612.
• Ben-Ali, W., Delphine, C., David, N., Christian, L., Annick, T. D., Emmanuel, B., Craig, B. F., Guiliano, S., Laurence, L. M., Eric, R., & Tahar, M. (2020). Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications. BMC Biotechnology, 20, 27. https://doi.org/10.1186/s12896-020-00637-1
• Cheng, Z. Z., Yang, Z. P., Jing, D. J., & Chen, H. P. (2012). Decolorization of 12 kinds of dye by the mycelium pellets of Trametes gallica under nonsterile condition. Mycosystema, 31(6), 878–889. Cab Digital Library+13ResearchGate+13IIPSeries+13
• Couto, S. R. (2007). Decolouration of industrial azo dye by crude laccase from Trametes hirsuta. Journal of Hazardous Materials, 148(3), 768–770. https://doi.org/10.1016/j.jhazmat.2007.06.123 Wiley Online Library+7PubMed+7ResearchGate+7
• Ellouze, M., & Sayadi, S. (2016). Whiterot fungi and their enzymes as a biotechnological tool for xenobiotic bioremediation. Intech, 17, 103–120.
• ElSayeh, I. (2010). Fungal biodegradation of textile dye. Egyptian Journal of Chemistry, 62(10), 1799–1813.
• Erum, S., & Ahmed, S. (2011). Comparison of dye decolorization efficiencies of indigenous fungal isolates. African Journal of Biotechnology, 10(17), 3399–3411.
• Feng, Y., Cui, J., Xu, B., Jiang, Y., Fu, C., & Tan, L. (2023). A potentially practicable halotolerant yeast Meyerozyma guilliermondii A4 for decolorizing and detoxifying azo dye and its possible halotolerance mechanisms. Journal of Fungi, 9, 851.
• FernándezRemacha, D., GonzálezRiancho, C., Osua, M. L., Arce, G. G., Montánchez, I., GarcíaLobo, J. M., EstradaTejedor, R., & Kaberdin, V. R. (2022). Analysis of laccaselike enzymes secreted by fungi isolated from a cave in northern Spain. MicrobiologyOpen, 11, e1279. https://doi.org/10.1002/mbo3.1279
• Fetyan, N. A. H., Abdelazeiz, A. Z., Ismail, I. M., & Shaban, S. A. (2016). Oxidative decolorization of direct blue 71 azo dye by Saccharomyces cerevisiae catalyzed by nano zerovalent iron. Annual Research & Review in Biology, 11(11), 1–12.
• Frisvad, J. C., & Samson, R. A. (2004). Polyphasic taxonomy of Penicillium subgenus Penicillium—A guide to identification of food and airborne terverticillate Penicillia and their mycotoxins. Studies in Mycology, 49, 1–173.
• Gavril, M., & Hodson, M. (2007). Investigation of the toxicity of the products of decoloration of amaranth by Trametes versicolor. Journal of Environmental Quality, 36, 1591–1598.
• Gharieb, M. M., Soliman, A. M., & Hassan, E. M. (2020). Biodegradation of the azo dye DR75 81 and reactive brilliant red X-3B by wild strains of yeasts Meyerozyma guilliermondii and Naganishia diffluens. International Journal of Current Microbiology and Applied Sciences, 9(3), 1243–1260.
• He, H., Chen, Y., Li, X., Cheng, Y., Yang, C., & Zeng, G. (2017). Influence of salinity on microorganisms in activated sludge processes: A review. International Biodeterioration & Biodegradation, 119, 520–527.
• Houbraken, J., Frisvad, J. C., & Samson, R. A. (2011). Taxonomy of Penicillium section Citrina. Studies in Mycology, 70, 53–138.
• Ikram, M., Naeem, M., & Zahoor, M. (2022). Biological degradation of the azo dye basic orange 2 by Escherichia coli: A sustainable and ecofriendly approach for the treatment of textile wastewater. Water, 14, 2043.
• Jafari, N., Soudi, M. R., & Kasra-Kermanshahi, R. (2014). Biodegradation perspectives of azo dye by yeasts. Microbiology, 83, 484–497.
• Kalyani, D. C., Telke, A. A., Dhanve, R. S., & Jadhav, J. P. (2009). Ecofriendly biodegradation and detoxification of reactive red 2 textile dye by newly isolated Pseudomonas sp. SUK1. Journal of Hazardous Materials, 163, 735–742.
• Kaushik, P., & Malik, A. (2009). Fungal dye decolorization: Recent advances and future potential. Environment International, 35(1), 127–141.
• Kilic, N. K., Nielsen, J. L., Yuce, M., & Donmez, G. (2007). Characterization of a simple bacterial consortium for effective treatment of wastewaters with reactive dye and Cr (VI). Chemosphere, 67, 826–831.
• Klatte, S., Schaefer, H., & Hempel, M. (2017). Pharmaceuticals in the environment – A short review on options to minimize the exposure of humans, animals and ecosystems. Sustainable Chemistry and Pharmacy, 5, 61–66.
• Li, N., Chen, G., Zhao, J., Yan, B., Cheng, Z., Meng, L., & Chen, V. (2019). Self-cleaning PDA/ZIF-67@PP membrane for dye wastewater remediation with peroxymonosulfate and visible light activation. Journal of Membrane Science, 591, 117341.
• Martorell, M. M., Ruberto, L. A. M., Fernández, P. M., Figueroa, L. I. C., & MacCormack, W. P. (2017). Bioprospection of cold-adapted yeasts with biotechnological potential from Antarctica. Journal of Basic Microbiology, 57(6), 504–516.
• Murshid, S., & Dhakshinamoorthy, G. P. (2021). Application of an immobilized microbial consortium for the treatment of pharmaceutical wastewater: Batch-wise and continuous studies. Chinese Journal of Chemical Engineering, 29, 391–400.
• Ozojiofor, U. O., Abdulsalami, M. S., Egbe, N. E., & Haroun, A. I. (2025). Biotechnological potential of marine-derived fungi for textile dye degradation via laccase-like activity. Suan Sunandha Science and Technology Journal, 12(2), 26-43.
• Ozojiofor, U. O., Abdulsalami, M. S., Egbe, N. E., Haroun, A. I., Hassan, A. U., & Onuh, K. (2025). Molecular docking of laccase from Trametes versicolor with ligand substrates from textile dyes and antibiotics impacting the environment. Science & Technology Asia, 30(2), 268–281.
• Parshetti, G. K., Kalme, S. D., Saratale, G. D., & Govindwar, S. P. (2007). Biodegradation of malachite green by Kocuria rosea MTCC 1532. Acta Chimica Slovenica, 54(4), 792–798.
• Phugare, S. S., Kalyani, D. C., Surwase, S. N., & Jadhav, J. P. (2011). Ecofriendly degradation, decolorization and detoxification of textile effluent by a developed bacterial consortium. Ecotoxicology and Environmental Safety, 74, 1288–1296.
• Pointing, S. B. (2001). Feasibility of bioremediation by white-rot fungi. Applied Microbiology and Biotechnology, 57(1–2), 20–33.
• Rai, H., Bhattacharyya, M. S., Singh, J., Bansal, T. K., Vats, P., & Banerjee, U. C. (2005). Removal of dyes from the effluent of textile and dyestuff manufacturing industry: A review of emerging techniques with reference to biological treatment. Critical Reviews in Environmental Science and Technology, 35(3), 219–238.
• Ren, S., & Osborne, W. (2020). Heavy metal determination and aquatic toxicity evaluation of textile dye and effluents using Artemia salina. Biocatalysis and Agricultural Biotechnology, 25, 101621.
• Revankar, M. S., & Lele, S. S. (2007). Synthetic dye decolorization by white rot fungus, Ganoderma sp. WR-1. Bioresource Technology, 98(4), 775–780.
• Rosales, E., Pazos, M., & Sanromán, M. A. (2011). Comparative efficiencies of the decolorization of leather dye by enzymatic and electrochemical treatments. Desalination, 278(3), 312–317.
• Saratale, R. G., Saratale, G. D., Chang, J. S., & Govindwar, S. P. (2011). Bacterial decolorization and degradation of azo dyes: A review. Journal of the Taiwan Institute of Chemical Engineers, 42(1), 138–157.
• Saratale, R. G., Saratale, G. D., Kalyani, D. C., Chang, J. S., & Govindwar, S. P. (2009). Enhanced decolorization and biodegradation of textile azo dye Scarlet R by using developed microbial consortium-GR. Bioresource Technology, 100(9), 2493–2500.
• Sarkar, S., Banerjee, A., Chakraborty, N., Soren, K., Chakraborty, P., & Bandopadhyay, R. (2020). Structural-functional analyses of textile dye degrading azoreductase, laccase and peroxidase: A comparative in silico study. Electronic Journal of Biotechnology, 43, 48–54.
• Saroj, S., Kumar, A., & Dastidar, M. G. (2014). Biodegradation of azo dyes by Penicillium oxalicum SAR-3 in aquatic environment. Environmental Technology, 35(13–16), 1636–1645.
• Singh, R. L., Singh, P. K., & Singh, R. P. (2015). Enzymatic decolorization and degradation of azo dyes – A review. International Biodeterioration & Biodegradation, 104, 21–31.
• Sumathi, S., & Manju, B. (2000). Uptake of reactive textile dye by Aspergillus foetidus. Enzyme and Microbial Technology, 27, 347–355.
• Telke, A. A., Kalyani, D. C., Jadhav, S. U., & Govindwar, S. P. (2010). Kinetics and mechanism of Reactive Red 141 degradation by a bacterial isolate Rhodococcus pyridinivorans NT2. Journal of Hazardous Materials, 186(1), 748–755.
• Trevizani, J. L. B., Nagalli, A., Passig, F. H., Carvalho, K. Q., Schiavon, G. J., & Model, A. N. L. (2018). Influence of pH and concentration on the decolorization and degradation of BR red azo dye by ozonization. Acta Scientiarum. Technology, 40(1), e35436.
• Vinodhkumar, T., Shamalaa, L. F., Maithilia, S. S., Ramanathan, V., & Sudhakar, S. (2013). Antibacterial properties of secondary metabolites from the endophytic marine algal bacterial population against chicken meat microbial pathogen. International Journal of Current Science, 5, 35–40.
• Wang, N., Chu, Y., Wu, F., Zhao, Z., & Xu, X. (2017). Decolorization and degradation of Congo red by a newly isolated white rot fungus, Ceriporia lacerata, from decayed mulberry branches. International Biodeterioration & Biodegradation, 117, 236–244.
• Willmott, N., Guthrie, J., & Nelson, G. (1998). The biotechnology approach to color removal from textile effluent. Review of Progress in Coloration and Related Topics, 114(2), 38–41.
• Yang, B., Feng, L. D., & Zhang, L. Y. (2012). Decolorization of wastewater containing reactive brilliant blue dye by laccase. Chinese Journal of Environmental Engineering, 6(10), 3514–3518.
• Yang, P., Shi, W., & Wang, H. (2016). Screening of freshwater fungi for decolorizing multiple synthetic dye. Brazilian Journal of Microbiology, 47, 828–834.
• Zhang, W., Zhang, D., Ling, H., & Liu, Y. (2013). Biodegradation of synthetic dyes by Penicillium oxalicum strain DSYD05. Environmental Science and Pollution Research, 20(7), 4601–4610.
• Zouari-Mechichi, H., Mechichi, T., Dhouib, A., Sayadi, S., Martínez, A. T., & Martínez, M. J. (2016). Laccase purification and characterization from Trametes trogii isolated in Tunisia: Decolourization of textile dye by the purified enzyme. Enzyme and Microbial Technology, 39, 141–148.
• Zubbair, N. A., Ajao, A. T., Adeyemo, E. O., & Adeniyi, D. O. (2020). Biodegradation of malachite green by white-rot fungus, Pleurotus pulminarious. Egyptian Academic Journal of Biological Sciences, 12(1), 79–90.