Investigation of microbial laccase mediated biodegradation of pharmaceuticals and toxicity assessments
Project no. 23407 · Status: Kapanmış (Yayın bekleniyor) · 2022
Summary
<p>In this thesis, the biodegradation potential of pharmaceuticals, including antiviral and antibiotic drugs, through the microbial laccase enzyme was investigated using two different local fungal isolates. Additionally, the aim was to evaluate the toxicity of the products formed during this process on prokaryotic and eukaryotic organisms and to develop environmentally friendly methods to remove pharmaceuticals without harming environmental systems. The laccase production capacities of the fungi Ganoderma adspersum A1 and Alternaria sp. D21, which are known to have the potential to produce various enzymes capable of degrading pharmaceutical compounds in their natural environments, were screened using four different indicator compounds, and their laccase activities were quantitatively measured. The two fungi selected as representatives of Ascomycota and Basidiomycota exhibit completely different adaptations to their environments. The differences in their environmental adaptation and adaptation's potential impact on laccase-mediated degradation processes were compared in this context. The terrestrial-origin Ganoderma adspersum A1 exhibited the highest laccase activity measured at 923.17 U/L. Following partial purification, this value increased by 2.5 times. On the other hand, marine-origin Alternaria sp. D21 showed an enzyme activity of 685 U/L. After conducting morphological and molecular identifications of the isolates, maximum drug tolerance levels were determined using pharmaceutical gradient tests. Following the determination of drug levels through harmaceutical gradient tests, degradation experiments were initiated to assess drug biodegradation using three approaches (Disk diffusion, Thin Layer Chromatography (TLC), and High Pressure Liquid Chromatography (HPLC)). The selected pharmaceuticals, oseltamivir, favipiravir, and ciprofloxacin, are heavily prescribed drugs that have been reported to increase in environmental concentrations in water systems following pandemics or outbreaks, raising concerns about potential toxic risks and the development of resistance. Disk diffusion tests were conducted to assess the decrease in antibiotic effectiveness of ciprofloxacin against four different test bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Staphylococcus epidermidis) following biodegradation. The results confirmed that the antibiotic zones determined by laccase-dependent biodegradation decreased in size. Using TLC analysis, the daily biodegradation of drugs was monitored. Accordingly, the TLC band indicating the presence of ciprofloxacin incubated with Alternaria sp. D21 isolate decreased significantly by the 5th day and completely disappeared by the 9th day. In contrast, in the study with Ganoderma adspersum A1 isolate, the drug remained stable throughout the days, with the TLC band showing relative reduction by the 15th day. Based on TLC monitoring, Ganoderma adspersum A1 isolate exhibited a higher potential for favipiravir degradation compared to Alternaria sp. D21 isolate. Ganoderma adspersum A1 isolate exhibited effective biodegradation potential starting from day 5, as determined by the relative reduction in the TLC band. For oseltamivir phosphate, it was observed that the drug remained stable. According to HPLC analysis of pharmaceutical residues, Alternaria sp. D21 degraded ciprofloxacin by 98.53%, favipiravir by 60.3%, and oseltamivir phosphate by 64.51%. Ganoderma adspersum A1, on the other hand, degraded ciprofloxacin by 47.51% and favipiravir by 66.03%, but did not show effective biodegradation potential for oseltamivir phosphate. The findings were consistent across all three methods.</p><p><br></p><p>When the toxicity profiles of metabolites generated during biodegradation were examined, it was found that they exhibit variable levels of prokaryotic toxicity. However, tests conducted on eukaryotic systems demonstrated that these metabolites are less toxic compared to control groups. Toxicity analyses were standardized and conducted using routine methods such as Microtox acute toxicity and Phytotoxicity assays. The findings indicate that laccase-producing fungi isolated in this study could be effectively utilized in biological wastewater treatment systems, thereby minimizing environmental risks. The use of laccase for the biodegradation of these pharmaceuticals should be considered a potential solution in environmental management strategies and should be further researched.</p>