Gallic acid production by some irradiated microbial isolates

Amira Gamal Zaki Mohamed


Abstract


As this study aimed to produce gallic acid (GA) with an economically and environmentally safe method, fungal isolates were used as a source of tannases which hydrolyze tannin compound in broth medium to obtain GA. In the present study, fungal isolates were obtained from dry tea samples. Two fungal isolates were selected as the most potent for GA production. Both fungal isolates gave larger hydrolysis zones on tannic acid agar (TAA) medium and higher GA production on tannic acid broth medium. Both isolates were identified according to their morphological characters (macroscopic and microscopic features) as Aspergillus niger and Aspergillus flavipes. A variety of fermentation conditions including the cultivation medium requirements and physical parameters were tested to enhance the production of GA by both Aspergillus species under submerged conditions. Optimum conditions as incubation period, incubation temperature, pH, shaking speed, inoculum size, substrate concentration, the best nitrogen and carbon sources were evaluated. Gamma irradiation studies were applied. The potentiality of both fungal isolates for further improvement of GA production was applied by mutagenesis through exposure to gamma rays. Only one mutant of Aspergillus flavipes was selected as the most potent obtained mutant for GA production. RAPD analysis was applied to wild and mutant strains of A. flavipes for evaluation the percentage of polymorphism and band pattern between both strains using ten primers. The produced GA was applied in nanotechnology field. GA was purified from cultured medium using ethyl acetate and was used for reduction of silver ions in silver nitrate solution under optimum condition to obtain silver nanoparticles (AgNPs). The synthesisized nanoparticles showed activity against tested human pathogenic bacteria, Candida albicans and three phytopathogenic fungi. The main experimental results can be summarized as follows: 1- Twenty six fungal isolates were recovered from five samples of dry tea. Sixteen isolates were selected as tannase producers by forming hydrolysis zone on TAA medium. 2- After screening, Aspergillus niger and Aspergillus flavipes were selected as the most potent tannase producers. 3- The initial production of GA by A. niger and A. flavipes was 293±9.55 and 234±7.79 mg/l, respectively under submerged fermentation. 4- After Optimization of cultural and nutritional conditions it was found that, 2 days incubation period with shaking at 120 rpm, 30oC incubation temperature, pH 5.0, 1 ml of (106 spore/ml) /50 ml medium, 1.5% TA alone in culture medium and sodium nitrate as nitrogen source were optimum for maximum GA yield by Aspergillus niger and Aspergillus flavipes (395±3.72 and 339±9.25 mg/l, respectively). 5- The optimized conditions resulted in 25.8% and 30.9% increase in GA production by A. niger and A. flavipes, respectively. 6- GA production by both Aspergillus species was found to be independent on fungal growth. 7- It was also noticed that higher tannic acid (TA) concentrations affected negatively the fungal growth due to its toxic effect on microbes. 8- Gamma rays at a dose of 0.5 KGy enhanced the production of GA by Aspergillus niger to 24.8%. In case of Aspergillus flavipes, GA production was enhanced by 55% at a dose of 1.0 KGy. 9- Two mutant strains (mutant 3 and mutant 4) of Aspergillus flavipes showed increase in GA yield than wild strain by 58.1% and 16%, respectively. All obtained mutants from Aspergillus niger showed lower GA yield than the control strain. Therefore, mutant 3 of Aspergillus flavipes was selected as the most potent mutant for GA production. 10- The percentage of polymorphism between wild and mutant strain (mutant 3 of Aspergillus flavipes) was 25.68% using RAPD analysis. 11- By observing the band pattern of both strains, it was noticed that 5 new bands appeared and other 6 bands disappeared in the mutant strain compared to wild strain of A. flavipes which showed that the radiation had an effect on fungal DNA. These results may explain the enhancing effect of gamma rays on GA-production by the mutant strain. 12- Partially purified extract of GA could reduce silver ions in silver nitrate solution and resulted in Ag NPs formation. The color of silver nitrate solution changed from colorless to dark brown. UV-visible spectra of the synthesized nanoparticles showed maximum absorbance peak at 407 nm. The mean size of the synthesized nanoparticles was 13nm±10nm using DLS analysis. 13- AgNPs were found to be active against all tested G-ve and G+ve pathogenic bacteria. Pseudomonas aeruginosa was the most sensitive one, while Staphylococcus aureus was the least sensitive one. MIC values for Pseudomonas aeruginosa, E. coli, Bacillus subtilis and Staphylococcus aureus were 0.53, 0.64, 0.85 and 1.07 µg/ml, respectively. 14- AgNPs were tested against three different Phytopathogenic fungi. Fusarium solani was the most sensitive tested fungus to AgNPs as its growth was completely inhibited at concentration of 53.5 µg/ml. A. niger was moderate sensitive and A. flavus was the least sensitive strain. 15- AgNPs also showed activity against human pathogenic Candida albicans. By increasing AgNPs concentration, the diameter of inhibition zone increased. At concentration of 42.8 µg/ml, the inhibition zone reached 43 mm.


Other data

Other Titles ﺇنتاج حمض الجاليك بواسطه بعض العزلات الميكروبيه المشععه
Issue Date 2015
URI http://research.asu.edu.eg/handle/12345678/27037


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