International Research Symposium on Pure and Applied Sciences (IRSPAS)

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    Production of certain extracellular enzymes by some bacteria and amplification of cellulase gene from Bacillus species
    (4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Jayasinghe, J. A. S. M.; Medhavi, P. I. H. R.; Magana-Arachchi, D. N.; Wanigatunge, R. P.; Herath, H. M.
    Bacteria have received attention, due to their ability to produce extracellular enzymes beneficial in various industries. In the present study, extracellular enzyme production by two thermophilic bacteria (Meiothermus ruber, Tepidimonas ignava) and eight other bacterial isolates (Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus aryabhattai, Pseudomonas stutzeri, Pseudomonas aeruginosa, Sphingomonas sp., Burkholderia lata) was investigated. Extracellular amylase, protease, pectinase and cellulase production was studied in vitro in media containing starch, skimmed milk, citric pectin and carboxymethylcellulose respectively, at 28 °C, 35 °C, 45 °C and 55 °C. Hydrolyzing Capacity Index (HCI) at day seven was calculated to identify the isolates, which hydrolyzed a substrate with minimal colony formation. Such isolates would have a higher potential in industrial applications. HCI values were analyzed using one-way ANOVA and Tukey’s multiple comparison tests. All isolates, except thermophilic M. ruber, produced at least one extracellular enzyme within 1-3 days. T. ignava, B. thuringiensis and P. aeruginosa produced amylases. All isolates except B. aryabhattai and M. ruber produced proteases. B. thuringiensis, Sphingomonas sp., B. amyloliquefaciens and P. stutzeri produced cellulases. Pectinases were produced only by B. lata. Thermophilic T. ignava produced amylases and proteases at 28 oC and 35 oC but did not produce any enzyme at 55 °C, the temperature of the Maha Oya hot springs from which it was isolated. B. amyloliquefaciens, P. stutzeri, P. aeruginosa, B. pumilus, Sphingomonas sp. and B. lata produced proteases, which were stable at higher temperatures; 45 °C and 55 °C. It was the only enzyme to be produced at those temperatures. According to the HCI values, B. thuringiensis and P. stutzeri were the most efficient degraders of starch and cellulose, respectively. P. stutzeri, Sphingomonas sp. and B. lata were the best protein degraders. A gene coding for glycoside hydrolase (a cellulase) was amplified from bacteria by PCR using primers designed for Bacillus licheniformis ATCC 14580. Although expected amplicon size was ~1683 bp, amplicons of apporiximately 500 bp, 600 bp and 1000 bp were generated from cellulase producing B. thuringiensis. According to the information available in NCBI, B. thuringiensis has glycoside hydrolase gene of 738 bp suggesting that those amplicons could also be some glycoside hydrolase genes of different lengths. This should be confirmed by DNA sequencing. PCR product was generated by the same primers for B. aryabhattai as well, although it did not produce cellulases in vitro. It could be due to non-expression of the particular gene at the experimental conditions used in this study. These Bacillus species are perceived as sources of purified cellulases and the particular genes would be useful also in transformation of other organisms for industrial purposes
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    Isolation and characterization of cellulose hydrolyzing bacteria for bioethanol production
    (4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Chandrarathne, B. M.
    Bioethanol is a renewable and cleaner liquid fuel alternative to fossil fuels. Bioethanol has gained growing interest over time, because it can provide an economical and environment friendly sustainable energy source. First generation bioethanol production used simple sugars produced by the sugar crops. However, utilization of sugar crops leads to a competition between food supply and energy production. Therefore, second generation bioethanol production has evolved, which uses cellulosic materials from agricultural biomass instead of sugar crops. However, cellulosic materials have to be hydrolysed prior to the fermentation. Enzymatic hydrolysis of cellulose into reducing sugars is catalysed by cellulases. This enzyme is produced by cellulose hydrolysing microorganisms. Therefore, the main objective of this study was to isolate and characterize cellulose hydrolysing bacteria that can be used in bioethanol production. Soil samples were collected from compost sites and from the top of the mat of fibrous roots at the center of the Bird’s nest ferns (Asplenium nidus). Bacterial strains were isolated from soil, using basal salt medium with filter papers as the only source of cellulose carbon. Streak plate technique was used to isolate single bacterial colonies. Isolated bacterial strain was characterized by colony morphology and Gram staining. Functional characterization was done by calculating the ratio between diameter of the clear zone and the diameter of bacterial colony in Congo red agar medium (cellulolytic index). Using DNS assay, the amount of reducing sugars generated during cellulosic material hydrolysis by bacteria-derived cellulase enzyme was measured. Co-culture system, which included isolated bacterial strain and dry baker yeast was optimized to ferment those reducing sugars to bioethanol. Production of bioethanol was measured by an Ebullio meter. Isolated bacterial strain produced 1.37 mg mL-1 concentration of reducing sugars and 23.67 mg mL-1 concentration of bioethanol. Further research is in progress to optimize isolated bacterial strain in bioethanol production