Browsing by Author "Edirisinghe, E. A. A. D."

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Isolation, identification and characterization of phosphate solubilizing bacteria from rhizosphere soil of Aloe vera and Vigna unguiculata
(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Chamikara, H.J.M.P.; Edirisinghe, E. A. A. D.
Phosphorus (P) is an essential element, which is next only to nitrogen, influencing plant growth and productivity. Unlike nitrogen, this element is not acquired through biochemical fixation, but comes from other sources to meet plant requirements. Phosphorus compounds in the soil can be categorized into three groups, including inorganic phosphate compounds, organic phosphate compounds of soil humus and organic/inorganic phosphate compounds associated with cells of living matter. Some of these are in insoluble form and some of them are in soluble form. Plants can absorb only the soluble part and soluble forms are infrequent than insoluble forms in soil. Phosphate Solubilizing Bacteria (PSB) can convert either insoluble organic phosphate or insoluble inorganic phosphate into soluble orthophosphates. They make phosphorus available for plants from insoluble organic and inorganic complex phosphorus sources by solubilization and mineralization. PSB were isolated from rhizosphere soil of Aloe vera and Vigna unguiculata, collected from Kuliyapitiya. Visual detection and even semi-quantitative estimation of the phosphate solubilization ability of microorganisms were carried out using serial dilution and spread plate method using Pikovskaya's Agar (PVK agar) medium. It showed clear zones around the microbial colonies in media containing insoluble mineral phosphates as the single P source. PVK agar plates were observed after incubating for 5 days and PSB strains were selected from the plates. Then selected strains were re-cultured in PVK agar medium. Then plates were observed after 5 days incubation and confirmed the PSB by clear zone formation. Morphological characterization of colonies was done by observing the appearance of colonies in PVK agar medium. Microscopic characterization was accomplished by Gram staining, motility test and endospore staining. Further biochemical tests and molecular biological tests were performed for further identification. Here, twenty-nine biochemical tests were carried out according to Cowan and Steel’s manual. And molecular biological 16S rRNA sequencing was carried out for the confirmation of identified strains from biochemical tests. According to the results obtained, isolated strains were identified as Pseudomonas aeruginosa, Achromobacter xylosoxidans from Aloe vera and Pseudomonas fluorescens, Bacillus subtilis from Vigna unguiculate.
Microbial enumeration assay of fermented products of cassava variety MU51
(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Perera, T. W. N. K.; Amarakoon, R.; Edirisinghe, E. A. A. D.
This study was carried out to investigate the effects of solid state fermentation of cassava variety MU51, on the qualitative properties and total viable microbial counts, of the fermented cassava products, developed by changing the fermentation lengths as two days and three days. The steps involved in the product development are peeling, washing, grating cassava roots into a mash, collecting the mash into sacks, dewatering and fermenting the mash under natural environmental conditions. The fermented, wet cakes obtained are further de-watered by oven-drying process to make fermented, dry products. These fermented dry cassava products appear cream-white in color and soft granular or powdered form in texture. Both products have a fermented smell and the pH value is 5. Microbial enumeration assay is carried out for the raw cassava, fermented wet cakes and fermented dry products, using plate count technique. Particular attention is given on mesophilic, aerobic and facultative anaerobic microorganisms. The bacterial count of raw cassava is (1.8-2.2) x10³ colony forming units per gram. For two days fermented wet cake, the bacterial count is (2.9-3.4) x10⁴ colony forming units per gram and for three days fermented wet cake, it is (2.8-3.3) x10⁴ colony forming units per gram. During fermentation, the bacterial count increases. Increase of acidity during fermentation and depletion of substrates could contribute to a slight decrease in the viable bacterial population, during later stage of fermentation. The fungal count of raw cassava is (6.3-8.6) x10² colony forming units per gram. Fungal count of two days fermented wet cake is 8.5x10² - 1.1x10³ colony forming units per gram and three days fermented wet cake is (1.1-1.3) x10³ colony forming units per gram. During fermentation, the fungal count increases as fungi are favored by the acidification of pulp and are benefited from the metabolites sourced from the growth of other microorganisms. Bacterial count of two days fermented dry product is (2.7-3.2) x10⁴ colony forming units per gram and three days fermented dry product is (2.6-3.0) x10⁴ colony forming units per gram. Fungal count of two days fermented dry product is (7.4-8.6) x10² colony forming units per gram and three days fermented dry product is 9.4x10² - 1.2x10³ colony forming units per gram. This plate count assay can be used as an index to evaluate the microbial content, in order to provide a microbiological specification for the fermented cassava products. Fermented cassava products would be an ideal option that reduces the post-harvest losses of raw cassava.
Soil Actinomycetes: potential source of novel antimicrobial substances
(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Gunasinghe, Y. H. K. I. S.; Edirisinghe, E. A. A. D.
Antimicrobial resistance has been recognized as an escalating problem that makes a huge impact on health and brings the economic outcomes down, through the increase of hospital costs, resource utilization and health care costs. Over-prescription of antibiotics and improper use of antimicrobials are currently described as the major causes for the development of resistant microbial strains. Thus, to overcome this issue, developing novel antimicrobial substances active against microorganisms resistant to currently used antimicrobials is equally important as refraining from over-prescription and improper use of them. Actinomycetes, dominant in soil have been recognized as one of the best antimicrobial compound producers. Therefore, in this study Actinomycetes were isolated from soil using the spread plate technique on Starch Casein Agar supplemented with Cyclohexamide and Nalidixic acid. Sixteen isolates were identified as the members of the order Actinomycetales by studying their morphological characteristics as described in the Bergey’s Manual of Systematic Bacteriology. They were screened for the production of antibacterial and antifungal agents against selected pathogens; Staphylococcus aureus, Escherichia.coli, Klebsiella pneumoniae, Listeria monocytogenes, Bacillus cereus, Aspergillus sp. and Candida sp., both on solid media and liquid media by culturing them in triplicate on Nutrient agar and Starch Casein Broth respectively. The screening on the solid medium revealed that two of the actinomycetes isolates produce antimicrobial compounds against S. aureus, while three of them were producing antimicrobial compunds against E. coli. Furthermore, three of them exhibited antimicrobial activity against K. pneumoniae, while L. monocytogenes was inhibited by two of them. Four of the isolates showed inhibitory effect against B. cereus, whereas six and two of the isolates exhibited antimicrobial activity against Aspergillus spp. and Candida spp. respectively. Through the screening carried out on the broth media in triplicates, it was found that three isolates produce antimicrobial compounds against S. aureus, three against E. coli, two against K pneumonia, four against L. monocytogenes, four against B. cereus, one against Aspergillus sp. and one against Candida sp. This revels that there is a noticeable difference in the pattern of antimicrobial compound production in solid and liquid media. This could mainly be due to the differences of composition of the media used or differences of the concentration of the antimicrobials in these two types of media. As the next step of this study, it is important to test these antibacterial substances against pathogenic organisms resistant to existing antimicrobials.