Browsing by Author "Magana-Arachchi, D. N."

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Detection of microcystins (cyanotoxin) in selected drinking water wells in the Gampaha district, Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Sinhapitiya, S. P. A.; Herath, H. M.; Bandara, W. M. S. N.; Rajapaksha, A. U.; Vithanage, M. S.; Magana-Arachchi, D. N.; Wanigatunge, R. P.
The prevalence of toxic cyanobacteria and various cyanotoxins, such as microcystins (MCs) in drinking water sources have gained considerable attention worldwide due to their potential health risk. Approximately 80% of the Sri Lankan rural population and 34% of the urban population rely on well water for their daily needs. Therefore, people could be exposed to cyanotoxins by consuming well water. This study investigated the presence of potentially toxic cyanobacteria and microcystin variants in the household well water samples collected from the 13 divisional secretariats in the Gampaha district, the second-most populated district in Sri Lanka. Twenty-six well water samples were collected, and physicochemical parameters such as temperature, salinity, pH, conductivity, and Total Dissolved Solids (TDS) were measured in triplicates, in situ using a multi-parameter. All samples met the Sri Lankan Standards (SLS) for drinking water in terms of pH, salinity, temperature and TDS. One well water sample collected from Wattala did not meet the SLS standards in terms of conductivity (829.00±2.05 μS/cm), indicating the well’s unsuitability for water consumption. Morphological identification of cultures originating from well water samples in cyano-specific BG11 medium indicated the presence of nine cyanobacterial genera, including Gloeobacter, Myxosarcina, Dermocarpa, Xenococcus, Synechococcus, Pseudanabaena, Chroococcus, Lyngbya and Geitlerinema. Among them, Synechococcus and Pseudanabaena are reported to be microcystin producers. The presence of extracellular MCs in water samples was detected by High-Performance Liquid Chromatography (HPLC). Extracellular MCs were extracted from water samples using 70% methanol. MC variants and their concentrations in each well water sample were determined using MC-RR-YR-LR standard mixture (Cat no. 33578; SIGMA ALDRICH) by comparing peak retention times and the area of the peaks. HPLC analysis revealed the presence of the microcystin variant MC-LR only in two water samples collected from wells located in the Divulapitiya and Katana areas with MC-LR concentrations of 195.1 μg/L and 278.3 μg/L, respectively. These values exceeded the standard guideline value of 1 μg/L for MC-LR set by the World Health Organization for drinking water. This indicates the potential health risk for consumers of those wells because there is a potential for hepatotoxic MCs to cause acute and chronic illnesses in humans.
Isolation and identification of microplastic-inhabiting bacteria sampled from a wastewater treatment plant in Gannoruwa, Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Marasinghe, M. G. S. B.; Welagedara, K.; Gunathilaka, H. M. S. A. S. T.; Magana-Arachchi, D. N.
Effluents from wastewater treatment plants may serve as point sources that release microplastic particles into aquatic environments. These microplastic particles may provide solid matrices to attach detrimental bacteria in wastewater and facilitate their dissemination. Our research aimed to identify bacterial genera by isolating culturable bacteria associated with microplastic particles sampled from the wastewater treatment plant in Gannoruwa, Sri Lanka. Samples were collected twice, first in November 2023 and again in January 2024. During each sampling, four wastewater samples and three solid waste samples were obtained for analysis. The wastewater samples were taken from four treatment sites: the inflow, oxidation tank, UV treatment section, and return sludge unit. The solid waste samples included sludge and sand debris separated from the fine screening unit. The wastewater sample (100 mL) was filtered through a 5 mm metal sieve and glass fiber filter (0.7 μm pore size, Whatman GF/F). The residue on the filter paper was examined for microplastic particles using a light microscope. Microplastics were picked manually using sterilized needles and washed with 0.85% sterile NaCl solution. The solid sample (10 g) was dissolved in sterile distilled water (100 mL), and the mixture was allowed overnight for solid particles to settle. The microplastics floating on the water surface were filtered and separated. The isolated microplastic particles from each sample were suspended in 0.85% sterile NaCl solution and vortexed at 2,500 rpm for 1 minute. The solution was shaken at 170 rpm for an hour and was diluted 10-fold. Next, 100 μl aliquots from the diluted suspension were plated on Luria-Bertani (LB) agar plates in duplicate using the spread plate technique and incubated at 37 °C for 24 hours. The culturing resulted in isolating 23 unique bacterial colony morphologies. Identification of bacterial genera of these isolated strains was based on colony morphologies, differential staining methods, and biochemical tests. Among the isolated bacterial strains, 41.66% belonged to the genus Staphylococcus, and over half of them showed the coagulase virulence factor. Genus Micrococcus, Bacillus, Acinetobacter, and Citricoccus represented 25.00%, 16.66%, 8.33%, and 4.16%, respectively. Most microplastic-inhabiting bacteria were found to be gram-positive cocci. The bacterial suspension derived from microplastic particles in return sludge samples had the highest average colony forming units (CFU) concentration of 490 CFU/mL, while the lowest count of 50 CFU/mL was observed from microplastic particles in the UV treatment section. This study indicated that Staphylococcus, Micrococcus, Bacillus, Acinetobacter, and Citricoccus were the common microplastic-inhabiting bacterial genera found in the particular wastewater treatment system, notably with Staphylococcus being the most abundant. Molecular biological bacterial identification is currently in progress.
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