International Research Symposium on Pure and Applied Sciences (IRSPAS)
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Item Screening of microcystin and microcystin-producing genes in Labugama and Kalatuwawa reservoirs in Sri Lanka(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) De Silva, I. U.; Manage, P. M.Microcystins (MCs) are the most widely studied carcinogenic cyanobacterial hepato- and neurotoxins. The MCs are synthesized by a non-ribosomal pathway through a multifunctional enzyme complex known as microcystin synthetase (mcy) encoded by the mcy gene clusters. The present study was aimed to screen MCs and MC-producing genes in Labugama and Kalatuwawa reservoirs which satisfies 60% of drinking water requirement for Colombo District. In the study, plankton and water samples were collected from the two reservoirs prior to water treatment process. The sampling was performed using a boat on the first week of each month from August to October in 2017 where three sampling locations were selected to collect water at each sampling time. Horizontal plankton samples were collected at 10 cm depth in each location using a 55 µm plankton net while the boat was moving. Water temperature and pH were measured on site using digital meters. Total inorganic nitrogen (N-NO3-, N-NO2-, N-NH3) and total phosphorous were determined using standard spectrophotometric methods, and cyanobacteria were identified under a light microscope using standard algae and cyanobacteria identification keys. Microcystis spp. were isolated and monocultures were prepared on cyanospecific BG 11 media from which the genomic DNA was extracted for the screening of MC-producing gene cluster in water samples using the Polymerase Chain Reaction (PCR). MCs were analysed by High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immunosorbent Assay (ELISA) respectively. Temperature of water samples ranged between 27.8 and 28 oC while pH fluctuated in a range between 7.2 and 7.5. Total inorganic nitrogen was recorded from 0.02 to 0.03 mg/L and total phosphorous fluctuated from 0.01 to 0.02 mg/L during the sampling period. Microcystis spp. was identified as dominant cyanobacteria in cell density range between 176 and 226 cells/mL for both reservoirs. Amazingly, MCs were not present at detectable levels following the HPLC method (detection limit 0.5 mg/L) and ELISA method (detection limit 0.1 µg/L). Also, the PCR amplification showed the absence of mcy cluster genes E, A and B in the water samples. Thus, the results of the study revealed that both Labugama and Kalatuwawa reservoirs have non toxic strains of cyanobacterium Microcystis spp. The results of the present study were supported HPLC, ELISA analysis and molecular analysis.Item Photocatalytic degradation of microcystin-LR using nanostructured rutile and coir fibre(Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Idroos, F. S.; Kottegoda, N.; Manage, P. M.Microcystins (MCs) are secondary metabolites of cyanobacteria, which tend to persist in the aquatic environment. Among 70 recorded analogues of MCs, Microcystin-LR (MC-LR) is the dominant and the most toxic cogener. Photocatalytic technology has been constantly recognized as a promising green approach in treating pollutants. The present study records the effective utilization of nanostructured rutile and coir fibre to treat MC-LR. Approximately 2 g of nanostructured rutile and coir fibre (100 nm) were coated in two separate glass slides and dipped in 100 µg/mL of filter sterilized lake water containing 50, 75 and 100 µg/mL of MC-LR. For both nanoparticles (rutile and coir fibre) at 50, 75 and 100 µg/mL of MC-LR concentrations, two experimental set ups (A-Exposed to sunlight, B-Exposed to 12 W UV light) and two control set ups (A1-Control exposed to sunlight, B1-Control exposed to 12 W UV light) were maintained. Sample aliquots of 1 mL was removed at every 30 minutes interval for a period of 3 hours. Subsequently, samples were subjected to freeze drying followed by reconstitution in 50% HPLC grade methanol and analyzed under PDA-HPLC to quantify the remaining MC-LR concentrations. Under the influence of UV light, nanostructured rutile showed, 100 % removal of MC-LR at 50 and 75 µg/mL within 1.5 hours and 2 hours respectively, whereas 87.4 ± 2.31% removal for 100 µg/mL of MC-LR was recorded at the end of 3 hours. When the same experiment was repeated by exposing to sunlight, MC-LR removal percentages were 77.29 ± 1.9 at 50 µg/mL, 36.4 ± 3.8 at 75 µg/mL and 19 ± 3.78 at 100 µg/mL. Moreover, when nanostructured coir fibre was used under 12W UV light, 100% removal of 50 µg/mL MC-LR, was evident at 2 hours, whereas 85.68 ± 9.4% for 75 µg/mL and 56.2 ± 4.37% for 100 µg/mL was observed at the end of 3 hours. At the exposure to sunlight, nanostructured coir particles showed 72.4 ± 2.3 at 50 µg/mL, 56.2 ± 8.2 at 75 µg/mL and 46.8 ± 6.98 at 100 µg/mL at the end of 3 hours. Two-way ANOVA confirmed that there is a significant difference in the MC-LR photocatalytic degradation ability of nanostructured rutile and coir fibre (P=0.02). Therefore, it could be concluded that nanostructured rutile is effective than coir fibre based nanoparticles in treating MC-LR contaminated water. Furthermore, UV exposure of both types of nanostructures can enhance photocatalytic degradation of MC-LR.Item Isolation, purification and structure elucidation of antimicrobial and bio-active compounds of soil fungi.(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Wijesekara, W. A. M. A.; Wijesinghe, K.D.; Kumara, K. G. N. P.; Manage, P. M.Soil fungi are considered as a rich source of bioactive compounds and antibiotics. They are able to produce a great variety of secondary metabolites characterized by a broad spectrum of properties of bioactive compounds including antiviral, antibacterial, antifungal, and anticancer. Therefore, the objective of the study was isolation, purification and structure elucidation of antimicrobial and bioactive compounds of soil fungi. In the present study, soil samples were collected from the Kelani River mouth, Sri Lanka and twenty nine fungi were isolated on potato dextrose agar. The antimicrobial activity of the methanol crude extract of fungi was tested against two human pathogenic Gram-positive bacteria (Bacillus sp., Staphylococcus aureus-ATCC 2593), two human pathogenic Gram-negative bacteria (Escherichia coli-ATCC 25922, 2785 and Salmonella typhi) and two human pathogenic fungi (Candida albicans and Candida tropicalis) using agar disc diffusion method. One fungal isolate was selected based on the diameter of inhibition zone to isolate and purify antimicrobial (antifungal and antibacterial) compound. The antioxidant property in the crude extract was evaluated using DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging test (Inhibitory Concentration, IC50 = 200.42 ppm). Secondary phytochemical screening for major classes of antibacterial compounds were done using TLC visualization reagent specific for major classes of antibacterial compounds. Structural elucidation of isolated compound was carried out using Gas Chromatography Mass Spectrometry (GC-MS) and Fourier-Transform InfraRed spectroscopy (FTIR). TLC plates of the crude extract developed using different solvent system showed different number of band with antimicrobial compounds as it is revealed by bioautochromatography, solvent system 1 ( Ethyl Acetate (EA): Methanol (Me): Water (Wa) (100:13.5:10) 6 bands against Bacillus sp. solvent system 2 ( EA :Toluene (93:7) ) 2 bands against Bacillus sp. and two bands with antifungal activity was recorded against C. albicans. Band with antimicrobial compounds on Preparative Thin Layer Chromatography (PTLC) was scraped and dissolved in methanol (HPLC) to further purify using Solid Phase Extraction (SPE) and High Performance Liquid Chromatography (HPLC). Antibacterial activity of each purified compounds, was verified using bioautochromatography. According to the secondary chemical screening, the compound with the highest antimicrobial activity was found to have contain Arbutin and Anthraquinone glycoside. The results of the present study revealed that the crude extracts of soil fungi had antibacterial activity against some human pathogenic bacteria and the soil fungi is a potential candidate to utilize as a source to produce antibiotics.