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Item Influence and optimization of growth conditions on phenol degradation by Pseudomonas aeruginosa(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Amirthalingam, S.; Dassanayaka, M. P.; Vivehananthan, K.With their relatively high toxicity, phenol and phenolics are water pollutants of major concern. The application of phenol biodegrading agents to treat phenol in wastewater has shown promising results. Pseudomonas aeruginosa is a well recorded phenol biodegrading bacteria. However, optimal growth parameters for efficient degradation of phenol may vary from one bacteria to another. Therefore, this study focused on testing the effect of growth conditions including strength and pH of the growth medium and inoculum size on phenol degradation by P. aeruginosa and to assess the phenol degradation efficiency by adding glucose as an additional carbon source. The bacterial strain of P. aeruginosa (MH031762) isolated from petroleum contaminated site in Sri Lanka was inoculated to Mineral Salt Media (MSM) with 1200 mgL-1 phenol under different growth conditions. Three-factor factorial design with three levels was used to determine the combination effect of strength (0.5x, 1x and 2x), pH (6.5, 7 and 7.5) of the medium and inoculum size (1%, 2% and 3%) on phenol degradation. Residual phenol in the medium inoculated with bacteria was measured using 4-aminoantipyrene spectrometric method under which the duration (in hours) it took for 50% phenol degradation was calculated. Under the growth conditions found to be as optimum, the biodegradation efficiency of phenol by P. aeruginosa was further tested by adding glucose as an additional carbon source at varying concentrations (0.1%, 0.25%, 0.50%, 0.75% and 1%). Pooled t-test was performed to test the phenol degradation efficiency in bio-stimulated media with glucose. According to the results, there was no combination effect by all the 3 factors together on phenol degradation by P. aeruginosa (P = 0.186). However, a significant impact was shown by the combination of the two factors, i.e. the strength and pH of the medium (P = 0.002). According to the Tukey’s test, quickest phenol degradation resulted in the double strength (2x) MSM with pH 7.5 (71.82 h). Among the 3 levels of inoculum sizes assessed, 3% showed the highest phenol degradation rate by showing the lowest time (64±4.5 h) to achieve degradation of the amount of phenol by 50%. Further, glucose in the level of 0.1% enhanced the phenol degradation rate, by degrading the same amount of phenol within 58.9±0.2 h. In conclusion, optimal growth conditions of P. aeruginosa for fast degradation of phenol were determined as 2x MSM, pH 7.5 with 3% inoculum size. Further, addition of 0.1% glucose in the medium enhanced the phenol degradation. The findings of this study provide valuable information for designing bioremediation protocols to treat phenol in industrial wastewater.Item Screening endophytic fungi of Macromitrium sp. for potential degradation of PAHs(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Ambadeniya, A.R.P.D.U.K.R.; Kannangara, S.; Abeywickrama, K.With rapid industrialization and urbanization, emission of air pollutants to the atmosphere has been increasing rapidly for several decades. Out of many pollutants, polyaromatic hydrocarbons (PAHs) take a prime advertence due to their toxicity, mutagenecity, carcinogenicity and long persistence in nature. Therefore, removal of these is one of the major cruxes that the modern world faces. In the present study, an effort was made to isolate and identify endophytic fungi in a moss (Macromitrium sp.) found in a polluted area (Sapugaskanda) and a less polluted area (Hettimulla), and to investigate their ability to degrade PAHs (naphthalene and phenanthrene). It was hypothesized that endophytes isolated from the moss can degrade PAHs and endophytes from polluted area have a higher ability to degrade PAHs compared to those isolated from the less polluted area. Moss plants from Sapugaskanda and Hettimulla area were used. Surface sterilized and trimmed moss plant pieces were placed on Malt Extract Agar and incubated for 10 days at room temperature. Percentage frequency of occurrence of each fungus grown was calculated. Utilization and degradation of PAHs by each of the fungus was assessed using a plate assay and a spectrophotometric analysis. Thirty six isolates were recovered from samples from Sapugaskanda area, 21 from Hettimulla and 6 were common to both areas. Highest frequency of occurrence was observed in Eupenicillium sp.2 (95.0%) in samples from Sapugaskanda and white sterile sp.7 (32.5%) for Hettimulla. Highest PAH utilization with the highest colony diameter, was recorded for Nigrospora oryzae for naphthalene (85.2 mm) and phenanthrene (59.5 mm). Almost all isolates from Hettimulla demonstrated low colony diameters. According to spectrophotometric analysis, highest degradation was observed with Penicillium oxalicum for naphthalene (98.60%) and Nigrospora oryzae against phenanthrene (98.02%). Almost all isolates in samples from Hettimulla area displayed poor degradation ability. The findings of the current study clearly reveal that Macromitrium sp. in Sapugaskanda harbours higher number of endophytic fungi than that in Hettimulla and most of them have a considerable ability to utilize and degrade PAHs in contrast to that in Hettimulla. It could be speculated that those endophytic fungi in Macromitrium sp. of Sapugaskanda, could be potential sources of fungal bioremediation. Further, they have potential practical application in removing PAHs from contaminated sites.Item Analysis of naphthalene and phenanthrene degradation catabolic genes of phyllosphere bacterial strains, Alcaligenes faecalis and Alcaligenes sp. DN25(2015) Kannangara, B.T.S.D.P.; Undugoda, L.J.S.; Sirisena, D.M.The bacterial strains, Alcaligenes feacalis and Alcaligenes sp. DN25 which were isolated from the phyllosphere of four ornamental plant species, Ixora chinensis, Ervatamia divaricata, Hibiscus rosa-sinensis and Amaranthus cruentus in five highly polluted sites in Sri Lanka, showed the highest phenanthrene and naphthalene degradation ability. Transformation and plasmid curing results of them revealed, naphthalene and phenanthrene degradation ability of these bacterial strains were plasmid encoded character. The occurrence of naphthalene specific (nahR and nahU) genes and phenanthrene specific (phnAc and phnG) genes of these catabolic plasmids were analyzed by PCR using degenerate primers. According to the amplification results, plasmids of Alcaligenes faecalis and Alcaligenes sp. DN25 harbour nahR, nahU and phnG genes but, lack of phnAc gene. RFLP and sequence data of nahU and nahR amplicons revealed, both of these genes were homologous to these two bacterial strains. But, phnG gene of two phenanthrene and naphthalene degrading phyllosphere bacterial strains was coexistence as two distinct copies of alleles.