Isolation Identification and Characterization of some Penicillium Isolates for Alpha Amylase Activity

Aninvestigation was carried out to determine the presence of amylase producing Penicillium species in the soils of Harwan area of Srinagar city. Two study sites were choosen from the area.Soil samples were collected from the study sites for the isolation o of Penicillium isolates. Pour plate method involving serial dilution was used for the isolation of fungi. The media used for isolation were Potato Dextrose Agar(PDA), Rose Bengal agar (RBA) and Malt Extract Agar (MEA). A total of 4 Penicillium isolates were obtained during the study, which showed positive alpha amylase activity, all belonging to division Ascomycota of kingdom fungi.The highest colony forming unit(cfu/g) of 3.5×103 was found for P.chrysogenum at site II in the month of August.Certain agricultural wastes like mustard oil cake,lin seed oil cake and wheat bran were used as crude ingredients in the submerged fermentation process for the production of amylases. The carbon and nitrogen sources were also altered in the culture media for the production of alpha amylases from the isolated Penicillium species. The results of this investigation show that P.chrysogenum has maximum activity of 550.4U/ml in medium with lin seed oil cake at pH 6 and incubation period of 6 days. P.purpurogenum showed maximum activity of 883.93U/ml infermented medium with mustard oil cake as crude substrateat pH6 incubation period of 6 days. P.caesi colum showed maximum activity of 834.8U/ml in medium with glucose as carbon sour ceat pH 9 and incubation period of 18 days. P.funiculosum showed maximum activity of 4254.4U/ml at pH 6 and incubation period of 18 days with mustard oil cake as crude substrate in the medium.The optimum conditions in the medium which were found best to promote the production of alpha amylase from Penicillium species were, when medium was maintained at pH 9 and incubation period of 18 days with glucose as carbon source followed by medium maintained at pH 9, 6 and incubation period of 18 days with yeast extract as nitrogen source and medium maintained at pH 6 and incubation period of 18 days with mustard oil cake in the medium

Bacterial Flora of Manasbal Lake, a Freshwater Ecosystem of Kashmir Valley

Micro-organisms have been used for a long time as indicators of water quality and total coliform bacteria have been commonly used to assess potential contamination of drinking and swimming water with pathogenic bacteria of intestinal origin coming from point source discharges, such as raw sewage, storm water, combined sewer overflows, effluents from wastewater treatment plants, industrial sources and non-point source discharge s, such as agriculture, forestry, wildlife, and urban run-off. The obtained data in this study reflect the importance of microbiological monitoring and reinf orce the need to implement environment protection programs, especially related to pathogenic species. The majority of bacteria isolated were recognized as human pathogens or potential human pathogens. The data was obtained by the bacteriological analysis of water sample taken from Manasbal Lake on monthly basis from four different microhabitats by plating the different dilutions on a solidified culture medium in petri dishes. After incubation the bacterial colonies were divided into different types according to some macromorphological features like appearance, shape, size, elevation, margin, colour and some micromorphological features with the isolated strains showing marked differences in these features. On the basis of these differences they were coded with numbers ranging from MBS01 to MBS52. The different recognizable colonies were streaked and restreaked on fresh media to obtain pure cultures. The selected purified colonies of various types were identified to genus or species level using biochemical tests. Total coliforms were enumerated using multiple tube fermentation technique with lactose broth as the presumptive medium and Eosine-Methylene-Blue agar medium as the confirmatory medium and Brilliant Green Bile broth for completed test. The developed colonies on plates were enumerated by digital Qubek colony counter and the bacterial load was assessed in terms of colony forming units (cfu/ml) revealing that the total monthly bacterial population increased from March to August and then decreased from September to December with peak bacterial population in the month of August at all sites. Moreover, the density of total culturable bacteria at site II (residential hamlets around) was significantly higher in all the months compared to other sites. The overall bacterial density was maximum at site II with a cfu/ml of 203x102 in the month of August and minimum at site III (central site) with a cfu/ml of 12x102 in the month of April. The total bacterial population was higher during warm temperature months than cold temperature months for all the four sites. As far as coliform count is concerned, all the water samples collected f rom the Lake were positive with respect to the coliform occurrence, with their proportion ranging between a minimum value of 4 MPN/100ml and a maximum value of 460 MPN/100ml. The highest proportion of these indicator organis ms was observed at site II. The category wise distribution of coliform count shows that about 95% samples lie in category II and III deeming the water unfit for drinking purposes, however, fit for bathing and swimming purposes. Moreover, the quality of water in some patches of the lake was very poor and unfit for any use. The water of the lake was characterised by a medium to high alkaline pH (ranging between 7.7 to 9.6) and temperature ranging between 9°C to 33.5°C. The overall Shannon diversity index (H) was highest at site I (Laar Kul) followed by site II, site III and site IV (outlet). The bacterial isolates were then tested for Gram’s reaction and subsequentl y examined under microscope for their cell shape revealing that 88.5% of the bacterial strains were Gram negative and 11.5% were Gram positive, out of which 34 strains (59.6%) were Gram negative bacillus, 12 strains (2 8.8%) were Gram negative cocci, 4 strains (7.6%) were Gram positive bacillus and 2 strains (3.8%) were Gram positive cocci. Among Gram-negative bacteria, b acillus was the most dominant genus isolated from all sites during all mo nths. A total of 19 bacterial strains, chosen arbitrarily were subjected to biochem ical tests like Citrate utilization, Glucose, Adonitol, Arabinose, Lactose, Sorbitol, Mannitol, Rhamnose, Sucrose, Urease, Lysine utilization, Ornithine ut ilization, H2S production, Phenylalanine Deamination, Nitrate utilization, Indole, Voges Proskauer’s and Methyl red revealed that 9 species viz Proteus spp. I, Proteus spp. II, Proteus spp. III, Escherichia coli, Klebsiella spp. II, Cedecea spp., Escherichia spp., Shigella spp. and Salmonella spp. belonged to Enterobacteriaceae family and 10 species viz Shigella spp. I, Shigella spp. II, Shigella spp. III, Enterobacter spp., Hafinia spp., Salmonella chloraesuis subspecies choleraesuis, Salmonella choleraesuis subspecies diarizonae, Vibrio sp p., Proteus spp. IV, and Klebsiella spp. I. to Gram negative rods. During the study Proteus spp. II occurred with a highest percentage occurrence of 14.67% and Shigella spp. I with a lowest percentage occurrence of 0.21%. Overall, the study allows us to conclude that the quality of lake water has deteriorated to the extent of being unfit for drinking purposes, though it is still fit for recreational and other uses. Hence, the lake calls for urgent restoration and eff ective management for its sustained existence and continued provisioning of various economic goods and ecosystem services

Antimicrobial and Antioxidant activities of Arnebia benthamii (Wall ex. G. Don Johnston) - A Critically Endangered Medicinal plant of Kashmir

Plants used in traditional medicines contain a vast array of substances that can be used to treat chronic and infectious diseases and the present study was carried out with the same intentto appraisethe possible medicinal value of Arnebia benthamii L. (Wall. ex G. Don) Johnston [Syn Macrotomia benthamii (Wall.) DC.] a threatened medicinal plant of Kashmir valley by examining its phyto-chemical constituents and evaluating the antimicrobial and antioxidant activity. Preliminary phytochemical screening was carried out to detect the presence of phyto-chemicals that add to the medicinal value of the plant. The antimicrobial assay was carried out by Disc diffusion and Micro-broth dilution methods. The crude plant extracts (both root as well as aerial parts) exhibited significant antimicrobial activity on majority of test organisms tested. The highest occurrence of secondary metabolites was of alkaloids and phenolic substances. The tannins and saponins were absent in all plant extracts. The total phenolic content that was quantitatively estimated showed highest presence in methanol extracts of the aerial parts. The antibacterial results revealed that methanol extracts of aerial parts were found to exhibit significant antibacterial activities against all tested bacterial strains except Staphylococcus aureus which was found resistant. The highest inhibition zone diameter (30mm) was recorded for Pseudomonas aeruginosa and Escherichia coli with respect to control i.e. Gentamycin (16 and21mm) followed by Salmonella typhimurium (28 mm). Chloroform extract also showed good inhibitory activity against all the tested bacterial strains likeE. coli (22mm) followed by Klebsella pnuemonie (20 mm) and P. aeruginosa (17mm). The aqueous extract didn’t show any inhibitory activity against the tested bacterial strains. The root extracts were separately tested for antibacterial activity against pathogenic strains and it was apparent from the results that they were less active than aerial part extracts. Among the strains tested, S. aureus was found susceptible to chloroform, ethyl acetate and methanol root extracts with an average inhibition zone diameter (10 mm) with respect to control and rest of extracts showed no activity against it. The Shigella flexneri was found susceptible to all the root extracts and the highest inhibition zone diameter was recorded for methanol (20mm) followed by ethanol (15 mm)against the control (Erythromicin 30 mm). Among all the strains, E. coli was highly susceptible towards the methanol root extracts followed by chloroform and ethyl acetate extract while the rest of the extracts did not exhibit any inhibitory activity. Most of the root extracts exhibit highest antibacterial activity against S. typhimurium compared to aerial parts. The screening served a s an indicator f or the selection of bacterial strains that displayed antibacterial activity for further testing to determine the MIC’s of plant extracts. Four bacterial strains S. fle xnerii, K. pneumonia, E. coli and P. aeruginosa were found viable for testing with the specific plant extracts. S. flexnerii was found highly susceptible towards methanol aerial part extract with MIC and MBC value of 200μg/ml and 750 μg/ml respectively as compared to reference (Erythromycin75 and 100 μg/ml) followed by methanol root extract { MIC 300 μg/ml and500 MBC μg/ml}. Methanol aerial part extract exhibited the MIC value 500 μg/ml and MBC 750 μg/ml against K. pneumonia. However the root extracts showed co mparatively higher value of MIC against the tested organism ranging f rom400-500μg/ml with respect to positive control (MIC-150 μg/ml).The MIC value for E . coli ranged between 150 to 500 μg/ml towards aerial part extracts w ith lowest MIC observed for methanol aerial part extract with respect to Erythromycin (175 μg/ml), and for root extract MIC value ranged between 225 -425 μg/ml. P. aeruginosa was found to be most susceptible towards all aerial part extracts with MIC value ranged between 75 μg/ml and 425 μg/ml and MBC value from 100 to750 μg/ml. All the fungal strains except Candida parapsilosis were more or less inhibited b y both aerial and root part extracts of the plant. The aqueous extract of both root as well as aerial parts did not show any antifungal activity. Asperigillus flavus was found to be highly susceptible to butanol aerial part extract with inhibition zone diameter (25mm) followed by ethyl acetate extract(22 mm) and chlorof orm extract (14 mm) compared to the control (Nystatin 35mm) used. The methanol aerial part extract showed highest inhibitory activity against Asperigill us versicolor with inhibition zone diameter (22 mm) and Acremonium spp (25 mm). The petroleum ether, ethyl acetate and chloroform extracts of aerial part did not exhibit any inhibitory activity against Candida spp . Only methanol extract followed by ethanol and butanol extract exhibited antif ungal activity against Candida spp. Among the Candida spp, the highest inhibition zone diameter (14 mm) of methanol extract was seen against C. albicans whereas C. parapsalosis was found completely resistant against the all aerial part extra cts. The ethanol and aqueous root extracts didn’t exhibit any inhibitory activity against any tested fungal strains. Meanwhile the ethyl acetate extract sho wed highest inhibition zone diameter(33mm) against C. albicans f ollowed by Acremonium spp (18mm).The methanol root extract showed good inhibitory activity with an average inhibition zone diameter (12mm) against A. versicolor, C. albicans and C. kruesie with respect to reference used. The four in-vitro tests i.e. DPPH radical scavenging action, riboflavin photo- oxidation method, hydroxyl scavenging and the lipid peroxidation assay for antioxidant activity were used. Together all the methods provide a better assessment of antioxidant properties and results revealed that inhibitory activity was concentration dependent. Free radical scavenging potential of aerial and root part extracts at different concentrations was tested by the DPPH method. Highest inhibition of 86% and 94% was recorded for both methanol extracts of aerial as well as root parts with respect to reference (Ascorbic acid 90%) at the higher concentration (300 μg/ml) followed by ethyl acetate (60%) and chloroform extract (63 %) of aerial parts. The superoxide radical scavenging activity of aerial parts was observed in the following order butanol extract (85%) >ethyl acetate (79%)> methanol extract (70%) however Petroleum ether, ethanol and aqueous extracts didn't exhibit any scavenging activity. The root extracts were also effective in scavenging the superoxide radicals and the order of scavenging was as butanol (91%)> methanol (88%)>aqueous (79%) extracts. As far as the protective effect of deoxyribose was concerned, the highest inhibition of radicals was observed for methanol extract of root part and aerial part (~85% inhibition). The ethanol extracts of both parts were found to be ineffective. The overall inhibition of FeSO4 induced lipid peroxidation was high in presence of positive control (ascorbic acid 95.78±1.0%) compared to the plant extracts of A. benthamii. However, the ethyl acetate extract of aerial parts (95 %) showed almost the same activity as compared to the reference antioxidant. The ethyl acetate (87%) of root parts also exhibited significant inhibitory activity followed by ethanol extracts (78%)