University Grant Commission MINOR RESEARCH PROJECT File No: F. 47-1221/14(WRO) Isolation of Xylanolytic Microorganisms from Marine Environment and Optimization of Xylanase Production Principal Investigator -Ms. Zamanat Fatima Syed

Executive Summary

Xylanases have applications in effluent treatment, ecofriendly bleaching of paper pulp, textile industry and food industry. Most of these applications demand stability of the enzyme under harsh environmental conditions. The present study was focused on isolation of xylanolytic bacteria capable of tolerating high salt concentration from marine environment, as the enzymes derived from such bacteria are known to be significantly tolerant to alkaline pH and high temperature. Enrichment and isolation of xyalanolytic bacteria was done using Han’s medium, Kapilan’s mineral medium and Oren’s Halophile medium containing 0.4% xylan and 7% NaCl. One hundred and two bacterial isolates were obtained, out of which, eleven isolates showing positive xylanase activity were shortlisted for further studies. These isolates were characterized with respect to salt tolerance, pH tolerance, morphological and cultural properties, 16srRNA sequencing and quantitative xylanase activity in the culture supernatant. Isolate no. Z69 (48) was eventually selected from the eleven isolates for xylanase production  based on its salt and pH tolerance and xylanase activity in the culture supernatant. The organism tolerated 14% NaCl and grew over pH range of 7 to 12.  It is an oxidase positive, Gram positive bacillus with terminal endospore. The Tm for the isolate is 72^oC and G+C content is 44.16%. 16srRNA sequence of Z69 (48) shares 95% homology with Gracilibacillus thailandensis. Hence, the isolate has been assigned a new strain no. viz;  Gracilibacillus thailandensis strain ZSKA 69.  16srRNA sequence of the organism has been deposited with DDBJ/GENBANK/EMBL. The accession no. is LC 274885. Xyalanse activity in the culture supernatant of Gracilibacillus thailandensis strain ZSKA 69 was determined by DNS method. Xylanase production was maximum in presence of beechwood xylan as the C source and tryptone as the nitrogen source. Optimum pH and temperature for production were 8 and 35^oC respectively. The yield of xylanase in culture supernatant increased by six times when the culture was shifted from static to shaker condition.  An inoculums size of 5% with incubation period of 72 hours was found to be optimum. KCl and LiCl  favoured xylanase production where as  HgCl₂ affected it negatively at the concentration of 2mM. MnCl₂, MgCl₂, ZnCl₂, CoCl₂, FeCl₃, AlCl₃, CuCl₂, CaCl₂ and NaMoO₄ did not have any significant effect on production of the enzyme, at the concentration of 2 mM. Optimal xylanase activity was observed at 60°C and pH of 8 as well as pH of 5. NaCl concentration did not show much effect on activity of the enzyme. The activity slightly improved at 8% concentration of NaCl. MnSO4, LiCl3, CoCl2 and CuSO4 positively affected the activity. HgCl2, EDTA and SDS decreased enzyme activity. Thus, xylanase produced by Gracilibacillus thailandensis strain ZSKA 69 is active over a wide range of pH and salinity. It is considerably tolerant to temperature too. Hence it appears promising for exploitation on industrial scale in detergent industry and in bleaching of paper pulp, as an alternate to chemical processes which release hazardous chemicals in the environment. This project has also provided an insight into bacterial biodiversity of saltpans located in coastal region of Mira Road-Virar in Thane District of Maharashtra. These saltpans are rapidly vanishing and loss of biodiversity is feared. The cultures isolated from saltpans have been preserved. Some of the cultures identified are Halomonas smyrnensis, Halobacillus blutaparonensis, Virgibacillus halodenitrifican