EXPLORING LIPID PRODUCTION IN LENTIBACILLUS SALARIUS NS12IITR USING AGRO- WASTES

Abstract

The hypersaline environment is inhabited by an array of microbes which have the potential to produce industrially important products. This study explored the biomass and lipid production potential of halophilic bacteria isolated from Sambhar Lake, Rajasthan. Total 13 halophilic strains were investigated for lipid production by growing under similar conditions in media containing glucose as a carbon source. Nile red staining and lipid content (% dry cell weight (DCW)) were used as screening parameters. Growth profile, lipid profile, hydrocarbon content, and hydrocarbon profile were also assayed. Since Nile red is a lipophilic dye thus it can stain both intracellular lipids and poly-hydroxybutyrate (PHB). Therefore, most of the halophilic strains showed positive results in staining. Thus lipid content (% of DCW) was used as selection criteria. Strains MSL-12 and MSL-8 which gave the highest lipid content (% of DCW) were selected in primary screening. These two strains were grown in media containing cellobiose and strain MSL-12 which gave the highest yield in glucose as well as cellobiose media was selected for further study. Sequencing and phylogenetic analysis of strain MSL-12 revealed that it belonged to the genus Lentibacillus. Effect of media parameters NaCl molarity, pH, temperature, and nitrogen sources were studied. Growth profile and lipid content (% of DCW) were used as screening parameters. The NaCl molarity and pH range did not show significant variation in growth and lipid content, whereas 37 C temperature was found optimum for growth and lipid production. For nitrogen source optimization several synthetic media with inorganic nitrogen sources were attempted but no media supported the growth of this halophilic isolate. Organic nitrogen sources yeast extract and peptone were also investigated, yeast extract was found to be best for growth and lipid production. Statistical media optimization was done using Design Expert 10 software. A fractional factorial design was used for screening the media components, the screened components were optimized using D-optimal design. Out of eight parameters, four parameters were found significant affecting growth and lipid production. The optimum media condition obtained by D-optimal design was used for further experimentation. In optimized medium, the isolate accumulated lipid up-to 18.9±0.45% of DCW. In addition, trans-esterification of extracted lipid yielded 77.6±5.56% of total esters as methyl ester of branched-chain fatty acids (BCFA’s). To assess the nature of extracted lipid, lipid sample was ii fractionated on the silicic acid column, which demonstrated that 49.03±1.35% of the total lipids was neutral in nature. Transesterification of the neutral lipid fraction yielded 60.62±4.88% of total esters as methyl ester of BCFA’s. Furthermore, the isolate produced hydrocarbons both extracellularly (C10–C30) and intracellularly (C15–C28). The concentration of BCFA and extracellular hydrocarbon synthesized by this strain is in close agreement with the yield reported from other bacteria. Interestingly, the strain was also able to efficiently valorize the inexpensive and widely available lignocellulosic biomass as feedstock for lipid production. Agro-wastes (wheat bran, rice bran, mango peel, and orange peel) were used. The dilute hydrochloric acid pretreatment of these agro-wastes was carried out at 121 C, 15 psi, for 30 and 60 min, to extract the fermentable sugars. The hydrolysis conditions giving highest amount of sugar from agro wastes in g/L: wheat bran (64.52+0.57), mango peel (55.64+1.14), rice bran (38.7+0.58), and orange peel (36.29+0.54) was used. These hydrolysates were further utilized as feedstock for the growth of the halophilic bacterium. The results showed, that all the agro waste hydrolysate supported growth and lipid production for Lentibacillus salarius NS12IITR, but wheat bran hydrolysate was found best with highest biomass (3.96+0.009) and lipid concentration (0.70+0.029) g/L, and also contained the highest percentage of branched-chain fatty acid methyl ester (81+4.72%). With doubling the sugar concentration of wheat bran hydrolysate from 10—20 g/L the biomass and lipid concentration were also almost doubled (6.22+0.04) and (1.26+0.051) g/L respectively. Further study was conducted, to replace sonication for cell disruption, with other economical and easy to scale up method. In this regard, first the sonication time was optimized to get the highest lipid content; 20 min of sonication gave the optimum lipid content of (26.6+2.26%). In the second part of this study different cell lysis methods were investigated. Among all the studied methods thermo-lysis at 85 C for 30 min along with osmotic shock at pH 2 was found most efficient, as lipid content (18.2+0.97%) was obtained which was comparable with lipid content obtained by sonication for 15 min (21.1+0.14%). The salient features of this strain are the production of lipid with a high percentage of BCFA and co-production of hydrocarbons. The conventional biodiesel suffers the common drawback of poor cold flow property and oxidative stability. Studies have demonstrated that introducing methyl branching can significantly improve biodiesel properties. Apart from the biodiesel industry the BCFA’s also have major applications in detergent, cosmetics, lubricant and pharmaceutical industries. In this regard, the Lentibacillus salarius NS12IITR could be a promising strain for BCFA’s production with the majority of its FAME as branched moieties.