European Journal of Biotechnology and Bioscience 22 European Journal of Biotechnology and Bioscience ISSN: 2321-9122 Impact Factor: RJIF 5.44 www.biosciencejournals.com Volume 2; Issue 6; November 2017; Page No. 22-26 Influence of temperature, pH variation and incubation period on single celled micro-organism Kulvinder Bajwa, Narsi R Bishnoi, Anita Kirrolia, Jyoti Sharma, Saloni Gupta Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar, Haryana, India Abstract The aim of the present study was to investigate effect of pH, temperature and incubation period on dry cellular weight and lipid content of Rhodococcus opacus and Gordonia alkanivorans. The influence of various temperatures (20-50°C) on biomass yield and lipid accumulation were reported, Rhodococcus opacus showed significantly higher biomass yield 1.41±0.041gL -1 at 37°C. Rhodococcus opacus and Gordonia sp. revealed significantly (p≤0.05) higher lipid accumulation (14.52%, 13.84%) respectively at 37°C. Similarly effect of pH on oleaginous bacteria also studied that Gordonia sp. exhibited significantly (P≤0.05) higher biomass 2.77±0.034 gL -1 at pH 6 whereas Rhodococcus opacus showed significantly higher biomass 2.45±0.042gL -1 at pH 7. Effect of incubation period showed lipid content significantly (P≤0.05) enhanced up to 120 hrs in Rhodococcus opacus (13.78 %) whereas Gordonia alkanivorans yielded increased biomass (14.08%) upto 72 hrs in both species. Keywords: oleaginous bacteria, lipid, biomass yield, biodiesel 1. Introduction Due to the expected depletion of fossil fuel sources in the near future, increasing attention is paid to the development of alternative sources of energy. Bioethanol and biodiesel (fatty acid alkyl ester, FAAE) are among others the most interesting organic compounds, currently satisfy about 90% of the biofuel market [1, 2] . Today the industrial production of biodiesel is restricted to the transesterification of fatty acids derived from oleaginous plants like rapeseed, oil palm and soya or from animal fats [3, 4] . Lipids are valuable compounds for versatile commercial applications and have the potential to replace fossil resources in many industrial processes. There has been made remarkable progress regarding the utilization of oils as sustainable alternative feedstock to generate a multitude of basic oleochemicals for organic synthesis, catalysis or biotechnology in the chemical industry [5, 6] . A major global objective, however, is the replacement of fossil fuels in the transportation sector and to date, biodiesel made from plant, animal or microbial oils is the predominant biofuel [7] . Utilization of oleaginous microorganisms as “micro-factories” for accumulation of single cell oils for biofuel (biodiesel) production has increased significantly to mitigate growing energy demands, resulting in efforts to upgrade industrial waste, such as second-generation lignocellulosic residues, into potential feedstocks [4] . Microbial triacylglycerol (TAG) share some properties with those from plants and animals but exhibit their own unique features [4, 8] . Another interesting applications of bacterial lipids is the potential production of renewable bio-based oleochemicals that are currently produced from petrochemicals or from plant lipids [9, 10] . The genus Rhodococcus is one of the most appropriate lipid producing microorganism due to its capability to accumulate mainly triacylglycerols (TAG) [10, 11] . It has been reported extensively that cultivation conditions such as temperature, pH, oxygen, C/N ratio, nitrogen resources, and concentration of trace elements and inorganic salt would have varied influence on oil accumulation. Bacillus subtilis cultured at temperatures between 30 and 45°C and within a pH range of 6.5 to 7.0, the endophyte Bacillus subtilis grew extremely well, and accumulated cellular lipid content of more than 20% [12] . The objective of this study was to investigate the effect of pH, temperature and incubation period on biomass yield and lipid content of oleaginous bacterial strains viz. Rhodococcus opacus and Gordonia alkanivorans. 2. Materials and Methods 2.1 Biomass Estimation Bacterial strains were cultured in MSM broth. Composition of MSM medium. Growth of bacterial isolates on MSM media was measured every 3 hours until 96 hours of the cultivation time and determined dry cell weight and optical density at 600 nm. There was a linear relationship between dry weight and OD 600 nm as linear regression equation. Standard linear regression curve prepared by dilution ranging between 0.2 to 1 [13] . y = 0.2425x + 0.2615, R² = 0.9923 2.2 Determination of growth and lipid content gravimetrically Lipid extraction was performed with modified Bligh and Dyer Protocol (Chloroform Methanol: Water) in ratio 1:2:08) respectively for bacterial cells cultivated in Minimal salt medium. Bacterial cells were collected by centrifugation at 5,000 rpm for 15 min. The cell pellet was washed with 40 mL of distilled water. The washed-cell pellet was freeze-dried, held in desiccator until constant mass was attained (usually 24 h) and weighed to estimate its dry cell weight, followed by extraction with a mixture of chloroform, methanol and water