Isolation, Screening, Characterization of Indigenous Oleaginous Bacteria: Evaluation of Various Carbon and Nitrogen Sources as Substrates for Single Celled Oil Producing Bacteria

Abstract

Aims: The study was aimed to, isolate, screen and characterize the heterotrophic lipid producing bacteria from various oil and fat contaminated sites. Additionally, the study was focused to evaluate the influence of some carbon and nitrogen sources on bacterial culture. Place and Duration of Study: The current study was carried out in the Department of Environmental Science and Engineering, Lab no. 211 (Bioenergy and bioremediation Lab) Guru Jambheshwar University of Science and Technology, Hisar. Duration of study from August 2014- January 2015. Methodology: Soil samples were collected from Hisar, Sirsa (Haryana) and waste water sludge from Guru Jambheshwar University of Science and Technology, Hisar. Isolation and purification of filamentous bacterial strains was done by simple plate streak plate method, followed by screening of bacterial strains by Sudan black/Nile Red dye. Genomic DNA was extracted from bacterial strain using Cetyl trimethyl ammonium bromide (CTAB) method. PCR product was sequenced by 16sRNA approach. In batch flasks study, effect of various carbon and nitrogen sources on lipid and biomass of Rhodococcus opacus and Gordonia alkanivorans were evaluated by using gravimetric Bligh and dyer method. Results: Filamentous bacterial strains were initially isolated using selective culture media, further these oleaginous bacterial strains were screened out on the basis of growth rate and lipid content (dcw%) and employed Nile red and Sudan black staining for detection of neutral lipids in cells. The biochemical behavior (biomass production, accumulation of total lipid) and substrate uptake by two oleaginous bacteria has been studied. Furthermore, Rhodococcus sp. and Gordonia sp. were cultivated under various carbon and nitrogen sources. Significant differences in the process of lipid accumulation and biomass yield as related to the carbon, nitrogen sources used were observed for both microorganisms. Although glucose containing MSM medium favours production of  biomass yield 1.81±0.026 gL-1 and 1.63±0.032 gL-1 with corresponding high lipid content 16.78%,17.05% in  Rhodococcus opacus as well as Gordonia alkanivorans respectively. Among Various tested nitrogen sources, Ammonium sulphate was found to be best nitrogen source for cultivation of Rhodococcus opacus and Gordonia alkanivorans (P≤0.05) indicating higher lipid content of 16.55%, 17.01%. Conclusion: Filamentous bacteria have capacity to accumulate substantial amount of oil. Nile Red and Sudan black staining dye was found to be effective method for prescreening of oleaginous bacteria. Glucose and Ammonium sulphate proved to be suitable carbon and nitrogen source for culturing of Rhodococcus opacus and Gordonia alkanivorans.