Studies on Biosurfactants Produced by Hydrocarbon Degrading Bacteria

Petroleum hydrocarbons are the most frequently occurring environmental contaminants because of their extensive use, found in numerous aquatic and terrestrial ecosystems. The present study is aimed in the study of biosurfactants and its role in hydrocarbon degradation. Twenty four bacterial isolates were isolated from soil samples collected from automobile workshops. These isolates were further screened for hydrocarbon degradation and biosurfactant production. After prelimnary screening, nine isolates SA3, SA6, SA7, SA8, SA10, SA12, SA14, SA18 and SA19 were selected for further analysis. Four of the isolates were identified through biochemical screening tests, and all the nine isolates were assessed for biosurfactant production through three screening tests: drop collapsing test, oil spreading technique and emulsification index tests. These screening procedures confirmed that SA3 and SA19 are the efficient strains for biosurfactant production. The presence of glycolipid and rhamnolipid biosurfactants is further supported by the results of TLC, SDS PAGE and FTIR analysis. These two strains were identified through 16SrRNA gene sequencing. Thus the present study made an attempt to isolate biosurfactants from bacteria that can be applied for degrading the petroleum hydrocarbon pollutants deposited in aquatic, marine or terrestrial habitats. Keyword: Glycolipids, Rhamnolipids, Biosurfactants, Orcinol, Hydrocarbon. INTRODUCTION The pollution of soil and water by industrial chemicals is a serious problem afflicting the modern world. Petroleum hydrocarbons are the most frequently occurring environmental contaminants because of their extensive use, in both aquatic and terrestrial ecosystems. Bacteria are particularly suitable for biodegradation application because of the wide variety of carbon sources or electron acceptors used by various strains [1, 2]. The main advantage of bioremediation is its flexibility in handling various composition waste types in both water and soil environments [3]. Petrophiles are very unique organisms that can naturally degrade large hydrocarbons and utilize them as a food source. Microorganisms degrade these compounds by using enzymes in their metabolism and can be useful in cleaning up contaminated sites. Petroleum hydrocarbon-degraders include Yokenella spp. , Alcaligenes spp. , Roseomonas spp. , Stenotrophomonas spp. , Acinetobacter spp. , Flavobacter spp. , Corynebacterium spp. , Streptococcus spp. , Providencia spp. , Sphingobacterium spp. , Capnocytophaga spp. , Moraxella spp. , Pseudomonas spp. , and Bacillus spp. [4, 5, 6]. Microorganisms produce surface active compounds either extracellularly or these compounds are attached to microbial cells [7]. The major classes of biosurfactants include glycolipids (Rhamnolipids, Trehalolipids, Sophrolipids), lipopeptides and lipoproteins, fatty acids, phospholipids, neutral lipids and polymeric biosurfactants [8]. Nowadays, biosurfactants are used in industries as a cosmetic and special chemical substances, food, pharmaceutics, agriculture, cleansers, enhanced oil recovery and bioremediation of oil-contaminated sites. Diverse functional properties namely, emulsification, wetting, foaming, cleansing, phase separation, surface activity and reduction in viscosity of crude oil, makes it feasible to utilize them for many application purposes [9]. MATERIALS AND METHODS Collection of sample: Soil samples were collected from four different mechanic workshops in Srivilliputtur and Sivakasi. Soil sample were collected from the ground surface to a depth of 10-20 cm from specific location within the workshops that had heavy spoilage of engine oil and transported to laboratory under sterile conditions. Isolation and enumeration of bacterial strains: The bacterial strains were isolated by the enrichment culture technique of the soil samples in minimal salt medium. Crude oil (5% w/v) was used as carbon source and incubated at 37°C on a rotator shaker (200 rpm) for 4 days. The bacterial colonies obtained were further purified on LuriaBertani (LB) agar plates. [10]. Screening of Hydrocarbon Degrading Bacteria -Hydrocarbon utilization test: The bacterial isolates were tested for their ability to utilize diesel oil (1%v/v) as sole source of carbon and energy for their growth. The overnight cultures of the bacterial isolates were kept for 24-48 hrs incubation at 37°C on mechanical shaker. Growth of the organisms was assayed at regular time intervals up to 15 days by measuring the optical density at 600nm [11]. Screening of Bio-surfactant Producing Organisms Detection and Quantification of Biosurfactants JOURNAL OF ADVANCED