Optimization of bio-surfactant production by Azo-rhizobium strain isolated from oil-contaminated soil

Bio-surfactants are amphiphilic molecules possessing both hydrophilic and hydrophobic moieties. They are surface active agents that are produced extracellularly or as a part of the cell membrane by bacteria, yeast, and fungi. In the present study, the screening and optimization of bio-surfactant production were carried out using oil-contaminated soil sample. The cultural, morphological and biochemical tests as well as 16s rRNA analysis identified the most efficient bio-surfactant producer as Azorhizobium strain. The preliminary screening of bio-surfactant production was done with the help of oil displacement test, drop collapse test and observed hemolysis on superimposed blood agar plates. Optimization studies revealed that 2% inoculum size of test strain (1.0 O.D 530 nm) can be exploited for bio-surfactant production using 2% coconut oil and ammonium nitrate with a C: N ratio of 20:1 in MSM medium (pH 8.5) and incubation conditions of 30 ˚C for 96 h. The crude yield of bio-surfactant produced was estimated as 2.5 g/L. Further purification of crude bio-surfactant was carried out using acid hydrolysis and rotary vacuum evaporator. The bio-surfactant thus obtained successfully reduced the surface tension of the medium from 59 mN/m to 38 mN/m with E24 60%. The characterization studies of the purified bio-surfactant carried out by FTIR analysis confirmed it to be lipopolysaccharide type of bio-surfactant. Thus our current study suggests useful application of bio-surfactant producing bacterial strain that may be helpful in the petroleum industry for the purpose of recovery of petroleum and other oils from oily sludge, cleaning of oil storage tanks and bioremediation of oil-contaminated sites.