Screening and characterization of rhamnolipids producing bacteria from hydrocarbon contaminated soils and studies on their plant growth promoting activities

Abstract

newline ABSTRACT newlineThe present study describes the isolation and characterization of rhamnolipids producing aerobic bacteria from soil contaminated with crude oils (Diesel, Petrol etc.). A total of 35 soil samples were collected from automobile workshops and petrol pumps in Solan and Mandi areas of Himachal Pradesh. The bacterial species were isolated on sterile minimal salt medium. The isolated bacteria species were grown in triplicate on minimal salt agar and were identified on the basis of morphological characteristics and biochemical tests. A total of 27 isolates were screened for biosurfactant production by, oil spreading technique, emulsification stability test, drop collapse method, lipase test, blood hemolysis test of which one isolate turned out to be negative. The bacterial species were identified as Pseudomonas spp. (n=6), Bacillus spp. (n=5), Enterobactor spp. (n=2), Streptococcus spp. (n=3), Serratia spp. (n=3), Staphylococcus spp. (n=2), Micrococcus spp. (n=3) and Kocuria spp.? (n=2). The rhamnolipids production was detected by Cetyl trimethyl ammonium bromide test. Only three isolates of Pseudomonas spp. were rhamnolipids producers. Rhamnolipids were quantified and their carbohydrate, protein and lipid contents were determined. In order to determine the functional groups present on the rhamnolipids the FTIR studies were conducted. Three among 26 isolates of were rhamnolipids producers, these were identified by conventional methods as Pseudomonas spp.. Following the standard protocol M7-A7 of Clinical and Laboratory Standard Institute (CLSI, 2008), for determining the susceptibility of 27 isolates to commonly used antibiotics by disc diffusion method was determined. The nucleotide sequencing of amplicons of 16s rDNA of these rhamnolipids producing isolates revealed that these isolates were P. aeruginosa as the amplicon of AB12 and OB2 isolates showed 99% sequence homology with P. aeruginosa, while AC3 isolate showed 97% homology with published NCBI sequences of P. aeruginosa and P. fluorescens. Partially purified and crude rhamnolipids showed inhibitory activity on the biosurfactant producing bacteria, isolates recovered from human urinary tract infection and some pathogenic fungi of plants and seeds. Minimum Inhibitory Concentration (MIC) range against biosurfactant producing isolates, uropathogens and plant and seed pathogenic fungi were 0.75- 3.0, 1.5-3.0 and 3-5 mg/ml respectively. These isolates were further evaluated for plant growth promoting properties (Production of IAA, ammonia, HCN, secretion of siderophores and phosphate solubalization). All the rhamnolipids producing P. aeruginosa isolates possessed these plant growth promoting activities.