Screening of different contaminated environments for polyhydroxyalkanoates-producing bacterial strains

Abstract

Total sixteen bacterial strains were isolated and purified from the samples collected from sugarcane molasses soil, sewage water and long-chain-hydrocarbon-contaminated area of the Punjab University, Lahore, Pakistan. Tolerance to different antibiotics was studied and strains showed multiple antibiotic resistance. All strains were characterized for Gram stain, biochemical reactions and polyhydroxyalkanoate (PHA) production. Total fourteen strains were Gram negative and two were Gram positive, while biochemically nine PHA producers showed affiliation to Pseudomonas, Enterobacter, Citrobacter, Bacillus and Escherichia. Screening for PHA production was done by Sudan black staining and nine out of sixteen strains exhibited PHA producing ability. PHA production was optimized for different growth parameters, like nitrogen concentration, pH and temperature. PHA extraction was done by solvent extraction method. Bacterial strains US1 and M1 accumulated up to 30% PHA of their cell dry weight on PHA extraction by solvent extraction method. Bacterial strain US1 was identified by 16S rRNA gene analysis as P. aeruginosa (DQ455691). PHA production was confirmed by PCR amplification of 500 bp fragment from PHA polymerase (Pha C) gene; five strains from nine PHA producers gave positive results on PCR. Pha C gene fragment of US1 was sequenced and submitted to Gene Bank under the accession number DQ455690. The amino acid sequence showed homology using the protein BLAST at 129–132 sites with different PHA synthases of the Pseudomonas sp.