Screening, Optimization and Extraction of Polyhydroxyalkanoates (PHAs) from Bacillus thuringienesis

Abstract

Aims: This study illustrated obtaining bacterial isolates capable to produce polyhydroxyalkanoates (PHA) under stressed conditions and also using cheap wastes for production process. Study Design: Two-step throughput screening of 50 bacterial isolates by lipophylic stain (Sudan black B) and flouroscence stain (Nile red) will be carried on, then the best condition for PHA production followed by identification of most potent isolate by molecular charterization will be investigated. Place and Duration of Study: The study were performed in physiological lab in botany & microbiology department in faculty of science, Al-azhar university and NRC from October 2012 until April 2014. Methodology: Bacterial isolates will be investigated using two different indicator stains, Sudan Black and Nile Red, for PHA productivity then the highly producing isolate will be identified. Factors controlling PHA production and stressed condition will be studied alternatively depends on increasing of PHA production which will be assayed quantitatively by converting PHA to crotonic acid. Biopolymer will be extracted using successive solvents. Result: Five soil samples were collected from different localities contaminated with industrial wastes and 50 isolates of bacteria were purified then screened for PHA production by using Sudan black B as a primary screening and confirmed by fluorescent Nile red staining. Eleven promising bacterial isolates were selected based on their PHA yields. Out of eleven natural promising isolates, BS11 was found to be the most efficient PHA producer and identified as Bacillus thuringiensis (KJ206079). The effect of different conditions on PHA produced by B. thuringiensis including carbon sources, nitrogen sources, incubation temperatures, pH and incubation periods were studied. The highest amount of PHA was obtained from this isolate using medium containing 30 g/l molasses, 0.8 g/L ammonium sulphate at pH 7.5 and incubation temp at 35ºC and after 72 hours. In conclusion, after the optimization of PHA synthesizing conditions, B. thuringiensis accumulate up to 69 % of its dry weight and can be employed for industrial production. Conclusion: The experiment indicate the efficiency of two-throughput screening systems targeting PHA producing isolates. Also optmiziation of PHA producing factors for the most potent producing strain, Bacillus thuringiensis (KJ206079), was increased from 2.5 g/L to 4.1 g/L and represent about 69% cell dry weight.