Biological and molecular strategies to improve PHB [poly-beta-hydroxybutyrate] production

The local isolate Bacillus megaterium GR149 grown on coconut water supplemented with 2% glucose and 0.5% ammonium chloride produces poly-beta-hydroxybutyrate (PHB), biopolymer that has the potential of being used as an alternative to petroleum-based plastics. One drawback, however, is its highly crystalline structure which makes the product somewhat hard and brittle. Manipulation of the growth medium is one method by which the composition of the polymer product can be modified. In this study, the effect of adding organic acids (lactic, butyric, propionic, valeric and succinic acid) to the basal coconut water medium was evaluated. Generally, the results showed that organic acid added at 3mM concentration has a detrimental effect on bacterial growth and, consequently, the yield of extracted PHB. In some cases, addition of the organic acid at 1mM concentration already resulted in decreased bacterial growth and PHB yield. The largest decrease in cell density was obtained when butyric acid was added to the medium. However, addition of 1mM and 3mM propionic acid resulted in a slight increase in PHB yield; the basal medium gave approximately 21% PHB yield compared to the supplemented media which gave 30% (1mM propionic acid) and 25% (3mM propionic acid) PHB yield. The PHB product from the propionic acid supplemented medium also appeared lighter in color and was less brittle compared to the control. Based on the results, it is recommended that additional studies be conducted using much lower organic acid concentrations (1mM). As a first step that would eventually lead to the cloning of the genes involved in PHB production, PCR amplification of these genes was carried out. Primers for the PHB genes were designed based on the published sequences of the phaPQRBC genes of Bacillus megaterium (McCool and Cannon, 1999; 2001). PCR conditions were optimized for each of the primer pairs and amplification products were visualized. Initial attempts at sequencing the purified PCR products were unsuccessful.