Dual biopolymer production and separation from cultures of Bacillus spp.

In the search of alternative new materials for biodegradable plastics, biopolymers provide attractive solutions with their vast range of applications. A challenge in industrial production of biopolymers is their high cost, and one approach to minimise the cost is expanding the number of valuable products obtained from a single batch.

The aim of this thesis was the dual production of biopolymers, P(3HB) and γ-PGA from cheap substrates with the view to lay grounds for a feasible, innovative, low cost production process. A common denominator between the two biopolymers focused in this thesis, (P(3HB) and γ-PGA), was that they both could be produced by Bacillus sp. One out of five strains screened, Bacillus subtilis OK2, was selected and the structures of both biopolymers produced were confirmed.

Subsequently, optimisation of the production medium via statistical optimisation tools, and scaling-up of the process from shaken flasks to fermenters were carried out. Statistical design tool Placket Burman (PB), (Design Expert 6.0), was used to determine the effect of medium components on γ-PGA and P(3HB) production and to identify the crucial medium components in production media. The outcome of PB analysis of dual polymer production did not match the PB analysis of single polymer production. Considering the complexity of the dual polymer production mechanism, central composite design was applied after the number of parameters was reduced from five to three. A medium composed of 20 g/L glucose, 1.5 g/L yeast extract, 2.4 g/L citric acid, 32 g/L glutamic acid and 12 g/L ammonium sulphate was identified as the dual polymer production medium. Using an inoculum medium different from the production medium proved to have a positive effect on the production. Consequently, 1 g/L P(3HB) and 0.4 g/L γ-PGA in shaken flasks and 0.6 g/L P(3HB) and 0.2 g/L γ-PGA in single batch fermenters were produced with the strain Bacillus subtilis OK2.

Selection of biowaste for the dual production was conducted using four biowastes; rapeseed cake, wheat bran, Spirulina powder and orange peel; using four pre-treatment methods, acid treatment, alkaline treatment, water infusion, and microwave exposure. γ-PGA production could not be detected when any of the waste materials was used as a sole medium component. Orange peel using water infusion pre-treatment was found to be the most suitable biowaste for the production of P(3HB). Bioreactor experiments showed that 1.24 g/L P(3HB) could be produced using orange peel as carbon source supplemented with yeast extract and citric acid. Dual polymer production using orange peel as carbon source proved to be more challenging as some of the ingredients in orange peel interfered with the dual production and inhibited production of both polymers. Although the different sugars in orange peel had a positive effect on production, pH control coupled with DOT control proved to be essential to overcome inhibition and 0.2 g/L of each polymer were produced in 79 h.

For the separation of the two polymers from the culture broth, magnetic field, floatation, and sedimentation methods were investigated. Exposure to magnetic field was found to be inhibitory for P(3HB) production. The use of floatation and sedimentation for the online separation of cells with and without polymer to facilitate a recycle strategy exhibited negative results. This was found to be due to cells undergoing cell lyses at the early stages of the fermentation releasing P(3HB) granules into the fermentation medium. The size distribution of these granules was identified. The results elicit the possibility of using cell auto-lysis behaviour for the separation of the two polymers from the culture broth leading to a reduction of costs.