Production of bacterial biopolymers from industrial fat-containing wastes

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology

Polyhydroxyalkanoates (PHAs) constitute a group of biobased and biodegradable polymers, which have been recognized as good substitutes for petroleum-based polymers in many applications. The large-scale production of PHAs is limited by the high cost of the most commonly used carbon sources (e.g. glucose, sucrose). However, the food industry generates large amounts of wastes, including fat-containing materials that can be used as low cost carbon sources for microbial cultivation, due their high carbon content. In this study, several bacterial strains (Cupriavidus necator, Comamonas testosteroni, Pseudomonas oleovorans, P. resinovorans, P. stutzeri, and P. citronellolis) were evaluated for their ability to grow and produce PHAs using fat-containing wastes generated by the food industry. The materials used in this study were mainly composed of free fatty acids, namely mystiric, oleic, linoleic and stearic acid. In the preliminary shake flask experiments, C. necator, C. testosteroni, P. oleovorans and P. citronellolis were able to grow and produce PHA polymer on margarine waste with the highest content. Those strains were selected for batch bioreactor experiments, wherein C. necator reached the highest polymer content (56%, wt/wt) and volumetric productivity (0.33 gPHA/L.h), Lower PHA contents were achieved by P. citronellolis and P. oleovorans (7.0 and 8.5%, wt/wt, respectively). However, in contrast with C. necator that synthesized polyhydroxybutyrate [P3(HB)], those strains produced medium chain length polyesters (mcl-PHA) containing monomers of 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD). C. necator was also cultivated in two different fed-batch strategies. The first cultivation achieved 76% (wt/wt) of P(3HB), while high cell densities were obtained in the second cultivation (48 g/L of active biomass concentration). Finally, the P(3HB) and mcl-PHA polymers had a glass transition temperature of 0.5–7.9ºC and -45.6, a melting point of 169.3–173.4ºC and 60.9ºC, and degree of crystallinity of 48.7–56.6% and 0.7%, respectively.