Production of polyhydroxyalkanoate copolymers from plant oil

Abstract

Polyhydroxyalkanoates (PHAs) are carbon storage polymers produced by a variety of bacteria. The model organism for studying PHA synthesis and accumulation is Ralstonia eutropha. This species can be used to convert renewable resources into PHA bioplastics, which can serve as biodegradable alternatives to traditional petrochemical plastics. A promising feedstock for PHA production is palm oil, a major agricultural product in Southeast Asia. Strains of R. eutropha were engineered to accumulate high levels of a PHA copolymer containing 3-hydroxybutyrate and 3-hydroxyhexanoate when grown on palm oil and other plant oils. This type of PHA has mechanical properties similar to those of common petrochemical plastics. The engineered strains expressed a PHA synthase gene from the bacterial species Rhodococcus aetherivorans I24. The amount of 3-hydroxyhexanoate in the PHA was controlled by modulating the level of acetoacetyl-CoA reductase (PhaB) activity in the engineered R. eutropha strains. Whole genome microarray studies were carried out to better understand R. eutropha gene expression during growth on plant oils. These results have provided insights that will allow for additional improvements to be made to the engineered strains. In order to study growth of R. eutropha strains on plant oils, fermentation methods were developed to grow the bacteria in oil medium and measure consumption of the carbon source. In one of these methods, the glycoprotein gum arabic was used to emulsify the plant oil. This emulsification reduced the lag phase in oil cultures and allowed representative samples to be taken early in experiments. High density palm oil fermentations were also carried out using unemulsified oil, which is more representative of industrial culture conditions. Techniques were developed for recovery of poly(3- hydroxybutyrate-co-3-hydroxyhexanoate) from R. eutropha biomass. Methyl isobutyl ketone was used to extract the PHA, and the polymer was precipitated from solution by addition of an alkane. A process model based on this procedure was developed for continuous recovery of PHA. The results described in this thesis include several advancements towards the goal of industrial PHA production from palm oil.