Lack of space for disposal and growing environmental concerns regarding non-biodegradable synthetic plastics has fuelled research into the development of eco-friendly biopolymer materials as sustainable alternatives to traditional petroleum derived polymers (Defra et al., 2006; Barnes et al., 2009; Hopewell et al., 2009). The cost of producing bioplastic has also been falling thanks to improved processes. Combining this with the increasing cost of crude oil has made bioplastic prices more competitive with regular plastics, although the cost is still a barrier to market uptake. The global demand for bioplastics is predicted to triple by 2015 to reach >1 million tons per year to represent a $2.9 billion market for opportunity and advancements (Adkins et al., 2012). Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely regarded as potential replacement biopolymers, are the polyhydroxyalkaonates (PHAs) (Koller et al., 2008).
In this thesis, PHA production by microbial populations in mixed cultures was investigated in order to obtain a better understanding of the process. A central theme of the thesis is examining the ways in which different organisms within a mixed culture contribute to the net PHA that has been produced by these organisms. We want to answer the question: do all the sub-populations in a mixed culture accumulate PHA in similar ways, so that the PHA content and PHA composition in all the organisms is similar? Secondly, we would like to address the question: in mixed cultures, how do the populations respond to various substrates and feeding strategies with regards to community dynamics and the accumulation of PHA?
A prominent aspect of the PhD was to assess the potential for using a buoyant density method to separate populations of PHA accumulating organisms in mixed cultures. The purpose was to try to understand why PHA in mixed cultures has such a broad chemical compositional distribution, containing broad blends of different poly(hydroxybutyrate-co-valerate) (PHBV) copolymers, as discovered and published recently by the UQ research group (Laycock et al., 2013b). The possible reasons are: (1) different populations in mixed cultures accumulate different copolymers, and/or (2) single populations accumulate a broad range of copolymers and/or (3) a single cell can accumulate a range of copolymers intracellularly. Following a long process of optimisation, Percoll buoyant density gradient separation was used to separate mixed cultures into two fractions. It was found that the PHA content in one of the III fractions was higher (24±0.6%) than the other (16±4.9%). It was also found that the intracellular PolyP granules assessed using transmission electron microscopies (TEM) were on average larger and that there were more of them per unit area of cell in the fraction with the higher PHA content. The community population was also found to be different in the separated fractions. But while differences in content and microbial composition were observed, the PHA composition in both fractions was found to be essentially the same (43 – 44 mol% HV), which shows that groups of microbial populations within mixed cultures don’t necessarily generate PHA with unique composition. However, it was also found that biomass separation in Percoll was influenced by factors other than simple PHA content, and that this separation technique was not a generically applicable approach to biomass fractionation based on PHA content.
The second aspect of this PhD was addressed through analysing the responses of biomass enriched on fermented whey permeate to different feeding strategies for PHA accumulation. It was found that the microbial community in the enrichment system was in a state of flux. Despite this flux in community, PHA accumulation in the biomass was very consistent, with very similar yields, final polymer contents and biomass growth rates. As expected, the polymer composition (mol% HV content) was primarily controlled by feed composition (ratio of acetic to propionic acids).