Production of polyhydroxyalkanoates in biological treatment of industrial wastewaters

In search of more environmentally friendly polymer materials, polyhydroxyalkanoates (PHAs) have been considered as promising candidates. PHAs exhibit a broad range of material properties, can be produced from renewable resources and are completely biodegradable. Nevertheless, high production costs associated with traditional PHA production based on fermentation by pure microbial cultures have so far limited broad application of these polymers. Therefore, alternative production strategies have been proposed based on the use of open mixed cultures that are selectively enriched by the imposed operating conditions. This does not require sterile conditions and allows for acclimation to various complex waste substrates leading to potential economic and environmental advantages. In this study, a process was developed for production of PHA by mixed cultures (activated sludge) treating industrial wastewaters allowing for PHA as a by-product from the wastewater treatment process. The process comprised of three stages, namely (1) anaerobic acidogenic fermentation to convert various organic matter into volatile fatty acids (VFA), which are preferred substrates for PHA production, (2) enrichment of PHA producing organisms while treating the wastewater under process conditions that were dynamic with respect to either carbon substrate or oxygen and (3) accumulation of PHA from the fermented effluent in the enriched biomass. Lab-scale experiments were conducted in which the process was optimized with respect to polymer productivity (PHA fraction of the biomass, PHA yield over substrate and rate of PHA production) utilizing different wastewaters (pulp/paper mill effluents, cheese whey and sugar cane molasses). Two different strategies for biomass enrichment, namely alternating high and low organic loading under aerobic conditions (feast and famine) versus alternating anaerobic-aerobic conditions, were evaluated and compared. Furthermore, strategies for the control of PHA monomer composition were developed. A paper mill effluent was treated with high (95 %) removal of organic matter (chemical oxygen demand) under feast and famine conditions with production of biomass containing up to 48 % of polymer. PHA was produced containing 3-hydroxybutyrate (3HB) and 61 mol % 3-hydroxyvalerate (3HV) with a yield of 0.66 C-mol PHA per C-mol VFA. By controlling the chemostat retention time (8-95 h) and pH (3.5-6) during acidogenic fermentation of the paper mill effluent and cheese whey, the composition of produced VFAs (mainly acetate, propionate and butyrate) was affected in such a way that the anticipated ratios between 3HB and 3HV would be affected in a broad range (23-100 mol-% 3HV). Alternating anaerobic-aerobic conditions resulted in the enrichment of glycogen accumulating organisms (GAOs) both with the paper mill effluent and fermented sugar cane molasses as substrate. As determined by fluorescence in situ hybridization, these cultures were dominated by Candidatus Competibacter phosphatis and organisms related to Defluviicoccus vanus. For GAOs treating the paper mill effluent, similar results with respect to PHA biomass content and yield were obtained as with the feast and famine enrichment strategy. For the first time, an open mixed culture was observed to produce PHA containing a medium chain length monomer. This culture was enriched in GAOs using molasses as substrate and exhibited a long-term drift towards production of increased amounts of 3-hydroxyhexanoate, up to 31 mol-% of the PHA. This suggests a broadening of the spectrum of biopolymers that can be produced from fermented waste by open mixed cultures. In order to optimize PHA accumulation in GAOs, the aerobic metabolism of GAOs in presence of VFAs was investigated. It was found that the fate of glycogen was highly dependent on the type of VFA being consumed. With the fermented molasses VFA mixture or synthetic acetate as substrate, glycogen was consumed which was not the case with propionate, butyrate or valerate as substrates. Polymers produced by GAOs were found to have weight average molecular weights between 350 000 and 900 000 g/mol and narrow weight distributions (polydispersity indexes around 2) despite the presence of a mixture of microorganisms. The melting temperature (89°C to 174°C) and melting enthalpy (0 to 82.1 J/g) were controlled in broad ranges by the monomer composition. Decomposition temperatures were between 277.2°C and 294.9°C and independent of monomer composition. Overall, production of PHA as a by-product in biological treatment of industrial wastewaters is feasible. Production of PHA from real wastes can be obtained with high yields and rates using open mixed cultures enriched under either feast and famine conditions or alternating anaerobic-aerobic conditions. Composition of the PHA produced from a wastewater can be affected both by acidogenic pretreatment conditions, which controls the VFA product distribution, and by the enrichment strategy