PhD Defence Mattéo Castiello

Abstract

Polyhydroxyalkanoates (PHA), biodegradable and biosourced polymers, constitute an attractive alternative to substitute in part conventional plastics derived from fossil fuels. In particular, the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) copolyester, whose thermomechanical properties are correlated to the 3HV content, has characteristics in line with industrial requirements. To limit production costs and make PHA more competitive, the use of non-axenic cultures is receiving increasing attention. They allow making use of complex substrates such as organic waste, even if they must first be converted into VFA by acidogenic fermentation.

In mixed microbial cultures (MMC), PHA production processes are generally carried out in two steps, with a step of enrichment in PHA-storing microorganisms, and a second step of PHA accumulation by the selected consortia. Although the majority of PHA production processes in MMC are executed in discontinuous mode, continuous processes are of real interest. It was previously demonstrated that applying a double carbon and phosphorus limitation in continuous culture ensured a robust enrichment of PHA producers. Nevertheless, this selection strategy has only been tested on acetate or acetate/butyrate, validating this strategy for PHB producers only.

In this context, this thesis sought to study and develop a process for the production of P(3HB-co-3HV) in a two-stage continuous reactor from mixed cultures from activated sludge. The reproducibility of the selection was tested with two mixtures integrating propionate (acetate/propionate and butyrate/propionate), emphasizing a systematic functional selection of PHA-producers. By comparing the results with previous studies carried out with other carbon substrates, the influence of the nature of the carbon source on the direction of the consumption of the substrate (between cell growth, PHA storage and reactions of maintenance) was evaluated.

A microbiological qualification of the selected consortia was performed through the study of the dynamics of selection and the identification of the dominant microbial genera (Acinetobacter, Malikia and Zoogloea). The influence of operating parameters (nature of the carbon source, level of phosphorus limitation, dilution rate) on microbial enrichment was assessed. In particular, it was shown that the relative abundances of the majority populations when fed with a mixture including propionate were largely conditioned by the degree of phosphorus limitation. In addition, the use of Monod’s model has contributed to a better understanding of the mechanisms that govern microbial competition .

The microbial consortia were also qualified on the kinetic aspect, particularly in terms of PHA accumulation potential, by means of cultures in batch mod under phosphorus-deficient conditions. This study in dynamic regime contributed to define the assimilable and preferential substrates. Based on the determination of the maximum specific rates of PHA production, combined with a kinetic model, the influence of the nature of the carbon substrate, the bacterial composition of the selected consortia and the physiological state of cells (i.e. the intracellular phosphorus content) on PHA accumulation capacity was depicted. Two classes of PHA producers could essentially be distinguished: poor producers (Acinetobacter) and overproducers (Zoogloea, Malikia).

Moreover, the effects of the application of an additional nutritional deficiency (nitrogen) on the kinetics of PHA production were evaluated in batch reactors and integrated into the kinetic model. Nitrogen deficiency has been shown to restrict PHA accumulation rates. The confrontation of the model with the experimental data obtained in the production stage of the two-stage continuous reactor has strengthened and improved the understanding of the mechanisms which govern the performance of PHA storage in continuous cultures. The model was then used as a prediction tool to define the optimal conditions for the process.

Finally, the influence of all the operating conditions was examined from the point of view of the monomeric composition of the P(3HB-co-3HV) copolymer. It was established that the 3HV content could be maximized by increasing the propionate fraction in the feed, rather associating it with acetate (instead of butyrate) and applying phosphorus deficiency exclusively.