Towards polyhydroxyalkanoates synthesis with mixed microbial communities: exploring the uncoupled feeding strategy

Plastic has addressed the needs of the society and revolutionised human lives. However, it comes from finite fossil sources destined for depletion and its massive production, intrinsic characteristics, and poor management have caused global consequences on various scales: from the Pacific Trash Vortex to microplastics invading human and animals bodies. In this context, bioplastics have emerged as a more sustainable alternative to fossil-based plastics, offering similar properties but with the advantage of being biodegradable or bio-based. This thesis focuses on the production of polyhydroxyalkanoate (PHA), a biodegradable and bio-based bioplastic produced from liquid waste using mixed microbial cultures (MMC) enriched with PHA accumulating bacteria. This process converts waste into a valuable product, promoting the transition from a linear to a circular economy; however, the technology is still immature, remaining costly and inefficient, requiring further research is needed to improve its feasibility and scalability. Specifically, energy consumption is still high, and productivity remains low, preventing PHA through MMC from competing with conventional plastic production. The selective techniques used to enrich PHA accumulating communities are inspired by the natural processes that favour such bacteria. In nature, PHA is a group of polymers produced by many bacteria as energy reserve in response to stress (e.g., osmotic stress) or unbalanced growth conditions. The feast an famine selection strategy alternates between phases when carbon is available (feast) and when it is absent (famine). If the famine phase is sufficiently long, it favours bacteria that store carbon during the feast to use it as an energy source during famine. When combined with nutrient limitation, this strategy further promotes accumulation over growth during the feast phase. This study has focused on uncoupled feeding strategy, where carbon is dosed at the beginning of the feast and nutrients at the beginning of the famine. This approach not only encourages PHA accumulation during feast but also enhances the growth of PHA accumulating bacteria by providing nutrients only after carbon has been consumed or converted into PHA. To further advantage these bacteria, an intermediate settling phase was introduced as physical selective pressure between feast and famine to remove residual organic carbon and eliminate non PHA accumulating bacteria. The experiments were conducted using sequencing batch reactors (SBRs), with an uncoupled feeding system of synthetic fermented wastewater, containing acetate and propionate. This mix of volatile fatty acids was chosen to potentially produce of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a higher quality polymer. The effect of salinity on the selection of PHA-accumulating bacteria was also explored, as osmotic stress can promote the growth of such organism. Most reactors operated under light salinity (5 g NaCl/L), except for one experiment under hypersaline conditions, at 107 g NaCl/L, the highest salinity ever applied to PHA production process. The aim of the experiment was to amplify criticalities and potential advantages. The microbial communities were selected from inocula acclimated to these salinities. Those cultivated at 5 g NaCl/L originated from activated sludge from a tannery wastewater treatment plant, while the hypersaline community was enriched from sediments and water collected from a salt pan. Monitoring was conducted at multiple levels: a rapid, daily approach using dissolved oxygen (DO) and pH measurements, and more comprehensive analyses of key compounds (PHA, biomass, nutrients and carbon) over individual cycles. Molecular analysis, including DNA extraction, amplification and sequencing, were crucial for understanding microbial dynamics and stabilisation times. Additionally, transmission electron microscopy (TEM) provided direct visualisation of PHA inclusions within bacteria cells. Initial experiments focused on varying potentially critical parameters, such as nutrient limitation (switching from nitrogen to phosphorus limitation) and famine duration, providing valuable insights into PHA accumulating bacteria selection. These parameters, relevant in “coupled” feeding, also proved important in the uncoupled system. The study further highlighted significant factors such as the organic loading rate (OLR), sludge retention time (SRT), and dissolved oxygen (DO). The activated sludge characteristics also emerged such as the presence of filamentous bacteria, biofilm formation, and various predators. Based on these findings, an experiment was conducted to reduce energy consumption and improve PHA accumulation, by minimising aeration and reducing predator activity during the famine phase. The experiment introduced a microaerophilic famine phase (0-1 mg DO/L) instead of traditional aerobic conditions. The PHA accumulation in this setup reached a maximum of 82% of the weight of biomass, surpassing the performance of the control reactor (55%). In terms of productivity, the two selective strategies were found to be comparable However, the quality of the polymer was found to be lower in microaerophilic conditions. Subsequently, an experiment under hypersaline conditions proved to be suitable for promoting the growth of a PHA accumulating community, with a maximum accumulation of 44% of the biomass weight. Overall, the results provide valuable insights for future applications, offering potential improvements to existing processes or avenues for new research. These findings could facilitate the transition from experimental research to practical implementation.