From cultivation to purification: bioprocess development for the production and recovery of tailored polyhydroxyalkanoates

Plastic pollution from littering and inefficient recycling systems is one of the biggest concerns of our time, posing a threat to the environment, wildlife, and human health. Large amounts of plastic enter landfills and oceans persisting for hundreds of years, harming animals or breaking down into microplastics. Bioplastics, such as polyhydroxyalkanoates (PHAs), are bio-based, biodegradable alternatives to petrochemical plastics. PHAs are polyesters synthesized by a variety of microorganisms as carbon and energy storage compounds. The model organism Ralstonia eutropha can accumulate up to 90 % of its cell dry weight as PHA and use various carbon sources, including sugars, organic acids and industrial waste like glycerol, CO2, food-and animal wastes. However, the most common PHA, polyhydroxybutyrate (PHB) is brittle with a melting temperature near thermal degradation. Thus, R. eutropha was engineered to produce the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] from oleaginous substrates, as medium-chain-length (mcl, 6 – 14 C atoms) comonomers lead to material characteristics resembling conventional plastics. Despite its potential, PHA is 4 – 6 times more expensive and produced at low capacity (100 k t year-1), representing less than 0.1 % of the global plastic production (400 million t in 2022). High costs stem from pure feedstocks, costly downstream processes, and low-efficiency cultivation processes, highlighting the need for high productivity processes for tailored, application driven PHA manufacturing. This thesis demonstrates tailoring the monomeric composition of P(HB-co-HHx) synthesized by Ralstonia eutropha Re2058/pCB113 in the range of 0 – 17 mol% HHx to modulate its materials characteristics by adjusting the ratio fructose to canola oil as feedstocks. Abiotic degradation is highly impacted by pH, with almost complete degradation at basic pH, and is slowed down with increasing HHx content. Elongation at break increased to 703 % at 14 mol% HHx while maintaining mechanical strength. Fed-batch cultivations at laboratory and pilot scale using three different substrate mixtures facilitated high cell densities (> 100 g L-1), PHA contents (> 60 wt%) and productivities (> 1 gPHA L-1 h-1) of tailored PHA. A C/N ratio of 22 g g -1 was critical for achieving high cell densities and triggering PHA accumulation. P(HB-co-HHx) was extracted in a two-step process using non-halogenated solvents (acetone and 2propanol), reaching yields and purities of up to 100 % and 88 – 100 %, respectively. Low-field nuclear magnetic resonance (NMR) was used to monitor extraction for the first time, providing extraction kinetics detecting dissolved PHA. Extraction of P(HB-co-HHx) from wet biomass is feasible using acetone and water as precipitant, with efficiency highly impacted by HHx content, making it suitable for high intracellular PHA contents with HHx fractions > 20 mol%. Lastly, mechanical pretreatment by high-pressure homogenization offers a solvent-free PHA recovery alternative, minimizing downstream process steps, eliminating the need for solvent recovery. Depending on the initial PHA content, high purity is achieved with additional polishing treatments lowering nitrogen or proteinaceous impurities. The developed fed-batch strategy provides P(HB-co-HHx) with varied monomer composition in kg-pilot scale to accelerate processing and application developments, bridging laboratory and industrial scales. The shift from halogenated to non-halogenated solvents enhances sustainability, while comparable results are achieved. Extraction monitoring in real-time by low-field NMR revealed extraction times < 20 min, compared to typical hour-long extractions, and is able to denote the extraction-end saving time and energy. Utilization of wet biomass containing PHA with high HHx content reduces time-, costand energy-intensive drying and separation steps. The final method, high pressure homogenization, is generally applicable regardless of PHA composition. Process integration possibilities were further discussed to improve the feasibility of PHA production to become competitive.