Development of circular bioprocesses and biorefineries for the production and biological recycling of poly(3-hydroxybutyrate)

This PhD thesis focuses on the development of integrated biorefineries and recycling strategies for the sustainable production of poly(3-hydroxybutyrate) (PHB). The research explores two complementary pathways: (i) the valorisation of brewers’ spent grain (BSG) through sequential fractionation and microbial fermentation for PHB production and (ii) the enzymatic and thermochemical depolymerization of bioplastics followed by the subsequent utilization of hydrolysates for circular production of new PHB. By integrating biorefinery processes with closed-loop recycling concepts, this work establishes a holistic circular strategy for the sustainable and resource-efficient production of PHB, highlighting its potential to replace fossil-based plastics in a climate-neutral materials framework. Alternative refining schemes of BSG were systematically evaluated to identify the most efficient integrated process combining protein extraction and PHB production. The optimal biorefinery configuration involved the sequential aqueous extraction of dextrins, acidic proteolysis for protein recovery and subsequent dilute nitric-acid pretreatment of the residual solids prior to enzymatic hydrolysis. This combined strategy maximized the overall recovery of value-added components, enabling efficient nutrient valorisation and high fermentable sugar yields. The enzymatic hydrolysis of the pretreated solids achieved 84.5% glucan and 70.5% hemicellulose conversion yields. Fed-batch bioreactor cultivation of Paraburkholderia sacchari using the resulting hydrolysate produced 86.7 g/L PHB with 65.5% (w/w) intracellular content, 0.32 g/g yield and 2.7 g/(L·h) productivity. In the integrated BSG-based biorefinery scenario, 1 kg of raw BSG could be converted into approximately 160.2 g protein-rich extract and 155.1 g PHB, illustrating a highly efficient and sustainable cascade process that couples protein recovery with microbial biopolymer synthesis. A plasma bubble reactor using air as the feeding gas was employed to enhance PHB production from BSG facilitating also the cascade fractionation of protein and lignin as value-added co-products. Plasma operating parameters (voltage, pretreatment duration, duty cycle, initial solids concentration) were evaluated with the highest overall conversion yield of glucan and hemicellulose (73%, 0.494 gsugars/gBSG) achieved for initial solids concentration of 150 g/L. SEM analysis confirmed significant morphological disruption of fiber bundles and partial delignification compared to untreated BSG. The produced BSG hydrolysate led to higher PHB production efficiency (130 g/L total dry weight, 74.4 gPHB/L, 0.32 g/g yield, 2.64 g/(L·h) productivity) than conventional hydrolysates produced by enzymatic hydrolysis alone and enzymatic hydrolysis combined with hydrothermal treatment. The protein and lignin losses in the remaining BSG solids were 24.1% and 29.2% respectively. When renewable electricity is employed, the global warming potential was estimated at 0.59 kg CO2-eq per kgPHB and the abiotic depletion potential fossil at 11.12 MJ per kgPHB for PHB production based on plasma treated BSG. In the proposed biorefinery concept, 1 kg BSG could be converted into 147.6 g PHB with a solid fraction containing 137.4 g protein and 98.4 g lignin that could be subsequently fractionated as value-added co-products. This study demonstrated that BSG plasma treatment can be used for the exploitation of the full potential of BSG for the production of PHB with improved environmental impact and minimal losses of protein and lignin. This PhD thesis has also evaluated the thermochemical depolymerization of commercial bioplastics – including pure poly(3-hydroxybutyrate) powder (PHBP), PHB-based compounded pellets (PHBBCP) and poly(lactic acid) (PLA) – followed by bioconversion of the resulting monomers into PHB by Paraburkholderia sacchari and Cupriavidus necator. Hydrothermal hydrolysates of PHBP and PHBBCP at 200°C and 5–6 h treatment duration resulted in 72.9 – 74.0% 3-hydroxybutyric acid (3HB) and 21.9 – 27.1% crotonic acid (CA) content, whereas PLA fully degraded into lactic acid at 140°C. Alkaline treatment of PHBP and PHBBCP resulted in higher CA:3HB ratio (0.43 – 0.55) than hydrothermal treatment (0.20 – 0.39). Shake-flask fermentations using PHBP (2.2 gPHB/L by P. sacchari) or PLA (3.5 gPHB/L by C. necator) hydrothermal hydrolysates showed higher polymer production than PHBBCP hydrothermal and PHBP alkaline hydrolysates. Hydrothermal co-hydrolysates of PLA and PHBP reduced polymer production (ca. 1.4 g/L) by both strains. Benchmark shake flask fermentations using succinic acid and 1,4-butanediol – representative monomers of poly(butylene succinate) – showed that P. sacchari efficiently converted succinic acid to PHB (2.1 g/L), whereas 1,4-butanediol led to low bacterial growth and polymer formation. Bioreactor fed-batch fermentations with P. sacchari showed that a pH-stat feeding strategy with a concentrated hydrothermal PHBP hydrolysate led to 8.2 gPHB/L (54.9% w/w intracellular content), 0.29 g/(L·h) productivity, 0.54 g/g yield and 356 kDa weight average molecular weight. Enzymatic depolymerization was established as a mild and selective recycling route for PHB samples, employing a recombinant PHB depolymerase from Schlegelella thermodepolymerans to hydrolyze pure and compounded PHB materials. Optimized conditions (60°C, pH 9, 30 mL/L enzyme loading) enabled up to 83.1% 3ΗΒ conversion yield when 50 g/L initial pure PHB powder (PHBP) concentration was used, while dilute nitric-acid pretreatment (0.1 – 0.5% HNO₃, 70°C and 90°C) significantly enhanced polymer accessibility and maintained high depolymerase activity achieving 3HB conversion yields of 88.6 – 90.1%. PHB-based blends showed slightly lower degradation efficiency (52.0%) and conversion yield (39.8%) at an initial solid concentration of 20 g/L, likely due to the interference of additives. Both degradation efficiency and conversion yield was significantly increased when HNO3 pretreatment of PHB-based blends was employed (ca. 87% and 17.5%, respectively). However, all enzymatic hydrolysates were consumed by Cupriavidus necator for the production of new PHB achieving up to 54.3% intracellular PHB accumulation, demonstrating an efficient and benign biological recycling pathway. This PhD thesis presents an integrated biorefinery and recycling approach for the sustainable production PHB in a circular manner. By coupling the valorisation of BSG with thermochemical and enzymatic recycling of post-consumer bioplastics, a closed-loop system was established for PHB synthesis and recovery. The developed processes demonstrate high conversion efficiencies and strong alignment with circular bioeconomy principles, supporting the transition towards sustainable and carbon-neutral polymer production.