Polyhydroxyalkanoates PHA biosynthesis from agro-food wastes: leading edge technologies for process monitoring and polymer tailoring

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Polyhydroxyalkanoate (PHA) biosynthesis from fruit waste at pilot scale: productivity maximisation and polymer tailoring

Publication . Matos, Mariana Campos de; Oehmen, Gilda; Reis, Maria D’Ascensão

Bio-based and biodegradable plastics are an ecological alternative to conventional petroleum-derived polyolefins. Polyhydroxyalkanoates (PHA) have drawn significant attention as one of the most promising biopolymers due to its biocompatibility and biodegradable character. In particular, the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) – P(3HB-co-3HV) – has exceptional thermal and mechanical properties, which can be manipulated by varying its monomeric composition. PHA production using non-aseptic mixed microbial cultures (MMCs) enables the use of waste/surplus feedstocks, contributing simultaneously to the implementation of a circular economy approach and to reduce the process operational costs associated to the traditional pure cultures’ PHA production. The MMC process usually comprises three steps: the acidogenic fermentation, the enrichment of a MMC in PHA-storers and finally the PHA production. The monomeric composition of the P(3HB-co-3HV) produced by MMCs largely depends on the fraction of each soluble fermentation product (SFP) produced in the first step of the process, which can be precursors of either 3-hydroxybutyrate (3-HB) or 3-hydroxyvalerate (3-HV). This Thesis project was focused on solving, at pilot scale, the main difficulties (at the production level) that currently prevent industrial implementation of the PHA production process by MMC: the high manufacturing costs associated to the low process performance and the ability to consistently manipulate and tailor the polymer composition (and thus its properties). A three-stage MMC process was implemented at pilot scale. Fruit waste (FW) was selected as feedstock, since it contains high concentration of carbon and is globally generated in large volumes, causing several environmental and economic problems. FW is a nutrient-deficient feedstock, thus enabling the implementation of an uncoupled carbon and nitrogen feeding strategy in the selection reactor. The first study conducted in this PhD assesses, for the first time, the impact of the sludge retention time (SRT) (2 and 4 days) and the organic loading rate (OLR) (from 2.6 to 14.5 gCOD.L-1.d-1) on the growth versus polymer storage dynamics of PHA-storers selected under the uncoupled feeding strategy. The results showed that, similar biomass volumetric productivities were achieved for each OLR tested, regardless the SRT. However, the culture selected at 4 d SRT showed superior specific storage rates and accumulation capacity resulting in a global PHA productivity (4.6 ± 0.3 g.L-1.d-1) that was 80% above that of 2 d SRT (2.6 ± 0.2 g.L-1.d-1 ). This study underlined the importance of achieving a good balance between culture growth and accumulation capacity to increase the viability of the PHA-producing process from wastes. The same pilot plant set-up and feedstock (FW) were used to incorporate different effective operating conditions in the three stages of the process with the objective of boosting the overall PHA production performance (namely, PHA content on biomass, global productivity and overall yield). The OLR and pH of the acidogenic reactor were successfully adjusted targeting a high fermentation yield (0.74 gCOD.gCOD-1) and the production of a fermentate highly enriched in butyrate (56.8%, gCOD-basis), resulting in enhanced PHA production steps. A well selected MMC was obtained as a result of uncoupling the carbon to the nitrogen feeding, and the biomass volumetric productivity attained the unprecedented value of 1.96 g.L-1.d-1 as a response to the high OLR (8.7 gCOD.L-1.d-1) imposed. The culture selected at the optimal OLR achieved a high storage yield (0.98 gCOD.gCOD-1), and the continuous feeding strategy led to a maximum PHA content of 80.5% (w/w) at the end of accumulation assays. The obtained global PHA productivity (8.1 g-PHA.L-1.d-1) and overall process yield (0.45 gCOD.gCOD-1) are the highest values reported for MMC using a real waste feedstock at pilot scale. Moreover, a P(3HB-co-3HV) copolymer with a 3-HV content of 0.24 (g-basis) and a molecular weight of 311 KDa was produced, making this material an ideal candidate for packaging applications, the largest market of plastic usage. Lastly, the possibility of tailoring the precursors that influence the P(3HB-co-3HV) composition was investigated, by controlling the operating pH (between 4.69 and 6.34) of a continuous upflow anaerobic sludge blanket (UASB) reactor fed with FW. The impact of the operating conditions imposed was assessed by evaluating the microbial community profile, the corresponding performance and the impact on polymer composition. The fermentation yield of the UASB was maintained quite stable (between 0.72 and 0.79 gCOD.gCOD-1) during the entire operational period. On the other hand, the 3-HV bioprecursors fraction in effluent was highly affected by pH, resulting in the production of P(3HB-co-3HV) copolymers with quite different monomeric compositions. Overall, the 3-HV content of the produced polymers varied from 0.16 to a maximum of 0.44 (gCOD-basis) when the pH of the acidogenic reactor increased from 4.69 to 5.92. Moreover, the end-stream SFP composition were similar for identical operational pH values tested in different periods, showing that despite the changes occurring in the system, the selected cultures were resilient and able to produce a consistent profile of fermentation products (and thus a constant PHA monomeric composition). Additionally, the IWA Anaerobic Digestion Model No. 1 (ADM1) was expanded to include the pathway of valerate production from lactate, aiming to dynamically predict the profile of the PHA bioprecursors produced. Calibration and validation procedures were done against data from two distinct pilot-scale UASB reactors fed with FW and describing different pH and OLR dynamics. The model was capable to predict the different PHA bioprecursors production in the UASB reactors with overall strong correlations with the experimental data for different OLRs over the pH range between 4.20 and 5.16, providing a useful tool for process optimisation and tailoring of the PHA monomer composition. This thesis highlights the importance of understanding in-depth the impact of key operating conditions on the PHA production process with MMC to increase its overall viability. Moreover, it shows that predicting and regulating the acidogenic process is essential to promote an adequate PHA bioprecursors composition production, which are both promising results towards the full-scale implementation of the PHA production from MMC.

2022-01-12Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

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OE

Número da atribuição

SFRH/BD/104767/2014