Dottorato in PROCESSI CHIMICI PER L'INDUSTRIA E PER
L'AMBIENTE

Titolo della tesi: Linking Microbial Electrochemical Technologies to Multi-Stage Processes for Polyhydroxyalkanoates Production with Mixed Microbial Cultures

Bio-based and fully biodegradable polymers (i.e., polyhydroxyalkanoates, PHAs) have been represented as the optimal candidates to replace petroleum-based polymers. PHAs are a family of linear polyesters stored by a wide range of microorganisms, and in the last decades, their production has been investigated to increase the spread of bio-plastic production. PHA production by mixed microbial cultures (MMCs) employs wastes or by-products as feedstocks, contributing simultaneously to the implementation of a circular economy approach. In addition, PHA-MMC production reduces the process operational costs associated with the traditional pure cultures-PHA production. The process scheme is represented by the combination of the acidogenic fermentation (AF) step and PHA selection and accumulation stages. The first step is diverted to the production of a mixture enriched into carboxylic acids (i.e., Volatile Fatty Acids, VFAs), which are the main precursors for PHA synthesis by MMCs. This Thesis project was focused on several goals including the evaluation of using agro-industrial by-products fermented at a pilot scale for the selection of PHA-storing microorganisms starting from an activated sludge. In addition, innovative approaches to improve and control each stage of the PHA-MMC process were evaluated. In particular, an enriched reground pasta (RP) fermented mixture was obtained by the pilot scale AF process and then fed in a Sequencing Batch Reactor (SBR) at a lab scale operated at an Organic Load Rate (OLR) of 2.125 gCODACIDS/Ld maintained under short hydraulic retention time (HRT; 0.5 days) and a sludge retention time of 1 day. The imposed parameters determined a good performance in terms of the PHA storage capacity, specifically the intracellular PHA content achieved the value of 22.3 ± 1 % wt/wt. This study underlined the feasibility of the PHA-MMC process involving a by-product derived from the food industry. The other object of this thesis focuses on the possibility of improving the performance of the MMC-PHA production, with main reference to the production of carboxylic acids and microbial selection, by using Microbial Electrochemical Technologies (MET). More in detail, Electro-Fermentation (EF) represents a niche of these technologies which consists of electrochemically controlling microbial fermentative metabolism with electrodes. First of all, cathodic EF (CEF) was investigated with MMCs, in order to assess the specific metabolic pathway involved and the role of the polarized electrode in the production of fatty acids. In our experiments, CEF (−700 mV vs. SHE) caused an increase in the acetate, propionate, and butyrate yields (0.90 ± 0.10, 0.22 ± 0.03, and 0.34 ± 0.05 (mol/mol), respectively) compared to the open circuit potential control experiments (OCPs) (0.42 ± 0.2, 0.15 ± 0.04 and 0.21 ± 0.001 (mol/mol), respectively). In parallel, the same approach but in anodic conditions (AEF) was involved to evaluate the accumulation of the PHA. In particular, the anode was investigated to determine the effect on the accumulation and composition of the stored biopolymer with a selected MMC into PHA-storing microorganisms. The AEF condition yielded a 3-fold increase in the PHA content (27.4 ± 9.9 %, wt/wt) compared to the control experiments. Interestingly, the application of the electrode polarization also resulted in higher hydroxy valerate (HV) content (relative to the total Poly-hydroxybutyrate-valerate, PBHV) compared to the control (54 ± 1 % vs. 44 ± 1 wt/wt). In the last, the effect of the anodic potential was also evaluated with an activated sludge imposed in anaerobic conditions in single chamber BES. The results showed that the flux of the electron derived from the oxidation of the organic substrate can be directed towards the accumulation of intracellular polymer (i.e., PHA). The maximum polymer storage yield was detected at 0 V and under an uncoupled carbon and nitrogen feeding strategy (0.13 ± 0.02 (mol- C PHB/mol-C acetate) confirming the hypothesis that the anode can replace the role of the oxygen under AEF. In conclusion, this thesis confirms the feasibility of using poorly explored agro-industrial by-products for the PHA-MMC process since a high PHA storage consortium was selected under the investigated conditions. In addition, highlights the importance of understanding in-depth the impact of the polarized electrode on the PHA production process with MMC to employ EF technologies. Furthermore, the possibility of obtaining a well-defined enriched fermented mixture regulating the acidogenic process with an electrode results in a promising approach for the full-scale implementation of the PHA production from MMC.