# Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste
## 1 Introduction
The existing literature provides extensive insights into PHA production and extraction at laboratory and pilot scales of 5, 10, or 200 L [25; 26; 27]. So far, there is a significant lack of literature focusing on PHA demonstrative scale production, especially in large fermenters, i.e., the technology has yet to be proven in a real environment, and it has not reached a sufficient readiness level. The transition from small-scale to industrial-scale fermenters introduces various challenges, including process stability, microbial community management, and optimization of operation conditions such as temperature, aeration, and pH control. Specifically, this study evaluates the production of scl-PHAs using MMC and OFMSW-derived VFAs as the primary carbon source in a 20 m\({}^{3}\) fermenter and the accumulation fermentation in 3 m\({}^{3}\). This research aims to optimize critical operational parameters to improve microbial performance and overall process efficiency. The microorganism's ability to store PHA intracellularly can be selected and enriched under aerobic dynamic feeding conditions, where the MMC are exposed to cyclic least and famine conditions (usually represented as ratio F/F) [28; 29; 30]. This F/F cycle is carried out under aerobic conditions where during short periods of time, the carbon source is easily accessible for MMC (feast phase) followed by long periods of time in the absence of a carbon source (famine phase) [28,30]. During the feast phase, the MMC is able to metabolize PHA from the carbon source, storing it intercellular in the form of granules. Once the carbon source has been consumed, the PHA-accumulating microorganisms use the previously stored PHA as an energy source to survive during famine phases. This accumulation capacity of the MMC is crucial for conferring them a significant competitive advantage over other non-PHA-accumulating MMC during the starvation phase [28,31]. Indeed, to achieve an adequate balance between the time lasted for the feast phase and the time lasted for the famine phase, i.e., the F/F ratio, is very important to assure a large number of PHA-storing MMC [32]. The employment of dynamic conditions (discontinuous feeding) together with an adequate balance between the F/F ratio during the enrichment phase lies in the presence of PHA-storing microorganisms in the activated sludge of wastewater treatment plants (WWTT) [33, 34, 35]. The feast phase must be sufficient to ensure complete substrate removal, while the famine phase must be prolonged enough to allow substantial consumption of the previously accumulated PHA [28,36,37] and to minimize the presence of MMC incapable of accumulating PHAs. Once the enrichment step of PHA production is completed and the fermentation broth is rich in PHA-accumulated microorganisms, the accumulation reaction takes place [38]. Its objective is to increase as much as possible the intracellular accumulation of the PHA in the MMC cells [28]. Some authors [28,39,40,41] have reported that the fed-batch strategy with MMC reaches a high scl-PHA productivity but, after the feeding cycles take place, the accumulated scl-PHA is largely consumed before the addition of a new pulse with a carbon source and, finally, the scl-PHA productivity decreases. On the one hand, the feast/famine (F/F) regime is effective in selecting PHA-accumulating microorganisms, but the implementation of this strategy at industrial scale presents several challenges. Thus, maintaining stable F/F cycles under fluctuating environmental and operational conditions can significantly impact microbial selection and PHA accumulation [28,30]. Additionally, optimizing the organic loading rate (OLR) and aeration parameters is crucial to prevent process inhibition while ensuring efficient substrate removal and biomass enrichment. The lack of precise control over temperature and pH can lead to process destabilization [42, 43, 44] (if the pH is outside the optimal range of 6.5 to 8), particularly during colder seasons.