# Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste
## 4 Discussion  
### Enrichment of scl-PHA-Accumulating Microorganisms  
The enrichment phase successfully selected an MMC capable of accumulating scl-PHAs under dynamic feast/famine (F/F) conditions. Steady-state conditions were established after approximately 65 cycles (Figure 2), characterized by stable total suspended solids (TSS), volatile suspended solids (VSS), and a consistent F/F ratio below 0.2 h/h. The biomass concentration (VSS) increased until reaching an organic loading rate (OLR) of 1 g COD/L-d (cycle 51), maintaining an average VSS concentration of \(\sim\)2 g/L. This stabilization of biomass concentration is particularly relevant in the context of PHA-accumulating microbial culture selection, where the F/F ratio plays a crucial role [47; 63; 64]. Studies have shown that low F/F ratios (\(<\)0.2) confer a competitive advantage to PHA  
Figure 9: Wet extracted scl-PHA concentrated in DMC (**a**), wet scl-PHA after DMC removal (**b**), dry scl-PHA (**c**), and grinded scl-PHA with a purity of 90% (**d**). accumulating microorganisms over non-accumulating bacteria [47,65]. A very low F/F ratio is indicative that the process could tolerate a higher OLR. The different shoulder shapes observed in the DO profile (Figure 3) may be attributed to varying rates of VFA consumption by MMC. The lack of temperature and pH control is the main limitation of the process studied. On the one hand, this approach simplifies the process control and requires auxiliary services for its further industrialization. On the other hand, it also introduces variability into process stability. A notable destabilization occurred between cycles 150 and 180, where TSS, VSS, and pH dropped significantly. This event coincided with a sharp decrease in the environmental temperature (Figure 4), likely reducing microbial growth rates and metabolic activity. Although the MMC exhibited resilience and was recovered after ~30 cycles, this adaptation period could carriage challenges at larger-scale applications where process stability is critical. Additionally, the inherent heterogeneity of the OFMSW used as the feedstock may have also contributed to fluctuations in substrate composition, further impacting microbial selection and process performance. It has been found that the variability in the composition of agri-food waste significantly influences the efficiency and yield of VFA and subsequent PHA production. According to [PERSON] et al. [66], the type and proportion of organic substrates present in the feedstock directly affect the metabolic pathways of microbial communities involved in fermentation. For instance, carbohydrate-rich wastes generally promote the formation of acetic and propionic acids, whereas lipid- and protein-rich residues favor the accumulation of butyric and valeric acids. Additionally, process parameters such as pH, retention time, and the presence of specific microbial consortia further modulate the VFA profile, ultimately impacting the composition of PHAs, including the proportion of HB and HV monomers in PHBV. The study highlights the importance of substrate selection and process optimization to achieve tailored VFA compositions, which can enhance the mechanical and thermal properties of PHBV bioplastics, ensuring their suitability for various industrial applications. Conversely, the VSS concentration remained stable (Table 3), although an increase in COD consumption was observed. The COD consumption rate progressively increased from 10.10 mg COD/L-min to 19.60 mg COD/L-min by cycle 133, suggesting improved microbial metabolic activity. However, the study did not explore the upper limit of OLR that the MMC could tolerate. Higher OLRs could enhance productivity but may also increase the risk of system overload, requiring further optimization [67]. [PERSON] et al. [42] studied PHA production with MMC using SBR. They reported COD consumption rates ranging from 21.8 to 33.45 mg COD/L-min.