# Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste
## 4 Discussion
### Enrichment of scl-PHA-Accumulating Microorganisms
The higher consumption rates were typically achieved with shorter cycle lengths (2 to 6 h) and increased feeding frequencies (4 to 12 times per day), demonstrating the influence of operational strategies on COD removal efficiency. In contrast, the present study that was conducted under real operational conditions, i.e., without synthetic VFA addition, recorded a lower consumption rate (19.56 mg COD/L.min), suggesting that feed composition and cycle duration significantly impact system performance. Temperature control has been identified as a key factor in optimizing PHA production by several authors [42,44]. The studies have shown that optimal temperatures (25-30 \({}^{\circ}\)C) enhance microbial activity, substrate uptake, and PHA accumulation. While previous research demonstrated improved performance under controlled temperature conditions [42, 43, 44], maintaining such temperature control at an industrial scale is energy-intensive, and as a result, it increases the cost of the final bioproduct. The observed microbial adaptation to environmental fluctuations in this study suggests that implementing modifications in other process variables, rather than strict temperature control, could be a more viable strategy for large-scale applications. The enhancement of the process's robustness and industrial scalability must be the center of future research. Thus, prioritizing the mitigation of temperature fluctuations should be examined by developing more efficient process control strategies, such as optimizing organic loading rates (OLR) and/or conducting comprehensive microbial community analyses. The implementation of precise environmental regulations, refining substrate feeding regimes, and leveraging advanced bioprocess monitoring techniques will be crucial for improving system stability and ensuring consistent biopolymer yields in large-scale operations. While temperature control may not be feasible in large-scale operations with MMC, adaptive strategies such as adjusting HRT or modifying feeding cycles during colder months could also be helpful in mitigating microbial activity loss. Correspondingly, further research should evaluate increasing OLRs beyond 19.6 mg COD/L-min to determine the threshold at which microbial performance is maximized without causing system instability. A more comprehensive study on microbial population dynamics during the enrichment phase may provide feedback on identifying key species responsible for scl-PHA accumulation and environmental stressors. Moreover, rather than implementing comprehensive pH and temperature control, partial regulation methodologies, such as passive insulation for temperature stabilization or intermittent buffering for pH control, could be further investigated to ensure economic and technical feasibility. Regarding pH control in the process, Figure 2 shows how the pH profile remained relatively stable throughout the enrichment cycles, fluctuating around 8-9 with minor variations. This stability suggests that the lack of pH control did not compromise microbial selection or PHA accumulation. The observed pH range aligns with the literature reports [42; 43; 44] indicating optimal conditions for PHA-accumulating bacteria (6.5-8.0), preventing the proliferation of non-accumulating species. A slight pH decreased around cycles 150-180 coincided with fluctuations in TSS and VSS, suggesting that environmental factors, such as temperature, may influence process stability. However, COD removal remained consistent, supporting the feasibility of operating without pH control in an industrial scenario. Despite its limitations, this study demonstrates that PHA-accumulating MMC can be enriched under real operational conditions without strict process control. However, ensuring consistency in biomass concentration and microbial performance remains a challenge.