# Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste
## 4 Discussion
### Accumulation of scl-PHA in Microorganisms
On the other hand, in this second sequence of experiments, several accumulations were performed by applying a COD feeding rate of 81.5 mg COD/L.min (accumulation batches from n\({}^{\circ}\) 32 to n\({}^{\circ}\) 37), obtaining an average scl-PHA in the biomass of 43.95 \(\pm\) 10.14 wt.%, in which two reached almost 60 wt.% of scl-PHA within the cells. However, higher COD feeding rates by applying 81.5 mg COD/L.min did not improve the scl-PHA storage by the cells (accumulation batches n\({}^{\circ}\) 38 and n\({}^{\circ}\) 39). These values agree with the ones obtained in other studies at lower scales (40-50 wt.%) [25; 48]. Thus, the scl-PHA production process upscaling has demonstrated to be technically feasible. However, further studies should explore a broader range of feeding rates to define the most effective balance between substrate availability, microbial growth, and PHA storage efficiency. In this context, comparative studies have studied the impact of different operational conditions and carbon sources on PHA production efficiency. Among them, [PERSON]. [71] investigated the production of PHAs using VFAs derived from anaerobic digestion of municipal waste as a carbon source, employing a mixed microbial culture in SBR (5 L), achieving similar results than those obtained in this study. The research focuses on utilizing a mixed microbial culture enriched through a feast/amine strategy to maximize PHA accumulation. The fermentation process was based on a VFA mixture, (primarily rich in hexanoic acid, 29.5 g/L). The maximum biomass concentration achieved during the production phase was 31.4% of VSS, which was lower than the maximum accumulation obtained in the present work. Regarding the PHA composition, a very similar ratio was achieved, 97.1% and 2.9% of PHB and PHV, respectively. On the one hand, [PERSON] et al. [72] were able to reach a laboratory scale of 2.4 g/L of scl-PHA in a mixed culture, where synthetic VFAs were used as a carbon source. In addition, a similar scl-PHA accumulation percentage was obtained by [PERSON] et al. [73] at pilot plant scale (120 L for enrichment fermentation) from a combined treatment of OFMSW and sewage sludge (46 wt.%). On the other hand, [PERSON] et al. [43] reached up to 78 wt.% of scl-PHA in the biomass by using an SBR for the enrichment process of 2 L of OFMSW. [PERSON] et al. [74] explored the use of acidogenic fermentation of food waste to produce VFAs as a cost-effective carbon source for PHA synthesis. In their work, a pure culture was used, _Pseudomonas pseudoflara_, which was tested under varying carbon concentrations (4-55 g/L), achieving the highest PHA yield (52.5%) at 19 g/L and obtaining an average PHB and PHV composition of 97.1 and 2.9, respectively. Regarding the PHB/PHV composition of the obtained scl-PHA, a consistent PHB/PHV ratio was observed across most of the accumulation batches (Figure 7). The average PHB and PHV compositions were 85.67 \(\pm\) 6.09 and 13.16 \(\pm\) 6.09%, respectively. For instance, [PERSON] et al. [75] obtained a scl-PHA with a PHB/PHV ratio of 53/47% from OFMSW by using a mixed microbial culture. The enrichment and accumulation processes were carried out in 800 and 300 mL reactors, respectively. Even though, from the accumulation batches from n\({}^{\circ}\) 1 to n\({}^{\circ}\) 10 a remarkably higher presence of PHV than PHB was observed, in comparison with the accumulation batches from n\({}^{\circ}\) 11 to n\({}^{\circ}\) 39. This may be due to the influence of aeration in the composition of PHA [76]. Nevertheless, from the accumulation batches from n\({}^{\circ}\) 11 to n\({}^{\circ}\) 39 the PHA composition was very similar, except for accumulation batch 13 (PHB/PHV = 44/57%).