# Urban Biorefinery Demonstration: Production of Polyhydroxyalkanoates from a Municipal Solid Waste
## 2 Materials and Methods  
### Enrichment Fermentation  
The MMC enrichment in scl-PHA accumulating bacteria was carried out in a 20 m\({}^{3}\) fermenter (Figure 1a, FS-20000 series, Bionet, Murcia, Spain) with a working volume of 15 m\({}^{3}\). The fermenter consists of the following main parts: fermenter reactor, stirring system able to reach up to 50 rpm (shaft with Rushton-type blades), vent condenser, instrumentation (transmitters for temperature, pressure, pH, and dissolved oxygen, with an air flow meter and upper-level switch for foams) and control system by the control unit. Process air was used as the auxiliary service with a capacity to reach up to 3600 Nm\({}^{3}\)/h. The auxiliary service is also equipped with a flowmeter, proportional control valve, and automatic on/off valve).  
Table 1 shows the operational conditions employed to grow PHA-accumulating MMC at the enrichment stage.  
The inoculum (50% _v/v_) was a mixture of aerobic sludge from the wastewater treatment plant of the municipality of Puertollano, Spain. The MMC was monitored at the end of each F/F cycle by means of total suspended solids (TSS), volatile suspended solids (VSS), pH, and chemical oxygen demand (COD). Oxygen saturation (pO\({}_{2}\)) was also monitored but during the whole F/F cycle. Steady conditions were considered to be reached at the enrichment fermenter once the TSS and VSS were stabilized, and the least/famine (F/F) ratio was lower than 0.2 h/h [39].  
The carbon source employed was a solution of VFAs. This solution, rich in VFAs, is the result of the pretreatment of the Organic Fraction of Municipal Solid Waste (OFMSW) selectively collected by URBASER in the municipality of Zaragoza, Spain in anaerobic  
\begin{table}
\begin{tabular}{c c} \hline \hline
**Enrichment** & \\ \hline Working volume (L) & 15,000 \\ HRT (d) & 5 \\ Cycle length (h) & 24 \\ Feeding period (min) & 30 \\ OLR (gCOD/L. d) & 1 \\ Aeration (m\({}^{3}\)/h) & 781 \\ Stirring (rpm) & 50 \\ pH & No control \\ Temperature & No control \\ \hline \hline \end{tabular}
\end{table}
Table 1: Operational condition of the enrichment fermenter.  
Figure 1: Fermenters of 20 (**a**) and 3 (**b**) m\({}^{3}\) employed for enrichment and accumulation, respectively.  
digestion (AD) [57]. Table 2 summarizes the average analytical results of the VFA-rich solution used as the carbon source.  
Prior to use, the VFA-rich digestate was clarified in a Alfa Laval Clara 80 centrifugation system (Alfa Laval AB, Lund, Sweden) to eliminate any solid material that could interfere with the subsequent PHA transformation process. Thus, TSS and VSS were reduced after clarification to a final concentration of 1.34 \(\pm\) 0.10 and 1.1 \(\pm\) 0.10, respectively. Nevertheless, the average daily VFA feeding composition to the MMC was as follows; acetic acid (5.60 g/L), propionic acid (1.37 g/L), isobutyric acid (0.33 g/L), butyric acid (11 g/L), isovaleric acid (1.12 g/L), valeric acid (1.60 g/L), hexanoic acid (10 g/L), and heptanoic acid (0.70 g/L). The soluble COD of the VFA solution was 47 \(\pm\) 7.61 g/L. Water dilution was needed to reduce the high VFA concentration of the original VFA-rich digestate solution. Thus, OLR was set to be 1 g COD per L of fermenter and day.  
During the commissioning of the bioprocess, OLR was initially set at 0.5 COD/L.d (cycles 1 to 13) and gradually increased. Therefore, OLR was set at 0.7 for cycles 14 to 21), at 0.9 for cycles 22 to 50, and finally it was set at 1 g COD/L.d for cycles 51 to 214. The C:N:P molar ratio in the medium was fixed at 1:0.15:0.05 adding NH\({}_{4}\)Cl and K\({}_{2}\)HPO\({}_{4}\). Allythiourea (10-15 mg/L) [58] was also supplied during the first 3 HRTs to avoid nitrifier bacteria growth and Antifoam 204 (Sigma, Saint Louis, MO, USA) was employed when foam formation was observed.