## 2 Materials and Methods
### Analytical Procedures  
The SBR's operation was followed through the online monitoring of the DO and the pH response, along all cycles, allowing to assess the least-phase length. Additionally, at least once a week, an entire cycle was closely monitored by collecting samples of the mixed liquor, along the cycle, in order to assess the substrates consumption, biomass growth, and the PHA production. Furthermore, when fast transient periods were expected after a perturbation was induced in the reactor, samples for biomass concentration determination were collected, each for 2-3 days. During the PHA accumulation fed-batch tests, samples were collected periodically during all the pulses in order to assess the substrates consumption and the PHA production. Biomass concentration, i.e., cells (X) plus PHA, was estimated as volatile suspended solids (VSS) concentration, determined by filtration, according to the standard methods [19]. Organic acids and ethanol were quantified in filtered samples (0.45 \(\upmu\)m) through high-performance liquid chromatography (HPLC), using a Merck-Hitachi chromatographer, equipped with an RI detector \begin{table}
\begin{tabular}{l c c c c c} \hline
**Periods** & **I** & **II** & **III** & **IV** & **V** \\ \hline \multicolumn{6}{l}{**Operational Parameters**} \\ \hline
**OLR** & 42 (4) & 45 (1) & 44 (4) & 43 (1) & 38 (4) \\
**fCW C/N ratio** & & & & & \\ (Cmol/Nmol) & 100/5 & 100/5 & 100/5 & 100/5 \\
**Supplemented C/N** & & & & \\
**ratio** (Cmol/Nmol) & & & & & \\
**Final C/N ratio** & & & & \\ (Cmol/Nmol) & 100/15 & 100/10 & 100/7.5 & 100/6.25 & 100/5 \\ \hline \end{tabular} The listed values are averages and associated standard errors of the mean (SEM), in brackets.  
\end{table} 
Table 2: Summary of the operational parameters applied to the selection SBR on the five different periods. and Agilent MetaCarb 87H pre-column and column (column temperature 30 \({}^{\circ}\)C, 0.001 M H\({}_{2}\)SO\({}_{4}\) eluent, flow rate 0.5 mL\(\cdot\)min\({}^{-1}\)). The HOrgs concentrations were calculated from the standard calibration curves (25-1000 mg\(\cdot\)L\({}^{-1}\) of each organic acid). PHAs were quantified by gas chromatography (GC) using a method adapted from [PERSON] et al. [18]. Briefly, a defined amount of lyophilised biomass was incubated for methanolysis, in a 1 mL acidic methanol (20% H\({}_{2}\)SO\({}_{4}\)) and extracted with chloroform (1 mL). The mixture was digested at 100 \({}^{\circ}\)C for 3.5 h. After the digestion step, the organic phase (methylated monomers dissolved in chloroform) was extracted and injected (2 \(\mu\)L) into a gas chromatograph, equipped with a flame ionization detector (Bruker 430-GC) and a BR-SWax column (60 m, 0.53 mm internal diameter, 1 mm film thickness, Bruker, Billerica, MA, USA), using helium as carrier gas, at 1.0 mL\(\cdot\)min\({}^{-1}\). The temperature regime started at 40 \({}^{\circ}\)C, increased to 100 \({}^{\circ}\)C at a rate of 20 \({}^{\circ}\)C\(\cdot\)min\({}^{-1}\), to 175 \({}^{\circ}\)C at a rate of 3 \({}^{\circ}\)C\(\cdot\)min\({}^{-1}\), and reached a final temperature of 220 \({}^{\circ}\)C at a rate of 20 \({}^{\circ}\)C\(\cdot\)min\({}^{-1}\), ensuring cleaning of the column after each injection. Injector and detector temperatures were 280 \({}^{\circ}\)C and 230 \({}^{\circ}\)C, respectively. Through the obtained chromatograms, it was confirmed that the PHA in the samples was solely constituted by PHBV polyhydroxybutyrate-hydroxyvalerate (PHBV). 3-HB and 3-HV monomers concentrations were determined through the use of two calibration curves (0.1-10 g\(\cdot\)L\({}^{-1}\)), one for each compound, using standards of a commercial PHBV (88/12 wt.HB/HV%, Sigma, St. Louis, MO, USA), and corrected using heptadecane as an internal standard (concentration of approximately 1 g\(\cdot\)L\({}^{-1}\)). Protein content in filtered samples (0.45 \(\upmu\)m) was determined spectrophotometrically, at 750 nm, by the alkaline copper method, as described by [PERSON] et al. [20].