## 2 Materials and Methods
### MMC Enrichment in PHA-Accumulating Organisms  
The PHA-accumulating cultures selection was accomplished in a 0.8 L working volume sequencing batch reactor (\(SBR\)), inoculated with a PHA-accumulating MMC acclimatised to fCW, and operated under similar conditions to those of the reactor, from which the inoculum was harvested [15]. Briefly, the SBR cycle length was 12 h, comprising four discrete phases, influent filling (5 min), aerobiosis (680 min), settling (20 min), and supernatant withdrawal (15 min). The HRT was kept at 1 day, and an OLR of 40 Cmmol L\({}^{-1}\cdot\)d\({}^{-1}\) was applied. The reactor was operated in a temperature-controlled room (23-25 \({}^{\circ}\)C). The SRT was kept at 4 days by purging 125 mL of mixed liquor at the end of each aeration phase (end of famine). Allythiourea was always added to the diluting mineral solution (10 mg\(\cdot\)L\({}^{-1}\)) to inhibit nitrification. Air was supplied through a fine bubble diffuser at an aeration rate of 1 vvm (1 L\(\cdot\)L\({}^{-1}\cdot\)min\({}^{-1}\)), controlled by a mass flow controller (GFC17A, Aalborg). Mixing was kept at 300 rpm during the influent filling and the aerobiosis phases. Proteins present in the fCW were shown to start precipitating at a pH higher than 8.8 (data not shown), thus, whenever the pH reached a value above 8.8, an automatic dosing of 1 M HCl was performed. The pH ranged from 8.0 to 8.8. Pumping (fill and withdraw), aeration, and mixing were automatically controlled by a software program developed within the research group, which also permitted online pH and dissolved oxygen concentration (DO) data acquisition, allowing the online monitoring of the biological process.  
In order to assess the possibility of not supplementing nitrogen in the selection reactor, the SBR was initially operated under a \"reference operation conditions\" (Period I), being supplemented in a similar manner as reported by [PERSON] et al. [15], i.e., a mineral solution of NH\({}_{4}\)Cl was added resulting in a supplemented C/N ratio of 100/10 (without taking into account the N present in the fCW, Table 2).  
\begin{table}
\begin{tabular}{l l} \hline \hline
**Collected fermented cheese whey** & \\ \hline
**Volume** & \\ (L) & 20 \\ \hline
**Time of AnMBR operation** & \\ (d) & 3 \\ \hline
**Substrates concentration** & \\ (Cmmol-HOrgs-L\({}^{-1}\)) & 128 (9) \\ (gCOD-HOrgs-L\({}^{-1}\)) & 4.6 (0.3) \\ \hline
**HOrgs** [HAc/HBu/HVa/HPr/HLa] & \\ (Cmol\%) & 46(1)/44(1)/5(1)/4(1)/0.3(0.2) \\ (gCOD\%) & 41(1)/49(1)/6(1)/4(1)/0.3(0.2) \\ \hline
**Ethanol concentration** & \\ (Cmmol-L\({}^{-1}\)) & 2.9 (0.4) \\ (gCOD-L\({}^{-1}\)) & 0.37 (0.05) \\ \hline
**Proteins concentration** & \\ (gProt-L\({}^{-1}\)) & 1.3 (0.3) \\ \hline
**Total-N concentration** & \\ (Nmmol-L\({}^{-1}\)) & 6 \\ \hline
**Ammonia-N concentration** & \\ (Nmmol-L\({}^{-1}\)) & 0.78 (0.11) \\ \hline
**Total-P** & \\ (Pmmol-L\({}^{-1}\)) & 3.1 (0.8) \\ \hline
**Phosphate phosphorus** & \\ (Pmmol-L\({}^{-1}\)) & 0.89 \\ \hline
**C/N/P ratio** & \\ (Cmol/Nmol/Pmol) & 100/5/3 \\ \hline \hline \end{tabular} The listed values are averages and associated standard errors of the mean (SEM), in brackets.  
\end{table}
Table 1: _Cont._The SBR was stably-operated for sixteen days, under the reference conditions. In the subsequent operational periods, the nutrients supplementation was gradually decreased by reducing the NH4 Cl concentration in the mineral solution. fCW with the organic acids (HOrgs) as carbon substrates, was diluted in a constant volume of a mineral solution, with varying concentrations of nitrogen (NH4 Cl), to impose the different C/N ratios, which were, to a half in Period II; to a quarter in Period III; to an eighth in Period IV; and finally in Period IV the SBR was operated with no external nutrients supplementation. The phosphorus present in the fCW, originated a stable ratio of 100/3 Cmol/Pmol, throughout the SBR operation.