## Results
### Culture's Dynamic Performance
However, due to the biomass concentration decrease, it stabilised at a higher value than in the previous operational periods (0.35 \(\pm\) 0.02, corresponding to least periods of ca. 3 h). Even though a community capable of using proteins as a source of nutrients was selected, its PHA storage capacity was lower than that of cultures selected in the two initial operational periods. This was probably related to the lower selective pressure which it was subjected to (higher F/f). Indeed, lower amounts of PHA were produced (12 and 14 wt.%, in the selection reactor, and in the accumulation assay, respectively, Table 3), and lower, specific PHA-production rates were attained (0.07 and 0.11 Cmol-PHA\(\cdot\)Cmol-X\({}^{-1}\).h\({}^{-1}\) in the selection reactor and in the accumulation assay, respectively, Table 3). Reductions of ca. 30% in the storage yield (0.35 and 0.52 Cmol-PHA\(\cdot\)Cmol-HOrgs\({}^{-1}\) in the selection reactor and in the accumulation assay, respectively, Table 3) and of ca. 50 % in the productivity on the accumulation assay were achieved. Regardless of the change in PHA-accumulating performance of the culture, the monomer composition of the produced PHA was approximately the same, in average 91(1)/9(1) wt.%HB/wt.%HV (Table 3). When the nutrients supplementation was decreased from one-quarter to one-eighth of the reference, i.e., from Period III to IV, the F/f increased sharply (from 0.35 \(\pm\) 0.02 in Period III to 1.84 \(\pm\) 0.15, corresponding to least periods of ca. 8 h in Period IV). However, this increase was not related to the biomass diminution, as the biomass concentration just slightly decreased (from 1.3 \(\pm\) 0.1 to 1.0 \(\pm\) 0.1 gVSS\(\cdot\)L\({}^{-1}\) from Periods III to IV), reaching a value close to that estimated for the condition when total nitrogen supplied to the system was used for cellular growth (1.2 gVSS\(\cdot\)L\({}^{-1}\)). Thus, the selected culture in Period IV was able to successfully retrieve most of the nitrogen present in the proteins. Encouraged by this observation, it was decided to completely remove the nitrogen supplementation, after only 7 days of operating under the conditions of Period IV. This was done, even though, the storage capacity of the selected culture drastically decreased, with low amounts of PHA being produced during the selection cycles (5 wt.%), the storage yield decreased to 0.23 Cmol-PHA\(\cdot\)Cmol-HOrgs\({}^{-1}\), and the specific storage rate decreased to 0.05 Cmol-PHA\(\cdot\)Cmol-X\({}^{-1}\).h\({}^{-1}\), in the selected SBR (Table 3). Comparing the typical cycles of operation in Period I and Period IV (Figure 2), the loss of PHA-storing capacity of the selected culture becomes clear. In the cycle of Period I (Figure 2a), the culture presented the expected behaviour of a culture enriched in PHA-storing organisms. During the least phase, most carbon substrates were mainly channelled towards storage (biomass and ammonia concentrations were almost invariable, during the least phase, while PHA concentration increased), indicating that the microorganisms were growth-limited. Then, during the famine-phase cells grew at the expense of the stored PHA (during the famine phase a clear decrease of PHA and ammonia, and an increase in biomass concentration were observed). In the cycle of Period IV (Figure 2b), cellular-growth occurred to a significant extent, during the least phase (from 0.87 to 1.09 gVSS-L\({}^{-1}\)), possibly indicating that the famine phase was not long enough to induce a selective pressure for PHA-accumulating organisms. The resulting short famine phase, in the consecutive cycles, was thus not enough to discard the non-accumulating organisms from the selected culture. Additionally, the specific protein consumption rate and the total amount of proteins consumed during the SBR cycle in Period I were lower (6.2 mgProt-gX\({}^{-1}\).h\({}^{-1}\) and 27.7 mgProt, Table 3) than in Period IV (11.6 mgProt-gX\({}^{-1}\).h\({}^{-1}\) and 76.2 mgProt respectively, Table 3).