## 4 Discussion  
The storage capacity of the cultures, evaluated by the PHA storage yield, tended to decrease progressively, as the nutrient supplementation was reduced (Table 3). In fact, when supplementation was reduced by half (from Period I to II), the storage yield had not significantly changed, which seems to indicate the accumulating organisms were not limited in nutrients, therefore they still dominated the microbial community. However, on the following reductions of nutrients supplementation (Periods III and IV), the PHA storage-yield decreased, while the protein consumption increased (\(\Delta\)Prot, Table 3). Indeed, there was an inverse linear correlation between the PHA storage-yield and the specific protein consumption (\(-\)q\({}_{\text{Prot}}\), Table 3). An escalation of events might have caused this. The decrease in an easily bioavailable nitrogen increased the reliance on proteins-nitrogen, and the consequent protein uptake. This was a slower process [34] which required more energy, increasing the least period (decreasing the selective pressure towards PHA accumulation), on one hand, and routing more carbon to be utilised in the catabolism instead of the anabolism, on the other hand, thus, reducing the PHA yield. Another possibility might be that the PHA-accumulating organisms, lacking the required enzymatic mechanisms, were unable to use the proteins as a nitrogen source, thus becoming growth-limited by nitrogen, and consequently, outcompeted by the non-accumulating organisms capable of using the proteins-nitrogen. The reported observations were supported by the relation between the specific PHA storage rate and the specific ammonia consumption (Figure 5), which additionally pointed to a minimum amount of ammonia-availability (ca. 8 Nmol-NH\({}_{3}\)-Cmol-X\({}^{-1}\)), from which the storage rate drastically decreased. Indeed, the decrease in the PHA-storing efficiency when the selection reactor was supplied with lower amounts of nutrients, is in accordance with the findings of [PERSON] et al. [5] who used sugar can molasses, a nutrients deficient feedstock. In the referred work, it was shown that the insufficient supply of a nitrogen source, in the selection reactor, led to a decrease in the PHA-storage capability of the selected MMC, over time. The loss of selectivity for the PHA-accumulating organisms might also be confirmed by the fractions of ammonia consumed, which were related to the biomass growth, during the least and famine periods. In a culture efficiently selected on the PHA-accumulating organisms, biomass growth occurred mainly during the famine phase of the FF cycle, at the expense of a stored polymer. Biomass growth, in the SBR cycles, could be indirectly monitored through the ammonia consumption, since nitrification was inhibited through the addition of allythiourea. The fraction of ammonia used during  
Figure 5: Relation between the specific PHA storage rate (\(q\)_PHA_) and the specific ammonia–nitrogen consumption (\(\Delta\)_NH\({}_{3}\)-N/X_), during the different periods (I–V). the least increased significantly as nitrogen supplementation was reduced (Period I to Period V) (Figure 6), particularly, after Period III, when the least phase became longer, suggesting an increase in growth on the external carbon, rather than in storage. These results confirm that the selective pressure for PHA-accumulating organisms was progressively lost from Period II to V. This decrease in the selective pressure could possibly be related to the significant decrease of the biomass concentration (Figure 1). Ultimately, the selection of the PHA-accumulating organisms, under the conditions tested in this study, was strongly affected by limiting the addition of nutrients.