## 5 Conclusions  
The objective of this study was to select a PHA-storing culture using cheese whey proteins as the sole nutrients source. Although a progressive selection process was applied to allow a gradual acclimatisation to the limiting ammonia conditions, the selected PHA-producing culture was not achieved. This implies that under the applied conditions, PHA-accumulating organisms could not hydrolyse/assimilate complex nitrogen compounds. The lack of this ability created the proliferation of a flanking-population capable of assimilating protein-nitrogen and overcoming the long periods of famine, with relation to HOrgs, in all probability, by also using protein-carbon.  
In conclusion, feedstocks limited in easily bioavailable nitrogen, as cheese whey-proteins, might require additional treatment steps which allow the partial hydrolysis of the nutrients source, or require nutrients supplementation for the efficient selection of PHA-producing cultures. Nevertheless, the cost-benefits analysis of having an additional step for protein hydrolysis or using a cheap external nitrogen source (e.g., urea), as well as the environmental impact of both strategies, need to be evaluated.  
Figure 6: Average fractions of ammonia–nitrogen (N–NH\({}_{3}\)) consumption in the least (light grey) and famine (dark grey) phases of the selected SBR, during the five different operational periods (I–V). The consumption percentages are based on the total consumed ammonia–nitrogen, during the entire cycle.  
**Supplementary Materials:** The following are available online at [[http://www.mdpic.com/2076-3417/8/10/1817/s1](http://www.mdpic.com/2076-3417/8/10/1817/s1)]([http://www.mdpic.com/2076-3417/8/10/1817/s1](http://www.mdpic.com/2076-3417/8/10/1817/s1)), Figure S1: Nile Blue staining (left) and bright field (right) images of biomass samples collected on Period I (a-d) and Period V (e-h) of operation. Stained cells (Nile Blue-positive) indicate the presence of intracellular PHA.  
**Author Contributions:** C.S.S.O. (Conceptualization, Methodology, Validation, Formal Analysis, Writing-Original Draft Preparation, Writing-Review & Editing, Visualization, Supervision); M.O.D.S. (Investigation, Data Curation, Formal Analysis); C.E.S. (Investigation); G.C. (Methodology, Writing-Original Draft Preparation, Writing-Review & Editing); M.A.M.R. (Conceptualization, Resources, Writing-Review & Editing, Project Administration, Funding Acquisition).  
**Funding:** The research leading to these results has received funding from the European Union's Horizon 2020 research and innovation programme through the YPACK project (grant agreement No. 773872). This work was further supported by the _Unidade de Ciencias Biomouclares Aplicadas_-UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-00772). [PERSON] further acknowledges _Fundacio para a Ciencia e a Tecnologia_-FCT for the financial support through the grant SFRH/BPD/88817/2012.  
**Acknowledgments:** The authors acknowledge Bel Portugal for supplying the sweet whey powder. The authors further acknowledge [PERSON] for the help with biomass samples preparation for FISH analysis, and [PERSON] for the help with samples preparation for GC analysis.  
**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.