## 3 Results and Discussion
### Homopolymer PHB Production from Acetic Acid
#### 3.1.2 Evaluation of Two-Phase and Three-Phase Fed-Batch Culture  
Different feeding strategies have been utilised for the production of PHA, including continuous feeding, exponential feeding, pH stat, DO stat, online measurement of substrate concentration, CO\({}_{2}\) evolution rate or alkali-addition monitoring coupled to a controlled feeding of carbon source [16]. Recently, [PERSON] et al. developed a new feeding strategy for organic acid fermentation by another _C. necator_ strain, using a pH-stat, feeding organic acids to maintain the pH, in combination with a DO-dependent feed (DO-stat) delivering organic acid salts [4]. The latter feeding strategy led to the best process performance for PHA production from a mixture of organic acids. Therefore, this feeding strategy was selected for the current study. Two production routes were evaluated in terms of process performance using the feeding strategy adopted by [PERSON] et al. [4]. The first process (i.e., two-phase fed-batch culture) consisted of biomass growth followed by PHB production induced by changing the pH-stat feed with a C/N = 10 to an acid feed solution without nitrogen (C/N = \(\infty\)). The second one (i.e., three-phase fed-batch culture) contained an intermediate phase to enable a low residual biomass growth rate for maintaining an active metabolism and increasing PHB productivity. This intermediate phase was induced by substituting the initial pH-stat solution with a solution with a high C/N (C/N = 90). The results of the two-phase fed-batch culture show that during the biomass growth phase (phase 1), a biomass concentration of 15 g/L was attained after 30 h that contained 10% PHB (Figure 2a). The average specific growth rate \(\mu\) was calculated as 0.152 L/h. After 30 h, nitrogen limitation was initiated to trigger PHB biosynthesis. After a short time, nitrogen in the culture broth was depleted, thereby triggering PHB synthesis (phase 2). The maximal biomass (DCM) and PHB concentrations were 40 g/L and 30 g/L, respectively, after 119 h, resulting in a PHB content of 75% and a PHB productivity of 0.257 g/L/h. Figure 2b shows the results of the three-phase fed-batch cultivation process. Since the main objective was the comparison of the key performance parameters of different PHA production processes using CO\({}_{2}\) as a feedstock, rather than to fully optimise the fermentation process in terms of PHA productivity, content and concentration, the first phase (from 0 to 30 h) was performed under conditions identical to the first stage of the two-phase fed-batch culture. Biomass was produced (16 g/L) with low PHA biosynthesis levels (8%). During the second phase (from 30 to 53 h), nitrogen availability was limited, and PHB concentration and content increased to 26 g/L and 61%, respectively. Finally, the third phase (from 53 to 118 h) allowed for the further increase of PHB percentage of the cells by stopping growth. The final biomass and PHB concentration were 60 g/L and 43 g/L, respectively. An overall PHB content and productivity of 72% and 0.365 g/L h, respectively, was attained. In both cases, acetic acid concentration was maintained below its minimal inhibitory value (3 g/L) over the duration of the process, and the applied feeding strategy was highly reproducible for the first phase (i.e., biomass production). Integrating an intermediary phase consisting of both biomass and PHB production outperformed the overall process performance in terms of biomass concentration, PHB concentration and PHB productivity. This finding is in accordance with [PERSON] et al., in which a fed-batch fermentation strategy was developed to overcome the toxic influence of organic acids as sole carbon source in high cell density cultures of another _C. necator_ strain H16 [4]. The higher anabolic activity of the cells during their exponential growth might lead to faster PHB production rates, since more reducing power (NADPH) is produced and the maximal specific PHB production rate is defined by the maximum specific rate of NADPH produced [26].