## 3 Results and Discussion
### Copolymer PHBV Production from Acetic Acid and Valeric Acid
#### 3.2.2 Evaluation of Three-Phase Fed-Batch Culture  
Since including an intermediate phase consisting of both biomass and PHB production outperformed the overall process performance for PHB production, the three-phase feeding strategy was also selected for PHBV production. The overall results are shown in Figure 4. During the biomass growth phase (from 0 to 29 h), cell mass concentration increased exponentially to 18 g/L with a PHB content of 10%. In the second phase (from 29 to 46 h), accumulation of 3 HV units started in the polymer chain as a result of the valeric acid cofeeding. A PHBV concentration and content of 22 g/L and 58%, respectively, was obtained. Equal amounts of 3 HB and 3 HV were synthesised, resulting in a 3 HV fraction of 50 mol%. During the last phase (from 46 to 119 h), PHBV percentage increased up to 78%. Although both acetic and valeric acid were fed as carbon source, biopolymer production was almost fully attributed to 3 HB synthesis. Indeed, as observed during the shake flask experiments, _C. necator_ favoured consumption of acetic acid over valeric acid during PHA production. A total 3 HB concentration of 35 g/L was obtained, while the 3 HV concentration amounted to 13 g/L, corresponding to a 3 HV fraction of 27 mol%. The final biomass and PHBV concentration were 65 g/L and 48 g/L, respectively. The PHBV productivity was 0.413 g/L/h. Acetic and valeric acid were maintained at low concentrations over the duration of the process (results not shown).  
Fed-batch cultivation for the production of PHBV by _C. necator_ has been investigated previously. Most studies have focused on propionic acid as a co-substrate due to its lower cost compared to valeric acid. However, in most of the cultivations, the 3 HV fraction in the copolymer was in the range of 5-30%. The superior convertibility of valeric acid to 3 HV units has been demonstrated in a few reports with carbohydrates as the main carbon source. [PERSON] et al. obtained, after 50 h, a high cell density culture of 120 g/L with glucose and valeric acid containing 75% of PHBV with a maximum 3 HV fraction of 20 mol% [36]. In another study, batch cultivation from fructose with one pulse feeding of 6 g/L valeric acid after 20 h resulted in the production of PHA containing a maximum of 54% HV units after 40 h [38]. However, at the end of the fermentation, 15 g/L CDM was obtained consisting of 50% PHBV with a 3 HV fraction of 33 mol%. Comparable maximum and final HV fraction were obtained in this study.  
Furthermore, the type of carbon source in the presence of valeric acid seems to affect PHBV monomer distribution. In the study by [PERSON] and [PERSON], a slight decrease of 3 HB concentration was observed with the initiation of the valeric acid pulse in the presence of fructose. The incorporation of 3 HB units in the polymeric chain started increasing again after the exhaustion of valeric acid,  
Figure 4: Fed-batch fermentation of _C. necator_ for PHBV production using acetic acid and valeric acid as carbon source. The accumulation of biomass (CDM), PHB, residual cell concentration (RCC), acetic acid and valeric acid concentration over time of fermentation are shown. A three-phase feeding strategy was applied.  
indicating the preferential consumption of valeric acid over fructose for _C. necator_. As a result, a block copolymer was most likely produced, consisting of 3 HB (from acetic acid) and 3 HV (from valeric acid) blocks of repeating units. In this study, simultaneous production of the monomers occurred during phase 2, which is indicative for the production of random PHBV [10]. Moreover, results showed that _C. necator_ favoured consumption of valeric acid over acetic acid during growth, while during PHA production, consumption of acetic acid was preferential. The aforementioned antagonism in metabolism (see Section 3.2.1) [4; 26] could thus be extended to acetic and valeric acid. By optimising the ratio of acetic to valeric acid in the feed during the last phase, the 3 HV fraction can probably be increased.