## 3 Results and Discussion
### Copolymer PHBV Production from Acetic Acid and Valeric Acid  
#### 3.2.1 Effect of Initial Acid Concentration and Composition  
For the production of copolymers such PHBV, addition of a co-substrate is required. In this study, valeric acid was chosen as a precursor for 3 HV units rather than propionic acid as a higher HV fraction can be obtained [36; 37; 38]. 3 HV synthesis from valeric acid does not involve the catabolism of the acid to  
Figure 2: Fed-batch fermentation of _C. necator_ for PHB production using acetic acid as carbon source. The accumulation of biomass (CDM), PHB, residual cell concentration (RCC) and acetic acid concentration over time of fermentation are shown. (**a**) Two-phase feeding strategy; (**b**) Three-phase feeding strategy. a shorter alkyl-CoA like this is the case for propionic acid, but rather its direct incorporation into the polymer via valeryl-CoA and its oxidation to 3-hydroxyvaleryl-CoA. Furthermore, decarboxylation of propionyl-CoA and loss of units with an odd number of carbons is reduced, but not totally eliminated, as the intermediary 3-hydroxyvaleryl-CoA can be degraded to propionyl-CoA and acetyl-CoA, which can be used for PHB synthesis [15; 36; 37; 39]. To determine the influence of the total acid concentration and the ratio of acetic acid to valeric acid on PHBV production of _C. necator_, a series of shake flask experiments were conducted which differed in the relative ratio of acetic and valeric acid added while keeping the total acid concentration constant at 3 and 6 g/L. The fraction of 3 HV in the resulting PHBV copolymer was dependent on the ratio of acetic acid to valeric acid and not on the total acid concentration (Figure 3). Indeed, similar 3 HB and 3 HV fractions were obtained at an acetic acid to valeric acid ratio of 1:1, irrespective of the total acid concentration. Furthermore, _C. necator_ favoured the consumption of valeric acid compared to acetic acid at ratio 1:1, while at higher concentrations of acetic acid, the opposite was observed. PHB production was also affected by total acid concentration (Table 2). As the objective of the study is to valorise CO\({}_{2}\) towards biopolymers via the production of acetic acid, it was decided to further proceed with the research using an acetic to valeric acid ratio of 2:1. In a search for inexpensive waste substrates to decrease PHA production costs, the use of organic acids--with acetic, propionic and butyric acid as the major fermentation products--recovered from anaerobically treated waste streams has been explored for PHA production by pure cultures [4; 24; 40; 41]. These studies acknowledged the toxic effect of short-chain volatile fatty acids. Furthermore, our results are in line with the literature results. [PERSON] and [PERSON] observed a decrease in the amount of biomass formed by _C. necator_ NRRL B 14690 when increasing the initial valeric acid concentration from 2 to 4 g/L in the fermentation broth [38]. [PERSON] and [PERSON] reported valeric acid concentrations above 2 g/L as toxic for _C. necator_ B-5786 [42]. Several authors observed the favoured consumption of longer organic acids over acetic acid for cell growth. However, [PERSON] et al. and [PERSON] et al. recently reported an antagonism in metabolism for acetic, propionic and butyric acid, which led to preferential consumption of butyric acid and propionic acid, depending on carbon fluxes directed to growth, as compared to fluxes going to PHA production [4; 26]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Total Acid Concentration (g/L)** & **C2:C5 Ratio** & **Acetic Acid (g/L/h)** & **Valeric Acid (g/L/h)** \\ \hline
3 & 1:1 & 0.047 & 0.108 \\
3 & 2:1 & 0.156 & 0.129 \\
3 & 4:1 & 0.243 & 0.074 \\
3 & 1:2 & 0.048 & 0.158 \\
6 & 1:1 & 0.095 & 0.151 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Consumption rate of acetic and valeric acid during PHB production of _C. necator_ in shake flask. Figure 3: Effect of ratio acetic to valeric acid on PHBV content of _C. necator_ in shake flask after 24 h of incubation.