## 3 Results and Discussion  
### Homopolymer PHB Production from Acetic Acid  
#### 3.1.1 Effect of Initial Acetic Acid Concentration  
Organic acids such as acetic acid exhibit a toxic effect on cell physiology when present above a certain concentration threshold. To determine the minimal inhibitory concentration of acetic acid on biomass growth of _C. necator_, a series of shake flask experiments were conducted which differed in the amount of acetic acid added. Figure 1 shows the effect of the initial acetic acid concentration on the cell growth of _C. necator_ in the shake flasks. The optimum concentration of acetic acid for cell growth was in the range of 3 g/L. The inhibitory effect of acetate on _C. necator_ became significant at a concentration above 3 g/L, as evidenced by the limited biomass growth and acetic acid consumption after 24 h of incubation.  
These observations are in accordance with previous studies [17; 18]. In these reports, a decline in specific growth rate of _C. necator_ under nutrient-rich conditions was observed with increase in acetate concentration following an exponential function. An inhibitory effect of acetic acid above 3 g/L at lower cellular density was reported and no growth was observed at a concentration of 6 g/L or above. The use of a high cell concentration, however, significantly increased the acetate tolerance of _C. necator_. [PERSON] et al., however, reported an optimum concentration of acetate for cell growth of 1 g/L and serious inhibition of growth by slight increase in acetate concentration [19]. Compared to [PERSON] and [PERSON] [17; 18] and our study, these authors used another _C. necator_ strain.  
The toxicity of carboxylates can be attributed to their undissociated lipophilic molecules that freely penetrate the cell membrane, dissociate and acidify the cytoplasm [20]. As a result, the gradient of protons through the membrane cannot be maintained, and the production of energy and the transport system dependent on this gradient are decoupled [21]. The dissociation also induces an anion accumulation, resulting in increased internal osmotic pressure of cells [22]. In response to the accumulation of fatty acids, microorganisms release free energy via ATPase and expel protons out of the cells in order to maintain the proton gradient. This results in an overall decline of microbial activity including reduced acid utilisation rate, growth rate and yield [23; 24]. At neutral pH, the acid is present in a dissociated form, and the passage through the cellular membrane is limited. Carboxylic acid anions affect cell growth in a variety of manners. Increased anion concentration has been shown to lead to an increased transport of potassium ions into the cell, which increases turgor pressure. To maintain a constant turgor pressure and cell volume, glutamate is transported out of the cell. This transport activity concomitantly disrupts the osmolarity of the cytoplasm, which in turn lowers the cell's growth potential and viability. In addition to this general anion effect, there are also effects specific to each carboxylic acid [25].  
Figure 1: Effect of the initial acetic acid concentration on the cell growth of _C. necator_ in shake flask after 24 h of incubation. Results are shown as average \(\pm\) standard deviation.