# Cocoa Bean Shell as Promising Feedstock for the Production of Poly(3-hydroxybutyrate) (PHB)
## 3 Results and Discussion
### Fermentation with B. firms
#### 3.2.2 Non-Centrifuged Medium  
The evolution of the concentration of PHB, total and reducing sugars, and bacterial growth during fermentation of the non-centrifuged hydrolysate (with CBS solids) is shown in Figure 4. As can be seen, the behaviour was completely different from the case with the centrifuged hydrolysate. During the first two days, a mild lag phase was observed that was accompanied by the moderate consumption of reducing sugars and the fast production of PHB. Then, a period of exponential growth was observed that extended until the 10 th day. The maximum specific rate of growth was calculated obtaining a value of 1.18 days\({}^{-1}\). This value is within the range given (0.2-2.1 days\({}^{-1}\)) when _B. firmus_ was used for aniline degradation [39]. During the exponential phase of growth, a fast consumption of sugars took place and the production of PHB continued more slowly achieving a maximum of PHB concentration of 20 g/L (107.5 mg PHB/g dry weight) after 8 days of fermentation. The simultaneous increase in both PHB concentration and biomass would confirm that PHB production is related in some way to the growth of _B. firmus_. In addition, PHB production ceased when bacterial growth approached the stationary phase. The consumption of sugars by the bacterium was much more noticeable in this case than in the fermentation of the centrifuged medium and, at the end of the experiment, the concentrations of total and reducing sugars were about half of the initial concentrations. It can be observed that after 10 days of fermentation, there was no consumption of sugars by _B. firmus_ and the PHB concentration began to decrease. [PERSON] et al. (2022) [40], who tested the use of soybean hull hydrolysed to obtain PHB, observed that after 48 h of fermentation, there was no sugar consumption by _C. necator_ and the concentration of PHB remained stable. They explained that this fact could be due to the microorganism's preference for a specific type of simple sugar. Thus, those sugars not consumed by microorganisms remained in the broth at the final stages of fermentation. It is necessary to consider that in this fermentation, as the CBS solids are in the broth, it seems quite possible that some sugars were being dissolved at the same time as the fermentation was being conducted with the participation of enzymes released by the bacterium. For this reason, the evolution of total suspended solids (TSS) was also followed during fermentation. TSS included the CBS solids and the microorganisms. However, considering the concentrations of _B. firmus_ during the fermentation and the average dry weight of bacterium, it was probed that the weight of the bacteria was always lower than that of 0.3% of the TSS measured, so it was considered that the TSS measured corresponded with the concentration of CBS solids. As can be seen in Figure 4A, the concentration of CBS solids decreased during all of the experiment. Therefore, sugars were dissolving from the solid at the same time as they were being consumed by the microorganism. To describe the transformations that occurred, a model based on irreversible first-order reactions was used [3]. It considers that CBS solids are dissolved by enzyme activity into non-reducing sugars that are subsequently hydrolysed to reducing sugars. Thus, \(S\) is the solid matter, \(D\) is the dissolved non-reducing-sugars (estimated as dissolved total  
Figure 4: Evolution of (**A**) the concentration of TSS (**x**), PHB (\(\blacklozenge\)), and bacteria viability (\(\blacktriangle\)), and (**B**) the concentration of total sugars (\(\bullet\)), reducing sugars (\(\blacksquare\)), and non-reducing sugars (\(\cdot\)) during fermentation of the non-centrifuged medium obtained from CBS hydrolyzed at 135 °C for 10 min. The symbols and dashed lines correspond to experimental data and continuous lines correspond to model results. sugars minus dissolved reducing sugars), and \(M\) is the dissolved reducing sugars that are being consumed by microorganisms.