# Cocoa Bean Shell as Promising Feedstock for the Production of Poly(3-hydroxybutyrate) (PHB)
## 3 Results and Discussion  
### Characterization of Hydrolysed Broths and CBS Solids  
In order to hydrolyze the complex carbohydrates and to extract as many fermentable sugars as possible, CBS was treated by means of the acid hydrothermal procedure [3] for 10 min at 135 \({}^{\circ}\)C. These conditions were selected according to previous tests carried out in order to maximize the extraction of fermentable sugars. According to the methodology previously described, two different hydrolyzed media were used as fermentation broths, one without CBS solids (centrifuged) and one with them (non-centrifuged). Therefore, just after the hydrothermal treatment, both media differ only in the solid content. Table 1 shows the composition of the two media used as fermentation broths in terms of the concentration of total suspended solids, soluble total and reducing sugars, total nitrogen, total phosphorus, and pH. In addition, the dry extract of the different batches of CBS employed in this work was quite similar, between 92.3 and 93.0%. At the beginning of fermentation (time 0), the concentrations of soluble total carbohydrates and reducing sugars in both media ranged between 34-36 and 27-32 g/L, respectively. Reducing sugars accounted for approximately 80% of the total sugars. As expected, the liquid fractions of both broths were quite similar regarding the amount of sugars and the total nitrogen concentration. However, the concentration of total phosphorus was slightly higher in the non-centrifuged medium with respect to the broth without solids. The composition of the hydrolyzed broths is similar to those described in the literature for other media used to obtain PHB by fermentation. For instance, [PERSON] and [PERSON] (2018) [26] employed a hydrolysate of pineapple peels with a concentration of fermentable sugars of 28 g/L for the production of PHB. Likewise, a broth with a concentration of 30 g/L of glucose and fructose, used for bioplastic production, was obtained by [PERSON] et al. (2019) [27] from palm-oil fruit treated by acid hydrolysis. PHB, a semicrystalline biopolyester that bacteria accumulate as a reserve material within their cells, is a secondary metabolite produced in response to environmental stress [20]. Specifically, it has been reported that this polymer is generated through a fermentation process under restricted growth conditions for nitrogen, phosphorus, sulfur and/or oxygen in the presence of an excess carbon source [28]. Hence, knowing the nitrogen and phosphorus concentrations in the growth media is fundamental. Some authors have reported that the optimized medium conditions to maximize PHB production were 22-30 g/L of carbon, 2 g/L of nitrogen, 0.5 g/L of phosphorous, and a pH of 7, values quite similar to those obtained in this study in the initial media [29; 30; 31]. Therefore, according to bacterial nutritional requirements reported in the literature data, the resulting broths from CBS hydrolysis seems to be an optimal fermentation medium for the production of PHB by _B. firms_. Another aspect that must be taken into account is the possible presence of inhibiting compounds of fermentation. Acetic acid, furfural, or 5-hydroxymethylfurfural (HMF) can be formed when severe conditions in hydrolysis processes are employed and these species may affect cell viability due to the physiological stress generated in bacteria [32]. For acetic acid, concentrations of 1-10 g/L have been reported to inhibit sugar fermentation by _Bacillus_, whereas it has been found that the growth of this genus was inhibited at concentrations of furfural and HMF between 0.05-5 g/L [33; 34]. In this work, both broths employed for fermentation contained initially concentrations of acetic acid and HMF of 2.8 and 0.03 g/L, respectively. Hence, the presence of these compounds could affect bacterial growth, which would affect the production of PHB. In the case of furfural, for both centrifuged and non-centrifuged media, the concentration of this inhibitor was below the detection limit (<1 mg/L).