# Biosynthesis of Polyhydroxyalkanoates from Defatted _Chlorella_ Biomass as an Inexpensive Substrate
## 3 Results and Discussion  
### PHA Production via Batch Cultivation in Shake Flasks  
The cell growth profile, PHA production, and substrate consumption by _B. megaterium_ ALA2 during the fermentation process are shown in Figure 1. The reducing sugar concentration in DCB HCl hydrolysate-supplemented media decreased with increasing biomass production, giving rise to a maximum DCW of 2.81 \(\pm\) 0.05 g/L at 72 h of cultivation. PHA production occurred concomitantly with cell growth and achieved a maximum of 0.84 \(\pm\) 0.06 g/L (29.7% of DCW). Lower DCW (1.80 g/L) and PHA (0.40 g/L, 21% of DCW) levels were previously reported by [PERSON] et al. [15] when _B. megaterium_ ALA2 was cultivated in minimal media supplemented with _Laminaria japonica_ hydrolysate. Such a difference in DCW and PHA yield can be explained by the difference in reducing sugar composition between the two hydrolysates. Previously, [PERSON] et al. [18] investigated the effect of different carbon sources, including maltose, fructose, Na-gluconate, glucose, lactose, xylose, and sucrose, on PHA accumulation by _B. megaterium_. The maximum production in terms of PHA was recorded in media supplemented with 2% glucose, which indicates the suitability of this carbon source for _B. megaterium_. Based on our previous study, glucose was identified as the main reducing sugar in DCB HCl hydrolysate [10], which may explain, in part, the higher level of PHA that accumulated inside _B. megaterium_ ALA2 cells in this study, compared with that produced from _Laminaria japonica_ hydrolysate [15] for which mannitol was identified as the main carbohydrate [19]. Figure 1: Time profile of optical density (OD) at 600 nm, dry cell weight (DCW), polyhydroxyalkanoate (PHA) accumulation, and substrate utilization by _Bacillus megaterium_ ALA2 using 0.3 N HCl defatted _Chlorella_ biomass (DCB) hydrolysate as the carbon source in a flask culture. Data are shown as mean values \(\pm\) standard deviation (\(n\) = 3). Figure 2 shows the cell growth profile, PHA production, and substrate consumption by _C. necator_ KCTC 2649 during batch cultivation in shake flasks. Reducing sugar consumption increased with increasing bacterial growth. PHA accumulation started during the exponential phase and gradually increased during the stationary phase, giving rise to a maximum polymer content of 7.51 \(\pm\) 0.20 g/L (75.4% of DCW) at 120 h of cultivation due to PHA accumulation inside bacterial cells. In fact, during the stationary phase, cells cease division but remain metabolically active and start accumulating PHA under the excess of carbon source and nitrogen-limiting conditions [20]. The PHA level decreased after this time due to PHA utilization for cell maintenance purposes. PHA accumulation by _C. necator_ in this work was significantly higher than that reported by [PERSON] et al. [15] when cultivating _C. necator_ NCIMB 11599 in a minimal medium supplemented with 2% of _L. japonica_ acid hydrolysate (1.58 g/L; 32% of DCW). Likewise, a PHA production of only 0.62 g/L was previously reported by [PERSON] et al. [21] when _C. necator_ was cultivated in a basal salt medium supplemented with waste rapeseed oil. Interestingly, the PHA content accumulated by _C. necator_ in this work was significantly higher than that produced by the same strain cultivated in a mineral salt medium supplemented with 1% glucose, for which a maximum PHA production of only 1.61 g/L (57.07% of dry cell weight) was recorded [22]. This result underlines the importance of using DCB hydrolysate as a promising low-cost substrate for industrial bioplastics production. The cell growth profile, PHA accumulation, carotenoid production, and substrate utilization by _H. mediterranei_ DSM 1411 cultivation during the fermentation process are shown in Figure 3. The reducing sugar content gradually decreased with time due to cell growth, denoting the ability of the cultivated strain to assimilate reducing sugars from HCl DCB hydrolysate.