# Cell Disruption and Hydrolysis of _Microchloropysis salina_ Biomass as a Feedback for Fermentation
## 3 Results and Discussion
### Enzymatic Hydrolysis of Disrupted Cells
Here, although the best results of 7.2 g L\({}^{-1}\) sugars (glucose and mannose) and 26% saccharification efficiency were reached with 15% cellulase dosing, the difference from 10% cellulase was not significant enough to consider it better with respect to price \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline
**Experiment** **\#** & **Degree of Cell** **Disruption, –** & **C1, g g\({}^{-1}\)** & **C2, g g\({}^{-1}\)** & **M\({}_{\star}\)** & **H\({}_{\star}\)** & **P\({}_{\star}\)** & **Glucose, g L\({}^{-1}\)** & **Mannose, g** **Efficiency, –** \\ \hline \multicolumn{10}{c}{Effect of autoclaving vs. antibiotic usage for sterilization} \\ \hline
1 & 75\% & 5.92\% & – & 0.12\% & – & – & 2.63 (\(\pm\)0.10) & 1.88 (\(\pm\)0.38) & 10.1\% (\(\pm\)0.9\%) \\ \hline
2 & 75\% & 5.92\% & – & 0.12\% & – & – & 2.45 (\(\pm\)0.08) & 2.05 (\(\pm\)0.05) & 10.1\% (\(\pm\)0.2\%) \\ \hline \multicolumn{10}{c}{Effect of cell disruption degree} \\ \hline  
\hline \end{tabular}
\end{table} 
Table 7: Enzymatic hydrolysis of mechanically disrupted _M. salina_ cells on a milliliter scale. Disruption degree of microalgae biomass prior to enzymatic hydrolysis, as well as the enzyme type and dosing (g enzyme per g CDW) used, are listed for each experiment. Names of the enzymes used are abbreviated. C1: Cellic® CTe3 HS; C2: Rohament® CEP; M: Rohalase® GMP; H: Hemicellulase from _A. niger_; P: Protamex®. CDW concentration of the initial biomass prior to mechanical cell disruption was 150 g L\({}^{-1}\) in all cases. Concentrations of glucose and mannose measured after the enzymatic hydrolysis were assumed to be representative of all sugars released and used as basis for the calculation of the saccharification efficiency. performance ratio. For hydrolysis of the biomass with a 78% cell disruption degree, adding 10% cellulase together with 0.12% manmanase yielded 6.9 g L\({}^{-1}\) sugars released and 25% saccharification efficiency. Thus, a cellulase dose of 10% was used in the enzyme mixture in further hydrolysis experiments. Table 7 also reveals the results of the experiment investigating the effect of mannanase dosing on saccharification efficiency. Using 10% cellulase only without mannanase, 9% of the carbohydrates of the microalgal biomass were solubilized. Adding 0.12% mannanase into the enzyme mixture increased the hydrolysis efficiency to 25%, and a further increase of mannanase dosing to 2.0% increased the efficiency further to 30%. Moreover, the sum of glucose and mannose released increased from 2.4 g L\({}^{-1}\) to 6.9 g L\({}^{-1}\) and further to 8.2 g L\({}^{-1}\), respectively. This indicates a possible synergy between the cellulase and mannanase mixes used, which led to improved glucose release with increased dosing of mannanase, even though the amount of mannose released remained the same. Scale-up of the enzymatic hydrolysis of mechanically disrupted _M. salina_ biomass to first 60 L, and, finally, to 200 L, was performed successfully. Figure 8 shows the results of the scale-up experiments. On both scales, a cellulase dosing of 10% of CDW was applied. Even though a higher mannanase dose (1.0%) was used on the 200 L scale, no improvement in saccharification efficiency was observed compared to the 60 L scale with 0.12% mannanase dosing. The final concentration of released glucose was 5.7-5.9 g L\({}^{-1}\), whereas 1.3-1.4 g L\({}^{-1}\) mannose was solubilized. Hence, the resulting saccharification efficiency was very similar, namely 22.2% and 22.6% in the 60 L and the 200 L scale processes, respectively. Nonetheless, saccharification efficiency achieved on the 200 L scale was 7% lower than the 30% recorded on the milliliter scale. Based on the sugar composition of _M. salina_ grown in a nutrient-replete medium in thin-layer cascade photobioreactors as reported by [PERSON] et al. [12], with an initial  
Figure 8: Enzymatic hydrolysis of mechanically disrupted _M. salina_ cells on 60 L (blue) and 200 L (white) scales. The degree of cell disruption was 78% in both experiments.