# Cell Disruption and Hydrolysis of _Microchloropysis salina_ Biomass as a Feedback for Fermentation
## 3 Results and Discussion
### Enzymatic Hydrolysis of Disrupted Cells  
Enzymatic hydrolysis of mechanically disrupted _M. salina_ cells was first investigated on a milliliter scale. Initially, the effects of the sterilization method and degree of cell disruption on the effectiveness of enzymatic hydrolysis were investigated, considering the impact on a subsequent scale-up. Then, the dosing of various enzymes was examined for increased saccharification efficiency, and the hydrolysis was scaled up to 200 L using the best-performing enzyme composition. Table 7 presents the experimental results for the enzymatic hydrolysis of mechanically disrupted _M. salina_ cells, showing the disruption degree of microalgal biomass prior to enzymatic hydrolysis, enzyme dosing used, the concentration of glucose and mannose released, and the saccharification efficiency achieved in experiments on a milliliter scale. First of all, the influence of the sterilization method and cell disruption degree on subsequent enzymatic hydrolysis of mechanically disrupted _M. salina_ cells was examined. As a sterilization method, autoclaving at 121 \({}^{\circ}\)C for 20 min was compared with the addition of antibiotics (each 100 mg L\({}^{-1}\) of kanamycin and tetracycline) to inhibit the growth of contaminants. These are labeled in Table 7 as experiments number 1 and 2, respectively. It was demonstrated that skipping the autoclaving step and using antibiotics instead did not affect hydrolysis efficiency, which reached 10% in both cases. Therefore, the addition of antibiotics was preferred to prevent contamination effects on biomass hydrolysis in the following experiments. The degree of cell disruption had a significant influence on the effectiveness of hydrolysis. An increase in initial cell disintegration degree from 70% to 92% increased saccharification efficiency from 10% to 16%. As anticipated, higher degrees of mechanical cell disruption at the beginning of enzymatic hydrolysis resulted in a higher hydrolysis efficiency since the carbohydrates in the cell wall become more exposed, making them more readily available for the enzymes. The effects of adding hemicellulose or protease to the enzyme mixture on hydrolysis efficiency were also investigated. Adding 1% hemicellulose to the enzyme mixture in addition to cellulase and mannanase improved biomass hydrolysis minimally, increasing the saccharification efficiency by 2% from 16% to 18%. Adding 1% protease to the enzyme mixture in addition to cellulase and mannanase increased hydrolysis efficiency only by 1% to 11%. Thus, the use of hemicellulose or protease in addition to the carbohydrates affected the effectiveness of enzymatic hydrolysis only negligibly, in contrast to the literature reports for other microalgae strains [50], and was therefore omitted in the following experiments. Next, two different cellulase mixes, namely Cellic(r) C Tec3 HS and Rohament(r) CEP, labeled as C1 and C2 in Table 7, respectively, were compared with respect to the saccharification efficiency. Only 2% of the carbohydrates were solubilized using a mannanase dose of 0.12% without cellulases. Using 5% Cellic(r) C Tec3 HS in addition to mannanase improved the saccharification efficiency to 10%, whereas with 5% Rohament(r) CEP, it increased to 12%. With Cellic(r) C Tec3 HS, more mannose was released, while Rohament(r) CEP solubilizedmore glucose. Due to its better performance than Cellic(r) CTe3 HS, as well as its easy-to-use powder form, Rohament(r) CEP was used in further enzymatic hydrolysis experiments. Then, the effect of cellulase dosing on saccharification efficiency was examined. For this, two sets of experiments with different initial cell disruption degrees are presented in Table 7. In the case of biomass with a 70% cell disruption degree, using 5% and 10% cellulase in addition to mannanase increased saccharification efficiency by 10% and 16%, respectively. Using biomass with a 78% cell disruption degree, dosing of 10% and 15% cellulase in addition to mannanase resulted in an efficiency increase of 16% and 18%, respectively.