# Cell Disruption and Hydrolysis of _Microchloropysis salina_ Biomass as a Feedback for Fermentation
## 3 Results and Discussion
### Enzymatic Hydrolysis of Disrupted Cells
Enzyme dosing is given as a mass ratio of CDW and adjusted to 10.0% cellulase (C2) and 0.12% or 1.00% mannanase (M) for hydrolysis on the 60 L, and the 200 L scales, respectively. (**a**) Final concentrations of glucose (\(\blacksquare\)) and mannose (\(\blacktriangle\)) on the 60 L scale and glucose (\(\square\)) and mannose (\(\triangle\)) on the 200 L scale; (**b**) saccharification efficiency (\(\upeta_{\text{Sac}}\)). CDW concentration of 250 g L\({}^{-1}\) and 100% saccharification efficiency, the resulting microalgae hydrolysate would have a 53.4 g L\({}^{-1}\) total concentration of glucose and mannose. However, the technical equipment used in the current study allowed for the processing of only 150 g L\({}^{-1}\) CDW containing microalgal biomass since the industrial HPH used for mechanical cell disruption did not allow for higher feed stream viscosity (\(\leq\)20 mPa s at a shear rate of 1000 s\({}^{-1}\)). Hence, calculating with an initial CDW concentration of 150 g L\({}^{-1}\) and 100% saccharification efficiency, the resulting microalgae hydrolysate would have a 32.1 g L\({}^{-1}\) total concentration of glucose and mannose. Nevertheless, the best results obtained in this experimental series were 8.2 g L\({}^{-1}\) on a milliliter scale and 7.2 g L\({}^{-1}\) on a 200 L scale. In this work, the maximum saccharification efficiency achieved was 30%, which corresponds to 77.3 mg released sugars per g CDW, very similar to the yield reported by [PERSON] and [PERSON] [52] using commercial enzyme mixtures on disrupted _M. salina_ biomass. Nevertheless, a 30% saccharification yield is way below the expected range of 47-90% [50], yielding lower sugar concentrations than what would be required for the use of the hydrolysate as a feedstock for fermentation. One solution to the unexpectedly low sugar content of the microalgae biomass hydrolysate would be concentrating the hydrolysate using an evaporator, which was not demonstrated in this study due to the lack of appropriate equipment for a large-scale application. It might be possible to improve the carbohydrate saccharification yield for high-density _M. salina_ biomass by adding other commercially available enzymes with various activities into the enzyme cocktail used. With the help of a growth inhibition screen, [PERSON] et al. [53] suggested that chitinase, lysozyme, pectinase, sulfatase, b-glucuronidase, and laminarinase could aid the enzymatic cell wall degradation of _Microchloropsis_ strains. Moreover, [PERSON] et al. [54] could improve the lipid extraction yield for autoclaved _M. oculata_ biomass by around 20% using Viscozyme (a multi-enzyme mixture containing a wide range of carbohydrates) and Proteinase K (an alkaline serine protease of fungal origin). However, the prospects of success at improving the effectiveness of enzymatic hydrolysis seem to be low with _M. salina_ cells grown in nutrient-replete medium, since these have a different macromolecular and cell wall composition [12; 55] and are apparently harder to disrupt and hydrolyze than the _M. salina_ cells grown in nutrient-limited medium [55; 56]. Applying nutrient limitation on the microalgae culture, on the other hand, would reduce biomass productivity.