# Cell Disruption and Hydrolysis of _Microchloropysis salina_ Biomass as a Feedback for Fermentation
## 3 Results and Discussion
### Chemical Hydrolysis of Undisrupted Cells  
Since the mechanical disruption of microalgae cells requires high energy consumption, enzymatic hydrolysis of undisrupted _M. salina_ cells with and without acid pretreatment was investigated first. Figure 7 shows the results of this experiment using the same enzyme dosing but with prior proteolysis (18 h) using proteases working at different pH optima of pH 3.5 and pH 7.0. The proteolysis was followed by autoclaving (at 121 \({}^{\circ}\)C for 20 min) at the specified pH for both sterilization and protease inactivation purposes. After autoclaving, the pH was adjusted to pH 5.0 for all batches, and the specified amounts of cellulase and mannanase mixtures were added for enzymatic hydrolysis (72 h). Since changes in reaction volume due to pH adjustments throughout the process were not negligible, sugar concentrations normalized to the initial reaction volume are presented in addition to the measured values. After the proteolysis and heat pretreatment at pH 3.5, around 2.0 g L\({}^{-1}\) glucose and 2.5-2.7 g L\({}^{-1}\) mannose were already released, which corresponded to a saccharification efficiency of 13%. In contrast, no sugar release was observed for proteolysis and heat pretreatment at pH 7.0. Then, the same enzymatic hydrolysis procedure with polysaccharide degrading enzymes was applied to all batches. For the batches that were pretreated at pH 3.5, final concentrations of 4.1-5.1 g L\({}^{-1}\) glucose and 1.3-1.6 g L\({}^{-1}\) mannose were achieved. On the other hand, for batches that were pretreated at pH 7.0, the final sugar concentrations were as low as 0.3-0.9 g L\({}^{-1}\) glucose and 0.2-0.3 g L\({}^{-1}\) mannose. Consequently, a significantly higher saccharification efficiency of 27-32% was achieved with acidic proteolysis and heat pretreatment combined with enzymatic hydrolysis, while it remained at 6-8% with proteolysis and heat pretreatment at a neutral pH. Most studies on the hydrolysis of microalgal biomass have either focused on product extraction, such as lipid separation, or have utilized already-extracted biomass containing disrupted cells [47,48]. Some have reported over 90% carbohydrate solubilization of mechanically or chemically disrupted microalgae cells after enzymatic hydrolysis [10,11,49]. Direct enzymatic hydrolysis of undisrupted microalgae cells, especially with conclusions on biomass saccharification, is uncommon in the literature. Few studies focusing on enzymatic disruption of microalgal cell walls have used diluted biomass with 1-10% dry weight, which would yield a very diluted hydrolysate with around 2-20 g L\({}^{-1}\) sugar [50]. In these studies, using cellulases or a combination of proteases and carbohydrates has accomplished saccharification yields of 47-96% with other microalgae species. Saccharification efficiency of 27-32% achieved with acidic hydrolysis followed by enzymatic hydrolysis was relatively low compared to literature reports for other microalgae strains mentioned above. [PERSON] et al. [17] determined that the cell wall of _M. gaditana_ contained around 14% algaenan, which is a non-hydrolysable biopolymer, presenting itself as an insoluble residue following severe acid and base hydrolysis, that is also well established in other species of _Microchloropsis_, including _M. salina_[51]. Thus, a relatively low saccharification efficiency is to be expected with _Microchloropsis_ species even after chemical hydrolysis. [PERSON] et al. [6] carried out a comparable study regarding saccharification of undisrupted _M. gaditana_ biomass with acidic hydrolysis followed by enzymatic treatment. They reported that 93 mg sugars per g dry weight (DW) were released after diluting the biomass with 7% (\(v/v\)) sulfuric acid and autoclaving (at 121 \({}^{\circ}\)C for 30 min). In this study, proteolysis and heat pretreatment at pH 3.5 (autoclaving at 121 \({}^{\circ}\)C for 20 min) released only 27-29 mg g\({}^{-1}\) DW sugars.