# Cell Disruption and Hydrolysis of _Microchloropysis salina_ Biomass as a Feedback for Fermentation
## 3 Results and Discussion
### Mechanical Cell Disruption  
#### 3.2.1 Ultrasonication  
Figure 2 shows the disruption of \(M\). _salina_ biomass by ultrasonication. Microalgal biomass with a solids content ranging between 10 and 25% (\(w/w\)) was ultrasonicated for 40 min (Figure 2a). Cell disruption progressed very similarly for solids content between 10 and 20%, reaching nearly 40% after 40 min of ultrasonication. Only for a solids content of 25% did the degree of cell disruption reach 12% after 20 min and did not increase with further treatment. Typically, during ultrasonication, there is visible jet stream formation in the liquid medium, which also helps mix the sample. In this experiment, this was also the case for all the biomass samples except for the one with a 25% solids content, meaning that this sample was not mixed sufficiently during ultrasonication. Moreover, it was noted for biomass with a solids content of 25% that a dry biomass layer formed on the surface of the sonotrode, which grew thicker over time. Thus, it was concluded that ultrasonication is not suited for the cell disruption of microalgal biomass with such a high solids content and viscosity since the forces that accomplish cell disruption by ultrasonication act mainly through the liquid medium, which was not sufficiently present in this case. Figure 1: (**a**) Elemental and (**b**) macromolecular composition of \(M\). _salina_ biomass grown in nutrient-replete ASW medium. Ash content was assumed to be 10.0% based on previous reports [12]. To determine if the disruption degree of _M. salina_ cells would further increase with increasing ultrasonication time, a subsequent experiment with microalgal biomass with a 15% solids content was performed for a total treatment duration of 90 min (Figure 2b). It was shown that the cell disruption efficiency increased to 60% after 60 min and to 70% after 90 min of ultrasonication. [PERSON] et al. [31] examined the effect of high-intensity ultrasound on lipid extraction from high-solids viscous slurries of the _Microchloropsis_ species. The extraction yields were similar with 12% and 20% solids content but decreased considerably for the 25% solids content of biomass. The same phenomenon was also observed in our study, confirming that there was an upper limit to the _M. salina_ biomass density between 20% and 25% solids content, after which the efficiency of cell disruption via ultrasonication decreased drastically. On the other hand, the disruption degree achieved at different biomass concentrations below 20% was very similar. [PERSON] et al. [31] suggested that such a decrease in efficiency could be due to an increased attenuation of ultrasound waves, which could result in less efficient cell rupture. This was attributed to the increase in the biomass viscosity and decrease in the speed of sound with the increasing solid content. In this study, the highest disruption efficiency of _M. salina_ cells achieved by ultrasonication was 70%, while 10-20% was reached after 10 min. This is in accordance with the literature values reported for ultrasonication at 20 kHz frequency for 10 min [32], whereas 60-90% disruption of the _Microchloropsis_ species after 10-20 min has also been reported for ultrasonication at much higher frequencies (1.0-4.3 MHz) [32,33]. For the ultrasonic disruption of _Microchloropsis oculata_ cells, [PERSON] et al. [34] reported increased effectiveness by a combination of high-frequency focused ultrasound (3.2 MHz) and low-frequency non-focused ultrasound (20 kHz) treatments. However, frequencies applied at a large scale are usually lower due to concerns about energy consumption [35]. The literature regarding microalgal cell disruption using ultrasonication has focused more on the extraction efficiency of lipids or other products after the treatment, mostly without direct statements about the actual disruption efficiency. Nevertheless, its efficiency depends on microalgae species, biomass concentration, and operational conditions such as temperature, time, and frequency [18].