# Cell Disruption and Hydrolysis of _Microchloropysis salina_ Biomass as a Feedback for Fermentation
## 3 Results and Discussion
### Mechanical Cell Disruption
#### 3.2.2 Bead Beating  
Disruption of _M. salina_ cells in a mixer mill was examined using beads of various materials and sizes. In this work, cell disruption experiments using a mixer mill were considered a preliminary exploration for a possible process transfer to an industrial-scale agitated media mill. Microalgal biomass with a solids content varying between 10 and 25% (\(w\)/\(w\)) was disrupted by bead milling for 40 min. Figure 3 shows the cell disruption degree over time using beads with 0.5-1.0 mm diameters. The beads were made of zirconium oxide and agate, which had very similar densities of 6.0-7.0 g mL\({}^{-1}\) (Table 2). As expected, the disruption efficiency increased with the declining biomass solids content and increasing milling time. The results were very similar for zirconium oxide and agate beads with the same diameter, which was to be expected since the densities of these two materials were quite similar. For the disruption of microalgae, but also specifically of _Microchloropsis_ species, by bead milling, a bead size of 0.3-0.6 mm has been reported to yield higher disruption efficiency than bead sizes over 1 mm [36; 37; 38; 39]. This was also the case for biomass with solids content greater than 15% in this study, with the disruption efficiency being higher with a smaller bead size of 0.5 mm than with 1.0 mm, although it stayed below 35% in all cases. Figure 3: Disruption of _M. salina_ biomass by bead milling using beads of various materials and sizes. Microalgal biomass with various solids content ranging between 10 and 25%, as indicated by the legend, was milled for 40 min. (**a**) Zirconium oxide (0.5 mm); (**b**) zirconium oxide (1.0 mm); (**c**) agate (1.0 mm). Error bars show the standard deviation of duplicate experiments. However, on the contrary, for a solids content of 10%, the highest degree of disruption achieved with 0.5 mm beads was 53% after 40 min, which was around 25% lower than the 76-78% achieved after the same milling duration with 1 mm beads. This appears to be in contrast with the existing literature reports cited above. Figure 4 shows the microalgal cell disruption using beads with a diameter between 2 and 5 mm. The beads were made of glass, stainless steel, and tungsten carbide, with material densities of 2.9 g mL\({}^{-1}\), 7.7 g mL\({}^{-1}\), and 15.0 g mL\({}^{-1}\), respectively. Using steel beads of 5 mm size, the disruption efficiency was almost independent of solids content and ranged between 46 and 54% for all biomass concentrations after 40 min. On the other hand, reducing the diameter of steel beads from 5 mm to 2 mm resulted in a remarkable increase in disruption degree. For biomass with a solids content of 10-20%, 87-97% of the cells were disrupted after 40 min. For a 25% solids content, only 67% of the cells were disrupted, which was still the highest disruption degree achieved with 25% solids content in this work. Using glass beads (3 mm) resulted in the lowest disruption efficiency of the dense _M. salina_ biomass, as expected, due to its low density (Figure 4d). However, with tungsten carbide beads (3 mm), which had the highest density among the tested materials, a lower disruption efficiency than with the 2 mm steel beads was measured (Figure 4c). Very high sample temperatures and cell agglomeration with visible clumps were observed during cell disruption with tungsten carbide beads. This was probably the result of much higher energy input at the same mixing frequency compared to the other materials tested due to the significantly higher density of tungsten carbide. Hence, the measurements may have been faulty and lower than the actual values due to clumped cell debris being counted as intact cells. All in all, a higher material density of the same-sized beads was  
Figure 4: Disruption of _M. salina_ biomass by bead milling using beads of various materials and sizes. Microalgal biomass with various solids content ranging between 10 and 25%, as indicated by the legend, was milled for 40 min.