# Cell Disruption and Hydrolysis of _Microchloropysis salina_ Biomass as a Feedback for Fermentation
## 3 Results and Discussion
### Phosphorus Elimination
#### 3.5.2 Phosphate Precipitation Using Microalgae Hydrolysate  
Initial experiments with microalgae hydrolysiset showed that a single-step process was not as effective as demonstrated with the phosphate buffer (see Table S1). Moreover, a single-step precipitation with a stoichiometric excess of iron over phosphorus would require uneconomically large amounts of FeCl\({}_{3}\). Hence, a stepwise reduction of the phosphorus level was developed. To prevent an unnecessary dilution of the hydrolysate, the precipitating agent FeCl\({}_{3}\) - 6 H\({}_{2}\)O and KOH for pH adjustment were added to the reaction mixture in solid form. Phosphate precipitation of the microalgal biomass hydrolysate was carried out on a 200 L scale. An Fe:P stoichiometric ratio of 1.2:1 was used in the first step, which was increased to 2:1 for the second step. As shown in Table 8, the initial phosphate concentration in the microalgae hydrolysate was 6.5 g L\({}^{-1}\), which was much higher than the concentration anticipated based on the elemental composition of _M. salina_ biomass (2\(-\)3 g L\({}^{-1}\)). Nonetheless, the phosphate content was successfully reduced to 83 mg L\({}^{-1}\) and then to 28 mg L\({}^{-1}\) after each step, with an overall phosphate depletion degree of 99.6%. Performing additional precipitation steps on a milliliter scale allowed a decrease of the phosphate concentration to around 25 mg L\({}^{-1}\), but not further (see Tables S1 and S2). In general, phosphorus limitation of microbial growth requires a very high C/P ratio around 3000-4000 g g\({}^{-1}\) to be present in the cultivation medium [5; 10]. To achieve this C/P ratio with a phosphate concentration of 25-100 mg L\({}^{-1}\), a sugar content of 61-326 g L\({}^{-1}\) would be required, which is significantly higher than the concentrations achieved in the biomass hydrolysate in this study. An alternative strategy would be to first concentrate the biomass hydrolysate to increase the sugar concentration, for instance, by evaporation, and then perform the \begin{table}
\begin{tabular}{c c c c} \hline \hline  & **Fe:P Ratio** & **Phosphate Concentration, mg L\({}^{-1}\)** & **Phosphate Depletion, \%** \\ \hline Hydrolysate & \(-\) & 6512.0 & \(-\) \\ Step 1 & 1.2 & 82.6 & 98.7 \\ Step 2 & 2.0 & 27.6 & 66.6 \\ \hline Final & \(-\) & 27.6 & 99.6 \\ \hline \hline \end{tabular}
\end{table} 
Table 8: Stepwise phosphate precipitation of microalgae hydrolysate on a 200 L scale. An Fe:P stoichiometric ratio of 1.2 and 2.0 was used in 1 st and 2 nd steps, respectively. The reaction mixture was centrifuged using a disc separator after each step. Precipitating agent FeCl\({}_{3}\) - 6 H\({}_{2}\)O and KOH for pH adjustment were added to the reaction mixture in solid form to prevent an unnecessary dilution of the hydrolysate. Figure 9: Variation of the Fe:P stoichiometric ratio for phosphate precipitation of a phosphate buffer using FeCl\({}_{3}\). (**a**) Phosphate concentration in the buffer solution and after precipitation with various Fe:P ratios; (**b**) degree of phosphate depletion with various Fe:P ratios. phosphorus elimination. In this way, the increased sugar concentration would make it possible to reach a higher C/P ratio at the same phosphate level. Using a very similar procedure with a Fe:P ratio of 1.5:1 on a different microalgae hydrolysate, [PERSON] et al. [10] successfully reduced the phosphate content of a microalgae hydrolysate by 99.7% from 1.62 g L\({}^{-1}\) to 10 mg L\({}^{-1}\). However, the biomass they used was pretreated in a different manner, which resulted in a much lower organics concentration in the biomass hydrolysate. [PERSON] et al. [24] suggested that there might be various reasons making it hard to achieve very low phosphorus levels in streams containing high amounts of organic matter, such as the formation of organic side products with Fe\({}^{3+}\) and other components, most importantly, charged organics (through ion-pairing and binding), also stating that the extent of these reactions is unknown.