## 3 Results and Discussion
### Effect of Preparation of M. oleifera Flocculant on the Harvesting Efficiency  
As the adjustment of process conditions and pH values was unable to increase the maximum harvesting efficiencies found of 81-86%, different preparation procedures for the _M. oleifera_ flocculant were tested to possibly reduce the amount of flocculant needed while improving the harvesting efficiency obtained. Initially, two parameters that have not been investigated before, i.e., method of grinding and powder granulometry, were assessed to understand their impact on the flocculation efficiency of MP.  
MP obtained both by manually grinding with a mortar and automatically milling with a ball mill were compared, and the results indicated that the milling process only slightly affected the flocculation efficiency of _M. oleifera_ (Figure 3). The flocculation efficiency at the lowest dose (100 mg L\({}^{-1}\)) was significantly reduced (\(p<0.05\)) by 13% when using the powder produced with the automatic mill, but the maximum harvesting efficiency was obtained when using doses higher than 200 mg L\({}^{-1}\). This result suggests that the proteins responsible for flocculation are well protected within the seed and are not easily subjected to physical denaturation, which is an advantage when upscaling the production of flocculant for microalgae harvesting from this seed. _Moringa_ seeds contain \(\sim\)35-40 oil, commercially called 'Ben oil' [44; 45], and it has been reported that the lipid cells surround the protein molecules [45], which could explain the observed resistance to denaturation.  
The powder produced with the automatic mill was then tested after sieving through three different grid sizes (1.00 mm, 0.60 mm, 0.15 mm) to assess whether a finer or coarser granulometry would positively impact the flocculation efficiency of the used powder. However, no significant difference was observed between the different granulometries at the end of the experiment (Figure S7). It may be that the proteins responsible for flocculation are highly soluble in the algal culture, and therefore even a coarser granulometry enables sufficient contact between the solution and the proteins to result in high harvesting efficiency.  
To further test the water solubility of the proteins responsible for flocculation in the _M. oleifera_ seed, the seed powder was dissolved in water, and the solution was filtered before being mixed with the algae suspension at different doses (Figure 4). A harvesting efficiency between 88% and 91%, the highest one observed in this study, was obtained at 2-3 g L\({}^{-1}\) of MP-equivalent. As a result, it was confirmed that the flocculant proteins were indeed water-soluble, and the previous dissolution possibly increased their ability to  
Figure 3: Effect of _M. oleifera_ seed powder prepared with different grinding methods, manually-ground powder (MMP) and automatically-ground powder (MAP), on the harvesting efficiency of a mixed culture of _Desmodesmus_ sp. and _C. vulgaris_ (500 mg L\({}^{-1}\)) after a 60-min sedimentation time. Control without the addition of flocculants was also carried out in the same conditions as the assays with the addition of flocculants. The rotation speed and time used were 150 rpm for 5 min, followed by 70 rpm for 20 min in an orbital shaker. Columns represent the average values, and error bars represent the standard deviations of triplicates. Different letters indicate significant differences between different dosages of _Moringa_ powders using two different grinding methods after a one-way ANOVA followed by a Tukey test (\(p<0.05\)).  
interact with the algal cells, improving the harvesting efficiency of the process. However, a higher dosage was needed to achieve similar flocculation as the one obtained with the seed powder. Previous studies have shown better protein extraction from the seed powder when using a saline solution than water [23; 46; 47].