# Application of Extracellular Polymeric Substances during Cultivation of Microalgae Biomass
## 3 Results and Discussion
The sedimentation efficiency was not improved in _Chlorella_ sp. when EPS was introduced at the end of cultivation, independently of the EPS concentration. However, the sedimentation efficiency of _T. subcoriformis_ and _A. platensis_ were improved. In the case of _T. subcoriformis_, the better sedimentation properties were not connected to higher EPS content, because in all variants of the experiments, the EPS content was similar. However, in _A. platensis_ biomass, the EPS content increased. The EPS content in the control biomass of microalgae/cyanobacteria and the biomass from the microalgae/cyanobacteria culture to which EPS of the activated sludge were added during cultivation did not differ. The use of mixotrophic conditions did not affect  
Figure 2: Microalgae/cyanobacteria growth during different strategies of EPS addition to cultivation: (**A**) biomass concentration of _T. subcoriformis_; (**B**) biomass concentration of _Chlorella_ sp. ; (**C**) biomass concentration of _A. platensis_. the improvement in the sedimentation properties and EPS content. It is well known that increased carbon availability during cultivation will increase the biomass concentration and decrease the chlorophyll content [30]. Moreover, mixotrophic cultivation also often has a positive effect on the \"production capacity\" of microalgal cells, e.g., _Chlorella_ sp. often has more oil in the cells, while _A. platensis_ has more phycocyanin in the cells [31]. In this study, in the EPS samples, the protein content, sugar content, and carbohydrate composition were determined (Table 2). In the EPS extracted from _T. subcordiformis_ and _Chlorella_ sp., the protein content was similar in variants of experiments. However, in the EPS extracted from _A. platensis_, the protein content increased. The positively charged amino groups in proteins can neutralize the negative charges of polysaccharides, uronic acid, and carboxylic acid in DNA, phosphate groups, etc. ; thus, proteins might effectively change the surface properties of the cell, inducing the formation of aggregates [13, 32, 33]. The higher content of proteins in _A. platensis_ could improve the sedimentation properties of biomass. In the EPS extracted from _T. subcordiformis_, sugar content has significantly increased in the variants of experiments where EPS were added at the end of microalgae growth (V2 and V3) compared to autotrophic cultivation (Table 2). Among the measured carbohydrates, the highest concentration was glucose, the concentration of which also increased in V2 and V3; however, it was not a significant increase. The higher content of sugars in the EPS might be caused by some other exopolysaccharide that also influenced the better sedimentation of _T. subcordiformis_ (Table 1). Exopolysaccharide produced by _Bacillus subtilis_ was used for harvesting _Nantochloropsis aculata_ and _Botryococcus braunii_ biomass [34]. The authors obtained the best flocculation with the dose of 5% \(v/v\) of exopolysaccharide and pointed out that harvesting efficiency and flocculant concentration are not linearly dependent. Polysaccharides were also connected to the formation of algal-bacterial biofilms. The sedimentation properties of biofilocs were better with a higher content of polysaccharides [35]. Similarly, polysaccharides were responsible for cell capture and aggregation during the rapid biogranulation of the microalgae _Ankistrodesmus falcatus_ var. _acicularis_[36]. The polysaccharide derived from _T. subcordiformis_ should be further investigated via extraction and analyses to determine its influence on the sedimentation properties. In the EPS extracted from _Chlorella_ sp., the sugar content increased in the variants to which EPS were added at the end of microalgae growth (V2 and V3) compared to autotrophic cultivation (Table 2). The main components of carbohydrates in the EPS extracted from _Chlorella_ sp. were glucose and mannose. Despite the increase in sugar content, the EPS content and sedimentation efficiency did not improve.