# Application of Extracellular Polymeric Substances during Cultivation of Microalgae Biomass
## 3 Results and Discussion  
The study tested different strategies used for the possible application of EPS for microalgae/cyanobacteria biomass. In strategy V1, the biomass was characterized by lower chlorophyll content compared to the control cultivation (Table 1). In this mixotrophic cultivation (V1), the chlorophyll content was significantly reduced by 72.5%, 71.4%, and 50% in the biomass of _T. subcordiformis_, _Chlorella_ sp., and _A. platensis_, respectively (\(p<0.05\)). Figure 1: The scheme of the experiment. The introduction of EPS during the cultivation in V1 caused microalgae/cyanobacteria to use polymers as an external carbon source. Photoautotrophic microorganisms use light as a source of energy for CO\({}_{2}\) assimilation and store light energy in the chemical bonds of organic molecules. Some of these microorganisms also have a capacity for heterotrophic growth or mixing heterotrophic and photoautotrophic growth in metabolic traits called photomixtrophic traits. The strategy for changing metabolic traits is used for fast acclimation to the environmental conditions of aquatic microorganisms. The environmental conditions that could modulate the metabolic traits and, therefore, growth of microorganisms include light intensity, temperature, and concentrations of different forms of carbon. The ability of microorganisms to change metabolic traits is poorly described in the literature and has remained rather undefined. It was found that among microalgae species, _Chorella_ sp. is capable of mixotrophic growth [21; 22]. The studies proved that higher biomass concentrations were observed under mixotrophic growth than under only photoautotrophic or heterotrophic growth. The presence of organic and inorganic carbon sources during mixotrophic conditions results in combining photosynthetic activity with high intracellular inorganic carbon concentrations created by glucose breakdown and the release of CO\({}_{2}\), despite the direct utilization of glucose-derived carbon skeletons for growth [22; 23]. During the proteomic analysis under different metabolic conditions in _Chorella_ sp. cultivation, it was observed that after feeding the photoautotrophic culture with glucose to start the mixotrophic metabolism, the chlorophyll content in biomass decreased immediately, and this decline lasted 8 h. During the next 24 h, the transition from the mixotrophic conditions to the photoautotrophic conditions occurred [22]. The present study showed slightly different results: the chlorophyll content in biomass did not increase in the two days after the addition of organic carbon (EPS). These differences might be the result of different sources and concentrations of organic carbon being added to cultivation. EPS consist of polysaccharides, proteins, nucleic acids, and lipids, which could exert a longer effect on cell metabolism than simple compounds, such as glucose. The presence of organic carbon sources inhibits the synthesis of pigments at the start of mixotrophy, causing the lower chlorophyll content [24]. Moreover, the reduction in the abundance of proteins from the thylakoid membranes and the downregulation of photosynthesis-related genes were reported under mixotrophic conditions [25]. Similar observations of lower chlorophyll content in the cultures with organic carbon addition were observed during cultivation of _Arthrospira platensis_[26] and _Tetraselmis subcondiformis_[14; 27].