# The Microstructures of _Tetradesmus obliquus_ Biofilms Formed Under Different Nutrient Supplies
## 4 Discussion  
The results of this study indicate that the microalgae biofilms cultured under some nutrients (i.e., BG11, BG11\({}^{+\text{glycerine}}\), BG11\({}^{+\text{urea}}\), and BG11\({}^{+\text{NH}_{4}\text{Cl}}\) media) possessed wrinkled surfaces and fewer inner pores, whereas the microalgae biofilms cultured with BG11\({}^{+\text{NaHCO}_{3}}\) and BG11\({}^{+\text{NaNO}_{3}}\) media had many internal pores. As shown in Figure 6, the cells cultured with BG11, BG11\({}^{+\text{glycerine}}\), BG11\({}^{+\text{urea}}\), and BG11\({}^{+\text{NH}_{4}\text{Cl}}\) media possessed a lower amount of C-(C, H) and a higher amount of C-(O, N), compared with the cells cultured under BG11\({}^{+\text{NaHCO}_{3}}\) and BG11\({}^{+\text{NaNO}_{3}}\) media. Generally, the C-(C, H) groups are hydrophobic, and the C-(O, N) groups are hydrophilic [34]. The results revealed that the cells cultured with BG11, BG11\({}^{+\text{glycerine}}\), BG11\({}^{+\text{urea}}\), and BG11\({}^{+\text{NH}_{4}\text{Cl}}\) media were less hydrophobic (or more hydrophilic), which made these cells disperse well in the liquid media of biofilms. Based on our previous studies, the interactions between hydrophilic cells were relatively weak [18]. Therefore, the formed biofilms were homogeneous inside and possessed fewer inner pores. Furthermore, we found that the cells cultured with BG11\({}^{+\text{NaHCO}_{3}}\) and BG11\({}^{+\text{NaNO}_{3}}\) media had a higher amount of C-(C, H) and a lower amount of C-(O, N), which revealed that these cells were more hydrophobic than those cultured under BG11, BG11+glycerine, BG11+urea, and BG11\({}^{+\text{NH}_{4}}\)Cl media. Our previous studies [23] indicated that the hydrophobic cells possessed high cell-cell interactions in water\(-\)based cultured media. As shown in Figure 6, it can be expected that the cells with high cell-cell interactions would tend to aggregate together, which may induce the formation of cell agglomerations. Due to the differences in cell viabilities at different locations of biofilms, these cell agglomerations may be intermittently distributed in biofilms, which would induce biofilms to possess many inner pores. The above results and analysis revealed that the cells cultured under different nutrients possessed different surface compositions. When the surface had more hydrophobic groups (e.g., C-(C, H), O=C\(-\)OR) and less hydrophilic groups (e.g., C-(O, N), -NHR), the cells would tend to aggregate together due to the high cell-cell interactions, and form biofilms with more internal pores. Similarly, [PERSON] et al. [18] demonstrated that biofilms cultured under white light exhibited higher porosity due to hydrophobic cell surfaces, whereas homogeneous biofilms under red/blue light correlated with hydrophilic cell surfaces. This parallels our observation that nutrient supplements like NaHCO\({}_{3}\)/NaNO\({}_{3}\) increase cell hydrophobicity and biofilm porosity, suggesting the role of surface properties of cells in biofilm structural modulation. In addition, [PERSON] et al. [22] found that low light intensity promoted porous biofilms to enhance nutrient/light transport, while high light intensity triggered compact structures to mitigate photodamage. Our study reveals that nutrient types may also drive structural adaptations of biofilms. Future studies need to be conducted to understand the relationship between the physiology of microalgae cells and the microstructure of biofilms cultured under different nutrients. In addition, it should be noted that the inner pores and microstructure of biofilms would affect the transport phenomena of light, gas (e.g., CO\({}_{2}\), O\({}_{2}\)), and nutrients in biofilms [35], and thus the growth of microalgae cells and their lipid productivity, which is a key factor in microalgae\(-\)based biofuel technology.