# The Microstructures of _Tetradesmus obliquus_ Biofilms Formed Under Different Nutrient Supplies
## 3 Results
### Surface Properties of Micoralgae Cells Cultured with Different Nutrients  
The results of surface characteristics and pore structure for the biofilms suggested that the surfaces of biofilms cultured under these different nutrients were all wrinkled with many folds, and no significant difference existed in the surface roughness of these biofilms, while the porosities for these biofilms were different. To understand the mechanism of forming biofilms with different porosities under these nutrient supplies, we further characterized the cell properties, including the cell morphology and surface compositions. Figure S3 indicates that under these nutrient conditions, all cells displayed a spindle\(-\)like  
Figure 3: The z\(-\)projection images (**a–f**) for the biofilms cultured under different nutrient supplies (BG11, BG11+glycerine, BG11+NaHCO\({}_{3}\), BG11+urea, BG11+NH\({}_{4}\)Cl, and BG11+NaNO\({}_{3}\)). (**g**) The determined biofilm porosity. The lowercase letters of “a, b, c, d” in (**g**) represent different significance groups. morphology, ranging from 7 to 9 \(\upmu\)m in length and 4 to 5 \(\upmu\)m in width, with no observable surface spines or appendages. The results suggested that there was no difference in the cell's shape and size. That is to say, the different porosities for these biofilms may not be due to the cell morphology. Furthermore, we characterized the cell surface compositions by using FTIR and XPS. Figure 4 shows the FTIR peak spectra for these cells cultured under different nutrients. We found that two new peaks (1280 and 835 cm\({}^{-1}\)) appeared on the cell surfaces cultured under BG11\({}^{+\text{NaHCO}_{3}}\). Generally, the peak at 1280 cm\({}^{-1}\) is associated with the C\(-\)O\(-\)C stretching band of the ethers or esters. However, because the extracellular polymers of algae typically contain polysaccharides, proteins, and lipids, and generally lack ether compounds, we assign this peak to the O\(=\)C\(-\)OR group (C\(-\)O\(-\)C stretching vibration). Additionally, the 835 cm\({}^{-1}\) peak can also be attributed to the C\(-\)H bending vibration. Since both the O\(=\)C\(-\)OR and C\(-\)H groups are hydrophobic, the results suggested that the cells cultured with BG11\({}^{+\text{NaHCO}_{3}}\) medium exhibit a higher abundance of hydrophobic groups compared to those cultured in BG11 medium. Moreover, a new peak (826 cm\({}^{-1}\)) appeared on the surface of cells cultured under BG11\({}^{+\text{NaNO}_{3}}\) medium, which was also related to the hydrophobic C\(-\)H group. In addition, the results indicated that the peak at 3274 cm\({}^{-1}\) for the cells cultured under BG11\({}^{+\text{NaNO}_{3}}\) was weaker than those cultured with BG11 medium. Generally, the peak at 3274 cm\({}^{-1}\) is related to the hydrophilic \(-\)NHR group (caused by N\(-\)H stretching vibration). Therefore, these results suggested that the cells cultured with BG11\({}^{+\text{NaNO}_{3}}\) medium had more hydrophobic groups and less hydrophilic groups than those cultured with the BG11 medium. The above FTIR results indicated that the cells cultured with BG11\({}^{+\text{NaHCO}_{3}}\) and BG11\({}^{+\text{NaNO}_{3}}\) media possessed more hydrophobic groups (i.e., C\(-\)H group and O\(=\)C\(-\)OR group) than those cultured with BG11 medium. To quantitatively characterize the hydrophobic groups on the cell surface, we further measured cell surface compositions using XPS. Figure S4 shows the XPS survey spectra for these microalgae cells. Figures S5 and S6 depict the carbon peaks (C 1s) and oxygen peaks (O 1s) obtained from Figure S4. Table S3 shows the contents of the specific functional groups in the C 1s and O 1s on the cell's surface. Notably, Figure 5 presents the contents of functional groups of C\(-\)(C, H) and C\(-\)(O, N) on microalgae cell surfaces. Figure 5a indicates that the contents of C\(-\)(C, H) on the surface of cells cultured with BG11, BG11\({}^{+\text{NaHCO}_{3}}\), and BG11\({}^{+\text{NaNO}_{3}}\) media were about 65%, 79%, and 75%, respectively.