# The Microstructures of _Tetradesmus obliquus_ Biofilms Formed Under Different Nutrient Supplies
## 1 Introduction  
Microalgae can use light, CO2, and nutrients to produce valuable organics via photosynthesis, such as lipid, protein, xanthophylls, and astaxanthin [1, 2, 3]. Due to their fast growth rate, low nutrient requirements, and high content of valuable biocompounds, microalgae have been considered one of the promising biological resources to produce biofuels and other biological products [4, 5, 6, 7]. Presently, microalgae are generally cultured in either a suspended system or a biofilm-based system. In a suspended system, microalgae are cultured suspendedly in large amounts of water containing nutrients [8]. In a biofilm-based culture system, microalgae aggregate together and form biofilms on substrates [9]. Previous studies suggested that, compared with suspended systems, biofilm-based systems had the advantages of high biomass productivity, low water consumption, and easy harvesting, thus attracting much interest in biotechnology and environmental engineering [10, 11, 12]. Therefore, it is important to develop efficient microalgae biofilm-based culture systems for promoting the microalgae industry. In the biofilm-based culture systems, microalgal cells are first inoculated onto substrates that are submerged in or floated on aqueous culture media; then, the cells grow and form biofilms by adhering to substrates and sticking to each other. Generally, due to the cell-cell interactions and the influence of culture conditions (i.e., hydrodynamics, substrates, temperature, gas, and nutrient supplies), the formed microalgae biofilms always possessed complex three\(-\)dimensional (3D) microstructures [13; 14; 15; 16]. The microstructure of microalgae biofilms influences the transport of light, gases, and nutrients within the biofilm, directly affecting cellular photosynthetic activity and lipid accumulation [17], which in turn impacts lipid productivity. Additionally, these microstructures also influence the rheological properties, apparent wettability, and overall life cycle of the biofilm, which critically affects microalgae biomass harvesting costs and the economic viability of microalgae\(-\)based biodiesel technologies [18; 19; 20; 15]. Therefore, it is essential to understand the microstructures of microalgae biofilms at different culture conditions to develop more efficient and sustainable microalgae biofilm-based culture systems. Recently, some researchers have studied the microstructures of microalgae biofilms under certain conditions. [PERSON] et al. [21] found that, at low shear stress, _Chlorella vulgaris_ formed heterogeneous and loose biofilms, while, at high shear stress, cells formed thick and compact biofilms. [PERSON] et al. [22] reported that the biofilms of _Chlorella_ sp. cultured at lower light intensities (20 and 50 \(\upmu\)mol\(\cdot\)m\({}^{-2}\cdot\)s\({}^{-1}\)) were more porous; in contrast, under higher light intensities (100, 200, and 400 \(\upmu\)mol\(\cdot\)m\({}^{-2}\cdot\)s\({}^{-1}\)), the biofilms presented no visible voids and were more compact. [PERSON] et al. [18] found that _Tetradesmus obliquus_ cultured under white light formed biofilms with many inner voids and high surface roughness; while, under red and blue lights, these cells formed homogeneous and compact biofilms with low porosity. [PERSON] et al. [23] reported that microalgae cultured on different agar substrates formed different biofilm microstructures by varying their cell surface properties and cell-cell interactions. The cells with low surface energy formed heterogeneous biofilms with many voids; whereas, cells with high surface energy formed flat biofilms with low porosity. The above results suggested that microalgae biofilms would present diverse 3D microstructures at different shear stress, lighting conditions, or substrates, which may be due to the external hydrodynamic conditions, cell surface properties, and cell-cell interaction.