## 3 Results and Discussion  
### Growth and Ultrastructure of C. reinhardtii  
The growth kinetics of _C. reinhardtii_ was determined during 30 days of cultivation for quality monitoring of _C. reinhardtii_ batch cultures to achieve a stable and steady quality of the biomass used in subsequent analysis (Figure 1A). Figure 1A shows cell growth number reached \(1.68\times 10^{7}\) cell/mL with a doubling time of 9.35 h. It was reported that the doubling time of _C. reinhardtii_ ranges between 7 and 17 h and it depends on many factors including strain type and growth condition (light duration and intensity, temperature, media nutrient, etc.) [35; 36].  
Four distinct phases were recognised during _C. reinhardtii_ growth, which resembles the expected microalgae growth curve when total cells are measured. The first phase is the lag or induction phase, which is characterised by a slight increase in cell density due to the time required for the cells' metabolism to adapt to the new environment such as enzymes level and metabolites required for cell division and photosynthesis. This phase lasted for around 2 days when cell number of _C. reinhardtii_ reached \(0.2\times 10^{7}\) cells/mL. The second phase is the logarithmic (exponential) phase when cell density increment follows a logarithmic function. This phase lasted for around 3 days with a maximum cell number of \(1.8\times 10^{7}\) cells/mL. The third phase is the early stationary phase, which starts when the media limiting factors and the cell division rate are balanced, resulting in a relatively constant cell density. The cells ranged from\(1.8\times 10^{7}\) to \(1.2\times 10^{7}\) cells/mL during the 5 days of this phase. The fourth phase is the late stationary or aged culture, which comprised the last 20 days of the cultivation. During this phase, the change in chemical and physical factors limits the growth and slows down the cell division pace. The cell number slightly decreased to a range between \(1.2\times 10^{7}\) and \(1.4\times 10^{7}\) (Figure 1A). Similar results for cell count was obtained throughout the growth curve using haemocytometer and spectrophotometer. This confirms the accuracy of the results as well as the methods used.  
Representative ultrastructure of TEM images are also presented in Figure 1B. The images revealed different cell sections with a detailed morphology and localisation of each intracellular cell organelles. The round-shaped images resulted from a section across the width, while the oval-like images represent a section across the length [37]. The sheet-like thylakoid membranes (TM) are the main component of the chloroplast, the organelle that occupies two-thirds of the cells grown under standard conditions, which is clear in Figure 1B, where chloroplasts occupy a significant part of the cell volume. The eyespot, as it appears in Figure 1B, is attached to the inner plastid membrane. In cells grown in nutrients replete media to stationary phase (Figure 1 Ba-c), all the cell organelles are observable in different sections. In addition, few starch granules were detected at the stationary phase, while no lipid bodies were identified. Conversely, both storage entities (starch granules and lipid bodies) are abundant at 30-days old cells, while TM are less noticeable (Figure 1 Bd-f). It is well established that lipid accumulation as TAG in microalgae increases as the cells age, and generally as a result of nutrient consumption during the stationary growth and is accompanied by a cessation in cell division [38].