Optimization of iron, phosphate, and salinity in nutrient medium using response surface methodology for enhancing biochemical composition in Chlorella sp. culture

Microalga Chlorella sp. is a highly scientifically and commercially attractive unicellular microorganism and has developed a stable industry as a nutritional supplement for people and animals. Various nutrient media have been used to grow Chlorella sp. cultures to enhance their growth, pigmentation, and lipid content. However, the optimal biochemical composition and density of Chlorella sp. cultures require an understanding of optimizing the nutrient medium to study their production. The present study aims to investigate the effects of iron, phosphate, and salinity concentrations by working synergically in a nutrient medium on the growth responses, pigments, and lipid accumulation of Chlorella sp. culture using a response surface methodology (RSM) approach. Using the result from the RSM software, a total of 18 experimental groups (E1 – E18) were evaluated, and one confirmation (C) study was conducted. The results revealed that the E7 experiment in Chlorella sp. culture provided the highest cell density and specific growth rate (SGR) with 176.00 × 10⁶ cells mL⁻¹ and 0.35 day⁻¹, respectively. Similarly, the E5 and E1 experiments produced the highest cell density of 166.10 × 10⁶ cells. mL⁻¹ and 152.13 × 10⁶ cells mL⁻¹, respectively. The SGR was also increased at 0.33 day⁻¹ in the E5 experiment and 0.33 day⁻¹ in the E1 experiment. Consequently, the culture of Chlorella sp. containing high iron and phosphate concentrations and lower salinity in a nutrient medium had the highest number of cells, SGR, and pigment accumulation (chlorophyll a and total carotenoid). In addition, the presence of high salinity concentrations reduces Chlorella sp. growth. However, the increase in the growth of Chlorella sp. culture did not indicate an increase in other biochemical compositions. In some cases, biochemical compositions are high due to nutritional limitations or stress factors. For example, in pigment accumulation, chlorophyll a pigment accumulation was increased in experiment E7 (51.57 μg mL⁻¹), while total carotenoid accumulation was increased in experiment E8 (20.68 μg mL⁻¹). In addition, increasing salinity concentration increased chlorophyll a and total carotenoid contents per cell, but decreased Chlorella sp. growth as shown in the E4 experiment, which achieved chlorophyll a levels of 1.38 pg cell⁻¹ and total carotenoid levels of 0.52 pg cell⁻¹. Furthermore, Chlorella sp. culture produces a higher lipid accumulation of 37.38 % in the E3 experiment. Hence, the results of this study contribute to understanding the optimal biochemical composition and cell growth of Chlorella sp. cultures.