Previous study has shown that Spirulina has a developed CO₂ enrichment mechanism (CCM), which can actively pump enough HCO₃ ⁻ into cells to improve intracellular CO₂ concentration and promote carbon utilization efficiency (Li et al., 2020). In this study, the used microalgae were mutagenic Spirulina platensis which was from Zhejiang University.

Mutagenic Spirulina platensis was initially precultured with Zarrouk medium. After 5 days of culture, the mutagenesis Spirulina platensis in the logarithmic growth phase was used as inoculum for the formal experiment.

The chemical absorbents selected in this paper were Na₂CO₃ and K₂CO₃, respectively, and the corresponding NaHCO₃ and KHCO₃ were formed after fully absorbing CO₂. .1 mol/L、0.2 mol/L and .3 mol/L NaHCO₃ and KHCO₃ were added to the basal medium as carbon sources, respectively, to investigate the effects of different carbon sources on the growth of mutagenic Spirulina in CAMC system, as shown in Table 1. Microalgae cultivation was preceded in 250 ml serum bottles with 200 ml culture medium. The incubator was placed at a constant temperature of 30°C ± 1°C and illuminated all day at a light intensity of 4,000 Lux. Three parallel experiments were performed in each group and shake the bottle at a set time each day. The biomass, pH, inorganic carbon and other experimental indexes were measured every 3 days.

The standard curve of mutagenic Spirulina biomass was obtained based on the linear relationship between dry weight and OD₅₆₀. Firstly, dilute the algae solution with deionized water to OD₅₆₀ = .2, .4, .6, .8, 1.0, and then measure 100 ml, respectively. The filter membrane with a pore size of .45 μm was dried in an oven at 105°C to a constant weight. Then the filter membrane was weighed and the weight was recorded. The measured 100 ml of algae solution was filtered through the membrane, and the membrane was again dried in an oven at 105°C to a constant weight and the weight was recorded. The difference between the two membrane weights was the dry weight of mutagenic Spirulina platensis biomass. The standard curve was as following: X b i o m a s s = 0.404 • O D 560 − 0.0016 , R 2 = 0.9988 (1)

Carbon fixation rate (P_CO2, mg/L/d) was calculated by referring to (Song et al., 2019c). P C O 2 m g / L / d = P b i o m a s s · C a l g a e · M C O 2 / M C (2) Hereinto, P_biomass was the biomass yield (mg/L/d), C_algae was the carbon content in mutagenic Spirulina, M_CO2 and M_C were the molar mass of CO₂ molecule and C atom, respectively.

Carbon utilization efficiency was calculated according to the method mentioned by (Ding et al., 2017): E C = 100 · C a l g a e · X b i o m a s s / 12 / 84 · D C (3)

E_C represented carbon conversion efficiency (%), X_biomass represented the biomass dry weight (g/L), D_C represented the consumption of NaHCO₃ or KHCO₃ (g/L), and C_algae represented the carbon content of mutagenic Spirulina platensis (%).

The lipid content was determined by Nile red staining. 2.55 ml algae solution was added with 450 μL dimethyl sulfoxide and 24 μL Nile red solution. It was placed in a darkroom at 30°C for 10 min, and 580 nm was selected as the excitation fluorescence detection wavelength (Bertozzini et al., 2011).

20 mg of mutagenic Spirulina powder was added to the test tube, and 4 ml of deionized water was added to the water bath at 100°C for 2 h. The supernatant was then filtered through a vacuum pump, 1 ml of the filtrate was taken and 4 ml of ethanol was added, and it was placed in a refrigerator at 4°C for 12 h. Finally, the content of polysaccharide was determined by phenol sulfuric acid method (Gasljevic et al., 2008).

Chlorophyll a and b and carotenoids of mutagenic Spirulina were determined by spectrophotometer. The detail determination method is as follows: 5 ml uniform algal liquid was centrifuged at 5,000 rpm for 10 min. Then, the supernatant was removed, 5 ml methanol (90%) was added, extracted at 4°C for 24 h, then centrifuged at 5,000 rpm for 10 min, the supernatant was taken, and the absorbance of the supernatant was measured at 665, 652 nm and 470 nm by UV–visible spectrophotometer. The contents of chlorophyll a and b and carotenoids in microalgae were calculated by using the Formulas (Eqs. 4–6). C h l o r o p h y l l   a   m g / L = 16.82   A 665   –   9.28   A 652 (4) C h l o r o p h y l l   b   m g / L = 36.92   A 652   –   16.54   A 665 (5) C c a r o t e n o i d   m g / L = 1000   A 470   –   1.91   C a   –   95.15   C b / 225 (6)

After the cultivation and harvesting, the algae powder was dried, and the algae powder was analyzed and measured. The proportion of N element in biomass N_algae (%) was obtained. The content of protein X_protein (mg/L) was calculated according to the relationship between nitrogen content and protein production multiplied by the coefficient 6.25. X_biomass (g/L) was the dry weight of mutagenic Spirulina platensis biomass at the end of the cycle (Li et al., 2020). X p r o t e i n = X b i o m a s s · N a l g a e · 6.25 (7)

One-way analysis of variance (ANOVA) was used for statistical analysis. Experimental results are expressed as mean ± standard error. The mean is based on parallel experiments and is within a 95% confidence interval.

The biomass dry weight curve was shown in Figure 1. The growth of mutagenic Spirulina in the CAMC system with different inorganic carbon concentrations were similar in the first 9 days, indicating that the three concentration ranges of NaHCO₃ and KHCO₃ (.1, .2, .3 mol/L) could maintain the growth of mutagenic Spirulina in the CAMC system. After the 9th day, the growth trend of mutagenic Spirulina began to show obvious changes. The biomass of mutagenic Spirulina with .1 mol/L and .2 mol/L NaHCO₃ and KHCO₃ tended to be stable, while the biomass of mutagenic Spirulina with .3 mol/L NaHCO₃ and KHCO₃ still increased rapidly. After 18 days of culture, the biomass dry weight of Na-.3 mol/L group was the highest, reaching 2.29 g/L, while that of K-.3 mol/L group was 2.00 g/L. Therefore, compared with K₂CO₃, Na₂CO₃ as a CO₂ chemical absorber was more conducive to the growth of mutagenic Spirulina in CAMC system, and the optimal concentration was .3 mol/L.

The change of pH in the CAMC was shown in Figure 2. HCO₃ ⁻ will enter cells by active transport under the action of transporters, and the pH of the solution will change with the consumption of HCO₃ ⁻. After HCO₃ ⁻ enters the cell, it will react with intracellular H⁺, leading to an increase in the concentration of OH⁻ in the internal environment of cell. H⁺ in extracellular solution will transmembrane neutralize OH⁻ in cells, leading to an increase in pH (Chi et al., 2013). This process involves one or more CA that facilitate the conversion between HCO₃ ⁻ and CO₂. Therefore, the pH of each group showed an upward trend during the first 9 days of culture. However, the pH of the CAMC system began to decrease on 9^th day. This could be caused by more CO₃ ^2- in the system, which absorbed CO₂ from the air. After 18 days of culture, the pH of different groups was stable between 8.5 and 10, but the range of pH change was relatively stable in the CAMC system with .3 mol/L HCO₃ ⁻.

Chlorophyll a can be used as an indicator to evaluate the photosynthetic capacity of microalgae. The change of chlorophyll a content of mutagenic Spirulina was shown in Figure 3. At the first 9 days of culture, the chlorophyll a content of mutagenic Spirulina increased with the growth of biomass. In the Na-.2 mol/L group, the chlorophyll a content of mutagenic Spirulina reached the maximum on the 9^th day, reaching 11.05 mg/L. After 9 days of culture, the content of chlorophyll a of mutagenic Spirulina showed a downward trend. This may be related to the change of solution pH, which affects the activity of pigment synthetase and the synthesis of chlorophyll a. After 15 days of culture, the chlorophyll-a content of mutagenic Spirulina decreased obviously, which may be caused by the insufficient nitrogen source in the medium. Under the condition of nitrogen limitation, the activities of enzymes involved in the degradation of organic nitrogen sources in mutagenic Spirulina were enhanced, which could degrade the nitrogen pigments in the cells to maintain their normal metabolism (Pancha et al., 2014; Depraetere et al., 2015).

The concentration of inorganic carbon in the solution will change with the growth of mutagenic Spirulina, and the content of inorganic carbon and carbon sequestration rate in the solution of each group was shown in Figure 4A. After 18 days of culture, the inorganic carbon content of Na-.1 mol/L group decreased from 1,200 mg/L to 970 mg/L, and the carbon utilization efficiency was 15.79%. The concentration of inorganic carbon in Na-.2 mol/L group decreased from 2,400 mg/L to 1998 mg/L, and the carbon utilization efficiency was 26.45%. The highest carbon utilization efficiency (26.71%) was obtained in Na-.3 mol/L group, and the inorganic carbon concentration decreased from 3,600 mg/L to 2,531 mg/L. The carbon utilization efficiency of K-.1 mol/L, K-.2 mol/L and K-.3 mol/L groups were 17.25%, 20.38% and 25.49%, respectively. This indicated that NaHCO₃ was more conducive to carbon utilization by mutagenic Spirulina in CAMC system.

The carbon fixation rate of mutagenic Spirulina in CAMC system was shown in Figure 4B. The carbon fixation rate of mutagenic Spirulina in Na-.1 mol/L, Na-.2 mol/L and Na-.3 mol/L groups were 87.83 mg/L/d, 128.55 mg/L/d and 230.36 mg/L/d, respectively. It was much higher than the 15 mg/L/d and 17 mg/L/d obtained by Song et al. who using NH₄HCO₃ as carbon source (Song C. et al., 2019). (Khoobkar et al., 2022) reported similar carbon fixation rate when cultivating Chlorella sp. in a photobioreactor. While the carbon fixation rate of mutagenic Spirulina in K-.1 mol/L, K-.2 mol/L and K-.3 mol/L groups were 86.58 mg/L/d, 113.74 mg/L/d and 153.41 mg/L. This indicated that the carbon fixation rate in NaHCO₃ system was higher than that in KHCO₃ system, which was also consistent with the dry weight of biomass.