Screening of mutant strains
The tolerance of microalgae to high CO2 concentration depends on its tolerance to low pH. A total of 43 viable mutants were screened by low pH medium in about 5000 micropores, named Ulothrix SDJZ-17-A1、SDJZ-17-A2……SDJZ-17-A43, and the wild strain was named SDJZ-17-WT. After Nile red staining of wild and mutant strains, they were detected in a fluorescence microplate reader, and the relative fluorescence intensity of each algae strain was calculated as shown in Fig. 1.
It can be seen from Fig. 1 that the fluorescence intensity of a total of 13 mutant strains exceeded that of the wild strain, and five strains with the highest fluorescence intensity were screened out, including A20 (1.73), A30 (1.47), A23 (1.39), A29 (1.29) and A6 (1.18). These five mutants can survive in a low pH medium and have high neutral lipid fluorescence intensity, and are selected as candidate algae strains for CO2 fixation and biodiesel production.
Genetic stability screening
The selected 5 mutants were continuously cultured for 5 generations under high concentration CO2 culture conditions in Section 1.3, and the biomass concentration and lipid content at 7 days of harvest were detected as shown in Fig. 2. By comparing their growth curves and lipid content under (15% v/v) conditions, it can be seen that A29 cannot maintain the genetic stability of normal growth under high CO2 concentration, and A6, A29 and A30 cannot maintain the genetic stability of oleaginous. Therefore, only A20 and A23 were retained as candidate mutant strains.
Growth characteristics
Comparing the growth curve shown in Fig. 3 with the detailed parameters shown in Table 2, it can be seen that the wild-type strain WT and the mutant strains A20 and A23 can grow normally in the air. There was no significant difference in Xmax、µmax and P among the three strains, but the µmax of A20 and A23 appeared on the second day, indicating that their adaptation period was slightly longer than that of WT, and the latter had almost no adaptation period when growing in the air. In order to overcome the problem of low CO2 concentration in the air, almost all algae activate the key enzyme of photosynthesis (Rubisco)-ribulose-1,5-bisphosphate carboxylase/oxygenase through CO2 concentration mechanisms (CCMs) to promote carbon assimilation [26, 27], WT, A20 and A23 are no exception. This indicates that the CCMs mechanism in A20 and A23 has not been destroyed, unlike Chlorella vulgaris SDEC-3M[28] and Synechococcus PCC7942[29], which have high CO2 demand characteristics due to the destruction of CCMs mechanism.
Table 2
The maximum biomass concentration (Xmax), maximum specific growth rate (µmax) and maximum biomass yield (Pmax) of Ulothrix SDJZ-17 wild type ( WT ) and mutants ( A20 and A23 ) cultured in air and 15% (v/v) CO2 for 7 days.
Microalgae strain | Xmax (mg L− 1) | µmax(d− 1) | Pmax (mg L− 1 d− 1) |
Air | 15% CO2 | Air | 15% CO2 | Air | 15% CO2 |
WT | 455.49 ± 13.6(7)b | 230.95 ± 13.49(6)a | 0.42 ± 0.06(1)b | 0.26 ± 0.01(4)a | 55.82 ± 2.49(6)c | 26.57 ± 2.25(6)a |
A20 | 426.34 ± 14.95(7)b | 582.94 ± 48.93(7)c | 0.4 ± 0.05(2)b | 0.43 ± 0.05(2)b | 51.57 ± 2.34(6)bc | 74.68 ± 3.33(6)d |
A23 | 413.66 ± 12.75(7)b | 625.14 ± 24.97(7)c | 0.4 ± 0.07(2)b | 0.57 ± 0.09(1)c | 48.93 ± 1.82(7)b | 81.26 ± 4.57(6)e |
Note : Each data represents the mean ± standard deviation from three independent cultures. Each value in the parentheses represents the time (d) when the parameter reaches its maximum. The 6 sets of data under the same parameters were marked with different letters, indicating that the results were significantly different by Duncan test ( p < 0.05 ). |
However, the growth of WT under 15% CO2 was significantly worse than that in air, and Xmax, µmax and P decreased by 49.30%, 37.83% and 52.39%, respectively, µmax appeared on the 4th day and required a long adaptation period. This should be caused by the decrease of enzyme activity caused by 'anesthesia' and acidification culture of microalgae cells under high CO2 concentration (i.e., growth under low CO2 concentration in air)[30]. Most wild Ulothrix are typically aerogenic and have low tolerance to CO2[31], and are therefore not generally considered as potential candidates for biomass and biofuel production.
Fortunately, the mutated mutant improved the cell's tolerance to CO2. The Xmax, µmax and Pmax of A20 and A23 grown under 15% CO2 conditions were significantly increased by 152.41% and 170.69%, 63.52% and 116.81%, 181.02% and 205.80%, respectively, compared with WT, and also increased by 40.92% and 170.69%, 7.44% and 116.81%, 40.92% and 170.69%, respectively, compared with their growth under air conditions. This shows that when A20 and A23 have high CO2 tolerance, a high concentration of CO2 provides a sufficient carbon source, balances the increase of pH value caused by microalgae growth, and omits many benefits such as CCMs process to reserve biomass and saves energy, which promotes the growth of microalgae[26, 28]. In fact, under the condition of high concentration of CO2, the growth rate of high CO2 tolerant microalgae is generally higher[26, 28, 30], and a higher culture economy is obtained. Moreover, in terms of biomass accumulation, A23 is better than A20.
It can also be seen from Table 2 that Pmax is mostly obtained on the 6th day. Therefore, as far as biomass production is concerned, under the experimental conditions, it can be harvested on the 6th day, not the longer the culture time, the better.
Production of biochemical components
Due to the randomness of the mutation, the biochemical components of the cells may have undergone synchronous changes. Microalgae are raw materials for food, fuel and other biochemical products.[32] The content and yield of its biological components are key parameters for predicting its economic potential.[32]As shown in Fig. 4 (1), there were significant differences in the total lipid content data of the six groups obtained by the three Ulothrix strains under air and 15% CO2 conditions. The total lipid content of A20 and A23 was significantly higher than that of WT, and the total lipid content obtained under 15% CO2 conditions was higher than that in air. The data of Fig. 4 (2) reflecting the yield obtained the same results. Among them, A20 obtained the highest total lipid content of 22.45 ± 1.09% under the condition of 15% CO2, which was 138.63% and 111.96% higher than that of WT under air and 15% CO2 conditions, respectively. In terms of yield, A20 also obtained the highest total lipid yield of 16.43 ± 2.33 mg L− 1 d− 1 under 15% CO2 conditions, which was 217.98% and 668.56% higher than that of WT under air and 15% CO2 conditions, respectively. These results indicate that the two Ulothrix mutants indeed enhance the tendency of lipid production, and high concentrations of CO2 can promote the tilt of intracellular carbon and energy to lipid synthesis[28, 33, 34]. The high concentration of CO2 adaptation and high biomass yield of the mutant further enhanced its lipid production capacity. The total lipid content and yield of some oleaginous green microalgae can reach 30% and 30 mg L− 1 d− 1, respectively[35]. Although A20 has a certain gap with the data, considering that Ulothrix strains with lipid content exceeding 10% have never been reported, the performance of A20 in lipid accumulation is still satisfactory. Moreover, this is data under stress conditions such as no nitrogen starvation, and its lipid production potential is still expected.
It can be seen from Fig. 4 (1) that although the lipid content of the Ulothrix wild strain is not high, it has rich carbohydrate and protein content. Although the mutant strain increased the lipid content at high CO2 concentration, there was no significant difference in carbohydrate content between the mutant strain and the wild strain. Although the protein content decreased, the difference was not as obvious as the lipid content. This shows that under the pressure of mutagenesis and high concentration of CO2, the mutant enhances the energy and carbon source required for lipid production, which is unrelated to carbohydrates, and partly reduces the energy and carbon source required for protein production. The high biomass yield of the mutant under high CO2 concentration made it obtain higher carbohydrate yield, A20 and A23 reached 23.08 ± 2.43 mg L− 1 d− 1 and 26.76 ± 1.29 mg L− 1 d− 1, respectively. Lipids and carbohydrates are raw materials for the production of two biofuels, biodiesel and bioethanol[3, 19, 34]. Therefore, A20 and A23 are more suitable as a composite feedstock for biodiesel and bioethanol production than as a single feedstock for biodiesel.
Photosynthetic efficiency
Light conversion efficiency (LCE) and maximum PSII quantum yield (Fv/Fm) are two important parameters for analyzing photosynthetic efficiency. As shown in Table 3, there was no significant difference in LCE and Fv/Fm between WT and A20 and A23 under air conditions, and their LCE was precisely the range of LCE distribution in most microalgae–4–9%[36]. The results showed that the mutation did not change the efficiency of photosynthesis under air conditions in both wild and mutant strains of Ulothrix SDJZ-17. However, under the condition of 15% CO2, the LCE and Fv/Fm of WT were significantly lower than those under air conditions, while the A20 and A23 were the opposite. Among them, A23 obtained the highest LCE (14.79 ± 0.48%) and Fv/Fm (71.04 ± 1.62%), respectively. The results obtained by A20 were slightly lower than those of A23, but there was no significant difference. The Fv/Fm value reflects the potential maximum quantum efficiency of PSII, which is related to the peripheral antenna complex. As described in Section 2.3, WT had poor tolerance to high CO2 concentration, which affected its photosynthetic efficiency. The mutant's peripheral antenna complex has undergone significant changes, resulting in high CO2 concentration tolerance. In an environment with a high concentration of CO2, algal cells can obtain sufficient carbon sources under low energy consumption, which greatly improves the efficiency of photosynthesis. The results showed that the effect of CO2 concentration on the photosynthetic efficiency of SDEC-2M was more significant than that of wild strain. Therefore, CO2 level plays an important role in improving the energy storage compounds, namely starch and lipid production of microalgae strains with high CO2 tolerance.
In addition, it can be observed from the data in Table 3 that the HHV of the algae strains at 15% CO2 level was higher than that in air culture, but the wild strains showed no significant difference. Previous studies have confirmed that HHV is highly correlated with lipid content. Therefore, this should be the result of algae cells cultured under high concentrations of CO2, which accumulated more lipids.
Table 3
High calorific value (HHV), light conversion efficiency (LCE) and maximum quantum yield of PSII (Fv/Fm) of Ulothrix SDJZ-17 wild type (WT) and mutant strains (A20 and A23) cultured in air and 15% (v/v) CO2 for 7 days.
Microalgae strain | HHV (kJ g− 1) | LCE (%) | Fv/Fm (%) |
Air | 15% CO2 | Air | 15% CO2 | Air | 15% CO2 |
WT | 19.19 ± 0.72a | 20.09 ± 0.06ab | 8.75 ± 0.3b | 3.37 ± 0.4a | 64.17 ± 2.53b | 56.74 ± 0.18a |
A20 | 21.2 ± 0.76b | 23.48 ± 0.85c | 8.94 ± 0.22b | 14.23 ± 1.26c | 64.32 ± 3.72b | 67.6 ± 2.79bc |
A23 | 20.09 ± 0.51ab | 22.5 ± 0.43c | 8.17 ± 0.45b | 14.79 ± 0.48c | 63.8 ± 3.46b | 71.04 ± 1.62c |
Note: Each data represents the mean ± standard deviation from three independent cultures. The 6 groups of data under the same parameters were marked with different letters, indicating that the results were significantly different by the Duncan test (p < 0.05). |