A microalgae capable of tolerating high concentrations of carbon dioxide and its application

Abstract

The present invention discloses a microalgae strain capable of tolerating high concentrations of carbon dioxide and its applications. The strain is named Coelastrum sp. GX03, with a deposit number of CCTCC NO: M20241314 and a date of June 20, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. This strain can tolerate high concentrations of carbon dioxide and exhibits strong CO2 fixation and lipid production, suggesting promising applications.

Description

Technical Field

[0001] The present invention relates to the field of biological treatment of pollutants, and in particular to a microalgae capable of tolerating high concentrations of carbon dioxide and applications thereof. Background Art

[0002] Microalgae have great potential in reducing carbon dioxide (CO2) emissions and utilizing it as a resource. Studies have shown that for every ton of microalgae biomass produced, approximately 1.8 tons of CO2 can be fixed. At the same time, the biomass produced by microalgae carbon fixation has high application value. Therefore, microalgae carbon fixation is considered to be a carbon capture, utilization, and storage technology (CCUS) with large-scale application prospects and economic feasibility. However, microalgae have limited tolerance to high concentrations of CO2. It is generally believed that CO2 concentrations exceeding 10% will be toxic to microalgae. However, the CO2 concentration in many waste gases exceeds this value. For example, the CO2 concentration in biogas ranges from 10% to 60%, and in industrial flue gas it even reaches 10% to 99%. Therefore, it is necessary to screen microalgae that can tolerate high concentrations of CO2 to fix CO2 in these pollution sources. Summary of the Invention

[0003] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a microalgae that can tolerate high concentrations of carbon dioxide.

[0004] Another object of the present invention is to provide applications of the microalgae that can tolerate high concentrations of carbon dioxide.

[0005] The purpose of the present invention is achieved by the following technical solution: a microalgae that can tolerate high concentrations of carbon dioxide is named Coelastrum sp. GX03, with a preservation number of CCTCC NO: M20241314 and a preservation date of June 20, 2024, and the preservation unit is the China Type Culture Collection located at Wuhan University, Wuhan, China.

[0006] The application of the microalgae capable of tolerating high concentrations of carbon dioxide in carbon fixation is to fix CO2 using the single-cell microalgae; preferably, the application comprises the following steps:

[0007] (1) culturing the microalgae capable of tolerating high concentrations of carbon dioxide to a logarithmic growth phase or a stationary phase to obtain a microalgae liquid;

[0008] (2) Cultivating the microalgae liquid obtained in step (1) in a CO2 environment to perform carbon fixation.

[0009] The culture conditions described in step (1) are preferably: temperature 25-30°C, light 6000-8000lx, and a light-dark time ratio of 8-16h:8-16h; more preferably: temperature 28°C, light 7000lx, and a light-dark time ratio of 16h:8h.

[0010] The high concentration of carbon dioxide refers to a CO2 concentration of preferably 50-70% (v / v), more preferably 60% (v / v). The concentration of CO2 is controlled by a mixed gas flow ratio device of pure CO2 and dry air.

[0011] The culture medium used in the culture in step (1) is preferably BG11 liquid culture medium;

[0012] The composition of BG11 liquid medium is as follows: K2HPO 4. 3H2O 0.04g / L, MgSO 4. 7H2O 0.075g / L, CaCl 2. 2H2O 0.036g / L, citric acid 0.006g / L, ammonium ferric citrate 0.006g / L, EDTA 0.001g / L, Na2CO3 0.02g / L, NaNO3 1.5g / L, trace element A5 1mL.

[0013] The composition of trace elements A5 is as follows: H3BO3 2.860g / L, NaMoO 4. 2H2O 0.021g / L, ZnSO4 . 7H2O0.222g / L, CuSO4 . 5H2O 0.079g / L, MnCl2 . 4H2O 1.810g / L, NiSO4 . 6H2O 0.479g / L.

[0014] The culture medium used in the culture described in step (2) is preferably a BG11 liquid culture medium without the addition of inorganic carbon (ie, without Na2CO3).

[0015] The concentration of the microalgae solution in step (2) is preferably OD 680 =0.1~0.4; more preferably OD 680 =0.3.

[0016] The culture conditions described in step (2) are preferably at 28.0±0.5°C, 10000±1000lx of light, and a day-night ratio of 24h:0h.

[0017] The concentration of CO2 in the CO2 environment described in step (2) is preferably 50-70% (v / v); more preferably 60% (v / v).

[0018] The microalgae capable of tolerating high concentrations of carbon dioxide are used in CO2 fixation and lipid production.

[0019] The present invention has the following advantages and effects compared to the prior art:

[0020] The present invention obtains a single-cell green algae through separation and purification. Studies have shown that it can tolerate an environment with a pH of 4.5 and a CO2 concentration of up to 60%, has strong carbon fixation and lipid production capabilities, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a photo of the screening experiment of algae strains that tolerate different pH values.

[0022] Figure 2 These are photos of the algae strain screening experiment that tolerates 60% CO2; among them, A is the initial state of the first batch of culture, B is the final state of the first batch of culture, C is the initial state of the third batch of culture, and D is the final state of the third batch of culture.

[0023] Figure 3 This is a graph showing the effect of 60% CO2 on the growth, pH, and chlorophyll fluorescence parameters of different microalgae; where A is the OD value, B is the pH value, C is the Fv / Fm value, and D is the Pi_Abs value.

[0024] Figure 4 is a picture of algae morphology; from left to right are a picture of BG11-agar solid plate and a picture of optical microscope. DETAILED DESCRIPTION

[0025] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0026] Example 1: Isolation, purification and identification of microalgae

[0027] (1) Isolation and purification of microalgae

[0028] GX01, GX02, and GX03 were sourced from surface soil in the Nandan mining area in Guangxi. HH01 was sourced from activated sludge from the Hanhai Wastewater Treatment Plant in Heshan, Guangdong Province.

[0029] Algae isolation and purification: 20 g of surface soil from the Nandan mining area in Guangxi and 20 mL of activated sludge from the Hanhai Wastewater Treatment Plant in Heshan, Guangdong, were added to 80 mL of BG11 liquid culture medium, respectively. The culture was pre-incubated in a light-incubator at 28.5°C, 4000 lx of light, and a 12 h:12 h light-dark ratio. After 10 days of incubation, the culture was shaken and 0.5 mL of the culture medium was spread onto five 9 cm diameter plates containing sterile BG11 agar solid medium. The plates were incubated upside down in the incubator under the above conditions. Once visible algae grew, they were carefully picked out and re-plated for microscopic examination. This process was repeated multiple times to obtain a single culture of microalgae. Finally, the microalgae were treated with a combination of five antibiotics: chloramphenicol, gentamicin, kanamycin, penicillin hydrochloride, and streptomycin, as well as cycloheximide. Before use, the stock solutions were prepared and filtered through a sterile, disposable 0.22 μm Millipore filter. The final concentration of each antibiotic was 0.1 mg / L, and the final concentration of cycloheximide was 5 mg / L. Algae were inoculated at a 1% (v / v) inoculum in BG11 liquid medium containing the six antimicrobial agents listed above for at least three consecutive inoculations, each incubation lasting six days. Finally, streaks were made onto 9 cm diameter surfaces of BG11 solid agar, PDA, and NA medium. After 20 days of incubation, the cells were checked for bacterial presence. If bacterial presence was detected, the purification steps were repeated until sterility was achieved.

[0030] Example 2: Screening for acid-resistant algae strains

[0031] The algae strains GX01, GX02, GX03 and HH01 were isolated and purified from Example 1, and acid-resistant algae strains were screened. The specific steps were as follows: the pH of the BG11 liquid culture medium was adjusted for culture screening. The initial OD 680 The value was 0.1, and the culture was carried out in a light incubator at a temperature of 28°C, a light intensity of 7000 lx, and a light-dark time ratio of 16h:8h. The results are shown in Table 1 and Figure 1 : All four algae strains survived at pH 7.6 and 4.5, but died at pH 3.

[0032] Table 1 Cultivation and screening of acid-resistant algae strains

[0033]

[0034] Note: Different letters in the same initial pH treatment indicate significant differences in the relevant parameter values ​​of different algae (p<0.05); Fv / Fm represents the maximum photochemical quantum yield of PSII.

[0035] Example 3: Screening of algae strains acclimated to 60% CO2 tolerance

[0036] According to the screening results, algae strains GX01, GX02, GX03 and HH01 can survive under the conditions of medium pH = 7.6 and pH = 4.5. The above four algae strains were selected for screening of algae strains tolerant to 60% CO2. Batch culture screening was carried out by bubbling 60% CO2 in carbon-free BG11 liquid medium. The initial OD 680 The value is 0.1, and the results are shown in Table 2 and Figure 2 The first batch of culture showed that Chlorella HH01 died, and 3 strains of algae remained. The second and third batches of screening culture were transferred from the previous batch as algae seeds.

[0037] Table 2 Screening of algae strains tolerant to 60% CO2 by three consecutive batch cultures

[0038]

[0039]

[0040] Note: Pi_Abs is the PSII performance index of algae.

[0041] Example 4: Evaluation of the Ability of Algae to Treat High Concentrations of CO2 and to Produce Bioenergy

[0042] Based on the screening results, algae strains GX01, GX02, and GX03 were selected for evaluation of their high-concentration CO2 treatment capacity and bioenergy production potential. The culture medium used was carbon-free BG11 medium, 400 mL per bottle was sterilized (121°C for 16 minutes), and then inoculated with algal cells (the concentration of algal cells in carbon-free BG11 medium was OD 680 =0.3) and simultaneously introduce 60% CO2. Culture in a light incubator for 8 days at a temperature of 28.0±0.5°C, a light intensity of 10,000±1,000 lx, a day / night ratio of 24h:0h, and shake 4-6 times a day.

[0043] OD 680 , pH value, Fv / Fm and Pi_Abs changes are shown in Figure 3 The results showed that GX01, GX02, and GX03 all achieved varying degrees of growth under 60% CO2 aeration conditions, with GX03 showing a higher growth rate. On day 8, its OD value was significantly higher than that of GX01 and GX02, indicating that this strain is more adaptable to high CO2 concentrations. Regarding pH, the continuous introduction of high CO2 concentrations caused a brief drop in the culture medium pH between days 0 and 2, followed by a gradual increase with algal biomass growth. After 8 days of culture, the pH stabilized at around 8, and the culture medium increased from slightly acidic to slightly alkaline.

[0044] The biomass, lipid production, and carbon fixation capacity of the three algae species under 60% CO2 aeration conditions are shown in Table 3. As shown in Table 3, under high CO2 conditions, the biomass yield and rate, lipid yield, and carbon fixation rate of algae GX03 were significantly higher than those of GX01 and GX02. Its lipid content was also significantly higher than that of GX02, but not significantly different from that of GX01.

[0045] Table 3 Algal biomass, lipid production and carbon fixation capacity under different treatments

[0046]

[0047]

[0048] Note: Different letters in the same column indicate significant differences (p<0.05), the same below.

[0049] The elemental composition, theoretical methane production potential, and calorific value of the three algae species under 60% CO2 aeration conditions are shown in Table 4. Table 4 shows that there were no significant differences in the C and H content and calorific value among the three algae species. GX03 had a significantly higher O content than GX01 and GX02, while its N and S content and theoretical methane production potential were significantly lower than those of GX01 and GX02. All three algae species had high theoretical methane production potential when producing algal biomass under high CO2 concentrations.

[0050] Table 4 Elemental composition (%) analysis, theoretical methane production potential (mL / g VS) and calorific value (MJ / kg)

[0051]

[0052] Example 5: Algae species identification

[0053] Through the above experiments, a strain of microalgae GX03 was selected that can tolerate high concentrations of CO2 and has strong CO2 fixation and lipid production capabilities. Figure 4 Shown are single-cell microalgae that can grow rapidly in BG11 medium.

[0054] Molecular identification: PCR amplification was performed using the 18S and ITS primer pairs shown below, followed by GenBank Blast comparison. The 18S results showed 100% similarity to the known algae strains Coelastropsis sp. YACCYB444 and Coelastrum sp. YACCYB414 (Table 5). ITS analysis showed the highest similarity, at 99.54%, to the known algae strain Coelastrum proboscideum. These results indicate that this algae belongs to the genus Coelastrum. Therefore, it was named Coelastrum sp. GX03, with a deposit number of CCTCC NO: M20241314 on June 20, 2024, with the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0055] 18S-F: 5'-GCGCGGTCATTAAATCAGTTATAG3';

[0056] 18S-R: 5'-CCCTTGTTACGATTTCTCTCCTTCCTC3';

[0057] ITS1: 5'-TCCGTAGGTGAACCTGCGG3';

[0058] ITS4: 5'-TCCTCCGCTTATTGATATGC3'.

[0059] 18S rDNA sequence:

[0060] TAGTAATTCTAGAGCTAATACGTGCGTAAATCCCGACTCCTGGAAGGGACGTATATATTAG

[0061] ATAAAAGGCCGACCGGACTTTGTCCGACCCGCGGTGAATCATGATATCTTCACGAAGCGC

[0062] ATGGCCTCGCGCCGGCGCTGTTCCATTCAAATTTCTGCCCTATCAACTTTCGATGGTAGGA

[0063] TAGAGGCCTACCATGGTGGTAACGGGTGACGGAGGATTAGGGTTCGATTCCGGAGAGGG

[0064] AGCCTGAGAAACGGCTACCACATCCAAGGAAGGCAGCAGGCGCGCAAATTACCCAATCC

[0065] TGATACGGGGAGGTAGTGACAATAAATAACAATATCGGGCATTTAATGTCTGGTAATTGGA

[0066] ATGAGTACAATCTAAATCCCTTAACGAGGATCCATTGGAGGGCAAGTCTGGTGCCAGCAG

[0067] CCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAGCTCGTAGT

[0068] TGGATTTCGGGTGGGTTCTAGCGGTCCGCCTATGGTGAGTACTGCTATGGCCTTCCTTTCT

[0069] GTCGGGGACGGGCTTCTGGGCTTCACTGTCCGGGACTCGGAGTCGACGTGGTTACTTTGA

[0070] GTAAATTAGAGTGTTCAAAGCAGGCTTACGCCAGAATACTTTAGCATGGAATAACACGATA

[0071] GGACTCTGGCCTATCTTGTTGGTCTGTAGGACCGGAGTAATGATTAAGAGGGACAGTCGG

[0072] GGGCATTCGTATTTCATTGTCAGAGGTGAAATTCTTGGATTTATGAAAGACGAACTACTGC

[0073] GAAAGCATTTGCCAAGGATGTTTTCATTAATCAAGAACGAAAGTTGGGGGCTCGAAGACG

[0074] ATTAGATACCGTCGTAGTCTCAACCATAAACGATGCCGACTAGGGATTGGCGAATGTTTTT

[0075] TTATGACTTCGCCAGCACCTTATGAGAAATCAAAGTTTTTGGGTTCCGGGGGGAGTATGGT

[0076] CGCAAGGCTGAAACTTAAAGGAATTGACGGAAGGGCACCACCAGGCGTGGAGCCTGCG

[0077] GCTTAATTTGACTCAACACGGGAAAACTTACCAGGTCCAGACATAGTGAGGATTGACAGA

[0078] TTGAGAGCTCTTTCTTGATTCTATGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGGTTG

[0079] CCTTGTCAGGTTGATTCCGGTAACGAACGAGACCTCAGCCTGCTAAATAGTCTCAGTTGC

[0080] TTTTTGCAGCTGGCTGACTTCTTAGAGGGACTATTGGCGTTTAGTCAATGGAAGTATGAGG

[0081] CAATAACAGGTCTGTGATGCCCTTAGATGTTCTGGGCCGCACGCGCGCTACACTGATGCAT

[0082] TCAACAAGCCTATCCTTGACCGAAAGGTCCGGGTAATCTTTGAAACTGCATCGTGATGGG

[0083] GATAGATTATTGCAATTATTAGTCTTCAACGAGGAATGCCTAGTAGGCGCAAGTCATCAGC

[0084] TTGCGTCGATTACGTCCCTGCCCTTTGTACACACCGCCCGTCGCTCCTACCGATTGGGTGT

[0085] GCTGGTGAAGTGTTCGGATTGGCAGCTTAGGGTGGCAACACCTCAGGTCTGCCGAGAAGTC

[0086] ITS sequence:

[0087] ACGGGCAGCTTGCCTGAGCTCAGGTCGAAAGTTTAGAACGTGCAAGCACGTTTCCTGCTT

[0088] GGCTCCTATCAAAGTCCACAAGCTCCAACTTCGTGTAGTCGGCAGAAGCCGTTGCTACCT

[0089] ATCCAGTTGAAGCCCATATCGGGTCCTTGATTAAGCCTCTACACTTCAGCCAACCCAACCC

[0090] AGAAAGGATTGGGAAAGCCAGATCCACCCCTAGGGCCAGCTGATAAGCTAACCTGCGATC

[0091] CAAAAGGAAAGAGGGGTGAGGGTGTAAACCGACGCTGAGGCAGACATGCTCTTGCCCG

[0092] AGGGCTCGAGCGCAATATGCGTTCAAAGATTCGATGGTTCACGGAATTCTGCAATTCACA

[0093] CTACGTATCGCATTTCGCTGCGTTCTTCATCGTTGCGAGAGCCAAGATATCCGTTGTTGAG<l

[0094] AGTTGTCTTTGGTTAGGATTGCCAGTTACTAGCAATCAAAACTTCAGAGTTTGGTTTTGAC

[0095] AGTGGTTAGCACTGGTGTATAGGCATGCCAGAGCGCCACTGATGCGGGCAGCAATGCTCG

[0096] CACCAGCTGCAGGCTGCAATCAGGACAAGCCCGATTACCCTGCTAGCAGCAAAGGCACG

[0097] GTACAACAGAGTTCACGTTGTGGTTTTAATAATTCAATGATCCTTCCGAGGTTCCCCCTCCCGGGAAAAGGGG。

[0098] Table 5 Blast analysis results of 18S rDNA and ITS of isolate

[0099]

[0100] In addition, according to the 18S rDNA identification results, the algae strains GX01, GX02 and HH01 were Desmodesmus sp., Chlamydomonas sp. and Chlorella sp., respectively.

[0101] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A carbon dioxide-tolerant microalgae, characterized by: The carbon dioxide-tolerant microalgae is named as Asteraceae ( Coelastrum sp.) GX03, the deposit number is CCTCC NO: M20241314, the deposit date is June 20, 2024, and the depository is the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

2. Use of the carbon dioxide-tolerant microalgae according to claim 1 in carbon fixation.

3. The use according to claim 2, characterized in that The following steps are included: (1) Cultivating the carbon dioxide-tolerant microalgae according to claim 1 to a logarithmic growth phase or a stationary phase to obtain a microalgae liquid; (2) Cultivating the microalgae liquid obtained in step (1) in a CO2 environment to perform carbon fixation.

4. The use according to claim 3, characterized in that: The culture conditions described in step (1) are as follows: culturing at 25-30°C, 6000-8000 lx of light, and a light-dark time ratio of 8-16h:8-16h; The culture medium used in the culture described in step (1) is BG11 liquid culture medium.

5. The use according to claim 4, characterized in that The culture conditions described in step (1) are a temperature of 28°C, a light intensity of 7000 lx, and a light-dark time ratio of 16h:8h.

6. The use according to claim 3, characterized in that: The concentration of the microalgae solution in step (2) is OD 680 = 0.1~0.

4.

7. The use according to claim 3, characterized in that: The culture conditions described in step (2) are 28.0±0.5°C, 10000±1000 lx of light, and a day-night ratio of 24h:0h.

8. The use according to claim 3, characterized in that: The concentration of CO2 in step (2) is 50-70% (v / v).

9. The use according to claim 8, characterized in that: The concentration of CO2 in step (2) is 60% (v / v).

10. Use of the carbon dioxide-tolerant microalgae according to claim 1 in CO2 fixation and lipid production.

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