Method for preparing deuterated compound by microalgae fermentation

Abstract

The method comprises the following steps: (1) preparing a domestication culture medium by taking deuterium water as a solvent, carrying out illumination culture by taking CO2 or CO2 mixed gas as a carbon source, and domesticating the microalgae for multiple times to obtain deuterium water-resistant microalgae; (2) transferring the deuterium-water-resistant microalgae into a growth culture medium taking deuterium water as a solvent, and continuously carrying out illumination culture on the microalgae by taking CO2 or CO2 mixed gas as a carbon source, so as to obtain the deuterium-water-resistant microalgae with high carbohydrate content; (3) hydrolyzing the deuterium-water-resistant microalgae with high carbohydrate content by using an acid hydrolysis method to obtain a deuterium-water-resistant microalgae hydrolysate; and (4) putting the deuterium-resistant water microalgae hydrolysate into a fermentation container, and adding fermentation microorganisms to obtain the deuterated compound. According to the method disclosed by the invention, various deuterated compounds can be prepared by taking the microalgae biomass as a substrate and combining a fermentation process, so that the production cost is reduced, the sustainable development is promoted, the production process is simplified, and the production efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of chemical preparation, specifically relating to a method for preparing deuterated compounds using microalgae fermentation. Background Technology

[0002] Deuterated compounds form isotope-labeled compounds through hydrogen / deuterium atom substitution, possessing both the biological activity of the parent molecule and unique isotope effects. Deuterium and hydrogen have highly similar atomic properties (e.g., a radius difference of only 0.005 Å), increasing the bond energy of the CD bond by 5–10 times compared to the CH bond, significantly slowing down the metabolic rate (reducing it by 6–10 times). This kinetic isotope effect makes them valuable in medicine (prolonging drug efficacy), agriculture (enhancing pesticide activity), and nuclear energy. However, large-scale preparation faces multiple challenges: synthesis requires high-temperature, high-pressure environments and precious metal catalysts, resulting in costs 3–5 times higher than conventional compounds; the process involves flammable and explosive deuterium sources (such as D2 gas), leading to high equipment complexity; and improper handling of byproducts can easily cause isotope pollution, hindering industrialization.

[0003] For deuterated compounds, deuterated succinic acid and deuterated ethanol, for example, do not produce background signals in H-NMR, giving them a significant advantage in analyzing other proton signals in the laboratory and making them commonly used in metabolic tracking and chemical mechanism analysis. However, the main synthetic methods for substances such as deuterated succinic acid and deuterated ethanol currently include catalytic deuteration hydrogenation and hydrogen-deuterium exchange. Catalytic hydrogenation is prone to producing large amounts of byproducts and excessive reduction, while hydrogen-deuterium exchange is expensive. Therefore, there is an urgent need for a stable, environmentally friendly, and low-cost method for preparing deuterated compounds. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing deuterated compounds using microalgae fermentation, which uses microalgae biomass as a substrate and combines fermentation processes to prepare a variety of deuterated compounds.

[0005] The technical solution adopted in this invention is as follows: A method for preparing deuterated compounds using microalgae fermentation includes the following steps: (1) Select microalgae species in the logarithmic growth phase, prepare acclimatization culture medium with deuterium water as solvent, use CO2 or CO2 mixed gas as carbon source, cultivate under light and subculture multiple times to acclimatize microalgae, so that microalgae can be propagated and grown in deuterium water environment, and grow to the middle and late stages of the logarithmic growth phase to obtain deuterium-tolerant microalgae. (2) Transfer the deuterium-tolerant microalgae obtained in step (1) to a growth medium with deuterium water as the solvent, and continue to cultivate the microalgae under light with CO2 or CO2 mixed gas as the carbon source to obtain deuterium-tolerant microalgae with high carbohydrate content. (3) Hydrolyze the deuterium-tolerant microalgae with high carbohydrate content obtained in step (2) using acid hydrolysis to obtain deuterium-tolerant microalgae hydrolysate; (4) The deuterium-tolerant microalgae hydrolysate obtained in step (3) is placed in a fermentation container and fermentation microorganisms are added to obtain the fermentation product deuterated compound.

[0006] Furthermore, in step (1), the microalgae species are either freshwater algae or marine algae. The freshwater algae are one of Spirulina, freshwater Chlorella, or fibrous algae, while the marine algae are one of Dunaliella salina or marine Chlorella.

[0007] The deuteration rate of the deuterated water in steps (1) and (2) is >99.8%.

[0008] Furthermore, in step (1), the CO2 mixed gas is one or more of air, nitrogen, and argon mixed with CO2.

[0009] Furthermore, the volume fraction of CO2 in the CO2 mixture is 0.01% to 10%.

[0010] Furthermore, the light source used in steps (1) and (2) is sunlight or artificial light, with a light intensity of 1000 Lux–300000 Lux; the microalgae culture temperature is 25~35℃.

[0011] Furthermore, the growth medium in step (1) is ordinary BG11 or Zarrouk medium.

[0012] Furthermore, in step (2), the growth medium is a modified BG11 or Zarrouk medium, and the sodium nitrate content in the medium is 5~30 mmol / L.

[0013] In the above technical solutions, to obtain microalgae with higher sugar concentrations, the composition of the culture medium BG11 or Zarrouk medium was optimized. The sodium nitrate content in the culture medium significantly affects the carbohydrate content produced by microalgae growth. Adjusting the sodium nitrate content can control the carbohydrate content produced by microalgae, but different microalgae species require different sodium nitrate concentrations. For freshwater Chlorella vulgaris, a sodium nitrate concentration of 7–10 mmol / L results in higher carbohydrate content and faster growth; for Spirulina, a sodium nitrate concentration of 8–12 mmol / L also results in higher carbohydrate content and faster growth.

[0014] Further, the acid hydrolysis method in step (3) specifically involves drying and grinding the deuterium-tolerant microalgae with high carbohydrate content obtained in step (2) to make microalgae powder, then adding the microalgae powder to dilute sulfuric acid with a mass concentration of 0.5~2.0%, with a solid-liquid ratio of 1:10~20, and reacting at 110~130℃ for 20~60 minutes.

[0015] Furthermore, the fermenting microorganism in step (4) is one of Yersinia lipophila, Saccharomyces cerevisiae, or Aspergillus niger.

[0016] Furthermore, the fermentation conditions in step (4) are set according to the fermentation temperature and fermentation environment required by the fermenting microorganisms.

[0017] The present invention has the following beneficial effects: (1) This invention provides a method for preparing deuterated compounds by microalgae fermentation, which enables the preparation of a variety of deuterated compounds by using microalgae biomass as a substrate and combining it with fermentation process. The reaction of this invention is carried out at room temperature and pressure, without the need for precious metal catalysts, thus reducing production costs. (2) The method of the present invention mainly includes the steps of domestication of deuterium-tolerant algae strains, production of microalgae carbohydrates and microalgae bio-fermentation. In this invention, by inoculating microalgae into deuterium water culture medium and domesticating them multiple times, the deuterium-tolerant microalgae are induced to efficiently integrate deuterium atoms in low-cost heavy water to obtain deuterium-tolerant algae strains, which can reduce the cost of expensive deuterium sources by more than 40%. The present invention also optimizes the microalgae culture conditions by inoculating the deuterium-tolerant algae strains into a modified culture medium containing deuterium water, so that the microalgae produce higher content of carbohydrates such as starch and cellulose during the growth process. Then, the high carbohydrate content microalgae are combined with the fermentation process, and fermentation is achieved at room temperature and pressure by introducing different fermentation microorganisms to obtain compounds such as deuterated succinic acid and deuterated ethanol. This realizes the direct preparation of various deuterated compounds from microalgae as raw materials, avoids complex chiral resolution, simplifies the production process, improves production efficiency, and the conversion rate of fermentation products can reach more than 90%, and the deuteration rate can reach more than 95%, with high conversion and deuteration rates. Attached Figure Description

[0018] To clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a flowchart of the present invention.

[0020] Figure 2The curves showing the changes in total sugar and deuterated succinic acid concentration over time during the preparation of deuterated succinic acid using microalgae in Example 1 of this invention are shown. Figure 3 The curves showing the changes in total sugar and deuterated ethanol concentration over time during the preparation of deuterated ethanol using microalgae in Example 2 of this invention are shown. Detailed Implementation

[0021] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0022] Example 1 Reference Figure 1 In this embodiment, deuterated succinic acid is prepared by microalgae fermentation. The specific steps are as follows: (1) Several freshwater Chlorella vulgaris in the logarithmic growth phase were taken as algal species. An acclimatization culture medium was prepared using deuterium water with a deuteration rate of 99.9% as the solvent. Specifically, it was a standard BG11 medium (potassium dihydrogen phosphate 40 mg / L, magnesium sulfate 75 mg / L, calcium chloride 36 mg / L, ferric ammonium citrate 6 mg / L, boric acid 2.86 g / L, manganese chloride 1.81 g / L, zinc sulfate 0.222 g / L, copper sulfate 0.079 g / L, sodium molybdate 0.390 g / L, cobalt nitrate 0.0494 g / L, and sodium nitrate content 17.6 mmol / L). A mixture of air and CO2 (with a CO2 volume concentration of 10%) was used as the carbon source, and the medium was prepared under a light intensity of 3000 nm. Chlorella vulgaris was cultured and domesticated multiple times under artificial light conditions of Lux and 30℃. The inoculation amount was 5% (i.e., 5 mL of microalgae solution was inoculated into every 100 mL of culture medium). Chlorella vulgaris was then propagated and grown in deuterium water environment until it reached the middle and late stages of the logarithmic growth phase, thus obtaining deuterium-tolerant Chlorella vulgaris. (2) A modified BG11 medium for the growth of Chlorella vulgaris was prepared using deuterated water with a deuteration rate of 99.9% as the solvent. The medium components were: potassium dihydrogen phosphate 40 mg / L, magnesium sulfate 75 mg / L, calcium chloride 36 mg / L, ferric ammonium citrate 6 mg / L, boric acid 2.86 g / L, manganese chloride 1.81 g / L, zinc sulfate 0.222 g / L, copper sulfate 0.079 g / L, sodium molybdate 0.390 g / L, cobalt nitrate 0.0494 g / L, and sodium nitrate 8 mmol / L. A mixture of air and CO2 (with a CO2 volume concentration of 5%) was used as the carbon source. The total bubbling flow rate was 0.5 L / min, and the light intensity was 3000 nm. Chlorella vulgaris was cultured under artificial light conditions of Lux and 30℃ with an inoculum of 5%. After 72 hours, deuterium-tolerant Chlorella vulgaris with high carbohydrate content was collected. (3) Hydrolyze the deuterium-tolerant common chlorella with high carbohydrate content obtained in step (2) using acid hydrolysis. Specifically, the deuterium-tolerant common chlorella with high carbohydrate content obtained in step (2) is dried and ground at 60°C to make microalgae powder. Then, the microalgae powder is added to dilute sulfuric acid with a mass concentration of 1% at a solid-liquid ratio of 1:15 and reacted at 121°C for 30 minutes to obtain deuterium-tolerant common chlorella hydrolysate. (4) Take 300 mL of the deuterium-tolerant Chlorella hydrolysate obtained in step (3) and put it into a 500 mL Erlenmeyer flask. Add the Yersinia lipolyticis seed culture to make the viable Yersinia lipolyticis count in the Erlenmeyer flask 3 × 10⁻⁶. 5 The concentration of the sample was 1 / mL, and then fermented at 36°C with the mixture stirred at 300 rpm to obtain the fermentation product, deuterated succinic acid.

[0023] The fermentation product obtained in Example 1 was tested, and the concentration of deuterated succinic acid reached 38.9 g / L on the 4th day, with a deuteration rate of 99.6%.

[0024] Example 2 This embodiment utilizes microalgae fermentation to prepare deuterated ethanol, and the specific steps are as follows: (1) Take dried Spirulina in the logarithmic growth phase as the algal strain, and use deuterium water with a deuteration rate of 99.9% as the solvent to prepare an acclimatization culture medium, specifically a common Zarrouk medium (A5 solution: potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.2 g / L, ferric sulfate 0.01 g / L, EDTA 0.001 g / L; B6 solution: calcium chloride 0.33 g / L, sodium sulfate 0.1 g / L, manganese sulfate 0.001 g / L, zinc sulfate 0.001 g / L, copper sulfate 0.0001 g / L; sodium nitrate content is 29.4 g / L). Spirulina was cultured and domesticated multiple times under artificial light conditions of 2000 Lux and 28℃, using a mixture of air, nitrogen and CO2 (with a CO2 volume concentration of 10%) as the carbon source. The inoculum was 5%, allowing Spirulina to expand and grow in a deuterium-tolerant water environment until it reached the mid-to-late logarithmic growth phase. (2) Zarrouk medium for the growth of Spirulina was prepared using deuterated water with a deuteration rate of 99.9% as the solvent (A5 solution: potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.2 g / L, ferric sulfate 0.01 g / L, EDTA 0.001 g / L; B6 solution: calcium chloride 0.33 g / L, sodium sulfate 0.1 g / L, manganese sulfate 0.001 g / L, zinc sulfate 0.001 g / L, copper sulfate 0.0001 g / L, sodium nitrate content 10 mmol / L). A mixture of air, nitrogen and CO2 (the volume concentration of CO2 in the mixture was 10%) was used as the carbon source, the total bubbling flow rate was 0.5 L / min, and the light intensity was 2000 ppm. Spirulina was cultured under artificial light conditions of Lux and 28°C with an inoculation amount of 5% to allow it to grow. After 96 hours, the deuterium-tolerant Spirulina with high carbohydrate content was collected. (3) Hydrolyze the deuterated water-tolerant spirulina with high carbohydrate content obtained in step (2) using acid hydrolysis. Specifically, the deuterated water-tolerant common spirulina with high carbohydrate content obtained in step (2) is dried and ground at 60°C to make microalgae powder. Then, the microalgae powder is added to dilute sulfuric acid with a mass concentration of 1.5% at a solid-liquid ratio of 1:20 and reacted at 121°C for 30 minutes to obtain deuterated water-tolerant spirulina hydrolysate. (4) Take 300 mL of the deuterium-tolerant Spirulina hydrolysate obtained in step (3) and put it into a 500 mL Erlenmeyer flask, and add the Saccharomyces cerevisiae seed culture so that the number of viable Saccharomyces cerevisiae in the Erlenmeyer flask is 3 × 10⁻⁶. 5 The fractions were collected at 36°C and fermented at 300 rpm to obtain the fermentation product, deuterated ethanol.

[0025] The fermentation product obtained in Example 2 was tested, and the concentration of deuterated ethanol reached 19.5 g / L on the second day, with a deuteration rate of 99.5%.

[0026] Comparative Example 1 This comparative example utilizes microalgae fermentation to prepare succinic acid, and the specific steps are as follows: (1) Take a number of freshwater Chlorella vulgaris in the logarithmic growth phase as algal species, and prepare ordinary BG11 medium (potassium dihydrogen phosphate 40 mg / L, magnesium sulfate 75 mg / L, calcium chloride 36 mg / L, ferric ammonium citrate 6 mg / L, boric acid 2.86 g / L, manganese chloride 1.81 g / L, zinc sulfate 0.222 g / L, copper sulfate 0.079 g / L, sodium molybdate 0.390 g / L, cobalt nitrate 0.0494 g / L, sodium nitrate content is 17.6 mmol / L) with a mixture of air and CO2 (the volume concentration of CO2 in the mixture is 10%) as carbon source, and the inoculum amount is 5%. Under artificial light conditions with a light intensity of 3000 Lux and a temperature of 30 degrees Celsius, the freshwater Chlorella vulgaris is cultured and expanded and grown until it reaches the middle and late logarithmic growth phase. (2) BG11 medium for the growth of Chlorella vulgaris was prepared using ordinary purified water as solvent. The medium composition was (potassium dihydrogen phosphate 40 mg / L, magnesium sulfate 75 mg / L, calcium chloride 36 mg / L, ferric ammonium citrate 6 mg / L, boric acid 2.86 g / L, manganese chloride 1.81 g / L, zinc sulfate 0.222 g / L, copper sulfate 0.079 g / L, sodium molybdate 0.390 g / L, cobalt nitrate 0.0494 g / L, sodium nitrate content 8 mmol / L). A mixture of air and CO2 (the volume concentration of CO2 in the mixture was 5%) was used as the carbon source. The total bubbling flow rate was 0.5 L / min. Chlorella vulgaris was cultured under artificial light conditions with a light intensity of 3000 Lux and 30℃. The inoculum size was 5%. After 72 hours, Chlorella vulgaris with high carbohydrate content was collected. (3) The common chlorella with high carbohydrate content obtained in step (2) is hydrolyzed by acid hydrolysis. Specifically, the common chlorella with high carbohydrate content obtained in step (2) is dried and ground at 60°C to make microalgae powder. Then, the microalgae powder is added to dilute sulfuric acid with a mass concentration of 1% at a solid-liquid ratio of 1:15 and reacted at 121°C for 30 minutes to obtain common chlorella hydrolysate. (4) Take 300 mL of the common Chlorella hydrolysate obtained in step (3) and put it into a 500 mL Erlenmeyer flask, and add the Yersinia lipolyticis seed culture so that the number of viable Yersinia lipolyticis cells in the Erlenmeyer flask is 3 × 10⁻⁶. 5The concentration of the sample was 1 / mL, and then fermented at 36°C with the mixture stirred at 300 rpm to obtain the fermentation product, deuterated succinic acid.

[0027] The fermentation product obtained from Comparative Example 1 was tested, and the concentration of deuterated succinic acid reached 40.1 g / L on the 4th day, with a deuteration rate of 0.0125%.

[0028] Comparative Example 2 This comparative example utilizes microalgae fermentation to prepare deuterated ethanol. The specific steps are as follows: (1) Take dried Spirulina in the logarithmic growth phase as the algal strain, and prepare ordinary Zarrouk medium (A5 solution: potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.2 g / L, ferric sulfate 0.01 g / L, EDTA 0.001 g / L; B6 solution: calcium chloride 0.33 g / L, sodium sulfate 0.1 g / L, manganese sulfate 0.001 g / L, zinc sulfate 0.001 g / L, copper sulfate 0.0001 g / L, sodium nitrate content is 29.4 mmol / L) with a mixture of air, nitrogen and CO2 (the volume concentration of CO2 in the mixture is 10%) as the carbon source. The inoculum amount is 5%. Spirulina is cultured and grown under artificial light conditions with a light intensity of 2000 Lux and 28℃ until it grows to the middle and late stages of the logarithmic growth phase. (2) Zarrouk medium for the growth of Spirulina was prepared using ordinary purified water as solvent. The medium composition was (A5 solution: potassium dihydrogen phosphate 0.1 g / L, magnesium sulfate 0.2 g / L, ferric sulfate 0.01 g / L, EDTA 0.001 g / L, B6 solution: calcium chloride 0.33 g / L, sodium sulfate 0.1 g / L, manganese sulfate 0.001 g / L, zinc sulfate 0.001 g / L, copper sulfate 0.0001 g / L, sodium nitrate content 10 mmol / L). A mixture of air, nitrogen and CO2 (the volume concentration of CO2 in the mixture was 10%) was used as the carbon source. The total bubbling flow rate was 0.5 L / min. Spirulina was cultured under artificial light conditions with a light intensity of 2000 Lux and 28℃. The inoculum size was 5%. After 96 hours, Spirulina with high carbohydrate content was collected. (3) The spirulina with high carbohydrate content obtained in step (2) is hydrolyzed by acid hydrolysis. Specifically, the ordinary spirulina with high carbohydrate content obtained in step (2) is dried and ground at 60°C to make microalgae powder. Then, the microalgae powder is added to dilute sulfuric acid with a mass concentration of 1.5% at a solid-liquid ratio of 1:20 and reacted at 121°C for 30 minutes to obtain spirulina hydrolysate. (4) Take 300 mL of the spirulina hydrolysate obtained in step (3) and put it into a 500 mL Erlenmeyer flask, and add the Saccharomyces cerevisiae seed culture so that the number of viable Saccharomyces cerevisiae in the Erlenmeyer flask is 3 × 10⁻⁶. 5 The concentration of ethanol was increased to 100 μL, and then fermented at 36°C with stirring at 300 rpm to obtain the fermentation product ethanol.

[0029] The fermentation product obtained from Comparative Example 2 was tested, and the ethanol concentration reached 20.2 g / L on the second day, with a deuteration rate of 0.0102%.

[0030] Comparative Example 3 The difference between this comparative example and Example 1 is that in this comparative example, when preparing deuterated succinic acid using deuterium-tolerant microalgae, the sodium nitrate content in the BG11 culture medium prepared in step (2) is 2 mmol / L, while all other aspects are the same.

[0031] The fermentation product obtained from Comparative Example 3 was tested, and the concentration of deuterated succinic acid reached 32.7 g / L on the 4th day, with a deuteration rate of 99.3%.

[0032] Comparative Example 4 The difference between this comparative example and Example 1 is that in this comparative example, when preparing deuterated succinic acid using deuterium-tolerant microalgae, the sodium nitrate content in the BG11 culture medium prepared in step (2) is 16 mmol / L, while all other aspects are the same.

[0033] The fermentation product obtained from Comparative Example 4 was tested, and the concentration of deuterated succinic acid reached 10.4 g / L on the 4th day, with a deuteration rate of 99.1%.

[0034] By comparing and analyzing Example 1 with Comparative Example 1 and Example 2 with Comparative Example 2, it can be found that the carbohydrate content of microalgae acclimated to deuterium water and grown in a deuterium-containing culture medium is not much different from that of unacclimated microalgae grown in a normal culture medium. This results in the final fermentation product concentration being basically the same, indicating that deuterium water has little effect on the carbohydrate content produced during the growth process of acclimated microalgae. Acclimated microalgae can be directly used for fermentation to produce deuterated compounds.

[0035] In addition, the fermentation process of Examples 1 and 2 above was continuously monitored, and the curves of the changes in total sugar and fermentation product concentration over time were obtained when preparing fermentation products using microalgae fermentation, as shown in the figures below. Figure 2 and Figure 3 As shown. From Figure 2 and Figure 3 As can be seen, the total sugar concentration gradually decreases while the fermentation product concentration gradually increases, until the total sugar is consumed and the fermentation product concentration stabilizes. Furthermore, from... Figure 2It can be seen that microalgae cultured in deuterium water can produce deuterated succinic acid, with a concentration of 38.9 g / L and a conversion rate of 91.5%; from Figure 3 It can be seen that microalgae cultured in deuterium water can produce deuterated ethanol with a concentration of 19.5 g / L and a conversion rate of 46.4%.

[0036] Furthermore, a comparative analysis of Examples 1, 3, and 4 reveals that the sodium nitrate content in the culture medium significantly affects the carbohydrate content produced by microalgae growth. The carbohydrate content in microalgae directly impacts the production of subsequent fermentation products, resulting in low succinic acid content in the fermentation products of Examples 3 and 4. Specifically, in Example 4, when the sodium nitrate content in the culture medium was 16 mmol / L, although *Chlorella vulgaris* grew rapidly, the carbohydrate content it produced was low. In Example 3, when the sodium nitrate content in the culture medium was 2 mmol / L, although *Chlorella vulgaris* grew slowly, it was conducive to carbohydrate production, but the carbohydrate content it produced was still slightly lower than that of Example 1. Additionally, although the final fermentation product of Example 3 still had a relatively high concentration of deuterated succinic acid, this was because the amount of dried microalgae powder and hydrolysate taken in steps (3) and (4) during the experiment was the same as in Example 1, and a sodium nitrate content of 2 mmol / L was conducive to carbohydrate production, so the final fermentation product concentrations of the two examples were similar. However, in actual production, the Chlorella in Comparative Example 3 grows slowly, and its total yield is very low under the same growth cycle.

[0037] The above are merely embodiments of the present invention and do not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing deuterated compounds using microalgae fermentation, characterized in that, Includes the following steps: (1) Select microalgae species in the logarithmic growth phase, prepare acclimatization culture medium with deuterium water as solvent, use CO2 or CO2 mixed gas as carbon source, cultivate under light and subculture multiple times to acclimatize microalgae, so that microalgae can be propagated and grown in deuterium water environment to obtain deuterium water tolerant microalgae. (2) Transfer the deuterium-tolerant microalgae obtained in step (1) to a growth medium with deuterium water as the solvent, and continue to cultivate the microalgae under light with CO2 or CO2 mixed gas as the carbon source to obtain deuterium-tolerant microalgae with high carbohydrate content. (3) Hydrolyze the deuterium-tolerant microalgae with high carbohydrate content obtained in step (2) using acid hydrolysis to obtain deuterium-tolerant microalgae hydrolysate; (4) The hydrolysate of deuterium-tolerant microalgae obtained in step (3) is placed in a fermentation container and fermentation microorganisms are added to obtain a deuterated compound as the fermentation product.

2. The method for preparing deuterated compounds by microalgae fermentation according to claim 1, characterized in that, In step (1), the microalgae species are either freshwater algae or marine algae. The freshwater algae are one of Spirulina, freshwater Chlorella, or fibrous algae, while the marine algae are one of Dunaliella salina or marine Chlorella.

3. The method for preparing deuterated compounds by microalgae fermentation according to claim 1, characterized in that, The deuteration rate of the deuterated water in steps (1) and (2) is >99.8%.

4. The method for preparing deuterated compounds by microalgae fermentation according to claim 1, characterized in that, In step (1), the CO2 mixed gas is one or more of air, nitrogen, and argon mixed with CO2.

5. The method for preparing deuterated compounds by microalgae fermentation according to claim 4, characterized in that, The volume fraction of CO2 in the CO2 mixture is 0.01% to 10%.

6. The method for preparing deuterated compounds by microalgae fermentation according to claim 1, characterized in that, The light source used in steps (1) and (2) is sunlight or artificial light, with a light intensity of 1000 Lux–300000 Lux; the microalgae culture temperature is 25~35℃.

7. The method for preparing deuterated compounds by microalgae fermentation according to claim 1, characterized in that, The growth medium in step (1) is ordinary BG11 or Zarrouk medium.

8. The method for preparing deuterated compounds by microalgae fermentation according to claim 1, characterized in that, In step (2), the growth medium is a modified BG11 or Zarrouk medium, and the sodium nitrate content in the medium is 5~30 mmol / L.

9. The method for preparing deuterated compounds by microalgae fermentation according to claim 1, characterized in that, The acid hydrolysis method in step (3) is as follows: the deuterium-tolerant microalgae with high carbohydrate content obtained in step (2) is dried and ground to make microalgae powder. Then, the microalgae powder is added to dilute sulfuric acid with a mass concentration of 0.5~2.0%, the solid-liquid ratio is 1:10~20, and the reaction is carried out at 110~130℃ for 20~60 minutes.

10. The method for preparing deuterated compounds by microalgal fermentation according to claim 1, characterized in that, The fermenting microorganism in step (4) is one of Yersinia lipophila, Saccharomyces cerevisiae, or Aspergillus niger.

Images