Method for preparing platform compound by microalgae fermentation

Abstract

The invention discloses a method for preparing a platform compound by utilizing microalgae fermentation, which comprises the following steps: (1) selecting microalgae species, and culturing the microalgae for expanding the species and growing by using CO2 or CO2 mixed gas as a carbon source under illumination; (2) transferring the microalgae obtained in the step (1) into a culture medium, and continuously carrying out illumination culture on the microalgae by taking CO2 or CO2 mixed gas as a carbon source, so as to obtain microalgae with high carbohydrate content; (3) hydrolyzing the microalgae with high carbohydrate content obtained in the step (2) by using an acid hydrolysis method to obtain microalgae hydrolysate; and (4) putting the microalgae hydrolysate obtained in the step (3) into a fermentation container, and adding fermentation microorganisms to obtain the fermentation chemicals. According to the method disclosed by the invention, various chemicals 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 platform compounds using microalgae fermentation. Background Technology

[0002] With the urgent global demand for sustainable development and carbon neutrality, utilizing biomass resources to replace fossil fuels in the production of high-value-added chemicals has become a core direction of industrial biotechnology. However, traditional biomass raw materials (such as sugar crops like corn and sugarcane, or lignocellulose) have significant limitations: for example, food crops occupy arable land resources and may lead to competition between food and land; lignocellulose requires complex and energy-intensive physical / chemical pretreatment (such as acid hydrolysis and enzymatic hydrolysis) to release fermentable sugars, resulting in poor economic viability; and plants have long growth cycles and limited photosynthetic carbon fixation efficiency (usually ≤1%), restricting production scale. Microalgae biomass is considered an ideal next-generation biorefining substrate due to its unique advantages. This is because microalgae have a short doubling time (hours) and an annual yield per unit area that can be more than 10 times that of terrestrial plants; they can be cultivated on a large scale in non-arable land (such as deserts and tidal flats) using seawater and wastewater, alleviating land pressure; and their photosynthetic efficiency (theoretical value 3%~8%) is significantly higher than that of higher plants, enabling them to directly fix industrial CO2 emissions.

[0003] Microbial technology, as an emerging biomanufacturing technology, has demonstrated significant technological innovation in compound production. Compared to traditional chemical synthesis methods that rely on high-temperature, high-pressure reactions, multi-step purification, and the use of precious metal catalysts, microbial technology can achieve directional transformation under ambient temperature and pressure conditions. Combining microalgae with microbial technology not only reduces raw material costs but also provides milder production conditions. For example, Chinese patent application 202411751798.3 discloses an algal fermentation cultivation method that directly utilizes microalgae fermentation to produce the natural product lutein; Chinese patent application 202410353552.4 discloses a fermentation system and its application in the co-cultivation of microalgae and yeast to produce oils, which physically isolates microalgae and yeast for co-cultivation, effectively utilizing the O2 and CO2 produced during the fermentation process to increase the biomass yield of yeast and microalgae oils. However, the above patents mainly utilize fermentation to produce and extract natural products from microalgae, and cannot obtain other types of chemicals, thus limiting the application of microalgae. Summary of the Invention

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

[0005] The technical solution adopted in this invention is as follows: A method for preparing platform compounds using microalgae fermentation includes the following steps: (1) Select microalgae in the logarithmic growth phase and place them in an aqueous culture medium. Use CO2 or CO2 mixture as the carbon source and cultivate microalgae under light to expand and grow them, so that the microalgae grow to the middle and late stages of the logarithmic growth phase. (2) Transfer the microalgae obtained in step (1) to a culture medium, use CO2 or CO2 mixed gas as a carbon source, and continue to cultivate the microalgae under light to obtain microalgae with high carbohydrate content; (3) The microalgae with high carbohydrate content obtained in step (2) are hydrolyzed by acid hydrolysis to obtain microalgae hydrolysate; (4) Place the microalgae hydrolysate obtained in step (3) into a fermentation container and add fermentation microorganisms to obtain fermentation chemicals.

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

[0008] Furthermore, the volume fraction of CO2 in the CO2 mixture is 0.01% to 10%.

[0009] Furthermore, the light source used in steps (1) and (2) is sunlight or an artificial light source with an illumination intensity of 1000 Lux to 300000 Lux.

[0010] Furthermore, the microalgae culture temperature in steps (1) and (2) is 25~35℃.

[0011] Furthermore, in step (2), the culture medium is a modified BG11 or Zarrouk medium, and the sodium nitrate content in the culture medium is 5~30 mmol / L.

[0012] In the above technical solutions, to obtain microalgae with higher sugar concentrations, the sodium nitrate content is modified based on the ordinary BG11 or Zarrouk medium to ensure that the microalgae produce more carbohydrates rather than oils or other byproducts during production. Furthermore, different microalgae require different sodium nitrate concentrations, which are selected according to the specific microalgae species during actual cultivation. 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.

[0013] Further, the acid hydrolysis method in step (3) specifically involves: drying and grinding the 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 (m / v, unit g / ml), and reacting at 110~130℃ for 20~60 min, preferably at 121℃ for 30 min.

[0014] Furthermore, the fermenting microorganism in step (4) is one of Yersinia lipolyticis, Saccharomyces cerevisiae, or Aspergillus niger.

[0015] Furthermore, the fermentation conditions in step (4) are set according to the fermentation temperature and fermentation environment required by the fermenting microorganisms.

[0016] The present invention has the following beneficial effects: (1) This invention provides a method for preparing platform compounds by microalgae fermentation, which enables the preparation of a variety of chemicals by using microalgae biomass as substrate and combining fermentation process. This not only reduces production costs and raw material costs by more than 40%, but also promotes sustainable development. In addition, the reaction of this invention is carried out at room temperature and pressure, without the need for precious metal catalysts, which further reduces production costs. (2) This invention first optimizes the microalgae production process to increase the content of carbohydrates such as starch and cellulose in the microalgae. Then, the microalgae with high carbohydrate content are combined with the fermentation process. By introducing different fermentation microorganisms, fermentation is achieved at room temperature and pressure to obtain a variety of fermentation chemicals. This invention realizes the direct preparation of a variety of platform compounds such as citric acid, ethanol, and succinic acid from microalgae as raw materials, avoiding complex chiral resolution, simplifying the production process, and improving production efficiency. Moreover, this invention directly utilizes microalgae fermentation to prepare platform compounds, achieving a product conversion rate of over 95%, which is high. Attached Figure Description

[0017] 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.

[0018] Figure 1 This is a flowchart of the present invention.

[0019] Figure 2 This is a curve showing the changes in total sugar and citric acid concentrations over time during the preparation of citric acid using microalgae in Example 1 of the present invention. Figure 3This is a curve showing the change of total sugar and citric acid concentrations over time when preparing citric acid using microalgae in Comparative Example 2 of this invention. Detailed Implementation

[0020] 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.

[0021] Example 1 Reference Figure 1 This embodiment utilizes microalgae fermentation to prepare citric acid, and the specific steps are as follows: (1) Take a number of freshwater Chlorella vulgaris in the logarithmic growth phase as algae seed and put them into ordinary water culture medium. Use air and CO2 mixed gas (the volume concentration of CO2 in the mixed gas is 10%) as carbon source. The inoculation amount is 5% (that is, 5 mL of microalgae solution is inoculated into every 100 mL of ordinary water culture medium). Chlorella vulgaris is cultured and expanded under artificial light conditions with a light intensity of 3000 Lux and 30℃, so that it grows to the middle and late stages of the logarithmic growth phase. (2) Prepare a modified BG11 medium with the following components: 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. Then, transfer 300 mL of the Chlorella salina solution obtained in step (1) to a reactor containing 30 L of medium. Introduce a mixture of air and CO2 (the volume concentration of CO2 in the mixture is 10%). The total bubbling flow rate is 0.5 L / min. Continue to cultivate Chlorella salina under artificial light conditions of 3000 Lux and 30°C. After 72 hours, collect Chlorella salina with high carbohydrate content. (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 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 2.5L of the common Chlorella hydrolysate obtained in step (3) and put it into a 3L Erlenmeyer flask, and add Aspergillus niger seed solution so that the number of viable Aspergillus niger in the Erlenmeyer flask is 3×10⁻⁶. 5The concentration of citric acid was increased to 1 / mL and then fermented at 36°C for 72 hours. During the fermentation process, the mixture was stirred at 300 rpm to obtain the fermentation product citric acid.

[0022] The fermentation product obtained in Example 1 was tested, and the citric acid concentration reached 41.1 g / L, with a citric acid conversion rate of 96.7%.

[0023] Example 2 This embodiment utilizes microalgae fermentation to prepare ethanol, and the specific steps are as follows: (1) Take the dry Spirulina in the logarithmic growth phase as the algal species, use the air and CO2 mixed gas (the volume concentration of CO2 in the mixed gas is 0.5%) as the carbon source, the inoculum amount is 5%, and the Spirulina is cultured and grown under artificial light conditions with a light intensity of 2000 Lux and 28 degrees Celsius, so that it grows to the middle and late stages of the logarithmic growth phase. (2) Prepare a modified 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 10 mmol / L); then transfer 300 mL of Spirulina algal solution obtained in step (1) to a reactor containing 30 L of medium, introduce a mixture of air and CO2 (the volume concentration of CO2 in the mixture is 0.5%), the total bubbling flow rate is 0.5 L / min, continue to culture Chlorella under artificial light conditions of 2000 Lux and 28 degrees Celsius, and collect Spirulina with high carbohydrate content after 72 hours; (3) The spirulina with high carbohydrate content obtained in step (2) is hydrolyzed by acid hydrolysis. Specifically, the spirulina with high carbohydrate content is first 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 1.5L of the spirulina hydrolysate obtained in step (3) and put it into a 3L 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 the sample was increased to 100 μL, and then fermented at 36°C for 72 h. During the fermentation process, the mixture was stirred at 300 rpm to obtain the fermentation product ethanol.

[0024] The fermentation product obtained in Example 2 was tested, and the ethanol concentration reached 19.8 g / L, and the ethanol conversion rate reached 47.5%.

[0025] Example 3 This embodiment utilizes microalgae fermentation to prepare succinic acid, and the specific steps are as follows: (1) Take a number of seawater Chlorella vulgaris in the logarithmic growth phase as algae seeds and put them into ordinary water culture medium. Use CO2 gas as carbon source and cultivate Chlorella vulgaris under artificial light conditions with light intensity of 5000 Lux and 30℃. The inoculation amount is 5% and let it grow to the middle and late logarithmic growth phase. (2) Prepare a modified BG11 medium with the following components: 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. Then, transfer 300 mL of Chlorella vulgaris solution obtained in step (1) to a reactor containing 30 L of medium, introduce CO2 gas, and maintain a total bubbling flow rate of 0.5 L / min. Continue to cultivate Chlorella vulgaris under artificial light conditions with a light intensity of 3000 Lux and a temperature of 30 degrees Celsius. After 72 hours, collect Chlorella vulgaris with high carbohydrate content. (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 is first dried and ground at 60°C to make microalgae powder. Then, the microalgae powder is added to 1% dilute sulfuric acid at a solid-liquid ratio of 1:15 and reacted at 115°C for 40 minutes to obtain common chlorella hydrolysate. (4) Take 2.5L of the common Chlorella hydrolysate obtained in step (3) and put it into a 3L 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. 5 The concentration of succinate / mL was increased, and the mixture was fermented at 36°C for 72 hours. During the fermentation process, the mixture was stirred at 300 rpm to obtain the fermentation product, succinic acid.

[0026] The fermentation product obtained in Example 3 was tested, and the succinic acid concentration reached 39.8 g / L, with a succinic acid conversion rate of 98.1%.

[0027] Comparative Example 1 This comparative example utilizes corn starch fermentation to prepare citric acid. The specific steps are as follows: (1) Dilute the 19wt% corn liquefaction solution with water to a total sugar concentration of about 45 g / L, so that its sugar concentration is similar to that of the microalgae hydrolysate in Example 1, and add culture medium, wherein the nitrogen source is ammonium sulfate 2.5 g / L; the inorganic salts are potassium dihydrogen phosphate 1.0 g / L and magnesium sulfate 0.25 g / L, and then sterilize at 115℃ for 15 min. (2) Take 2.5L of the diluted corn liquefaction solution and culture medium obtained in step (1) and put them into a 3L Erlenmeyer flask, and add Aspergillus niger seed solution to make the viable count in the Erlenmeyer flask 3×10⁻⁶. 5 The concentration of citric acid was increased to 1 / mL and then fermented at 36°C for 72 hours. During the fermentation process, the mixture was stirred at 300 rpm to obtain the fermentation product citric acid.

[0028] The fermentation product obtained from Comparative Example 1 was tested, and the concentration of citric acid reached 40.2 g / L, with a citric acid conversion rate of 89.3%.

[0029] Comparative Example 2 This comparative example utilizes microalgae fermentation to prepare citric acid, and the specific steps are as follows: (1) Take a number of freshwater Chlorella vulgaris in the logarithmic growth phase as algae seeds and put them into ordinary water culture medium. Use air and CO2 mixed gas (the volume concentration of CO2 in the mixed gas is 10%) as carbon source. The inoculum amount is 5%. Under artificial light conditions with a light intensity of 3000 Lux and 30℃, Chlorella vulgaris is cultured for expansion and growth, so that it grows to the middle and late stages of the logarithmic growth phase. (2) Prepare ordinary BG11 medium, the medium components are (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); then transfer 300 mL of ordinary Chlorella algal solution obtained in step (1) to a reactor containing 30 L of medium, introduce a mixture of air and CO2 gas (the volume concentration of CO2 in the mixture is 10%), the total bubbling flow rate is 0.5 L / min, continue to cultivate ordinary Chlorella under artificial light conditions of light intensity of 3000 Lux and 30℃, and collect ordinary Chlorella with high carbohydrate content after 72 hours; (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 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 2.5L of the common Chlorella hydrolysate obtained in step (3) and put it into a 3L Erlenmeyer flask, and add Aspergillus niger seed solution so that the number of viable Aspergillus niger in the Erlenmeyer flask is 3×10⁻⁶. 5 The concentration of citric acid was increased to 1 / mL and then fermented at 36°C for 72 hours. During the fermentation process, the mixture was stirred at 300 rpm to obtain the fermentation product citric acid.

[0030] The fermentation product obtained from Comparative Example 2 was tested, and the citric acid concentration reached 12.2 g / L, with a citric acid conversion rate of 96.9%.

[0031] Comparative Example 3 The difference between this comparative example and Example 1 is that the sodium nitrate content in the culture medium used in step (2) of this comparative example is 2 mmol / L, while all other aspects are the same.

[0032] The fermentation product obtained from Comparative Example 3 was tested, and the citric acid concentration reached 35.7 g / L, with a citric acid conversion rate of 97.3%.

[0033] It should be noted that in Comparative Example 2, when the sodium nitrate content in the culture medium was 17.6 mmol / L, although the growth rate of Chlorella was fast, the content of carbohydrates produced was low. In Comparative Example 3, when the sodium nitrate content in the culture medium was 2 mmol / L, although the growth of Chlorella was slow, it was conducive to carbohydrate production, but the content of carbohydrates produced was still slightly lower than that in Example 1. In addition, although the final concentration of fermentation product in Comparative Example 3 was still relatively high, this was because the amount of microalgae powder and hydrolysate taken in steps (3) and (4) of the experiment was the same as that in Example 1, and the sodium nitrate content of 2 mmol / L was conducive to carbohydrate production, so the difference in the final concentration of fermentation product between the two was small. However, in actual production, the growth of Chlorella in Comparative Example 3 was slow, and its total yield was very low under the same growth cycle.

[0034] By comparing and analyzing Example 1 and Comparative Example 1 above, it is shown that microalgae biomass can replace traditional corn starch in the production of citric acid; and the microalgae biomass hydrolysate can be directly fermented without adding an additional nitrogen source. By comparing and analyzing Example 1, Comparative Example 2, and Comparative Example 3 above, it is shown that optimizing the composition of the microalgae culture medium can increase the content of fermentable carbohydrates produced in the microalgae, thereby increasing the yield of citric acid. In addition, the fermentation process of Example 1 and Comparative Example 2 was continuously monitored, and the curves of the changes in total sugar and citric acid concentration over time during the preparation of citric acid by microalgae fermentation were obtained, as shown 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 citric acid concentration gradually increases as fermentation progresses. Once the total sugar is consumed, the citric acid concentration stabilizes. Furthermore, because the microalgae in Comparative Example 2 produce low levels of carbohydrates, their citric acid production is also relatively low.

[0035] The above are merely embodiments of the present invention and do not limit the scope of the patent. Any equivalent modifications made based on the content of this specification, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing platform compounds using microalgae fermentation, characterized in that, Includes the following steps: (1) Select microalgae in the logarithmic growth phase and place them in an aqueous culture medium. Use CO2 or CO2 mixture as the carbon source and cultivate microalgae under light for propagation and growth. (2) Transfer the microalgae obtained in step (1) to a culture medium, use CO2 or CO2 mixed gas as a carbon source, and continue to cultivate the microalgae under light to obtain microalgae with high carbohydrate content; (3) The microalgae with high carbohydrate content obtained in step (2) are hydrolyzed by acid hydrolysis to obtain microalgae hydrolysate; (4) Place the microalgae hydrolysate obtained in step (3) into a fermentation container and add fermentation microorganisms to obtain fermentation chemicals.

2. The method for preparing platform compounds using 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 platform compounds using 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.

4. The method for preparing platform compounds using microalgae fermentation according to claim 3, characterized in that, The volume fraction of CO2 in the CO2 mixture is 0.01% to 10%.

5. The method for preparing platform compounds using microalgae fermentation according to claim 1, characterized in that, The light source used in steps (1) and (2) is sunlight or artificial light, with an intensity of 1000 Lux to 300000 Lux.

6. The method for preparing platform compounds using microalgae fermentation according to claim 1, characterized in that, The microalgae culture temperature in steps (1) and (2) is 25~35℃.

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

8. The method for preparing platform compounds using microalgae fermentation according to claim 1, characterized in that, The acid hydrolysis method in step (3) is as follows: the 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 min.

9. The method for preparing platform compounds using microalgae fermentation according to claim 1, characterized in that, The fermenting microorganism in step (4) is one of Yersinia lipophila, Saccharomyces cerevisiae, or Aspergillus niger.

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