Method for increasing oil content of microalgae by using traditional Chinese medicine residue water extract and application

Abstract

The invention discloses a method for increasing the oil content of microalgae by using a traditional Chinese medicine residue water extract and application of the method. The method comprises the following steps: mixing traditional Chinese medicine residues with water according to a specific ratio, boiling, filtering, adding into a microalgae culture medium, and culturing for 5-7 days. The method disclosed by the invention has the advantages that (1) abundant plant active ingredients in the water extract of the traditional Chinese medicine residues are utilized to regulate the metabolic pathway of the microalgae, promote the grease synthesis of the microalgae and remarkably improve the grease content of the microalgae; (2) according to the method, a specific microalgae variety is selected, and a traditional Chinese medicine residue water extract is added in the culture process, so that the optimal accumulation effect of the microalgae grease is realized, and the grease production efficiency and sustainability are ensured; (3) on one hand, the method obviously improves the grease content of microalgae cells, and on the other hand, realizes resource reutilization of the traditional Chinese medicine residues, thereby reducing the waste treatment pressure in the traditional Chinese medicine industry; and (4) the method provides a new idea for development and application of sustainable biological energy sources such as biodiesel.

Description

Technical Field

[0001] The invention belongs to the technical field of biological resource utilization, and relates to an improved microalgae cultivation method, specifically a method and application of increasing the microalgae oil content by using a water extract of traditional Chinese medicine residues. Background Art

[0002] Microalgae is an emerging source of bioenergy, with the advantages of fast growth, high oil content and no occupation of arable land. However, the large-scale production of biodiesel using microalgae is still in the research and experimental stage and has not yet been commercialized. Finding suitable oil-producing microalgae germplasm resources and reducing production costs while improving the purity and efficiency of large-scale cultivation are important issues that need to be solved in the process of commercial production of microalgae biodiesel.

[0003] Microalgae with high oil content are the basis for producing biodiesel. In the prior art, the oil content of most microalgae accounts for 20-50% of the cell dry weight, such as: Chlorella and Scenedesmus obliquus, which have been cultivated on a large scale, have oil contents of 28-32% and 22-29%, respectively. Therefore, how to increase the oil content in microalgae cells has become a key issue restricting the large-scale production of microalgae biodiesel. At present, the ways to increase the oil content of microalgae include genetic modification, nutritional starvation induction, chemical additive stimulation and other methods. However, these methods have the defects of complex operation, high cost and unfriendly environment in practical applications. Therefore, developing an economical, efficient and environmentally friendly method to increase the oil content of microalgae has become a technical problem that needs to be solved urgently.

[0004] Traditional Chinese medicine residue is waste generated during the processing of traditional Chinese medicine. Its main components are plant fiber, protein, carbohydrates and a small amount of active ingredients. For a long time, traditional Chinese medicine residue has been directly treated as waste or used for composting and feed, but it has not been fully utilized. However, there has been no report on the use of traditional Chinese medicine residue water extract to increase the oil content of microalgae. Summary of the invention

[0005] Technical problem to be solved: In order to overcome the shortcomings of the existing technology, by adding water extract of traditional Chinese medicine residues during the microalgae cultivation process, on the one hand, the oil content of microalgae cells can be significantly improved, and on the other hand, the resource recycling of traditional Chinese medicine residues can be achieved, thereby reducing the waste treatment pressure in the traditional Chinese medicine industry; in view of this, the present invention provides a method and application of using water extract of traditional Chinese medicine residues to increase the oil content of microalgae.

[0006] Technical solution: A method for increasing the oil content of microalgae by using water extract of traditional Chinese medicine residues, the method comprising the following steps:

[0007] S1. Preparation of water extract of Chinese medicinal residues

[0008] The Chinese medicine residue from Jiangsu Zhongxing Pharmaceutical Co., Ltd. was dried and mixed with water at a weight volume ratio of g:mL=1:10-20, boiled for 30-60 minutes, and filtered through a 200-mesh filter to obtain a Chinese medicine residue water extract;

[0009] S2. Microalgae cultivation

[0010] The Chinese medicine residue water extract prepared in S1 was evenly mixed with TAP culture medium at a volume ratio of 1:1-2, and added to microalgae for cultivation for 5-7 days. After the cultivation, algae mud was obtained by centrifugation at 5000-8000 rpm for 5-8 minutes at room temperature, and algae powder was further obtained by freeze drying to determine the lipid content of microalgae cells.

[0011] Preferably, the source of the Chinese medicinal residues in S1 includes residual medicinal residues from the production of Shenmei Yangwei Granules, Wushicha Granules or Shenqi Jianwei Granules, and the above Chinese medicinal residues are all from Jiangsu Zhongxing Pharmaceutical Co., Ltd.

[0012] Preferably, the microalgae in S2 include Chlorela sp. and Scenedesmus sp.

[0013] Preferably, the composition of TAP medium in S2 is: 0.38 g / L NH 4 Cl, 0.10 g / L MgSO 4 7H 2 O, 0.05 g / L CaCl 2 ·2H 2 O, 0.11 g / L K 2 HPO 4 , 0.06g / L KH 2 PO 4 , 0.05 g / L EDTA disodium salt, 22 mg / L ZnSO 4 7H 2 O, 11 mg / L H 3 BO 3 , 5mg / L MnCl 2 ·4H 2 O, 5 mg / L FeSO 4 7H 2 O, 1.5 mg / L CoCl 2 6H 2 O, 1.5 mg / L CuSO 4 ·5H 2 O, 1 mg / L (NH 4 ) 6 Mo 7 O 24 ·4H 2O, 2.42 g / L tromethamine, 1 mL / L acetic acid.

[0014] Preferably, the microalgae culture conditions in S2 are: temperature 25±1°C, light intensity 60±5 μmol photons / m 2 / s, the photoperiod was 12 h light / 12 h dark, and the shaking speed was set at 140 ± 10 rpm.

[0015] Preferably, the method for determining the lipid content of microalgae cells in S2 comprises the following steps:

[0016] a. Weigh the algae powder to a weight of g1, add chloroform-methanol-water with a volume ratio of 1:2:0.8, and extract the oil in the algae powder by ultrasonic;

[0017] b. Centrifuge the extract from step a, collect the supernatant, and repeat the extraction until the algae powder turns grayish white;

[0018] c. Combine the supernatants, add chloroform and water, adjust the volume ratio of chloroform-methanol-water to 1:1:0.9, centrifuge and take the lower layer of liquid;

[0019] d. Blow dry the liquid obtained in step c to a constant weight, and the weight is g2. The formula for the oil content in the algae cells is: oil content = g2 ÷ g1 × 100%.

[0020] Preferably, the method increases the oil content of Chlorella by more than 52%, and increases the oil content of Scenedesmus obliquus by more than 34%.

[0021] Application of any of the above methods in large-scale production of biodiesel using microalgae.

[0022] Application of any of the above methods in realizing resource recycling of traditional Chinese medicine residues.

[0023] Application of any of the above methods in developing sustainable bioenergy.

[0024] Beneficial effects: (1) The method of the present invention utilizes the rich plant active ingredients in the water extract of traditional Chinese medicine residue to regulate the metabolic pathway of microalgae, promote the oil synthesis of microalgae, and significantly increase the oil content of microalgae; (2) The method selects specific microalgae species and adds the water extract of traditional Chinese medicine residue during the cultivation process to achieve the best accumulation effect of microalgae oil, thereby ensuring the efficiency and sustainability of oil production; (3) On the one hand, the method significantly increases the oil content of microalgae cells, and on the other hand, it realizes the resource reuse of traditional Chinese medicine residue, thereby reducing the waste treatment pressure in the traditional Chinese medicine industry; (4) The method provides a new idea for the development and application of sustainable bioenergy such as biodiesel. DETAILED DESCRIPTION

[0025] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0026] Examples 1-24

[0027] The method for increasing the oil content of microalgae by using the water extract of traditional Chinese medicine residues comprises the following steps, and the specific parameters are shown in Table 1:

[0028] 1. Preparation of water extract from Chinese medicine residues

[0029] The residues left in the production of Chinese patent medicines such as Shenmei Yangwei Granules, Wushicha Granules or Shenqi Jianwei Granules are mixed with water at a weight-volume ratio of g:mL=1:10-20, heated to boiling, and kept for 30-60 minutes. Then, filter with a 200-mesh filter to obtain the Chinese medicine residue water extract. This step ensures that the active ingredients in the residue are extracted to provide nutrition for subsequent microalgae cultivation.

[0030] 2. Preparation of mixed culture medium

[0031] The Chinese medicine residue water extract obtained in step 1 was mixed evenly with the TAP medium at a volume ratio of 1:1-2 to obtain a mixed medium. The specific composition of the TAP medium used was: 0.38 g / L NH 4 Cl, 0.10 g / L MgSO 4 7H 2 O, 0.05 g / L CaCl 2 ·2H 2 O, 0.11 g / L K 2 HPO 4 , 0.06g / L KH 2 PO 4 , 0.05 g / L EDTA disodium salt, 22 mg / L ZnSO 4 7H 2 O, 11 mg / L H 3 BO 3 , 5mg / L MnCl 2 ·4H 2 O, 5 mg / L FeSO 4 7H 2 O, 1.5 mg / L CoCl 2 6H 2 O, 1.5 mg / L CuSO 4 ·5H 2 O, 1 mg / L (NH 4) 6 Mo 7 O 24 ·4H 2 O, 2.42 g / L tromethamine, and 1 mL / L acetic acid. This mixed culture medium provides comprehensive nutrition for the growth of microalgae.

[0032] 3. Microalgae cultivation

[0033] Chlorella sp. or Scenedesmus sp. was inoculated into the mixed culture medium for 5 to 7 days. The culture conditions were set as follows: temperature 25±1°C, light intensity 60±5 μmol photons / m 2 / s, the photoperiod was 12 h light / 12 h dark, and the shaker speed was set at 140 ± 10 rpm to ensure that the microalgae grew under ideal conditions.

[0034] 4. Determination of oil content

[0035] After the culture is completed, centrifuge at room temperature at 5000-8000 rpm for 5-8 minutes to obtain algae mud, which is further freeze-dried to obtain algae powder, and the oil content in the algae cells is determined. The method for determining the oil content is as follows:

[0036] Weigh a certain amount of algae powder and add chloroform-methanol-water (volume ratio 1:2:0.8) for ultrasonic extraction.

[0037] Centrifuge, collect the supernatant, and repeat the extraction until the algal powder turns off-white.

[0038] The supernatants were combined, chloroform and water were added, and the volume ratio of chloroform, methanol and water was adjusted to 1:1:0.9. After centrifugation, the lower layer of liquid was taken and placed in a clean glass bottle.

[0039] Use nitrogen blower to blow dry to constant weight.

[0040] The oil content in the algae cells was calculated according to the formula (oil content = oil mass / microalgae dry weight × 100%).

[0041] 5. Effect evaluation

[0042] The increase rate of oil content in algae cells compared with the control group was used as an indicator to judge the effect of using the water extract of Chinese medicine residues to increase the oil content of microalgae in this embodiment. The specific data are shown in Table 1. As shown in Table 1, the oil content in Chlorella cells is 43.0-55.4%, which is 52.48-96.45% higher than that in the control group; the oil content in Scenedesmus obliquus cells is 32.1-42.3%, which is 34.87-77.73% higher than that in the control group. The above data show that this method has a significant effect of increasing oil production.

[0043] Comparative Example 1

[0044] The difference between this comparative example and the embodiment is that the Chlorella was inoculated into the TAP medium and cultured for 7 days without adding the Chinese medicine residue water extract.

[0045] Comparative Example 2

[0046] The difference between this comparative example and the embodiment is that: Scenedesmus obliquus was inoculated into TAP medium and cultured for 7 days without adding the aqueous extract of traditional Chinese medicine residues.

[0047] Table 1 Beneficial effects obtained by the embodiments of the present invention

[0048]

Claims

1. A method for increasing the oil content of microalgae by using water extract of traditional Chinese medicine residues, characterized in that: The method comprises the following steps: S1. Preparation of water extract of Chinese medicinal residues Mix the dried Chinese medicine residue and water at a weight volume ratio of g:mL=1:10-20, boil for 30-60 minutes, and filter through a 200-mesh filter to obtain a Chinese medicine residue water extract; S2. Microalgae cultivation The Chinese medicine residue water extract prepared in S1 was evenly mixed with TAP culture medium at a volume ratio of 1:1-2, and added to microalgae for cultivation for 5-7 days. After the cultivation, algae mud was obtained by centrifugation at 5000-8000 rpm for 5-8 minutes at room temperature, and algae powder was further obtained by freeze drying to determine the lipid content of microalgae cells.

2. The method for increasing the oil content of microalgae by using water extract of Chinese medicinal residues according to claim 1, characterized in that: The sources of the Chinese medicinal residues in S1 include the residual residues from the production of Shenmei Yangwei Granules, Wushicha Granules or Shenqi Jianwei Granules.

3. The method for increasing the oil content of microalgae by using water extract of Chinese medicinal residues according to claim 1, characterized in that: The microalgae in S2 include Chlorella sp. and Scenedesmus sp.

4. The method for increasing the oil content of microalgae by using water extract of traditional Chinese medicine residues according to claim 1, characterized in that: The composition of TAP medium in S2 is: 0.38g / L NH4Cl, 0.10g / L MgSO4·7H2O, 0.05g / L CaCl2·2H2O, 0.11g / L K2HPO4, 0.06g / L KH2PO4, 0.05g / L EDTA disodium salt, 22mg / L ZnSO4·7H2O, 11mg / L H3BO3, 5mg / L MnCl2·4H2O, 5mg / L FeSO4·7H2O, 1.5mg / L CoCl2·6H2O, 1.5mg / L CuSO4·5H2O, 1mg / L (NH4)6Mo7O 24 4H2O, 2.42 g / L of tromethamine, 1 mL / L of acetic acid.

5. The method for increasing the oil content of microalgae by using water extract of traditional Chinese medicine residues according to claim 1, characterized in that: The microalgae culture conditions in S2 were: temperature 25±1℃, light intensity 60±5μmol photons / m 2 / s, the photoperiod was 12 h light / 12 h dark, and the shaking speed was set at 140 ± 10 rpm.

6. The method for increasing the oil content of microalgae by using water extract of traditional Chinese medicine residues according to claim 1, characterized in that: The method for determining the lipid content of microalgae cells in S2 comprises the following steps: a. Weigh the algae powder to a weight of g1, add chloroform-methanol-water with a volume ratio of 1:2:0.8, and extract the oil in the algae powder by ultrasonic; b. Centrifuge the extract from step a, collect the supernatant, and repeat the extraction until the algae powder turns grayish white; c. Combine the supernatants, add chloroform and water, adjust the volume ratio of chloroform-methanol-water to 1:1:0.9, centrifuge and take the lower layer of liquid; d. Blow dry the liquid obtained in step c to a constant weight, and the weight is g2. The formula for the oil content in the algae cells is: oil content = g2 ÷ g1 × 100%.

7. The method for increasing the oil content of microalgae by using water extract of traditional Chinese medicine residues according to claim 1, characterized in that: The method can increase the oil content of Chlorella by more than 52%, and increase the oil content of Scenedesmus obliquus by more than 34%.

8. Use of the method according to any one of claims 1 to 7 in large-scale production of biodiesel using microalgae.

9. Application of the method according to any one of claims 1 to 7 in realizing resource recycling of traditional Chinese medicine residues.

10. Use of the method according to any one of claims 1 to 7 in developing sustainable bioenergy.

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