A microalgae expansion and mixed culture medium and its application

Abstract

The present invention relates to the field of microalgae biotechnology, and in particular to a microalgae expansion and mixed culture medium and its application. The microalgae expansion and mixed culture medium comprises the following components in parts by weight: 2-4 parts of a pH buffer, 0.1-0.4 parts of a phosphorus source, 2-5 parts of a carbon source, 0.01-0.1 parts of a growth factor, 0.3-0.54 parts of a nitrogen source, and 0.075-0.237 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; and the initial pH value of the microalgae expansion and mixed culture medium is 7.0. The present invention uses a pH buffer to stabilize the pH value of the culture solution to increase the growth rate of microalgae polyculture; then, tryptone is used to replace the complex trace elements in the general culture medium, and NH4Cl is used as the nitrogen source. The designed microalgae polyculture culture medium has the advantages of simple ingredients and high microalgae biomass, and the culture medium is more suitable for polyculture of microalgae under the condition of high-concentration organic carbon source, and can further obtain microalgae products with higher abundance by increasing the addition amount of the organic carbon source, and is very suitable for industrialized cultivation of microalgae.

Description

Technical Field

[0001] The invention relates to the field of microalgae biotechnology, and in particular to a microalgae expansion and mixed culture medium and application thereof. Background Art

[0002] Microalgae is a general term for a class of single-cell photosynthetic microorganisms. Microalgae have become an attractive alternative to traditional terrestrial plants for the production of biofuels and other high-value products, while alleviating the pressure on arable land, carbon dioxide emissions and global warming. However, achieving high productivity of microalgae products remains a major challenge for the commercial application of microalgae. Mixed culture fermentation of microalgae refers to a cultivation mode in which microalgae use both organic matter and light for growth. Due to the advantages of both photosynthetic autotrophic growth and heterotrophic growth, mixed culture fermentation of microalgae can be a method to increase the growth rate and product yield of microalgae. Microalgae such as Chlorella, Chlamydomonas, Nannochloropsis, and Scenedesmus obliquus have been found to grow in mixed culture. The advantages of mixed culture fermentation technology in the production of biodiesel and bioproducts by microalgae have been widely studied.

[0003] However, the existing microalgae polyculture medium is mostly designed in the form of autotrophic medium plus organic carbon source, which has the disadvantages of complex culture components and low microalgae biomass yield. Therefore, in order to realize the industrial application of microalgae polyculture fermentation, it is necessary to design a special microalgae polyculture medium to simplify the medium formula, reduce the medium cost and increase the microalgae yield. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a microalgae expansion mixed culture medium and its application, the culture medium has the advantages of simple ingredients, low cost and high microalgae yield, and has broad industrial application prospects.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present invention is as follows: In the first aspect, the present invention provides a microalgae expansion culture mixed culture medium, comprising the following components in parts by weight: 2-4 parts of a pH buffer, 0.1-0.4 parts of a phosphorus source, 2-5 parts of a carbon source, 0.01-0.1 parts of a growth factor, 0.3-0.54 parts of a nitrogen source and 0.075-0.237 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; and the initial pH value of the microalgae expansion culture mixed culture medium is 7.0.

[0006] The expansion culture and mixed culture medium of the present invention contains essential elements for the growth of microalgae, including a phosphorus source, a carbon source, a growth factor, a nitrogen source, and an inorganic salt; at the same time, a pH buffer is added to stabilize the pH value of the culture solution to increase the growth rate of the microalgae mixed culture. Bis-Tris has a relatively strong neutral pH buffering capacity and can be added as a pH buffer. In the culture of microalgae, the pH changes with the change of the biomass of the algae, affecting the growth of the algae. The culture medium is added with Bis-Tris to play a pH buffering role in this process, stabilize the pH value of the culture solution and increase the growth rate of the microalgae mixed culture; K2HPO4 is used as a phosphorus source of the culture medium; sodium acetate is used as a carbon source of the culture medium to jointly promote the growth of the microalgae biomass. The pH stability of the culture medium can be improved by increasing the concentration of K2HPO4 and using Bis-Tris with strong neutral pH buffering capacity. Most microalgae can use acetic acid for mixed culture growth, while only a small part of microalgae can use glucose, so the culture medium of the present invention has a wider adaptability to the cultivation of microalgae.

[0007] The applicant has found through a large number of experiments that, at this weight, the final pH value of the microalgae expansion mixed culture medium is stable at 8.0-8.5; microalgae cultured in this medium can have a good biomass, and is suitable for the growth of different microalgae and microalgae mixed culture under high concentration organic carbon source conditions.

[0008] Preferably, the weight portion of Bis-Tris is 4 parts; the weight portion of K2HPO4 is 0.4 parts; and the weight portion of sodium acetate is 2 parts.

[0009] Under the specific weight of the present invention, the biomass OD of microalgae is 680 The value reaches 5.0 or above.

[0010] Preferably, in the above-mentioned microalgae expansion and mixed culture medium, the growth factor is trypsin; and the nitrogen source is NH4Cl.

[0011] The use of trypsin to replace the complex trace elements contained in most commercially available culture media can simplify the composition of the culture medium while ensuring the biomass of the microalgae; the use of NH4Cl as a nitrogen source is more suitable for the mixed culture growth of microalgae.

[0012] Preferably, in the above-mentioned microalgae expansion and mixed culture medium, the weight portion of the trypsin is 0.1 parts; the weight portion of the NH4Cl is 0.54 parts.

[0013] Preferably, in the above-mentioned microalgae expansion mixed culture medium, the inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0014] Preferably, the weight portion of the MgSO4·7H2O is 0.05-0.15 parts; the weight portion of the CaCl2·2H2O is 0.02-0.072 parts; and the weight portion of the ferric citrate is 0.005-0.015 parts.

[0015] More preferably, the weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0016] The preparation method of the above-mentioned microalgae expansion mixed culture medium is as follows: the culture medium components are weighed according to the above-mentioned weight parts, dissolved in water, and sterilized to obtain the culture medium. The mixed culture medium preparation method of the present invention is simple, easy to use, suitable for the cultivation of various microalgae, can reduce the cultivation workload, increase the microalgae yield, and provide a guarantee for the industrial production of microalgae.

[0017] In a second aspect, the present invention provides the use of the above-mentioned microalgae expansion mixed culture medium in microalgae mixed culture.

[0018] The above-mentioned microalgae expansion mixed culture medium is suitable for microalgae that can be grown in mixed culture.

[0019] Preferably, the microalgae is any one of Chlorella, Chlamydomonas, Nannochloropsis, and Scenedesmus obliquus.

[0020] More preferably, the Chlorella is Pseudochlorella kessleri; and the Chlamydomonas is Chlamydomonas reinhardtii.

[0021] The beneficial effects of the present invention are:

[0022] The microalgae polyculture medium designed by the present invention has the advantages of simple ingredients and high microalgae biomass, and the medium is more suitable for polyculture of microalgae under conditions of high concentration of organic carbon source, and can further obtain microalgae products with higher abundance by increasing the amount of organic carbon source added, and is very suitable for industrial cultivation of microalgae. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :The final OD of Pseudochlorella kessleri in polyculture in BG-11, BG-11-A, BG-11-AT and BG-11-AB medium 680 Value and pH value, **p < 0.05;

[0024] Figure 2 :The final OD of Pseudochlorella kessleri in polyculture in BG-11-AB, B-ABP and B-ABPAm medium 680 Value, **p<0.05;

[0025] Figure 3:The final OD of Chlorella kessleri in polyculture in B-ABPAm medium with different K2HPO4 contents 680 Value, **p<0.05;

[0026] Figure 4 :The final OD of Pseudochlorella kessleri and Chlamydomonas reinhardtii co-cultured in TAP and B-ABPAm medium 680 Value and pH value, **p < 0.05;

[0027] Figure 5 : Final biomass of Pseudochlorella kessleri grown in TAP and B-ABPAm media with different acetate concentrations, **p<0.05. DETAILED DESCRIPTION

[0028] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] The algae species used in the following experiments: Parachlorella kessleri FACHB-4 and Chlamydomonas reinhardtii FACHB-265 were purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences.

[0030] Embodiment 1:

[0031] An embodiment of the microalgae expansion and mixed culture medium of the present invention comprises the following components in parts by weight: 4 parts of a pH buffer, 0.1 parts of a phosphorus source, 2 parts of a carbon source, 0.1 parts of a growth factor, 0.54 parts of a nitrogen source, and 0.116 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; and the initial pH value of the microalgae expansion and mixed culture medium is 7.0.

[0032] The growth factor is trypsin; the nitrogen source is NH4Cl.

[0033] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0034] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0035] Embodiment 2:

[0036] An embodiment of the microalgae expansion and mixed culture medium of the present invention comprises the following components in parts by weight: 4 parts of a pH buffer, 0.2 parts of a phosphorus source, 2 parts of a carbon source, 0.1 parts of a growth factor, 0.54 parts of a nitrogen source, and 0.116 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; and the initial pH value of the microalgae expansion and mixed culture medium is 7.0.

[0037] The growth factor is trypsin; the nitrogen source is NH4Cl.

[0038] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0039] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0040] Embodiment 3:

[0041] An embodiment of the microalgae expansion and mixed culture medium of the present invention, hereinafter referred to as B-ABPAm medium, comprises the following components in parts by weight: 4 parts of pH buffer, 0.4 parts of phosphorus source, 2 parts of carbon source, 0.1 parts of growth factor, 0.54 parts of nitrogen source and 0.116 parts of inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; the initial pH value of the microalgae expansion and mixed culture medium is 7.0.

[0042] The growth factor is trypsin, the nitrogen source is NH4Cl, and the inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0043] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0044] Embodiment 4:

[0045] An embodiment of the microalgae expansion and mixed culture medium of the present invention comprises the following components in parts by weight: 4 parts of a pH buffer, 0.4 parts of a phosphorus source, 3 parts of a carbon source, 0.1 parts of a growth factor, 0.54 parts of a nitrogen source, and 0.116 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; and the initial pH value of the microalgae expansion and mixed culture medium is 7.0.

[0046] The growth factor is trypsin; the nitrogen source is NH4Cl.

[0047] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0048] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0049] Embodiment 5:

[0050] An embodiment of the microalgae expansion and mixed culture medium of the present invention comprises the following components in parts by weight: 4 parts of a pH buffer, 0.4 parts of a phosphorus source, 4 parts of a carbon source, 0.1 parts of a growth factor, 0.54 parts of a nitrogen source, and 0.116 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; and the initial pH value of the microalgae expansion and mixed culture medium is 7.0.

[0051] The growth factor is trypsin; the nitrogen source is NH4Cl.

[0052] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0053] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0054] Embodiment 6:

[0055] An embodiment of the microalgae expansion and mixed culture medium of the present invention comprises the following components in parts by weight: 4 parts of a pH buffer, 0.4 parts of a phosphorus source, 5 parts of a carbon source, 0.1 parts of a growth factor, 0.54 parts of a nitrogen source, and 0.116 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; and the initial pH value of the microalgae expansion and mixed culture medium is 7.0.

[0056] The growth factor is trypsin; the nitrogen source is NH4Cl.

[0057] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0058] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0059] Comparative Example 1:

[0060] A comparative example of the microalgae expansion and mixed culture medium of the present invention, hereinafter referred to as BG-11 medium, comprises the following components in parts by weight:

[0061] 0.004 parts of phosphorus source, 0.002 parts of carbon source, 1.5 parts of nitrogen source and 0.0116 parts of inorganic salt; the phosphorus source is K2HPO4; the carbon source is Na2CO3; after mixing and stirring the above components evenly, the initial pH value of the microalgae expansion culture medium is adjusted to 7.0 with acid.

[0062] The nitrogen source is NaNO3.

[0063] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0064] The weight portion of the MgSO4·7H2O is 0.007 parts; the weight portion of the CaCl2·2H2O is 0.004 parts; and the weight portion of the ferric citrate is 0.0006 parts.

[0065] The culture medium also includes the following components in parts by weight:

[0066] 0.0006 parts of citric acid, 0.0001 parts of EDTANa2, 0.000286 parts of H3BO3, 0.000186 parts of MnCl2·4H2O, 0.000022 parts of ZnSO4·7H2O, 0.000039 parts of Na2MoO4·2H2O, 0.000008 parts of CuSO4·5H2O, and 0.000005 parts of Co(NO3)2·6H2O.

[0067] Comparative Example 2:

[0068] A comparative example of the microalgae expansion and polyculture medium of the present invention, hereinafter referred to as BG-11-A medium, comprises the following components in parts by weight:

[0069] 0.004 parts of phosphorus source, 2.002 parts of carbon source, 1.5 parts of nitrogen source and 0.0116 parts of inorganic salt; the phosphorus source is K2HPO4; the carbon source is Na2CO3 and sodium acetate; the weight portion of Na2CO3 is 0.002 parts; the weight portion of sodium acetate is 2 parts; after mixing and stirring the above components evenly, use acid to adjust the initial pH value of the microalgae expansion culture medium to 7.0.

[0070] The nitrogen source is NaNO3.

[0071] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0072] The weight portion of the MgSO4·7H2O is 0.007 parts; the weight portion of the CaCl2·2H2O is 0.004 parts; and the weight portion of the ferric citrate is 0.0006 parts.

[0073] The culture medium also includes the following components in parts by weight:

[0074] 0.0006 parts of citric acid, 0.0001 parts of EDTANa2, 0.000286 parts of H3BO3, 0.000186 parts of MnCl2·4H2O, 0.000022 parts of ZnSO4·7H2O, 0.000039 parts of Na2MoO4·2H2O, 0.000008 parts of CuSO4·5H2O, and 0.000005 parts of Co(NO3)2·6H2O.

[0075] Comparative Example 3:

[0076] A comparative example of the microalgae expansion and polyculture medium of the present invention, hereinafter referred to as BG-11-AT medium, comprises the following components in parts by weight:

[0077] 2.42 parts of pH buffer, 0.004 parts of phosphorus source, 2.002 parts of carbon source, 1.5 parts of nitrogen source and 0.0116 parts of inorganic salt; the pH buffer is Tris-base; the phosphorus source is K2HPO4; the carbon source is Na2CO3 and sodium acetate; the weight portion of Na2CO3 is 0.002 parts; the weight portion of sodium acetate is 2 parts; after mixing and stirring the above components evenly, use acid to adjust the initial pH value of the microalgae expansion culture mixed culture medium to 7.0.

[0078] The nitrogen source is NaNO3.

[0079] The inorganic salts include MgSO4·7H2O, MgSO4·7H2O and ferric citrate.

[0080] The weight portion of the MgSO4·7H2O is 0.007 parts; the weight portion of the CaCl2·2H2O is 0.004 parts; and the weight portion of the ferric citrate is 0.0006 parts.

[0081] The culture medium also includes the following components in parts by weight:

[0082] 0.0006 parts of citric acid, 0.0001 parts of EDTANa2, 0.000286 parts of H3BO3, 0.000186 parts of MnCl2·4H2O, 0.000022 parts of ZnSO4·7H2O, 0.000039 parts of Na2MoO4·2H2O, 0.000008 parts of CuSO4·5H2O, and 0.000005 parts of Co(NO3)2·6H2O.

[0083] Comparative Example 4:

[0084] A comparative example of the microalgae expansion and mixed culture medium of the present invention, hereinafter referred to as BG-11-AB medium, comprises the following components in parts by weight:

[0085] 4 parts of pH buffer, 0.004 parts of phosphorus source, 2.002 parts of carbon source, 1.5 parts of nitrogen source and 0.0116 parts of inorganic salt; the pH buffer is Bis-base; the phosphorus source is K2HPO4; the carbon source is Na2CO3 and sodium acetate; the weight portion of Na2CO3 is 0.002 parts; the weight portion of sodium acetate is 2 parts; after mixing and stirring the above components evenly, use acid to adjust the initial pH value of the microalgae expansion culture mixed culture medium to 7.0.

[0086] The nitrogen source is NaNO3.

[0087] The inorganic salts include MgSO4·7H2O, MgSO4·7H2O and ferric citrate.

[0088] The weight portion of the MgSO4·7H2O is 0.007 parts; the weight portion of the CaCl2·2H2O is 0.004 parts; and the weight portion of the ferric citrate is 0.0006 parts.

[0089] The culture medium also includes the following components in parts by weight:

[0090] 0.0006 parts of citric acid, 0.0001 parts of EDTANa2, 0.000286 parts of H3BO3, 0.000186 parts of MnCl2·4H2O, 0.000022 parts of ZnSO4·7H2O, 0.000039 parts of Na2MoO4·2H2O, 0.000008 parts of CuSO4·5H2O, and 0.000005 parts of Co(NO3)2·6H2O.

[0091] Comparative Example 5:

[0092] A comparative example of the microalgae expansion and polyculture medium of the present invention, hereinafter referred to as B-ABP medium, comprises the following components in parts by weight:

[0093] 4 parts of pH buffer, 0.004 parts of phosphorus source, 2 parts of carbon source, 0.1 parts of growth factor, 1.5 parts of nitrogen source and 0.0116 parts of inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; after mixing and stirring the above components evenly, the initial pH value of the microalgae expansion culture mixed culture medium is adjusted to 7.0 with acid.

[0094] The growth factor is trypsin; the nitrogen source is NaNO3.

[0095] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0096] The weight portion of the MgSO4·7H2O is 0.007 parts; the weight portion of the CaCl2·2H2O is 0.004 parts; and the weight portion of the ferric citrate is 0.0006 parts.

[0097] Comparative Example 6:

[0098] A comparative example of the microalgae expansion and polyculture medium of the present invention, hereinafter referred to as TAP medium, comprises the following components in parts by weight:

[0099] 2.42 parts of pH buffer, 0.0019 parts of phosphorus source, 2 parts of carbon source, 0.375 parts of nitrogen source and 0.1505 parts of inorganic salt; the pH buffer is Tris base; the phosphorus sources are K2HPO4 and KH2PO4; the carbon source is sodium acetate; the weight part of K2HPO4 is 0.0015 parts; the weight part of KH2PO4 is 0.0004 parts; after mixing and stirring the above components evenly, the initial pH value of the microalgae expansion culture mixed culture medium is adjusted to 7.0 with acid.

[0100] The nitrogen source is NH4Cl.

[0101] The inorganic salts include MgSO4·7H2O, MgSO4·7H2O and ferric citrate.

[0102] The weight portion of the MgSO4·7H2O is 0.1 part; the weight portion of the CaCl2·2H2O is 0.05 part; and the weight portion of the ferric citrate is 0.0005 part.

[0103] The culture medium also includes the following components in parts by weight:

[0104] Na2EDTA·2H2O 0.005 parts, ZnSO4·2H2O 0.0022 parts, MnCl2·4H2O 0.0005 parts, CuSO4·5H2O 0.00016 parts, (NH4)6Mo7O 24 ·4H2O 0.00011 parts, CoCl2·6H2O 0.00016 parts, H3BO3 0.00114 parts.

[0105] Comparative Example 7:

[0106] A comparative example of a microalgae expansion and mixed culture medium of the present invention comprises the following components in parts by weight: 4 parts of a pH buffer, 0.04 parts of a phosphorus source, 2 parts of a carbon source, 0.1 parts of a growth factor, 0.54 parts of a nitrogen source, and 0.116 parts of an inorganic salt. The pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; after the above components are mixed and stirred evenly, an acid is used to adjust the initial pH value of the microalgae expansion and mixed culture medium to 7.0.

[0107] The growth factor is trypsin; the nitrogen source is NH4Cl.

[0108] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0109] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0110] Comparative Example 8:

[0111] A comparative example of a microalgae expansion culture mixed culture medium of the present invention comprises the following components in parts by weight: 4 parts of a pH buffer, 0.8 parts of a phosphorus source, 2 parts of a carbon source, 0.1 parts of a growth factor, 0.54 parts of a nitrogen source and 0.116 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; after the above components are mixed and stirred evenly, an initial pH value of the microalgae expansion culture mixed culture medium is regulated to 7.0 with acid.

[0112] The growth factor is trypsin; the nitrogen source is NH4Cl.

[0113] The inorganic salts include MgSO4·7H2O, CaCl2·2H2O and ferric citrate.

[0114] The weight portion of the MgSO4·7H2O is 0.07 parts; the weight portion of the CaCl2·2H2O is 0.04 parts; and the weight portion of the ferric citrate is 0.006 parts.

[0115] Test example

[0116] 1. Determination of microalgae biomass

[0117] 1. Effects of different culture media on microalgae biomass

[0118] (1) Using Parachlorella kessleri FACHB-4 as the microalgae species, it was first enriched and cultured in BG-11 medium. When the OD of the culture medium of Parachlorella kessleri reached 680=1.5, the seed solution of Pseudochlorella kesleri was obtained.

[0119] (2) The seed solution of Pseudochlorella kesleri obtained in step (1) was inoculated into the culture medium prepared in Comparative Examples 1-3 at a volume ratio of 2%, and the culture was placed in a light incubator for culture under the conditions of 30°C and 6000 Lux full illumination. The OD of the culture solution was measured by a spectrophotometer. 680 Value, by culture medium OD 680 The value reflects the biomass of Pseudochlorella kjeldahlii, and the pH value of the culture solution is measured by a pH meter. The OD 680 The results of pH value and Figure 1 shown.

[0120] (3) The obtained Chlorella vulgaris seed solution was inoculated into the culture medium prepared in Example 3 (B-ABPAm culture medium), Comparative Example 4 (BG-11-AB culture medium), and Comparative Example 5 (B-ABP culture medium) at a volume ratio of 2%, and the culture was placed in a light incubator for culture under the conditions of 30°C and 6000 Lux full illumination. The OD of the culture solution was measured by a spectrophotometer. 680 Value, by culture medium OD 680 The value reflects the biomass of Pseudochlorella kjeldahlii. The OD value of the culture medium after 3 days of cultivation is 680 The measurement results are as follows Figure 2 shown.

[0121] from Figure 1 It can be seen that when the BG-11-A medium of Comparative Example 2 was used to culture microalgae, the final OD of Pseudochlorella kessleri was 680 =0.97, which is not much increased compared with the BG-11 medium in comparative example 1 when growing autotrophically. The pH value of the BG-11-A medium in comparative example 1 was detected, and it was found that its final pH value was 10.41. Therefore, too high a pH value would affect the growth rate of Pseudo-Chlorella kessleri in mixed culture. The BG-11-AT medium in comparative example 3 was used to culture microalgae, and the final pH value of the medium was 9.4, which was significantly lower than the pH value in comparative example 2. The final Pseudo-Chlorella kessleri biomass OD 680 =3.67, compared with the culture medium without pH buffer in Comparative Example 1 and Comparative Example 2, the biomass of microalgae was greatly increased. This shows that the addition of pH buffer can increase the growth rate of microalgae polyculture by stabilizing the pH value of the culture solution.

[0122] The BG-11-AB medium of comparative example 4 was used to culture microalgae, and the final pH value of the medium was 8.58, which was significantly lower than that of comparative example 3 in which Tris-base was added as a buffer, indicating that Bis-Tris with the same molecular weight as a pH buffer has a stronger neutral pH buffering capacity than Tris-base, which further improves the growth rate of Pseudo-Chlorella kessleri in mixed culture. 680 =4.33.

[0123] The difference between the B-ABPAm medium of Example 3 and the BG-11-AB medium of Comparative Example 4 is that the medium of Example 3 uses tryptone instead of the multiple trace elements of Comparative Example 4. The difference between the B-ABPAm medium of Example 3 and the B-ABP medium of Comparative Example 5 is that the medium of Example 1 uses NH4Cl as the nitrogen source instead of NaNO3 of Comparative Example 5. Figure 2 It can be seen that when the B-ABP medium of Comparative Example 2 was used to culture microalgae, the final OD of Pseudochlorella kessleri was 680 =4.31, which is equivalent to the culture result of BG-11-AB medium in comparative example 4. However, when the microalgae were cultured in B-ABPAm medium in example 3, the final OD of Pseudochlorella kelleri was 680 =4.66, and the biomass is significantly greater than that of the microalgae cultured in the culture medium of Comparative Examples 4 and 5, indicating that the addition of tryptone in the present invention to replace the multiple trace elements in the culture medium of Comparative Example 4 can achieve the purpose of simplifying the culture medium components, and ammonia nitrogen is a nitrogen source more suitable for the mixed culture growth of microalgae.

[0124] 2. Effect of phosphate concentration on microalgae biomass

[0125] The seed solution of Pseudochlorella kelleri was inoculated into the culture medium of Examples 1-3, Comparative Examples 7 and 8 at a volume ratio of 2%, and cultured in a light incubator under the following conditions: 30°C, 6000 Lux full illumination. The OD of the culture solution was measured by a spectrophotometer. 680 Value, by culture medium OD 680 The value reflects the biomass of Pseudochlorella kjeldahlii. The OD value of the culture medium after 3 days of cultivation is 680 The measurement results are as follows Figure 3 shown.

[0126] From the culture medium OD 680 The measured results show that the weight of K2HPO4 is within the range of the present invention (0.1-0.5), and the OD 680 The biomass reached the maximum value when the weight of K2HPO4 was 0.4 parts. 680 =5.59. The weight of K2HPO4 in Comparative Example 7 is lower than the range of the present invention, OD680 Only about 4.6; K2HPO4 in Comparative Example 8 exceeds the weight range of the present invention, OD 680 Therefore, the final B-ABPAm medium formula was determined to be 4 parts of Bis-Tris, 2 parts of sodium acetate, 0.54 parts of NH4Cl, 0.4 parts of K2HPO4, 0.07 parts of MgSO4·7H2O, 0.04 parts of CaCl2·2H2O, 0.006 parts of ferric citrate, and 0.1 parts of tryptone.

[0127] 2. Microalgae Adaptive Cultivation Experiment

[0128] 1. Adaptability test of mixed culture of different algae

[0129] Parachlorella kessleri FACHB-4 and Chlamydomonas reinhardtii FACHB-265 were used as microalgae species. They were first enriched and cultured in BG-11 medium. When the algae solution OD 680 =1.5, the microalgae seed liquid was obtained.

[0130] The obtained Chlorella vulgaris seed solution was inoculated into the B-ABPAm medium of Example 3 and the TAP medium of Comparative Example 6 at a volume ratio of 2%, and cultured in a light incubator under the following conditions: 30°C, 6000 Lux full illumination. The OD of the culture solution was measured by a spectrophotometer. 680 Value, by culture medium OD 680 The value reflects the biomass of microalgae, and the pH value of the culture solution is measured by a pH meter. The OD value of the culture solution after 3 days of cultivation is 680 The measurement results of pH value are as follows Figure 4 shown.

[0131] TAP medium is the most widely used microalgae co-culture medium. By comparing the culture results of different microalgae cultured using TAP and B-ABPAm medium, it can be seen that when microalgae are cultured using the B-ABPAm medium of Example 3, the final pH value of the culture solution is lower than that when the TAP medium of Comparative Example 6 is used for culture, indicating that the B-ABPAm medium has stronger pH stability.

[0132] When cultured using the B-ABPAm medium of Example 3, the final OD of Pseudochlorella kessleri 680 =5.62, which is 13.55% higher than that of the TAP medium in Comparative Example 6, indicating that the B-ABPAm medium is more suitable for the mixed growth of Pseudomonas kessleri.

[0133] When Chlamydomonas reinhardtii was cultured using the B-ABPAm medium of Example 3, the final OD680 =4.95, which is 13.89% higher than that of the TAP medium in comparative example 6. This shows that the B-ABPAm medium is more suitable for microalgae cultivation than the traditional TAP medium, and is more universal. Moreover, compared with the TAP medium in comparative example 6, which requires 15 different reagents to prepare, the B-ABPAm medium in Example 3 only requires 8 reagents, and the medium composition is greatly simplified, which is more conducive to the industrial production of microalgae.

[0134] 2. Polyculture test of microalgae under high carbon source concentration

[0135] (1) Parachlorella kessleri FACHB-4 (purchased from the freshwater algae bank of the Chinese Academy of Sciences) was used as the microalgae species and was first enriched and cultured in BG-11 medium. When the OD of the culture medium of Parachlorella kessleri reached 680 =1.5, the seed solution of Pseudochlorella kesleri was obtained.

[0136] (2) The sodium acetate in the TAP culture medium of comparative example 6 was prepared into 2 parts, 3 parts, 4 parts, and 5 parts respectively.

[0137] (3) The seed solution of Pseudochlorella kesleri was inoculated into the TAP medium with different sodium acetate contents and the microalgae expansion mixed culture medium of Examples 3-6 at a volume ratio of 2%, and the Pseudochlorella kesleri culture was placed in a light incubator for cultivation under the following culture conditions: 30° C. and 6000 Lux full illumination.

[0138] (4) Measure the OD of the culture medium 680 Value, by OD 680 The value reflects the biomass of Pseudochlorella kessleri. The OD value after 3 days of cultivation is 680 The value result is Figure 5 shown.

[0139] From the culture medium OD 680 The values ​​of the measured results show that the TAP medium prepared with different amounts of sodium acetate prepared above to culture microalgae shows a trend of increasing first and then decreasing with the increase of the sodium acetate content in the medium. The highest biomass is obtained when the weight portion of sodium acetate is 3 parts, and its OD 680 =6.41. This phenomenon shows that TAP medium is not suitable for mixed culture of microalgae under high carbon source concentration, and the production efficiency of microalgae cannot be further increased by increasing the organic carbon source in the medium.

[0140] When the microalgae high-abundance expansion culture medium of Examples 3-6 was used for culture, the final biomass of Pseudochlorella kelleri increased with the increase of the number of sodium acetate portions. When the sodium acetate portion was 5 portions, the final OD 680=8.41, which is 132.5% of the TAP medium of Comparative Example 6. This result shows that the culture medium of Examples 3-6 of the present invention is more suitable for mixed culture of microalgae under high concentration of organic carbon source conditions than the TAP medium of Comparative Example 6, and can obtain a higher abundance of microalgae products by increasing the amount of organic carbon source added, which is more conducive to the industrial production of microalgae. When the amount of sodium acetate is the same, the biomass of microalgae cultured in the B-ABPAm medium of the present invention is higher than that in the TAP medium.

[0141] The above experiments show that the B-ABPAm medium, a high-abundance microalgae culture medium of the present invention, has simple ingredients, a simple preparation method, is easy to use, and is suitable for the cultivation of a variety of microalgae, which can reduce the cultivation workload and increase the yield of microalgae. From the perspective of economic benefits, the B-ABPAm medium is selected to cultivate microalgae, which can reduce costs while ensuring the yield of microalgae; in addition, the concentration of organic carbon sources is increased on the basis of the B-ABPAm medium, which can also promote the increase of microalgae biomass, providing a guarantee for the industrial production of microalgae.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A microalgae expansion and polyculture medium, characterized in that: The invention comprises the following components in parts by weight: 2-4 parts of a pH buffer, 0.1-0.4 parts of a phosphorus source, 2-5 parts of a carbon source, 0.01-0.1 parts of a growth factor, 0.3-0.54 parts of a nitrogen source and 0.075-0.237 parts of an inorganic salt; the pH buffer is Bis-Tris; the phosphorus source is K2HPO4; the carbon source is sodium acetate; the growth factor is tryptone; the nitrogen source is NH4Cl; the inorganic salt is a combination of MgSO4·7H2O, CaCl2·2H2O and ferric citrate; in the inorganic salt, the weight portion of MgSO4·7H2O is 0.05-0.15 parts, the weight portion of CaCl2·2H2O is 0.02-0.072 parts, and the weight portion of ferric citrate is 0.005-0.015 parts; and the initial pH value of the microalgae expansion culture and mixed culture medium is 7.

0.

2. The microalgae expansion and polyculture medium according to claim 1, characterized in that: The weight portion of the Bis-Tris is 4 parts; the weight portion of the K2HPO4 is 0.4 parts; and the weight portion of the sodium acetate is 2 parts.

3. The microalgae expansion and polyculture medium according to claim 1, characterized in that: The weight portion of the tryptone is 0.1 part; the weight portion of the NH4Cl is 0.54 part.

4. The microalgae expansion and polyculture medium according to claim 1, characterized in that: Among the inorganic salts, the weight portion of MgSO4·7H2O is 0.07 parts, the weight portion of CaCl2·2H2O is 0.04 parts, and the weight portion of ferric citrate is 0.006 parts.

5. The use of the microalgae expansion mixed culture medium according to any one of claims 1 to 4 in microalgae mixed culture, characterized in that: The microalgae is Chlorella or Chlamydomonas.

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