A method for preserving liquid algae-bacteria composite microbial agent

Abstract

The invention discloses a method for preserving a liquid algae-bacteria composite microbial agent, wherein glucose, urea, potassium dihydrogen phosphate and magnesium sulfate are added to the liquid algae-bacteria composite microbial agent, ethyl paraben, natamycin, uniconazole and glycerol are added after dissolution, pH is adjusted, and a composite microbial agent preservative solution is obtained, which is stored in the dark. The preservation method of the invention utilizes the synergistic effect between the various components to effectively improve the survival rate of the liquid algae-bacteria composite microbial agent. The invention is suitable for preserving the liquid algae-bacteria composite microbial agent, and provides a basis for further application of the liquid algae-bacteria composite microbial agent in production and life.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a method for preserving a microbial agent, in particular to a method for preserving a liquid algae-bacteria composite microbial agent. Background Art

[0002] Microalgae can absorb nutrients such as nitrogen and phosphorus and produce oxygen for bacterial growth through photosynthesis. At the same time, bacteria degrade large-molecule organic matter into carbon dioxide and low-molecular compounds for use by microalgae. Algae-fungus composite microbial agents can effectively enhance soil fertility, promote vegetation's absorption of nutrients, and improve crop resistance. Therefore, the algae-fungus symbiotic system has huge application potential in agricultural agents and soil improvement.

[0003] At present, the research on liquid algae-bacteria composite microbial inoculants is developing rapidly, but most of them are ready-to-use and there is very little research on the preservation methods of composite inoculants. The limitations of the preservation of algae-bacteria composite microbial inoculants hinder the further application and development of algae-bacteria co-cultivation technology. In order to meet the needs of research and production, it is necessary to develop an ideal preservation method to make up for the shortcomings of existing technologies and lay the foundation for the widespread application of algae-bacteria co-cultivation technology. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preserving a liquid algae-fungus composite microbial agent with a simple process and easy operation, so as to solve the problem of difficulty in preserving the liquid algae-fungus composite microbial agent in the prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for preserving a liquid algae-bacteria composite microbial agent comprises the following steps: adding glucose, urea, dipotassium hydrogen phosphate and magnesium sulfate to the liquid algae-bacteria composite microbial agent, adding ethyl paraben, natamycin, uniconazole and glycerol after dissolving, adjusting the pH to obtain a composite microbial agent preservative solution, and preserving the solution away from light.

[0007] As a limitation, the algae species in the liquid algae-bacteria composite microbial agent is Chlorella pyrenoidosa, and the concentration of the algae species in the composite microbial agent preservation liquid is 2.0 to 2.5×10 7 / mL, the strain is Bacillus subtilis, and the strain concentration in the composite microbial agent preservation solution is 7.0-7.6×10 8 CFU / mL.

[0008] As another limitation, in the liquid composite microbial agent preservation solution, the glucose concentration is 5.0-7.0 g / L, the urea concentration is 0.8-1.0 g / L, the dipotassium hydrogen phosphate concentration is 2.0-3.0 g / L, and the magnesium sulfate concentration is 0.9-1.1 g / L.

[0009] As a third limitation, in the liquid composite microbial agent preservation solution, the concentration of ethyl paraben is 0.4-0.6 g / L, the concentration of natamycin is 0.1-0.2 g / L, the concentration of uniconazole is 0.10-0.12 mg / L, and the concentration of glycerol is 1.0-1.5 g / L.

[0010] As a fourth limitation, the pH adjuster is citric acid, L-lactic acid or acetic acid;

[0011] The pH of the composite microbial agent preservation solution is 6.5-7.5.

[0012] As a fifth limitation, the storage temperature is 4-10°C.

[0013] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0014] ① In the preservation method of the liquid algae-bacteria composite microbial agent provided by the present invention, glucose, urea, dipotassium hydrogen phosphate and magnesium sulfate are added to provide basic nutrient elements for the growth of algae and bacteria, wherein magnesium ions are an important component of microalgae chlorophyll and participate in the synthesis of some enzymes;

[0015] ② In the preservation method of the liquid algae-bacteria composite microbial inoculant provided by the present invention, the addition of uniconazole can effectively delay the growth of algae cells, ethyl paraben plays an antibacterial role, and natamycin increases the permeability of the cell membrane, making it easier for ethyl paraben to enter the cell to exert its efficacy, and has a synergistic effect with ethyl paraben, effectively reducing the amount of antibacterial agent, maintaining the stability of the number of inoculant cells, and using glycerol as a protective liquid to enhance the tolerance of algae cells to changes in environmental temperature;

[0016] ③ In the storage method of the liquid algae-bacteria composite microbial agent provided by the present invention, weak acid is used for regulation to keep the mixed liquid stable; and photosynthesis is inhibited by placing it away from light to extend the storage time.

[0017] The preservation method of the present invention is suitable for preserving liquid algae-bacteria composite microbial inoculant, and provides a basis for further application of the liquid algae-bacteria composite microbial inoculant in production and life. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below through specific embodiments. It should be understood that the described embodiments are only used to explain the present invention, and are not intended to limit the present invention.

[0019] Example 1 A method for preserving a liquid algae-bacteria composite microbial agent

[0020] This embodiment includes the following steps performed in sequence:

[0021] S1. C. pyrenoidosa and Bacillus subtilis were co-cultured to a cell concentration of 2.4×10 7 / mL and 7.2×10 8 CFU / mL, and liquid algae-bacteria composite culture solution was obtained;

[0022] S2. Add the following ingredients to the liquid algae-bacteria composite culture medium of step S1 to maintain the final concentrations of: glucose 5.0 g / L, urea 0.8 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate 0.9 g / L, so that the added ingredients are fully dissolved;

[0023] S3. Add ethylparaben, natamycin, uniconazole and glycerol to step S2 to make the final concentrations 0.5 g / L, 0.1 g / L, 0.10 mg / L and 1.0 g / L respectively;

[0024] S4. Add citric acid to adjust the pH end point to 7.0;

[0025] S5. After being stored at 4°C in the dark for 6 months, the number of algal and bacterial cells was measured respectively. Compared with the initial cell concentration, the survival rate of Chlorella pyrenoidosa was 90.1%, and the survival rate of Bacillus subtilis was 92.4%.

[0026] Comparative Example 1:

[0027] Chlorella pyrenoidosa and Bacillus subtilis were co-cultured to a cell concentration of 2.4×10 7 / mL and 7.2×10 8 CFU / mL, and a liquid algae-bacteria composite culture solution was obtained; after being stored at 4°C in the dark for 6 months without adding additives or protective agents, the number of algae and bacteria cells were measured respectively. Compared with the initial cell concentration, the survival rate of microalgae was 6.2%, and the survival rate of Bacillus subtilis was 35.4%.

[0028] Comparative Example 2:

[0029] S1. C. pyrenoidosa and Bacillus subtilis were co-cultured to a cell concentration of 2.4×10 7 / mL and 7.2×10 8 CFU / mL, and liquid algae-bacteria composite culture solution was obtained;

[0030] S2. Add the following ingredients to the liquid algae-bacteria composite culture medium of step S1 to maintain the final concentrations of: glucose 5.0 g / L, urea 0.8 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate 0.9 g / L, so that the added ingredients are fully dissolved;

[0031] S3. Add citric acid to adjust the pH end point to 7.0;

[0032] S4. After being stored at 4°C in the dark for 6 months, the number of algal and bacterial cells was measured respectively. Compared with the initial cell concentration, the survival rate of Chlorella pyrenoidosa was 45.4%, and the survival rate of Bacillus subtilis was 61.3%.

[0033] Comparative Example 3:

[0034] S1. C. pyrenoidosa and Bacillus subtilis were co-cultured to a cell concentration of 2.4×10 7 / mL and 7.2×10 8 CFU / mL, and liquid algae-bacteria composite culture solution was obtained;

[0035] S2. Add ethylparaben, natamycin, uniconazole and glycerol to make the final concentrations 0.5g / L, 0.1g / L, 0.10mg / L and 1.0g / L respectively;

[0036] S3. Add citric acid to adjust the pH end point to 7.0;

[0037] S4. After being stored at 4°C in the dark for 6 months, the number of algal and fungal cells was measured. Compared with the initial algal and fungal concentrations, the survival rates of Chlorella pyrenoidosa and Bacillus subtilis were 33.2% and 62.4%, respectively.

[0038] Comparative Example 4:

[0039] S1. C. pyrenoidosa and Bacillus subtilis were co-cultured to a cell concentration of 2.4×10 7 / mL and 7.2×10 8 CFU / mL, and liquid algae-bacteria composite culture solution was obtained;

[0040] S2. Add the following ingredients to the liquid algae-bacteria composite culture medium of step S1 to maintain the final concentrations of: glucose 5.0 g / L, urea 0.8 g / L, potassium dihydrogen phosphate 2.0 g / L, magnesium sulfate 0.9 g / L, so that the added ingredients are fully dissolved;

[0041] S3. Ethyl paraben, natamycin, uniconazole and glycerol were added to step S2 to make the final concentrations 0.5 g / L, 0.1 g / L, 0.10 mg / L and 1.0 g / L, respectively, without adjusting the pH;

[0042] S4. After being stored at 4°C in the dark for 6 months, the number of algal and fungal cells was measured. Compared with the initial algal and fungal concentrations, the survival rates of Chlorella pyrenoidosa and Bacillus subtilis were 73.5% and 86.1%, respectively.

[0043] It can be seen from the survival rates of microalgae and Bacillus subtilis in Example 1 and Comparative Examples 1 to 4 that the preservation method of the present invention can effectively improve the survival rate of microorganisms in the liquid algae-bacteria composite microbial inoculant.

[0044] Examples 2 to 9: Method for Preserving Liquid Algae-Bacteria Composite Microbial Agents

[0045] Examples 2 to 9 are respectively a method for preserving a liquid algae-bacteria composite microbial agent, and their steps are basically the same as those of Example 1, and the only difference is the amount of raw materials used and the process parameters. For details, see Table 1:

[0046] Table 1 List of process parameters in Examples 2 to 9

[0047]

[0048]

Claims

1. A method for preserving a liquid algae-bacteria composite microbial agent, It is characterized in that The preservation method comprises the following steps: adding glucose, urea, potassium hydrogen phosphate and magnesium sulfate to a liquid algae-bacteria composite microbial agent, adding ethyl paraben, natamycin, uniconazole and glycerol after dissolving, adjusting the pH to obtain a composite microbial agent preservation solution, and preserving the solution away from light; The algae species in the liquid algae-bacteria composite microbial inoculant is Chlorella pyrenoidosa, and the bacterial species is Bacillus subtilis; In the liquid composite microbial agent preservation solution, the glucose concentration is 5.0-7.0 g / L, the urea concentration is 0.8-1.0 g / L, the dipotassium hydrogen phosphate concentration is 2.0-3.0 g / L, the magnesium sulfate concentration is 0.9-1.1 g / L, the ethyl paraben concentration is 0.4-0.6 g / L, the natamycin concentration is 0.1-0.2 g / L, the uniconazole concentration is 0.10-0.12 mg / L, and the glycerol concentration is 1.0-1.5 g / L; The pH of the composite microbial agent storage solution is 6.5 to 7.5; The storage temperature is 4-10 ℃.

2. The method for preserving the liquid algae-bacteria composite microbial agent according to claim 1, It is characterized in that The algae concentration in the liquid algae-bacteria composite microbial agent is 2.0 to 2.5×10 7 / mL, the bacterial concentration is 7.0~7.6×10 8 CFU / mL.

3. A method for preserving the liquid algae-bacteria composite microbial agent according to claim 1 or 2, It is characterized in that The pH regulator is citric acid, L-lactic acid or acetic acid.

Images