Novel microbial inoculum dry powder capable of efficiently maintaining strain activity and preparation method of novel microbial inoculum dry powder

Abstract

The invention discloses novel bacterial agent dry powder capable of efficiently maintaining strain activity and a preparation method of the novel bacterial agent dry powder. The preparation method comprises the following specific steps: (1) selecting five dominant strains, namely A.faecalis S1, Bacillus megaterium XN1, Bacillus subtilis XN2, Raoultella sp.XY-1 and Pandoraea sp.XY-2, which are capable of efficiently degrading tetracycline, and carrying out ultrasonic treatment on the dominant strains, so as to obtain a strain A for efficiently degrading tetracycline; (2) performing amplification culture on the strain obtained in the step (1), and then preparing a composite flora according to the ratio of 1: 1: 2: 3: 2; (3) mixing the composite flora obtained in the step (2) with a phyllostachys pubescens substrate according to the ratio of 1: 5, 1: 10, 1: 15 and 1: 20 to obtain different microbial agents, detecting the effects of the microbial agents respectively, and determining the optimal ratio; and (4) adding 10% of a trehalose solution into the microbial agent with the optimal proportion obtained in the step (3), mixing, carrying out low-temperature vacuum filtration, and carrying out vacuum freeze drying, so as to prepare the microbial agent dry powder. The microbial inoculum dry powder prepared by the invention can effectively improve the activity and stability of the strain. In addition, the removal rate of tetracycline in tetracycline (60 mg / L) type wastewater by the dry powder of the microbial agent is 89.31%, and the microbial agent has a good application prospect in environmental treatment of tetracycline type wastewater.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a novel dry powder of a bacterial agent capable of efficiently maintaining the activity of bacterial strains and a preparation method thereof. Background Art

[0002] In the context of the "dual carbon" goal, people are paying more and more attention to environmental protection and sustainable development. Biological control technology has the advantages of no harm, short repair cycle, low cost, and easy operation, and is increasingly widely used in agriculture, industry, and urban environmental governance. However, during the cultivation, storage, transportation, and use of microorganisms, the number of live bacteria is greatly reduced and the activity declines during use due to environmental factors and the metabolism of the microorganisms themselves. Maintaining the activity of strains and extending the shelf life have always been technical difficulties in this field. At present, the methods for maintaining the activity of strains mainly include low-temperature freezing, vacuum freeze drying, high osmotic protective agents, and the application of colloidal vesicles. However, these methods may be environmentally unfriendly, costly, or have a greater impact on the activity of strains in maintaining the activity of strains. Therefore, in view of the drawbacks in maintaining the activity of strains, it is necessary to develop a new type of sewage treatment bacterial agent that can efficiently maintain the activity of strains to improve the vitality and stability of strains. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings and defects mentioned in the above background technology, and to provide a dry powder of bacterial agent and a preparation method thereof that can effectively reduce the adverse effects of environmental factors on the activity of bacterial strains and maintain the activity of bacterial strains for a long time, so as to protect the bacterial strains at low temperatures while ensuring that the bacterial agent has a high activity recovery rate after long-term storage.

[0004] The technical problem solved by the present invention is achieved by adopting the following technical scheme: a dry powder of bacterial agent capable of efficiently maintaining the activity of bacterial strains and a preparation method thereof, with particular reference to the main ingredients, carriers and drying and preservation. The main ingredient adopts a synthetic functional bacterial community, which has a stronger adaptability to the impact of the environment during the biotechnology production process, and avoids the influence of environmental factors on vitality to a certain extent. The carrier is a bamboo matrix, which has the advantages of high affinity, good stability and continuous provision of slow-acting carbon sources. The low-temperature drying method reduces the metabolic rate of microbial strains, puts the life activities of bacteria in a semi-permanent dormant state, prolongs the life of the strains and allows the strains to maintain their original properties.

[0005] In the present invention, the main material is to construct a functional bacterial community, which is mainly an artificially created functional bacterial community containing multiple dominant strains A.faecalis S1, Bacillus megaterium XN1, Bacillus subtilis XN2, Raoultella sp.XY-1 and Pandoraea sp.XY-2, which is relatively simple and highly controllable. Compared with a single pure culture, a functional bacterial community can better adapt to a changeable environment and achieve more complex functions.

[0006] In the present invention, the carrier is mainly a bamboo matrix, which not only has good stability but also can continuously provide a slow-acting carbon source for the bacteria.

[0007] In the present invention, the drying method mainly adopts low-temperature vacuum filtration combined with vacuum freeze-drying technology. The quick-drying process can make the bacteria dormant. The prepared bacterial agent dry powder not only has low water content and long preservation time, but also has high activity and is suitable for industrial application.

[0008] The present invention also provides a method for preparing a dry powder of a microbial agent, which is characterized by comprising the following process steps:

[0009] (1) The dominant strains A.faecalis S1, Bacillus megaterium XN1, Bacillus subtilis XN2, Raoultella sp.XY-1 and Pandoraea sp.XY-2 are cultured separately, and the number of viable cells of each strain is not less than 10 9 cfu / mL.

[0010] (2) The strain obtained in step (1) is expanded and cultured, and then mixed evenly in a ratio of 1:1:2:3:2 to prepare a composite bacterial community.

[0011] (3) The composite bacterial community obtained in step (2) is uniformly mixed with the carrier bamboo matrix in a ratio of 1:5, 1:10, 1:15, and 1:20 (m / v) to prepare different microbial agents, and their effects are tested to determine the optimal ratio.

[0012] (4) Add 10% trehalose solution to the microbial inoculum obtained in the optimal ratio in step (3), and then transfer it to a vacuum freeze dryer to remove moisture from the bacteria by adjusting the temperature and pressure. After the freeze drying process, a dry powder of the inoculum can be obtained. The dry powder of the inoculum has a low water content and can improve the storage stability and activity of the strain under appropriate storage conditions.

[0013] Beneficial effects of the present invention:

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The dry powder of the bacterial agent prepared by the bacterial agent and the method can effectively maintain the vitality and vitality of the bacterial strain and avoid obvious loss during storage and application. In addition, under various environmental conditions, the dry powder of the bacterial agent can stably preserve the bacterial strain and ensure its feasibility of long-term storage.

[0016] (2) In the present invention, the bacterial species used are functional bacterial communities constructed from multiple dominant strains, which interact with each other and complement each other in function. At the same time, they have stronger adaptability to environmental impacts during biotechnology production. Compared with the dry powder of bacterial agent prepared from a single pure culture, the effect was improved by 17.31% in the treatment of tetracycline (60 mg / L) wastewater.

[0017] (3) In the present invention, the bamboo matrix is ​​used as a carrier, which not only effectively maintains the activity of the bacteria, but also provides a slow-acting carbon source for the bacteria, and is environmentally friendly. In practical applications, the bacterial agent prepared by this method increases the activity of the bacteria by 15%.

[0018] (4) In the present invention, the preparation of the bacterial agent can be completed in a short time by using low-temperature vacuum filtration and vacuum freeze-drying technology. The preservation effect on the activity of the bacterial strain is good, and the long-term storage stability of the bacterial agent can be ensured. After the obtained bacterial agent is stored for 60 days, the number of viable bacteria can reach 10 8 cfu / mL or above.

[0019] (5) The dry powder of the bacterial agent of the present invention has a high degradation ability for tetracycline in tetracycline (60 mg / L) wastewater, with a removal rate of more than 89.31% and a COD removal rate of 90.43%. After the dry powder of the bacterial agent was stored at low temperature for 100 days, the survival rate of the strain was 76.37%, the removal rate of tetracycline was 69.41%, and the removal rate of COD was 70.34%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The removal efficiency of tetracycline by dry powder of bacterial agent prepared by mixing composite bacterial community with different masses of bamboo matrix.

[0021] Figure 2 The survival rate and performance of the dry powder of the bacterial agent stored for different times. The horizontal axis is the storage time, the vertical axis (black) is the survival rate of the strain, the vertical axis (red) is the tetracycline removal rate, and the vertical axis (blue) is the COD removal rate. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific examples. The strains used in the present invention are the dominant strains specifically screened by our company; the reagents used in the present invention, unless otherwise specified, are purchased from a biochemical store; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0023] Example 1: Preparation and preservation of bacterial agent dry powder

[0024] A method for preparing a microbial agent comprises the following steps:

[0025] (1) Five dominant strains of tetracycline that are highly efficient in degrading tetracycline, A.faecalis S1, Bacillus megateriumXN1, Bacillus subtilis XN2, Raoultella sp.XY-1 and Pandoraea sp.XY-2, were selected and cultured separately for plate confrontation experiments. The five strains were inoculated in equal amounts and cultured separately. When the strains were cultured to the mid-logarithmic phase, the OD values ​​were prepared. 600 =1.0±0.05 bacterial suspension; mixed in different proportions to obtain a composite bacterial community. The composite bacterial community was inoculated into 100 mL of LB medium containing 60 mg / L tetracycline at a total inoculation amount of 2%, and cultured in the dark at 30°C and 150 r / min. After 12 days of culture, the changes in tetracycline in the culture medium were detected to obtain the strain combination with the best tetracycline degradation effect. By means of microbial combination, a co-culture system with better effect on the biodegradation of tetracycline antibiotics was constructed.

[0026] The composition of LB medium is: 5 g / L sodium chloride, 10 g / L tryptone, and 5 g / L yeast extract.

[0027] Preparation of 60 mg / L tetracycline culture medium: Add an appropriate amount of tetracycline stock solution (20 mg of tetracycline standard in 1 mL of methanol, fully dissolved, filtered with a 0.22 μm organic filter membrane, prepared and used immediately) to the sterilized LB culture medium to make the initial tetracycline concentration 60 mg / L.

[0028] (2) A. faecalis S1, Bacillus megaterium XN1, Bacillus subtilis XN2, Raoultella sp. XY-1 and Pandoraea sp. XY-2 were cultured separately on LB medium for 18 h. The pH value of the medium was 7.0, the temperature was 30°C, and the shaking speed was 150 rpm. The above bacterial solutions were mixed at a volume ratio of 1:1:2:3:2 to obtain the composite bacterial community.

[0029] (3) Wash, dry and polish the bamboo, then pass it through a 100-mesh sieve. Sterilize the pretreated bamboo with high-pressure steam at 115°C for 30 min. After cooling, mix the composite bacterial community obtained in step (2) with the carrier bamboo matrix at a ratio of 1:5, 1:10, 1:15, and 1:20 (m / v) to obtain different microbial agents. Test their effects and determine the optimal ratio to be 1:10 (m / v) (e.g. Figure 1 as shown).

[0030] (4) Add 10% trehalose solution cryoprotectant to the optimal proportion of microbial inoculant obtained in step (3) above and mix well. After low-temperature vacuum filtration, vacuum freeze-drying is performed to obtain the microbial inoculant dry powder. The product is packed in an aluminum foil bag and stored in a cool and dry place.

[0031] Preparation of 10% trehalose solution: weigh 10 g of trehalose and place it in a 100 mL beaker, then add 80 mL of deionized water, stir with a glass rod to dissolve, then make up to the volume in a 100 mL volumetric flask to prepare a 10% mass volume percentage trehalose solution, and then sterilize at 115°C for 20 min.

[0032] Example 2: Activity verification of bacterial agent dry powder

[0033] Put 500 g of dry powder of the inoculant into an aluminum foil bag and store it in a cool, dry place. Take a sample every 10 days, dissolve 5 g in 50 mL of 30°C clean water and stir to dissolve. After fully dissolved, add it with tetracycline (60 mg / L) wastewater at a ratio of 5% (v:v). Measure the tetracycline content in the water every 10 days at room temperature, and calculate the microbial content in the water with a hemocytometer. Use high performance liquid chromatography to quantify the residual concentration of tetracycline, and the COD is determined by the potassium dichromate method.

[0034] pass Figure 2 It can be seen that after the dry powder of the microbial agent was stored for 10 days, the survival rate of the strain remained almost unchanged. When used to treat wastewater containing tetracycline (60 mg / L), the removal rate of tetracycline was as high as 89.31%, and the removal rate of COD was as high as 90.43%. After the dry powder of the microbial agent was stored for 30 days, the survival rate of the strain decreased slightly, but within 35 days to 60 days, the cell number of the microbial agent still reached 10 8 cfu / mL, which meets the technical indicators of agricultural microbial agents. After the microbial agent dry powder was stored for 100 days, the survival rate of the strain was 76.37%, the removal rate of tetracycline was 69.41%, and the removal rate of COD was 70.34%. Therefore, due to its good characteristics and ability to maintain constant vitality, it is expected to become a microbial agent dry powder with excellent performance.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, change or equivalent substitution made within the spirit and scope of the present invention shall be included in the protection scope of the present invention.

Claims

1. A novel dry powder of sewage treatment bacterial agent that can efficiently maintain bacterial activity and a preparation method thereof, wherein the characteristic steps are: (1) Select 5 dominant strains that are highly efficient in degrading tetracycline and culture them separately. The number of viable cells of each strain should be no less than 10 9 cfu / mL.

2. (2) The strain obtained in step (1) is expanded and cultured, and mixed in a ratio of 1:1:2:3:2 to prepare a composite bacterial community.

3. (3) The composite bacterial community obtained in step (2) is uniformly mixed with the carrier in a ratio of 1:5, 1:10, 1:15, and 1:20 (m / v) to prepare different microbial agents, and their effects are tested to determine the optimal ratio.

4. (4) Add a cryoprotectant to the microbial inoculum of the optimal ratio obtained in step (3), then remove excess liquid by low-temperature vacuum filtration, and transfer it to a vacuum freeze dryer to remove moisture from the microorganisms by adjusting the temperature and pressure. After the freeze-drying process is completed, the dry powder of the microbial inoculum can be obtained.

5. The sewage treatment bacterial agent dry powder and the preparation method thereof according to claim 1, characterized in that: The dominant strains mainly include A. faecalis S1, Bacillus megaterium XN1, Bacillus subtilis XN2, Raoultella sp. XY-1 and Pandoraea sp. XY-2.

6. The sewage treatment bacterial agent dry powder and the preparation method thereof according to claim 1, characterized in that: The bacterial community carrier is a bamboo matrix based on biomass materials.

7. The sewage treatment bacterial agent dry powder and the preparation method thereof according to claim 1, characterized in that: The optimal ratio of the composite bacterial community to the carrier bamboo matrix is ​​1:10 (m / v).

8. The sewage treatment bacterial agent dry powder and the preparation method thereof according to claim 1, characterized in that: The low temperature cryoprotectant is 10% trehalose solution.

9. A bacterial agent dry powder according to claims 1-5, characterized in that: The bacterial agent dry powder can be used for the bioremediation of tetracycline (60 mg / L) wastewater, and has a high removal rate for tetracycline and COD, which are 89.31% and 90.43%, respectively. After the bacterial agent dry powder was stored at low temperature for 100 days, the survival rate of the strain was 76.37%, the removal rate of tetracycline was 69.41%, and the removal rate of COD was 70.34%.

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