Method for activating treatment of steel slag by using multi-element microorganisms

Abstract

The invention relates to the technical field of solid waste recovery and application, in particular to a method for activating treatment of steel slag by using polyelement microorganisms, and the treatment method comprises the following steps: crushing and grinding steel slag, taking a soil sample, and separating and purifying the soil sample to obtain the polyelement microorganisms; the method comprises the following steps: domesticating and screening high-activity microorganisms in a liquid culture medium containing steel slag particles, culturing the steel slag particles in a fermentation tank filled with thalli, carrying out ball milling on the cultured steel slag particles to obtain steel slag powder, culturing bacterial liquid in an industrial fermentation tank, and circularly spraying the bacterial liquid on the steel slag powder from top to bottom, and drying and crushing the treated steel slag powder to obtain a microbial steel slag cementing material, manufacturing a mortar test piece, carrying out compression resistance test on the sample by using an electronic universal testing machine, and carrying out comparison verification with a cement mortar test piece prepared under the same condition. According to the method, carbonate deposition in the steel slag is induced by microorganisms to generate the gelling property, so that the gelling activity of the steel slag is improved, the pollution is less, and the method is simple.

Description

Technical Field

[0001] The invention relates to the technical field of solid waste civil engineering material application, and in particular to a method for activating and treating steel slag using multi-microorganisms. Background Art

[0002] In my country, the steel industry, as a pillar industry, plays an irreplaceable role in economic development and production life. Steel slag is the molten slag discharged during the steelmaking process and is one of the main solid wastes in the smelting industry. Every ton of crude steel produced will produce 0.1 to 0.15 tons of steel slag. With the growth of steel production capacity, a large amount of steel slag is difficult to use and remains in situ. The resulting environmental pollution problem is becoming increasingly severe, seriously endangering the lives and health of residents. With the development of technology, the resource utilization of steel slag has gradually received attention. The main ways to utilize steel slag resources are internal recycling and external utilization. Internal recycling is mainly used as a solid solution of sintered ore. The slag is mainly used as a desulfurization agent and a solvent in blast furnaces or cupolas; the external utilization is mainly as a raw material for cement, concrete, road materials, water and soil remediation materials, etc. The large-scale utilization of steel slag is still mainly concentrated in civil engineering such as construction and roads. There are many discussions on the treatment and disposal of steel slag, but the overall utilization of steel slag is relatively low. At present, the utilization rate of steel slag in my country is less than 20%, and most of the steel slag is in a state of simple storage and random discharge. These large amounts of accumulated steel slag will occupy precious land resources and cause serious pollution to the water around the slag yard. At the same time, the open-air accumulated steel slag will also generate a lot of dust, causing haze weather and endangering human health.

[0003] Therefore, the efficient, safe and stable utilization of steel slag has become a bottleneck for the sustainable development of the steel industry. The development and application of clean and effective treatment processes and large-scale steel slag products are of great significance to promoting the circular economy development of the steel industry. However, free calcium oxide and free magnesium oxide are commonly found in existing steel slag. These substances can react with water and expand in volume, which will cause stability problems of steel slag during use. This problem makes it impossible for steel slag to be used on a large scale in civil engineering materials. Although methods such as mechanical grinding and chemical excitation can solve the stability problem to a certain extent, there are still problems such as environmental pollution, energy waste and long reaction time. Summary of the invention

[0004] The object of the present invention is to provide a method for treating steel slag by activating it with multi-microorganisms to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for treating steel slag by using multi-microorganism activation, comprising the following steps:

[0006] S1: Use a jaw crusher to coarsely crush the steel slag raw material, and use a vibrating screen to screen the coarsely crushed steel slag raw material to obtain steel slag particles with a particle size not greater than 5 mm;

[0007] S2: mixing the steel slag particles with the liquid culture medium at a solid-liquid mass ratio of 5% to 25% to prepare a steel slag particle suspension;

[0008] S3: Obtain soil samples and separate and purify them to obtain multi-microorganisms, including silicate lysing bacteria and mineralization-inducing mixed bacteria, and culture them in a suspension of steel slag particles to screen out domesticated silicate lysing bacteria and domesticated mineralization-inducing mixed bacteria, among which the enzyme activity of domesticated silicate lysing bacteria is ≥0.05U / min, and the mineralization deposition rate of domesticated mineralization-inducing bacteria is ≥5×10 -8 mM·min -1 / cell;

[0009] S4: placing the steel slag particles in a fermentation tank filled with domesticated silicate lysing bacteria or domesticated mineralization inducing mixed bacteria for cultivation and adding a grinding aid during the cultivation period, and ball milling the cultured steel slag particles with a ball mill to obtain steel slag powder with a particle size of less than 75 μm;

[0010] S5: Use an industrial fermentation tank to cultivate a bacterial solution of domesticated silicate lysing bacteria or domesticated mineralization induced mixed bacteria, spray the cultured bacterial solution on the steel slag powder from top to bottom in a circular manner, use a constant temperature blast drying oven to maintain a constant temperature of 40-60 degrees to dry the treated steel slag powder to a constant weight, use a ball mill to crush the dried steel slag powder to make its particle size less than 75um, and finally obtain a microbial steel slag cementitious material with good gelling properties;

[0011] S6: Use microbial steel slag cementitious material to make mortar specimens, use an electronic universal testing machine to test the compressive strength and flexural strength of the specimens to determine their cementing activity, and compare the compressive strength and flexural strength with those of cement mortar specimens prepared under the same conditions;

[0012] S7: Use the plate colony counting method to detect the indicators of multiple microorganisms in microbial slag cementitious materials.

[0013] Furthermore, in S3, the specific steps of separating and purifying the silicate lysate are as follows:

[0014] S31a: Obtain 1.0 g of soil sample, add it to a test tube containing 9.0 ml of sterile culture medium, incubate it for 3 days, take the supernatant and inoculate it into a conical flask containing sterile culture medium, the inoculation ratio is 1:100, and incubate it in a constant temperature shaking incubator at a constant temperature of 30 degrees and an shaking frequency of 120 rpm for 3 to 5 days;

[0015] S32a: Use sterile deionized water sterilized at 121℃ for more than 20min to dilute the culture supernatant to 103 ~10 8 times, taking 1.0 ml of bacterial solution from the dilution at each dilution degree respectively and mixing it with the culture medium added with 1.5% to 3.0% agar, so as to prepare a solid culture medium plate, and then placing it in a sterile constant temperature incubator to maintain a constant temperature of 25°C to 35°C for 3 to 5 days. After the colony growth in the solid culture medium plate is completed, using an inoculation loop or an inoculation needle to pick out silicate lysate colonies with regular morphology and full growth, and inoculating them again into a conical flask containing culture medium for subculture, so as to obtain a silicate lysate with an activity preservation rate of ≥95%, wherein the culture medium components are: 10 g / L sucrose, 0.2 g / L potassium hydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.005 g / L ferric chloride, 0.7 g / L calcium carbonate, and the pH value of the culture medium is 7.2.

[0016] Furthermore, in S3, the specific steps of separating and purifying the mineralization-inducing mixed bacteria are as follows:

[0017] S31b: Obtain 1.0 g of soil sample, add it to a test tube containing 9.0 ml of sterile culture medium, incubate it for 3 days, take the supernatant and inoculate it into a conical flask containing sterile culture medium, the inoculation ratio is 1:100, and incubate it in a constant temperature shaking incubator at a constant temperature of 30 degrees and an shaking frequency of 120 rpm for 3 to 5 days;

[0018] S32b: Use sterile deionized water sterilized at 121℃ for more than 20min to dilute the culture supernatant to 10 3 ~10 8 times, 1.0 ml of the dilution at each dilution multiple is taken and mixed with the culture medium added with 1.5% to 3.0% agar to prepare a solid culture medium plate, and then placed in a sterile constant temperature incubator to maintain a constant temperature of 25°C to 35°C for 3 to 5 days, and then the mineralization-induced mixed bacteria colonies with better growth are picked out and inoculated into a conical flask containing the culture medium for subculture to obtain a mineralization-induced mixed bacteria with a mineralization activity preservation rate of ≥95%, wherein the culture medium components are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the pH value of the culture medium is 7.0.

[0019] Furthermore, in S3, the mineralization-inducing mixed bacteria is a mixed bacteria strain mainly composed of mineralization-inducing bacteria, wherein the mineralization-inducing bacteria account for no less than 80%, and the remaining mixed bacteria are one or more species of Bacillus pasteurianus, Bacillus pseudofirmus, Bacillus cohnii, Bacillus colloidus or carbonic anhydrase bacteria. The specific steps of screening the domesticated silicate lysing bacteria and the domesticated mineralization-inducing mixed bacteria are as follows:

[0020] S33: The silicate lysing bacteria and mineralization-inducing mixed bacteria were inoculated into the steel slag suspension respectively, and then cultured in a constant temperature shaking incubator at 28±5℃ for 3 days. 0.5 ml of the corresponding bacterial solution was drawn into the culture dish using a pipette, and then the drawn bacterial solution was evenly spread to every corner of the culture dish using a coating rod;

[0021] S34: Select the microbial colonies with complete morphology and full growth and re-cultivate them. After repeated subculturing for 5 to 10 times, isolate the acclimated silicate lysis bacteria with enzyme activity ≥ 0.05 U / min and mineralization deposition rate ≥ 5×10 -8 mM·min -1 / cell's acclimation and mineralization-induced mixed bacteria.

[0022] Furthermore, when the bacteria in the fermentation tank in S4 are domesticated silicate lysozyme, the corresponding bacteria in the cultured bacterial liquid in S5 are domesticated silicate lysozyme or domesticated mineralization induced mixed bacteria. When the bacteria in the fermentation tank in S4 are domesticated mineralization induced mixed bacteria, the corresponding bacteria in the cultured bacterial liquid in S5 are domesticated mineralization induced mixed bacteria.

[0023] Furthermore, the mass ratio of the steel slag particles in the S4 to the acclimated silicate lysate or the acclimated mineralization induced mixed bacteria solution is 1:0.25-1.30, and the culture time is 24-72 hours.

[0024] Furthermore, the grinding aid in S4 is compounded with triethanolamine, ethylene glycol and deionized water in a mass ratio of 7:3:10, and stirred with a stirrer for 30 minutes after compounding.

[0025] Furthermore, the spraying frequency of the bacterial liquid in S5 is 1 to 3 times / d, the spraying cycle is 1 to 2 days, and the drying temperature in S5 is 40 to 80 degrees Celsius.

[0026] Furthermore, the mortar specimen in S6 is prepared from 450g of microbial steel slag cementitious material, 1350g of standard sand and 225g of water, and the size of the mortar specimen is 40mm×40mm×160mm. The prepared mortar specimens are divided into two groups, each group including three mortar specimens, one group is cured for 7d in a standard concrete curing box or a standard concrete curing room at a constant temperature of 20°C and a humidity of 95%, and the other group is cured for 28d under the same conditions. The compressive strength and flexural strength of the cured specimens are tested using an electronic universal testing machine, and the compressive strength and flexural strength are compared with those of cement mortar specimens prepared under the same conditions.

[0027] Furthermore, in S7, before the plate colony counting method is performed, the prepared microbial steel slag gelling material is crushed by a crusher so that its particle size is ≤5 mm, the microbial steel slag gelling material particles are dissolved in water and suspended and soaked for 2 to 4 hours, and then the upper clear liquid is taken to plate count, and sterile deionized water is used as a control group to ensure that the environment is sterile. The specific steps of the plate colony counting method are as follows:

[0028] S71: Under a sterile environment, take 25 ml of the supernatant of the microbial steel slag cementitious material and the mineralization-induced mixed bacterial solution and place them in a 500 ml conical flask, make up to 250 ml with sterilized deionized water, and place 3 to 5 sterilized glass beads in the flask, and shake to obtain a uniform dilution of 1:10;

[0029] S72: Use a 1 ml sterile pipette to draw 1 ml of the 1:10 dilution into a test tube, add 9 ml of sterile deionized water, and shake well to obtain a 1:100 dilution;

[0030] S73: Repeat the above steps and dilute to 10 8 times, replace a new sterile pipette each time it increases, and select 10 6 , 10 7 , 10 8 Make three dilutions, and at the same time as making 10-fold incremental dilutions, take 1 ml of the bacterial solution at the corresponding dilution and place it in a culture dish, and make 3 plates for each dilution;

[0031] S74: After the culture medium solidifies, turn the culture dish over and place it in a constant temperature shaking incubator to maintain a constant temperature of 30 degrees for 48 hours. After the culture is completed, select the plates with a colony count in the range of 30 to 300 as valid plates for counting, calculate the number of colonies in the plate culture dish, and obtain the number of bacteria per milliliter of bacterial solution based on the corresponding dilution.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention provides a method for treating steel slag by utilizing multi-microorganism activation, wherein microorganisms are utilized to induce carbonate deposition in steel slag to produce gelling properties, thereby improving the gelling activity of steel slag. The method for catalyzing the generation of stable carbonate minerals based on the enzymatic hydrolysis and mineralization of mineralizing microorganisms is efficient and specific, has low pollution, is simple in method, and has environmentally friendly products and reaction processes. The application of microbial technology reduces the cost of chemical reagents, stimulates the activity of steel slag, and increases the hydration rate. Multi-microorganisms are used to treat steel slag, and specific microbial systems are used to react to solve the different contradictions in the application process of steel slag, thereby improving the stability of steel slag. The advantages of targetedness, high efficiency, and pollution-free are not possessed by traditional strategies.

[0034] 2. The present invention provides a method for activating and treating steel slag using multi-microorganisms. Through the application of microbial solid waste cementitious materials, a new type of cement developed with industrial secondary products such as steel slag has certain application prospects in this regard. The activity characteristics and cementing properties of the steel slag cementitious materials can meet its service requirements in most scenarios, and the accelerated carbon capture and production effect of microorganisms is also more conducive to protecting the environment and saving resources.

[0035] 3. The present invention provides a method for treating steel slag using multi-microorganisms for activation. The multi-microorganisms used to treat and activate steel slag are mainly used through the synergy and optimization between different reaction systems, thereby improving the reaction efficiency, reducing the reaction time, and optimizing the production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In the following description of the invention, it should be noted that the terms "upper", "lower", "left", "right", "inner", "outer", etc., indicating directions or positional relationships, are based on directions or positional relationships shown in the drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction or be constructed and operated in a specific direction. The term "connection" only indicates the connection between devices and has no special meaning.

[0039] In addition, the technical fields and installation methods involved in the embodiments of the present invention described below can be combined with each other as long as there is no conflict between them.

[0040] Specific embodiment 1, as Figure 1 A method for treating steel slag by using multi-microorganism activation comprises the following steps:

[0041] S1: Use a jaw crusher to coarsely crush the steel slag raw material, and use a vibrating screen to screen the coarsely crushed steel slag raw material to obtain steel slag particles with a particle size not greater than 5 mm;

[0042] S2: mixing the steel slag particles with the liquid culture medium at a solid-liquid mass ratio of 5% to 25% to prepare a steel slag particle suspension;

[0043] S3: Obtain soil samples and separate and purify them to obtain multi-microorganisms, including silicate lysing bacteria and mineralization-inducing mixed bacteria, and culture them in a suspension of steel slag particles to screen out domesticated silicate lysing bacteria and domesticated mineralization-inducing mixed bacteria, among which the enzyme activity of domesticated silicate lysing bacteria is ≥0.05U / min, and the mineralization deposition rate of domesticated mineralization-inducing bacteria is ≥5×10 -8 mM·min -1 / cell;

[0044] S4: placing the steel slag particles in a fermentation tank filled with domesticated silicate lysing bacteria or domesticated mineralization inducing mixed bacteria for cultivation and adding a grinding aid during the cultivation period, ball milling the cultivated steel slag particles with a ball mill for a ball milling time of not less than 10 hours and a ball mill speed of not less than 150 revolutions per minute, to obtain steel slag powder with a particle size of less than 75 μm after ball milling;

[0045] S5: Use an industrial fermentation tank to cultivate a bacterial solution of domesticated silicate lysing bacteria or domesticated mineralization inducing mixed bacteria, spray the cultivated bacterial solution on the steel slag powder from top to bottom in a cycle, use a constant temperature blast drying oven to maintain a constant temperature of 40-60 to dry the treated steel slag powder to a constant weight, because the steel slag raw material is crushed and ball-milled to make its particle size less than 75um, a small amount of steel slag powder will agglomerate during the spraying process, in order to avoid the agglomeration problem, the sprayed steel slag powder is secondary crushed, and the dried steel slag powder is crushed with a ball mill to make its particle size less than 75um, the ball milling time is not less than 10h, and the ball mill speed is not less than 150 rpm, and finally a microbial steel slag cementitious material with good gelling properties is obtained;

[0046] S6: Use microbial steel slag cementitious material to make mortar specimens, use an electronic universal testing machine to test the compressive strength and flexural strength of the specimens to determine their cementing activity, and compare the compressive strength and flexural strength with those of cement mortar specimens prepared under the same conditions;

[0047] S7: Use the plate colony counting method to detect the indicators of multiple microorganisms in microbial slag cementitious materials.

[0048] Further, in S3, the specific steps of separating and purifying the silicate lysate are as follows:

[0049] S31a: Obtain 1.0 g of soil sample, add it to a test tube containing 9.0 ml of sterile culture medium, incubate it for 3 days, take the supernatant and inoculate it into a conical flask containing sterile culture medium, the inoculation ratio is 1:100, and incubate it in a constant temperature shaking incubator at a constant temperature of 30 degrees and an shaking frequency of 120 rpm for 3 to 5 days;

[0050] S32a: Use sterile deionized water sterilized at 121℃ for more than 20min to dilute the culture supernatant to 10 3 ~108 times, taking 1.0 ml of bacterial solution from the dilution at each dilution degree respectively and mixing it with the culture medium added with 1.5% to 3.0% agar, so as to prepare a solid culture medium plate, and then placing it in a sterile constant temperature incubator to maintain a constant temperature of 25°C to 35°C for 3 to 5 days. After the colony growth in the solid culture medium plate is completed, using an inoculation loop or an inoculation needle to pick out silicate lysate colonies with regular morphology and full growth, and inoculating them again into a conical flask containing culture medium for subculture, so as to obtain a silicate lysate with an activity preservation rate of ≥95%, wherein the culture medium components are: 10 g / L sucrose, 0.2 g / L potassium hydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.005 g / L ferric chloride, 0.7 g / L calcium carbonate, and the pH value of the culture medium is 7.2.

[0051] Further, in S3, the specific steps of separating and purifying the mineralization-inducing mixed bacteria are as follows:

[0052] S31b: Obtain 1.0 g of soil sample, add it to a test tube containing 9.0 ml of sterile culture medium, incubate it for 3 days, take the supernatant and inoculate it into a conical flask containing sterile culture medium, the inoculation ratio is 1:100, and incubate it in a constant temperature shaking incubator at a constant temperature of 30 degrees and an shaking frequency of 120 rpm for 3 to 5 days;

[0053] S32b: Use sterile deionized water sterilized at 121℃ for more than 20min to dilute the culture supernatant to 10 3 ~10 8 times, 1.0 ml of the dilution at each dilution multiple is taken and mixed with the culture medium added with 1.5% to 3.0% agar to prepare a solid culture medium plate, and then placed in a sterile constant temperature incubator to maintain a constant temperature of 25°C to 35°C for 3 to 5 days, and then the mineralization-induced mixed bacteria colonies with better growth are picked out and inoculated into a conical flask containing the culture medium for subculture to obtain a mineralization-induced mixed bacteria with a mineralization activity preservation rate of ≥95%, wherein the culture medium components are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the pH value of the culture medium is 7.0.

[0054] Further, in S3, the mineralization-inducing mixed bacteria is a mixed bacteria strain mainly composed of mineralization-inducing bacteria, wherein the mineralization-inducing bacteria account for no less than 80%, and the remaining mixed bacteria are one or more species of Bacillus pasteurianus, Bacillus pseudofirmus, Bacillus cohnii, Bacillus colloidus or carbonic anhydrase bacteria. The specific steps of screening the domesticated silicate lysing bacteria and the domesticated mineralization-inducing mixed bacteria are as follows:

[0055] S33: The silicate lysing bacteria and mineralization-inducing mixed bacteria were inoculated into the steel slag suspension respectively, and then cultured in a constant temperature shaking incubator at 28±5℃ for 3 days. 0.5 ml of the corresponding bacterial solution was drawn into the culture dish using a pipette, and then the drawn bacterial solution was evenly spread to every corner of the culture dish using a coating rod;

[0056] S34: Select the microbial colonies with complete morphology and full growth and re-cultivate them. After repeated subculturing for 5 to 10 times, isolate the acclimated silicate lysis bacteria with enzyme activity ≥ 0.05 U / min and mineralization deposition rate ≥ 5×10 -8 mM·min -1 / cell's acclimation and mineralization-induced mixed bacteria.

[0057] Furthermore, when the bacteria in the fermentation tank in S4 are domesticated silicate lysozyme, the corresponding bacteria in the cultured bacterial liquid in S5 are domesticated silicate lysozyme or domesticated mineralization induced mixed bacteria. When the bacteria in the fermentation tank in S4 are domesticated mineralization induced mixed bacteria, the corresponding bacteria in the cultured bacterial liquid in S5 are domesticated mineralization induced mixed bacteria.

[0058] Furthermore, the mass ratio of the steel slag particles in S4 to the domesticated silicate lysate or the domesticated mineralization induced mixed bacterial solution is 1:0.25-1.30, and the culture time is 24-72 hours.

[0059] Furthermore, the grinding aid in S4 is compounded with triethanolamine, ethylene glycol and deionized water in a mass ratio of 7:3:10, and stirred with a stirrer for 30 minutes after compounding.

[0060] Furthermore, the spraying frequency of the bacterial solution in S5 is 1 to 3 times / d, the spraying cycle is 1 to 2 days, and the drying temperature in S5 is 40 to 80 degrees Celsius.

[0061] Furthermore, the mortar specimens in S6 were prepared from 450 g of microbial steel slag cementitious material, 1350 g of standard sand and 225 g of water. The size of the mortar specimens was 40 mm × 40 mm × 160 mm. The prepared mortar specimens were divided into two groups, each group including three mortar specimens. One group was cured for 7 days in a standard concrete curing box or a standard concrete curing room at a constant temperature of 20°C and a humidity of 95%, and the other group was cured for 28 days under the same conditions. The compressive strength and flexural strength of the cured specimens were tested using an electronic universal testing machine, and the compressive strength and flexural strength were compared with those of cement mortar specimens prepared under the same conditions.

[0062] Further, in S7, before the plate colony counting method is performed, the prepared microbial steel slag cementitious material is crushed by a crusher so that its particle size is ≤5 mm, the microbial steel slag cementitious material particles are dissolved in water and suspended and soaked for 2 to 4 hours, and then the upper clear liquid is taken to plate count, and sterile deionized water is used as a control group to ensure that the environment is sterile. The specific steps of the plate colony counting method are as follows:

[0063] S71: Under a sterile environment, take 25 ml of the supernatant of the microbial steel slag cementitious material and the mineralization-induced mixed bacterial solution, respectively, and place them in a 500 ml conical flask, dilute to 250 ml with sterilized deionized water, and place 3 to 5 sterilized glass beads in the flask, and shake to obtain a uniform dilution of 1:10;

[0064] S72: Use a 1 ml sterile pipette to draw 1 ml of the 1:10 dilution into a test tube, add 9 ml of sterile deionized water, and shake well to obtain a 1:100 dilution;

[0065] S73: Repeat the above steps and dilute to 10 8 times, replace a new sterile pipette each time it increases, and select 10 6 , 10 7 , 10 8 Make three dilutions, and at the same time as making 10-fold incremental dilutions, take 1 ml of the bacterial solution at the corresponding dilution and place it in a culture dish, and make 3 plates for each dilution;

[0066] S74: After the culture medium solidifies, turn the culture dish over and place it in a constant temperature shaking incubator to maintain a constant temperature of 30 degrees for 48 hours. After the culture is completed, select the plates with a colony count in the range of 30 to 300 as valid plates for counting, calculate the number of colonies in the plate culture dish, and obtain the number of bacteria per milliliter of bacterial solution based on the corresponding dilution.

[0067] When a method of treating steel slag by using multi-microorganism activation is used in a specific application, steel slag produced by a steel company in Taiyuan is used, and the raw steel slag is coarsely crushed by a jaw crusher, and the coarsely crushed raw steel slag is screened by a vibrating screen to obtain steel slag particles with a particle size of no more than 5 mm. 1.0 g of local soil sample is taken and added to a test tube containing 9.0 ml of sterile culture medium. After static cultivation for 3 days, the supernatant is inoculated into a conical flask containing sterile culture medium. The inoculation ratio is 1:100. The constant temperature of 30 degrees is maintained in a constant temperature shaking incubator, and the shaking frequency is 120 rpm for 3 days. It is diluted step by step with sterile deionized water to 10 3 ~10 8times, take 1.0 ml of the dilution at each dilution multiple and mix it with the culture medium added with 1.5% to 3.0% agar to make a solid culture medium plate, then place it in a sterile constant temperature incubator and keep it at 25℃ to 35℃ for 3 days, use an inoculation loop or an inoculation needle to pick out the silicate lysing bacteria and mineralization induced mixed bacteria with regular morphology and full growth, and inoculate them again into the conical bottle containing the culture medium for subculture, and obtain the silicate culture medium with an activity preservation rate of ≥95%. The mineralization-induced mixed bacteria with a lysis and mineralization activity preservation rate of ≥95% are as follows: the culture medium components for separating silicate lysis are: 10g / L sucrose, 0.2g / L potassium hydrogen phosphate, 0.2g / L magnesium sulfate heptahydrate, 0.005g / L ferric chloride, 0.7g / L calcium carbonate, and the pH value of the culture medium is 7.2; the culture medium components for separating mineralization-induced mixed bacteria are: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, and the pH value of the culture medium is 7.2;

[0068] The mineralization-induced mixed bacteria and silicate lysing bacteria were acclimated and cultured in a culture medium with a solid-liquid ratio of 5% between steel slag particles and liquid culture medium. The silicate lysing bacteria and mineralization-induced mixed bacteria were inoculated into the steel slag suspension, respectively, and cultured at a constant temperature of 28±5℃ in a constant temperature shaking incubator for 3 days. A pipette was used to draw 0.5 ml of the corresponding bacterial solution into a culture dish, and then the absorbed bacterial solution was evenly spread to every corner of the culture dish with a coating rod. The colonies with stronger activity were selected for re-culture. After repeated passage for 5 times, the acclimated silicate lysing bacteria with enzyme activity ≥0.05U / min and the mineralization deposition rate ≥5×10 -8 mM·min -1 / cell's acclimation and mineralization induced mixed bacteria;

[0069] The steel slag particles are placed in a fermentation tank filled with an acclimated silicate lysate and cultured for 24 hours, wherein the mass ratio of the steel slag particles to the acclimated silicate lysate is 1:0.25. During the culture period, a grinding aid prepared by mixing triethanolamine, ethylene glycol and deionized water is added, and the distribution mass ratio is 7:3:10. The cultured steel slag particles are ball-milled with a ball mill to obtain steel slag powder with a particle size of less than 75 um. The acclimated mineralization-inducing mixed bacteria are cultured in an industrial fermentation tank, and the cultured bacterial solution is sprayed on the steel slag powder from top to bottom in a cycle, with a spraying frequency of 3 times / d and a spraying cycle of 2d. At the same time, the substrate recovery liquid is collected and circulated for spraying. The treated steel slag powder is dried to a constant weight by a constant temperature blast drying oven maintained at a constant temperature of 60°C, and the dried steel slag powder is crushed with a ball mill to make its particle size less than 75 um, and finally a microbial steel slag cementitious material with good gelling properties is obtained.

[0070] 450g of microbial steel slag cementitious material, 1350g of standard sand and 225g of water were used to prepare 40mm×40mm×160mm mortar specimens. The prepared mortar specimens were divided into two groups, each group including three mortar specimens. One group was cured for 7d in a standard concrete curing box or a standard concrete curing room under the conditions of 20℃ constant temperature and 95% humidity, and the other group was cured for 28d under the same conditions. The compressive strength and flexural strength of the cured specimens were tested by an electronic universal testing machine, and the arithmetic mean of the compressive strength test value and the flexural strength test value obtained in each group was taken as the test result. Two groups of cement mortar specimens were prepared under the same conditions for the biological steel slag mortar specimens, each group included three cement mortar specimens, and the compressive strength and flexural strength of the cement mortar specimens after curing were tested using an electronic universal testing machine. The arithmetic mean of the compressive strength test value and the flexural strength test value obtained in each group was taken as the test result. The test values ​​of the microbial steel slag mortar specimens and the cement mortar specimens made in the same batch in each group were compared. The compressive strength and flexural strength of the mortar specimens prepared using microbial steel slag cementitious material reached 95% of those of ordinary cement mortar specimens, which can basically replace the application scenarios of ordinary Portland cement.

[0071] The test results of the microbial steel slag cementitious material and the ordinary cementitious material control group are shown in Table 1:

[0072] Table 1 Comparison of strength values ​​of microbial steel slag mortar specimens and cement mortar specimens in Example 1

[0073]

[0074] Under a sterile environment, take 25 ml of the supernatant of the microbial slag cementitious material and the mineralization-induced mixed bacterial solution and place them in a 500 ml triangular flask, dilute to 250 ml with sterilized deionized water, and place 3 to 5 sterilized glass beads in the bottle. After shaking, a 1:10 uniform dilution is obtained. Use a 1 ml sterile pipette to draw 1 ml of the 1:10 dilution into a test tube, and then add 9 ml of sterilized deionized water. After shaking, a 1:100 dilution is obtained. Repeat the above steps to continuously dilute to 10 8 times, replace a new sterile pipette each time it increases, and select 10 6 , 10 7 , 10 8Three times dilutions were prepared to observe and count the number of colonies. At the same time as preparing 10-fold incremental dilutions, 1 ml of the bacterial solution at the corresponding dilution was aspirated and placed in a culture dish. Three plates were made for each dilution. After the culture medium solidified, the culture dish was turned over and placed in a constant temperature shaking incubator at 30 degrees for 48 hours. After the culture was completed, the plates with colony counts in the range of 30 to 300 were selected as valid plates for counting. The average number of colonies in the valid plate culture dish was calculated. According to the corresponding dilution, the final colony count was 2.30×10 10 CFU / mL.

[0075] Example 2: Silicate lysing bacteria were acclimated and cultured in a culture solution with a solid-liquid ratio of 5% between steel slag particles and liquid culture medium, and the culture was maintained at a constant temperature of 28±5°C in a constant temperature shaking incubator for 3 days. 0.5 ml of bacterial solution was taken out with a pipette and put into a culture dish, and then the taken bacterial solution was evenly spread to all corners of the culture dish with a coating rod, and silicate lysing colonies with strong activity were selected for re-cultivation. After repeated passage for 7 times, acclimated silicate lysing bacteria with an enzyme activity of ≥0.05U / min were isolated;

[0076] The steel slag particles are placed in a fermentation tank filled with an acclimated silicate lysate and cultured for 24 hours, wherein the mass ratio of the steel slag particles to the acclimated silicate lysate is 1:0.50. During the culture period, a grinding aid prepared by mixing triethanolamine, ethylene glycol and deionized water is added, and the distribution mass ratio is 7:3:10. The cultured steel slag particles are ball-milled with a ball mill to obtain a steel slag powder with a particle size of less than 75 um. The acclimated silicate lysate is cultured in an industrial fermentation tank, and the cultured bacterial solution is cyclically sprayed on the steel slag powder from top to bottom, with a spraying frequency of 2 times / d and a spraying cycle of 1d. At the same time, the substrate recovery liquid is collected and cyclically sprayed. The treated steel slag powder is dried to a constant weight by a constant temperature blast drying oven maintained at a constant temperature of 60°C, and the dried steel slag powder is crushed with a ball mill to make its particle size less than 75 um, and finally a microbial steel slag gelling material with good gelling properties is obtained.

[0077] A plurality of 40 mm × 40 mm × 160 mm mortar specimens were prepared using 450 g of microbial steel slag cementitious material, 1350 g of standard sand and 225 g of water. The prepared mortar specimens were divided into two groups, each group including three mortar specimens. One group was cured for 7 days in a standard concrete curing box or a standard concrete curing room under the conditions of a constant temperature of 20°C and a humidity of 95%, and the other group was cured for 28 days under the same conditions. The compressive strength and flexural strength of the cured specimens were tested using an electronic universal testing machine. The arithmetic mean of the compressive strength test value and the flexural strength test value obtained in each group was taken as the test result. Two groups of cement mortar specimens were prepared under the same conditions for the microbial steel slag mortar specimens, each group included three cement mortar specimens, and the compressive strength and flexural strength of the cured cement mortar specimens were tested using an electronic universal testing machine. The arithmetic mean of the compressive strength test value and the flexural strength test value obtained in each group was taken as the test result. The test values ​​of the microbial steel slag mortar specimens and the cement mortar specimens made in the same batch in each group were compared. The compressive strength and flexural strength of the mortar specimens prepared using microbial steel slag cementitious material reached 85% of those of ordinary cement mortar specimens, which can basically replace the application scenarios of ordinary Portland cement.

[0078] The test results of the microbial steel slag cementitious material and the control group of ordinary cementitious material are shown in Table 2:

[0079] Table 2 Comparison of strength values ​​of microbial slag mortar specimens and cement mortar specimens in Example 2

[0080]

[0081] Other settings are the same as those in Example 1.

[0082] Example 3: The mineralization-inducing mixed bacteria were cultured with a culture solution having a solid-liquid ratio of 5% between steel slag particles and liquid culture medium, and the culture was maintained at a constant temperature of 28±5°C in a constant temperature shaking incubator for 3 days. A pipette was used to draw 0.5 ml of the bacterial solution into a culture dish, and then the drawn bacterial solution was evenly spread to every corner of the culture dish with a coating rod. The mineralization-inducing mixed bacteria colonies with strong activity were selected and cultured again. After repeated passage for 7 times, the mineralization-inducing mixed bacteria that could grow in large quantities in the steel slag environment and had a mineralization deposition rate of ≥5×10 -8 mM·min -1 / cell's acclimation and mineralization induced mixed bacteria;

[0083] The steel slag particles were placed in a fermentation tank filled with a mineralization-inducing mixed bacterial solution and cultured for 24 hours, wherein the mass ratio of the steel slag particles to the mineralization-inducing mixed bacterial solution was 1:1.20, and a grinding aid prepared by mixing triethanolamine, ethylene glycol and deionized water was added during the culture period, with a distribution mass ratio of 7:3:10. The cultured steel slag particles were ball-milled with a ball mill to obtain steel slag powder with a particle size of less than 75 um. The mineralization-inducing mixed bacteria were cultured and domesticated in an industrial fermentation tank, and the cultured bacterial solution was sprayed on the steel slag powder from top to bottom in a cycle, with a spraying frequency of 3 times / d and a spraying cycle of 1d, and the substrate recovery liquid was collected and circulated for spraying, and the treated steel slag powder was dried to a constant weight by a constant temperature blast drying oven maintained at a constant temperature of 60°C, and the dried steel slag powder was crushed with a ball mill to make its particle size less than 75 um, and finally a microbial steel slag cementitious material with good gelling properties was obtained;

[0084] A plurality of 40 mm × 40 mm × 160 mm mortar specimens were prepared using 450 g of microbial steel slag cementitious material, 1350 g of standard sand and 225 g of water. The prepared mortar specimens were divided into two groups, each group including three mortar specimens. One group was cured for 7 days in a standard concrete curing box or a standard concrete curing room under the conditions of a constant temperature of 20°C and a humidity of 95%, and the other group was cured for 28 days under the same conditions. The compressive strength and flexural strength of the cured specimens were tested using an electronic universal testing machine. The arithmetic mean of the compressive strength test value and the flexural strength test value obtained in each group was taken as the test result. Two groups of cement mortar specimens were prepared under the same conditions for the microbial steel slag mortar specimens, each group included three cement mortar specimens, and the compressive strength and flexural strength of the cement mortar specimens after curing were tested using an electronic universal testing machine. The arithmetic mean of the compressive strength test value and the flexural strength test value obtained in each group was taken as the test result. The test values ​​of the microbial steel slag mortar specimens and the cement mortar specimens made in the same batch in each group were compared. The compressive strength and flexural strength of the mortar specimens prepared using microbial steel slag cementitious material reached 90% of those of ordinary cement mortar specimens, which can basically replace the application scenarios of ordinary Portland cement.

[0085] The test results of the microbial steel slag cementitious material and the control group of ordinary cementitious material are shown in Table 3:

[0086] Table 3 Comparison of strength values ​​of microbial slag mortar specimens and cement mortar specimens in Example 3

[0087]

[0088] Under a sterile environment, take 25 ml of the supernatant of the microbial slag cementitious material and the mineralization-induced mixed bacterial solution and place them in a 500 ml triangular flask, dilute to 250 ml with sterilized deionized water, and place 3 to 5 sterilized glass beads in the bottle. Shake to obtain a 1:10 uniform dilution. Use a 1 ml sterile pipette to draw 1 ml of the 1:10 dilution, put it into a test tube, add 9 ml of sterilized deionized water, shake well to obtain a 1:100 dilution, repeat the above steps, and continuously dilute to 10 8 times, replace a new sterile pipette each time it increases, and select 10 6 , 10 7 , 10 8 Three times dilutions were prepared to observe and count the number of colonies. At the same time as preparing 10-fold incremental dilutions, 1 ml of the bacterial solution at the corresponding dilution was aspirated and placed in a culture dish. Three plates were made for each dilution. After the culture medium solidified, the culture dish was turned over and placed in a constant temperature shaking incubator at 30 degrees for 48 hours. After the culture was completed, the plates with colony counts in the range of 30 to 300 were selected as valid plates for counting. The average number of colonies in the valid plate culture dish was calculated. According to the corresponding dilution, the final colony count was 1.89×10 9 CFU / mL.

[0089] Other settings are the same as those in Example 1.

[0090] The microbial steel slag cementitious material prepared in this embodiment can be applied to the preparation of cushion, filler, cement and concrete materials. Through the application of microbial solid waste cementitious materials, the new cement developed with industrial secondary products such as steel slag has certain application prospects in this regard. The activity characteristics and gelling properties of steel slag cementitious materials can meet its service requirements in most scenarios, and the accelerated carbon capture and carbon production effect of microorganisms is also more conducive to protecting the environment and saving resources. The use of multi-microorganisms to treat and activate steel slag is mainly through the synergy and optimization between different reaction systems, thereby improving the reaction efficiency, reducing the reaction time, and optimizing the production process. When the method is used, microorganisms are used to induce carbonate deposition in steel slag to produce gelling properties, thereby improving the gelling activity of steel slag. Based on the enzymatic hydrolysis and mineralization of mineralizing microorganisms, the method of catalyzing the generation of stable carbonate minerals is efficient and specific, less polluting, simple in method, and environmentally friendly in product and reaction process. The application of microbial technology reduces the cost of chemical reagents, stimulates the activity of steel slag, increases the hydration rate, and improves the stability of steel slag. The use of multiple microorganisms to treat steel slag, targeting the different contradictions in the application process of steel slag, uses a specific microbial system to react and solve the problems. Its advantages such as targetedness, high efficiency and pollution-free are not available in traditional strategies.

Claims

1. A method for treating steel slag by using multi-microorganism activation, characterized in that: The following steps are involved: S1: Use a jaw crusher to coarsely crush the steel slag raw material, and use a vibrating screen to screen the coarsely crushed steel slag raw material to obtain steel slag particles with a particle size not greater than 5 mm; S2: mixing the steel slag particles with the liquid culture medium at a solid-liquid mass ratio of 5% to 25% to prepare a steel slag particle suspension; S3: Obtain soil samples and separate and purify them to obtain multi-microorganisms, including silicate lysing bacteria and mineralization-inducing mixed bacteria, and culture them in a suspension of steel slag particles to screen out domesticated silicate lysing bacteria and domesticated mineralization-inducing mixed bacteria, among which the enzyme activity of domesticated silicate lysing bacteria is ≥0.05U / min, and the mineralization deposition rate of domesticated mineralization-inducing bacteria is ≥5×10 - 8 mM·min -1 / cell; S4: placing the steel slag particles in a fermentation tank filled with domesticated silicate lysing bacteria or domesticated mineralization inducing mixed bacteria for cultivation and adding a grinding aid during the cultivation period, and ball milling the cultured steel slag particles with a ball mill to obtain steel slag powder with a particle size of less than 75 μm; S5: Use an industrial fermentation tank to cultivate a bacterial solution of domesticated silicate lysing bacteria or domesticated mineralization induced mixed bacteria, spray the cultured bacterial solution on the steel slag powder from top to bottom in a circular manner, use a constant temperature blast drying oven to maintain a constant temperature of 40-60 degrees to dry the treated steel slag powder to a constant weight, use a ball mill to crush the dried steel slag powder to make its particle size less than 75um, and finally obtain a microbial steel slag cementitious material with good gelling properties; S6: Use microbial steel slag cementitious material to make mortar specimens, use an electronic universal testing machine to test the compressive strength and flexural strength of the specimens to determine their cementing activity, and compare the compressive strength and flexural strength with those of cement mortar specimens prepared under the same conditions; S7: Use the plate colony counting method to detect the indicators of multiple microorganisms in microbial slag cementitious materials.

2. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: In S3, the specific steps of separating and purifying silicate lysate are as follows: S31a: Obtain 1.0 g of soil sample, add it to a test tube containing 9.0 ml of sterile culture medium, incubate it for 3 days, take the supernatant and inoculate it into a conical flask containing sterile culture medium, the inoculation ratio is 1:100, and incubate it in a constant temperature shaking incubator at a constant temperature of 30 degrees and an shaking frequency of 120 rpm for 3 to 5 days; S32a: Use sterile deionized water sterilized at 121℃ for more than 20min to dilute the culture supernatant to 10 3 ~10 8 times, taking 1.0 ml of bacterial solution from the dilution at each dilution degree respectively and mixing it with the culture medium added with 1.5% to 3.0% agar, so as to prepare a solid culture medium plate, and then placing it in a sterile constant temperature incubator to maintain a constant temperature of 25°C to 35°C for 3 to 5 days. After the colony growth in the solid culture medium plate is completed, using an inoculation loop or an inoculation needle to pick out silicate lysate colonies with regular morphology and full growth, and inoculating them again into a conical flask containing culture medium for subculture, so as to obtain a silicate lysate with an activity preservation rate of ≥95%, wherein the culture medium components are: 10 g / L sucrose, 0.2 g / L potassium hydrogen phosphate, 0.2 g / L magnesium sulfate heptahydrate, 0.005 g / L ferric chloride, 0.7 g / L calcium carbonate, and the pH value of the culture medium is 7.

2.

3. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: In S3, the specific steps of separating and purifying the mineralization-inducing mixed bacteria are as follows: S31b: Obtain 1.0 g of soil sample, add it to a test tube containing 9.0 ml of sterile culture medium, incubate it for 3 days, take the supernatant and inoculate it into a conical flask containing sterile culture medium, the inoculation ratio is 1:100, and incubate it in a constant temperature shaking incubator at a constant temperature of 30 degrees and an shaking frequency of 120 rpm for 3 to 5 days; S32b: Use sterile deionized water sterilized at 121℃ for more than 20min to dilute the culture supernatant to 10 3 ~10 8 times, 1.0 ml of the dilution at each dilution multiple is taken and mixed with the culture medium added with 1.5% to 3.0% agar to prepare a solid culture medium plate, and then placed in a sterile constant temperature incubator to maintain a constant temperature of 25°C to 35°C for 3 to 5 days, and then the mineralization-induced mixed bacteria colonies with better growth are picked out and inoculated into a conical flask containing the culture medium for subculture to obtain a mineralization-induced mixed bacteria with a mineralization activity preservation rate of ≥95%, wherein the culture medium components are: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the pH value of the culture medium is 7.

0.

4. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: In S3, the mineralization-inducing mixed bacteria is a mixed bacteria strain mainly composed of mineralization-inducing bacteria, wherein the mineralization-inducing bacteria account for no less than 80%, and the remaining mixed bacteria are one or more species of Bacillus pasteurianus, Bacillus pseudofirmus, Bacillus cohnii, Bacillus colloidus or carbonic anhydrase bacteria. The specific steps of screening the acclimated silicate lysing bacteria and the acclimated mineralization-inducing mixed bacteria are as follows: S33: The silicate lysing bacteria and mineralization-inducing mixed bacteria were inoculated into the steel slag suspension respectively, and then cultured in a constant temperature shaking incubator at 28±5℃ for 3 days. 0.5 ml of the corresponding bacterial solution was drawn into the culture dish using a pipette, and then the drawn bacterial solution was evenly spread to every corner of the culture dish using a coating rod; S34: Select the microbial colonies with complete morphology and full growth and re-cultivate them. After repeated subculturing for 5 to 10 times, isolate the acclimated silicate lysis bacteria with enzyme activity ≥ 0.05 U / min and mineralization deposition rate ≥ 5×10 -8 mM·min -1 / cell's acclimation and mineralization-induced mixed bacteria.

5. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: When the bacteria in the fermentation tank in S4 are domesticated silicate lysozyme, the corresponding bacteria in the cultured bacterial liquid in S5 are domesticated silicate lysozyme or domesticated mineralization induced mixed bacteria. When the bacteria in the fermentation tank in S4 are domesticated mineralization induced mixed bacteria, the corresponding bacteria in the cultured bacterial liquid in S5 are domesticated mineralization induced mixed bacteria.

6. The method for treating steel slag by using multi-microorganism activation according to claim 5, characterized in that: The mass ratio of the steel slag particles in the S4 to the acclimated silicate lysate or the acclimated mineralization induction mixed bacteria solution is 1:0.25-1.30, and the culturing time is 24-72 hours.

7. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: The grinding aid in S4 is compounded with triethanolamine, ethylene glycol and deionized water in a mass ratio of 7:3:10, and stirred with a stirrer for 30 minutes after compounding.

8. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: The spraying frequency of the bacterial liquid in S5 is 1 to 3 times / d, the spraying cycle is 1 to 2 days, and the drying temperature in S5 is 40 to 80 degrees Celsius.

9. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: The mortar specimen in S6 is prepared from 450g of microbial steel slag cementitious material, 1350g of standard sand and 225g of water. The size of the mortar specimen is 40mm×40mm×160mm. The prepared mortar specimens are divided into two groups, each group including three mortar specimens. One group is cured for 7d in a standard concrete curing box or a standard concrete curing room at a constant temperature of 20°C and a humidity of 95%, and the other group is cured for 28d under the same conditions. The compressive strength and flexural strength of the cured specimens are tested using an electronic universal testing machine, and the compressive strength and flexural strength are compared with those of cement mortar specimens prepared under the same conditions.

10. The method for treating steel slag by using multi-microorganism activation according to claim 1, characterized in that: In S7, before the plate colony counting method is performed, the prepared microbial steel slag gelling material is crushed by a crusher so that its particle size is ≤5 mm, the microbial steel slag gelling material particles are dissolved in water and suspended and soaked for 2 to 4 hours, and then the upper clear liquid is taken to plate count, and sterile deionized water is used as a control group to ensure that the environment is sterile. The specific steps of the plate colony counting method are as follows: S71: Under a sterile environment, take 25 ml of the supernatant of the microbial steel slag cementitious material and the mineralization-induced mixed bacterial solution and place them in a 500 ml conical flask, make up to 250 ml with sterilized deionized water, and place 3 to 5 sterilized glass beads in the flask, and shake to obtain a uniform dilution of 1:10; S72: Use a 1 ml sterile pipette to draw 1 ml of the 1:10 dilution into a test tube, add 9 ml of sterile deionized water, and shake well to obtain a 1:100 dilution; S73: Repeat the above steps and dilute to 10 8 times, replace a new sterile pipette each time it increases, and select 10 6 , 10 7 , 10 8 Make three dilutions, and at the same time as making 10-fold incremental dilutions, take 1 ml of the bacterial solution at the corresponding dilution and place it in a culture dish, and make 3 plates for each dilution; S74: After the culture medium solidifies, turn the culture dish over and place it in a constant temperature shaking incubator to maintain a constant temperature of 30 degrees for 48 hours. After the culture is completed, select the plates with a colony count in the range of 30 to 300 as valid plates for counting, calculate the number of colonies in the plate culture dish, and obtain the number of bacteria per milliliter of bacterial solution based on the corresponding dilution.

Images