Roadbed microbial modifier and preparation method and application thereof

Abstract

According to the application, firstly, target microorganisms are domesticated through five times of iterative culture, a culture medium comprises glucose, calcium chloride, calcium hydroxide, urea, monopotassium phosphate and sodium chloride, and infectious microbes extracted from roadbed field soil are added in the last step for mixed culture. And centrifuging or filtering to separate out calcium carbonate precipitate, and when the precipitation amount reaches 30-80g / L, extracting clear supernate as a microbial improver. In the application process, the modifier is mixed with brown sugar, urea and monopotassium phosphate and cultured, then the modifier is applied to roadbed construction, and the stability and drainage performance of a roadbed are finally improved by mixing the modifier with filler and carrying out mechanical leveling and compaction. The method particularly pays attention to gradual domestication of microorganisms and specific condition control of field application so as to ensure the effectiveness and applicability of the modifier.

Description

Technical Field

[0001] The invention belongs to the technical field of roadbed engineering, and in particular relates to a roadbed microbial improver and a preparation method and application thereof. Background Art

[0002] Microbial induced mineralization (MICP) technology uses microorganisms to precipitate calcium carbonate in the soil to improve soil properties and enhance foundation stability. However, the application of traditional MICP technology is limited, mainly because it requires strict sterile conditions and complex culture processes, which not only increases costs but also prolongs the construction period. Traditional technology usually requires the use of specialized equipment in a laboratory environment to culture microorganisms, which need to be sterilized in situ before the cultured microorganisms can be applied to on-site construction. The ideal MICP modifier should be able to grow and reproduce under non-sterile conditions and adapt to different temperatures, humidity and soil types, thereby improving its robustness and applicability in various complex construction environments. In actual roadbed construction sites, microorganisms are usually in an aerobic environment, which means that they can carry out metabolic activities through aerobic respiration, thereby producing the carbon dioxide needed to promote the precipitation of calcium carbonate. More importantly, the traditional method mainly uses urease in an anaerobic environment to promote the conversion of urea and CaCl 2 The reaction generates calcium carbonate, which cannot be used in open roadbed environments; and directly adding calcium carbide and urea will increase costs significantly.

[0003] In addition, the nitrogen source (such as urea) required by traditional MICP mineralization technology may cause the release of toxic and irritating gases such as ammonia when mixed with roadbed soil or under the action of microorganisms, which is not conducive to construction safety and environmental protection. Therefore, the development of an environmentally friendly, safe and low-cost roadbed microbial improver and its supporting preparation and construction method is crucial to promote the application of MICP technology in roadbed engineering. Summary of the invention

[0004] The purpose of the embodiment of the present invention is to provide a roadbed microbial improver to achieve effective operation under non-sterile conditions, reduce construction difficulty and cost, promote calcium oxide solidification of roadbed soil through microorganisms, and improve soil strength;

[0005] The second object of the present invention is to provide a construction method of a roadbed microbial improver;

[0006] The third object of the present invention is to provide an application of a roadbed microbial improver.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is a method for preparing a roadbed microbial improver, which comprises preparing an acclimation medium to gradually acclimate the target microorganisms, adding miscellaneous bacteria extracted from the roadbed soil at the last acclimation for mixed culture; collecting the generated CaCO 3 Sedimentation, separating the precipitate by centrifugation or filtration, and measuring the amount of precipitation; when the amount of precipitation reaches the target range, extracting the clear culture fluid in the upper half of the mixed culture acclimation culture fluid; and obtaining the microbial improver.

[0008] Furthermore, the gradual acclimatization included five iterative cultures, and the culture medium parameters were as follows: glucose 5-10 g / L, CaCl 2 0-12g / L, Ca(OH) 2 0-35g / L, urea 4-6g / L, potassium dihydrogen phosphate 0.8-1.2g / L, sodium chloride 4-6g / L.

[0009] Furthermore, during the five iterative cultivations, CaCl 2 The concentration gradually decreases; Ca(OH) 2 The concentration was gradually increased; urea was introduced only in the first acclimatization culture; Ca(OH) 2 Concentrations are not introduced in the first acclimatization culture.

[0010] Furthermore, the specific steps of the gradual domestication are:

[0011] S1011. Sterilize the prepared acclimatization medium with high pressure steam for 20 to 30 minutes;

[0012] S1012, waiting for the culture medium to cool to 25-30°C;

[0013] S1013, inoculate the pasteurized culture medium with the sporosarcina strain, and culture in a shake flask for 24 to 48 hours until the microbial density in the culture medium reaches OD 600 =0.8~1.5;

[0014] S1014, subculture the culture medium every 24 to 48 hours;

[0015] S1015. In the last acclimatization culture, add the bacteria extracted from the roadbed soil and mix them with the target microorganisms. Regularly monitor the growth and concentration of the microorganisms in the culture solution. When the concentration of the bacterial solution reaches OD 600 =0.8~1.5 when the acclimation is completed.

[0016] Furthermore, the target precipitation amount ranges from 30 to 80 g / L.

[0017] Furthermore, in S1015, the content of miscellaneous bacteria after training is ≤20%.

[0018] A roadbed microbial improver prepared by the above-mentioned method for preparing a roadbed microbial improver.

[0019] An application of a roadbed microbial improver comprises the following steps:

[0020] S2. On-site cultivation of roadbed microbial improvers:

[0021] S201. Add brown sugar, urea and potassium dihydrogen phosphate in mass fractions of 9% to 11%, 0.4% to 0.6% and 0.4% to 0.6% respectively into a ton barrel, and the balance is water, and the water is aerated for 3 hours or left to stand for 3 days;

[0022] S202. After sufficient stirring, add the roadbed microbial improver solution in the barrel at a liquid temperature of 25-30°C and a pH value of 5-9, and culture for 3-5 days. Monitor the concentration of the solution and keep it ready for use after it reaches the on-site requirements.

[0023] S3. Use roadbed microbial improver to construct the roadbed:

[0024] S301, turning over and drying the roadbed filler, and controlling the moisture content of the roadbed filler;

[0025] S302, spreading lime and glucose powder in a mass ratio of 3 to 5:1, wherein the amount of lime spread is 3% to 7% of the mass of the roadbed filler; then, mixing twice with a rotary tiller or a lime-soil mixer to evenly mix the lime and glucose powder with the roadbed filler, and each mixing depth should be 3 to 5 cm to the next layer; during the mixing process of the filler and the culture medium components, spray the bacterial solution obtained in S202;

[0026] S303. After the bacterial liquid and filler are evenly mixed, they are leveled with a grader. After each layer is initially leveled, ensure that a certain road arch is formed; a large-tonnage vibratory roller is used to roll the leveled filling surface; when rolling, the straight section is from both sides to the middle, and the small radius curve section is from the inside to the outside, and the longitudinal forward and backward method is used. The horizontal joints overlap by 0.4 to 0.5m, and the longitudinal rolling wheel tracks overlap by 0.4 to 0.5m. The driving speed of the roller is controlled within 4km / h. Static pressure is used for the initial compaction, and then it is changed to vibration compaction. The number of compaction times is determined by experiments; where the machine cannot reach the corners or the compaction machine cannot meet the requirements, a frog-type rammer is used for processing.

[0027] Furthermore, in S301, for sandy soil, the moisture content is controlled at 8% to 12%; for silty soil, the moisture content is controlled at 12% to 16%; for clay soil, the moisture content is controlled at 16% to 20%.

[0028] Furthermore, the mixing ratio of the bacterial liquid spray in S302 is 1.5 to 3.5 L / m 3 .

[0029] The beneficial effects of the present invention include the following aspects:

[0030] (1) Reduce costs and increase efficiency. Traditional MICP technology usually requires the cultivation of microorganisms under strict laboratory conditions and the use of expensive equipment and raw materials, such as in-situ sterilization of microbial culture tanks, which greatly increases costs. In contrast, the preparation method of the present invention is simple, does not require complex aseptic conditions, uses common culture equipment and simple culture media, and significantly reduces preparation costs. In addition, through rapid preparation and use on site, time and labor costs are saved, and the economic benefits of the project are improved.

[0031] (2) Easy to operate and adapt to on-site construction needs. The preparation process is simple and clear and can be completed under ordinary laboratory conditions. The construction site is easy to operate and does not require strict sterilization conditions, which lowers the technical threshold and improves the cultivation efficiency. During the construction process, a rotary tiller is used to quickly turn the culture material and the roadbed soil, and combined with spraying equipment to complete the full mixing of the improved soil, bacterial solution and air, providing a reaction environment with sufficient oxygen, improving construction efficiency and promoting the mineralization of MICP.

[0032] (3) Green, environmentally friendly, safe and reliable. The microbial improver developed by the present invention uses natural soil and environmentally friendly culture medium components, avoiding the environmental pollution and ecological risks that may be caused by traditional chemical agents. During the construction process, no harmful chemicals need to be added, which reduces the negative impact on the environment and meets the demand of modern society for green and environmentally friendly construction. In addition, the microbial improver of the present invention does not release toxic gases during use, ensuring the safety and health of construction workers.

[0033] (4) Improve the stability and structural strength of the roadbed. MICP technology precipitates calcium carbonate in the soil through microorganisms, effectively improving the physical and mechanical properties of the soil. The precipitation of calcium carbonate fills the soil pores, improves the compactness and compressive strength of the soil, and makes the roadbed more stable and durable. This method is not only suitable for the reinforcement of new roadbeds, but can also be used for the repair and reinforcement of old roadbeds, extending the service life of the roadbed and reducing subsequent maintenance costs.

[0034] (5) Sustainable development. The technical solution of the present invention is in line with the concept of sustainable development. By utilizing natural microorganisms and environmentally friendly culture conditions, it effectively promotes the rational use and protection of soil resources. It reduces resource consumption and carbon emissions during the construction process, thus contributing a new solution to green engineering construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 It is a flow chart of the preparation of green roadbed microbial improver and its construction plan;

[0037] Figure 2 1 is a graph showing the results of a field dynamic modulus of resilience test of the roadbed of Example 1 of the present invention;

[0038] Figure 3 is a curve diagram showing the relationship between the survival rate of the strains and time in Example 1 of the present invention;

[0039] Figure 4 This is a graph showing the results of the on-site dynamic modulus of resilience test of the roadbed of Comparative Example 1;

[0040] Figure 5 is a curve diagram showing the relationship between the survival rate of the strains and time in Comparative Example 1;

[0041] Figure 6 This is a graph showing the results of the on-site dynamic modulus of resilience test of the roadbed of Comparative Example 2;

[0042] Figure 7 It is the curve diagram of the relationship between the survival rate of the strains and time in Comparative Example 2. DETAILED DESCRIPTION

[0043] 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.

[0044] like Figure 1 This embodiment provides a method for preparing a roadbed microbial improver, which specifically includes the following steps:

[0045] S1. Preparation of green roadbed microbial improver:

[0046] Example 1

[0047] S101, prepare acclimation medium, the culture parameters of the medium are shown in Table 1, gradually reduce the concentration of CaCl2, increase Ca(OH) 2concentration, and in the fifth acclimation, miscellaneous bacteria extracted from the roadbed soil were added for mixed culture.

[0048] Table 1 Acclimation medium configuration (g / L)

[0049]

[0050]

[0051] The specific bacterial culture process is as follows:

[0052] S1011, dissolving the components of the acclimation medium in deionized water to obtain the target concentration for the first acclimation, and sterilizing the acclimation medium by high pressure steam in an automatic sterilizer for 20 minutes;

[0053] S1012, waiting for the culture medium to cool to a temperature suitable for microbial growth (25-30°C);

[0054] S1013, inoculate an appropriate amount of the pasteurized culture medium (initial bacterial solution), and culture in a shake flask for 48 hours until the microbial density OD in the culture medium reaches 600 =1.15;

[0055] The Sporosarcina pasteurii strain in this embodiment has been deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number of CGMCC No.1.3687 and the deposit date of June 18, 2004. In addition, the strain also has other deposit numbers, including ATCC 11859, CCM 2056, NCIB 8841 and NCTC 4822.

[0056] S1014. Every 48 hours, take a portion of the culture solution for subculture, that is, prepare the second to fifth culture media according to the components in Table 1, and gradually increase the Ca(OH) 2 The concentration is then inoculated with the domesticated strain for domestication and cultivation;

[0057] S1015. In the fifth domestication, add miscellaneous bacteria extracted from the roadbed soil and culture them together with the target microorganisms. Regularly monitor the growth and concentration of the microorganisms in the culture medium to ensure that the microorganisms gradually adapt and proliferate in the culture medium, with the content of miscellaneous bacteria ≤10%.

[0058] In this embodiment, to ensure the dominance of the target microorganisms during the domestication process, the content of the initially introduced bacteria should be controlled within 10% of the total amount, and regular monitoring should be performed during the culture period to ensure that the final bacteria content does not exceed 20% of the total amount.

[0059] S102. Through five times of domestication and mixed culture of various bacteria, the interaction between the target microorganisms and the natural microorganisms in the local soil can be promoted to enhance their adaptability and growth ability. The concentration of the bacterial solution is determined by a spectrophotometer to reach OD 600 =1.15, and the formed colonies were counted using the plate counting method to determine that the content of foreign bacteria was less than 20%.

[0060] In the traditional microbial induced calcium carbonate precipitation (MICP) technology, a pure culture method of a single strain is generally used, which requires extremely strict aseptic conditions and complex culture processes, increases operating costs and prolongs the construction period of the project. In addition, in a non-sterile environment, a single strain is susceptible to contamination by foreign microorganisms, resulting in a decrease in its biological activity and treatment efficiency. Moreover, a single microorganism often has high requirements for the growth environment and is difficult to adapt to the actual and changing conditions of the construction site. If non-target microorganisms (miscellaneous bacteria) are introduced, unpredictable biochemical reactions may be triggered, thereby posing a threat to the quality and safety of the project. This embodiment proposes an improved solution, that is, a proper amount of miscellaneous bacteria is purposefully introduced during the domestication and culture stage, and co-cultured with the target microorganism to enhance the environmental adaptability and adversity resistance of the target microorganism. In this process, by continuously monitoring the dynamic changes of the microbial community, it is ensured that the target microorganism can occupy a dominant position in the competition, thereby ensuring its effective performance in on-site construction. In order to prevent the potential negative effects of miscellaneous bacteria, this method strictly controls its proportion to no more than 20% to enhance the function of the target microorganism without causing adverse consequences. This optimization strategy effectively solves the uncertainty problem caused by the introduction of miscellaneous bacteria and achieves high stability and high efficiency of MICP technology under complex construction conditions. Experiments show that while maintaining efficient calcium carbonate precipitation and soil improvement effects, this embodiment has improved adaptability and construction efficiency under non-sterile conditions compared to traditional technologies, and is particularly suitable for on-site construction environments with complex and changeable conditions.

[0061] Collect the generated CaCO 3 The precipitate is separated by centrifugation or filtration, and its mass is weighed to determine whether it reaches the target precipitation amount (in this embodiment, the CaCl 2 and Ca(OH) 2 The target precipitation amount determined by the mass conversion should be controlled within the range of 30-80 g / L) to evaluate the stability of the generated CaCO3 precipitation. After the above domestication requirements are met, the upper half of the clear culture fluid is extracted from the domestication culture fluid as the preferred bacterial fluid for on-site expansion production.

[0062] In this embodiment, when CaCl 2 The concentration exceeds 15g / L or Ca(OH) 2When the concentration of CaCl exceeds 30 g / L, it may lead to excessive precipitation (over 80 g / L), resulting in large particle crystallization of the precipitated material, affecting the uniformity of soil pores and the metabolic activity of microorganisms; 2 The concentration is lower than 5g / L or Ca(OH) 2 The concentration of Ca is lower than 10 g / L, which will result in insufficient precipitation (lower than 30 g / L). 2+ and OH - ions are not enough to generate enough calcium carbonate, which affects the consolidation effect of the soil. - Too high concentration, Ca 2+ will be too fast with CO 3 2- Combined to form excessive CaCO 3 particles, affecting the uniformity of precipitation; when the pH value is lower than 7.0, OH - Insufficient concentration, CaCO 3 The amount of CO generated by microbial metabolism is reduced, and the ideal precipitation effect cannot be achieved. 2 Too much CaCO 3 If the amount of precipitation is too large, exceeding 80g / L, coarse calcium carbonate particles will be formed, affecting soil stability. When the concentration of bacterial solution is lower than 1.0, the number of microorganisms is insufficient, and CO 2 The amount of calcium carbonate generated is low, and it is impossible to produce enough calcium carbonate precipitation, which affects the roadbed reinforcement effect. When the temperature exceeds 35°C, the microbial metabolism is too active, and CO 2 If the amount of generation is too high, the precipitation amount may exceed the target value of 80g / L, resulting in uneven precipitation; when the temperature is below 25℃, the microbial metabolism slows down, CO 2 Insufficient generation, calcium carbonate precipitation is less than 30g / L, affecting the soil reinforcement effect. In summary, when the precipitation exceeds 80g / L, excessive precipitation will lead to large particles of CaCO 3 The formation and filling are uneven, which may block the soil pores and reduce the drainage capacity of the roadbed, while affecting the further metabolism of microorganisms and the overall stability of the soil; when the precipitation amount is less than 30g / L, the insufficient precipitation amount cannot fully solidify the soil, affecting the strength and bearing capacity of the roadbed, and cannot meet the construction requirements. It is necessary to readjust the reaction conditions or increase the input of reactants, which will extend the construction period.

[0063] In this embodiment, CaCl 2 As a calcium source, it can induce the precipitation of calcium carbonate, and its concentration is gradually reduced to adapt to different acclimation stages; Ca(OH) 2 As a step-wise calcium source to improve microbial fitness and CaCO 3The production efficiency of microorganisms is improved; urea is only used in the first domestication to reduce the risk of ammonia production and provide the necessary nitrogen source; potassium dihydrogen phosphate: as a stable phosphorus source to ensure the normal metabolism of microorganisms; sodium chloride is used to maintain a certain osmotic pressure to prevent osmotic imbalance of microbial cells.

[0064] Example 2

[0065] S101, prepare acclimation medium, the culture parameters of the medium are shown in Table 2, gradually reduce CaCl 2 concentration, increase Ca(OH) 2 concentration, and in the fifth acclimation, miscellaneous bacteria extracted from the roadbed soil were added for mixed culture.

[0066] Table 2 Acclimation medium configuration (g / L)

[0067] Domestication stage 1st time 2nd time 3rd 4th 5th glucose 10 10 10 10 10 <![CDATA[CaCl 2 ]]> 12 5 2.5 1 0 <![CDATA[Slaked lime Ca(OH) 2 > 0 5 10 20 35 Urea 6 0 0 0 0 Potassium dihydrogen phosphate 1.2 1.2 1.2 1.2 1.2 Sodium chloride 6 6 6 6 6

[0068] The specific bacterial culture process is as follows:

[0069] S1011, dissolving the components of the acclimation medium in deionized water to obtain the target concentration for the first acclimation, and sterilizing the acclimation medium by high pressure steam in an automatic sterilizer for 30 minutes;

[0070] S1012, waiting for the culture medium to cool to a temperature suitable for microbial growth (25-30°C);

[0071] S1013, inoculate an appropriate amount of pasteurized sporosarcina strain (initial bacterial solution) into the sterile culture medium, and culture in a shake flask for 36 hours until the microbial density OD in the culture medium reaches 600 =1.5;

[0072] S1014. Every 36 hours, a portion of the culture medium was taken for subculture, that is, the second to fifth culture mediums were prepared according to the components in Table 2, and the Ca(OH) 2 The concentration is then inoculated with the domesticated strain for domestication and cultivation;

[0073] S1015. In the fifth domestication, add miscellaneous bacteria extracted from the roadbed soil and culture them together with the target microorganisms. Regularly monitor the growth and concentration of the microorganisms in the culture medium to ensure that the microorganisms gradually adapt and proliferate in the culture medium, with the content of miscellaneous bacteria ≤10%.

[0074] S102. Through five times of domestication and mixed culture of various bacteria, the interaction between the target microorganisms and the natural microorganisms in the local soil can be promoted to enhance their adaptability and growth ability. The concentration of the bacterial solution is determined by a spectrophotometer to reach OD 600 =1.5, and the formed colonies were counted using the plate counting method to determine that the content of foreign bacteria was less than 20%.

[0075] Collect the generated CaCO 3 Sedimentation, separation of the precipitate by centrifugation or filtration, and weighing its mass to determine whether it has reached the target precipitation amount. After the above domestication requirements are met, the upper half of the clear culture fluid is extracted from the domestication culture fluid as the preferred bacterial fluid for on-site expansion production.

[0076] Example 3

[0077] S101, prepare acclimation medium, the culture parameters of the medium are shown in Table 3, gradually reduce CaCl 2 concentration, increase Ca(OH) 2 concentration, and in the fifth acclimation, miscellaneous bacteria extracted from the roadbed soil were added for mixed culture.

[0078] Table 3 Acclimation medium configuration (g / L)

[0079] Domestication stage 1st time 2nd time 3rd 4th 5th glucose 7.5 7.5 10 10 10 <![CDATA[CaCl 2 ]]> 12 6 3 1 0 <![CDATA[Slaked lime Ca(OH) 2 > 0 5 10 17.5 35 Urea 4 0 0 0 0 Potassium dihydrogen phosphate 0.8 0.8 0.8 0.8 0.8 Sodium chloride 4 4 4 4 4

[0080] The specific bacterial culture process is as follows:

[0081] S1011, dissolving the components of the acclimation medium in deionized water to obtain the target concentration for the first acclimation, and sterilizing the acclimation medium by high pressure steam in an automatic sterilizer for 25 minutes;

[0082] S1012, waiting for the culture medium to cool to a temperature suitable for microbial growth (25-30°C);

[0083] S1013, inoculate an appropriate amount of pasteurized sporosarcina strain (initial bacterial solution) into the sterile culture medium, and culture in a shake flask for 24 hours until the microbial density OD in the culture medium reaches 600 =0.8;

[0084] S1014. Every 24 hours, take a portion of the culture solution for subculture, that is, prepare the second to fifth culture media according to the components in Table 2, and gradually increase the Ca(OH) 2 The concentration is then inoculated with the domesticated strain for domestication and cultivation;

[0085] S1015. In the fifth domestication, add miscellaneous bacteria extracted from the roadbed soil and culture them together with the target microorganisms. Regularly monitor the growth and concentration of the microorganisms in the culture medium to ensure that the microorganisms gradually adapt and proliferate in the culture medium, with the content of miscellaneous bacteria ≤10%.

[0086] S102. Through five times of domestication and mixed culture of various bacteria, the interaction between the target microorganisms and the natural microorganisms in the local soil can be promoted to enhance their adaptability and growth ability. The concentration of the bacterial solution is determined by a spectrophotometer to reach OD 600=0.8, and the colonies formed were counted using the plate counting method to determine that the content of foreign bacteria was less than 20%.

[0087] Collect the generated CaCO 3 Sedimentation, separation of the precipitate by centrifugation or filtration, and weighing its mass to determine whether it has reached the target precipitation amount. After the above domestication requirements are met, the upper half of the clear culture fluid is extracted from the domestication culture fluid as the preferred bacterial fluid for on-site expansion production.

[0088] Comparative Example 1

[0089] The difference from Example 1 is that in the fifth acclimation, the bacteria extracted from the roadbed soil were not added; the remaining steps were the same as in Example 1.

[0090] like Figure 4 , Figure 5 Comparison of the dynamic rebound modulus test results shows that the dynamic rebound modulus value of the roadbed is significantly lower than that of the embodiment, which is manifested by large fluctuations in the overall value and fails to meet the design requirements. This indicates that the stability and strength of the soil cannot be effectively improved without the introduction of foreign bacteria. Comparison of the relationship between the survival rate of the strains and time shows that the survival rate of the strains in Comparative Example 1 is significantly lower than the results in the embodiment. The failure to introduce foreign bacteria results in poor adaptability of the microorganisms in the soil, a rapid decrease in the number of strains, and a low survival rate. This is because the target microorganisms lack competitive domestication with the foreign bacteria in the on-site soil, resulting in a weakened survival ability.

[0091] The failure to add foreign bacteria limits the adaptability and growth potential of microorganisms. In practical applications, the target microorganisms will have to face competitive pressure from indigenous microorganisms. However, due to the lack of corresponding foreign bacteria coexistence training during the domestication process, these microorganisms are difficult to effectively colonize in a complex natural environment and perform their intended functions, especially the microbial-induced calcium carbonate precipitation (MICP) process is affected, which in turn has an adverse effect on the mechanical properties and overall stability of the roadbed materials.

[0092] Comparative Example 2

[0093] The difference from Example 1 is that after the fifth acclimation, the culture is carried out until the content of miscellaneous bacteria reaches 50%.

[0094] like Figure 6 , Figure 7Comparison of the dynamic rebound modulus test results shows that the dynamic rebound modulus of the roadbed in Example 2 is low and fluctuates greatly, failing to meet the design standard. Compared with Example 1, the strength and stability of the soil are obviously insufficient; the curve of the relationship between the survival rate of the strains and time shows that the survival rate of the strains in Example 2 shows a significant downward trend, indicating that the target microorganisms fail to grow and proliferate effectively. The content of 50% of the miscellaneous bacteria is too high, which inhibits the normal growth of the target microorganisms, resulting in too fierce competition in the microbial population, and the metabolism of the target microorganisms and CaCO 3 The amount of generation is reduced.

[0095] When the content of foreign bacteria in the soil is too high, the competitiveness of the target microorganisms will be inhibited, which in turn affects their normal metabolic activities and the formation of calcium carbonate precipitation. Excessive concentration of foreign bacteria prevents the target microorganisms from establishing a dominant population in the soil ecosystem, thus having a negative impact on the microbial induced calcium carbonate precipitation (MICP) process. Ultimately, this inferior state in ecological competition will lead to the failure of the roadbed material to obtain sufficient strength and stability.

[0096] This embodiment also provides an application method of the roadbed microbial improver, which is as follows:

[0097] S2. On-site cultivation of green roadbed microbial improver: In a 50-liter barrel with a stirrer, add 9-11% brown sugar, 0.4-0.6% urea, and 0.4-0.6% potassium dihydrogen phosphate by mass, respectively, and the balance is tap water (aerated for 2-4 hours or left to stand for 2-4 days) or well water. Use a stirrer to fully mix the water in the barrel and the added ingredients. In a suitable environment (temperature of 25-30°C, pH of 5-9), add 500 mL of the preferred bacterial solution, culture in an open environment for 3-5 days, and monitor whether the concentration of the bacterial solution meets the on-site requirements (such as OD 600 =0.8~1.5, which can be set according to actual needs) to ensure that the concentration of microorganisms is suitable for on-site roadbed construction.

[0098] When the on-site culture is in winter, a heating rod should be placed in the barrel to heat the bacterial solution to the required temperature, and an appropriate amount of warm water should be added to adjust the pH value of the bacterial solution to between 6.5 and 7.5 to promote the rapid growth of the bacterial solution. At the same time, it is also necessary to regularly sample and measure the OD of the bacterial solution. 600 values ​​to understand the progress of microbial growth and adjust culture conditions as needed.

[0099] In some possible embodiments, during winter cultivation, first, the temperature of the culture solution needs to be maintained at 28°C to 30°C by a heating rod to ensure that the microorganisms are in a suitable growth temperature range; secondly, the amount of brown sugar, urea, potassium dihydrogen phosphate or agricultural fertilizer added needs to be slightly increased by 5%-10%. Properly increasing the concentration of nutrients can compensate for the adverse effects of low temperature on microbial growth, but when adjusting the amount of nutrients added, care should be taken to avoid excessive increase to avoid nutrient waste or burden on microorganisms; thirdly, the culture time is extended by 24 to 48 hours to ensure that the microorganisms reach the required concentration and activity. The extension of the culture time should be adjusted according to the monitoring results of the OD600 value to ensure that the quality of the final bacterial solution meets the construction requirements; finally, the stirring speed is appropriately increased by 10% to 20%. By appropriately increasing the stirring speed, the uniform distribution of nutrients and microorganisms can be ensured, thereby promoting the growth of microorganisms.

[0100] S3. Construction method of green roadbed microbial improver for roadbed treatment: After the roadbed filler is turned over and dried to reach the appropriate moisture content, lime and glucose powder are spread in a mass ratio of 3 to 5:1; the amount of lime spread is 3% to 7% of the roadbed filler mass, and the corresponding amount of glucose powder is 1 / 3 to 1 / 5 of the lime mass. The specific spreading amount should be adjusted according to the roadbed soil conditions and design requirements to ensure the strength and stability of the roadbed.

[0101] In this embodiment, lime is mainly used for solidification and stabilization of roadbed and provides a calcium source; glucose powder mainly provides a carbon source for microorganisms and supports their metabolic activities; when the ratio of lime to glucose powder is 3:1, although the proportion of glucose is relatively increased, the generation rate of calcium carbonate is still controlled, which is suitable for environments with good soil conditions. At this ratio, while retaining the activity of microorganisms, the alkaline reaction of the soil is moderately enhanced; when the ratio of lime to glucose powder is 5:1, the lime content is higher and the generation rate of calcium carbonate is accelerated, which is suitable for roadbed projects with poor soil conditions or high strength requirements. In this case, lime provides a stronger alkaline environment, but does not inhibit the growth of microorganisms, and helps to quickly improve the structural strength of the soil.

[0102] Use a rotary tiller or soil mixer to mix evenly. During the mixing process of the filler and culture medium components, spray high-concentration bacterial solution, where the bacterial solution mixing ratio is 1.5-3.5L / m 3 , between 1.5 and 2.0 L / m 3 In this lower ratio range, the amount of bacterial solution used is relatively small, which is suitable for situations where the soil conditions are good or the bacterial solution cost is high. At this ratio, the basic calcium carbonate precipitation effect can be achieved, the structural strength of the soil can be enhanced, and the material cost and construction complexity can be reduced. In the range of 2.0-3.5L / m 3In this higher ratio range, the amount of bacterial solution used increases, which is suitable for construction environments with poor soil conditions or high requirements for reinforcement effects. This ratio can significantly increase the amount of calcium carbonate generated, thereby greatly improving the compressive strength and stability of the soil, and is suitable for high-demand engineering projects; when the bacterial solution mixing ratio exceeds 3.5L / m 3 It may lead to unnecessary waste of materials and increase construction costs. At the same time, too much bacterial solution may lead to excessive microbial density, which in turn inhibits the normal metabolism of microorganisms and affects the precipitation efficiency of calcium carbonate. When the bacterial solution mixing ratio is lower than 1.5L / m 3 This may lead to insufficient calcium carbonate precipitation, affect the effect of soil solidification, and even fail to achieve the soil stability and strength required by the design. 3 ~3.5L / m 3 The range provides flexible adjustment space for different soil conditions and construction requirements, ensuring that the best microbial activity and calcium carbonate precipitation effects can be achieved in various environments. The proportion setting within this range takes into account both economy and sufficient calcium carbonate production to achieve the ideal roadbed reinforcement effect. The moisture content of the filler after mixing should be controlled within the range of ±2% of the optimal moisture content (the optimal moisture content in this embodiment is determined by indoor compaction test. For sandy soil, the optimal moisture content range is 10% ± 2%; for silty soil, the optimal moisture content range is 14% ± 2%; for clay soil, the optimal moisture content range is 18% ± 2%). After the bacterial liquid and filler are evenly mixed, a grader is used to level them, and a large-tonnage vibratory roller is used to roll the leveled fill surface to meet the compaction requirements of the roadbed construction. The specific construction method is as follows:

[0103] S301, turning over and drying, moisture content judgment: Use a plow and a rotary tiller to turn over and dry the soil. If the moisture content of the soil is high, the plow should be used multiple times to turn up the lower soil, and the rotary tiller should be used to break up the soil clumps. Before spreading lime and glucose powder, the moisture content of the soil should be controlled within 5 percentage points greater than the optimal moisture content. For soils with too high a moisture content, use a plow to turn over and dry the soil, and for soils with too low a moisture content, sprinkle water to moisten the soil. Since lime and glucose powder have water-absorbing properties, they can absorb moisture from the soil, thereby reducing the moisture content of the soil. Especially for high liquid limit soils, it can effectively reduce the humidity of the roadbed filler, shorten the turning over and drying time of the roadbed filler, and improve construction efficiency.

[0104] S302. Spread the culture medium components and bacterial solution: First, spread the lime and glucose powder in a ratio of 3 to 5:1. Since the bacterial solution is not sprayed in advance, the lime and glucose powder in the mixed soil will not trigger the MICP reaction. This can avoid the MICP reaction before the roadbed is filled, and achieve a controllable effect on the solidification process. Then, use a rotary tiller or ash-soil mixer to mix twice to mix the culture material and filler evenly. The mixing depth each time should be 3 to 5 cm to the next layer. During mixing, a dedicated person follows the mixer to control the mixing quality, check the mixing depth and uniformity at any time, and cooperate with the mixer operator to make adjustments. At the same time, after the filler and culture medium components are mixed, spray a high-concentration bacterial solution, in which the bacterial solution mixing ratio is 2L / m 3 (The mixing ratio of bacterial solution should be controlled at 1.5L / m 3 ~3.5L / m 3 When the ratio exceeds 3.5L / m 3 First, it will accelerate the precipitation of calcium carbonate, but it will cause competition for nutrients between microorganisms, inhibit the growth and metabolic activities of some microorganisms, and reduce the overall precipitation efficiency; secondly, too much bacterial liquid will not significantly improve the efficiency of calcium carbonate production, but will cause unnecessary waste of materials and increase construction costs; finally, excessive CaCO 3 Sedimentation may lead to blockage of soil pores, affecting the permeability and drainage capacity of the roadbed, thereby reducing the stability of the roadbed. 3 First, the number of microorganisms in the bacterial solution is insufficient to provide sufficient CO 2 to promote the precipitation of calcium carbonate, resulting in CaCO 3 The amount of calcium carbonate generated is reduced, and the amount of precipitation may be lower than the expected 30g / L, which cannot achieve effective reinforcement effect; secondly, calcium carbonate precipitation is not enough to fill soil pores, and cannot significantly improve the compressive strength of the soil, which may lead to insufficient bearing capacity of the roadbed; finally, too low concentration of bacterial solution may lead to uneven soil reinforcement effect, especially in large-scale construction, insufficient precipitation reaction in local areas, affecting the stability and durability of the overall roadbed. ), so that the moisture content of the filler after mixing meets the roadbed design requirements of the optimal moisture content ± 2%. The advantage of this approach is that it utilizes the metabolic characteristics of Bacillus, so that the organic carbon source (such as glucose) is completely oxidized to CO 2 It also avoids the reliance on nitrogen sources such as urea in traditional MICP methods, thereby reducing environmental risks and operational complexity.

[0105] S303, shaping and rolling: After the bacterial liquid and filler are mixed evenly, they are leveled with a grader. After each layer is initially leveled, a certain road arch is formed to facilitate drainage. Manual leveling is used for corners that cannot be reached by machinery. A large-tonnage vibrating roller is used to roll the leveled fill surface. When rolling, the straight section is from both sides to the middle, and the small radius curve section is from the inside to the outside, and the longitudinal forward and backward method is used. The horizontal joints overlap by 0.4 to 0.5m, and the longitudinal rolling wheel tracks overlap by 0.4 to 0.5m. The driving speed of the roller is controlled within 4km / h. Static pressure is used for the initial compaction, and then it is changed to vibration compaction. The number of compaction passes is determined by experiments. For corners that cannot be reached by machinery or where the compaction machinery cannot meet the requirements, a frog-type rammer is used for processing, which should achieve no leakage and no dead angle to ensure uniform rolling.

[0106] In this embodiment, after the number of rolling passes meets the requirements, each compaction layer should be tested for compaction, and the test frequency is every 1000m 2 No less than 2 points.

[0107] In this embodiment, in order to balance the soil density and the activity of the microbial community, the compaction degree should be controlled within a moderate range to ensure that the strength requirements of the roadbed are met without excessive compaction, which affects the activity of the microbial community. Generally, for soil using MICP technology, the compaction degree can be controlled between 85% and 95% of the maximum dry density. This range can provide sufficient strength while maintaining an appropriate porosity to ensure the effective supply of oxygen and nutrients. At the same time, a slower rolling speed (such as within 4 km / h) helps to avoid excessive damage to the soil structure and reduce interference with the microbial environment. The number of rolling times needs to be determined based on field tests to avoid excessive rolling times that lead to excessive compaction of the soil, thereby significantly reducing the pores and oxygen in the soil available for microbial survival. In terms of compaction technology, static pressure is used in the initial compaction stage, which helps to initially stabilize the soil structure without significantly affecting the activity of the microbial community. Vibration compaction may have a greater impact on the microbial community in the soil. The vibration frequency and compaction times need to be controlled to avoid excessive mechanical stress on the microorganisms. After the compaction degree meets the requirements, the next layer can be filled and rolled until it meets the requirements. When compacting in layers, the compaction degree of each layer should meet the bearing capacity of the roadbed and avoid over-compacting. Bacterial liquid can also be sprayed after each layer is compacted to replenish the bacterial flora that may be damaged by compaction. During the construction process, the survival rate and activity of the bacterial flora should be tested regularly, such as by OD 600 Values ​​or microbial counts are tested and the construction process is adjusted to ensure the smooth progress of the MICP process.

[0108] S4. Performance and microbial activity detection of improved roadbed. After the roadbed is completed, a portable drop-weight deflectometer is used to test the dynamic rebound modulus of the roadbed top surface (the bacterial solution is that of Example 1). Figure 2As shown, it is ensured that it meets the requirements of roadbed design. At the same time, 10g of soil samples were taken at the time of just filling, 24h after filling, 36h after filling, 48h after filling, and 72h after filling, and the number of microorganisms in the soil after the corresponding time period was determined by plate counting method a2, a3, a4, a5, and a6. The number of strains before and after filling was compared, the survival rate of the strains was calculated, and the relationship curve between the survival rate of the strains and time was plotted, as shown in the figure. Figure 3 As shown, the changes in bacterial activity were obtained.

[0109] Each embodiment in this specification is described in a related manner, and the same or similar parts between the embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, for the system embodiment, since it is basically similar to the method embodiment, the description is relatively simple, and the relevant parts can be referred to the partial description of the method embodiment.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A method for preparing a roadbed microbial improver, characterized in that: An acclimation culture medium is configured to gradually acclimate the target microorganisms, and in the last acclimation, miscellaneous bacteria extracted from the soil at the roadbed site are added for mixed culture; the generated CaCO3 precipitate is collected, the precipitate is separated by centrifugation or filtration, and the precipitate amount is measured; when the precipitate amount reaches the target range, the clear culture fluid in the upper half of the mixed culture acclimation culture fluid is extracted; and a microbial improver is obtained.

2. The method for preparing a roadbed microbial improver according to claim 1, characterized in that: The stepwise acclimatization included five iterative cultures, and the specific culture medium parameters were as follows: glucose 5-10 g / L, CaCl2 0-12 g / L, Ca(OH)2 0-35 g / L, urea 4-6 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, and sodium chloride 4-6 g / L.

3. The method for preparing a roadbed microbial improver according to claim 2, characterized in that: During the five iterative cultivation processes, the CaCl2 concentration gradually decreased; the Ca(OH)2 concentration gradually increased; urea was only introduced in the first acclimation cultivation; and the Ca(OH)2 concentration was not introduced in the first acclimation cultivation.

4. The method for preparing a roadbed microbial improver according to any one of claims 1 to 3, characterized in that: The specific steps of the gradual domestication are: S1011. Sterilize the prepared acclimatization medium with high pressure steam for 20 to 30 minutes; S1012, waiting for the culture medium to cool to 25-30°C; S1013, inoculate the pasteurized culture medium with the sporosarcina strain, and culture in a shake flask for 24 to 48 hours until the microbial density in the culture medium reaches OD 600 =0.8~1.5; S1014, subculture the culture medium every 24 to 48 hours; S1015. In the last acclimatization culture, add the bacteria extracted from the roadbed soil and mix them with the target microorganisms. Regularly monitor the growth and concentration of the microorganisms in the culture solution. When the concentration of the bacterial solution reaches OD 600 =0.8~1.5 when the acclimation is completed.

5. The method for preparing a roadbed microbial improver according to claim 1, characterized in that: The target precipitation amount ranges from 30 to 80 g / L.

6. The method for preparing a roadbed microbial improver according to claim 4, characterized in that: In the S1015, the content of miscellaneous bacteria after training is ≤20%.

7. A roadbed microbial improver prepared by the method for preparing a roadbed microbial improver according to any one of claims 1 to 6.

8. The use of a roadbed microbial improver as claimed in claim 7, characterized in that: The following steps are involved: S2. On-site cultivation of roadbed microbial improvers: S201. Add brown sugar, urea and potassium dihydrogen phosphate in mass fractions of 9% to 11%, 0.4% to 0.6% and 0.4% to 0.6% respectively into a ton barrel, and the balance is water, and the water is aerated for 2-4 hours or left to stand for 2-4 days; S202. After sufficient stirring, add the roadbed microbial improver solution in the barrel at a liquid temperature of 25-30°C and a pH value of 5-9, and culture for 3-5 days. Monitor the concentration of the solution and keep it ready for use after it reaches the on-site requirements. S3. Use roadbed microbial improver to construct the roadbed: S301, turning over and drying the roadbed filler, and controlling the moisture content of the roadbed filler; S302, spreading lime and glucose powder in a mass ratio of 3 to 5:1, wherein the amount of lime spread is 3% to 7% of the mass of the roadbed filler; then, mixing twice with a rotary tiller or a lime-soil mixer to make the lime, glucose powder and roadbed filler evenly mixed, and each mixing depth should be 3 to 5 cm to the next layer; during the mixing process of the filler and the culture medium components, spraying the bacterial solution obtained in S202; S303. After the bacterial liquid and filler are evenly mixed, they are leveled with a grader. After each layer is initially leveled, ensure that a certain road arch is formed; a large-tonnage vibratory roller is used to roll the leveled filling surface; when rolling, the straight section is from both sides to the middle, and the small radius curve section is from the inside to the outside, and the longitudinal forward and backward method is used. The horizontal joints overlap by 0.4 to 0.5m, and the longitudinal rolling wheel tracks overlap by 0.4 to 0.5m. The driving speed of the roller is controlled within 4km / h. Static pressure is used for the initial compaction, and then it is changed to vibration compaction. The number of compaction times is determined by experiments; where the machine cannot reach the corners or the compaction machine cannot meet the requirements, a frog-type rammer is used for processing.

9. The use of a roadbed microbial improver according to claim 8, characterized in that: In S301, for sandy soil, the moisture content is controlled at 8% to 12%; for silty soil, the moisture content is controlled at 12% to 16%; for clay soil, the moisture content is controlled at 16% to 20%.

10. The use of a roadbed microbial improver according to claim 8, characterized in that: The mixing ratio of bacterial liquid spray in S302 is 1.5~3.5L / m 3 .

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