WO2023123082A1

WO2023123082A1 - Preparation method for double-bacteria microbial agent, and soil remediation method

Info

Publication number: WO2023123082A1
Application number: PCT/CN2021/142592
Authority: WO
Inventors: 郭丽莉; 刘亚茹; 高艳丽; 李书鹏; 王祺; 熊静; 郝弟; 李嘉晨
Original assignee: 北京建工环境修复股份有限公司
Priority date: 2021-12-29
Filing date: 2021-12-29
Publication date: 2023-07-06

Abstract

A double-bacteria microbial agent comprising carbonate mineralization bacteria and carbonic anhydrase producing bacteria, a preparation method for the double-bacteria microbial agent, and a use in desertified soil remediation. Carbonate mineralization bacteria and carbonic anhydrase producing bacteria in the double-bacteria microbial agent work in conjunction and are applied to desertified soil remediation. The stabilizing rate of the system is increased, and the stabilizing time is greatly shortened compared with that in the case of only using carbonate mineralization bacteria. CO2 is used as one of carbon sources, such that emission of CO2 is reduced and the use amount of urea is reduced; in addition, the method is non-toxic, harmless, free of secondary pollution, and low in cost, the double-bacteria microbial agent is simple to prepare, and the soil gets more fertile due to the urea left in the soil in the stabilizing process.

Description

A kind of preparation method of double bacteria agent and a kind of soil remediation method

technical field

The invention relates to the field of soil remediation, in particular to a preparation method of a dual-bacteria agent and a soil remediation method.

Background technique

Due to human activities, such as deforestation, random digging, overloading and overgrazing, etc., soil desertification is becoming more and more serious. The resulting grassland degradation, soil erosion, vegetation coverage reduction, and frequent sandstorms seriously threaten human beings. living space. The control of desertified soil has received more and more attention worldwide, and it is urgent to "control sand and stop desertification".

Desertification soil control methods can be divided into three categories: physical methods, chemical methods and biological methods. The implementation process of physical methods is more complicated and requires a lot of manpower and material resources. The chemical method is costly and has the problem of causing secondary pollution. Biological methods are divided into plant methods and microbial methods. The restoration process of plant methods has a long cycle and slow results. In recent years, Microbially Induced Carbonate Precipitation (MICP) technology has received extensive attention in the control of desertified soils due to its advantages of low cost, sustainability, and no secondary pollution. MICP technology is based on a series of biochemical reactions of carbonate mineralizing bacteria, that is, cations such as Ca ²⁺ and Mg ²⁺ adsorbed on microbial extracellular polymers and CO ₃ ^2- produced during microbial metabolism form calcium carbonate crystals , These calcium carbonate crystals fill the gaps between soil particles, increase the compactness of the soil and the friction and bonding between soil particles, and improve the aggregation of the soil. MICP technology can form a hard shell on the surface of sandy soil that can effectively resist wind and water erosion. However, it takes a long time to fix desertified soil with carbonate mineralizing bacteria alone under natural conditions, and a large amount of urea is required as a substrate, which limits the application of carbonate mineralizing bacteria in desertification soil remediation.

Contents of the invention

Therefore, the technical problem to be solved by the present invention is to overcome the defects that the existing MICP technology is slow to fix under natural conditions and needs to use a large amount of urea as a substrate, thereby providing a dual-bacteria agent and its application in desertification soil restoration.

For this reason, the present invention adopts following technical scheme:

The invention provides a dual-bacteria agent, the dual-bacteria agent includes carbonate mineralizing bacteria and carbonic anhydrase-producing bacteria, the carbonic anhydrase-producing bacteria in the dual-bacteria agent The live bacteria ratio is 1-5:1.

The present invention also provides a preparation method of the above-mentioned dual-bacteria agent, which comprises separately fermenting the carbonate mineralizing bacteria and the carbonic anhydrase-producing bacteria to obtain a bacterial liquid, and then mixing the bacterial liquid evenly to obtain the dual-bacterial agent.

Further, the method for cultivating the carbonate mineralizing bacteria is to pick a single colony of the carbonate mineralizing bacteria into the first culture medium, and culture the activated bacteria at 25-35°C and 200-250rpm overnight to obtain Overnight culture: absorb the overnight culture and add it to the first medium whose volume is 100-200 times its volume, and then culture it at 25-35° C. at 200-250 rpm for 24 hours to obtain a carbonate mineralizing bacteria liquid.

The cultivation method of the carbonic anhydrase-producing bacteria is as follows: pick a single colony of the carbonic anhydrase-producing bacteria to the second culture medium, place the activated bacteria overnight at 25-35°C and 200-250rpm to obtain an overnight culture; The overnight culture is added to the second culture medium whose volume is 100-200 times its volume, and cultured at 25-35° C. at 200-250 rpm for 24 hours to obtain a carbonic anhydrase-producing bacteria liquid.

Preferably, the first medium contains 20 g of yeast extract, 10 g of ammonium sulfate, and 15.748 g of Tris per liter, and the pH is adjusted to 9 with dilute hydrochloric acid;

The second medium contains 10 g of peptone, 5 g of yeast powder, and 10 g of sodium chloride per liter.

Further, use the first medium to dilute the carbonate mineralizing bacteria solution to OD ₆₀₀ =1, use the second medium to dilute the carbonic anhydrase-producing bacteria solution to OD ₆₀₀ =1, and then dilute the carbonate mineral The volume ratio of the bacteria liquid and the carbonic anhydrase-producing bacteria liquid is 1-5:1 and mixed evenly

The present invention also provides the application of the above-mentioned dual-bacteria agent, which is applied to desertification soil remediation.

The present invention also provides a method for remediating desertification soil, comprising the following steps:

S1: Mix the above-mentioned dual-bacteria agent with the soil to be repaired and let it stand to obtain the first mixed soil;

S2: adding fixative solution to the first mixed soil, and obtaining the second mixed soil after mixing;

S3: Slowly add cementing liquid to the second mixed soil, and obtain the third mixed soil after mixing;

S4: Steps S1-S3 are repeated 1-5 times to obtain repaired soil.

Further, the fixative in step S2 is a calcium chloride solution with a concentration of 0.01M-0.05M;

The cementing liquid in step S3 is a mixed solution of calcium chloride and urea, and the molar concentration of the calcium chloride and urea is the same, which is 0.5M-1.5M.

Preferably, in step S1, the volume ratio of the dual-bacteria agent to the soil to be repaired is 1-1.5:1, and the standing time is 2-5h;

In step S2, the volume ratio of the fixative to the soil to be repaired is 0.1-0.3:1;

In step S3, the volume ratio of the cementing liquid to the soil to be repaired is 5-10:1.

The technical solution of the present invention has the following advantages:

(1) In the dual-bacteria agent provided by the present invention, the carbonate mineralizing bacteria can secrete urease in a large amount, and urease can decompose molecules such as urea in the environment to form carbonate ions, which can be metabolized into calcium carbonate precipitation through its growth Create a suitable alkaline environment, and at the same time, the microorganisms are negatively charged, and will adsorb positively charged calcium ions and other cations, causing Ca ²⁺ to be enriched, and carbonate ions and calcium ions form carbonic acid with carbonate mineralizing bacteria as crystallization nuclei. Calcium and magnesium carbonate crystallization, the induced calcium carbonate crystallization plays a bridging role around the loose gravel and between particles, improving the mechanical properties of the medium;

Carbonic anhydrase-producing bacteria can accelerate the dissolution rate of CO ₂ in the system, provide enough carbonate ions for carbonate mineralizing bacteria, and accelerate the effect of carbonate mineralizing bacteria while utilizing carbon dioxide in the environment.

(2) The present invention applies the dual-bacteria agent to desertification soil remediation. Compared with the process of immobilizing desertification soil with carbonate mineralizing bacteria only, the dual-bacteria system greatly improves the curing rate of the system. Under these conditions, the soil with more than 2 times the strength of the single-bacteria system can be obtained. The carbonic anhydrase-producing bacteria use part of the carbon dioxide produced in the atmosphere and by the carbonate mineralizing bacteria to provide Licarbonate ions for the curing system. Compared with the single-bacteria curing, Using CO ₂ as part of the carbon source reduces CO ₂ emissions and reduces the amount of urea used.

(3) In the desertification soil remediation of the present invention, the fixative is used before the cementation fluid, so that the microbial cells are fixed on the surface of the soil particles and are not easily washed away by the subsequent cementation fluid.

(4) The desertification soil restoration provided by the present invention is non-toxic, harmless, and has no secondary pollution, low cost, simple preparation of the dual-bacteria agent, and the urea left in the soil during the solidification process also makes the soil more fertile.

Description of drawings

In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the specific implementation or description of the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

Fig. 1 is a diagram of the metabolic relationship of the dual-bacteria agent in the process of soil remediation in a specific embodiment of the present invention.

Detailed ways

The following examples are provided in order to further understand the present invention better, are not limited to the best implementation mode, and do not limit the content and protection scope of the present invention, anyone under the inspiration of the present invention or use the present invention Any product identical or similar to the present invention obtained by combining features of other prior art falls within the protection scope of the present invention.

The carbonate mineralizing bacterium used in the present invention is Bacillus Pasteur, purchased from the American Culture Collection, numbered ATCC 11859;

The carbonic anhydrase-producing bacterium used in the present invention is an engineering bacterium E-22b-IRLS, and its preparation method is described in the master's thesis of the School of Chemical Engineering, Tianjin University, "Research on Carbonic Anhydrase Displayed on Cell Surface and Its Enzymatic Properties".

If no specific experimental steps or conditions are indicated in the examples, it can be carried out according to the operation or conditions of the conventional experimental steps described in the literature in this field. The reagents or instruments used, whose manufacturers are not indicated, are all commercially available conventional reagent products.

As shown in Figure 1, when the dual-bacteria agent provided by the present invention is applied to the soil remediation process, the carbonate mineralizing bacteria can secrete a large amount of urease, which can decompose molecules such as urea in the environment to form carbonate ions, and pass Its growth and metabolism are calcium carbonate precipitation to create a suitable alkaline environment. At the same time, the microorganisms are negatively charged and will absorb positively charged calcium ions and other cations, resulting in the enrichment of Ca ²⁺ . Carbonate ions and calcium ions form carbonate minerals. The bacteria form calcium carbonate and magnesium carbonate crystals as crystallization nuclei, and the induced calcium carbonate crystals act as bridges around the loose gravel and between particles, improving the mechanical properties of the medium; at the same time, the carbonic anhydrase-producing bacteria can accelerate the CO ₂ The dissolution rate in the system provides sufficient carbonate ions for the carbonate mineralizing bacteria, and accelerates the solidification rate of desertified soil while utilizing carbon dioxide in the environment.

Example 1

Present embodiment provides a kind of antibacterial agent, preparation method is as follows:

(1) Preparation of single bacteria liquid:

Pick a single colony of carbonate mineralizing bacteria into 5mL of the first medium, and place it at 30°C and 220rpm to cultivate the activated bacteria overnight to obtain an overnight culture; pipette the overnight culture into 5 bottles of the first medium, Bottle 100mL, cultured at 30°C and 220rpm for 24h to obtain carbonate mineralizing bacteria liquid; the first culture medium contained 20g of yeast extract, 10g of ammonium sulfate, and 15.748g of Tris per liter, and the pH was adjusted with dilute hydrochloric acid to 9;

Pick a single colony of carbonic anhydrase-producing bacteria into 5mL of the second medium, and place it at 30°C and 220rpm to cultivate the activated bacteria overnight to obtain an overnight culture; pipette the overnight culture into 5 bottles of the second medium, each bottle 100 mL, cultured at 30° C. and 220 rpm for 24 hours to obtain a carbonic anhydrase-producing bacteria liquid; each liter of the second culture medium contained 10 g of peptone, 5 g of yeast powder, and 10 g of sodium chloride.

(2) Preparation of double bacteria agent:

Use the first medium to dilute the carbonate mineralizing bacteria solution to OD ₆₀₀ =1, use the second medium to dilute the carbonic anhydrase-producing bacteria solution to OD ₆₀₀ =1, and then dilute the carbonate mineralizing bacteria The liquid and the carbonic anhydrase-producing bacteria liquid are mixed uniformly at a volume ratio of 1:1 to obtain a dual-bacteria agent.

(1) Take the above-mentioned dual-bacteria agent and the soil to be repaired in a volume ratio of 1:1 and mix it for 3 hours to obtain the first mixed soil;

(2) Add 0.05M calcium chloride solution to the first mixed soil, the volume ratio of the calcium chloride solution and the soil to be repaired is 0.2:1, and the second mixed soil is obtained after mixing;

(3) Add calcium chloride and urea concentration to the second mixed soil and be the mixed solution of 1M, the volume ratio of described mixed solution and the soil to be repaired is 8:1, obtain the third mixed soil after mixing;

(4) Steps (1)-(3) were repeated every 48 hours for a total of 4 times to obtain the restored desertified soil.

Example 2

(1) Preparation of single bacteria liquid:

Pick a single colony of carbonate mineralizing bacteria into 5mL of the first medium, and place it at 25°C and 250rpm to cultivate the activated bacteria overnight to obtain an overnight culture; draw the overnight culture into 10 bottles of the first medium, Bottle 100mL, cultured at 25°C and 200rpm for 24h to obtain carbonate mineralizing bacteria liquid; the first culture medium contained 20g of yeast extract, 10g of ammonium sulfate, and 15.748g of Tris per liter, and the pH was adjusted with dilute hydrochloric acid to 9;

Pick a single colony of carbonic anhydrase-producing bacteria into 5mL of the second medium, and place it at 25°C and 200rpm to cultivate the activated bacteria overnight to obtain an overnight culture; draw the overnight culture into 10 bottles of the second medium, each bottle 100 mL, cultured at 25° C. and 200 rpm for 24 hours to obtain a carbonic anhydrase-producing bacteria liquid; each liter of the second culture medium contained 10 g of peptone, 5 g of yeast powder, and 10 g of sodium chloride.

(2) Preparation of double bacteria agent:

Use the first medium to dilute the carbonate mineralizing bacteria solution to OD ₆₀₀ =1, use the second medium to dilute the carbonic anhydrase-producing bacteria solution to OD ₆₀₀ =1, and then dilute the carbonate mineralizing bacteria The liquid and the carbonic anhydrase-producing bacteria liquid are mixed uniformly at a volume ratio of 5:1 to obtain a dual-bacteria agent.

(1) Take the above-mentioned dual-bacteria agent and the soil to be repaired in a volume ratio of 1.5:1 and mix and let it stand for 5 hours to obtain the first mixed soil;

(2) Add 0.01M calcium chloride solution to the first mixed soil, the volume ratio of the calcium chloride solution and the soil to be repaired is 0.3:1, and the second mixed soil is obtained after mixing;

(3) Add calcium chloride and urea concentration to the second mixed soil and be the mixed solution of 0.5M, the volume ratio of described mixed solution and soil to be repaired is 10:1, obtain the third mixed soil after mixing;

(4) Steps (1)-(3) were repeated every 48 hours for a total of 3 times to obtain the restored desertified soil.

Example 3

(1) Preparation of single bacteria liquid:

Pick a single colony of carbonate mineralizing bacteria into 5mL of the first medium, and place it at 35°C and 200rpm to cultivate the activated bacteria overnight to obtain an overnight culture; pipette the overnight culture into 7 bottles of the first medium, Bottle 100mL, cultured at 35°C and 200rpm for 24h to obtain carbonate mineralizing bacteria liquid; the first medium contains 20g of yeast extract, 10g of ammonium sulfate, and 15.748g of Tris per liter, and the pH is adjusted with dilute hydrochloric acid to 9;

Pick a single colony of carbonic anhydrase-producing bacteria into 5mL of the second medium, and place it at 35°C and 200rpm to cultivate the activated bacteria overnight to obtain an overnight culture; pipette the overnight culture into 7 bottles of the second medium, each bottle 100 mL, cultured at 35° C. and 200 rpm for 24 hours to obtain a carbonic anhydrase-producing bacteria liquid; each liter of the second culture medium contained 10 g of peptone, 5 g of yeast powder, and 10 g of sodium chloride.

(2) Preparation of double bacteria agent:

Use the first medium to dilute the carbonate mineralizing bacteria solution to OD ₆₀₀ =1, use the second medium to dilute the carbonic anhydrase-producing bacteria solution to OD ₆₀₀ =1, and then dilute the carbonate mineralizing bacteria The liquid and the carbonic anhydrase-producing bacteria liquid are mixed uniformly at a volume ratio of 3:1 to obtain a dual-bacteria agent.

(1) Take the above-mentioned dual-bacteria agent and the soil to be repaired in a volume ratio of 1:1 and mix it for 2 hours to obtain the first mixed soil;

(2) Add 0.04M calcium chloride solution to the first mixed soil, the volume ratio of the calcium chloride solution and the soil to be repaired is 0.1:1, and the second mixed soil is obtained after mixing;

(3) Add calcium chloride and urea concentration to the second mixed soil and be the mixed solution of 1.5M, the volume ratio of described mixed solution and the soil to be repaired is 5:1, obtain the third mixed soil after mixing;

(4) Steps (1)-(3) were repeated every 48 hours for a total of 5 times to obtain the restored desertified soil.

Comparative example 1

Compared with Example 1, this Comparative Example 1 only uses the carbonate mineralizing bacteria liquid, and does not use the carbonic anhydrase-producing bacteria liquid, and the rest of the steps are the same as in Example 1.

Comparative example 2

Compared with Example 1, this Comparative Example 2 only uses the carbonic anhydrase-producing bacterium liquid, and does not use the carbonic anhydrase-producing bacterium liquid, and the rest of the steps are the same as in Example 1.

Test case

Respectively take 8 soil samples to be repaired from the same source, use the double-bacteria agent obtained in Example 1 and the single-bacteria agent obtained in Comparative Examples 1 and 2 as repair reagents, and carry out according to the desertification soil repair method provided by the present invention Restoration experiments are recorded as experimental group, comparison group 1 and comparison group 2, and steps (1)-(3) of the desertification soil restoration method are recorded as a batch of grouting, and each sample is filled with a batch of grouting every 48 hours. A total of 4 grouting. From the second day after the grouting started, two samples were taken from the experimental group and the control group every 48 hours to test their compressive strength and shear strength, and the average value of the results was taken. The results are shown in Table 1:

Table 1 Experimental results of experimental group and comparison group

As can be seen from the above table, under the same grouting conditions, compared with Comparative Example 1 in Example 1, under the same time and urea consumption, the compressive strength and shear strength of Example 1 are the same as those of Comparative Example 1 More than 2 times, and the compressive strength and shear strength in Comparative Example 2 cannot be compared with Example 1. It shows that to obtain the same strength of soil, the technical solution in Example 1 of the present application can greatly save time and reduce the consumption of urea compared with the comparative example.

Apparently, the above-mentioned embodiments are only examples for clear description, rather than limiting the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in different forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. And the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims

A dual-bacteria agent, characterized in that, said dual-bacteria agent comprises carbonate mineralizing bacteria and carbonic anhydrase-producing bacteria, and in said dual-bacteria agent, carbonate mineralizing bacteria and carbonic anhydrase-producing bacteria The live bacteria ratio is 1-5:1.
The preparation method of the dual-bacteria agent according to claim 1, characterized in that it comprises fermenting the carbonate mineralizing bacteria and the carbonic anhydrase-producing bacteria respectively to obtain the bacterial liquid, and then mixing the bacterial liquid evenly to obtain the dual-bacterial agent Bacteria.
The preparation method according to claim 2, wherein the method for cultivating the carbonate mineralizing bacteria is to pick a single colony of the carbonate mineralizing bacteria to the first culture medium and place it at 25-35°C for 200 Cultivate the activated bacteria overnight at -250rpm to obtain an overnight culture; absorb the overnight culture and add it to the first medium whose volume is 100-200 times its volume, and then cultivate it at 25-35°C for 24 hours at 200-250rpm to obtain carbonate Mineralizing bacteria liquid.
The preparation method according to claim 3, characterized in that, the cultivation method of the carbonic anhydrase-producing bacteria is to pick a single colony of the carbonic anhydrase-producing bacteria to the second culture medium and place it at 25-35°C at 200-250rpm Cultivate the activated bacteria overnight under certain conditions to obtain an overnight culture; absorb the overnight culture and add it to the second medium whose volume is 100-200 times its volume, and then culture it at 25-35°C at 200-250rpm for 24 hours to obtain carbonic anhydrase-producing bacteria bacteria liquid.
The preparation method according to claim 4, wherein the first culture medium contains 20 g of yeast extract, 10 g of ammonium sulfate, and 15.748 g of Tris per liter, and the pH is adjusted to 9 with dilute hydrochloric acid;

The second culture medium contains 10 g of peptone, 5 g of yeast powder and 10 g of sodium chloride per liter.
The preparation method according to claim 5, characterized in that the carbonate mineralizing bacteria solution is diluted to OD600=1 using the first medium, and the carbonic anhydrase-producing bacteria solution is diluted to OD600 =1 using the second medium. OD 600 =1, then mix the carbonate mineralizing bacteria liquid and the carbonic anhydrase-producing bacteria liquid at a volume ratio of 1-5:1 evenly.
The application of the double-bacteria agent according to claim 1 or the double-bacteria agent prepared by the preparation method according to any one of claims 2-6 is characterized in that it is applied to desertification soil remediation.
A kind of desertification soil remediation method is characterized in that, comprises the steps:

S1: Mix the dual-bacteria agent according to claim 1 or the dual-bacteria agent prepared by the preparation method described in any one of claims 2-6 with the soil to be repaired and let it stand to obtain the first mixed soil;

S2: adding fixative solution to the first mixed soil, and obtaining the second mixed soil after mixing;

S3: adding cementation liquid to the second mixed soil, and obtaining the third mixed soil after mixing;

S4: Steps S1-S3 are repeated 1-5 times to obtain repaired soil.
The repair method according to claim 8, characterized in that the fixative in step S2 is a calcium chloride solution with a concentration of 0.01M-0.05M;

The cementing liquid in step S3 is a mixed solution of calcium chloride and urea, and the molar concentration of the calcium chloride and urea is the same, which is 0.5M-1.5M.
The restoration method according to claim 8 or 9, characterized in that, in step S1, the volume ratio of the dual-bacteria agent to the soil to be repaired is 1-1.5:1, and the standing time is 2-5h;

In step S2, the volume ratio of the fixative to the soil to be repaired is 0.1-0.3:1;

In step S3, the volume ratio of the cementing liquid to the soil to be repaired is 5-10:1.