Preparation method and application of Pseudochrobacterium insaccharolyticum and preparation thereof

Abstract

The present invention belongs to the field of microbial technology, and specifically discloses a preparation method and application of a Pseudochrobactrum asaccharolyticum and a preparation thereof, wherein the Pseudochrobactrum asaccharolyticum is deposited in the Guangdong Microbial Culture Collection Center with a deposit number of GDMCC NO: 62528 and a deposit date of July 7, 2022, and is named Pseudochrobactrum asaccharolyticum Z11. The Pseudochrobactrum asaccharolyticum Z11 screened by the present invention has the ability to produce alkali and has a large tolerance to Cd(II), provides a new strain resource for cadmium passivation alkali-producing microorganisms, and can be used to repair cadmium-contaminated soil.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a preparation method and application of Pseudochryobacterium insaccharolyticum and a preparation thereof. Background Art

[0002] Cadmium (Cd) is a soft, sulfur-loving heavy metal with a blue-white metallic luster. Cadmium is considered to be one of the most biologically toxic heavy metal elements in the natural environment. The toxicity of its compounds is greater than that of its single substance. Cadmium compounds can be enriched in organisms and enter the human body through the food chain to cause chronic poisoning. Cadmium that stays in the human body can damage the renal tubules and glomeruli in the kidneys, and can replace calcium ion deposition in the bones to cause rickets. Cadmium-containing compounds are pathogenic, carcinogenic, and mutagenic. At present, all cadmium-related or derived compounds are classified as carcinogens. Cadmium was originally abundant in zinc mines as a compound, and it was not easy for humans to come into contact with it. With the development of lead and zinc mining and smelting industries, cadmium stored underground was developed and then entered the environment in which people live. The control of soil cadmium pollution is a global problem. Once the soil is contaminated by cadmium, the removal of cadmium and soil remediation will be a long-term process that requires a lot of manpower, financial resources and material resources.

[0003] The treatment methods for cadmium pollution include physical, chemical and biological methods. The main bioremediation methods include plant remediation, microbial remediation and microbial-plant joint remediation. Microorganisms mainly secrete acid radical ions, organic acids and other substances with strong affinity for heavy metals to dissolve, complex and chelate the exchangeable heavy metals in the environment to form insoluble and difficult to dissolve heavy metals in other forms (organic binding, iron and manganese oxide binding, etc.); or absorb heavy metal ions into cells through adsorption, transmembrane transport and other methods, enrich them in cells, and inhibit the migration of heavy metals in the soil; microorganisms with redox effects can also convert highly toxic heavy metals into low-toxic heavy metals through redox reactions. Microorganisms commonly used to treat cadmium pollution mainly include bacteria, fungi and algae, among which bacteria are the majority. The use of microorganisms for soil heavy metal pollution has the advantages of good environmental compatibility, no secondary pollution, low cost and high efficiency.

[0004] The mechanism of microbial treatment of cadmium pollution includes adsorption, mineralization and the use of bacterial consortium to repair cadmium pollution. The factors affecting microbial treatment of cadmium pollution include microbial factors, pH, temperature, microbial carriers, other metal elements and plants.

[0005] Compared with Western countries, my country has a shorter research time on microbial agents, but so far my country has achieved many research results in the preparation and application of microbial agents. There are two main ways to prepare microbial agents: adsorption and embedding. The two preparation methods are roughly the same, generally including three steps: obtaining target microorganisms, strain culture and detection, and strain immobilization.

[0006] Cadmium pollution in mining areas is currently the most important challenge facing China's environmental management. Excessive cadmium emissions will not only pollute water sources and soil, but also endanger human health through enrichment in the food chain. The toxicity of heavy metal cadmium mainly comes from the effective cadmium that is easily absorbed by organisms. Microbial treatment of cadmium pollution is highly efficient, compatible, and has no secondary pollution.

[0007] Microorganisms mainly convert the effective cadmium in the soil into an organic state, an iron-manganese oxide-bound state, and a residual state that are difficult to be used by organisms through various methods such as adsorption, mineralization, and chelation, thereby preventing the flow of cadmium in the material cycle and reducing the impact of cadmium on organisms. However, on the one hand, the ability of microorganisms to produce alkali is not strong, and the strains that passivate cadmium have a weak passivation effect on cadmium. On the other hand, the strains used for microbial remediation are mainly single strains. When a single microorganism is applied to the soil, there are shortcomings such as slow remediation efficiency and the growth of other microorganisms in the soil will be affected. The use of microbial carriers for remediation can not only promote the adaptation of microorganisms to the new growth environment, but also adsorb toxic and harmful substances in the soil, which can promote the remediation of cadmium-contaminated soil in farmland. Therefore, in recent years, screening new strains with high passivation effects on cadmium and preparing bacterial agents to repair soil pollution and enhance soil fertility has become a research hotspot. Summary of the invention

[0008] In view of the above-mentioned shortcomings, the present invention provides a preparation method and application of Pseudocobacillus insaccharolyticus and its preparation. The Pseudocobacillus insaccharolyticus screened by the present invention has the ability to produce alkali and has a large tolerance to Cd(II), providing a new strain resource for cadmium-passivated alkali-producing microorganisms.

[0009] In order to achieve the above-mentioned object, the present invention provides a Pseudochrobactrum asaccharolyticum, which is deposited in Guangdong Microbial Culture Collection Center (GDMCC for short, address of the deposit unit: 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, Postal Code: 510070), with a deposit number of GDMCC NO: 62528, a deposit date of July 7, 2022, and is named Pseudochrobactrum asaccharolyticum Z11.

[0010] Based on the same inventive concept, the present invention also provides the use of the above-mentioned Pseudochalobacterium insaccharolyticum in cadmium passivation.

[0011] Based on the same inventive concept, the present invention also provides the use of the above-mentioned Pseudochalobacterium insaccharolyticum in repairing cadmium-contaminated soil.

[0012] Based on the same inventive concept, the present invention also provides a microbial alkali-producing preparation suitable for the remediation of cadmium-contaminated soil, comprising the above-mentioned Pseudocobacillus insaccharolyticus and an organic carrier.

[0013] According to one aspect of the present invention, the organic carrier includes any one of wheat bran, sawdust, charcoal, charcoal ash, and plant ash.

[0014] Based on the same inventive concept, the present invention also provides a method for preparing a microbial alkali-producing preparation suitable for cadmium-contaminated soil remediation using the above-mentioned Pseudocobacillus insaccharolyticus, and the method includes an embedding method and an adsorption method.

[0015] According to one aspect of the present invention, the embedding method comprises the following steps:

[0016] Step 1: inoculate the Z11 strain into a sterile LB liquid medium for culture activation to obtain an activated Z11 strain; inoculate the activated Z11 strain into a sterile LB medium, and after the culture strain reaches a stable period, dispense into a centrifuge tube, and obtain Z11 bacteria after centrifugation; take an organic carrier and add it to the LB liquid medium, and sterilize it for standby use; wherein the organic carrier includes any one of wheat bran, sawdust, charcoal, charcoal ash, and plant ash;

[0017] Step 2: Add the above Z11 bacteria to the sterilized LB liquid culture medium containing the carrier, and add 4% sodium alginate solution, and shake at a constant temperature until fully mixed to obtain a mixture;

[0018] Step 3: Use a syringe to draw up the mixture and squeeze it out of the needle in the form of droplets to a concentration of 2% CaCl 2 The solution was continuously shaken and stirred, and fixed at a speed of 180 r / min and a temperature of 30°C for 2 h. The embedded fixed bacterial agent particles were filtered out, and the obtained particles were rinsed three times with a 0.9% NaCl solution, freeze-dried for 48 h to obtain the embedded fixed bacterial agent, which was placed in a 4°C refrigerator for future use.

[0019] According to one aspect of the present invention, the adsorption method comprises the following steps:

[0020] Step 1: inoculate the Z11 strain into a sterile LB liquid culture medium for culture activation to obtain an activated Z11 strain; inoculate the activated Z11 strain into a sterile LB culture medium, and after the culture strain reaches a stable period, dispense into a centrifuge tube, and obtain Z11 bacteria after centrifugation; take an organic carrier and add it to the LB liquid culture medium, and sterilize it for standby use; wherein the organic carrier includes any one of wheat bran, sawdust, charcoal, charcoal ash, and plant ash;

[0021] Step 2: Add the above Z11 bacteria into the sterilized LB liquid culture medium containing the carrier to obtain a sample after inoculation;

[0022] Step 3: Incubate the inoculated samples at a constant temperature of 150 r / min and 30°C. When the optimal adsorption time is reached, divide the samples into centrifuge tubes and centrifuge them at 1500 r / min for 10 min. Discard the supernatant. Repeat the centrifugation three times and discard the supernatant. Wash the bottom solid with 0.85% NaCl solution and freeze-dry in vacuum for 24 hours to obtain the finished product. Store it in a 4°C refrigerator for later use.

[0023] Based on the same inventive concept, the present invention also provides the use of an alkali-producing preparation prepared by any of the above-mentioned preparation methods for preparing a microbial alkali-producing preparation suitable for remediation of cadmium-contaminated soil in remediation of cadmium-contaminated soil.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention conducted morphological and 16S rRNA identification on the isolated strain, and the results showed that the strain belonged to Pseudochrobactrum asaccharolyticum sp. It was named Z11, and the maximum pH value of its growth medium was measured to be 8.84 (the initial pH value was 6.0);

[0026] (2) The maximum tolerance concentration of strain Z11 of the present invention to Cd(II) is 70 mg / L (60 μM), the growth period is 50 h, the temperature is 30° C., the initial pH is 6, peptone is used as the carbon source, and yeast extract is used as the nitrogen source, the passivation efficiency of the strain to Cd(II) can reach 68.5%;

[0027] (3) The insoluble Ochrobactrum saccharolyticum and its preparation can repair cadmium-contaminated soil. Through cytotoxicity test calculation, it is known that the inhibition rate of the strain is positively correlated with the change of Cd(II) concentration. When the Cd(II) concentration is 600μM, the inhibition rate is the maximum, and the inhibition rate is 81%. The Hill equation calculates that the half lethal dose (LD) 50 of the bacteria is 0.216mM, and the maximum tolerance concentration of the strain to Cd(II) is about 10 times the maximum cell toxicity concentration; by comparing the Raman spectra of Cd(II) with different concentration gradients, it can be known that when the Cd(II) concentration is ≤60μM, the signals of protein, amino acid, lipid and sugar are basically unchanged. When the Cd(II) concentration reaches 600μM, the signals of protein and amino acid are stronger than those of lipid and sugar, which is mainly related to protein denaturation, and the peak of ribonucleic acid (882cm -1), which may be related to bacterial fragmentation, indicating that Cd(II) is highly toxic to cells when the growth of the strain is inhibited at a concentration of 600 μM, further indicating that the maximum tolerance concentration of Cd(II) is 60 μM; by comparing the infrared spectra of Cd(II) at different concentration gradients, it can be seen that with the increase of Cd(II) concentration, the heavy metal Cd(II) is used for -PO in the cell wall of bacteria Z11. 4 3- The complexation between -C=O, -NH and other groups of Z11 changed the charge distribution in the above groups, which confirmed that Z11 bacteria mainly adsorbed Cd(II) by complexation, reducing the effectiveness of Cd(II); In summary, the mechanism of action of this strain with Cd(II) was mainly through the complexation between the active groups of the bacterial cell wall (such as -C=O, -NH, etc.) and Cd(II), thereby inhibiting the mobility of heavy metals;

[0028] (4) Using five cheap materials, namely wheat bran, sawdust, charcoal, charcoal ash and plant ash, as carriers, the bacterial agent was prepared by adsorption fixation and embedding fixation; when the carrier was wheat bran, it promoted the growth and alkali production of the strain, and when the carrier was sawdust, it promoted the growth of the strain;

[0029] (5) The bacterial agent with wheat bran as carrier was added to cadmium-contaminated soil, and the effects of different dosages of the strain and soil moisture content on the strain's alkali production were tested. The results showed that the optimal dosage of the bacterial agent was 3 g / kg, and the optimal soil moisture content was about 60%, which effectively improved the acidity and alkalinity of the soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a picture of the single clone of strain Z11 screened in Example 1 of the present invention after streak plate culture;

[0031] Figure 2 This is the agarose gel electrophoresis diagram of strain Z11 screened in Example 1 of the present invention;

[0032] Figure 3 This is the phylogenetic tree of strain Z11 screened in Example 1 of the present invention;

[0033] Figure 4 This is the growth curve of the strain under Cd(II) stress described in Example 2 of the present invention;

[0034] Figure 5 This is the pH value change curve of the strain under Cd(II) stress described in Example 2 of the present invention;

[0035] Figure 6This is a routine toxicity test of the strain Z11 cells described in Example 3 of the present invention; wherein (a) is the absorbance change of the bacterial solution at 571 nm; (b) is the absorbance change of the bacterial solution at 601 nm;

[0036] Figure 7 The change between the cell inhibition rate of strain Z11 and the Cd(II) concentration described in Example 3 of the present invention;

[0037] Figure 8 This is a Raman spectrum stacking diagram of strain Z11 cells treated with different Cd(II) concentrations in Example 3 of the present invention;

[0038] Fig. 9 This is an infrared spectrum stacking diagram of strain Z11 cells treated with different Cd(II) concentrations in Example 3 of the present invention;

[0039] Fig.10 The effect of the bacterial agent on the growth and alkali production of the strain when the carrier of Example 4 of the present invention is wheat bran; wherein (a) is the effect of the bacterial agent on the growth of the strain; (b) is the effect of the bacterial agent on the alkali production of the strain;

[0040] Fig.11 The effect of the bacterial agent on the growth of the strain and the alkali production effect when the carrier of Example 4 of the present invention is sawdust; wherein (a) is the effect of the bacterial agent on the growth of the strain; (b) is the effect of the bacterial agent on the alkali production effect of the strain;

[0041] Fig.12 The salting-out pH values ​​of the bacterial agents added to the soil in two ways prepared in Example 4 of the present invention; wherein (a) is the salting-out pH value of the embedded fixed bacterial agent added to the soil; and (b) is the salting-out pH value of the adsorbed fixed bacterial agent added to the soil. DETAILED DESCRIPTION

[0042] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0043] It should be noted that the preparation of the culture medium and solution that may be involved in this application is as follows:

[0044] Preparation of 5mol / L NaOH solution: weigh 1000g of sodium hydroxide solid, add a small amount of water to dissolve, transfer the solution to a 500ml volumetric flask, wash the beaker three times, transfer the washing solution to the volumetric flask, add deionized water to the mark, mix well and set aside.

[0045] Preparation of 0.2mol / L hydrochloric acid solution: Measure 16.8mL of 36.5% concentrated hydrochloric acid, pour into a beaker, dilute with deionized water, transfer to a volumetric flask, wash the beaker three times, transfer the washing solution into a volumetric flask, add deionized water to the mark, mix well and set aside.

[0046] Preparation of broth culture medium: Weigh 10 g of tryptone, 5 g of sodium chloride, and 3 g of beef extract respectively, dissolve them in 950 mL of deionized water, and then adjust the pH value of the solution with 5 mol / L NaOH solution. Transfer to a 1 L volumetric flask to make up the volume, shake well, and dispense into conical flasks. Sterilize at 121 ° C for 20 min.

[0047] Preparation of nutrient medium: Weigh 1 g beef extract, 2 g yeast extract, 5 g peptone, and 5 g NaCl respectively, dissolve them in 950 mL deionized water, adjust the pH value of the solution with 5 mol / L NaOH solution, transfer to a 1 L volumetric flask, shake well, and dispense into conical flasks, and sterilize at 121 ° C for 20 min.

[0048] Preparation of LB liquid culture medium: Weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride respectively, dissolve them in 950 mL of deionized water, adjust the pH value of the solution with 5 mol / L NaOH solution, transfer to a 1 L volumetric flask, shake well, and dispense into conical flasks, and sterilize at 121 ° C for 20 min.

[0049] Preparation of LB solid culture medium: The steps are the same as those for liquid culture medium. After the LB liquid culture medium is fixed to volume and mixed evenly, it is dispensed into conical flasks, 2% agar is added, and sterilized at 121°C for 20 min.

[0050] Preparation of normal saline: Weigh 9 g of sodium chloride, put it in a beaker, add deionized water, stir to dissolve, transfer the solution to a 1L volumetric flask, make up to volume, mix well, dispense into conical flasks, and sterilize at 121°C for 20 min.

[0051] Example 1

[0052] Isolation, screening and identification of a non-saccharolytic Pseudochribacterium saccharolyticum:

[0053] (1) Sample collection: Collect No. 1-8 pig excrement samples from different regions, seal them in ziplock bags, and place them in a 0-4℃ refrigerator for later use.

[0054] (2) Enrichment: Weigh equal amounts of samples from pig excrement samples No. 1 to 8 and add them to conical flasks. Mix each sample with an equal amount of LB medium and enrich them in a constant temperature incubator at 30°C and 150 r / min for 4 days. During this period, weigh the conical flasks every day and add sterile water to promote microbial growth.

[0055] (3) Preliminary screening: From the enriched sample, take 1 mL of the enrichment solution into 9 mL of sterile saline and dilute it to 10-1, 10-2, 10-3, and 10-4 in sequence. Make three replicates for each dilution gradient. Take 0.1 mL from each dilution gradient and spread it on LB solid culture medium. For eight samples and four dilutions, spread three culture dishes for each dilution and number them. Culture them in a 30°C constant temperature incubator for 4 days. The colony distribution on the solid culture medium is obvious.

[0056] (4) Purification: The single colony obtained by culture on the plate was inoculated into a sterilized LB solid medium with an inoculation needle for streak pure culture, and then placed in a constant temperature incubator at 30°C for 4 days for purification culture.

[0057] (5) Rescreening: Use an inoculation needle to pick up the purified colonies and inoculate them into sterilized LB medium for cultivation. Set up another group, which only contains sterilized LB medium, as a blank control. Weigh the bacterial solution every day and add sterile water. Use an ultraviolet spectrophotometer to measure the absorbance of the strain at 600nm (OD600) every 4 hours. After the strain grows to the stable period, use pH test paper to measure the pH value of the bacterial solution. If the pH value of the bacterial solution is greater than 7, it is an alkali-producing strain; if the pH value does not change or decreases, it is not an alkali-producing strain. Select the strain with alkali-producing ability, further test the pH value of the bacterial solution with a pH meter, and select the bacterial solution with the largest pH value. This bacterium is the strain with the strongest alkali-producing ability.

[0058] (6) Purification: The alkaline-producing strains obtained by repeated screening were activated and cultured. A portion of the bacterial solution was taken with an inoculation needle and inoculated onto the surface of a sterilized LB solid culture medium by streaking. Each strain of alkaline-producing bacteria was streaked three times and cultured in a 30°C constant temperature incubator for 3 days. If a single colony without foreign bacteria was observed on the solid culture medium, the purification was considered successful. Otherwise, the purification steps were repeated until a single colony was observed by streaking. The purified single strain was stored in glycerol and stored in a -80°C refrigerator.

[0059] The strain with the best alkali production effect screened above was inoculated into LB solid medium. After 4 days of plate culture, the morphological characteristics of the colonies were observed, and three parallel samples were selected and sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for molecular biological identification. The specific steps are as follows:

[0060] PCR product detection and purification: Take a 1.5ml centrifuge tube, add 200μL pretreatment solution and three glass beads, then add an appropriate amount of bacterial sample, and put it in a grinder to grind until it is fully ground. Add 20μL ProteinaseK solution, mix well, and place at 37℃ for 30-60min. 3.1.3 Add 200μL lysis solution, invert and mix thoroughly, and place at 70℃ for 10min. Add 200μL anhydrous ethanol, invert and mix thoroughly, and centrifuge briefly to remove droplets on the inner wall of the tube cap. Pass through the adsorption column, wash once with washing solution, and wash twice with rinsing solution. Place the adsorption column at room temperature for 5-10 minutes to thoroughly dry the residual rinsing solution in the adsorption material. Transfer the adsorption material to a new centrifuge tube, and drop 50-100μL ddH 2 O, place at room temperature for 5 to 10 minutes, centrifuge at 12000r / min for 2 minutes, and collect the solution into a centrifuge tube.

[0061] PCR amplification reaction system: premix 15 μL, primer-F (10p) 1 μL, primer-R (10p) 1 μL, template (ng / μL) 1 μL, ddH 2 O12μL, total 30μL. The amplification program was set to 96℃5min, 96℃20s, X℃20s, 72℃30s, 72℃10min, and then naturally decreased to 16℃. The cycle was set to 35 cycles. When amplifying 16S, X=62; when amplifying 18S, X=56.

[0062] 3 μL PCR product was tested on 1.0% agarose gel to observe the band characteristics. PCR product purification was performed according to the standard operation procedure of magnetic bead purification, using the principle that magnetic beads can adsorb or release charged substances, adsorb DNA in high-salt, low-pH solution, and release DNA in low-salt, high-pH solution, thereby achieving the purpose of separating and purifying DNA products.

[0063] Sequencing: The purified PCR product is tested on the sequencing machine.

[0064] The sequencing results were compared with NCBI-BLAST to determine the species of Z11, and the phylogenetic tree was constructed using MEGA software.

[0065] Results and Discussion:

[0066] (1) Isolation, screening and identification of alkaline-producing bacteria

[0067] Pig excrement was collected from different regions, and single colonies were obtained through enrichment, screening, and purification. The pH value of the culture medium during the growth of the purified single strain was detected using pH test paper. It was found that the pH value of 16 strains increased during the growth process, and they had the ability to produce alkali. Further measurement by pH meter showed that the 16 strains could increase the pH value of the LB culture medium with an initial pH value of 6 to 6.43, 6.66, 6.63, 6.37, 7.92, 8.04, 7.78, 8.27, 8.03, 8.41, 8.84, 7.77, 8.07, 8.16, and 8.32, respectively. The strain with the strongest alkali production ability, which could increase the pH value of the LB culture medium to 8.84, was selected and named Z11. Figure 1 As shown, the colonies of strain Z11 on LB solid culture medium are small, light yellow, with neat and smooth edges, uniform and opaque texture, a bulge in the middle and flat around, and easy to pick up.

[0068] Identified by morphology and 16S rRNA, Figure 2 As shown in the agarose gel electrophoresis diagram of Z11, M is 5000, 3000, 2000, 1000, 750, 500, 250, and 100 bp from top to bottom. Based on the BLAST results, the phylogenetic tree was constructed using MEGA software. The results showed that Z11 was most closely related to Pseudochrobactrum asaccharolyticum sp. Its phylogenetic tree is shown in Figure 3 .

[0069] Example 2

[0070] Resistance of Pseudochryobacillum alkaligenes Z11 to Cd(II):

[0071] The Z11 strain was inoculated into LB liquid culture medium with Cd(II) concentrations of 0 mg / L and 70 mg / L (maximum tolerance concentration of Cd(II)), and a blank control was set. Three parallel samples were set for each group. The initial OD600 value was 0.098 measured by UV spectrophotometer. Thereafter, the bacterial solution was sampled every 5 hours and the OD600 was measured until the value stabilized. Figure 4As shown, the growth cycle of strain Z11 in the control group and the test group is about 55h (the growth curve of the strain mainly includes the lag period of bacteria adapting to the new environment, the exponential period of bacteria exponential growth, the stable period after the number of bacteria reaches a plateau, and the decline period when the number of live bacteria continues to decrease). The control group is in a lag period of 0-2h, and the lag period of the control group is shorter. The control group enters the exponential period after 2h. From 2-40h, the concentration of the bacterial solution increases rapidly. This period is the exponential period. At 40h, the maximum bacterial solution concentration is 2.33. From 40-55h, the bacterial solution concentration no longer increases and remains constant. This period is the stable period. The Z11 strain in the experimental group with a Cd(II) concentration of 70 mg / L was in a lag phase from 0 to 10 h. The lag phase of the experimental group was longer than that of the control group, mainly because of the stress of Cd(II). At 10 h, it entered the exponential phase, and at 10-45 h, the bacterial solution concentration grew at the fastest rate, with the OD600 value increasing from 0.1 to 1.97. At 45-55 h, the strain growth slowed down and entered a stable phase, still maintaining a high bacterial solution concentration, with the OD600 fluctuating around 2.03. Because the Z11 strain in the experimental group was stressed by Cd(II), the OD600 value of each period was lower than that of the control group under the condition of a Cd(II) concentration of 70 mg / L. Secondly, the changes in each period lagged behind those of the control group. Figure 5 As shown, the initial pH value of the control group and the experimental group was 6.03. The pH value of the bacterial solution increased continuously over time. In the 10-40h period, the pH value increased fastest and reached the maximum value at 40h. The pH values ​​of the control group and the experimental group were 8.85 and 8.28, respectively. This was mainly because the strain entered the exponential growth period in the 10-40h period, the nutritional conditions in the environment were sufficient, the strain grew and multiplied in large quantities, and continuously produced alkaline substances, thereby increasing the environmental pH value. In the 40-55h period, the pH value of the bacterial solution tended to stabilize. Compared with the control group, the experimental group contained a higher concentration of Cd(II), which inhibited the life activities of the strain, thereby reducing the strain's ability to produce alkali. The pH values ​​of the experimental group at each time period were lower than those of the control group. Figure 4 and Figure 5 It can be seen that the growth of microorganisms and the alkali production effect are positively correlated, so the optimal growth and alkali production conditions of microorganisms can be determined by measuring the growth of the strain.

[0072] After testing, it was found that the passivation efficiency of Z11 strain for Cd(II) could reach 68.5% when the maximum tolerance concentration of Cd(II) was 70 mg / L (60 μM), the optimal temperature was 30°C, the growth period was 50 h, the initial pH was 6, peptone was used as the carbon source, and yeast extract was used as the nitrogen source.

[0073] Example 3

[0074] Study on the passivation characteristics of cadmium by alkaline-producing Pseudochalobacterium saccharolyticum Z11:

[0075] Cytotoxicity assay:

[0076] (1) Cell culture: The strain Z11 was activated and inoculated onto LB solid medium, cultured at a constant temperature, and then inoculated into 100 mL LB liquid medium and cultured in a constant temperature oscillator at 30°C and 150 rpm until the late exponential growth stage.

[0077] (2) Conventional toxicity test: Weigh Cd(NO 3 ) 2 ·4H 2 O, put Cd(NO 3 ) 2 ·4H 2 O was added to the cell culture medium in the late exponential growth period. The Cd(II) concentrations in the cell culture medium after addition reached 6 μM, 60 μM, and 600 μM, respectively. 3 ) 2 ·4H 2 O's bacterial solution was set as the control group, and three parallels were set for each concentration. The strain was exposed to different concentrations of Cd(II) for 6 hours, and 180μL of the bacterial mixture (bacterial solution + Cd(II)) was transferred to a transparent 96-well microplate, and 20μL of PrestoBlue cell activity reagent was added to the well. The experiment was repeated four times for each concentration. When the PrestoBlue cell activity reagent entered the living cells, the PrestoBlue cell activity reagent was reduced from resorcinol to resorcinol. It was placed in an ELISA instrument for measurement, and the absorbance values ​​at 571nm (resorcinol) and 601nm (resorcinol) were compared. Finally, the Hill equation was used to calculate the median lethal dose of Cd(II) to strain Z11.

[0078] Infrared spectrum analysis of Z11 strain after adsorption and accumulation of cadmium:

[0079] The weighed Cd(NO 3 ) 2 ·4H 2 O was added to the cell culture medium in the late exponential growth period, and different Cd(II) concentration gradients were set, namely 6 μM, 60 μM, and 600 μM. 3 ) 2 ·4H 2 The bacterial solution of Cd(II) was set as the control group, and three parallel experiments were set for each concentration. After exposure to different Cd(II) concentrations for 6 h, 40 mL of the bacterial mixture was centrifuged at 6400 r / min for 10 min and washed three times with magnesium sulfate solution. The cell pellets were collected and centrifuged at 10000 r / min for 3 min, and the cells were collected and sent to Central South University for infrared spectroscopy testing.

[0080] The specific steps of infrared testing are as follows: 10 μL of collected biomass is dispersed on the gold surface, dried at room temperature for 20 min, and then analyzed. -1 Resolution, 60 scans, scanning range 400-4000cm -1 Each exposure condition was performed three times, and the total data set corresponded to 4000 spectra.

[0081] Raman spectroscopy analysis of Z11 strain after adsorption and accumulation of cadmium:

[0082] The weighed Cd(NO 3 ) 2 ·4H 2 O was added to the cell culture medium in the late exponential growth period, and different Cd(II) concentration gradients were set to 6μM, 60μM, and 600μM. The bacterial solution without Cd(II) concentration of 0 was set as the control group, and three parallel experiments were set for each concentration. After exposure to different Cd(II) concentrations for 6h, 40mL of the bacterial mixture was centrifuged at 6400r / min for 10min and washed 2-3 times with magnesium sulfate sterile solution. The cell pellets were collected in a centrifuge tube and centrifuged at 10000r / min for 3min. The centrifuged bacteria were preserved and sent to Central South University for Raman spectroscopy testing.

[0083] The specific steps of Raman testing are as follows: take 10 μL of each collected biological sample, evenly spread it on the surface of the slide, and air-dry it for later use; scan each sample with a Raman spectrometer at an excitation wavelength of 785 nm, with a scanning range of 400-1800 cm -1 ,Each sample was scanned three times at different locations, and the total data set corresponded to 1800 spectra.

[0084] Since the Raman spectrum test process is affected by laser stability, temperature fluctuations, and baseline offset, for biological samples with weak Raman signals, it is necessary to repeat the test on the sample and process the obtained Raman spectrum to reduce the impact of environmental factors on the analysis results. For the obtained Raman spectrum data, this section uses three processing steps of averaging, smoothing, and debasing in sequence. By averaging the Raman spectrum data of three groups of repeated tests, the data fluctuation caused by accidental factors can be effectively reduced. In order to eliminate the background noise in the Raman spectrum, the Savitaky-Golay convolution smoothing method is used to perform polynomial least squares fitting on the spectrum data to achieve spectrum smoothing. For the same biological sample, although the peak shape of the measured Raman spectrum is similar, the intensity of the spectral signal may fluctuate greatly. Therefore, the absolute intensity of the Raman spectrum is of great value, and its relative intensity is used more, so it is necessary to deduct the baseline of the spectrum.

[0085] Experimental results and discussion:

[0086] Cytotoxicity assay

[0087] After PrestoBlue cell viability reagent enters living cells, PrestoBlue cell viability reagent (retrocyanin) can be reduced to resorcinol, and the reduction rate of the detection reagent can reflect the number of active cells. When the amount of reduced resorcinol increases, the number of living cells will increase. When the remaining resorcinol increases, that is, the reduced resorcinol decreases, the number of living cells will be small, and then the toxic effect of Cd(II) on cells can be concluded. Figure 6 As shown in (a) and 6(b), as the concentration of Cd(II) increases, the absorbance at 571nm decreases continuously, that is, the generated resorcinol decreases continuously, while the absorbance at 601nm increases continuously, that is, the remaining amount of cell viability detection reagent (heavy cyanin) increases with the increase of Cd(II) concentration. Therefore, the reduction rate of cell viability detection reagent (heavy cyanin) decreases with the increase of concentration. From this result, it can be seen that as the concentration of Cd(II) increases, Cd(II) inhibits the activity of strain Z11 cells, enhances the toxic effect on strain Z11, reduces the alkali production effect of the strain, and inhibits the effect of the strain on passivating Cd(II).

[0088] Using the data from the above routine cell tests ( Figure 6 ), and draw Figure 7 is the relationship between Cd(II) concentration and strain inhibition rate, Figure 7 It can be seen that as the concentration of Cd(II) increases, the inhibition rate of Cd(II) on bacteria also increases. The results show that when 0.01mM Cd(II) is added, the inhibition on strain Z11 is small. When the concentration is 600μM Cd(II), the inhibition is significantly enhanced, with a maximum of 81%. The strain inhibition rate is positively correlated with the change in Cd(II) concentration. The Hill equation calculated that the median lethal dose (LD)50 of the bacteria is 0.216mM. The maximum tolerance concentration of Cd(II) for this strain is about 10 times the maximum toxic concentration of the cell.

[0089] Analysis of Raman spectra of Z11 bacteria after adsorption and accumulation of cadmium

[0090] The specific Raman spectra of four groups of different Cd(II) concentrations after averaging, smoothing, and debasing were made into a stacking diagram (e.g. Figure 8 As shown in Figure 2, a comparative analysis was conducted. Figure 8 It can be seen that, intuitively, the peak shapes of the Raman spectra of bacteria treated with different Cd(II) concentrations are similar, showing the following peaks: 1656cm -1 (amide I), 1572 cm-1 (Tryptophan), 1394cm -1 (amide II), 1350 cm -1 (polysaccharide), 1166cm -1 (lipid), etc. This indicates that in the Raman spectrum of bacteria, the signals of proteins and amino acids are stronger than those of lipids and sugars. -1 (amide I), 1572 cm -1 (Tryptophan), 1394cm -1 The relative intensities of the three (amide II) become stronger as the Cd(II) concentration increases, which can be explained by protein denaturation. When the Cd concentration reaches 600 μM, the Raman spectrum shows peaks of RNA and ribonucleic acid (882 cm -1 ), which may be related to bacterial fragmentation.

[0091] Analysis of infrared spectra of Z11 bacteria after adsorption and accumulation of cadmium

[0092] In order to determine the changes in the functional groups of Z11 bacteria under different Cd(II) concentrations, the infrared spectra of bacteria with Cd(II) concentrations of 6μM, 60μM, and 600μM were analyzed by stacking diagrams. The absorption peaks and changes of bacteria under different Cd(II) concentrations are shown in Figure 2. Fig. 9 As shown. Fig. 9 It can be seen that the main absorption peaks of the bacteria without Cd(II) are: 1079cm -1 (-PO 4 3- )、1550cm -1 (-NH, -CH), 1639cm -1 (-C=O stretching vibration peak, cell surface protein amide bands Ⅰ and Ⅱ), 2322 cm -1 (Alkynyl or cyano characteristic vibration), 2920cm -1 (Asymmetric stretching vibration of -CH of cell surface protein). The addition of Cd(II) will combine with the charged groups on the bacterial cell membrane, causing the characteristic peaks of the infrared spectrum to change. As the concentration of Cd(II) increases, the absorption peak of the phosphate group at 1079 cm -1 , C=O- stretching vibration peak 1639cm -1 and (-NH, -CH) 1550cm -1 The phenomenon indicates that the heavy metal Cd(II) may be through the -PO in the cell wall of strain Z11. 4 3-The complexation of -C=O, -NH and other groups changed the charge distribution in the above groups, thus confirming that Z11 bacteria adsorbed Cd(II) mainly by complexation, thereby reducing the effectiveness of free Cd(II).

[0093] Example 4

[0094] Preparation method and application of a preparation containing insoluble sugar pseudo-Ochrobacterium:

[0095] Preparation method of preparation containing insoluble sugar pseudo-Ochrobacterium:

[0096] Preparation of bacterial agent by embedding method:

[0097] Take 100uL of the activated Z11 strain (inoculated in the sterilized LB liquid medium, cultured and activated), take 50μL of the activated Z11 strain and inoculate it in multiple sterilized 50mL LB medium, culture at the same time for 48h, and after the culture strain reaches the stable period, divide it into 50mL centrifuge tubes, centrifuge at a speed of 5000r / min for 15min, repeat twice, and obtain Z11 bacteria. Weigh 0.5g of sawdust, wheat bran, charcoal, charcoal ash, and plant ash with a particle size (100 mesh) and add them to 25mL LB liquid medium. Set up three parallel experiments for each carrier and a blank control experiment without bacteria. Sterilize at 121℃ for 20min. Add the Z11 bacteria to the sterilized LB liquid medium containing the above carrier to obtain a sterilized sample.

[0098] Measure 25 mL of 4% sodium alginate solution and add it to each sterilized sample. The mixed sample was shaken at a constant temperature of 150 r / min and 30°C for 40 minutes to fully mix. After mixing, the mixture was drawn up with a syringe and squeezed out from the needle in the form of droplets to a concentration of 2% CaCl 2 The solution was continuously shaken and stirred to prevent the droplets from sticking together. After preparation, it was fixed at a speed of 180 r / min and a temperature of 30°C for 2 h, the embedded fixed bacterial agent particles were filtered out, the obtained beads were rinsed 3 times with a 0.9% NaCl solution, freeze-dried for 48 h to obtain the embedded fixed bacterial agent, and stored in a 4°C refrigerator for later use.

[0099] Preparation of bacterial agent by adsorption method:

[0100] (1) Determine the optimal adsorption time

[0101] Weigh 0.5g sawdust, wheat bran, charcoal, charcoal ash, and plant ash respectively, add them to 50mL LB liquid culture medium, sterilize and set aside. Take 100μL of the activated strain, inoculate it into the sterilized LB liquid culture medium, culture for 48h, until the strain grows steadily, divide it into 50mL centrifuge tubes, centrifuge it at a speed of 5000r / min for 15min, repeat twice, and obtain Z11 bacteria. Add the collected bacteria to the above-mentioned sterilized LB liquid culture medium containing the carrier, and set three parallels and one control for each test carrier. After the inoculation, the sample is cultured at a constant temperature of 150r / min and 30℃ for 28h, and samples are taken every 4h. The sample is centrifuged at a speed of 1500r / min for 10min, and the supernatant is discarded. Repeat the centrifugation three times, collect the bottom solid obtained by centrifugation, use 0.85% NaCl solution to wash the bottom solid, collect 5mL of supernatant and make the volume to 50mL. The solution was placed at 600 nm to measure its absorbance, and the optimal adsorption time was determined by the time corresponding to the maximum OD600 value.

[0102] (2) Preparation of bacterial agents by adsorption and fixation

[0103] Repeat the above activation and centrifugation process to obtain the Z11 strain in the stable phase. Weigh 0.5g of sterilized sawdust and wheat bran respectively, add them to 50mL of sterilized LB liquid culture medium, add the bacteria obtained by centrifugation to the LB liquid culture medium containing the carrier, and set up three parallels for each test carrier and a blank control without bacteria. After the inoculation, the sample is cultured at a constant temperature of 150r / min and 30°C. When the optimal adsorption time is reached, the sample is divided into centrifuge tubes and centrifuged at a speed of 1500r / min for 10min, and the supernatant is discarded. Repeat the centrifugation three times, and discard the supernatant. Use 0.85% NaCl solution to wash the bottom solid, vacuum freeze-dry for 24h, and obtain the finished product, which is placed in a 4°C refrigerator for storage.

[0104] Results and Discussion:

[0105] The activated strains are embedded and fixed with sodium alginate to ensure that the live bacteria in the prepared microbial agent particles are easy to preserve and can also protect the strains to the greatest extent during the application process. The microbial agent with wheat bran and sawdust as carriers is light yellow, which is mainly related to the color of the carrier, and the microbial agent particles are relatively uniform; the microbial agent with charcoal as the carrier is black in appearance and the microbial agent particles are uniform.

[0106] Effect of bacterial agents on strains when the carrier is wheat bran

[0107] Fig.10(a) is the effect of wheat bran carrier on the growth of strain Z11. The results show that the OD600 values ​​of the experimental group with the addition of the bacterial agent and the control group with only the Z11 strain increased. During the period of 0-48h, the OD600 value of the control group continued to increase, and the number of strains continued to increase. During the period of 48-120h, the number of strains remained stable, with a maximum value of 2.05; while the OD600 value of the experimental group did not change during the period of 0-24h. During the period of 24-96h, the OD600 value of the experimental group increased, the number of strains increased, and the maximum value was 2.26; and the concentration of the bacterial solution in the experimental group when the strain growth reached the stable period was significantly greater than that of the control group. This is mainly because wheat bran can provide organic matter for the growth of strains and increase the number of strains.

[0108] Fig.10 (b) is the effect of wheat bran on the pH of the culture medium during the growth of strain Z11. The results show that at 0h, the initial pH values ​​of the experimental group and the blank control with only wheat bran carrier added were relatively high, 6.56 and 6.60 respectively, while the pH value of the control group with only Z11 strain was 6, mainly due to the increase in environmental pH value caused by the addition of wheat bran. During the period of 0-120h, the pH value of the blank control group fluctuated around 6.50; during the period of 0-48h, the environmental pH value of the control group with only Z11 added continued to increase, and during the period of 48-120h, the pH value reached a stable value, with a maximum value of 8.30; during the period of 0-96h, the pH value of the experimental group showed an increasing trend, and during the period of 96-120h, the pH value tended to be stable, and the maximum pH value reached 8.54.

[0109] Depend on Fig.10 (a) and Fig.10 (b) It can be seen that wheat bran can promote the growth of strains and the alkali production effect when used as a test carrier; if only wheat bran is added, the pH value of the environment will also increase. The number of strains and the alkali production efficiency of the strains are positively correlated. When the number of strains reaches a stable period, the pH value of the environment where the strains grow will also remain relatively stable.

[0110] Effects of inoculants on strains when sawdust is used as carrier

[0111] Depend on Fig.11 (a) The effect of sawdust carrier on the growth of strain Z11 can be obtained. The OD600 values ​​of the experimental group with added bacterial agent and the control group with only added strain Z11 increased. The OD600 value of the experimental group did not change during the 0-24h period. Compared with the control group in the same time period, the bacterial agent with sawdust as a carrier had a delayed effect on the initial growth of the strain. During the 24-96h period, the strain growth rate increased, the OD600 value of the experimental group increased, and the number of strains increased. During the 96-120h period, the bacterial solution concentration gradually stabilized, and the maximum value could reach 2.25. The bacterial solution concentration of the experimental group was significantly greater than that of the control group when the strain growth reached the stable period. Secondly, a relatively large bacterial solution concentration could still be maintained during the stable period.

[0112] Depend on Fig.11 (b) The effect of sawdust on the pH of the culture medium during the growth of strain Z11 was known. The results showed that at 0h, the initial pH values ​​of the experimental group and the blank control group with only sawdust carrier added were higher, 6.61 and 6.69, respectively. It can be seen that the addition of sawdust can increase the pH value of the environment. During the period of 0-120h, the pH value of the blank control group fluctuated around 6.70; the pH value of the experimental group showed an increasing trend during the period of 0-96h, and the pH value tended to be stable during the period of 96-120h, with the maximum pH value reaching 8.26. Compared with the control group with only bacterial solution added, when the strain growth was stable, the control group and the experimental group had roughly the same effect on the pH value of the strain growth environment.

[0113] Depend on Fig.11 (a) and Fig.11 (b) It can be seen that when sawdust is used as the test carrier, the growth of the strain can be promoted, but its promoting effect on alkali production is not obvious.

[0114] Effect of the dosage of microbial agents on soil acidity and alkalinity

[0115] Take the bacterial agent prepared with wheat bran as the carrier and add it to the soil at the dosage of 3g / kg, 5g / kg, and 7g / kg respectively to ensure the soil moisture. Weigh the conical flask every 48 hours, add deionized water regularly, shake well, and stir regularly. Take samples every 7 days, perform salting out and water precipitation to determine the soil pH value. The results of salting out and water precipitation are as follows: Fig.12 As shown in the figure, the bacterial agent was prepared by adsorption fixation and embedding fixation, and the pH value of the salt precipitation was added to the soil at different concentrations. It can be seen from the figure that compared with the pH value of the experimental group with a dosage of 3g / kg, 5g / kg, and 7g / kg, the pH value in the soil increased significantly. As time went by, the pH value continued to increase, but with the increase in the dosage, the effect of increasing the pH value did not change much. This is mainly because with the increase of time, the number of strains in the experimental groups with different dosages increased, secreting alkaline substances, causing the pH value in the soil to rise. Therefore, when the dosage is 3g / kg, the pH value increases the most. Continuing to increase the dosage, the pH change range decreases instead, indicating that beyond this range, the increase in dosage has no significant effect, and the optimal dosage range is 3g / kg.

[0116] Application of microbial agents in remediation of cadmium-contaminated soil

[0117] Determination of available cadmium content in soil before and after restoration

[0118] Weigh 5.00g of the ground and air-dried cadmium-contaminated soil samples after screening, place them in 100mL conical flasks, add 25.00mL DTPA extractant, set up 3 parallel experiments, set up 2 blank control experiments for each batch of samples, and shake at a constant temperature of 30°C and a speed of 180r / min for 2h. After extraction, filter, discard the filtrate that initially passed through the filter paper, filter the filtrate again, collect and dilute the filtrate, transfer it to a centrifuge tube, and use ICP-MS to determine the effective cadmium content of the sample. The extraction and content determination of effective cadmium in the remediated soil are the same as above.

[0119] Take the bacterial agent prepared with wheat bran as the carrier, under the condition of the optimal dosage of 3g / kg and soil water holding capacity of 60%, ensure soil moisture, weigh the conical flask every 48h, add deionized water regularly, shake well, and stir. Take samples every 7d until the 35th day, a total of 5 samples. The remediation results are shown in Table 1. As of the 35th day, the maximum remediation rate of the bacterial agent for effective cadmium was 62.6%.

[0120] Table 1:

[0121] Time (day) 0 7 14 21 28 35 Effective cadmium content (mg / kg) 1.23 1.21 1.01 0.94 0.63 0.46 Repair rate (%) 0 1.63 17.89 32.5 48.8 62.6

[0122] Example 5

[0123] Application of Z11 bacteria in remediation of cadmium-contaminated soil

[0124] Determination of available cadmium content in soil before and after restoration

[0125] Weigh 5.00g of the ground and air-dried cadmium-contaminated soil samples after screening, place them in 100mL conical flasks, add 25.00mL DTPA extractant, set up 3 parallel experiments, set up 2 blank control experiments for each batch of samples, and shake at a constant temperature of 30°C and a speed of 180r / min for 2h. After extraction, filter, discard the filtrate that initially passed through the filter paper, filter the filtrate again, collect and dilute the filtrate, transfer it to a centrifuge tube, and use ICP-MS to determine the effective cadmium content of the sample. The extraction and content determination of effective cadmium in the remediated soil are the same as above.

[0126] Prepare 300 mL of LB culture medium, divide it into 3 equal parts, and put them into 3 250 mL conical flasks respectively. Inoculate 1 mL of Z11 bacterial solution. After culturing for 24 hours, add the above 3 parts of soil into Z11 bacterial culture medium respectively, place them in a constant temperature shaking incubator and shake them for 48 hours. Let them stand and filter. Collect the filtered soil and place it in a constant temperature drying oven to dry. After the soil weighs constant, use DTPA extractant to extract the effective cadmium in the soil. The extraction method is the same as above.

[0127] The measured results showed that the effective cadmium content in the soil before restoration was 1.23 mg / kg, and the effective cadmium content in the soil after restoration was 0.61 mg / kg, and the passivation rate of effective cadmium reached 50.4%, indicating that the Z11 bacteria of the present application can restore cadmium-contaminated soil.

[0128] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A pseudochlorobacterium insaccharolyticum, characterized in that The pseudochlorobacterium insaccharolyticum is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC NO: 62528 and a deposit date of July 7, 2022, and is named Pseudochrobactrum asaccharolyticum Z11.

2. Use of the Pseudochalobacterium insaccharolyticum as claimed in claim 1 in cadmium passivation.

3. A use of the Pseudochalobacterium insaccharolyticum as claimed in claim 1 in remediating cadmium-contaminated soil.

4. A microbial alkali-producing preparation suitable for remediation of cadmium-contaminated soil, characterized in that: The invention comprises the insugar-degrading Pseudocobacillus sp. according to claim 1 and an organic carrier.

5. The microbial alkali-producing preparation suitable for cadmium-contaminated soil remediation according to claim 4, characterized in that: The organic carrier includes any one of wheat bran, sawdust, charcoal, charcoal ash and plant ash.

6. The method for preparing a microbial alkali-producing preparation suitable for cadmium-contaminated soil remediation by using Pseudochrobacterium insaccharolyticum according to claim 1, characterized in that: The method includes an embedding method and an adsorption method.

7. The method for preparing a microbial alkali-producing preparation suitable for cadmium-contaminated soil remediation by using Pseudochrobacterium insaccharolyticum according to claim 6, characterized in that: The embedding method comprises the following steps: Step 1: inoculate the Z11 strain into a sterilized LB liquid medium for culture activation to obtain an activated Z11 strain; inoculate the activated Z11 strain into a sterilized LB medium, and after the culture strain reaches a stable period, dispense into a centrifuge tube, and obtain Z11 bacteria after centrifugation; take an organic carrier and add it to the LB liquid medium, and sterilize it for standby use; wherein the organic carrier includes any one of wheat bran, sawdust, charcoal, charcoal ash, and plant ash; Step 2: Add the above Z11 bacteria to the sterilized LB liquid culture medium containing the carrier, and add 4% sodium alginate solution, shake at a constant temperature until fully mixed, to obtain a mixture; Step 3: Use a syringe to absorb the mixture and squeeze it out from the needle in the form of droplets into a 2% CaCl2 solution, shake and stir continuously, and fix it at a speed of 180 r / min and a temperature of 30°C for 2 h. Filter out the embedded fixed bacterial agent particles, rinse the obtained particles with a 0.9% NaCl solution for 3 times, freeze-dry for 48 h to obtain the embedded fixed bacterial agent, and store it in a 4°C refrigerator for later use.

8. The method for preparing a microbial alkali-producing preparation suitable for cadmium-contaminated soil remediation by using Pseudochrobacterium insaccharolyticum according to claim 6, characterized in that: The adsorption method comprises the following steps: Step 1: inoculate the Z11 strain into a sterilized LB liquid medium for culture activation to obtain an activated Z11 strain; inoculate the activated Z11 strain into a sterilized LB medium, and after the culture strain reaches a stable period, dispense into a centrifuge tube, and obtain Z11 bacteria after centrifugation; take an organic carrier and add it to the LB liquid medium, and sterilize it for standby use; wherein the organic carrier includes any one of wheat bran, sawdust, charcoal, charcoal ash, and plant ash; Step 2: Add the above Z11 bacteria into the sterilized LB liquid culture medium containing the carrier to obtain a sample after inoculation; Step 3: After the inoculation, the sample was cultured at a constant temperature of 150 r / min and 30°C. When the optimal adsorption time was reached, the sample was divided into centrifuge tubes and centrifuged at 1500 r / min for 10 min. The supernatant was discarded. The centrifugation was repeated three times and the supernatant was discarded. The bottom solid was washed with 0.85% NaCl solution and vacuum freeze-dried for 24 hours to obtain the finished product, which was stored in a 4°C refrigerator for later use.

9. Use of the alkali-producing preparation prepared by the method for preparing a microbial alkali-producing preparation suitable for remediation of cadmium-contaminated soil by Pseudocobacillus insaccharolyticus according to any one of claims 6 to 8 in remediation of cadmium-contaminated soil.

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