Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention provides a complex microbial inoculant for cadmium treatment and a preparation method and application thereof, the complex microbial inoculant comprises biochar, hydroxyapatite and a gas-producing klebsiella inoculant, and in the complex microbial inoculant, the surface of the biochar is loaded with a large amount of gas-producing klebsiella and phosphate ions. When the prepared complex microbial inoculant is applied to cadmium treatment, the cadmium fixation effect is excellent, meanwhile, environmental pollution is avoided, and both environmental benefits and economic benefits are considered.

Description

Technical Field

[0001] The invention relates to the field of heavy metal treatment, and in particular to a composite bacterial agent and a preparation method and application thereof. Background Art

[0002] The main sources of cadmium pollution are wastewater discharged from electroplating, mining, smelting, dyes, batteries and chemical industries. Cadmium in the human body is absorbed from the external environment after birth, and mainly enters the body through food, water and air and accumulates. Cadmium is highly toxic. Air and food contaminated by cadmium are seriously harmful to the human body, and it is metabolized slowly in the human body. Japan has had "Itai-itai disease" due to cadmium poisoning. It is recognized as one of the most toxic heavy metals in the world. The problem of cadmium pollution is becoming more and more serious, and cadmium pollution control is imminent.

[0003] Soluble phosphate compounds have been widely used to repair heavy metal pollution and have achieved high fixation efficiency, but they are not only more expensive than sparingly soluble phosphate compounds, but are also more likely to cause other environmental problems such as eutrophication of water bodies, soil acidification, and plant nutrient imbalance. However, the ability of phosphorus-containing materials to supply free phosphate ions is closely related to their passivation efficiency and sedimentation rate of heavy metals. The low solubility of insoluble phosphate compounds will limit the fixation efficiency of heavy metals.

[0004] Based on the above, it is urgent to develop a phosphate cadmium-solidifying material that has good cadmium-solidifying effect and is environmentally friendly. Summary of the invention

[0005] The main purpose of the present invention is to provide a composite bacterial agent and a preparation method and application thereof, so as to solve the technical problem that it is difficult to balance the cadmium fixation effect and environmental benefits in common phosphate cadmium fixation materials.

[0006] To achieve the above-mentioned purpose, the present invention provides a composite bacterial agent for cadmium treatment, which comprises biochar, hydroxyapatite and gas-producing Klebsiella agent. In the composite bacterial agent, the surface of the biochar is loaded with a large amount of gas-producing Klebsiella and hydroxyapatite, wherein a small number of the hydroxyapatites are directly attached to the biochar, and most of the hydroxyapatites are adsorbed on the cell surface of the gas-producing Klebsiella.

[0007] The present invention provides a method for preparing a composite bacterial agent, comprising the steps of:

[0008] The gas-producing Klebsiella and biochar are mixed in a phosphate-solubilizing medium for cultivation to obtain a solid-liquid mixture; wherein the phosphate-solubilizing medium contains 0.3% to 0.7% hydroxyapatite by dry weight;

[0009] The solid-liquid mixture is centrifuged and purified, the solid mixture is collected, and the solid mixture is freeze-dried to obtain the composite bacterial agent.

[0010] Furthermore, the aerogenes Klebsiella is an activated and expanded cultured aerogenes Klebsiella species, and the preparation of the aerogenes Klebsiella comprises the following steps:

[0011] Activation: taking out the frozen Klebsiella aeruginosa strains and thawing them, transferring the strains to the first LB liquid culture medium, and activating the culture by shaking on a shaker at a constant temperature;

[0012] Expanding the culture: transferring the activated Klebsiella aeruginosa bacterial liquid to a second LB liquid culture medium, and expanding the culture in a constant temperature shaker to obtain the Klebsiella aeruginosa; wherein the volume of the second LB liquid culture medium is twice the volume of the first LB liquid culture medium.

[0013] Furthermore, the volume ratio of the added amount of the biochar to the phosphate-solubilizing medium is 3-7 mg:1 mL, the added amount of the aerogenes Klebsiella to the inoculation ratio of the phosphate-solubilizing medium is 0.9-1.1:90-110 mL, and the OD600 range of the bacterial liquid of the aerogenes Klebsiella is 0.9-1.1.

[0014] Furthermore, the biochar includes coconut shell biochar, and the particle size of the biochar is no greater than 50 meshes.

[0015] Furthermore, in the step of mixing gas-producing Klebsiella and biochar in a phosphate-solubilizing medium for culturing, the modified oscillation conditions of the attachment culture are 28-32° C., 150-160 rpm / min, and the time is 70-75 hours.

[0016] Furthermore, the formula of the phosphate-solubilizing medium includes, by mass fraction: 10 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of sodium chloride, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate, 0.036 g of ferrous sulfate heptahydrate, 0.03 g of manganese sulfate monohydrate, 5 g of hydroxyapatite, 1000 mL of distilled water, and the pH is adjusted to 7.0 with 5 mol / L NaOH solution or 5 mol / L hydrochloric acid solution.

[0017] Furthermore, after centrifuging and purifying the solid-liquid mixture, the step of collecting the solid mixture specifically includes removing the supernatant after centrifuging at a speed of 8000-8500 rpm for 4-6 minutes to obtain the solid mixture.

[0018] The present invention also provides an application of any one of the preparation methods described above in the treatment of cadmium-containing wastewater.

[0019] The effects of the present invention are as follows:

[0020] The present invention provides a composite bacterial agent, which is composed of biochar, hydroxyapatite and gas-producing Klebsiella agent, and the loose and porous surface of the biochar is loaded with a large amount of gas-producing Klebsiella and phosphate ions. Due to the unique genes and their expression of gas-producing Klebsiella, gas-producing Klebsiella can use a special coupling mechanism of phosphorus metabolism and release to dissolve insoluble phosphates - in the early stage of the bacterial reproduction process, it will quickly produce high-yield D-gluconic acid and acetic acid to dissolve hydroxyapatite and produce phosphate to achieve sufficient precipitation of heavy metals.

[0021] Since the present invention utilizes the periodic metabolism of Klebsiella aerogenes to digest insoluble phosphate hydroxyapatite, it continuously provides phosphate to the heavy metal environment, which solves the technical problem of low supply capacity of insoluble phosphate in common technologies, and at the same time avoids the partial failure caused by the insolubility of insoluble phosphates, achieves a significant improvement in heavy metal sedimentation efficiency and sedimentation rate, and has excellent cadmium fixation effect.

[0022] In addition, the microbial induced phosphate precipitation (MIPP) formula is expressed as:

[0023] Polymer organic phosphorus / insoluble phosphate————→PO4 3-

[0024] M x+ +Cell→Cell-M x+

[0025] Cell-M x+ +PO4 3- →Cell-(M)3(PO4) x

[0026] In comparison, the general mechanism of phosphate mineralization of heavy metals in common technologies is as follows:

[0027] M X+ (aq)+PO4 3- (aq)→M(PO4)OH↓

[0028] M(PO4)OH is easily released into the environment due to chemical reactions in an acidic environment, while Cell-(M)3(PO4) x The crystallinity is high and the stability is strong. The microbial mineralization products will be tightly bound to the functional groups on the surface of microorganisms. The dissolution rate after 7 days of leaching in a solution with a pH of 3 is about 0.06%, and the biological effectiveness of heavy metals is greatly reduced, which solves the technical problem of secondary pollution caused by M(PO4)OH leaching and re-dissolution in common technologies.

[0029] The present invention utilizes Klebsiella aeruginosa and hydroxyapatite to modify biochar. Biochar, as a porous adsorptive carrier, provides abundant sites for the aggregation of heavy metal ions, the co-cultivation of bacterial colonies, and the attachment of phosphate ions, further ensuring the high stability of Cell-(M)3(PO4) x Targeted formation.

[0030] Specifically, biochar has a large specific surface area and contains many nutrients and organic matter, which provide a habitat and nutrients for Klebsiella aeruginosa to enhance its attachment and metabolism on the surface of biochar. Based on the adsorption characteristics of biochar, heavy metal ions with high biological effectiveness are aggregated on the surface of biochar, which helps Klebsiella aeruginosa induce phosphate precipitation of heavy metal ions; at the same time, phosphate ions are aggregated on the surface of biochar and rarely scatter when entering cadmium-containing water bodies, effectively avoiding environmental problems such as eutrophication of water bodies, soil acidification, and plant nutrient imbalance that occur when soluble phosphate compounds in common technologies enter cadmium-containing systems.

[0031] In summary, the present invention solves the technical problem that it is difficult to take both the cadmium fixation effect and the environmental benefit into consideration in the conventional technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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 the structures shown in these drawings without paying creative work.

[0033] Figure 1 is a scanning electron microscope image of the composite bacterial agent prepared in Example 1 of the present invention;

[0034] Figure 2 This is the XRD analysis diagram of the coconut shell biochar used in Example 1 of the present invention;

[0035] Figure 3 This is the XRD analysis diagram of the composite bacterial agent prepared in Example 1 of the present invention;

[0036] Figure 4 FTIR images of the control group and the composite bacterial agent in Example 1 of the present invention;

[0037] Figure 5 The cadmium content of Example 2 of the present invention varies with reaction time;

[0038] Figure 6 The figure is a comparison chart of cadmium removal rates of the control group and the experimental group in Example 2 of the present invention;

[0039] Figure 7 This is an XRD data analysis diagram of the solid cadmium product of Example 2 of the present invention;

[0040] Figure 8 This is a comparison chart of cadmium content in soil samples of Example 3 of the present invention;

[0041] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0042] 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.

[0043] It should be noted that all directional indications (such as up, down, etc.) in the embodiments of the present invention are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0044] In addition, in the present invention, the descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features.

[0045] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that they can be implemented by ordinary technicians in the field. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0046] A composite bacterial agent is composed of biochar, hydroxyapatite and gas-producing Klebsiella agent. In the composite bacterial agent, a large amount of gas-producing Klebsiella and phosphate ions are loaded on the loose and porous surface of the biochar.

[0047] Hydroxyapatite, biochar, Klebsiella aeruginosa, soluble phosphate compounds, and sparingly soluble phosphate compounds all have a certain ability to fix cadmium, but the application effect is not good:

[0048] Hydroxyapatite is a poorly soluble phosphate compound, the main component of which is calcium apatite, and its chemical formula is often written as Ca 10(PO4)6(OH)2. It has a certain adsorption capacity for cadmium. In an aqueous solution environment, hydroxyapatite can remove cadmium through mechanisms such as ion exchange. Specifically, when the cadmium ion concentration is low, the adsorption of cadmium by hydroxyapatite mainly occurs on the surface of hydroxyapatite, and the adsorption sites are limited. As the cadmium concentration increases, the adsorption process becomes more complicated, and cadmium can be precipitated by co-precipitation. Regardless of the cadmium concentration, the effect of hydroxyapatite is limited by its low solubility.

[0049] Biochar contains a large number of pores and rich functional groups, with a large specific surface area and strong stability. It has been widely used in soil remediation, carbon fixation and wastewater treatment. The adsorption of cadmium by biochar mainly includes physical adsorption and chemical adsorption. Among them, physical adsorption mainly comes from the high porosity, large specific surface area and electrostatic force of biochar: biochar has a rich pore structure, including micropores, mesopores and macropores. These pores provide a large number of adsorption sites for cadmium, thereby increasing the contact area between cadmium and biochar. At the same time, the surface of biochar carries an electric charge, which can generate electrostatic attraction with cadmium ions with opposite charges, thereby achieving physical adsorption.

[0050] Chemical adsorption mainly comes from the presence of abundant functional groups on the surface of biochar, such as hydroxyl, carboxyl and amine groups. These functional groups can form chemical bonds such as coordination bonds or ionic bonds with cadmium ions, thereby firmly fixing the cadmium ions on the biochar. In addition, the negative charge on the surface of biochar can undergo cation exchange with cadmium ions, replacing the cadmium ions on the surface of biochar. However, biochar is light in weight, has small powder particles and a single composition, which affects its ability to adsorb cadmium and organic pollutants, and its recovery in water and soil is also limited.

[0051] When Klebsiella aeruginosa is directly used for cadmium treatment, its solidification product is a stable combination of the microbial cells and cadmium ions. Limited by the number of microbial cells and the stability of the combination, its solidification effect is general and there is a risk of secondary pollution. Cell lysis during microbial apoptosis will cause some effective cadmium adsorbed by microbial cells to be released back into the environment. In the early logarithmic growth period of Klebsiella aeruginosa (referring to the 3rd to 12th hour after entering the hydroxyapatite-Klebsiella aeruginosa-biochar system), there are few phosphorus sources in the system, and the main phosphorus source is hydroxyapatite. At this time, the yield of phosphate is very high. Subsequently, Klebsiella aeruginosa enters a stable period, and the main phosphorus source includes phosphorus produced by cell apoptosis. At this time, the yield of phosphate is reduced; when Klebsiella aeruginosa is directly inoculated into contaminated water or soil, the supply of phosphate is not stable enough and there is a certain loss.

[0052] Soluble phosphate compounds have been widely used to remediate heavy metal pollution, but they are not only more expensive than sparingly soluble phosphate compounds, but are also more likely to cause other environmental problems such as water eutrophication, soil acidification, and plant nutrient imbalance.

[0053] The application of insoluble phosphates does not pose the above environmental threats, but the low solubility in the compound will limit the efficiency of heavy metal fixation. The ability of phosphorus-containing materials to supply free phosphate ions is closely related to their efficiency in passivating heavy metals.

[0054] Based on the above, the present invention provides a composite bacterial agent, which is composed of biochar, hydroxyapatite and gas-producing Klebsiella agent, and the loose and porous surface of the biochar is loaded with a large number of gas-producing Klebsiella and phosphate ions. Due to the unique genes and their expression of gas-producing Klebsiella, gas-producing Klebsiella can use a special coupling mechanism of phosphorus metabolism and release to dissolve insoluble phosphates - in the early stage of the bacterial reproduction process, it will quickly produce high-yield D-gluconic acid and acetic acid to dissolve hydroxyapatite and produce phosphate to achieve sufficient precipitation of heavy metals.

[0055] Since the present invention utilizes the periodic metabolism of Klebsiella aerogenes to digest insoluble phosphate hydroxyapatite, it continuously provides phosphate to the heavy metal environment, which solves the technical problem of low supply capacity of insoluble phosphate in common technologies, and at the same time avoids the partial failure caused by the insolubility of insoluble phosphates, achieves a significant improvement in heavy metal sedimentation efficiency and sedimentation rate, and has excellent cadmium fixation effect.

[0056] In addition, the microbial induced phosphate precipitation (MIPP) formula is expressed as:

[0057] Polymer organic phosphorus / insoluble phosphate————→PO4 3-

[0058] M x+ +Cell→Cell-M x+

[0059] Cell-M x+ +PO4 3- →Cell-(M)3(PO4) x

[0060] In comparison, the general mechanism of phosphate mineralization of heavy metals in common technologies is as follows:

[0061] M X+ (aq)+PO4 3- (aq)→M(PO4)OH↓

[0062] M(PO4)OH is easily released into the environment due to chemical reactions in an acidic environment, while Cell-(M)3(PO4) x The crystallinity is high and the stability is strong. The microbial mineralization products will be tightly bound to the functional groups on the surface of microorganisms. The dissolution rate after 7 days of leaching in a solution with a pH of 3 is about 0.06%, and the biological effectiveness of heavy metals is greatly reduced, which solves the technical problem of secondary pollution caused by M(PO4)OH leaching and re-dissolution in common technologies.

[0063] The present invention utilizes hydroxyapatite-modified biochar and co-cultured Klebsiella gas-producing. Biochar, as a porous adsorptive carrier, provides abundant sites for the polymerization of heavy metal ions, co-culture of bacterial colonies, and attachment of phosphate ions, further ensuring the high stability of Cell-(M)3(PO4) x Targeted formation.

[0064] Specifically, biochar has a large specific surface area and contains many nutrients and organic matter, which provide a habitat and nutrients for Klebsiella aeruginosa to enhance its attachment and metabolism on the surface of biochar. Based on the adsorption characteristics of biochar, heavy metal ions with high biological effectiveness are aggregated on the surface of biochar, which helps Klebsiella aeruginosa induce phosphate precipitation of heavy metal ions; at the same time, phosphate ions are aggregated on the surface of biochar and rarely scatter when entering cadmium-containing water bodies, effectively avoiding environmental problems such as eutrophication of water bodies, soil acidification, and plant nutrient imbalance that occur when soluble phosphate compounds in common technologies enter cadmium-containing systems.

[0065] In summary, the present invention solves the technical problem that it is difficult to take both the cadmium fixation effect and the environmental benefit into consideration in the conventional technology.

[0066] The present invention also provides a method for preparing a composite bacterial agent, comprising the steps of:

[0067] S1. Mixing gas-producing Klebsiella and biochar in a phosphate-solubilizing medium for attachment culture modification to obtain a solid-liquid mixture; wherein the phosphate-solubilizing medium contains 0.3% to 0.7% hydroxyapatite by dry weight.

[0068] The biochar includes coconut shell biochar, the particle size of the biochar is no more than 50 meshes, and the grinding and screening specifically includes: grinding the obtained biochar through a 50-mesh standard sieve, and storing it in a light-proof and dry place for future use.

[0069] Klebsiella aerogenes is Klebsiella aerogenes Wn, and its Latin classification name is Klebsiella aerogenes. The strain was deposited in the China General Microbiological Culture Collection on July 22, 2021, with the deposit number CGMCC No.22889, named Klebsiella aerogenes Wn, abbreviated as Wn bacteria.

[0070] In some embodiments, the ratio of the amount of biochar added to the phosphate-solubilizing medium is 3-7 mg:1 mL, the ratio of the amount of Klebsiella aeruginosa added to the inoculation ratio of the phosphate-solubilizing medium is 1:100 mL, and the OD600 of the bacterial liquid of Klebsiella aeruginosa ranges from 0.9 to 1.1.

[0071] Calculated by mass fraction, the formula of the phosphate-solubilizing medium includes: 10g of glucose, 0.5g of ammonium sulfate, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate, 0.036g of ferrous sulfate heptahydrate, 0.03g of manganese sulfate monohydrate, 5g of hydroxyapatite, 1000mL of distilled water, and the pH is adjusted to 7.0 with 5mol / L NaOH solution or 5mol / L hydrochloric acid solution.

[0072] In some embodiments, the attachment modification culture is cultured under shaking conditions of 28-32° C., 150-160 rpm / min, for 72 hours.

[0073] S2. The solid-liquid mixture is centrifuged and purified, the solid mixture is collected, and the solid mixture is freeze-dried to obtain the composite bacterial agent.

[0074] In some embodiments, the Klebsiella aerogenes is an activated and expanded Klebsiella aerogenes strain, and the preparation of the Klebsiella aerogenes comprises the steps of:

[0075] Activation: The frozen Klebsiella aeruginosa strains are taken out and thawed, and transferred to the first LB liquid culture medium, and activated and cultured by shaking on a shaker at a constant temperature.

[0076] Alternatively, the bacterial solution can be inoculated into LB solid culture medium by the plate streak method or the dilution plate method, and then placed in a constant temperature incubator for activation culture.

[0077] In some embodiments, the Klebsiella aeruginosa strain may be a -80°C glycerol frozen strain, and may be thawed using 25°C room temperature water.

[0078] Exemplarily, the activation culture by shaking in a constant temperature shaker is specifically: shaking culture at 30° C. and 150 rpm / min for 48 hours.

[0079] Expanding the culture: transferring the activated Klebsiella aeruginosa bacterial liquid to a second LB liquid culture medium, and expanding the culture in a constant temperature shaker to obtain the Klebsiella aeruginosa.

[0080] Alternatively, a single colony from the activated plate may be picked and transferred to LB liquid culture medium, cultured in a constant temperature shaker, and then taken out and subcultured to increase bacterial activity and bacterial liquid concentration.

[0081] Exemplarily, the activation culture in the constant temperature incubator is specifically: incubating in a constant temperature incubator at 30° C. for 72 hours.

[0082] In some embodiments, the volume of the second LB liquid culture medium is twice the volume of the first LB liquid culture medium.

[0083] Illustratively, the volume of the second LB liquid culture medium may be 100 ml, and the volume of the first LB liquid culture medium may be 50 ml.

[0084] In some embodiments, after centrifuging and purifying the solid-liquid mixture, the step of collecting the solid mixture specifically includes removing the supernatant after centrifuging at a speed of 8000-8500 rpm for 4-6 minutes to obtain the solid mixture.

[0085] It should be noted that LB liquid / solid culture medium, NBRIP phosphate solubilization medium, coconut shell biochar, and all glass instruments involved were sterilized in a 100Kpa, 121°C high-pressure steam autoclave for 15 to 20 minutes before use.

[0086] The present invention also provides an application of any one of the preparation methods described above in the treatment of cadmium-containing wastewater.

[0087] The cadmium removal rate of eutrophic cadmium-containing sewage can reach more than 99.98% when the biochar composite gas-producing Klebsiella bacterial agent is used to treat the cadmium-containing sewage; the cadmium removal rate of cadmium-contaminated soil can reach 69.01%.

[0088] The biochar-composite gas-producing Klebsiella agent of the present invention has a rice cadmium control effect in the soil.

[0089] For further understanding of the present invention, examples are given below:

[0090] Example 1

[0091] A composite bacterial agent of hydroxyapatite-modified biochar and gas-producing Klebsiella mainly includes preliminary preparation and preparation method, wherein the preliminary preparation includes:

[0092] (1) Prepare LB liquid medium: weigh 25 g of LB Borth medium and dissolve it in 1000 mL of distilled water;

[0093] (2) Prepare phosphate-dissolving medium with the following formula: 10 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g potassium chloride, 0.3 g magnesium sulfate, 0.036 g ferrous sulfate heptahydrate, 0.03 g manganese sulfate monohydrate, 5 g hydroxyapatite, 1000 mL distilled water, and adjust the pH to 7.0 with 5 mol / L NaOH solution or 5 mol / L hydrochloric acid solution; the brand of hydroxyapatite used is McLean;

[0094] (3) LB liquid / solid culture medium, NBRIP phosphate solubilization medium, coconut shell biochar, and all glass instruments involved were sterilized in a 100 KPa, 121 °C high pressure steam sterilizer for 15 min before use.

[0095] The preparation method comprises the following steps:

[0096] (1) Biochar preparation: Wash and dry the coconut shell biochar, crush it using a ball mill, and grind it through a 50-mesh sieve for later use;

[0097] (2) Activation of bacterial strains: After taking out the frozen bacterial strains, thaw them with 25°C room temperature water. After thawing, inoculate the bacterial solution into 50 mL of LB liquid culture medium and activate the culture by shaking at a constant temperature on a shaker for 48 hours.

[0098] (3) Expansion culture: Transfer the activated bacterial solution to 100 mL of LB liquid medium and culture in a constant temperature shaker at 30°C and 150 rpm / min for 48 h;

[0099] (4) Adhesion modification culture: Take the bacterial solution after expansion culture in a sterile clean bench and transfer it to 100 mL of phosphate-solubilizing medium, add 0.5 g of the prepared biochar, and shake it in a constant temperature shaker at 30°C and 150 rpm / min for 72 hours to perform attachment culture modification;

[0100] (5) Removing the bacterial agent: After adding biochar attachment culture, the solid-liquid mixture was centrifuged at 8000 rpm for 4 to 6 min, the supernatant was removed, and the composite bacterial agent was freeze-dried. After SEM sample pretreatment, SEM, XRD and FTIR analysis were performed.

[0101] The scanning electron microscope image of the composite bacterial agent prepared in Example 1 is shown in Figure 1 , it can be seen from the figure that Klebsiella aerogenes was successfully loaded.

[0102] XRD analysis of coconut shell biochar is shown in Figure 2 It can be seen from the figure that the biochar before and after cadmium adsorption has no crystal phase except the carbon peak; the XRD analysis diagram of the composite bacterial agent is shown in Figure 3 , it can be seen that hydroxyapatite was successfully loaded on biochar; the FTIR images of the control group and the composite bacterial agent are shown in Figure 4, can see 600cm -1 The significantly increased phosphate functional group absorption peak at the surface of the biochar indicates that the functional groups on the biochar surface have been modified; the above analysis results show that hydroxyapatite has successfully modified the biochar.

[0103] Example 2

[0104] The invention discloses an application of a composite bacterial agent to fix heavy metal cadmium in water.

[0105] Prepare cadmium mother solution: weigh 20.317 g of cadmium chloride and dissolve it in 500 mL of ultrapure water. Stir until dissolved. Transfer to a 1 L volumetric flask and make up to the mark. The cadmium concentration of the solution is 10 g / L. Transfer to a 1 L glass bottle, label it and set aside.

[0106] Prepare solid cadmium experimental solution: In order to make the bacterial agent work better, add 10g of glucose, 0.5g of ammonium sulfate, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate, 0.036g of ferrous sulfate heptahydrate, 0.03g of manganese sulfate monohydrate, and 1mL of cadmium mother liquor into 500mL of ultrapure water, transfer to a 1L volumetric flask to make up to volume, and dispense 100mL into 250mL conical flasks for later use.

[0107] The composite bacterial agent obtained in Example 1 was added to the experimental solution. Gas-producing Klebsiella could not grow and reproduce without a phosphorus source. Therefore, a control group in which only hydroxyapatite was added and a control group in which only coconut shell biochar was added were set up. The conical flask, the biochar in the control group and the hydroxyapatite were sterilized in a high-pressure steam autoclave at 100 Kpa and 121 ° C for 15 min, and then added in equal amounts on an ultra-clean workbench. The mixture was then placed in a shaker and cultured at 30 ° C and 150 rpm / min. Samples were taken at regular intervals, and after filtering through a 0.22 μm filter head, the cadmium content in the solution was detected by ICP-OES or ICP-MS, and the final solid cadmium mineralization product was detected by XRD scanning.

[0108] The cadmium content changes with reaction time. Figure 5 As shown in the figure, the cadmium removal rate reached 99.84% on the 7th day and 99.98% on the 35th day. The comparison of cadmium removal rates between the control group and the experimental group is shown in the figure Figure 6 As shown; the XRD data analysis diagram of the solid cadmium product is as follows Figure 7 As shown, the final product is Ca2+ tightly bound to Klebsiella aerogenes. 3.9 (Ca 4.7 Cd 0.7 )(PO)4(OH) 1.8 .

[0109] Example 3

[0110] Application of a composite bacterial agent to fix heavy metal cadmium in soil.

[0111] Prepare cadmium mother solution: weigh 20.317 g of cadmium chloride and dissolve it in 500 mL of ultrapure water. Stir until dissolved. Transfer to a 1 L volumetric flask and make up to the mark. The cadmium concentration of the solution is 10 g / L. Transfer to a 1 L glass bottle, label it and set aside.

[0112] Prepare the experimental nutrient solution: add 10 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of sodium chloride, 0.3 g of potassium chloride, 0.3 g of magnesium sulfate, 0.036 g of ferrous sulfate heptahydrate, 0.03 g of manganese sulfate monohydrate, and 1 mL of cadmium mother liquor into 500 mL of ultrapure water, transfer to a 1 L volumetric flask to make up the volume, and sterilize in a high-pressure steam autoclave at 100 KPa and 121 °C for 15 min, then cool for use.

[0113] Preparation of experimental soil samples: farmland soil samples with severe cadmium pollution were collected from Hongsheng Rice Planting Professional Cooperative in Heshan District, Yiyang. After air-drying, they were ground through a 10-mesh sieve. 10 g of soil sample was placed in each sealed breathable glass culture bottle, and sterilized in a 100 Kpa, 121 °C high-pressure steam autoclave for 30 min and cooled for use.

[0114] In addition to the experimental group with compound bacterial agent, blank control groups were set up without any treatment, only biochar, only hydroxyapatite, and only gas-producing Klebsiella inoculated. In order to exclude the influence of other microorganisms, the experiment was carried out in a clean bench. 10mL of nutrient solution was added to each experimental bottle to simulate the actual farmland environment, and then compound bacterial agent, biochar, etc. were added respectively. The culture bottle was placed in a 30℃ constant temperature greenhouse. After standing for 10 days, the soil sample was taken out for air drying, grinding, and 10 mesh sieve. 1mol / L MgCl2 solution was added at a soil-liquid ratio of 1:8, and the cadmium content was detected by ICP-OES after shaking for 2h at 25℃ and 180rpm / min.

[0115] The comparison chart of cadmium content in soil samples is shown in the figure below. Figure 8 As shown, it can be seen that the cadmium removal rate of the T1 group treated by the present invention is the highest, and the cadmium removal rate of the composite bacterial agent of the present invention can reach 69.01% under soil conditions.

[0116] The above technical solutions of the present invention are only preferred embodiments of the present invention, and the patent scope of the present invention is not limited thereto. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A composite bacterial agent for cadmium treatment, characterized in that: The composition includes biochar, hydroxyapatite and gas-producing Klebsiella agent. In the composite bacterial agent, a large amount of gas-producing Klebsiella and hydroxyapatite are loaded on the surface of the biochar, wherein a small amount of the hydroxyapatite is directly attached to the biochar, and a large amount of the hydroxyapatite is adsorbed on the cell surface of the gas-producing Klebsiella.

2. A method for preparing a composite bacterial agent, characterized in that: Includes steps: The gas-producing Klebsiella and biochar are mixed in a phosphate-solubilizing medium for cultivation to obtain a solid-liquid mixture; wherein the phosphate-solubilizing medium contains 0.3% to 0.7% hydroxyapatite by dry weight; The solid-liquid mixture is centrifuged and purified, the solid mixture is collected, and the solid mixture is freeze-dried to obtain the composite bacterial agent.

3. The preparation method according to claim 2, characterized in that: The aerogenes Klebsiella is an activated and expanded cultured aerogenes Klebsiella species, and the preparation of the aerogenes Klebsiella comprises the following steps: Activation: taking out the frozen Klebsiella aeruginosa strains and thawing them, transferring the strains to the first LB liquid culture medium, and activating the culture by shaking on a shaker at a constant temperature; Expanding the culture: transferring the activated Klebsiella aeruginosa bacterial liquid to a second LB liquid culture medium, and expanding the culture in a constant temperature shaker to obtain the Klebsiella aeruginosa; wherein the volume of the second LB liquid culture medium is twice the volume of the first LB liquid culture medium.

4. The preparation method according to claim 2, characterized in that: The volume ratio of the added amount of the biochar to the phosphate-solubilizing medium is 3-7 mg:1 mL, the added amount of the aerogenes Klebsiella to the inoculation ratio of the phosphate-solubilizing medium is 0.9-1.1:90-110 mL, and the OD600 range of the bacterial liquid of the aerogenes Klebsiella is 0.9-1.

1.

5. The preparation method according to claim 2, characterized in that: The biochar includes coconut shell biochar, and the particle size of the biochar is no greater than 50 meshes.

6. The preparation method according to claim 2, characterized in that: In the step of mixing gas-producing Klebsiella and biochar in a phosphate-solubilizing medium for culturing, the modified attachment culture has shaking conditions of 28-32° C., 150-160 rpm / min, and a time of 70-75 hours.

7. The preparation method according to claim 6, characterized in that: Calculated by mass fraction, the formula of the phosphate-solubilizing medium includes: 10g of glucose, 0.5g of ammonium sulfate, 0.3g of sodium chloride, 0.3g of potassium chloride, 0.3g of magnesium sulfate, 0.036g of ferrous sulfate heptahydrate, 0.03g of manganese sulfate monohydrate, 5g of hydroxyapatite, 1000mL of distilled water, and the pH is adjusted to 7.0 with 5mol / L NaOH solution or 5mol / L hydrochloric acid solution.

8. The preparation method according to claim 2, characterized in that: The step of collecting the solid mixture after centrifugation for purification includes removing the supernatant after centrifugation at a speed of 8000-8500 rpm for 4-6 minutes to obtain the solid mixture.

9. Use of the preparation method according to any one of claims 2 to 8 in the treatment of cadmium-containing wastewater.

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