Preparation method and application of decontamination-growth-promoting immobilized microbial inoculum

Abstract

The invention provides a preparation method and application of a decontamination-growth-promoting immobilized microbial agent, belongs to the technical field of biology, and can improve the immobilization effect and survival rate of plasticizer degrading bacteria and plant root growth-promoting bacteria so as to improve the soil remediation effect. According to the method, plasticizer degrading bacteria and plant root growth-promoting bacteria are immobilized on an immobilization carrier to obtain an immobilization microbial inoculum, the plasticizer degrading bacteria comprise a combination of at least five bacteria selected from bacillus glutamicum, rodokia, bacillus, Acinetobacter picepei, Gordonia phthalate and corynebacterium brassicae according to any ratio, and the plant root growth-promoting bacteria comprise at least five bacteria selected from bacillus glutamicum, rodokia, bacillus, Acinetobacter picepei, Gordonia phthalate and corynebacterium brassicae according to any ratio. The plant root system growth-promoting bacteria comprise a combination of at least three of pantoea, bacillus mucilaginosus, paenibacillus passiflora, bacillus subtilis, paenibacillus and bacillus amyloliquefaciens according to any ratio.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a preparation method and application of a pollution-eliminating and growth-promoting immobilized bacterial agent. Background Art

[0002] Phthalic Acid Esters (PAEs) are a class of plasticizers and additives mainly used in plastic films and products. They are also endocrine disruptors with carcinogenic, teratogenic and mutagenic effects. They have attracted widespread attention due to their high toxicity, persistence and bioaccumulation.

[0003] For soil contaminated by PAEs, soil remediation can be achieved through microbial degradation to promote plant growth. Specifically, plant rhizosphere growth-promoting bacteria can be fixed on a carrier through immobilization technology, and the microorganisms on the carrier can be used to remediate the soil and promote plant growth.

[0004] However, in actual applications, the existing organic pollution disinfectants have low survival rates of microorganisms due to the unsatisfactory immobilization effect of the carrier on the microorganisms, resulting in poor effects on soil remediation and promotion of plant growth. Summary of the invention

[0005] The present invention provides a preparation method and application of a pollution-removing and growth-promoting immobilized bacterial agent, which can improve the immobilization effect and survival rate of plasticizer-degrading bacteria and plant root growth-promoting bacteria, thereby improving the soil remediation effect.

[0006] Immobilized materials have a large specific surface area and pore structure. Through the immobilization operation, bacteria can be adsorbed on the surface or inside of the material, thereby improving the survival rate of bacteria.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a decontamination-promoting immobilized bacterial agent, comprising: preparing a composite bacterial community, wherein the composite bacterial community comprises a combination of plasticizer-degrading bacteria and plant root growth-promoting bacteria in any ratio. The plasticizer-degrading bacteria comprises a combination of at least five of Bacillus glutamicum, Rhodokia, Bacillus, Acinetobacter piti, Gordonia phthalic acid, and Corynebacterium brassicae in any ratio, and the plant root growth-promoting bacteria comprises a combination of at least three of Pantoea, Bacillus mucilaginosus, Paenibacillus Barcelona, ​​Bacillus subtilis, Paenibacillus, and Bacillus amyloliquefaciens in any ratio. An immobilized carrier of the composite bacterial community is prepared. The composite bacterial community is fixed on the immobilized carrier of the composite bacterial community to obtain an immobilized bacterial agent.

[0009] It can be understood that any combination of the above-mentioned plasticizer degrading bacteria and plant root growth promoting bacteria in any ratio does not include a ratio of zero, and any combination of the above-mentioned at least five bacteria in any ratio and any combination of the above-mentioned at least three bacteria in any ratio do not include a ratio of zero.

[0010] In an implementation of the first aspect, plasticizer-degrading bacteria are activated to obtain activated plasticizer-degrading bacteria, and the activated plasticizer-degrading bacteria include at least the following five types: activated Bacillus glutamicum, activated Rhodokia, activated Bacillus, activated Acinetobacter piti, activated Gordonia phthalic acid, and activated Corynebacterium brassicae. Plant root growth-promoting bacteria are activated to obtain activated plant root growth-promoting bacteria, and the activated plant root growth-promoting bacteria include at least the following three types: activated Pantoea, activated Bacillus colloidus, activated Paenibacillus Barcelona, ​​activated Bacillus subtilis, activated Paenibacillus, and activated Bacillus amyloliquefaciens. The activated plasticizer-degrading bacteria and the activated plant root growth-promoting bacteria are cultured and fermented respectively to obtain the fermentation liquid of each bacteria. The fermentation liquid of each bacteria contained in the composite bacterial community is mixed to obtain a liquid composite bacterial community.

[0011] In an implementation of the first aspect, the activated plasticizer-degrading bacteria and the activated plant root growth-promoting bacteria are cultured and fermented respectively to obtain a fermentation liquid of each bacteria, including:

[0012] According to the inoculation amount of 1% to 5% by volume, the activated Gordonia phthalic acid, the activated Corynebacterium brassicae, the activated Pantoea and the activated Paenibacillus are inoculated into a tryptone soybean culture medium respectively; the activated Paenibacillus barcelonae, the activated Bacillus subtilis and the activated Bacillus amyloliquefaciens are inoculated into a nutrient broth culture medium respectively; the activated Bacillus mucilaginosus is inoculated into a potassium bacterial culture medium; the activated Bacillus glutamicum, the activated Rhodoxia, the activated Bacillus and the activated Acinetobacter piti are inoculated into an LB culture medium respectively.

[0013] The activated Bacillus glutamicum, the activated Rhodoxia, the activated Bacillus, the activated Acinetobacter piti, the activated Gordonia phthalic acid, the activated Corynebacterium brassicae, the activated Pantoea, the activated Bacillus colloids, the activated Paenibacillus Barcelona, ​​the activated Bacillus subtilis, the activated Paenibacillus and the activated Bacillus amyloliquefaciens are cultured to the logarithmic growth phase to obtain the fermentation broth of Gordonia phthalic acid, the fermentation broth of Corynebacterium brassicae, the fermentation broth of Pantoea, the fermentation broth of Paenibacillus, the fermentation broth of Paenibacillus Barcelona, ​​the fermentation broth of Bacillus subtilis, the fermentation broth of Bacillus amyloliquefaciens, the fermentation broth of Bacillus glutamicum, the fermentation broth of Rhodoxia, the fermentation broth of Bacillus and the fermentation broth of Acinetobacter piti.

[0014] In one implementation of the first aspect, the formula of the tryptone soy medium includes: tryptone, soytone, and NaCl in a mass ratio of 15.0:5.0:5.0. The formula of the nutrient broth medium includes: peptone, beef extract, and NaCl in a mass ratio of 10.0:3.0:5.0. The formula of the potassium bacterial medium includes: sucrose, K2HPO4, yeast extract, and MgCl2 in a mass ratio of 10.0:0.5:0.4:0.2. The formula of the LB medium includes: peptone, yeast extract powder, and NaCl in a mass ratio of 10.0:5.0:10.0.

[0015] In one implementation of the first aspect, a liquid composite bacterial community is obtained by mixing the fermentation broth of each bacterium, including: mixing the fermentation broths of any five bacteria in the plasticizer-degrading bacteria according to a volume ratio of 0.8-1.2: 0.8-1.2: 0.8-1.2: 0.8-1.2 to obtain a plasticizer-degrading composite bacterial fermentation broth. Mixing the fermentation broths of any three bacteria in the plant root growth-promoting bacteria according to a volume ratio of 0.8-1.2: 0.8-1.2: 0.8-1.2 to obtain a plant root growth-promoting composite bacterial fermentation broth. Mixing the plasticizer-degrading composite bacterial fermentation broth with the plant root growth-promoting composite bacterial fermentation broth according to a volume ratio of 0.9-1.1: 0.9-1.1 to obtain a liquid composite bacterial community.

[0016] In one implementation of the first aspect, preparing an immobilized carrier of a composite bacterial flora comprises: drying, crushing and sieving rice straw, soybean straw, sawdust and rice husk respectively to obtain rice straw powder, soybean straw powder, sawdust powder and rice husk powder. Mixing the rice straw powder, soybean straw powder, sawdust powder and rice husk powder in a mass ratio of 4-5:4-5:1-3:1-3 to obtain a mixture, adding diatomaceous earth at 5% by weight of the mixture and mixing well to obtain an immobilized carrier of a composite bacterial flora.

[0017] In an implementation of the first aspect, the composite bacterial community is fixed on an immobilized carrier of the composite bacterial community to obtain an immobilized bacterial agent, including: mechanically mixing and fixing the liquid composite bacterial community and the immobilized carrier of the composite bacterial community according to a bacterial material ratio of 15 to 20 mL: 1 g to obtain the immobilized bacterial agent.

[0018] In a second aspect, the present invention provides an application of the pollution-eliminating and growth-promoting immobilized bacterial agent prepared by the above-mentioned preparation method, and the pollution-eliminating and growth-promoting immobilized bacterial agent is applied to repair plasticizer-contaminated soil and / or promote the growth of plants in plasticizer-contaminated soil.

[0019] In an implementation of the second aspect, the application achieves the repair of plasticizer-contaminated soil and / or promotes plant growth in plasticizer-contaminated soil by adding the immobilized bacterial agent prepared by the above-mentioned preparation method to plasticizer-contaminated soil.

[0020] The beneficial effects of the present invention are:

[0021] The present invention constructs a composite bacterial community through twelve kinds of bacteria including Bacillus glutamicum, Rhodokia, Bacillus, Acinetobacter piti, Gordonia phthalic acid, Corynebacterium brassicae, Pantoea, Bacillus mucilaginosus, Paenibacillus barcelonae, Bacillus subtilis, Paenibacillus and Bacillus amyloliquefaciens among plant root growth-promoting bacteria; at the same time, an immobilized carrier is prepared by using rice straw, corn straw, wood chips and rice husk, and then the composite bacterial community is loaded on the immobilized carrier by using adsorption immobilization operation, so as to prepare an immobilized bacterial agent, which has better practicality and extensiveness compared with immobilization using other materials.

[0022] The composite immobilization material prepared by the present invention has good mechanical strength, providing good attachment conditions for the bacterial community, and its raw materials (rice straw, corn straw, wood chips, rice husks) can provide nutrition for it, thereby improving the survival rate of the bacterial community, so as to achieve long-term and efficient utilization of the immobilized bacterial agent.

[0023] The immobilized bacterial agent prepared by the present invention has a strong degradation effect on plasticizers in the soil, and can achieve a removal effect of 77.19%-87.37% in plasticizer-contaminated soil with a total plasticizer (T-PAEs) concentration of 2.853 mg / kg, a removal rate of 83.56%-88.50% for DBP, and a removal rate of 75.16%-94.30% for DEHP.

[0024] The immobilized bacterial agent prepared by the present invention contains plant root growth-promoting bacteria, which have strong abilities of dissolving phosphorus, potassium, fixing nitrogen, producing IAA (indole-3-acetic acid) and producing siderophores, and can promote the development of plant roots and stems and leaves by synthesizing plant hormones (such as indole-3-acetic acid); can enhance the absorption and utilization of nutrients (such as nitrogen, phosphorus and potassium) in the soil by plants, and can directly convert nitrogen into a form available to plants through nitrogen fixation, thereby promoting the growth of plants in plasticizer-contaminated soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a flow chart of a method for preparing a pollution-eliminating and growth-promoting immobilized bacterial agent provided in an embodiment of the present application;

[0026] Figure 2 is a scanning electron microscope image of the bacterial agent 1 provided in the embodiment of the present application;

[0027] Figure 3 is a scanning electron microscope image of the bacterial agent 2 provided in the embodiment of the present application;

[0028] Figure 4 is a scanning electron microscope image of the bacterial agent 3 provided in the embodiment of the present application;

[0029] Figure 5 is a scanning electron microscope image of the bacterial agent 4 provided in the embodiment of the present application;

[0030] Figure 6 It is a schematic diagram of the plant height of water spinach in the greenhouse water spinach potted experiment provided in the embodiment of the present application;

[0031] Figure 7 It is a schematic diagram of the root length of water spinach in the greenhouse water spinach potted experiment provided in the embodiment of the present application;

[0032] Figure 8 This is a schematic diagram of the dry weight of water spinach in the greenhouse water spinach potted experiment provided in the embodiment of the present application;

[0033] Fig. 9 It is a schematic diagram of the removal rate of PAEs in plasticizer-contaminated soil in the greenhouse water spinach pot experiment provided in the examples of the present application. DETAILED DESCRIPTION

[0034] In the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0035] Phthalic Acid Esters (PAEs) are a type of plasticizer, among which dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethyl)hexyl phthalate (DEHP), and dioctyl phthalate (DnOP) are typical PAEs. PAEs can enter the soil environment through sewage irrigation and the use of plastic films. Due to their high n-octanol-water partition coefficient (Kow), they are easily adsorbed by organic matter and clay minerals in the soil, and easily accumulated in the soil, causing soil pollution. They can then be absorbed by crops and enter the human body through the food chain, endangering human health.

[0036] Plant growth promoting rhizobacteria (PGPR) refers to a type of bacteria that lives freely in the soil or is attached to plant roots and can promote plant growth and its absorption and utilization of mineral nutrients; it can activate soil organic and inorganic nutrients, decompose organic matter, release nutrients, and increase the effectiveness of nutrients; it promotes healthy plant growth through multiple mechanisms such as fixing nitrogen, secreting plant hormones, enhancing disease resistance, and improving soil conditions.

[0037] At present, plasticizer-degrading bacteria and plant rhizosphere growth-promoting bacteria can be immobilized by immobilization technology to obtain immobilized bacterial agents, and the immobilized bacterial agents can be applied to the contaminated soil to achieve the remediation of contaminated soil. Existing immobilization methods include adsorption, cross-linking, embedding and covalent bonding; adsorption as the main immobilization method has the advantages of simplicity, easy operation and low cost; the currently widely used immobilization technology mainly uses polymer carriers (sodium alginate) and inorganic carriers (biochar).

[0038] Although there are some immobilization means in the prior art, the existing organic pollution decontamination bacteria agents have low survival rates of microorganisms and poor soil remediation effects in actual applications due to unsatisfactory immobilization effects. In order to solve the above problems, the embodiments of the present application provide a preparation method and application of a decontamination-promoting immobilized bacteria agent, which uses plasticizer-degrading bacteria and plant root growth-promoting bacteria: Bacillus glutamicum, Rhodoxia, Bacillus, Acinetobacter piti, Gordonia phthalic acid, Corynebacterium brassicae, Pantoea, Bacillus mucilaginosus, Paenibacillus Barcelona, ​​Bacillus subtilis, Paenibacillus and Bacillus amyloliquefaciens, a total of twelve bacteria, to construct a composite bacterial community in proportion, and load the composite bacterial community onto an immobilized carrier to prepare an immobilized bacterial agent, which can improve the immobilization effect and survival rate of plasticizer-degrading bacteria and plant root growth-promoting bacteria, thereby improving the soil remediation effect.

[0039] For example, Figure 1As shown, a method for preparing a decontamination-promoting immobilized bacterial agent provided in an embodiment of the present application includes S101-S103.

[0040] S101, prepare composite bacterial flora;

[0041] The composite bacterial community includes plasticizer-degrading bacteria and plant root growth-promoting bacteria; the composite bacterial community includes a combination of plasticizer-degrading bacteria and plant root growth-promoting bacteria in any ratio; the plasticizer-degrading bacteria include a combination of at least five of Bacillus glutamicum, Rhodoxia, Bacillus, Acinetobacter piti, Gordonia phthalic acid, and Corynebacterium brassicae in any ratio, and the plant root growth-promoting bacteria include a combination of at least three of Pantoea, Bacillus mucilaginosus, Paenibacillus barcelonae, Bacillus subtilis, Paenibacillus spp., and Bacillus amyloliquefaciens in any ratio;

[0042] It can be understood that the combination of the above-mentioned plasticizer degrading bacteria and plant root growth promoting bacteria in any ratio does not include a ratio of zero, and the combination of any ratio of the above-mentioned at least five bacteria and the combination of any ratio of the above-mentioned at least three bacteria do not include a ratio of zero; for example, when the ratio of the above-mentioned plasticizer degrading bacteria and plant root growth promoting bacteria is 0:1, the above-mentioned composite bacterial community only includes plant root growth promoting bacteria, and when the ratio of the above-mentioned plasticizer degrading bacteria and plant root growth promoting bacteria is 0:0, the above-mentioned composite bacterial community does not exist; when the ratio of the above-mentioned at least five bacteria is 0:0:1:1:1, the above-mentioned at least five bacteria only include three bacteria; when the ratio of the above-mentioned at least three bacteria is 0:0:1, the above-mentioned at least three bacteria only include one bacteria; it can be seen that the above-mentioned ratios are not reasonable, and therefore, the above-mentioned three combinations do not include a ratio of zero;

[0043] The above-mentioned plasticizer-degrading bacteria were purchased from the China Industrial Microbiological Culture Collection Center, the China Type Culture Collection Center and the China General Microbiological Culture Collection Center; the specific collection numbers are: Gordonia phthalic acid (CICC24107), Corynebacterium brassicae (CICC24108), Acinetobacter pituitii (CICC10911); Rhodoxia (CCTCC: M2015056); Bacillus glutamicum (CCTCC M20221850); Bacillus (CGMCC NO.26265); the above-mentioned plant root growth-promoting bacteria were purchased from the China Agricultural Microbiological Culture Collection Center; the specific collection numbers are: Pantoea (ACCC60226), Bacillus mucilaginosus (ACCC19749), Paenibacillus Barcelona (ACCC19468), Bacillus subtilis (ACCC19743), Paenibacillus (ACCC60252) and Bacillus amyloliquefaciens (ACCC60428);

[0044] It is understandable that the above-mentioned plasticizer degrading bacteria and the above-mentioned plant root growth promoting bacteria may also adopt other bacterial species in the prior art, and are not limited to the twelve bacterial species given above;

[0045] Specifically, Pantoea, Paenibacillus, and Bacillus mucilaginosus have strong nitrogen fixation, siderophore production, phosphorus solubilization, and IAA (indole-3-acetic acid) production capabilities; Bacillus subtilis has the ability to solubilize phosphorus and potassium, which can promote the growth of crop roots; Paenibacillus Barcelona has nitrogen fixation; Bacillus amyloliquefaciens has the ability to fix nitrogen and produce IAA (indole-3-acetic acid);

[0046] Optionally, S101 includes S1011-S1013;

[0047] S1011, activating plasticizer-degrading bacteria to obtain activated plasticizer-degrading bacteria; activating plant root growth-promoting bacteria to obtain activated plant root growth-promoting bacteria;

[0048] Among them, the activated plasticizer degrading bacteria include at least the following five types: activated Bacillus glutamicum, activated Rhodoxia, activated Bacillus, activated Acinetobacter piti, activated Gordonia phthalic acid, activated Corynebacterium brassicae. The activated plant root growth-promoting bacteria include at least three types: activated Pantoea, activated Bacillus colloids, activated Paenibacillus Barcelona, ​​activated Bacillus subtilis, activated Paenibacillus, activated Bacillus amyloliquefaciens;

[0049] In one application scenario, single colonies of Bacillus glutamicum, Rhodoxia, Bacillus, Acinetobacter piti, Gordonia phthalimide, Corynebacterium brassicae, Pantoea, Bacillus mucilaginosus, Paenibacillus Barcelona, ​​Bacillus subtilis, Paenibacillus and Bacillus amyloliquefaciens were picked from a solid plate and inoculated into 20 ml of LB liquid medium (sterilized for 20 min at 121 ° C), and cultured under shaking conditions of 30 ° C and 150 r / min for 24 h to obtain a bacterial suspension, and each bacterial suspension was centrifuged at 8000 r / min for 5 min, the supernatant was removed, 10 ml of MSM solution was added, and the bacteria were re-shaken, and the above operation was repeated 2-3 times, and the bacteria were collected, and the single bacterial liquid of each of the above twelve bacteria was obtained after the bacterial suspension was conditioned with MSM solution to OD600 = 0.9-1.1;

[0050] Among them, the formula of LB liquid culture medium is: yeast extract (Yeast extract): 5.0g; tryptone (Tryptone): 10.0g; sodium chloride (NaCl): 10.0g; add ultrapure water to 1L, adjust pH = 7.0, and sterilize at 121°C for 20 minutes; LB solid culture medium is based on LB liquid culture medium and then add 1.5% (W / V) agar powder; the above-mentioned MSM (MinimalSalt Medium) solution is a basic salt culture medium. Since the basic salt culture medium belongs to a common culture medium in the technical field, the formula of the basic salt culture medium is not described in detail in the present application embodiment;

[0051] S1012, culturing and fermenting the activated plasticizer-degrading bacteria and the activated plant root growth-promoting bacteria respectively to obtain fermentation liquid of each bacteria;

[0052] In the embodiment of the present application, the activated Gordonia phthalic acid, the activated Corynebacterium brassicae, the activated Pantoea and the activated Paenibacillus were inoculated into a tryptone soybean medium at an inoculation volume ratio of 1% to 5%; the activated Paenibacillus Barcelona, ​​the activated Bacillus subtilis and the activated Bacillus amyloliquefaciens were inoculated into a nutrient broth medium; the activated Bacillus mucilaginosus was inoculated into a potassium bacterial culture medium; the activated Bacillus glutamicum, the activated Rhodoxia, the activated Bacillus and the activated Acinetobacter piti were inoculated into LB medium respectively;

[0053] Respectively culturing activated Bacillus glutamicum, activated Rhodokia, activated Bacillus, activated Acinetobacter piti, activated Gordonia phthalic acid, activated Corynebacterium brassicae, activated Pantoea, activated Bacillus colloids, activated Paenibacillus Barcelona, ​​activated Bacillus subtilis, activated Paenibacillus and activated Bacillus amyloliquefaciens at 28-32° C. and 160-200 r / min to a logarithmic growth phase to obtain fermentation broth of Gordonia phthalic acid, fermentation broth of Corynebacterium brassicae, fermentation broth of Pantoea, fermentation broth of Paenibacillus, fermentation broth of Paenibacillus Barcelona, ​​fermentation broth of Bacillus subtilis, fermentation broth of Bacillus amyloliquefaciens, fermentation broth of Bacillus colloids, fermentation broth of Bacillus glutamicum, fermentation broth of Rhodokia, fermentation broth of Bacillus and fermentation broth of Acinetobacter piti;

[0054] The formula of the tryptone soy medium includes: tryptone, soytone, and NaCl in a mass ratio of 15.0:5.0:5.0;

[0055] The formula of the nutrient broth medium includes: peptone, beef extract, and NaCl in a mass ratio of 10.0:3.0:5.0; the formula of the potassium bacterial medium includes: sucrose, K2HPO4, yeast extract, and MgCl2 in a mass ratio of 10.0:0.5:0.4:0.2; the formula of the LB medium includes: peptone, yeast extract powder, and NaCl in a mass ratio of 10.0:5.0:10.0;

[0056] S1013, mixing the fermentation broth of each bacteria included in the composite bacterial community to obtain a liquid composite bacterial community;

[0057] In the embodiment of the present application, the liquid composite bacterial community includes a plasticizer-degrading composite bacterial fermentation liquid and a plant root growth-promoting composite bacterial fermentation liquid;

[0058] Specifically, the fermentation liquids of any five kinds of the plasticizer-degrading bacteria are mixed according to a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2:0.8-1.2:0.8-1.2 to obtain a plasticizer-degrading composite bacteria fermentation liquid;

[0059] Optionally, the volume ratio may be 1:1:1:1:1, or 0.8:0.8:1.2:1.2:1.2, or 1.2:1.2:1.2:0.8:0.8, which is not limited in the embodiments of the present application;

[0060] Mixing fermentation liquids of any three kinds of the plant root growth-promoting bacteria in a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2 to obtain a plant root growth-promoting composite bacteria fermentation liquid;

[0061] Optionally, the volume ratio may be 1:1:1, or 0.8:1.2:1.2, or 1.2:0.8:0.8, which is not limited in the embodiment of the present application;

[0062] The plasticizer-degrading composite bacteria fermentation liquid and the plant root growth-promoting composite bacteria fermentation liquid are mixed at a volume ratio of 0.9 to 1.1:0.9 to 1.1 to obtain a liquid composite bacterial community;

[0063] Optionally, the volume ratio may be 1:1, 0.9:1.1, or 1.1:0.9, which is not limited in the embodiment of the present application;

[0064] It can be understood that the fermentation liquid of any five bacteria mentioned above refers to the fermentation liquid of five non-repetitive bacteria selected from plasticizer-degrading bacteria. The selection combination of the fermentation liquid of any five bacteria mentioned above is shown in Table 1. The embodiment of the present application does not limit the selection of any five bacteria mentioned above.

[0065] Table 1: Example 1 of selection combination of fermentation broth

[0066]

[0067] Note: “√” means the fermentation broth of this bacteria is selected as one of the fermentation broths of any five bacteria mentioned above;

[0068] The fermentation liquid of any three bacteria mentioned above refers to the fermentation liquid of three non-repetitive bacteria selected from plant root growth-promoting bacteria. The selection combination of the fermentation liquid of any three bacteria mentioned above is shown in Table 2. The embodiment of the present application does not limit the selection of the fermentation liquid of any three bacteria mentioned above;

[0069] Table 2: Example 2 of selection combination of fermentation broth

[0070]

[0071]

[0072] Note: “√” means the fermentation broth of this bacteria is selected as one of the fermentation broths of any three bacteria mentioned above;

[0073] S102, preparing an immobilized carrier of a composite bacterial flora;

[0074] Optionally, S102 includes S1021-S1022;

[0075] S1021, drying, crushing and sieving rice straw, soybean straw, sawdust and rice husk respectively to obtain rice straw powder, soybean straw powder, sawdust powder and rice husk powder;

[0076] S1022, mixing rice straw powder, soybean straw powder, sawdust powder and rice husk powder in a mass ratio of 4-5:4-5:1-3:1-3 to obtain a mixture, adding 5% by weight of diatomaceous earth relative to the weight of the mixture, mixing well, sterilizing at 121° C. for 20 min, cooling, and drying to room temperature to obtain an immobilized carrier of the composite bacterial flora;

[0077] It should be noted that the embodiment of the present application does not limit the execution order of the above S101 and S102. S101 may be executed first and then S102, or S102 may be executed first and then S101, or S101 and S102 may be executed simultaneously.

[0078] S103, fixing the composite bacterial community on an immobilized carrier of the composite bacterial community to obtain an immobilized bacterial agent;

[0079] In the embodiment of the present application, the liquid composite bacterial community and the immobilized carrier of the composite bacterial community are mechanically mixed and fixed at a bacterial-to-material ratio of 15 to 20:1 to obtain an immobilized bacterial agent;

[0080] Specifically, according to a bacterial-to-material ratio of 15 to 20:1, the liquid composite bacterial community and the immobilized carrier of the composite bacterial community are mechanically mixed and immobilized in an oscillating incubator at 28 to 32°C and a rotation speed of 160 to 200 r / min for 36 to 48 hours. The obtained material is centrifuged and filtered at a rotation speed of 1000 r / min for 5 minutes, and rinsed with physiological saline; the above operation is repeated three times, and after removing the supernatant, the solid material is taken out and placed in a 30°C oven for drying.

[0081] The present application also provides an application of the decontamination-promoting immobilized bacterial agent prepared by the above preparation method in repairing plasticizer-contaminated soil and promoting plant growth in plasticizer-contaminated soil, that is, applying the decontamination-promoting immobilized bacterial agent to repair plasticizer-contaminated soil and / or promote the growth of plants in plasticizer-contaminated soil. Exemplarily, the application achieves repairing plasticizer-contaminated soil and promoting plant growth in plasticizer-contaminated soil by adding the immobilized bacterial agent prepared by the above preparation method to plasticizer-contaminated soil.

[0082] Among them, plasticizer pollutants in plasticizer-contaminated soil include six PAEs: dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP), di (2-ethyl)hexyl phthalate (DEHP), and dioctyl phthalate (DnOP).

[0083] In one application scenario, four liquid composite bacterial communities (composite bacterial community 1, composite bacterial community 2, composite bacterial community 3, composite bacterial community 4) are prepared.

[0084] The activated plasticizer-degrading bacteria in composite bacterial community 1 include Bacillus glutamicum, Rhodoxella, Bacillus, Acinetobacter pituitii, and Gordonia phthalic acid, and the plant root growth-promoting bacteria include Pantoea, Bacillus mucilaginosus, and Bacillus subtilis.

[0085] The activated plasticizer-degrading bacteria in composite bacterial community 2 include Bacillus glutamicum, Rhodoxella, Bacillus, Acinetobacter pituitii, and Gordonia phthalic acid, and the plant root growth-promoting bacteria include Bacillus mucilaginosus, Bacillus pasteurianus, and Bacillus spp.

[0086] The activated plasticizer-degrading bacteria in composite bacterial community 3 include Bacillus glutamicum, Rhodoxella, Acinetobacter pituitii, Gordonia phthalic acid, and Corynebacterium brassicae, and the plant root growth-promoting bacteria include Pantoea, Bacillus mucilaginosus, and Bacillus subtilis.

[0087] The activated plasticizer-degrading bacteria in composite bacterial community 4 include Bacillus glutamicum, Rhodoxia, Acinetobacter pituitarius, Gordonia phthalic acid and Corynebacterium brassicae, and the plant root growth-promoting bacteria include Bacillus mucilaginosus, Bacillus pasteurianus and Bacillus spp.

[0088] Among them, the plant root growth-promoting bacteria in the above-mentioned composite bacterial community 1, composite bacterial community 2, composite bacterial community 3 and composite bacterial community 4 are compounded according to the principle of complementary growth-promoting abilities, so that the compounded composite bacterial community has the ability to solubilize phosphate, dissolve potassium, fix nitrogen, produce IAA (indole-3-acetic acid) and produce iron carriers.

[0089] The above-mentioned liquid composite bacterial communities (composite bacterial community 1, composite bacterial community 2, composite bacterial community 3, composite bacterial community 4) were mixed with immobilized carriers at a bacterial-material ratio of 15:1, and immobilized in an oscillating incubator at 30°C and a rotation speed of 200 r / min for 48 hours; the materials were then centrifuged and filtered at a rotation speed of 1000 r / min for 5 minutes, rinsed with physiological saline, and the above operation was repeated 3 times. After removing the supernatant, the solid material was taken out and placed in a 30°C oven for drying; bacterial agent 1 (corresponding to the above-mentioned composite bacterial community 1), bacterial agent 2 (corresponding to the above-mentioned composite bacterial community 2), bacterial agent 3 (corresponding to the above-mentioned composite bacterial community 3), and bacterial agent 4 (corresponding to the above-mentioned composite bacterial community 4) were obtained.

[0090] The scanning electron microscopy image of bacterial agent 1 is as follows Figure 2 As shown; the scanning electron microscope image of bacterial agent 2 is as shown Figure 3 As shown; the scanning electron microscope image of bacterial agent 3 is as follows Figure 4 As shown; the scanning electron microscope image of bacterial agent 4 is as shown Figure 5 shown.

[0091] Depend on Figure 2 to Figure 5 It can be seen that immobilized materials have a large specific surface and pore volume, which can provide a good living space for microorganisms and are conducive to their growth and reproduction. Rough surfaces can usually provide more attachment points and a larger surface area, making it easier for microorganisms to attach. In addition, tiny depressions or pores on the surface can form a microenvironment, promote the accumulation of water and nutrients, and thus improve the attachment and growth of microorganisms.

[0092] In the above application scenario, water spinach is used as a plant in the soil contaminated by plasticizers. Optionally, other plants can also be used as plants in the soil contaminated by plasticizers, and the present application embodiment is not limited thereto.

[0093] The specific application process is described below.

[0094] 1. Incubation of water spinach seeds:

[0095] The water spinach seeds were soaked in 3% H2O2 solution to sterilize the surface, then rinsed with sterile water and placed in a 30°C constant temperature incubator for 48 hours. Water spinach seeds with consistent saturation were selected as test seeds.

[0096] 2. Greenhouse water spinach potted experiment:

[0097] The test soil was collected from the surface layer (0-20cm) of a vegetable field contaminated by plasticizers; the soil pH value was 6.38±0.11, and the soil bulk density was 1.37N / m 3 ; The soil samples were air-dried and then sieved through a 20-mesh sieve for later use; the above-prepared decontamination-promoting immobilized bacterial agent was added to a 400g soil sample pot at a dosage of 5%, and 6 water spinach seeds with the same saturation were selected and planted in the treated pots. During the growth of the plants, the plants were watered regularly and the positions of the pots were randomly exchanged every 2 days. During the experiment, the temperature was set at 25℃ during the day and 20℃ at night, and the relative humidity was 50-60%; each treatment had 3 parallels.

[0098] 30 days after the water spinach inoculation, the plants were collected, the plant surface was thoroughly washed with sterile water, and the plants were placed on the surface of filter paper to dry. The plant height (e.g. Figure 6 ), root length (as shown), Figure 7 At the same time, the fresh plant samples were placed in a freeze dryer and freeze-dried for 72 hours to obtain their dry weight (as shown in Figure 8 The specific data of plant height, root length and dry weight of each experimental group are shown in Table 3. Figure 6 to Figure 8 a, b, and c represent the significant differences among the groups.

[0099] Table 3: Plant height, root length and dry weight data

[0100] Treatment Group Plant height(cm) Root length (cm) Dry weight (mg) blank 20.13±2.8c 5.61±0.99b 59.83±8.95b Sterilization materials 24.58±4.29bc 5.66±1.27b 61.11±5.09b Bacterial Agent 1 30.52±6.34a 7.74±2.77a 85±10.93a Bacterial Agent 2 26.19±5.19ab 7.33±1.52a 65±8.89b Bacterial Agent 3 25.15±4.26bc 6.41±1.41ab 65.5±11.3b Bacterial Agent 4 27.22±5.13ab 6.98±1.24ab 64.25±2.47b

[0101] Note: The letters (a, b, c) in Table 3 indicate the significant differences among the groups.

[0102] As can be seen from Table 3, the height, root length and dry weight of the water spinach plants in the treatment group with the anti-pollution and growth-promoting immobilized bacterial agent were significantly improved compared with the water spinach plants without the application of bacterial agents and the water spinach plants with the inactivated bacterial agent. Among them, the application of bacterial agents 1 and 2 had a significant effect on promoting the growth of water spinach.

[0103] 3. Determination of plasticizer content in plasticizer-contaminated soil

[0104] After the greenhouse water spinach pot experiment (the total plasticizer T-PAEs concentration before the experiment was 2.853 mg / kg), the soil samples were placed in a freeze dryer for drying, 2 g of the prepared soil sample was placed in a 20 mL glass screw bottle, 15 mL of a 1:1 volume ratio of n-hexane-acetone solution was added, the bottle cap was tightly closed and placed on a vortex oscillator for 0.5 to 1 min, and after sufficient oscillation, it was transferred to an ultrasonic cleaner and extracted in a water bath at 100 Hz and room temperature for 45 min. The sample was then placed in a centrifuge and centrifuged at 2000 r / min for 10 min to take the supernatant; the supernatant was then poured into an active The column was eluted with 10 mL of n-hexane twice beforehand, the upper layer of the column was 2 g of anhydrous sodium sulfate, the lower layer was 2 g of silica gel (300 mesh), a 100 mL rotary evaporation bottle was placed under the column, and after elution, a total volume of 15 mL of a n-hexane-acetone solution with a volume ratio of 9:1 was added three times; the extract and the eluent were collected in a 100 mL rotary evaporation bottle, and then the collected liquid was concentrated to dryness on a rotary evaporator at 40° C. and 100 r / min, and 2 mL of methanol was transferred to rinse and make up the volume; then poured into a syringe with a 0.22 μm organic phase filter, filtered into a 2 mL liquid phase vial, and analyzed using HPLC-UV.

[0105] Chromatographic conditions: LC-20AT high performance liquid chromatograph (equipped with SPD-2A ultraviolet detector) was used. The detection time was 40 min, and the injection volume of the injection system was 20 μL; the separation system used acetonitrile-water as the mobile phase, the initial flow rate was 1.0 mL / min, and gradient elution was used to separate PAEs. The chromatographic column was a Φ4.6×250 mm Inertsil ODS-P liquid chromatography column, and the column temperature was 40°C; the detection system used an ultraviolet detector and turned on the dual wavelength detection mode, which were 205 nm and 225 nm respectively.

[0106] The final result is as follows Fig. 9 As shown by Fig. 9 It can be seen that the immobilized bacterial agent prepared by the present invention has a strong degradation effect on plasticizers in the soil. For plasticizer-contaminated soil with an initial total plasticizer (T-PAEs) concentration of 2.853 mg / kg, a total plasticizer (T-PAEs) removal effect of 77.29%-87.37% can be achieved, and a removal rate of 83.56%-88.50% can be achieved for DBP in the soil, and a removal rate of 75.16%-94.30% can also be achieved for DEHP.

[0107] In summary, the immobilized bacterial agent prepared in the examples of the present application can effectively degrade PAEs in plasticizer-contaminated soil, and can also promote the growth of plants in plasticizer-contaminated soil.

[0108] It should be noted that the various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a pollution-eliminating and growth-promoting immobilized bacterial agent, characterized in that: include: A composite bacterial community is prepared, wherein the composite bacterial community includes a combination of plasticizer-degrading bacteria and plant root growth-promoting bacteria in any ratio; the plasticizer-degrading bacteria include a combination of at least five of Bacillus glutamicum, Rhodokia, Bacillus, Acinetobacter piti, Gordonia phthalic acid, and Corynebacterium brassicae in any ratio, and the plant root growth-promoting bacteria include a combination of at least three of Pantoea, Bacillus mucilaginosus, Paenibacillus barcelonae, Bacillus subtilis, Paenibacillus spp., and Bacillus amyloliquefaciens in any ratio; preparing an immobilized carrier of the composite bacterial community; The composite bacterial community is fixed on an immobilization carrier of the composite bacterial community to obtain an immobilized bacterial agent.

2. The method according to claim 1, characterized in that The composite bacterial flora preparation comprises: Activating the plasticizer-degrading bacteria to obtain activated plasticizer-degrading bacteria, wherein the activated plasticizer-degrading bacteria include at least five of the following: activated Bacillus glutamicum, activated Rhodoxia, activated Bacillus, activated Acinetobacter pituitii, activated Gordonia phthalic acid, and activated Corynebacterium brassicae; Activating the plant root growth-promoting bacteria to obtain activated plant root growth-promoting bacteria, wherein the activated plant root growth-promoting bacteria include at least the following three types: activated Pantoea, activated Bacillus mucilaginosus, activated Paenibacillus barcelonae, activated Bacillus subtilis, activated Paenibacillus sp., and activated Bacillus amyloliquefaciens; Cultivating and fermenting the activated plasticizer-degrading bacteria and the activated plant root growth-promoting bacteria respectively to obtain fermentation liquid of each bacteria; The fermentation liquids of the bacteria contained in the composite bacterial consortium are mixed to obtain a liquid composite bacterial consortium.

3. The method according to claim 1, characterized in that The activated plasticizer-degrading bacteria and the activated plant root growth-promoting bacteria are cultured and fermented respectively to obtain fermentation liquid of each bacteria, including: According to the inoculation amount of 1-5% by volume, the activated Gordonia phthalic acid, the activated Corynebacterium brassicae, the activated Pantoea and the activated Paenibacillus are inoculated into the tryptone soybean culture medium respectively; the activated Paenibacillus barcelonae, the activated Bacillus subtilis and the activated Bacillus amyloliquefaciens are inoculated into the nutrient broth culture medium respectively; the activated Bacillus mucilaginosus is inoculated into the potassium bacterial culture medium; the activated Bacillus glutamicum, the activated Rhodoxia, the activated Bacillus and the activated Acinetobacter piti are inoculated into the LB culture medium respectively; The activated Bacillus glutamicum, the activated Rhodoxia, the activated Bacillus, the activated Acinetobacter piti, the activated Gordonia phthalic acid, the activated Corynebacterium brassicae, the activated Pantoea, the activated Bacillus colloids, the activated Paenibacillus Barcelona, ​​the activated Bacillus subtilis, the activated Paenibacillus and the activated Bacillus amyloliquefaciens are cultured to the logarithmic growth phase to obtain the fermentation broth of Gordonia phthalic acid, the fermentation broth of Corynebacterium brassicae, the fermentation broth of Pantoea, the fermentation broth of Paenibacillus, the fermentation broth of Paenibacillus Barcelona, ​​the fermentation broth of Bacillus subtilis, the fermentation broth of Bacillus amyloliquefaciens, the fermentation broth of Bacillus glutamicum, the fermentation broth of Rhodoxia, the fermentation broth of Bacillus and the fermentation broth of Acinetobacter piti.

4. The method according to claim 3, characterized in that The formula of the tryptone soy culture medium includes: tryptone, soytone, and NaCl in a mass ratio of 15.0:5.0:5.0; The formula of the nutrient broth medium includes: peptone, beef extract, and NaCl in a mass ratio of 10.0:3.0:5.0; The formula of the potassium bacterial culture medium includes: sucrose, K2HPO4, yeast extract, and MgCl2 in a mass ratio of 10.0:0.5:0.4:0.2; The formula of the LB culture medium includes: peptone, yeast extract powder and NaCl in a mass ratio of 10.0:5.0:10.

0.

5. The method according to claim 2, characterized in that The method of mixing the fermentation broths of the bacteria contained in the composite bacterial community to obtain a liquid composite bacterial community comprises: Mixing fermentation broths of any five kinds of the plasticizer-degrading bacteria in a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2:0.8-1.2:0.8-1.2 to obtain a plasticizer-degrading composite bacteria fermentation broth; Mixing fermentation liquids of any three kinds of the plant root growth-promoting bacteria in a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2 to obtain a plant root growth-promoting composite bacteria fermentation liquid; The plasticizer-degrading composite bacteria fermentation liquid and the plant root growth-promoting composite bacteria fermentation liquid are mixed at a volume ratio of 0.9-1.1:0.9-1.1 to obtain a liquid composite bacterial community.

6. The method according to claim 1 or 2, characterized in that: The method for preparing the immobilized carrier of the composite bacterial community comprises: After drying, crushing and sieving rice straw, soybean straw, sawdust and rice husk respectively, rice straw powder, soybean straw powder, sawdust powder and rice husk powder are obtained; The rice straw powder, soybean straw powder, sawdust powder and rice husk powder are mixed in a mass ratio of 4-5:4-5:1-3:1-3 to obtain a mixture, and 5% diatomaceous earth is added and mixed to obtain an immobilized carrier of the composite bacterial flora.

7. The method according to claim 2, characterized in that The method of fixing the composite bacterial community on an immobilized carrier of the composite bacterial community to obtain an immobilized bacterial agent comprises: The liquid composite bacterial community and the immobilized carrier of the composite bacterial community are mechanically mixed and fixed according to a bacterial material ratio of 15-20 mL: 1 g to obtain the immobilized bacterial agent.

8. An application of the decontamination-promoting immobilized bacterial agent prepared by the method according to any one of claims 1 to 7, characterized in that: The decontamination-growth-promoting immobilized bacterial agent is used to repair plasticizer-contaminated soil and / or promote the growth of plants in plasticizer-contaminated soil.

Images