Immobilized microbial agent with carbon sequestration and decontamination capabilities as well as preparation method and application of immobilized microbial agent

Abstract

The invention provides an immobilized microbial agent with carbon sequestration and decontamination capabilities as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The immobilized microbial agent is prepared by taking bacillus glutamicum, bacillus, haematococcus, carbon sequestration ammoxidation archaea and carbon sequestration bacterium acetobacter xylinum as raw materials and taking charcoal as a carrier, and has efficient degradation capacity on dimethyl phthalate, diethyl phthalate, dibutyl phthalate, butyl benzyl phthalate and the like; and the soil carbon sequestration capability can be enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to an immobilized bacterium agent with carbon fixation and pollution elimination capabilities, a preparation method thereof, and an application thereof. Background Art

[0002] In the face of a complex climate environment, measures need to be taken to formulate a green development strategy. Achieving carbon neutrality means achieving a balance between greenhouse gas emissions and carbon sinks, which is a crucial step towards the goal of keeping the global average surface temperature within 2 °C of the pre-industrial level. In addition to focusing on structural reforms and energy conservation and emission reduction in human society, it is necessary to attach importance to the carbon neutrality role of natural ecosystems. Soil carbon storage exceeds 80% of the global terrestrial carbon storage and is the largest carbon pool in terrestrial ecosystems. Minor changes in its carbon budget will significantly affect the atmospheric CO2 concentration. As an important part of terrestrial ecosystems, the farmland ecosystem has dual characteristics of a carbon source and a carbon sink. Regulating carbon fixation and emission reduction in farmland soil is of great significance for ensuring food security and achieving "carbon neutrality". Currently, the main carbon fixation methods include chemical carbon fixation, physical carbon fixation, and biological carbon fixation. In biological carbon fixation, in addition to the carbon fixation of plants, the assimilation and synthesis of soil microorganisms play a positive role in the accumulation of the soil carbon pool, and microbial residue carbon is an important component of the soil stable organic carbon pool. In addition, as a key driving factor in the soil carbon cycle, using microorganisms to fix CO2 is a way with low energy consumption, economic feasibility, and green pollution-free, which can provide new ideas for enhancing soil carbon fixation potential and reducing atmospheric CO2 concentration. According to different nutritional modes, carbon-fixing microorganisms are divided into heterotrophic microorganisms and autotrophic microorganisms. Heterotrophic microorganisms use organic carbon compounds as carbon sources and achieve carbon fixation through carboxylation reactions in their own metabolic processes; autotrophic microorganisms use CO2 as the sole carbon source and use photosynthesis or chemosynthesis to convert CO2 into their own cell substances to achieve photosynthetic CO2 fixation. Among them, ammonia-oxidizing archaea (AOA) grow chemoautotrophically using CO2 as a carbon source. AOA can couple the carbon fixation process with ammonia oxidation. First, electrons are obtained from NH 4+ and are transferred through the electron transport chain to convert the chemical energy stored in NH 4+ into the energy currency ATP, and then through the 3-hydroxypropionate / 4-hydroxybutyrate cycle (3-HP / 4-HB cycle), ATP is consumed to fix CO2 and synthesize organic matter. Related research shows that AOA is more energy-efficient in fixing carbon through the cycle than any other such pathway, which provides an explanation for its greater competitive advantage under oligotrophic conditions.

[0003] Phthalates are a type of environmental hormone compound, often used as plasticizers in plastics, resins, and rubber products. The addition amount in plastic products is generally 20 - 60%. The extensive use of agricultural films in agricultural production has led to the entry of phthalates into the protected agriculture environment, where they migrate and transform, causing pollution to the protected agriculture environment. There have been many studies on the pollution status of phthalates in soil, sediment, water bodies, and food packaging, etc., but there are few systematic studies on the pollution characteristics of phthalates in the protected agriculture environment, and there is a lack of attention to the environmental risks caused by phthalates in the protected agriculture environment. Through methods such as literature research, surface investigation sampling, and laboratory sample analysis, etc., analyze the current pollution status of phthalate organic pollutants in the protected agriculture environment from aspects such as pollutant sources, distribution characteristics, and bioaccumulation; use the research results and analysis data of risk assessment to evaluate the environmental health risks of phthalates in the protected agriculture environment, and point out the deficiencies in the current research on phthalate pollution in the protected agriculture environment. The research results show that the average total content of the six priority-controlled phthalates in the soil of protected vegetable fields is 0.47 - 8.80 mg·kg -1 , and in some areas, the total amount of the six phthalates in the surface soil of protected vegetable fields is as high as 30 mg·kg -1 ; the phthalate pollutants in the protected agriculture environment mainly include diethyl phthalate (DEP), dibutyl phthalate (DnBP), bis(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DnOP).

[0004] The environmental pollution and food safety problems caused by phthalates (PAEs) have attracted global attention. The environmental pollution problem cannot be ignored. By investigating the pollution status of 6 priority-controlled PAE pollutants in farmland soil, analyzing their sources, and focusing on elaborating the absorption and accumulation characteristics of different types of crops for PAE compounds and the biological toxicity effects of PAE pollutants. Atmospheric deposition, agricultural films, application of sludge, and sewage irrigation are the main sources of PAEs in agricultural soil. Different crops have significant differences in the absorption, accumulation, and distribution characteristics of PAEs. PAEs not only affect soil quality, crop growth, and physiological and biochemical properties, but also have a bioaccumulation effect. There are still deficiencies in the current research on PAEs. The scope of PAE pollution investigation can be expanded, the poisoning mechanism of PAEs on crops can be deeply revealed, and in-situ remediation technologies for PAE-polluted soil can be mainly developed. Summary of the Invention

[0005] The technical problem solved by the present invention is: to provide an immobilized bacterium agent with carbon fixation and pollution elimination capabilities, its preparation method and application. This immobilized bacterium agent can efficiently remove PAEs in the environment and increase the soil carbon fixation amount.

[0006] To solve the above problems, the technical solution of the present invention is as follows: The present invention provides an immobilized bactericide with carbon fixation and pollution elimination capabilities. The immobilized bactericide is prepared from a bacterial suspension obtained by mixing the bacterial solutions of Glutamicibacter, Bacillus, Rhodobacter, ammonia-oxidizing archaea, and Acetobacter xylinum in equal volumes as raw materials, using biochar as a carrier. The solid-liquid ratio of the biochar to the bacterial suspension is 1-2 g: 20 mL.

[0007] Note: The above method can fully exert the functional characteristics of each specific strain in carbon fixation and pollution elimination through the synergistic action of multiple specific strains, improving the overall treatment efficiency; biochar as a carrier can not only provide a stable attachment environment for the strains, enhance their activity and stability, but also has a certain ability to adsorb pollutants, further strengthening the pollution elimination effect. At the same time, this solution helps to achieve the dual goals of carbon fixation and pollutant removal, and has broad application prospects and important value in the field of environmental protection governance.

[0008] The present invention also provides a preparation method of the above immobilized bactericide, including the following steps: S1. Respectively take 500 μL of Glutamicibacter, Bacillus, and Rhodobacter strains and transfer them to 100 mL of LB medium each, and culture them for 12-48 h at a temperature of 30 °C and an oscillation speed of 100-200 rpm. Then centrifuge them at a speed of 8000 rcf in a centrifuge for 2-10 min to obtain the cultured Glutamicibacter bacterial solution, Bacillus bacterial solution, and Rhodobacter bacterial solution; then transfer 500 μL of the Acetobacter xylinum strain to 100 mL of acetic acid bacteria medium and statically culture it for 12-48 h at 30 °C. Then centrifuge it at a speed of 8000 rcf in a centrifuge for 2-10 min to obtain the cultured Acetobacter xylinum bacterial solution; S2. Wash the cultured Glutamicibacter bacterial solution, Bacillus bacterial solution, Rhodobacter bacterial solution, and Acetobacter xylinum bacterial solution twice with inorganic salt medium, and then adjust the OD 600 value to 1.0 to obtain a preparation, and temporarily store it for later use at an environmental temperature of 4 °C; take the Nitrososphaera viennensis strain and continuously culture it in the dark in a constant temperature incubator at 42 °C until the logarithmic growth phase is reached to obtain a single bacterial solution of ammonia-oxidizing archaea, and temporarily store it at a temperature of 4 °C. S3. Mix the obtained Glutamicibacter bacterium solution, Bacillus bacterium solution, Rhodobacter bacterium solution, Acetobacter xylinum bacterium solution, and ammonia-oxidizing archaea bacterium solution in an equal-volume ratio to obtain a microbial community suspension. Mix biochar and the microbial community suspension at a solid-liquid ratio of 1 - 20 g : 1 mL, place it in a constant-temperature shaking incubator, culture it at 30°C at a rotation speed of 150 - 200 rpm for 12 - 48 h, and then take it out. Subsequently, centrifuge it at a rate of 8000 rpm in a centrifuge for 5 - 10 min to obtain a supernatant and a lower-layer solid. Discard the supernatant, then wash the lower-layer solid with a sterilized inorganic salt medium, and centrifuge it for 5 - 10 min again and discard the supernatant; repeat the process of washing and discarding the supernatant 2 - 3 times to obtain a solid to be dried. Place the solid to be dried in a sterilized oven and dry it at a temperature of 25 - 30°C to obtain an immobilized bacterium agent.

[0009] Note: The above preparation method can adopt a differential culture method according to the characteristics of different strains to ensure the good growth and reproduction of various strains and guarantee the activity and quality of the bacterium solution; by means of cleaning and adjusting the OD 600 value (the OD 600 value refers to the absorbance value of a certain solution at a wavelength of 600 nm) and other steps to standardize the treatment of the bacterium solution, making the states of each bacterium solution unified, which is conducive to subsequent precise mixing; mixing the bacterium solutions in an equal-volume ratio to construct a microbial community suspension to ensure a reasonable microbial community structure and give full play to the synergistic effect; mixing biochar and the microbial community suspension for culture at a specific solid-liquid ratio to allow the strains to fully adhere to the biochar carrier, and subsequent operations such as centrifugation, washing, and drying further purify and immobilize the strains, which not only effectively removes impurities but also enables the strains to stably adhere to the carrier. The finally prepared immobilized bacterium agent has high activity and a stable structure, and has good efficacy and reliability in applications such as carbon fixation and pollution elimination.

[0010] Further, the method for adjusting the OD 600 value of bacteria to 1.0 is as follows: If the OD 600 value of bacteria is greater than 1.0, dilute the bacterium solution with MSM until the OD 600 value of bacteria is 1.0; if the OD 600 value of bacteria is less than 1.0, concentrate the bacterial cells by centrifugation and then resuspend them until the OD 600 value of bacteria is 1.0.

[0011] Note: Through the above method, the bacterium solutions with different initial concentrations can be quickly and accurately adjusted to a unified OD 600 value, providing a bacterium solution with consistent concentration and stable state for subsequent experiments or applications, and guaranteeing the reliability of experimental results and the consistency of application effects.

[0012] The present invention also provides an application of the immobilized bacterium agent, using the immobilized bacterium agent for degrading phthalate esters and increasing the soil carbon fixation amount.

[0013] Furthermore, the phthalate ester is dimethyl phthalate and / or diethyl phthalate and / or dibutyl phthalate and / or butyl benzyl phthalate and / or (2-ethyl)hexyl phthalate and / or di-n-octyl phthalate.

[0014] Note: The above immobilized microbial agent has a wide degradation spectrum, can act on a variety of phthalate ester pollutants, effectively cope with pollution problems in complex environments, helps to comprehensively improve the soil environmental quality, and has broad application prospects and significant environmental benefits in the treatment of soil pollution and the improvement of soil carbon sequestration capacity.

[0015] Furthermore, the immobilized microbial agent is added to the soil contaminated with phthalate ester and left standing for 24 - 72 h.

[0016] Note: The above application method is simple to operate, does not require complex equipment and additional energy consumption, reduces the application cost while ensuring the degradation efficiency, is suitable for large-scale soil pollution remediation projects, and has good economic efficiency and operability.

[0017] Furthermore, the application amount of the immobilized microbial agent is 1 - 5% of the soil quality.

[0018] Note: Within 72 hours after adding 1 - 5% of the soil quality of the solid microbial agent, six phthalate esters are significantly degraded. The degradation rates of short-chain phthalate esters all exceed 98%, and the degradation rates of long-chain phthalate esters can also reach more than 75%, indicating that the solid microbial agent has high degradation ability for six PAEs. And indicators such as total soil carbon and organic matter increase by about 10%, indicating that the solid microbial agent has the effect of increasing the soil carbon sequestration amount.

[0019] The beneficial effects of the present invention are as follows: The present invention provides a solid functional microbial agent with the ability to degrade phthalate esters and fix carbon dioxide. The microorganisms in the microbial agent can use dimethyl phthalate, diethyl phthalate, dibutyl phthalate, butyl benzyl phthalate, (2-ethyl)hexyl phthalate and di-n-octyl phthalate as the sole carbon source and energy source for growth and reproduction. Under the conditions of pot experiments, the solid microbial agent can almost completely degrade 50 mg / L of mixed phthalate esters in the soil, and the degradation rates all exceed 75%. And indicators such as total soil carbon and organic matter increase by about 10%. The application of the present invention has great prospects in the biological treatment of environmental pollutants. Description of the Drawings

[0020] Figure 1 It is a graph of the degradation data of phthalate esters by the solid microbial agent in Example 1; Figure 2 For Example 213 C / 12 Percentage of C atoms, data graph of the amount of assimilated CO2 in SOC; Figure 3 Data graph of the change in organic matter content after the application of the solid microbial agent in Example 3; Figure 4 Data graph of the degradation effect in the soil after the application of the solid microbial agent in Example 3; Figure 5 Data graph of the phthalate content in lettuce after the application of the solid microbial agent in Example 3; Figure 6 SEM images of the surfaces of rice charcoal (a, b), corn charcoal (c, d), and wheat charcoal (e, f) magnified 500 times in the examples. Detailed implementation manners

[0021] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "the", and "said" used in the embodiments of the present invention and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise. "Plural" generally includes at least two.

[0023] Example 1: This example is an immobilized microbial agent with carbon fixation and pollution elimination capabilities. The immobilized microbial agent is prepared from Glutamicibacter, Bacillus, Rhodobacter, ammonia-oxidizing archaea, and Acetobacter xylinum as raw materials and biochar as a carrier; The preparation method of the above-mentioned immobilized microbial agent includes the following steps: S1. Respectively take 500 μL of Glutamicibacter, Bacillus, and Rhodobacter strains and transfer them to 100 mL of LB medium each, and culture them for 24 h in an environment with a temperature of 30 °C and an oscillation speed of 150 rpm. Then centrifuge at a speed of 8000 rcf for 5 min in a centrifuge to obtain the cultured Glutamicibacter liquid, Bacillus liquid, and Rhodobacter liquid; then transfer 500 μL of the Acetobacter xylinum strain to 100 mL of acetic acid bacteria medium, and statically culture it for 24 h at 30 °C. Then centrifuge at a speed of 8000 rcf for 5 min in a centrifuge to obtain the cultured Acetobacter xylinum liquid; S2. After washing the cultured liquid of Glutamicibacter, Bacillus, Rhodobacter, and Acetobacter xylinum with the inorganic salt medium twice, adjust the OD 600 value of the liquid to 1.0 to obtain a preparation, and temporarily store it for later use at an environmental temperature of 4°C; Take the Nitrososphaera viennensis strain and continuously culture it in the dark in an incubator at 42°C until the logarithmic growth phase to obtain a single strain liquid of ammonia-oxidizing archaea, and temporarily store it at a temperature of 4°C for later use; Use the glycerol preservation method for preservation; among them, the operation steps of the glycerol preservation method are: mix the bacterial community with glycerol with a volume concentration of 30% at a volume ratio of 1:1, and store it in a refrigerator at -80°C for later use; The above inorganic salt medium is composed of 1.5 g / L of (NH4)2SO4, 0.5 g / L of KH2PO4, 1.91 g / L of K2HPO4·3H2O, 0.5 g / L of NaCl, and 0.2 g / L of MgSO4·7H2O; the pH is 7.0; the above LB medium includes the following components: 5.0 g of yeast extract, 10.0 g of tryptone, and 10.0 g of sodium chloride; add 1 L of ultrapure water and adjust the pH to 7.0, sterilize at 121°C for 20 - 25 min. The acetic acid bacteria medium includes the following components: 5.0 g of polypeptone, 5.0 g of yeast extract, 5.0 g of glucose, 5.0 g of mannitol, 1.0 g of MgSO4·7H2O, 5.0 mL of ethanol, add 1 L of ultrapure water and adjust the pH to 6.6 - 7.0, sterilize at 121°C for 20 - 25 min, and then add ethanol after sterilization is completed; The above Acetobacter xylinum was purchased from Beijing Bio-win Biotechnology Co., Ltd. and preserved in the China General Microbiological Culture Collection Center (CGMCC); the isolation source is wild fruits on the mountain; the normal culture time of the strain is about 2 days, and there will be a lag period of 5 - 7 days after the freeze-dried strain is revived or transferred again after being placed in the refrigerator for a long time. Determine the culture time according to the actual cellulose film production situation; the collection number of Rhodobacter is CCTCC M2015056; the collection number of Glutamicibacter is CCTCC M20221850; the collection number of Bacillus is CGMCC No.16233; the collection number of ammonia-oxidizing archaea is DSM 26422 = JCM 19564; The inorganic salt medium and LB medium prepared according to the above formula can better process the microbial liquid and effectively reduce the presence of inclusions, making the degradation rate of the solid microbial agent higher; S3. Add the ammonia-oxidizing archaea single-strain bacterial liquid to the above-mentioned preparation according to the equal-volume ratio of the glutamic acid bacillus bacterial liquid, the bacillus bacterial liquid, the rhodobacter sphaeroides bacterial liquid, the acetobacter xylinum bacterial liquid, and the ammonia-oxidizing archaea single-strain bacterial liquid to obtain a microbial suspension. Mix the biochar and the microbial suspension according to the solid-liquid ratio of 1 g:10 mL, place it in a constant-temperature shaking incubator, cultivate it at 30 °C at a rotation speed of 180 rpm for 24 h, then take it out, and then centrifuge it at a rate of 8000 rpm in a centrifuge for 8 min to obtain a supernatant and a lower-layer solid. Discard the supernatant, then wash the lower-layer solid with a sterilized inorganic salt medium, and centrifuge it for 8 min again and discard the supernatant; repeat the process of washing and discarding the supernatant 3 times to obtain the solid to be dried. Place the above-mentioned solid to be dried in a sterilized oven and dry it at a temperature of 28 °C to obtain an immobilized bactericide; The biochar is wheat / corn / rice straw biochar prepared at 600 °C; The biochar has a relatively high specific surface area. Selecting wheat straw, a farmland waste, to burn into biochar for characterization data is better. The biochar can provide a habitat and nutrients for microorganisms, improve the microbial living environment, and enhance the activity of functional bacteria. At the same time, the free radicals of the biochar burned at 600 °C are qualified and the yield is relatively high (the surface electron micrographs of the biochars obtained from the three straws in the present invention are as shown in Figure 6 ), and the degradation rate of the obtained immobilized bactericide is also relatively high; Using the method of gradient heating (specifically, for example, the method of gradient heating is that before the temperature is less than 200 °C, the heating rate is 15 °C / min; when the temperature is in the range of 200-400 °C, the heating rate is 8 °C / min, and when the temperature is in the range of 400-600 °C, the heating rate is 4 °C / min) to prepare the carbonized crude product can make the crushed straw gradually melt, and the biochar can be mechanically mixed into the straw in layers, making the mixture between the crushed straw and the biochar in the furnace more uniform; at the same time, the gradient heating can ensure that the raw materials in the crucible can be heated and melted step by step, and can effectively avoid damage caused by too high temperature during one-time heating; The immobilized carrier prepared by the above method is more environmentally friendly and low-cost; The method for adjusting the OD 600 value of the bacteria to 1.0 is as follows: If the OD 600 value of the bacteria is greater than 1.0, dilute the bacterial liquid with MSM until the OD 600 value of the bacteria is 1.0; If the OD 600 value of the bacteria is less than 1.0, concentrate the bacterial cells by centrifugation and then resuspend them until the OD 600 value of the bacteria is 1.0; An LC-20AT high performance liquid chromatograph (equipped with an SPD-2A ultraviolet detector) was used, the detection time was 40 min, and the injection volume of the injection system was 20 μL; in the separation system, acetonitrile-water was used 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 chromatographic column, and the column temperature was 40 °C; in the detection system, an ultraviolet detector was used for detection, and the dual-wavelength detection mode was turned on, which were 205 nm and 225 nm respectively; 0.1 g of the prepared carbon-fixing and pollution-reducing solid bacterial agent was added to 20 mL of MSM culture solution containing PAEs at a concentration of 50 mg / L (i.e., containing 50 mg / L DMP, 50 mg / L DEP, 50 mg / L DBP, 50 mg / L BBP, 50 mg / L DEHP, and 50 mg / L DnOP). Without adding the solid bacterial agent as a control, and the pH was adjusted to 7.0. Each group had three replicates. The samples were taken at 1 d, 3 d, 5 d, and 7 d after culturing in a constant temperature shaker at 30 °C and 150 rpm for 7 d. 40 mL of chromatographically pure methanol was added to the taken conical flask, and the mixture was ultrasonically shaken in a water bath for 1 h; after the ultrasonic treatment, it was vortexed and shaken well, and the supernatant was filtered through a 0.22 μm organic phase filter membrane and transferred into a 2 mL brown liquid phase vial, and then detected by a high performance liquid chromatograph; The degradation effect of the carbon-fixing and pollution-reducing solid bacterial agent on six kinds of mixed PAEs is as Figure 1 shown; Figure 1 In it, LMW-PAEs represents low molecular weight plasticizers DMP, DEP, DBP, HMW-PAEs represents high molecular weight plasticizers BBP, DEHP, DnOP, Total-PAEs represents total plasticizers. With the passage of time, the removal rate of high molecular weight plasticizers gradually increases, while the degradation rate of low molecular weight plasticizers has been stable above 90% from the beginning; During the above performance measurement process, DMP is the abbreviation of phthalic acid ester, DEP is the abbreviation of diethyl phthalate, DBP is the abbreviation of dibutyl phthalate, BBP is the abbreviation of butyl benzyl phthalate, DEHP is the abbreviation of bis(2-ethylhexyl) phthalate, and DnOP is the abbreviation of di-n-octyl phthalate.

[0024] Example 2: The solid bacterial agent obtained by using the wheat straw prepared by the method of Example 1 of the present invention as a raw material to prepare activated carbon was used to treat the soil to verify the carbon fixation ability of the solid bacterial agent; The experimental device was a 100 mL serum bottle. Sterile water was added to the soil until the water content was 60%, and three parallels were set for each treatment; among them, six kinds of mixed PAEs were added to the tested soil to make the final concentration 50 mg / kg; each device contained 10 g of soil; 13The CO2 content was 5%. After 90% of the CO2 was consumed, the top of the serum bottle was first rinsed with pressurized synthetic air (20% O2, 80% N2) for 1 min to maintain aerobic conditions, and then CO2 was added. 13 The microcosm culture system was placed at 30 °C for cultivation. The gas in the serum bottle was renewed every 7 days. All soil samples were collected at 7 d, 14 d, and 21 d for freeze-drying. The dried samples were immediately stored at -20 °C for subsequent analysis. The freeze-dried samples were measured for the δ 13 C values and SOC of the labeled and unlabeled soils for subsequent 13 C / 12 C atomic number percentage, the amount of assimilated CO2 in SOC, and the carbon sequestration efficiency analysis; The results are as Figure 2 shown; Figure 2 Among them, CK is the control group (i.e., the soil without adding any microbial agents), F1 is the microbial liquid treatment group (i.e., the soil only added with microbial liquid and without adding biochar treatment), F2 is the inactivated microbial agent treatment group (the soil treated with inactivated immobilized microbial agent), SD is the rice straw immobilized microbial agent treatment group (the soil treated with the combination of biochar prepared from rice straw and microbial liquid), XM is the wheat straw immobilized microbial agent treatment group (the soil treated with the combination of biochar prepared from wheat straw and microbial liquid), and YM is the corn straw immobilized microbial agent treatment group (the soil treated with the combination of biochar prepared from corn straw and microbial liquid); Among the treatment groups, the inactivated microbial agent F1 had the worst effect, indicating that microorganisms play an important role in carbon sequestration. The overall effect of the free microbial liquid was inferior to that of the microbial agent treatment, probably because the loose and porous structure of the biochar material provided sufficient attachment sites for the growth and function of microorganisms, which was beneficial to the expression of microbial functions. The effects of the three microbial agent treatment groups were: wheat ≈ corn > rice, which may be related to the different structures and element contents of different straw materials itself. The above experiments showed that the carbon sequestration and pollution reduction solid microbial agent prepared by the preparation method of the present invention could reduce the stress of six mixed PAEs on Nitrososphaeraviennensis and achieve a good carbon sequestration effect.

[0025] Example 3: This example is about the application of a group of solid functional microbial agents with phthalate degradation ability and carbon dioxide fixation ability. 500 g of farmland soil that had been pre-polluted and aged for one month was added to the pot, and the straw immobilized solid microbial agent was applied at a rate of 1%. Italian lettuce was planted, and the total cultivation period was 50 days. During this period, soil and vegetable samples were collected to measure the contents of six mixed PAEs in the soil and vegetables, and the physical and chemical properties indexes and enzyme activity indexes of the soil were measured. The treatment groups included: the tested soil CK, the bacterial community F1, and the inactivated bacterial agent F2; The implementation groups included: the immobilized bacterial agent (rice 1:10, the solid-liquid ratio of biochar obtained from rice straw to the bacterial community suspension was 1 g:10 mL) SD1, the immobilized bacterial agent (wheat 1 g:10 mL) XM1, the immobilized bacterial agent (corn 1 g:10 mL) YM1, the immobilized bacterial agent (rice 1 g:20 mL) SD2, the immobilized bacterial agent (wheat 1 g:20 mL) XM2, and the immobilized bacterial agent (corn 1 g:20 mL) YM2; The implementation results were as Figure 3 、 Figure 4 、 Figure 5 shown. Figure 3 Among them, the SOC in the XM2 and YM2 treatment groups was the highest, indicating that they had the best carbon sequestration ability. There was little difference among the other treatment groups, and there was no significant difference; Figure 4 The treatment group with the best degradation effect was selected: the XM2 treatment group. Its 40-day degradation rate of six phthalates was relatively high. The degradation rate of low-molecular-weight phthalates could exceed 90%, and the degradation rate of high-molecular-weight phthalates could also exceed 70%; Figure 5 In it were the remaining phthalate contents in the soil at 20, 30, and 40 days. The XM2 group could reduce the remaining phthalate content to about 4 mg / kg, showing a certain advantage compared with other treatment groups.

[0026] Example 4: The difference from Example 1 was that the application method of the solid bacterial agent was: The solid bacterial agent was inoculated into the potted soil contaminated with phthalate, and the application amount was 3% of the soil quality.

[0027] Example 5: The difference from Example 4 was that the application method was: The solid bacterial agent was inoculated into the soil contaminated with phthalate, and the application amount of the preparation was 5% of the soil quality.

[0028] Example 6: The difference from Example 1 was that the above immobilized bacterial agent was added to the soil contaminated with phthalate and left to stand for 48 h; the application amount of the above immobilized bacterial agent was 1% of the soil quality.

[0029] Example 7: The difference from Example 1 was that in S1, it was cultured in an environment with a shaking speed of 100 rpm for 48 h, and then centrifuged at a speed of 8000 rcf in a centrifuge for 2 min to obtain the cultured Glutamicibacter liquor, Bacillus liquor, and Rhodobacter liquor; then 500 μL of the strain Acetobacter xylinum was transferred to 100 ml of the acetic acid bacteria medium and statically cultured at 30 °C for 12 h, and then centrifuged at a speed of 8000 rcf in a centrifuge for 2 min to obtain the cultured Acetobacter xylinum liquor; In S3, according to the equal - volume ratio of Glutamicibacter sp. bacterial liquid, Bacillus sp. bacterial liquid, Rhodobacter sphaeroides bacterial liquid, Acetobacter xylinum bacterial liquid, and ammonia - oxidizing archaea single - strain bacterial liquid, the ammonia - oxidizing archaea single - strain bacterial liquid was added to the above - mentioned preparation to obtain a microbial community suspension. The biochar was mixed with the microbial community suspension according to a solid - to - liquid ratio of 1 g:1 mL, and placed in a constant - temperature shaking incubator. After culturing at 30 °C at a rotation speed of 150 rpm for 12 h, it was taken out. Subsequently, it was centrifuged at a rate of 8000 rpm in a centrifuge for 5 min to obtain a supernatant and a lower - layer solid. The supernatant was discarded, and then the lower - layer solid was washed with a sterilized inorganic salt medium, and then centrifuged for 5 min and the supernatant was discarded. The process of washing and discarding the supernatant was repeated 2 times to obtain the solid to be dried. The above - mentioned solid to be dried was placed in a sterilized oven and dried at a temperature of 25 °C to obtain the immobilized microbial agent.

[0030] Example 8: The difference from Example 1 is that in S1, it was cultured for 12 h in an environment with a shaking speed of 200 rpm, and then centrifuged at a rotation speed of 8000 rcf in a centrifuge for 10 min to obtain the cultured Glutamicibacter sp. bacterial liquid, Bacillus sp. bacterial liquid, and Rhodobacter sphaeroides bacterial liquid; then 500 μL of Acetobacter xylinum strain was transferred to 100 ml of acetic acid bacteria medium and statically cultured at 30 °C for 48 h, and then centrifuged at a rotation speed of 8000 rcf in a centrifuge for 10 min to obtain the cultured Acetobacter xylinum bacterial liquid. In S3, according to the equal - volume ratio of Glutamicibacter sp. bacterial liquid, Bacillus sp. bacterial liquid, Rhodobacter sphaeroides bacterial liquid, Acetobacter xylinum bacterial liquid, and ammonia - oxidizing archaea single - strain bacterial liquid, the ammonia - oxidizing archaea single - strain bacterial liquid was added to the above - mentioned preparation to obtain a microbial community suspension. The biochar was mixed with the microbial community suspension according to a solid - to - liquid ratio of 20 g:1 mL, and placed in a constant - temperature shaking incubator. After culturing at 30 °C at a rotation speed of 200 rpm for 48 h, it was taken out. Subsequently, it was centrifuged at a rate of 8000 rpm in a centrifuge for 10 min to obtain a supernatant and a lower - layer solid. The supernatant was discarded, and then the lower - layer solid was washed with a sterilized inorganic salt medium, and then centrifuged for 10 min and the supernatant was discarded. The process of washing and discarding the supernatant was repeated 2 - 3 times to obtain the solid to be dried. The above - mentioned solid to be dried was placed in a sterilized oven and dried at a temperature of 25 - 30 °C to obtain the immobilized microbial agent.

[0031] Example 9: The difference from Example 1 is that the above - mentioned immobilized microbial agent was added to the soil contaminated with phthalate esters and left standing for 72 h.

[0032] Example 10: The difference from Example 1 is that the above - mentioned immobilized microbial agent was added to the soil contaminated with phthalate esters and left standing for 24 h.

[0033] Comparative Example 1: Different from Example 1, four free microorganisms (equal amounts of Glutamicibacter, Bacillus, Rhodobacter sphaeroides, Gordonia phthalatica (Gram-positive bacteria)) with high PAEs degradation ability were combined with Gluconacetobacter for the determination of the degradation ability of six mixed PAEs (dimethyl phthalate, diethyl phthalate, dibutyl phthalate, butyl benzyl phthalate, (2-ethyl)hexyl phthalate, di-n-octyl phthalate); it was found in the experiment that the degradation effect was lower than that of the combination in Example 1, and the reason might be that too many microorganisms with the same metabolic pathway led to mutual competition and affected the functional expression.

[0034] Comparative Example 2: Different from Example 1, in step S3, the biochar prepared by carbonizing corn at 400 °C was selected as the immobilization carrier; the carbon fixation and pollution removal effect of the microbial agent was lower than that under the carbonization condition of 600 °C. This might be because the carbonization temperature was relatively low, the biochar structure was incomplete, and there were few attachment sites for microorganisms, affecting the colonization and functional expression of microorganisms.

[0035] Comparative Example 3: Different from Example 1, in step S3, the biochar prepared by carbonizing rice at 800 °C was selected as the immobilization carrier. The carbon fixation and pollution removal effect of the microbial agent was lower than that under the carbonization condition of 600 °C. This might be because the carbonization temperature was too high, the biochar structure was damaged, the carbonization degree was severe, and there were few attachment sites for microorganisms, affecting the colonization and functional expression of microorganisms.

[0036] Comparative Example 4: Different from Example 1, in step S3, the microbial suspension was added according to the solid-liquid ratio of 1 g:30 mL; the carbon fixation and pollution removal effect was not good. It was speculated that the reason was that the ratio was too high, the bacterial concentration was too large, there was mutual competition, and the nutrition was scarce, resulting in a low survival rate of the strains and difficulty in exerting the carbon fixation and pollution removal ability.

[0037] Comparative Example 5: Different from Example 1, in the preparation method of the immobilization carrier, the biochar material was rinsed 4 times with ultrapure water and then dried; the carbon fixation and pollution removal effect was not good. It was speculated that the reason was that some strains that had not been fully attached to the pores of the biochar material were washed away during rinsing, resulting in a decrease in the microbial density and a reduction in the carbon fixation and pollution removal effect.

Claims

1. An immobilized microbial agent with carbon fixation and pollution elimination capabilities, characterized in that, The immobilized microbial agent is prepared by using a microbial suspension obtained by mixing the bacterial solutions of Glutamicibacter, Bacillus, Rhodobacter, ammonia-oxidizing archaea, and Acetobacter xylinum in equal volumes as raw materials and using biochar as a carrier. The solid-liquid ratio of the biochar to the microbial suspension is 1-2 g: 20 mL.

2. The preparation method of an immobilized microbial agent with carbon fixation and pollution elimination ability according to claim 1, characterized in that, It includes the following steps: S1. Respectively take 500 μL of the strains of Glutamicibacter, Bacillus, and Rhodobacter and transfer them to 100 mL of LB medium each. Cultivate them at a temperature of 30 °C and an oscillation speed of 100-200 rpm for 12-48 h, and then centrifuge them in a centrifuge at a speed of 8000 rcf for 2-10 min to obtain the cultured Glutamicibacter bacterial solution, Bacillus bacterial solution, and Rhodobacter bacterial solution. Then transfer 500 μL of the strain of Acetobacter xylinum to 100 mL of acetic acid bacteria medium and statically cultivate it at 30 °C for 12-48 h, and then centrifuge it in a centrifuge at a speed of 8000 rcf for 2-10 min to obtain the cultured Acetobacter xylinum bacterial solution; S2. After washing the cultured liquid of Glutamicibacter, Bacillus, Rhodobacter, and Acetobacter xylinum twice with an inorganic salt medium, adjust the OD value of the liquid to 1.0 and store it for standby at an environmental temperature of 4°C; 600 value to 1.0 and store it for standby at an environmental temperature of 4°C; Take the Nitrososphaera viennensis strain and continuously cultivate it in the dark in a constant temperature incubator at 42 °C until the logarithmic growth phase to obtain the ammonia-oxidizing archaea bacterial solution, and temporarily store it at 4 °C for standby; S3. Mix the Glutamicibacter bacterial solution, Bacillus bacterial solution, Rhodobacter bacterial solution, Acetobacter xylinum bacterial solution, and ammonia-oxidizing archaea bacterial solution obtained in S2 in equal volumes to obtain a microbial suspension. Mix the biochar and the microbial suspension according to the solid-liquid ratio of 1-2 g: 20 mL, and place them in a constant temperature shaking incubator. Under the condition of 30 °C, cultivate them at a speed of 150-200 rpm for 12-48 h and then take them out. Subsequently, centrifuge them in a centrifuge at a speed of 8000 rpm for 5-10 min to obtain the supernatant and the lower-layer solid. Discard the supernatant, then wash the lower-layer solid with sterilized inorganic salt medium, and then centrifuge for 5-10 min and discard the supernatant; Repeat the process of washing and discarding the supernatant 2-3 times to obtain the solid to be dried. Place the solid to be dried in a sterilized oven and dry it at a temperature of 25-30 °C to obtain the immobilized microbial agent.

3. The preparation method of an immobilized microbial agent with carbon fixation and pollution elimination ability as described in claim 2, characterized in that, The method for adjusting the OD of the bacterial solution 600 value to 1.0 is as follows: If the OD of the bacterial solution 600 value is greater than 1.0, dilute the bacterial solution with MSM until the OD of the bacterial solution 600 value is 1.0; if the OD of the bacterial solution 600 value is less than 1.0, concentrate the bacterial cells by centrifugation and then resuspend them until the OD of the bacterial solution 600 value is 1.

0.

4. The preparation method of an immobilized microbial agent with carbon fixation and pollution elimination ability according to claim 2, characterized in that, The biochar raw material is one of rice straw, corn straw, and wheat straw.

5. Use of an immobilized microbial agent with carbon fixation and pollution elimination capabilities as described in claim 1, characterized in that, The immobilized microbial agent is used to degrade phthalate esters and increase soil carbon sequestration.

6. Use of an immobilized microbial agent with carbon fixation and pollution elimination capabilities as described in claim 5, characterized in that, The phthalate ester is dimethyl phthalate and / or diethyl phthalate and / or dibutyl phthalate and / or butyl benzyl phthalate and / or (2-ethyl) hexyl phthalate and / or di-n-octyl phthalate.

7. Use of an immobilized bacterium agent with carbon fixation and pollution elimination capabilities according to claim 6, characterized in that, Add the immobilized microbial agent to the soil contaminated with phthalate esters and let it stand for 24-72 h.

8. Use of an immobilized microbial agent with carbon fixation and pollution elimination capabilities according to claim 7, characterized in that, The application amount of the immobilized microbial agent is 1-5% of the soil quality.

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