Preparation and Application of a Composite Bacterial Agent with the Functions of Efficient Nitrogen Fixation, Acid Regulation and Simultaneous Arsenic Methylation

Abstract

The present invention belongs to the field of microbial technology and discloses a microbial combination, including Paraclostridium benzoelyticum YH01 and Clostridium sporogenes LHA6. The microbial combination provided by the present invention can symbiotically cooperate, can not only carry out anaerobic arsenic methylation, but also accelerate the reduction and dissolution of adsorbed and bound arsenic, continuously provide As(III) substrate for the arsenic methylation process, improve the methylation efficiency of arsenic, and reduce the toxicity of arsenic in the environment. At the same time, it can efficiently generate clean energy H2, which can be used for the development and application of new energy, and has great application potential in aspects such as improving soil fertility, producing new energy H2, increasing soil pH, and remediating arsenic-contaminated soil (such as arsenic-contaminated paddy fields and wetland environments), and acidic soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to the preparation and application of a composite microbial agent with the functions of efficient nitrogen fixation, acid regulation and synchronous arsenic methylation. Background Art

[0002] Arsenic is a toxic metalloid element widely distributed in nature and is one of the carcinogenic and teratogenic substances recognized by the World Health Organization. In water and soil environments, inorganic arsenic mainly exists, including arsenate (As(V)) and arsenite (As(III)), among which As(III) is 60 times more toxic than As(V) and more than 100 times more toxic than methylarsenic. As(V) in soil is mainly adsorbed on iron oxides. The anaerobic environment in wetlands and paddy fields accelerates the reduction and dissolution of iron oxides, resulting in the release of As(V). Through the food chain, arsenic continuously accumulates in the human body, posing a serious risk to human health. The traditional way to reduce the bioavailability of arsenic is to promote the oxidation of As(III) to As(V) under anaerobic conditions. This method can reduce the mobility of arsenic to a certain extent, but still does not relieve the toxicity of arsenic and does not reduce the proportion of inorganic arsenic. To address this technical bottleneck, a new regulation strategy is proposed: promoting the reduction and methylation of arsenic, and simultaneously promoting the volatilization of organic arsenic to reduce the total arsenic and inorganic arsenic content. Therefore, how to promote the reduction of As(V) and the methylation of As(III) is the main research difficulty in this field.

[0003] Meanwhile, arsenic-contaminated paddy fields are often accompanied by production obstacles such as soil acidification and low soil fertility, which significantly affect the productivity of cultivated land in China. In the process of regulating the migration of arsenic, being able to simultaneously improve soil acidification and solve the problem of soil fertility obstacles is of great significance for achieving efficient and sustainable soil improvement and treatment.

[0004] Microbial nitrogen fixation technology can improve the nitrogen nutrient in soil, reduce the input of chemical fertilizers, and improve soil quality. Microorganisms such as Clostridium utilize N2 in the air to be converted into NH3 under the action of nitrogenase (Reaction formula 1). This nitrogen fixation reaction is also a hydrogen evolution reaction. The key enzyme for nitrogen fixation is [Mo-Fe], and the key enzymes for hydrogen production are [Fe-Fe], [Ni-Fe] and [Fe] hydrogenases. Microorganisms reduce protons to produce hydrogen through the electron transfer of these enzymes. The active centers of these enzymes are metal ions, so adding iron minerals is beneficial to nitrogen fixation and hydrogen production. In addition, natural organic matter materials such as woody peat and biochar can act as electron shuttles to promote electron transfer and also contribute to nitrogen fixation and hydrogen production. Methionine is a methyl donor. Using composite woody peat modified with methionine as the carrier of the microbial agent is beneficial to the colonization of the microbial agent, and promotes the reduction and methylation of arsenic, and simultaneously promotes the nitrogen fixation and hydrogen production process of the microbial agent, and is expected to solve problems such as the risk control of arsenic pollution in paddy fields and wetland environments and the improvement of cultivated land fertility.

[0005] N2 + 8H + + 8e - + 16ATP → 2NH3 + H2 + 16ADP + 16Pi (1)

[0006] In view of the above problems, the present invention is specifically proposed. Summary of the Invention

[0007] An object of the first aspect of the present invention is to provide a microbial combination.

[0008] An object of the second aspect of the present invention is to provide a compound bactericide.

[0009] An object of the third aspect of the present invention is to provide a preparation method of the compound bactericide of the second aspect of the present invention.

[0010] An object of the fourth aspect of the present invention is to provide the application of the microbial combination of the first aspect of the present invention and the compound bactericide of the second aspect of the present invention.

[0011] An object of the fifth aspect of the present invention is to provide a method.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] In the first aspect of the present invention, a microbial combination is provided, including Paraclostridium benzoelyticum YH01 and Clostridium sporogenes LHA6; wherein the taxonomic name of Paraclostridium benzoelyticum YH01 is Paraclostridium benzoelyticum, and it was deposited at the Guangdong Provincial Microbial Culture Collection Center on May 30, 2024, with the deposit number: GDMCC No: 64705.

[0014] In the second aspect of the present invention, a compound bactericide is provided, including at least one of the microbial combination of the first aspect of the present invention, the culture of the microbial combination of the first aspect of the present invention, the culture supernatant of the microbial combination of the first aspect of the present invention, or the extract of the microbial combination of the first aspect of the present invention.

[0015] The active ingredients of the above bactericide can be the above-mentioned Paraclostridium benzoelyticum YH01 and Clostridium sporogenes LHA6, the cultures (such as fermentation products) of the above-mentioned Paraclostridium benzoelyticum YH01 and Clostridium sporogenes LHA6. The active ingredients of the above bactericide can also contain other biological components or non-biological components. Those skilled in the art can determine other active ingredients of the bactericide according to the effect of the bactericide.

[0016] The term "culture" refers to the collective name for liquid or solid products (all substances in the culture vessel, fermentation products) with a microbial population after artificial inoculation and cultivation. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms or can contain a certain amount of culture medium, metabolites or other components produced during the cultivation process.

[0017] In the above-mentioned bacterial agent, in addition to the active ingredient, the bacterial agent also contains a carrier. The carrier can be a commonly used and biologically inert carrier in the field (such as microorganisms, agriculture). The carrier can be a solid carrier or a liquid carrier; the solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, bean flour and starch; the polymer compound can be polyvinyl alcohol and / or polyglycol; the liquid carrier can be an organic solvent or water; the organic solvent can be decane and / or dodecane.

[0018] The composite bacterial agent is anaerobically cultured in an environment containing inorganic pentavalent arsenic to simultaneously achieve the functions of nitrogen fixation, hydrogen production and arsenic methylation, and can be applied to the improvement of acidic soil, the enhancement of cultivated land fertility, and the remediation of arsenic-polluted paddy fields and wetland environments.

[0019] In some embodiments of the present invention, the carrier includes woody peat.

[0020] In some embodiments of the present invention, the woody peat is methionine-modified woody peat.

[0021] In some embodiments of the present invention, the methionine-modified woody peat is prepared by the following method: after drying the woody peat raw material, it is crushed and sieved through a 70-90 mesh sieve, the sieved woody peat is put into a 200-300 mesh mesh bag, placed in a 1-2 mM methionine solution and soaked for adsorption for 4-8 h, preferably 6 h, taken out and dried in the air, and then the methionine-modified woody peat is obtained.

[0022] In some embodiments of the present invention, the composite bacterial agent further includes a cell immobilization embedding material and an adhesive.

[0023] In some embodiments of the present invention, the cell immobilization embedding material includes at least one of sodium alginate, sodium carboxymethylcellulose, gelatin, chitosan, potassium alginate, polyvinyl alcohol, chitin, agar.

[0024] In some preferred embodiments of the present invention, the cell immobilization material is sodium alginate.

[0025] In some preferred embodiments of the present invention, divalent and higher metal ions Cu2+ 、 Fe 3+ 、 Ca 2+ and Zn 2+ at least one in the salt solution, such as calcium chloride.

[0026] In some embodiments of the present invention, the composite microbial agent is in the shape of spherical particles with a diameter of 2 - 3 mm.

[0027] The composite microbial agent provided by the present invention mainly contains Paraclostridium benzoelyticum with the function of synchronous arsenic reduction during nitrogen fixation and hydrogen production, and Sporocladium bacterium with the function of synchronous anaerobic arsenic methylation during fermentation hydrogen production. The two strains coexist symbiotically. After co-fermenting the two strains, they are colonized on methionine composite woody peat, and mixed with a cell immobilization material (such as sodium alginate) and subjected to a cross-linking reaction to prepare a gel microbial agent composite with the function of nitrogen fixation, hydrogen production and arsenic methylation by cell immobilization and embedding. Among them, the As(III) excreted by Paraclostridium benzoelyticum after intracellular reduction of As(V) can serve as a reaction substrate for Sporocladium bacterium with arsenic methylation function, promoting the methylation process of inorganic arsenic, thereby reducing the bioavailability and biotoxicity of arsenic. In addition, Paraclostridium benzoelyticum also has a nitrogen fixation effect, which can increase the nitrogen content in paddy fields and wetland environments, and can use protons to produce hydrogen synchronously, generating hydrogen energy while alleviating soil acidification, and has potential application value in acidic soil improvement, arsenic-polluted paddy fields and wetland environment restoration and treatment.

[0028] The third aspect of the present invention provides a preparation method of the composite microbial agent of the second aspect of the present invention, including the following steps:

[0029] Inoculate the microbial combination of the first aspect of the present invention into a culture medium containing woody peat for colonization culture to obtain a composite microbial liquid;

[0030] Mix the composite microbial liquid with a cell immobilization material, drop it into a cross-linking agent solution, and carry out a cross-linking reaction to obtain a composite microbial agent.

[0031] In some embodiments of the present invention, the culture medium is a TSB culture medium, and its main formula is 16 - 18 g / L of tryptone, 2 - 4 g / L of soy peptone, 4 - 6 g / L of sodium chloride, 2 - 3 g / L of potassium dihydrogen phosphate, 2 - 3 g / L of glucose, and the balance is water, with a pH value of 6.5 - 7.2; preferably: 17 g / L of tryptone, 3 g / L of soy peptone, 5 g / L of sodium chloride, 2.5 g / L of potassium dihydrogen phosphate, 2.5 g / L of glucose, and the balance is water, with a pH value of 7.0 ± 0.2.

[0032] In some embodiments of the present invention, the conditions for the colonization culture are to culture at 28 - 35 °C for 20 - 30 h; preferably culture at 30 °C for 24 h.

[0033] In some embodiments of the present invention, the colonization culture is anaerobic culture.

[0034] In some embodiments of the present invention, in the medium, the inoculation amounts of Paraclostridium benzoelyticum YH01 and Clostridium sporogenes LHA6 are 1% to 3%, preferably 2%.

[0035] In some embodiments of the present invention, the cell immobilization embedding material is a sodium alginate solution with a mass fraction of 2% to 5%; preferably a sodium alginate solution of 3%.

[0036] In some embodiments of the present invention, the volume ratio of the compound bacterial solution to the cell immobilization embedding material is 1:1 to 2.

[0037] In some embodiments of the present invention, the crosslinking agent solution is a calcium chloride solution with a mass fraction of 1% to 5%; preferably a calcium chloride solution of 2%.

[0038] In some embodiments of the present invention, the time of the crosslinking reaction is 4 to 10 h; preferably 4 to 8 h.

[0039] In some embodiments of the present invention, stirring is carried out during the dropping process, and the rotation speed is 3 to 10 mL / min, preferably 5 to 7 mL / min.

[0040] The fourth aspect of the present invention provides the application of the microbial combination of the first aspect of the present invention and the compound bactericide of the second aspect of the present invention in at least one of (1) to (15):

[0041] (1) Reducing arsenic;

[0042] (2) Preparing a product for reducing arsenic;

[0043] (3) Producing hydrogen;

[0044] (4) Preparing a product for producing hydrogen;

[0045] (5) Nitrogen fixation;

[0046] (6) Preparing a product for nitrogen fixation;

[0047] (7) Methylation of arsenic;

[0048] (8) Preparing a product for arsenic methylation;

[0049] (9) Remediation of polluted environment;

[0050] (10) Preparing a product for remediating arsenic-polluted environment;

[0051] (11) Preparing a soil conditioner;

[0052] (12) Reducing the content of inorganic arsenic in plants;

[0053] (13) Preparing a product for reducing the inorganic arsenic content in plants;

[0054] (14) Improving the fertility of cultivated land;

[0055] (15) Preparing a product for improving the fertility of cultivated land.

[0056] In some embodiments of the present invention, the environment described in (9) to (10) is an arsenic - polluted environment.

[0057] In some embodiments of the present invention, the arsenic includes inorganic pentavalent arsenic or inorganic trivalent arsenic.

[0058] In some embodiments of the present invention, the inorganic pentavalent arsenic is arsenic acid or arsenate, and the inorganic trivalent arsenic is arsenous acid or arsenite.

[0059] In some embodiments of the present invention, the concentration of the pentavalent arsenic or trivalent arsenic is 0 - 3 mmol / L.

[0060] In some embodiments of the present invention, the environment includes but is not limited to water bodies, soil, wetlands, and sediments.

[0061] In some embodiments of the present invention, the reduction product of the reduced arsenic is As(III), such as arsenous acid or arsenite.

[0062] In some embodiments of the present invention, the arsenic methylation is to convert inorganic arsenic into monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and / or trimethylarsine (TMA).

[0063] In some embodiments of the present invention, the nitrogen fixation is to convert nitrogen gas into the inorganic nitrogen source NH4 required by itself, symbiotic strains, and crops. + 。

[0064] In some embodiments of the present invention, the hydrogen production refers to the ability to produce hydrogen using protons in the environment, alleviating soil, water body, and wetland acidification.

[0065] In some embodiments of the present invention, the plants include crops, such as rice, Chinese cabbage, etc.

[0066] The fifth aspect of the present invention provides a method, which includes using the microbial combination of the first aspect of the present invention and the compound bactericide of the second aspect of the present invention to treat the sample to be treated.

[0067] The method includes at least one of a1) to a7):

[0068] a1) A method for reducing arsenic;

[0069] a2) A method for producing hydrogen;

[0070] a3) A method for nitrogen fixation;

[0071] a4) A method for arsenic methylation;

[0072] a5) A method for the remediation of polluted environment;

[0073] a6) A method for reducing the content of inorganic arsenic in plants;

[0074] a7) A method for improving the fertility of cultivated land.

[0075] In some embodiments of the present invention, the sample to be treated includes but is not limited to arsenic-polluted water, arsenic-polluted soil, plants, inorganic pentavalent arsenic, inorganic trivalent arsenic, and the like.

[0076] The beneficial effects of the present invention are:

[0077] The microbial consortium provided by the present invention can symbiotically cooperate, can not only perform anaerobic arsenic methylation, but also accelerate the reduction and dissolution of adsorbed and bound arsenic, continuously provide As(III) substrate for the arsenic methylation process, improve the methylation efficiency of arsenic, and reduce the toxicity of arsenic in the environment. At the same time, it can efficiently generate clean energy H2, which can be used for the development and application of new energy, and has great application potential in aspects such as improving soil fertility, producing new energy H2, increasing soil pH, and remediating arsenic-polluted soil (such as arsenic-polluted paddy fields and wetland environments).

[0078] The composite microbial agent provided by the present invention can effectively consume soil protons to produce hydrogen, alleviate soil acidification, and improve soil fertility and productivity by nitrogen fixation and ammonia production. When used for soil remediation, it can effectively increase nitrogen nutrients in the soil and reduce the application amount of chemical fertilizers by a certain proportion. At the same time, it can effectively supplement the energy consumption and carbon emissions in the process of arsenic pollution remediation in soil, sewage or sludge, and has application potential in carbon-neutral arsenic pollution remediation.

[0079] Specifically, Clostridium paraputrificum consumes protons in the medium to produce hydrogen while fixing nitrogen in the soil, thereby effectively increasing the inorganic nitrogen in the soil and alleviating soil acidification. Compared with soil acidification control technologies such as lime and biochar, this technology has low energy consumption, low cost of manual spreading, and has long-term effectiveness and can continuously increase soil fertility. Once this bacterium colonizes in the soil, it can effectively fix nitrogen and reduce acid; this strain has strong arsenic reduction ability and arsenic tolerance, can quickly reduce As(V) and excrete As(III) at the same time, grows synergistically with Clostridium sporogenes that uses As(III) as a substrate for arsenic methylation, and can efficiently fix nitrogen to provide nitrogen for Clostridium sporogenes, improve its arsenic methylation efficiency, and the two symbiotically cooperate, effectively reducing the arsenic pollution risk in soil and agricultural products.

[0080] Woody peat is a natural organic matter with humus as the main component. It has a large number of active functional groups such as carboxyl, alcohol hydroxyl and phenolic hydroxyl groups, so it has high reactivity and thus affects the migration and transformation process of pollutants. Woody peat contains certain amounts of phosphorus, potassium elements and organic carbon, and is a natural organic fertilizer. Inputting it into farmland can improve the fertility of the farmland.

[0081] Methionine is an effective methyl donor that can effectively promote the methylation reaction of arsenic. When it is compounded with woody peat and used as the carrier of the strain, it can increase the ecological sites of the strain and improve the loading capacity of the strain. The composite bacterium agent prepared therefrom can effectively regulate the methylation process of arsenic, reduce the proportion of inorganic arsenic in the soil and agricultural products, realize the regulation of the arsenic pollution risk of agricultural products, and at the same time efficiently reduce acid, increase the soil pH, increase the soil organic matter and nitrogen nutrient content. Description of the Drawings

[0082] Figure 1 It is the co-culture growth curve of the composite bacterium agent.

[0083] Figure 2 It is the arsenate transformation in the TSB medium added with arsenate.

[0084] Figure 3 It is the hydrogen production amount of the composite bacterium agent in the TSB medium added with arsenate.

[0085] Figure 4 It is the scanning electron micrograph of the composite bacterium agent.

[0086] Figure 5 It is the spherical morphology diagram of the methionine-woody peat composite gel bacteria.

[0087] Figure 6 It is the methyl arsenic content diagram of the rice in the pot experiment of cadmium-arsenic co-polluted soil.

[0088] Figure 7 It is the expression abundance diagram of the arsenic methylation gene arsM after the soil is inoculated with the composite bacterium agent.

[0089] Figure 8 It is the expression abundance diagram of the nitrogen fixation gene nifH after the soil is inoculated with the composite bacterium agent.

[0090] Figure 9 It is the concentration diagram of inorganic arsenic As(III) in the pore water of the cadmium-arsenic co-polluted paddy field experiment.

[0091] Figure 10 It is the concentration diagram of inorganic arsenic As(V) in the pore water of the cadmium-arsenic co-polluted paddy field experiment.

[0092] Figure 11 It is the inorganic arsenic content diagram of the rice in the cadmium-arsenic co-polluted paddy field experiment.

[0093] Figure 12 This is the soil pH change diagram of the acidic soil improvement experiment.

[0094] Figure 13 Experimental pore water NH4 for acidic soil improvement + Concentration graph.

[0095] Figure 14 Graph of total nitrogen and alkaline nitrogen content in acidic soil improvement experiment. DETAILED DESCRIPTION

[0096] The present invention is further described in detail below through specific examples.

[0097] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0098] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0099] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0100] Paraclostridium benzoelyticum YH01 was isolated from arsenic-contaminated paddy field soil and was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province on May 30, 2024, with the deposit number: GDMCC No: 64705.

[0101] Clostridium sporogenes LHA6 was deposited at the Guangdong Microbiological Culture Collection Center (GDMCC) at Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province on January 14, 2022, with the deposit number: GDMCC No: 62212. The bacterium has been disclosed in the Chinese invention patent CN114480230A.

[0102] Example 1 Composite bacterial agent collaborative culture test

[0103] Paraclostridium benzoelyticum YH01 (preservation number: GDMCC No: 64705) and Clostridium sporogenes LHA6 (preservation number: GDMCC No: 62212) were respectively inoculated into 50 mL of high-temperature sterilized LB medium for rejuvenation, with an inoculation amount of 1% for both. After aeration with high-purity nitrogen, a butyl rubber stopper was quickly pressed on and an aluminum cap was pressed on to maintain an anaerobic environment, and then placed in an incubator and statically cultured at 30 °C for 12 h until the logarithmic phase (OD 600 = 1.0). 1 mL of each bacterial liquid was taken and inoculated into a vial containing 500 mL of high-temperature sterilized TSB medium for co-culture, and at the same time, only one bacterial liquid was used as a control. 50 μM arsenate solution was added to the TSB medium (main components: 17 g / L of tryptone, 3 g / L of soy peptone, 5 g / L of sodium chloride, 2.5 g / L of potassium dihydrogen phosphate, 2.5 g / L of glucose, the balance being water, pH value 7.0 ± 0.2). After aeration with high-purity nitrogen for 40 min to remove the oxygen in the bottle, the cap was quickly pressed on and placed in an incubator at 30 °C for static culture for 36 h. 1 mL of bacterial liquid was taken with a syringe every 6 h to measure its OD 600 . At 36 h of culture, 2 mL of bacterial liquid was taken, and after high-speed centrifugation, it was filtered through a 0.22 μm filter membrane to obtain the supernatant. The concentrations of arsenate, arsenite, and methylarsenic in the liquid were determined by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS, PerkinElm); in addition, 1 mL of the headspace gas in the vial was taken and the H2 concentration was measured by a high-performance gas chromatograph (Agilent 7820A, USA).

[0104] As Figure 1 shown, the logarithmic phase of the growth of the composite bacterial community in the TSB medium was 10 - 18 h. The arsenic transformation efficiency during the co-culture of the composite bacterial community was as Figure 2 shown. Compared with the sterilized control group (sterilized TSB medium containing 50 μM arsenate without bacteria), when inoculated with Paraclostridium benzoelyticum (YH01) alone, all the added arsenate was reduced to arsenite. When inoculated with Clostridium sporogenes (LHA6) alone, no arsenite and methylarsenic were detected. When inoculated with the composite bacterial liquid of YH01 + LHA6, arsenite and methylarsenic (MMA (monomethylarsenic) and DMA (dimethylarsenic)) were detected, and the arsenic methylation efficiency reached 31.0%. The H2 production in the culture system was as Figure 3 shown. When inoculated with Paraclostridium benzoelyticum (YH01) or Clostridium sporogenes (LHA6) alone, the hydrogen production amounts reached 4401.3 Pa and 4218.4 Pa respectively. When inoculated with the composite bacterial liquid, the hydrogen production amount reached 4861.4 Pa, which was increased by 10.5% and 15.2% respectively compared with the single bacterial liquid.

[0105] Example 2 Preparation of Methionine Woody Peat Composite Gel Bacterial Balls

[0106] First, the rejuvenated Paraclostridium benzoelyticum YH01 (preservation number: GDMCC No: 64705) and Clostridium sporogenes LHA6 (preservation number: GDMCC No: 62212) were expanded in TSB medium. Prepare 500 mL of 30 mM PIPS buffer, adjust the pH to 7.0, add 1 / 4 dose of TSB medium, stir and dissolve it, then pour it into a 1 L vial. After aerating and deoxygenating with high-purity nitrogen for 1 h, plug in a butyl rubber stopper, press on an aluminum cap, and place it in an autoclave for sterilization for 20 min. After cooling, inoculate 1 mL of each of the two rejuvenated bacterial solutions (OD is 0.1) into the vial on a clean bench and co-culture them. Incubate in a constant temperature incubator at 30 °C for 24 h to obtain 500 mL of composite bacterial solution.

[0107] Prepare methionine composite woody peat: Weigh 100 g of woody peat raw material, dry it in an oven at 60 °C, take it out, grind it, sieve it (80 mesh), then put it into a 200-mesh mesh bag, and soak it in a 1 L, 2 mM methionine solution for adsorption for 6 h. Take it out and dry it to obtain methionine composite woody peat.

[0108] Weigh 7.5 g of methionine composite woody peat into a centrifuge tube, dispense 500 mL of TSB medium into a 1 L vial. The above materials are put into an autoclave for sterilization for 20 min. On a clean bench, add the cooled methionine composite woody peat into the above medium. After aerating and deoxygenating with high-purity nitrogen for 40 min, plug in a butyl rubber stopper and press the aluminum cap tightly to maintain an anaerobic state. Inoculate 10 mL of the above-prepared composite bacterial solution into the medium, and incubate it in a constant temperature incubator at 30 °C for 24 h and then take it out.

[0109] The SEM image of the methionine woody peat-loaded composite bactericide's microscopic morphology is as Figure 4 shown. Rod-shaped bacteria (YH01 and LHA6) are adsorbed on the surface of woody peat or colonized in the pores of woody peat.

[0110] Mix 500 mL of the above-mentioned complex bacterial solution loaded onto methionine woody peat with 500 mL of a 5% sodium alginate (SA) solution, and continuously introduce nitrogen to remove the oxygen in the container. Use a magnetic stirrer to continuously stir to mix the bacterial solution and the SA solution evenly. Additionally, prepare 500 mL of a 3% calcium chloride solution by mass and place it in a plastic bottle. Place it on a magnetic stirrer and stir, and continuously introduce high-purity nitrogen to remove the oxygen in the solution; use a peristaltic pump to evenly drip the above-mentioned complex bacterial solution and SA mixture into the calcium chloride solution continuously protected by nitrogen, and set the peristaltic pump drip rate to 5 mL / min. The mixture undergoes a cross-linking reaction during stirring to form gel bacteria balls with uniform particles ( Figure 5 as shown).

[0111] After measurement, the particle diameter of the composite gel bacteria balls is between 2 and 3 mm, the forming rate is 98%, and the wet weight of the composite gel bacteria balls prepared from 500 mL of the complex bacterial solution is 960 g.

[0112] Example 3 Evaluation of the pot experiment effect of methionine woody peat composite gel bacteria balls

[0113] A rice pot experiment was carried out on the acidic cadmium-arsenic composite polluted soil in a certain village in Chongyi County, Jiangxi Province. Six treatments were set up, namely control (CK), methionine composite woody peat treatment, single strain (YH01) treatment, single strain (LHA6) treatment, free composite bactericide treatment (MB), and methionine woody peat composite gel bacteria balls (WPB), with 3 replicates for each treatment. The initial soil pH was 5.36, the total Cd content was 1.772 mg / kg, the total As content was 56.1 mg / kg, the organic matter content was 20.7 g / kg, and the hydrolyzable nitrogen content was 113.6 mg / kg. Rice and soil samples were collected at the maturity stage, and the methyl arsenic content in the rice, the relative expression abundances of the arsenic methylation functional gene and the nitrogen fixation gene in the soil microorganisms were analyzed.

[0114] As Figure 6 shown, compared with the control, the proportion of inorganic arsenic decreased by 23.4% in the treatment with the single strain (LHA6) with arsenic methylation function, and the proportion of inorganic arsenic in the rice decreased by 44.5% in the treatment with the composite gel bacteria balls. The composite gel bacteria balls treatment significantly reduced the inorganic arsenic content in the rice and reduced the toxicity of arsenic in the rice; the relative expression abundances of the nitrogen fixation gene and the arsenic methylation gene in the soil microorganisms also increased by a certain proportion, as Figure 7 and Figure 8As shown, compared with the control, the copy number of the arsenic methylation functional gene arsM in the soil treated with the composite gel bacteria balls increased by 94.4%. Compared with the free composite microbial agent, the copy number of the arsenic methylation functional gene arsM in the soil treated with the composite gel bacteria balls increased by 45.5%. Compared with the treatment with a single strain (LHA6), the copy number of the arsM gene increased by 50.4%. In addition, the copy number of the nitrogen-fixing microbial nifH gene in the soil treated with the composite gel bacteria balls also increased significantly. Compared with the control, the copy number of the nifH gene increased by 64.8%. Compared with the treatment with the free composite microbial agent, the copy number of the nitrogen-fixing microbial functional gene nifH in the soil increased by 25.4%. Compared with the treatment with a single strain (LHA6), the copy number of the nitrogen-fixing microbial functional gene nifH in the soil increased by 58.1%, and the expression abundance of the nitrogen-fixing gene increased significantly.

[0115] Example 4 Effect of Methionine Woody Peat Composite Gel Bacteria Balls on the Improvement of Arsenic-Contaminated Soil

[0116] A plot experiment was carried out in a slightly arsenic-contaminated paddy field in Boluo County, Huizhou City. The initial soil pH was 5.78, and the total As content was 49.3 mg / kg, belonging to a single arsenic-contaminated soil. The area of the field plots was uniformly set to 30 m 2 , and six treatments were set up, namely control (CK), methionine composite woody peat treatment, single strain (YH01) treatment, single strain (LHA6) treatment, free composite microbial agent treatment (MB), and methionine woody peat composite gel bacteria balls (WPB), with 3 replicates for each treatment. The application rate of methionine woody peat was 150 kg per mu, the application rate of the single microbial agent was 10 kg per mu, and the application rates of the free composite microbial agent and the composite gel bacteria balls were both 20 kg per mu. They were evenly spread into the paddy field after plowing and harrowing, and then harrowed evenly. Pesticides could not be applied within 30 days after the application of the microbial agent to prevent the reduction of the activity of the microbial community. Pore water samples and rice samples at the mature stage were collected during the critical growth period of rice, and the concentrations of inorganic arsenic As(III) and As(V) in the pore water of each treatment were measured.

[0117] After measurement, the concentrations of inorganic arsenic As(III) and As(V) in the pore water of each treatment were as Figure 9 and Figure 10As shown in the figure, within the first 60 days of rice growth, soil-bound arsenic was rapidly released into the pore water. Especially on the 20th day of flooding, the concentrations of As(III) and As(V) in the control treatment reached 3201.6 mg / L and 802.3 mg / L respectively. In the treatment with methionine and woody peat, the concentrations of As(III) and As(V) in the pore water decreased to 3070.4 mg / L and 783.5 mg / L respectively, showing decreases of 4.3% and 2.3% respectively. In the treatment with a single arsenic-methylating functional bacterium LHA6, the concentrations of As(III) and As(V) in the pore water decreased to 3013.9 mg / L and 789.3 mg / L respectively, with the decline rates reaching 5.8% and 1.6% respectively. In the treatment with a free compound microbial agent, the concentrations of As(III) and As(V) decreased to 2963.5 mg / L and 689.6 mg / L respectively, with the decline rates reaching 7.4% and 14.0% respectively. While in the treatment with compound gel microspheres, the concentrations of As(III) and As(V) in the pore water decreased to 2253.4 mg / L and 537.1 mg / L respectively, with the decline rates reaching 29.6% and 33.1% respectively, and the inorganic arsenic in the pore water decreased significantly. In addition, through detection, the proportion of inorganic arsenic in the rice treated with compound gel microspheres also decreased significantly. As Figure 11 shown, compared with the control, the treatment with compound gel microspheres decreased by 29.1%, significantly reducing the toxicity of arsenic in rice. While in the treatments with free compound microbial agent and single strain (LHA6), the inorganic arsenic in rice decreased, but there was no significant difference compared with the control group.

[0118] Example 5 Evaluation of the improvement effect of methionine and woody peat compound gel microspheres on acidic soil

[0119] A field experiment was carried out on a certain acidic soil in Zhujizhen, Nanxiong, Shaoguan. The soil pH was 5.32 and the total nitrogen content was 1.32 g / kg. The experiment set up 6 treatments: control (CK), treatment with methionine compound woody peat, treatment with a single strain (YH01), treatment with a single strain (LHA6), treatment with a free compound microbial agent (MB), and treatment with methionine and woody peat compound gel microspheres (WPB), with 3 replicates for each treatment. Methionine and woody peat was applied at 150 kg / mu, and compound gel microspheres were applied at 20 kg / mu. They were spread into the flooded paddy field after plowing and harrowing, and transplanting was carried out 2 - 3 days later. Pesticides could not be applied within one month after transplanting. Yield measurement was carried out at the rice maturity stage, and soil and rice samples were collected to analyze soil pH, soil total nitrogen and ammonium nitrogen content, and ammonia nitrogen content in the pore water.

[0120] As Figure 12 shown, on the 20th day and 30th day of rice growth, the soil pH of the treatment groups with compound gel microspheres increased significantly, from the initial 5.39 to 6.67 and 6.53 respectively, and finally stabilized at 5.82, an increase of 0.43 units. The ammonia nitrogen concentration in the soil pore water also increased; As Figure 13As shown, the ammonia nitrogen concentration in the pore water increased after the application of methionine woody peat, single strain (YH01), free compound bacterial agent, and composite gel bacteria balls. Especially for the treatment with composite gel bacteria balls, between the 20th and 120th days, the ammonia nitrogen concentration in the pore water was between 22.5 and 55.6 mg / L, which was 9.1 - 24.6 mg / L higher than that of the control treatment at the same time. The nitrogen fixation effect of the composite gel bacteria balls was more significant, and the activity of Clostridium paraputrificum (YH01) in the composite gel bacteria balls was higher. After colonizing in the soil, it utilized nitrogen in the air to synthesize NH4 + , effectively increasing the ammonia nitrogen in the pore water. In addition, the total nitrogen and available nitrogen contents of the soil were analyzed. As Figure 14 shown, compared with the control, the total nitrogen in the soil increased by 24.6% and the available nitrogen in the soil increased by 27.0% after the application of the composite gel bacteria balls. The total nitrogen and available nitrogen contents in the soil increased significantly.

[0121] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A microbial combination comprising Clostridium parabenzoicum ( Paraclostridium benzoelyticum )YH01 and Clostridium sporogenes ( Clostridium sporogenes )LHA6; wherein the taxonomic name of Clostridium parabenzoic acidolyticum YH01 is Paraclostridium benzoelyticum , deposited in Guangdong Microbial Culture Collection Center on May 30, 2024, with the deposit number: GDMCC No: 64705; the taxonomic name of the Clostridium sporogenes LHA6 is Paraclostridium benzoelyticum , deposited in Guangdong Provincial Microbiological Culture Collection Center on January 14, 2022, with the deposit number GDMCCNo: 62212.

2. A composite bacterial agent comprising the microbial combination according to claim 1 and / or a culture of the microbial combination according to claim 1.

3. The composite bacterial agent according to claim 2, characterized in that: The composite bacterial agent also includes a carrier, and the carrier includes woody peat.

4. The composite bacterial agent according to claim 3, characterized in that: The woody peat is methionine-modified woody peat.

5. The composite bacterial agent according to claim 3, characterized in that: The composite bacterial agent also includes a cell immobilization embedding material and a cross-linking agent.

6. The composite bacterial agent according to claim 5, characterized in that: The cell fixation embedding material comprises at least one of sodium alginate, sodium carboxymethyl cellulose, gelatin, chitosan, potassium alginate, polyvinyl alcohol, chitin and agar.

7. The composite bacterial agent according to claim 5, characterized in that: The crosslinking agent includes divalent and higher metal ions Cu 2+ , Fe 3+ , Ca 2+ and Zn 2+ At least one of the salt solutions.

8. The method for preparing the composite bacterial agent according to any one of claims 5 to 7, comprising the following steps: Inoculating the microbial combination according to claim 1 into a culture medium containing woody peat for colonization culture to obtain a composite bacterial solution; The composite bacterial solution is mixed with the cell immobilization material, and then added dropwise to the crosslinking agent solution to undergo a crosslinking reaction to obtain a composite bacterial gel ball, namely, a composite bacterial agent.

9. The preparation method according to claim 8, characterized in that: The cross-linking reaction time is 4 to 10 hours.

10. Use of the microbial combination according to claim 1 or the composite bacterial agent according to any one of claims 2 to 7 in at least one of (1) to (15): (1) Reduction of arsenic; (2) preparing a product of reduced arsenic; (3) Hydrogen production; (4) preparing hydrogen-producing products; (5) Nitrogen fixation; (6) Preparation of nitrogen-fixing products; (7) Arsenic methylation; (8) preparing arsenic methylation products; (9) Remediation of polluted environment; (10) Preparing products for remediation of arsenic-contaminated environments; (11) preparing soil conditioner; (12) Reduce the content of inorganic arsenic in plants; (13) preparing products for reducing the content of inorganic arsenic in plants; (14) Improving the fertility of cultivated land; (15) Preparation of products for improving fertility of cultivated land; The environment described in (9) to (10) is an arsenic-contaminated environment.

11. The use according to claim 10, characterized in that: The arsenic includes inorganic pentavalent arsenic or inorganic trivalent arsenic.

12. A method comprising treating a sample to be treated using the microbial combination of claim 1 and the composite bacterial agent of any one of claims 2 to 7; The method comprises at least one of a1) to a7): a1) a method for reducing arsenic; a2) a method for producing hydrogen; a3) a method for fixing nitrogen; a4) a method for arsenic methylation; a5) A method for remediating a polluted environment; a6) A method for reducing the content of inorganic arsenic in plants; a7) A method for improving the fertility of cultivated land; The environment described in a5) is an arsenic-contaminated environment.

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