Complex microbial inoculant as well as preparation method and application thereof

Abstract

The invention provides a complex microbial inoculant and a preparation method and application thereof, the complex microbial inoculant comprises modified magnetic nanoparticles and a microbial inoculant, the ratio of the volume of the microbial inoculant to the mass of the modified magnetic nanoparticles is 10 ml / mg to 800 ml / mg, and the modified magnetic nanoparticles are prepared by mixing magnetic nanoparticles and an adhesive. The complex microbial inoculant shows magnetism under an external magnetic field and can be efficiently recycled, the formula of the complex microbial inoculant can be simplified, the complex microbial inoculant can be used for degrading organic matters, the usage amount of the complex microbial inoculant can be reduced, and the cost can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial agent preparation, and specifically relates to a composite bacterial agent and a preparation method and application thereof. Background Art

[0002] With the rapid development of agriculture, a large amount of straw has been produced, which contains a large amount of organic matter. By processing straw with fermentation technology, agricultural waste can be converted into biogas energy, and the generated biogas residue and biogas liquid can be reused as organic fertilizer, which has positive significance for alleviating energy shortages and reducing environmental pressure. Composite bacterial agents play an important role in improving straw degradation efficiency and product quality. However, in the process of straw fermentation, existing composite bacterial agents have the problems of high formula complexity and large demand for bacterial sources, which leads to increased operating costs and reduced processing efficiency of straw fermentation.

[0003] Therefore, how to provide a composite bacterial agent that can be efficiently recycled and utilized, simplify the composite bacterial agent formula, and reduce production costs is a technical problem that needs to be urgently solved in this field. Summary of the invention

[0004] The present invention provides a composite bacterial agent, which can be efficiently recycled after being put into culture, and the formula of the composite bacterial agent is simplified, thereby reducing production costs.

[0005] The present invention also provides a method for preparing the composite bacterial agent, which realizes efficient recovery and recycling of the composite bacterial agent.

[0006] The present invention also provides an application of a composite bacterial agent in degrading organic matter, wherein the composite bacterial agent can improve the efficiency of organic matter degradation and reduce costs.

[0007] In a first aspect, the present invention provides a composite bacterial agent, the composite bacterial agent comprising: modified magnetic nanoparticles and microbial agents;

[0008] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 10 ml / mg-800 ml / mg;

[0009] The modified magnetic nanoparticles are prepared by mixing magnetic nanoparticles and a binder.

[0010] The composite bacterial agent as described above, wherein the mass ratio of the magnetic nanoparticles to the binder is 1:(0.5919-0.6324); and / or,

[0011] The adhesive includes at least one of polyethylene, sodium alginate, and ethylene-vinyl acetate copolymer.

[0012] The composite bacterial agent as described above, wherein the microbial agent comprises Gordonia, Williamsia, and Methanosarcina mazei;

[0013] The ratio of the total number of bacteria of Gordonia, Williamsia and Methanosarcina mazei in each milliliter of microbial inoculant is (75-80):(3-4):1.

[0014] The composite bacterial agent as described above, wherein the particle size of the modified magnetic nanoparticles is 0.15nm-0.25nm; and / or,

[0015] The magnetic nanoparticles include ferroferric oxide.

[0016] In a second aspect, the present invention provides a method for preparing the composite bacterial agent according to the first aspect, comprising the following steps:

[0017] 1) mixing magnetic nanoparticles and a binder to obtain modified magnetic nanoparticles;

[0018] 2) Mixing the modified magnetic nanoparticles and a microbial agent to obtain the composite agent.

[0019] The preparation method as described above, wherein the preparation method of the magnetic nanoparticles comprises: mixing a divalent iron source, a trivalent iron source and a precipitant, performing a coprecipitation reaction, separating a precipitate, and obtaining magnetic nanoparticles;

[0020] The molar ratio of the ferrous iron source, the ferric iron source and the precipitant is 1:2:(25-30); and / or,

[0021] The precipitating agent includes NaOH.

[0022] The preparation method as described above, wherein the preparation method of the microbial agent comprises the following steps:

[0023] 1) inoculating Gordonia, Williamsia and Methanosarcina mazei into three liquid culture media for fermentation, respectively, to obtain Gordonia fermentation broth, Williamsia fermentation broth and Methanosarcina mazei fermentation broth;

[0024] 2) separating the Gordonia fermentation broth, the Williamsia fermentation broth and the Methanosarcina mazei fermentation broth to obtain Gordonia cells, Williamsia cells and Methanosarcina mazei cells;

[0025] 3) Mixing Gordonia cells, Williamsia cells and Methanosarcina mazei cells and dispersing them in sterile water to obtain the microbial agent.

[0026] The preparation method as described above, wherein the fermentation culture temperature is 22°C-35°C; and / or,

[0027] The pH value of the liquid culture medium is 7-7.4.

[0028] The preparation method as described above, wherein the concentration of Gordonia in the Gordonia fermentation broth is 3.458×10 8 CFU / mL-3.822×10 8 CFU / mL; and / or,

[0029] The concentration of Williamsella in the Williamsella fermentation broth is 6.536×10 7 CFU / mL-7.224×10 7 CFU / mL; and / or,

[0030] The concentration of Methanosarcina mazei in the Methanosarcina mazei fermentation broth is 4.864×10 8 CFU / mL-5.376×10 8 CFU / mL.

[0031] In a third aspect, the present invention provides an application of the composite bacterial agent described in the first aspect in degrading organic matter.

[0032] The composite bacterial agent of the present invention comprises modified magnetic nanoparticles and microbial agents. The composite bacterial agent exhibits magnetism under an external magnetic field, can be efficiently recycled, and can simplify the composite bacterial agent formula. The composite bacterial agent can be used to degrade organic matter, and can reduce the usage of the composite bacterial agent and reduce costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 TEM image of the modified magnetic nanoparticles in Example 1 of the present invention;

[0034] Figure 2 HRTEM image of the modified magnetic nanoparticles in Example 1 of the present invention;

[0035] Figure 3 is the X-ray diffraction pattern of the modified magnetic nanoparticles in Example 1 of the present invention;

[0036] Figure 4 is the hysteresis loop of the modified magnetic nanoparticles in Example 1 of the present invention;

[0037] Figure 5 Graph showing the magnetic functionalization efficiency of the composite bacterial agent in Examples 1-10 of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In a first aspect, the present invention provides a composite bacterial agent, which includes modified magnetic nanoparticles and microbial agents; the mass ratio of the volume of the microbial agents to the modified magnetic nanoparticles is 10ml / mg-800ml / mg, for example, it can be 10ml / mg, 100ml / mg, 150ml / mg, 200ml / mg, 250ml / mg, 300ml / mg, 400ml / mg, 450ml / mg, 500ml / mg, 600ml / mg, 700ml / mg, 800ml / mg, preferably 100ml / mg-400ml / mg; the modified magnetic nanoparticles are prepared by mixing magnetic nanoparticles and a binder.

[0040] The composite microbial agent of the present invention can be efficiently recycled, the composite microbial agent formula is simplified, and the composite microbial agent formula is avoided from leaking. The magnetic nanoparticles are fixed on the surface of the microbial agent by an adhesive, so that the composite microbial agent exhibits magnetism under an external magnetic field. The microbial agent with metabolic activity will gradually lose the modified magnetic nanoparticles on its surface during the division and proliferation process, and has weak magnetism, while the inert microbial agent does not divide and proliferate, and the modified magnetic nanoparticles are still fixed on its surface, and has strong magnetism. When an external magnetic field is applied, the inert microbial agent that still maintains magnetism will be attracted by the external magnetic field, while the microbial agent with strong metabolic activity will be less affected by the external magnetic field due to the weakening or loss of magnetism. Therefore, the inert microbial agent and the microbial agent with strong metabolic activity can be separated by adjusting the intensity and direction of the external magnetic field without affecting the biological activity of the microbial agent. At the same time, by controlling the volume of the microbial agent and the mass ratio of the modified magnetic nanoparticles, the modified magnetic nanoparticles are firmly adsorbed on the surface of the microbial agent.

[0041] In some embodiments, the mass ratio of the magnetic nanoparticles to the binder is selected so that the modified magnetic nanoparticles are better adsorbed on the surface of the microbial agent, and the mass ratio of the magnetic nanoparticles to the binder is 1:(0.5919-0.6324).

[0042] For example, the mass ratio of the magnetic nanoparticles to the binder may be 1:0.5919, 1:0.6, 1:0.61, 1:0.62, 1:0.63 or 1:0.6324, etc.

[0043] Furthermore, the adhesive includes at least one of polyethylene, sodium alginate, and ethylene-vinyl acetate copolymer, which can further enhance the bonding between the magnetic nanoparticles and the microbial agent.

[0044] In the present invention, the microbial agent includes Gordonia, Williamsia, and Methanosarcina mazei, wherein Gordonia and Williamsia are used to degrade complex organic compounds (including hydrocarbons, aromatic compounds, rubber, and petroleum products), and Methanosarcina mazei is a methanogen that converts organic matter into methane through methanogenesis (methanogenesis) in an anaerobic environment.

[0045] The total bacterial count of Gordonia, Williamsia and Methanosarcina mazei in each milliliter of the microbial agent is (75-80): (3-4): 1, so that the composite agent can degrade organic matter more comprehensively through synergistic effects, thereby improving the degradation efficiency of organic matter.

[0046] Illustratively, the total bacterial count ratio of Gordonia, Williamsia and Methanosarcina mazei per milliliter of microbial inoculant can be 75:3:1, 76:3:1, 77:3:1, 78:3:1, 79:3:1, 80:3:1, 75:4:1, 76:4:1, 77:4:1, 78:4:1, 79:4:1 or 80:4:1, etc.

[0047] According to the technical solution of the present invention, the particle size of the modified magnetic nanoparticles is 0.15nm-0.25nm, for example, it can be 0.15nm, 0.17nm, 0.19nm, 0.21nm, 0.23nm or 0.25nm, so that the modified magnetic nanoparticles can better adhere to the surface of the microbial inoculant and avoid the modified magnetic nanoparticles falling off the surface of the microbial inoculant.

[0048] In some embodiments, the magnetic nanoparticles include ferroferric oxide, which has good biocompatibility and low toxicity, and has a simple preparation process and low production cost.

[0049] In a second aspect, the present invention provides a method for preparing the composite bacterial agent according to the first aspect, comprising:

[0050] 1) mixing magnetic nanoparticles and a binder to obtain modified magnetic nanoparticles;

[0051] 2) Mixing the modified magnetic nanoparticles and microbial agents to obtain a composite agent.

[0052] In some embodiments, in step 1), after the magnetic nanoparticles and the binder are mixed, ultrasonic treatment is performed for 10 min-20 min to fully mix the magnetic nanoparticles and the binder to avoid agglomeration of the magnetic nanoparticles and promote uniform coating of the binder on the surface of the magnetic nanoparticles. The precipitate is then separated by centrifugation, and the precipitate is washed 3-6 times to remove impurities in the precipitate, and then dried to obtain modified magnetic nanoparticles.

[0053] In some embodiments, in step 2), the modified magnetic nanoparticles and the microbial agent are mixed and shaken for 15 min to 25 min to allow the modified magnetic nanoparticles and the microbial agent to fully contact and combine to obtain a composite agent.

[0054] The preparation method of the composite bacterial agent of the present invention realizes the efficient recovery and recycling of the composite bacterial agent, and has a simple process and is convenient for industrial production.

[0055] According to the technical solution of the present invention, the preparation method of magnetic nanoparticles includes: mixing a divalent iron source, a trivalent iron source and a precipitant, performing a coprecipitation reaction, separating a precipitate, and obtaining magnetic nanoparticles.

[0056] The divalent iron source in the present invention includes FeSO 4 、FeCl 2 , ferric iron sources include FeCl 3 、Fe(NO 3 ) 3 . In some embodiments, the ferrous iron source, the ferric iron source and the precipitant are respectively prepared into a ferrous iron source solution, a ferric iron source solution and a precipitant solution, and then the ferrous iron source solution, the ferric iron source solution and the precipitant solution are mixed to carry out a coprecipitation reaction. After the coprecipitation reaction is completed, the precipitate is separated from the supernatant by centrifugation or magnetic separation to separate the precipitate, and then the precipitate is washed with water 3-6 times until the pH of the washed supernatant is 7, effectively removing impurities in the precipitate to obtain magnetic nanoparticles. Furthermore, by adjusting the molar ratio of the ferrous iron source, the ferric iron source and the precipitant to obtain magnetic nanoparticles with a more uniform particle size, the molar ratio of the ferrous iron source, the ferric iron source and the precipitant is 1:2:(25~30).

[0057] Illustratively, the molar ratio of the ferrous iron source, the ferric iron source, and the precipitant may be 1:2:25, 1:2:26, ​​1:2:27, 1:2:28, 1:2:29, 1:2:30.

[0058] Furthermore, the precipitant includes NaOH, which can promote the rapid progress of the co-precipitation reaction.

[0059] In the present invention, the preparation method of the microbial agent comprises the following steps:

[0060] 1) inoculating Gordonia, Williamsia and Methanosarcina mazei into liquid culture medium for fermentation, respectively, to obtain Gordonia fermentation broth, Williamsia fermentation broth and Methanosarcina mazei fermentation broth;

[0061] 2) separating the Gordonia fermentation broth, the Williamsia fermentation broth and the Methanosarcina mazei fermentation broth to obtain Gordonia cells, Williamsia cells and Methanosarcina mazei cells;

[0062] 3) Mixing Gordonia cells, Williamsia cells and Methanosarcina mazei cells and dispersing them in sterile water to obtain the microbial agent.

[0063] Specifically, the preparation method of the microbial agent of the present invention comprises:

[0064] 1) Gordonia, Williamsia and Methanosarcina mazei are inoculated into three liquid culture media for fermentation, respectively, to obtain Gordonia fermentation broth, Williamsia fermentation broth and Methanosarcina mazei fermentation broth;

[0065] 2) separating and treating the fermentation liquid of Gordonia to obtain Gordonia bodies, separating and treating the fermentation liquid of Williamsia to obtain Williamsia bodies, and separating and treating the fermentation liquid of Methanosarcina mazei to obtain Methanosarcina mazei bodies;

[0066] 3) Mixing Gordonia cells, Williamsia cells and Methanosarcina mazei cells and dispersing them in sterile water to obtain the microbial agent.

[0067] The liquid culture medium can provide the necessary environment and nutritional support for the growth and metabolism of Gordonia, Williamsia and Methanosarcina mazei. The present invention does not limit the formula components of the liquid culture medium, and the formula components of the liquid culture medium may include tryptone, tryptic peptone, yeast extract, sodium chloride, potassium chloride, potassium chloride, urea, glycerol, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sterile water. The preparation method of the liquid culture medium comprises: dissolving the formula components of the liquid culture medium in water, adjusting the pH value to 7.0 with NaOH solution, and then diluting to 1L with water to obtain a liquid culture medium.

[0068] In some embodiments, in step 1), the fermentation culture can be carried out in a shaker or fermenter at 180 r / min-230 r / min, which helps to increase the solubility of oxygen in the liquid culture medium and ensure that the microorganisms obtain sufficient oxygen to support their metabolic activities. By stirring or shaking, the nutrients and microorganisms in the liquid culture medium can be evenly distributed to prevent local nutrient depletion or accumulation of metabolites.

[0069] In some embodiments, in step 2), when the number of Gordonia in the Gordonia fermentation broth, Williamsia in the Williamsia fermentation broth, and Methanosarcina mazei in the Methanosarcina mazei fermentation broth increases exponentially (the growth curve of the bacteria is monitored using a spectrophotometer), indicating that the bacteria are in a stage of rapid growth and division at a constant rate, the Gordonia fermentation broth, the Williamsia fermentation broth, and the Methanosarcina mazei fermentation broth are respectively centrifuged at 12000 r / min for 10 min, and the bacterial bodies at the bottom are collected to obtain Gordonia bodies, Williamsia bodies, and Methanosarcina mazei bodies.

[0070] In some embodiments, in step 3), Gordonia, Williamsia, and Methanosarcina mazei are mixed and dispersed in sterile water to obtain a microbial suspension. The OD600 value of the microbial suspension is adjusted to 1.00 with sterile water so as to obtain repeatable and comparable results in subsequent experiments. The OD600 value refers to the optical density measured at a wavelength of 600 nanometers, which is used to calculate the concentration of microorganisms. The microbial suspension is then centrifuged at 6500 r / min for 10-15 minutes to separate the precipitate, and the precipitate is dissolved in water with the same volume as the microbial suspension to remove impurities on the surface of Gordonia, Williamsia, and Methanosarcina mazei to obtain a microbial agent.

[0071] In some embodiments, the fermentation culture temperature is 22°C-35°C, so that Gordonia, Williamsella and Methanosarcina mazei have higher reproduction efficiency and growth rate; the pH value of the liquid culture medium is 7-7.4, which can promote the growth of Gordonia, Williamsella and Methanosarcina mazei.

[0072] According to the technical solution of the present invention, the concentration of Gordonia in the Gordonia fermentation broth is 3.458×10 8 CFU / mL-3.822×10 8 CFU / mL, the concentration of Williamsella in the Williamsella fermentation broth was 6.536×10 7 CFU / mL-7.224×10 7 CFU / mL, the concentration of Methanosarcina mazei in the fermentation broth was 4.864×10 8 CFU / mL-5.376×10 8 CFU / mL.

[0073] In a third aspect, the present invention provides an application of the composite bacterial agent described in the first aspect in degrading organic matter.

[0074] The present invention uses the composite bacterial agent to degrade organic matter, which can improve the degradation efficiency of the organic matter into usable resources and reduce the dosage of the composite bacterial agent.

[0075] Specifically, the composite bacterial agent of the present invention can be used to degrade materials containing organic matter. For example, in one embodiment, the materials containing organic matter can be corn stalks, soybean stalks, pineapple stalks, and the available resources can be biofertilizers, biofuels, etc.

[0076] Hereinafter, the present invention will be further described in detail through specific examples.

[0077] The sources of raw materials used in the examples and comparative examples of the present invention are as follows:

[0078] Gordonia (Gordonia alkanivorans): CGMCC1.944 Gordonia alkanivorans;

[0079] Williamsia (Pinnamonella Williamsia): CGMCC4.7271 Williamsia sterculiae;

[0080] Methanosarcinamazei (Methanosarcinamazei): CGMCC1.5193Methanosarcinamazei;

[0081] Tryptone: Thermo Fisher Scientific;

[0082] Yeast extract: Thermo Fisher Scientific;

[0083] Polyethylene: MacLean, H875017.

[0084] Preparation Example 1

[0085] The microbial agent of this preparation example is prepared by the following steps:

[0086] 1) Gordonia, Williamsia and Methanosarcina mazei were inoculated into liquid culture medium respectively, and fermented and cultured at 28°C and 180 r / min until the concentration of Gordonia in the fermentation broth of Gordonia was 3.64×108 CFU / mL and the concentration of Williamsia in the fermentation broth of Williamsia was 6.88×10 7 CFU / mL, the concentration of Methanosarcina mazei in the fermentation broth of Methanosarcina mazei was 5.12x108CFU / mL;

[0087] 2) centrifuging the fermentation broth of Gordonia, the fermentation broth of Williamsia and the fermentation broth of Methanosarcina mazei at 12000 r / min for 10 min respectively, collecting the bacterial cells at the bottom to obtain Gordonia cells, Williamsia cells and Methanosarcina mazei cells;

[0088] 3) Gordonia cells, Williamsia cells and Methanosarcina mazei cells were dispersed in sterile water at a mass ratio of 800:40:10 to obtain a cell suspension, and the OD600 value of the cell suspension was adjusted to 1.00 with sterile water to obtain a microbial inoculum.

[0089] The preparation method of the liquid culture medium includes: adding 12g of tryptone, 5g of yeast extract, 10g of NaCl and 5mL of 40wt% sterile urea aqueous solution to 950ml of deionized water, mixing evenly, adjusting the pH value to 7.0 with 5mol / L NaOH solution, and then making the volume to 1L with deionized water to obtain the liquid culture medium.

[0090] Example 1

[0091] The composite bacterial agent of this embodiment is prepared by the following steps:

[0092] 1) Add 1 mL of 1 mol / L FeCl 2 Solution, 2mL concentration of 1mol / L FeCl 3 The solution was mixed with 25 mL of a 1 mol / L NaOH solution to perform a coprecipitation reaction, and the precipitate was separated and dissolved back in water, washed until the pH of the supernatant was 7, and the supernatant was separated to obtain magnetic nanoparticles, which still contained a small amount of water (the water content was less than 1 wt % of the magnetic nanoparticles, and the water floated on the surface of the magnetic nanoparticles) and were in a flowable state;

[0093] 2) 5 mL of magnetic nanoparticles were placed in 45 mL of polyethylene, mixed, ultrasonically treated for 10 min, centrifuged, and the solid component was separated, then washed with water three times, and dried at 25° C. for 30 min to obtain modified magnetic nanoparticles;

[0094] 3) 100 mL of the microbial agent in Preparation Example 1 was centrifuged at 6500 r / min for 10 min to separate the precipitate, and the precipitate was dissolved back in 100 mL of deionized water, followed by adding 10 mg of modified magnetic nanoparticles and shaking on an oscillator for 15 min to obtain the composite microbial agent;

[0095] Among them, the magnetic nanoparticles are ferroferric oxide, the mass ratio of magnetic nanoparticles and binder is 1:0.6, the particle size of the modified magnetic nanoparticles is 0.15nm-0.25nm, the total bacterial count of Gordonia, Williamsia and Methanosarcina mazei per milliliter of microbial agent is 30:1.5:0.38, and the mass ratio of the volume of the microbial agent and the modified magnetic nanoparticles is 10ml / mg.

[0096] Example 2

[0097] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 1L of the microbial agent in Preparation Example 1 is centrifuged at 6500 r / min for 10 min to separate the precipitate, the precipitate is dissolved back in 1L of deionized water, and then 10 mg of modified magnetic nanoparticles are added to obtain the composite bacterial agent;

[0098] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 100 ml / mg.

[0099] Example 3

[0100] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 1.5 L of the microbial agent in Preparation Example 1 is centrifuged at 6500 r / min for 10 min to separate the precipitate, the precipitate is back dissolved in 1.5 L of deionized water, and then 10 mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15 min to obtain the composite bacterial agent.

[0101] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 100 ml / mg.

[0102] Example 4

[0103] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 3 L of the microbial agent in Preparation Example 1 is centrifuged at 6500 r / min for 10 min to separate the precipitate, the precipitate is dissolved back in 3 L of deionized water, and then 10 mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15 min to obtain the composite bacterial agent.

[0104] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 300 ml / mg.

[0105] Example 5

[0106] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 3.5 L of the microbial agent in Preparation Example 1 is centrifuged at 6500 r / min for 10 min to separate the precipitate, and the precipitate is dissolved back in 3.5 L of deionized water, and then 10 mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15 min to obtain the composite bacterial agent.

[0107] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 350 ml / mg.

[0108] Example 6

[0109] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 4 L of the microbial agent in Preparation Example 1 is centrifuged at 6500 r / min for 10 min to separate the precipitate, the precipitate is dissolved back in 4 L of deionized water, and then 10 mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15 min to obtain the composite bacterial agent.

[0110] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 400 ml / mg.

[0111] Example 7

[0112] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 4.5 L of the microbial agent in Preparation Example 1 is centrifuged at 6500 r / min for 10 min to separate the precipitate, and the precipitate is dissolved back in 4.5 L of deionized water, and then 10 mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15 min to obtain the composite bacterial agent.

[0113] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 450 ml / mg.

[0114] Example 8

[0115] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 5 L of the microbial agent in Preparation Example 1 is centrifuged at 6500 r / min for 10 min to separate the precipitate, the precipitate is back dissolved in 5 L of deionized water, and then 10 mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15 min to obtain the composite bacterial agent.

[0116] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 500 ml / mg.

[0117] Example 9

[0118] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 6L of the microbial agent in Preparation Example 1 is centrifuged at 6500r / min for 10min to separate the precipitate, the precipitate is dissolved back in 6L of deionized water, and then 10mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15min to obtain the composite bacterial agent.

[0119] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 600 ml / mg.

[0120] Example 10

[0121] The preparation method of the composite bacterial agent in this embodiment is basically the same as that in Example 1, except that: 8L of the microbial agent in Preparation Example 1 is centrifuged at 6500r / min for 10min to separate the precipitate, the precipitate is back dissolved in 8L of deionized water, and then 10mg of modified magnetic nanoparticles is added, and the mixture is placed on an oscillator for 15min to obtain the composite bacterial agent.

[0122] The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 800 ml / mg.

[0123] Application Examples 1-10:

[0124] 200 g of the composite bacterial agent in Example 1-10 was added to 500 ml of the pre-treated fermentation liquid of pineapple straw and treated at 25° C. for 48 h.

[0125] The pretreated fermentation liquid of pineapple straw is composed of 100g pineapple straw and 450ml ultrapure water.

[0126] Performance Testing

[0127] (1) Surface morphology and structure: The surface morphology and internal microstructure of the modified magnetic nanoparticles in Example 1 were observed using a transmission electron microscope (TEM, Jeol-2100f, Tokyo, Japan). Figure 1 ,Depend on Figure 1 It can be seen that the modified magnetic nanoparticles in Example 1 are aggregated together due to magnetization to form modified magnetic nanoparticle aggregates with a diameter of 50nm-100nm, which are much smaller than individual cells and can be attached to the cell surface; the lattice fringes of the modified magnetic nanoparticles can be observed using a high-resolution transmission electron microscope (HRTEM, Jeol-2100f, Tokyo, Japan), and the results are shown in Figure 2 ,Depend on Figure 2 It can be seen that the peaks of each characteristic peak are sharp and the half-peak width is small, indicating that the compound on the surface of the prepared modified magnetic nanoparticles is well crystallized;

[0128] (2) Crystallinity: The crystallinity of the modified magnetic nanoparticles in Example 1 was measured using an X-ray powder diffractometer (XRD-600, Dojin Seisakusho). During the measurement, nickel-filtered copper kα was used as the X-ray source, and the scanning range was 10-80° 2θ. The results are shown in Table 1. Figure 3 ,Depend on Figure 3 It can be seen that the peaks of each characteristic peak in the spectrum are sharp and have a small half-peak width, indicating that the compound on the surface of the prepared material is well crystallized;

[0129] (3) Magnetic properties: The magnetic characteristics of the modified magnetic nanoparticles in Example 1 were measured using a vibrating sample magnetometer (VSM, Lake Shore Cryotronics, Ltd, 8600 series). The results are shown in Figure 4 ,Depend on Figure 4 It can be seen that when an external magnetic field exists, the modified magnetic nanoparticles have a strong magnetic response.

[0130] (4) Magnetic functionalization efficiency of composite bacterial agents: A permanent magnet was placed close to the outer wall of a container containing the composite bacterial agents in Examples 1-10, and the mixture was allowed to stand for 10 min to allow the composite bacterial agents to be attracted to the container wall by the magnetic field. The supernatant was removed, and the concentrations of the microbial agents in the supernatant and the composite bacterial agents after magnetization (copies / mL) were measured using a real-time fluorescent quantitative polymerase chain reaction instrument (Roche, LightCycler 480II);

[0131] The magnetic functionalization efficiency of the composite microbial agent = (the concentration of the microbial agent in the supernatant / the concentration of the microbial agent in the composite microbial agent after magnetization) × 100%, the results are shown in Figure 5 and Table 1, by Figure 5 It can be seen that when the volume of the microbial agent and the mass ratio of the modified magnetic nanoparticles is 100ml / mg-400ml / mg, the composite bacterial agent has excellent magnetic response ability and is more easily manipulated by an external magnetic field, thereby achieving rapid and effective separation and recovery of the composite bacterial agent.

[0132] (5) Recovery rate of composite bacterial agent: The composite bacterial agent prepared in Examples 1-10 was added to the pretreatment fermentation liquid of pineapple straw during preparation, and fermentation was performed. Then, a circular electromagnet was placed in the fermentation liquid and allowed to stand for 15 min to recover the composite bacterial agent. The mixture was centrifuged at 7000 rpm / min for 5 min, and the supernatant was removed. The mass of the composite bacterial agent before recovery and the mass of the composite bacterial agent after recovery were measured using an analytical balance. The results are shown in Table 1.

[0133] Recovery rate of composite bacterial agent 1 = (mass of composite bacterial agent after recovery 1 / mass of composite bacterial agent before recovery) × 100%;

[0134] The mass of the composite bacterial agent after recovery refers to the mass of the composite bacterial agent attracted by the circular electromagnet after the circular electromagnet is placed in the fermentation liquid and allowed to stand for 15 minutes. The mass of the composite bacterial agent before recovery refers to the mass of the composite bacterial agent put in before the fermentation treatment.

[0135] Recovery rate of composite bacterial agent 2 = (mass of composite bacterial agent after recovery 2 / mass of composite bacterial agent before recovery) × 100%;

[0136] The mass 2 of the composite bacterial agent after recovery refers to the mass of the composite bacterial agent attracted by the circular electromagnet after the circular electromagnet is placed in the fermentation liquid and left to stand for 2 hours. The mass of the composite bacterial agent before recovery refers to the mass of the composite bacterial agent put in before the fermentation treatment.

[0137] Table 1

[0138]

[0139]

[0140] From the analysis of Table 1, it can be seen that the composite bacterial agent prepared in the present invention exhibits magnetism under an external magnetic field and can be efficiently recycled.

[0141] 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite bacterial agent, characterized in that: The composite bacterial agent comprises: modified magnetic nanoparticles and microbial agents; The volume ratio of the microbial agent to the mass ratio of the modified magnetic nanoparticles is 10 ml / mg-800 ml / mg; The modified magnetic nanoparticles are prepared by mixing magnetic nanoparticles and a binder.

2. The composite bacterial agent according to claim 1, characterized in that The mass ratio of the magnetic nanoparticles to the binder is 1:(0.5919-0.6324); and / or, The adhesive includes at least one of polyethylene, sodium alginate, and ethylene-vinyl acetate copolymer.

3. The composite bacterial agent according to claim 1 or 2, characterized in that: The microbial agents include Gordonia, Williamsia, and Methanosarcina mazei; The ratio of the total number of bacteria of Gordonia, Williamsia and Methanosarcina mazei in each milliliter of microbial inoculant is (75-80):(3-4):

1.

4. The composite bacterial agent according to any one of claims 1 to 3, characterized in that: The particle size of the modified magnetic nanoparticles is 0.15nm-0.25nm; and / or, The magnetic nanoparticles include ferroferric oxide.

5. A method for preparing the composite bacterial agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) mixing magnetic nanoparticles and a binder to obtain modified magnetic nanoparticles; 2) Mixing the modified magnetic nanoparticles and a microbial agent to obtain the composite agent.

6. The preparation method according to claim 5, characterized in that: The preparation method of the magnetic nanoparticles comprises: mixing a divalent iron source, a trivalent iron source and a precipitant, performing a coprecipitation reaction, separating a precipitate, and obtaining the magnetic nanoparticles; The molar ratio of the ferrous iron source, the ferric iron source and the precipitant is 1:2:(25-30); and / or, The precipitating agent includes NaOH.

7. The preparation method according to claim 5 or 6, characterized in that: The preparation method of the microbial agent comprises the following steps: 1) inoculating Gordonia, Williamsia and Methanosarcina mazei into three liquid culture media for fermentation, respectively, to obtain Gordonia fermentation broth, Williamsia fermentation broth and Methanosarcina mazei fermentation broth; 2) separating the Gordonia fermentation broth, the Williamsia fermentation broth and the Methanosarcina mazei fermentation broth to obtain Gordonia cells, Williamsia cells and Methanosarcina mazei cells; 3) Mixing Gordonia cells, Williamsia cells and Methanosarcina mazei cells and dispersing them in sterile water to obtain the microbial agent.

8. The preparation method according to claim 7, characterized in that: The fermentation culture temperature is 22°C-35°C; and / or, The pH value of the liquid culture medium is 7-7.

4.

9. The preparation method according to claim 7 or 8, characterized in that: The concentration of Gordonia in the Gordonia fermentation broth is 3.458×10 8 CFU / mL-3.822×10 8 CFU / mL; and / or, The concentration of Williamsella in the Williamsella fermentation broth is 6.536×10 7 CFU / mL-7.224×10 7 CFU / mL; and / or, The concentration of Methanosarcina mazei in the Methanosarcina mazei fermentation broth is 4.864×10 8 CFU / mL-5.376×10 8 CFU / mL.

10. Use of the composite bacterial agent according to any one of claims 1 to 4 in degrading organic matter.

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