A microbial agent adsorption carrier, a solid microbial agent, and a preparation method thereof

Abstract

The present application relates to the field of microbial inoculant, in particular to a microbial inoculant adsorption carrier, a solid microbial inoculant and a preparation method thereof, wherein zeolite powder modified by nano-enzyme, diatomite modified by PEG400 and straw powder are mixed to prepare the microbial inoculant adsorption carrier, the solid microbial inoculant is prepared by adsorbing aerobic denitrifying bacteria on the microbial inoculant adsorption carrier, the porosity of the microbial inoculant adsorption carrier is greater than 95%, the specific surface area is greater than or equal to 140 m 2 / g, and the space and oxygen environment are provided for bacterial reproduction and survival, the storage time of the prepared solid microbial inoculant is long, and the survival rate of microorganisms is still higher than 90% after being stored at 4 DEG C for 180 days.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial inoculant, in particular to a microbial inoculant adsorption carrier, a solid microbial inoculant and a preparation method thereof. BACKGROUND

[0002] As an efficient and environmentally friendly biological agent, microbial inoculant has been widely used in agriculture, environmental protection, biological engineering and other fields. It usually exists in liquid form, but there are many inconveniences in storage, transportation and use of liquid inoculant, such as easy inactivation of bacteria due to environmental factors, high packaging and transportation costs, and complex operation during use. Therefore, adsorbing liquid inoculant onto solid material to prepare solid inoculant has become an important development direction.

[0003] The key to the preparation of solid inoculant is to select a suitable adsorption carrier. A kind of nitrosation inoculant and its preparation method in Chinese patent CN114686471A includes nitrous acid bacteria and an adsorption carrier. The adsorption carrier includes cross-linked chitosan embedded with calcium carbonate and heterotrophic bacteria. Although the survival rate of nitrosation bacteria is improved by using heterotrophic bacteria-chitosan carrier, the microporous structure does not meet the special needs of aerobic denitrifying bacteria, and the specific surface area of the carrier is low, the adsorption capacity is low, and the compatibility of the carrier-bacteria is poor. Chinese patent CN109095623A discloses a kind of microcarrier for improving the culture density of aerobic nitrifying bacteria and nitrosation bacteria and its preparation method. Cross-linked chitosan microspheres are added into a solution of negatively charged polymer sodium alginate, and the microcarrier is prepared by surface charge modification of cross-linked chitosan. A layer of negatively charged shell is formed on the surface of the microcarrier, which eliminates the influence of chitosan on the growth of nitrifying bacteria, but reduces the electrostatic adsorption of microorganisms. In fact, it is not conducive to the attachment and growth of negatively charged nitrifying bacteria on the carrier. Chinese patent CN114438069A discloses a kind of double microcarrier for culturing nitrifying bacteria, which is composed of chitosan-calcium carbonate and heterotrophic bacteria. The bacteria are loaded by neutralizing the positive charge of chitosan with heterotrophic bacteria. The adsorption process is complex, and the stability of the bacteria is not mentioned.

[0004] The existing solid inoculant carrier has low adsorption efficiency and poor stability, and a new type of adsorption carrier needs to be developed to realize efficient adsorption of bacteria. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a kind of microbial inoculant adsorption carrier, solid microbial inoculant and its preparation method. The zeolite powder modified by nanoenzyme, the diatomite modified by PEG400 and the straw powder are mixed to prepare the adsorption carrier. The efficient and stable solid microbial inoculant is prepared by adsorbing the aerobic denitrifying bacteria, so as to meet the high performance requirements of microbial inoculant in the fields of agriculture, environmental protection and biological engineering.

[0006] The present application achieves the above-mentioned purposes by the following technical solutions:

[0007] The present application discloses a microbial agent adsorption carrier, which is composed of nano-enzyme zeolite powder (25%-35%), PEG400-diatomic earth (35%-40%) and PEG400-straw powder (25%-35%), and the sum of the three is 100%. The nano-enzyme is one of platinum nanoparticles, pure ferroferric oxide nanoparticles and nickel nanoparticles.

[0008] Further, the preparation method of the nano-enzyme zeolite powder is as follows: the zeolite powder is dispersed in 1%-2% dilute acetic acid solution, stirred uniformly, and fully dispersed to form a uniform zeolite powder solution; the nano-enzyme is dispersed in water to form a nano-enzyme dispersion liquid with a concentration of 25-100 μg / mL; the zeolite powder solution and the nano-enzyme dispersion liquid are mixed in a mass ratio of 3-7:1, fully stirred, and spray dried to obtain nano-enzyme modified zeolite powder.

[0009] Further, the preparation methods of the PEG400-diatomic earth and the PEG400-straw powder are as follows:

[0010] The diatomic earth and the straw powder are respectively dispersed in an appropriate amount of acetic acid solution, stirred and dispersed uniformly, then PEG400 is added and stirred uniformly, a crosslinking agent glutaraldehyde is added to the obtained solution, and a crosslinking reaction is carried out at 20-30℃. After the reaction is completed, the mixture is washed to remove unreacted PEG400 and the crosslinking agent, and dried at 95-105℃ to obtain PEG400-diatomic earth and PEG400-straw powder.

[0011] The present application discloses a solid microbial agent, which is composed of the microbial agent adsorption carrier and aerobic denitrifying bacteria liquid, and the mass ratio of the two is 1:8-12. The concentration of the aerobic denitrifying bacteria liquid agent is 1.5×10 8 -1.8×10 8 CFU / mL.

[0012] The present application discloses a preparation method of the solid microbial agent, which comprises the following steps:

[0013] The nano-enzyme zeolite powder, the PEG400-diatomic earth and the PEG400-straw powder are mixed according to the mass ratio, and the aerobic denitrifying bacteria liquid agent is added for adsorption. The adsorption temperature is 20-28℃, and the adsorption time is 1-3 hours. The solid microbial agent is obtained by separation.

[0014] Further, the calculation formula of the adsorption efficiency is as follows: adsorption efficiency=(C0-C e ) / C0×100%, wherein C0 is the initial concentration of the microorganisms in the aerobic denitrifying bacteria liquid agent before adsorption, and Ce The residual concentration of microorganisms in the aerobic denitrifying bacteria liquid inoculum after adsorption.

[0015] Further, the preparation process of the aerobic denitrifying bacteria liquid inoculum is as follows: the aerobic denitrifying bacteria separated from the MABR device is inoculated in a liquid culture medium and activated for 40-50 h, the number of microorganisms is 1.0 at OD600, and the aerobic denitrifying bacteria liquid inoculum is obtained.

[0016] Further, the cell of the aerobic denitrifying bacteria liquid inoculum is Paracoccus, the Latin name is Paracoccus , and is purchased from Shanghai Collection and Preservation Center of Microorganisms with the item number NBRC102.

[0017] The microbial inoculum adsorption carrier provided by the application is mixed with the aerobic denitrifying bacteria liquid inoculum to form a composite carrier with a multi-stage pore structure, high porosity and high adsorption rate, because: after the modification of PEG400 on diatomite, the nanoscale pores of diatomite are connected to form interconnected pores, thereby improving the loading capacity; after the modification of PEG400 on straw powder, the straw powder forms ordered arranged large pores, thereby improving the specific surface area and porosity; meanwhile, the hydroxyl groups on the surfaces of PEG400-diatomite and PEG400-straw powder form hydrogen bonds, thereby enhancing the hydrophilicity and adsorption capacity of diatomite and straw powder. The catalytic active sites of nano-enzyme and the hydroxyl groups and silicon-aluminum-oxygen skeleton on the surface of zeolite powder form ionic bonds or coordination bonds, thereby improving the mechanical strength of the surface of zeolite, and thus increasing the stability of zeolite powder.

[0018] After the composite carrier is mixed with the aerobic denitrifying bacteria liquid, the hydroxyl groups on the surfaces of PEG400-diatomite and PEG400-straw powder can form hydrogen bonds with the hydroxyl groups or silicon-oxygen bonds on the surface of nano-enzyme-zeolite powder, thereby further enhancing the connection among the three, wherein the large pores of PEG400-straw powder provide cell proliferation space, the interconnected pores of PEG400-diatomite guarantee oxygen transmission, and nano-enzyme-zeolite powder catalytically degrades cell metabolites, so that the concentration of toxic substances in the local microenvironment is reduced, and the activity preservation time of bacteria is improved. The spatial synergistic effect of nano-enzyme-zeolite powder, PEG400-diatomite and PEG400-straw powder not only increases the number of active sites, but also promotes the diffusion of bacteria in the interior of zeolite powder, diatomite and straw powder, thereby significantly improving the adsorption efficiency and bacterial survival rate.

[0019] The advantages and beneficial effects of the application are as follows:

[0020] The prepared adsorption carrier has the advantages of high porosity (porosity > 95%), high adsorption efficiency (> 77%) and high specific surface area (specific surface area ≥ 140 m² / g), and after being mixed with the aerobic denitrifying bacteria liquid inoculum, space and oxygen environment can be provided for bacterial reproduction and survival, the prepared solid microbial inoculum has a long storage time, and the survival rate of microorganisms is still higher than 90% after being stored at 4℃ for 180 days. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 SEM images of diatomite before and after PEG400 modification.

[0022] Figure 2 SEM images of straw powder before and after PEG400 modification.

[0023] Figure 3 SEM images of zeolite powder before and after nano-enzyme modification. DETAILED DESCRIPTION

[0024] The concept and the generated concept and the generated technical effects of the present application will be described below in conjunction with examples, so as to fully understand the purpose and effect of the present application.

[0025] Example 1

[0026] A solid microbial inoculum is composed of an adsorption carrier and an aerobic denitrifying bacteria liquid inoculum with a mass ratio of 1:10, and the adsorption carrier is composed of nano-enzyme-zeolite powder 30%, PEG400-diatomite 40% and PEG400-straw powder 30%.

[0027] The preparation method of the solid microbial inoculum comprises the following steps:

[0028] (1) Collecting a biofilm sample containing aerobic denitrifying bacteria from an MABR system, placing the biofilm sample in sterile water, oscillating and dispersing to obtain a suspension containing aerobic denitrifying bacteria, gradient diluting the suspension, and taking a 10 5 times diluted liquid to be coated on a BTB culture medium, culturing the coated culture medium under aerobic conditions (2 mg / L), selecting a single clone strain that turns blue on the BTB culture medium, repeatedly streaking and purifying until a purified single clone strain is obtained, and there are no miscellaneous bacteria and companion bacteria around, obtaining aerobic denitrifying bacteria, inoculating the separated aerobic denitrifying bacteria into an LB liquid culture medium, culturing at 30℃ and 120 rpm for 48 h on a shaker, obtaining an aerobic denitrifying bacteria liquid inoculum, and the initial concentration of the aerobic denitrifying bacteria liquid inoculum is 1.65×10 8 CFU / mL.

[0029] (2) The zeolite powder is dispersed in a 2% dilute acetic acid solution, stirred uniformly, and fully dispersed to form a uniform zeolite powder solution; the nano-enzyme (pure ferroferric oxide nanoparticles, purchased from Zhongke Leiming (Beijing) Technology Co., Ltd., product number ZKJ112-5) is dispersed in water to form a nano-enzyme dispersion liquid with a concentration of 80 μg / mL; the zeolite powder solution and the nano-enzyme dispersion liquid are mixed in a mass ratio of 5:1, fully stirred, and spray dried to obtain the nano-enzyme-zeolite powder. The SEM images of the zeolite powder before and after modification by the nano-enzyme are shown in FIGS. Figure 3

[0030] The diatomite 50 g and the straw powder 50 g are respectively dispersed in the acetic acid solution 150 mL, stirred and dispersed uniformly, then the PEG400 is stirred uniformly, and the crosslinking agent glutaraldehyde is added to the obtained solution, and the crosslinking reaction is carried out at 25°C for 3 hours. After the reaction is completed, the mixture is washed to remove the unreacted PEG400 and the crosslinking agent, and dried at 105°C to obtain the PEG400-diatomite and the PEG400-straw powder. The SEM images of the diatomite before and after modification by the PEG400 and the straw powder before and after modification by the PEG400 are shown in FIGS. Figure 1 Figure 2

[0031] (3) The nano-enzyme-zeolite powder 30%, the PEG400-diatomite 40%, and the PEG400-straw powder 30% are mixed, the adsorption carrier is added to the container containing the aerobic denitrifying bacteria liquid inoculum to fully contact and perform the adsorption experiment, the adsorption time is 2 hours, the adsorption temperature is 25°C, the weight ratio of the adsorption carrier to the aerobic denitrifying bacteria liquid inoculum is 1:10, and the adsorption carrier and the aerobic denitrifying bacteria liquid inoculum are separated by filtration to prepare the solid microbial inoculum.

[0032] The residual concentration of the microorganism in the aerobic denitrifying bacteria liquid inoculum after adsorption is 2.8×10 7 CFU / ml, the calculation formula of the adsorption efficiency is: adsorption efficiency = (C0-C e ) / C0×100%, wherein C0 is the initial concentration of the microorganism in the aerobic denitrifying bacteria liquid inoculum before adsorption, C e is the residual concentration of the microorganism in the aerobic denitrifying bacteria liquid inoculum after adsorption, and the adsorption rate is calculated to be 82.5%.

[0033] Example 2

[0034] A solid microbial inoculum is composed of an adsorption carrier and an aerobic denitrifying bacteria liquid inoculum in a mass ratio of 1:10, and the adsorption carrier is composed of nano-enzyme-zeolite powder 35%, PEG400-diatomite 38%, and PEG400-straw powder 27%. The initial concentration of the aerobic denitrifying bacteria liquid inoculum is 1.7×10​​​8 CFU / ml.

[0035] The preparation method of the solid microbial inoculant is the same as that in Example 1, and the residual concentration of microorganisms in the aerobic denitrifying bacteria liquid inoculant after adsorption is determined to be 3.5 x 10 7 CFU / ml, and the adsorption rate of the formula is 79.4%.

[0036] Example 3

[0037] A solid microbial inoculant is composed of an adsorption carrier and an aerobic denitrifying bacteria liquid inoculant in a mass ratio of 1:10, and the adsorption carrier is composed of nano-enzyme-zirconite powder 25%, PEG400-diatomic earth 40%, and PEG400-straw powder 35%. The initial concentration of the aerobic denitrifying bacteria liquid inoculant is 1.8 x 10 8 CFU / ml.

[0038] The preparation method of the solid microbial inoculant is the same as that in Example 1, and the residual concentration of microorganisms in the aerobic denitrifying bacteria liquid inoculant after adsorption is determined to be 4 x 10 7 CFU / ml, and the adsorption rate of the formula is 77.8%.

[0039] Comparative Example 1

[0040] The difference from Example 1 is that the adsorption carrier is composed of nano-enzyme-zirconite powder 40%, PEG400-diatomic earth 35%, and PEG400-straw powder in a mass ratio of 25%.

[0041] The residual concentration of microorganisms in the aerobic denitrifying bacteria liquid inoculant after adsorption is determined to be 6.3 x 10 7 CFU / ml, and the adsorption rate of the formula is 61.8%.

[0042] Comparative Example 2

[0043] The difference from Example 1 is that the adsorption carrier is composed of nano-enzyme-zirconite powder 20%, PEG400-diatomic earth 30%, and PEG400-straw powder 50% as the adsorption material of the microbial inoculant.

[0044] The residual concentration of microorganisms in the aerobic denitrifying bacteria liquid inoculant after adsorption is determined to be 4.8 x 10 7 CFU / ml, and the adsorption rate of the formula is 70.9%.

[0045] Comparative Example 3

[0046] The difference from Example 1 is that the adsorption carrier is composed of EDTA-zirconite powder 30%, PEG400-diatomic earth 40%, and PEG400-straw powder 30%.

[0047] The residual concentration of microorganisms in the aerobic denitrifying bacteria liquid inoculant after adsorption was 5.2 x 10 7 CFU / ml, and the adsorption rate of the formula was 71.1%.

[0048] Comparative Example 4

[0049] The difference from Example 1 is only that the diatomite and straw powder are acid modified. The specific steps are: (1) Take an appropriate amount of diatomite or straw powder, mix it with a 14 mol / L sulfuric acid solution, and stir at 45-80 ℃ for 1-3 hours. (2) After the reaction is completed, filter out the reaction liquid, and wash the diatomite with a large amount of deionized water until it is neutral to remove residual acid and impurities. (3) Dry the washed diatomite at 100-120 ℃ for 12 hours to obtain acid-modified diatomite and acid-modified straw powder. The adsorption carrier is composed of 30% nano-enzyme-zeolite powder, 40% acid-modified diatomite, and 30% acid-modified straw powder.

[0050] The residual concentration of microorganisms in the aerobic denitrifying bacteria liquid inoculant after adsorption was 6.8 x 10 7 CFU / ml, and the adsorption rate of the formula was 62.2%.

[0051] Comparative Example 5

[0052] The difference from Example 1 is only that the adsorption carrier uses only nano-enzyme-zeolite powder.

[0053] Comparative Example 6

[0054] The difference from Example 1 is only that the adsorption carrier uses only PEG400-diatomite.

[0055] Comparative Example 7

[0056] The difference from Example 1 is only that the adsorption carrier uses only PEG400-straw powder.

[0057] Comparative Example 8

[0058] The difference from Example 1 is only that the adsorption carrier uses only 60% PEG400-straw powder and 40% nano-enzyme-zeolite powder.

[0059] Comparative Example 9

[0060] The difference from Example 1 is only that the adsorption carrier uses only 60% PEG400-straw powder and 40% PEG400-diatomite.

[0061] Comparative Example 10

[0062] The difference from Example 1 is only that the adsorption carrier uses only 40% nano-enzyme-zeolite powder and 60% PEG400-diatomite.

[0063] Performance test

[0064] The porosity and specific surface area of the solid microbial inoculants prepared in the examples and comparative examples were tested, and the test data are shown in Table 1. The test method was as follows: (1) porosity: a. The dry mass m of the sample to be tested was weighed after cleaning d . b. The sample was completely immersed in water and soaked for 1-2 hours to ensure that the pores were filled with water. c. After soaking, the sample was taken out and the excess water on the surface was wiped off with filter paper, and the saturated mass m s was weighed again. d. The porosity was calculated according to the formula: porosity (%) = (m s -m d ) / m s x 100. (2) Specific surface area: a. The sample to be tested was ground into powder and dried to constant weight in a drying oven to remove water and impurities. b. The dried sample was placed in a sample tube of a specific surface area analyzer and degassed. c. After degassing, the sample tube was placed in the analyzer, and the specific surface area was calculated.

[0065] The microbial activity of the solid microbial inoculants prepared in the examples and comparative examples was tested after being stored at 4℃ for 180 days, and the microbial survival rate is shown in Table 1.

[0066] Table 1

[0067]

[0068] As can be seen from the data in Table 1, by modifying the zeolite powder, diatomite and straw powder and controlling the use ratio of each material after modification, the porosity of the bacterial carrier is more than 95%, the specific surface area is more than 145 m² / g, and the bacterial survival rate is more than 91% after being stored at 4℃ for 180 days. This is because the macropores of PEG400-straw powder provide space for bacterial proliferation, the interconnected pores of PEG400-diatomite ensure oxygen transmission, and the nanometer enzyme-zeolite powder catalytically degrades bacterial metabolites, reducing the concentration of toxic substances in the local microenvironment and improving the activity preservation time of bacteria. The synergistic effect of nanometer enzyme-zeolite powder, PEG400-diatomite and PEG400-straw powder not only increases the number of active sites, but also promotes the diffusion of bacteria in the zeolite powder, diatomite and straw powder, thereby significantly improving the adsorption efficiency.

[0069] Comparative examples 1-3 changed the amount of material used, which weakened the synergistic effect of the pore structure of the three materials, and did not maximize the use, resulting in a decrease in porosity, specific surface area and bacterial survival rate. This shows that the optimal ratio can improve the adsorption capacity and mass transfer efficiency of the material and provide greater adsorption capacity and catalytic activity.

[0070] Comparative Example 4 The diatomite and straw powder are modified by acid, which can remove part of impurities and increase the specific surface area, but at the same time, the surface hydroxyl, carboxyl and other functional groups are also removed, and the reduction of these functional groups will lead to the decrease of the adsorption capacity of diatomite and straw powder.

[0071] Comparative Examples 5-7 Only one or two modified materials are used, and the prepared adsorption carrier has a large decrease in porosity, specific surface area and bacterial survival rate, which shows that only one or two modified materials cannot maximize the space synergy of the bacterial carrier, so that the porosity and specific surface area cannot reach the optimal effect, and the biocompatibility cannot be significantly improved.

[0072] In summary, it can be seen that only nano-enzyme-zeolite powder, PEG400-diatomite and PEG400-straw powder can synergistically increase the number of active sites at the optimal ratio, promote the diffusion of bacteria inside the zeolite powder, diatomite and straw powder, and significantly improve the adsorption efficiency and survival rate.

[0073] The above is only the preferred embodiment of the present application, and it should be pointed out that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. A microbial agent adsorption carrier, characterized in that, The adsorption carrier is composed of 25%-35% nanozyme-zeolite powder, 38%-40% PEG400-diatomite, and 27%-35% PEG400-straw powder, and the sum of the three is 100%. The nanozyme is pure iron oxide nanoparticles. The method for preparing the nanoenzyme-zeolite powder is as follows: zeolite powder is dispersed in a 1%-2% dilute acetic acid solution and stirred until uniformly dispersed to obtain a zeolite powder solution; nanoenzyme is dispersed in water to form a nanoenzyme dispersion with a concentration of 25-100 μg / mL; the zeolite powder solution and the nanoenzyme dispersion are mixed in a mass ratio of 3-7:1, stirred thoroughly, and spray-dried to obtain nanoenzyme-zeolite powder. The preparation methods of PEG400-diatomaceous earth and PEG400-straw powder are as follows: diatomaceous earth and straw powder are dispersed in acetic acid solution and stirred until evenly dispersed. Then, PEG400 is added and stirred until evenly dispersed. Glutaraldehyde, a crosslinking agent, is added to the resulting solution. A crosslinking reaction is carried out at 20-30℃. After the reaction is completed, the mixture is washed to remove unreacted PEG400 and crosslinking agent, and then dried at 95-105℃ to obtain PEG400-diatomaceous earth and PEG400-straw powder. The microbial agent adsorption carrier has a porosity of 95.6-96.1%, an adsorption efficiency of 77.8-82.5%, and a specific surface area of ​​145.3-156.2 m². 2 / g, when mixed with aerobic denitrifying bacteria liquid inoculant, provides space and oxygen environment for bacterial reproduction and survival. The prepared solid microbial inoculant has a long shelf life, with a microbial survival rate of 91.2-92.3% after 180 days of storage at 4℃.

2. A solid microbial inoculant, characterized in that, It consists of a microbial inoculant adsorbent carrier as described in claim 1 and an aerobic denitrifying bacteria liquid in a mass ratio of 1:8-12, wherein the concentration of the aerobic denitrifying bacteria liquid inoculant is 1.5 × 10⁻⁶. 8 -1.8×10 8 CFU / mL.

3. The method for preparing solid microbial inoculant as described in claim 2, characterized in that, Includes the following steps: Nano-enzyme-zeolite powder, PEG400-diatomaceous earth, and PEG400-straw powder were mixed in a certain proportion, and then aerobic denitrifying bacteria liquid inoculant was added for full contact and adsorption. The adsorption temperature was 20-28℃ and the adsorption time was 1-3 hours, and solid microbial inoculant was obtained by separation.

4. The method for preparing solid microbial inoculant according to claim 3, characterized in that, The preparation process of the aerobic denitrifying bacteria liquid inoculant is as follows: aerobic denitrifying bacteria isolated from the MABR device are inoculated into a liquid culture medium and activated for 40-50 h. The number of microorganisms is 1.0 at OD600, thus obtaining the aerobic denitrifying bacteria liquid inoculant.

5. The method for preparing solid microbial inoculant according to claim 3, characterized in that, The aerobic denitrifying bacteria liquid inoculum consists of *Paracococcus*, whose Latin name is... Paracoccus .

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