阅读说明：本技术 一种固定化菌剂及其制备方法和应用 (Immobilized microbial inoculum and preparation method and application thereof ) 是由 陈惠明 蒋自胜 翟德勤 余艳鸽 郑茂盛 李金波 孙小玲 于 2019-08-05 设计创作，主要内容包括：本发明涉及菌剂固定化技术领域,具体公开一种固定化菌剂及其制备方法和应用。所述固定化菌剂,包含不动杆菌和固定化载体。所述的固定化菌剂的制备方法,至少包括以下步骤：将所述聚乙烯醇溶于蒸馏水中,得到第一溶液；将所述天然高分子凝胶载体溶于蒸馏水中,得到第二溶液,并将所述第二溶液与所述第一溶液混合、灭菌,得到第三溶液；将灭菌后的活性炭加入到菌悬液中,静置吸附,将吸附后的菌悬液加入到所述第三溶液中,混合均匀,得到第四溶液；将所述第四溶液滴加到CaCl<Sub>2</Sub>的饱和硼酸溶液中,在3-5℃的条件下反应,过滤、洗涤、干燥得到固定化菌剂。本发明以包埋法制备固定化菌剂,方法简便,条件温和,稳定性好。(The invention relates to the technical field of microbial inoculum immobilization, and particularly discloses an immobilized microbial inoculum, and a preparation method and application thereof. The immobilized microbial inoculum comprises acinetobacter and an immobilized carrier. The preparation method of the immobilized microbial inoculum at least comprises the following steps: dissolving the polyvinyl alcohol in distilled water to obtain a first solution; dissolving the natural polymer gel carrier in distilled water to obtain a second solution, mixing the second solution with the first solution, and sterilizing to obtain the natural polymer gel carrierTo a third solution; adding sterilized activated carbon into the bacterial suspension, standing for adsorption, adding the adsorbed bacterial suspension into the third solution, and uniformly mixing to obtain a fourth solution; dropwise adding the fourth solution into CaCl 2 Reacting in saturated boric acid solution at 3-5 deg.C, filtering, washing, and drying to obtain immobilized bacteria agent. The invention prepares the immobilized microbial inoculum by an embedding method, and has the advantages of simple method, mild condition and good stability.)

1. An immobilized microbial inoculum, which is characterized in that: the Acinetobacter and the immobilized carrier are contained, the Acinetobacter is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number of the Acinetobacter is CGMCC NO. 16620.

2. The immobilized inoculant according to claim 1, wherein: the nucleotide sequence of the 16S rDNA of the acinetobacter has a sequence shown as SEQ ID NO. 1.

3. The immobilized inoculant according to claim 1, wherein: the immobilized bacteria agent adopts an embedding method to load bacteria in the bacteria suspension of the acinetobacter onto the immobilized carrier.

4. The immobilized inoculant according to claim 3, wherein: the mass ratio of the immobilized carrier to the bacterial suspension is 0.8-1.2:0.8-2, the immobilized carrier comprises polyvinyl alcohol and a natural polymer gel carrier in a mass ratio of 8-10:1-2, and the natural polymer gel carrier is sodium alginate or agar.

5. The immobilized inoculant according to claim 3, wherein: the bacterial suspension is obtained by culturing the acinetobacter to logarithmic growth phase, and the OD600 of the bacterial suspension is 0.60-0.63.

6. A method for producing the immobilized fungicide according to any one of claims 4 to 5, characterized in that: at least comprises the following steps:

step a, dissolving the polyvinyl alcohol in distilled water to obtain a first solution;

b, dissolving the natural polymer gel carrier in distilled water to obtain a second solution, mixing the second solution with the first solution, and sterilizing to obtain a third solution;

step c, adding the sterilized activated carbon into the bacterial suspension, standing for adsorption, adding the adsorbed bacterial suspension into the third solution, and uniformly mixing to obtain a fourth solution;

d, dropwise adding the fourth solution into CaCl₂Reacting in saturated boric acid solution at 3-5 deg.C, filtering, washing, and drying to obtain immobilized bacteria agent.

7. The method for preparing an immobilized microbial inoculum according to claim 6, wherein: in the step a, adding the polyvinyl alcohol into distilled water, and heating for 50-70min at the temperature of 85-95 ℃ to obtain the first solution with the concentration of 8-10 wt%; and/or

In the step b, adding the natural polymer gel carrier into distilled water, and heating for 15-25min at the temperature of 75-85 ℃ to obtain a second solution with the concentration of 1-2 wt%; and/or

In the step b, the sterilization conditions are as follows: the sterilization temperature is 120-: 15-25 min.

8. The method for preparing an immobilized microbial inoculum according to claim 6, wherein: in the step c, the solid-to-liquid ratio of the activated carbon to the bacterial suspension is 1: 5-6; and/or

In the step c, the standing adsorption time is 15-25 min.

9. The method for preparing an immobilized microbial inoculum according to claim 6, wherein: in step d, the CaCl₂In saturated boric acid solution of (1)₂The concentration of (A) is 3-5 wt%; and/or

In the step d, the reaction time is 22-26 h; and/or

In the step d, the drying temperature is 25-35 ℃.

10. The use of the immobilized microbial agent of claims 1-5 in the field of nitrogen-containing sewage treatment.

Technical Field

The invention relates to the technical field of microbial inoculum immobilization, in particular to an immobilized microbial inoculum and a preparation method and application thereof.

Background

The rapid development of industry and agriculture and the production activities of human beings bring serious pollution to the water environment. The exceeding of nitrogen is the most common pollution problem in water body pollution, which can cause eutrophication of water body, cause mass propagation of algae, reduce dissolved oxygen in the water body and cause black and odorous water body, thereby not only endangering the water safety of human beings, but also seriously influencing the ecological balance of the water body; in addition, excessive nitrogen discharge can cause the nitrogen balance to be destroyed, thereby causing the water body to lose the self-cleaning function. Therefore, the sewage rich in nitrogen can be discharged into the natural environment after the sewage is subjected to denitrification treatment and reaches the standard.

Biological denitrification is a denitrification method widely applied at present and has the advantages of good treatment effect, stable and reliable treatment process, convenient operation and management and the like. The traditional biological denitrification is completed by utilizing the aerobic autotrophic nitrification of nitrifying bacteria and the anaerobic heterotrophic denitrification of denitrifying bacteria, and the biological denitrification needs to provide two reactors with oxygen and oxygen respectively to carry out nitrification and denitrification respectively so as to achieve the aim of denitrification. Therefore, the reactor occupies a large space, the operation cost is high, and the process is complicated.

Meanwhile, because the biological denitrifying bacteria are easy to degrade and inactivate, how to solve the problem of preservation of the biological denitrifying bacteria is the first problem in production. With the development of immobilized microbial cell technology in the rise of 20 th 70 s, a new way for cell concentration and starter preservation is opened up. However, immobilization is not applicable to all microorganisms, and some strains may even have reduced activity after immobilization.

Disclosure of Invention

Aiming at the problems of high cost, complex process, reduction of strain activity by an immobilization process and the like of the existing biological denitrification, the invention provides an immobilized microbial inoculum.

And a preparation method of the immobilized microbial inoculum.

And the application of the immobilized microbial inoculum.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

an immobilized bacteria agent comprises acinetobacter and an immobilized carrier, wherein the acinetobacter is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, and the preservation number of the acinetobacter is CGMCC NO. 16620.

The immobilized microbial inoculum provided by the invention has the following advantages:

the invention saves the acinetobacter in the form of the immobilized microbial inoculum, solves the problem that the acinetobacter is difficult to preserve, ensures that the acinetobacter has higher physiological activity and stability, is beneficial to keeping high viable bacteria concentration and improving the speed of degrading nitrogen-containing sewage when in use, and has the advantages of convenient application, separation and repeated use.

The Acinetobacter provided by the invention is an aerobic denitrifying bacterium, the Acinetobacter is obtained by enrichment screening through a biological culture means, the Acinetobacter has the characteristic of playing a role in denitrification under aerobic conditions, and the aerobic denitrifying bacterium strain is adopted to replace the conventional anaerobic denitrifying bacterium, so that the nitrification and the denitrification can be carried out in one reaction when the biological denitrification treatment of sewage is carried out, thereby simplifying the sewage treatment process, reducing the capital construction operation cost and improving the biological denitrification effect, and the Acinetobacter has good application prospect in the aspect of nitrogen-containing sewage treatment.

Specifically, the newly discovered Acinetobacter is named Acinetobacter sp.A3, the strain has an excellent aerobic denitrification function, nitrate nitrogen can be used as a unique nitrogen source for metabolism, and the nitrate nitrogen is converted into a gaseous product through denitrification under the aerobic condition so as to achieve the aim of denitrification. The acinetobacter discovered in the embodiment of the invention is screened from urban black and odorous water, and is a denitrification strain with high-efficiency aerobic denitrification capability, which can be applied to urban black and odorous water treatment; in the process of carrying out sewage treatment on the acinetobacter, the aerobic reactor and the anaerobic reactor which are respectively provided with the nitrobacteria and the denitrifying bacteria can be combined into a one-step aerobic reactor which is simultaneously provided with the nitrobacteria and the acinetobacter, so that the sewage treatment process is simplified, the capital construction operation cost is reduced, and the biological denitrification effect is improved.

Preferably, the nucleotide sequence of 16S rDNA of Acinetobacter has a sequence as shown in SEQ ID No. 1.

The 16S rDNA gene sequence of the acinetobacter provided by the invention is shown as follows:

SEQ ID NO.1：

GGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATTCTGATCCGCGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGATCGGCTTTTTGAGATTAGCATCCTATCGCTAGGTAGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATGATGACTTGACGTCGTCCCCGCCTTCCTCCAGTTTGTCACTGGCAGTATCCTTAAAGTTCCCGACATTACTCGCTGGCAAATAAGGAAAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTATGTAAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTTACTATGTCAAGGCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAGTCTTGCGACCGTACTCCCCAGGCGGTCTACTTATCGCGTTAGCTGCGCCACTAAAGCCTCAAAGGCCCCAACGGCTAGTAGACATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCATGCTTTCGCACCTCAGCGTCAGTGTTAGGCCAGATGGCTGCCTTCGCCATCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCATCCTCTCCCACACTCTAGCTAACCAGTATCGAATGCAATTCCCAAGTTAAGCTCGGGGATTTCACATTTGACTTAATTAGCCGCCTACGCGCGCTTTACGCCCAGTAAATCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACAGAGTTAGCCGGTGCTTATTCTGCGAGTAACGTCCACTATCTCTAGGTATTAACTAAAGTAGCCTCCTCCTCGCTTAAAGTGCTTTACAACCATAAGGCCTTCTTCACACACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCGGATCATCCTCTCAGACCCGCTACAGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCGACTTAGGCTCATCTATTAGCGCAAGGTCCGAAGATCCCCTGCTTTCTCCCGTAGGACGTATGCGGTATTAGCATTCCTTTCGAAATGTTGTCCCCCACTAATAGGCAGATTCCTAA。

the length of the sequence is 1282bp, and the accession number of the gene sequence in GenBank is MK 163532. The gene sequence of the strain is uploaded to GenBank for comparison, and the comparison result shows that: the gene sequence of the strain has the highest similarity with the sequence of Acinetobacter. Therefore, the strain was judged to belong to Acinetobacter (Acinetobacter) in combination with morphological characteristics of the bacteria, and was named Acinetobacter sp.a 3.

Specifically, the strain Acinetobacter sp.a3 of the present example was deposited at 24/10/2018 in the common microorganism center of the china committee for culture collection of microorganisms (CGMCC), address: the preservation number of the Xilu-Shi is CGMCC NO. 16620.

Preferably, the immobilized bacteria agent is used for loading the bacteria suspension of the acinetobacter onto the immobilized carrier by an embedding method.

The application adopts an embedding method to directly utilize the acinetobacter to carry out loading, removes the complicated extraction step of the acinetobacter, and has convenient manufacture and low cost.

Preferably, the mass ratio of the immobilized carrier to the bacterial suspension is 0.8-1.2:0.8-2, the immobilized carrier comprises polyvinyl alcohol and a natural polymer gel carrier in a mass ratio of 8-10:1-2, and the natural polymer gel carrier is sodium alginate or agar.

Preferably, one of sodium alginate or agar is compounded with polyvinyl alcohol to fix the acinetobacter, the sodium alginate or the agar can enable the immobilized bacteria agent to be easy to form balls and have good mass transfer performance, the polyvinyl alcohol can increase the mechanical strength of the immobilized bacteria, if the ratio of the polyvinyl alcohol to the natural polymer gel carrier is too high, the internal structure of the immobilized bacteria agent is compact, the matrix transfer is influenced, and the activity of the acinetobacter is reduced; if the ratio of polyvinyl alcohol to the natural polymer gel carrier is too small, the particles are easily formed, but the mechanical strength is reduced.

Preferably, the bacterial suspension is a bacterial suspension obtained by culturing the acinetobacter to a logarithmic growth phase, and the OD600 of the bacterial suspension is 0.60-0.63.

The growth speed of the microorganism in the logarithmic growth phase is the maximum, the number of cells is increased exponentially, and the selection of the bacterial suspension in the logarithmic growth phase can ensure that the prepared immobilized microbial inoculum has high growth speed and high biological activity.

And measuring the absorbance value of the bacterial liquid at the wavelength of 600nm by using an ultraviolet spectrophotometer, recording as OD600, and verifying the content of the acinetobacter in the bacterial liquid by using the OD600 value so as to control whether the bacterial liquid is in a logarithmic growth phase.

Furthermore, the invention also provides a preparation method of the immobilized microbial inoculum. The preparation method at least comprises the following steps:

step a, dissolving the polyvinyl alcohol in distilled water to obtain a first solution;

b, dissolving the natural polymer gel carrier in distilled water to obtain a second solution, mixing the second solution with the first solution, and sterilizing to obtain a third solution;

step c, adding the sterilized activated carbon into the bacterial suspension, standing for adsorption, adding the adsorbed bacterial suspension into the third solution, and uniformly mixing to obtain a fourth solution;

d, dropwise adding the fourth solution into CaCl₂Reacting in saturated boric acid solution at 3-5 deg.C, filtering, washing, and drying to obtain immobilized bacteria agent.

Compared with the prior art, the preparation method of the immobilized microbial inoculum provided by the invention has the following advantages:

the immobilized microbial inoculum is prepared by an embedding method, and the method is simple, mild in condition and good in stability; the natural polymer gel carrier is first cross-linked with PVA to raise balling performance and mechanical strength, and then the natural polymer gel carrier and CaCl are utilized₂Calcium ions in the saturated boric acid solution form a chelate gel which is insoluble in water,thus, the microorganism is fixed in the gel, and the biological toxicity of the gelling agent boric acid to the microorganism in the immobilization process is reduced and the biological activity of the immobilized particles is improved by utilizing the characteristics of the natural polymer gel carrier.

Preferably, in step a, the polyvinyl alcohol is added into distilled water and heated at a temperature of 85-95 ℃ for 50-70min to obtain the first solution with a concentration of 8-10 wt%.

Preferably, the polyvinyl alcohol is dissolved under conditions such that the polyvinyl alcohol is more fully dissolved and the dissolution rate is increased.

Preferably, in step b, the natural polymer gel carrier is added into distilled water and heated at 75-85 ℃ for 15-25min to obtain a second solution with the concentration of 1-2 wt%.

Preferably, the natural polymer gel carrier is dissolved under conditions that allow the natural polymer gel carrier to be dissolved more sufficiently and increase the dissolution rate of the solvent.

Preferably, in step b, the sterilization conditions are: the sterilization temperature is 120-: 15-25 min.

And the third solution is sterilized, so that other bacteria are not introduced in the later process, the activity of the acinetobacter is further influenced, and even the effect of the microbial inoculum on treating the nitrogen-containing sewage is influenced.

Preferably, in step c, the solid-to-liquid ratio of the activated carbon to the bacterial suspension is 1: 5-6.

Preferably, in step c, the standing adsorption time is 15-25 min.

By utilizing the characteristics of porous active carbon, large specific surface area and strong adsorption capacity, after the active carbon is added into the bacterial suspension, acinetobacter is promoted to move to the active carbon, so that microorganisms are enriched on the surface of the active carbon, and a foundation is provided for preparing the immobilized microbial inoculum.

Preferably, in step d, the CaCl₂Middle CaCl of saturated boric acid solution₂The concentration is 3-5 wt%.

CaCl₂Saturated boric acid solution as cross-linking agent reacts with natural polymer gel carrier, if the concentration is low, CaCl is added₂The content is insufficient, the immobilized bacteria agent is easy to break and can not be reused, and the concentration is high, CaCl₂Too much content can affect the mass transfer of the immobilized microbial inoculum.

Preferably, in step d, the reaction time is 22-26 h.

Preferably, in step d, the drying temperature is 25-35 ℃.

Furthermore, the invention also provides application of the immobilized microbial agent in the field of nitrogen-containing sewage treatment.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a graph showing denitrification performance of an immobilized fungicide provided in example 1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.