阅读说明：本技术 寡养单胞菌的固定化方法及其制备的组合物 (Immobilization method of stenotrophomonas and composition prepared by same ) 是由 刘晓玲 高红杰 徐瑶瑶 王冠颖 宋晨 宋楠楠 吕纯剑 于 2019-12-09 设计创作，主要内容包括：本发明属于环境治理领域,涉及一株寡养单胞菌(Stenotrophomonas sp.)sp3的固定化方法及制备的组合物,所述固定化方法包括,将寡养单胞菌sp3接入液体培养基中,在28～32℃下,以130～150r/min的转速进行吸附固定,固定化初始pH值为7.0-9.0,吸附时间为37.5～40h,以200mL液体培养基计,菌体接种量为5.0％～10.0％,载体添加量为0.5～2.0g；其中,所述寡养单胞菌sp3的保藏编号为CGMCC No.18394。利用本发明的固定化方法,能够高效固定寡养单胞菌sp3,固定化的菌剂能够明显提高寡养单胞菌氧化黑臭水体中的S<Sup>2-</Sup>。(The invention belongs to the field of environmental management, and relates to an immobilization method of Stenotrophomonas sp3 and a prepared composition, wherein the immobilization method comprises the steps of inoculating Stenotrophomonas sp3 into a liquid culture medium, carrying out adsorption immobilization at the temperature of 28-32 ℃ and the rotating speed of 130-150 r/min, wherein the initial immobilized pH value is 7.0-9.0, the adsorption time is 37.5-40 h, the inoculation amount of thalli is 5.0-10.0% and the addition amount of a carrier is 0.5-2.0 g based on 200mL of the liquid culture medium; wherein the preservation number of the stenotrophomonas sp3 is CGMCC No. 18394. By the immobilization method of the present invention, it is possible toThe stenotrophomonas sp3 is efficiently immobilized, and the immobilized microbial inoculum can obviously improve the S in the stenotrophomonas black-smelly water body 2‑ 。)

1. A method of immobilizing stenotrophomonas sp3, comprising:

inoculating stenotrophomonas sp3 into a liquid culture medium, performing adsorption fixation at the temperature of 28-32 ℃ and the rotating speed of 130-150 r/min, wherein the initial immobilized pH value is 7.0-9.0, the adsorption time is 37.5-40 h, the inoculation amount of thalli is 5.0% -10.0% and the addition amount of a carrier is 0.5-2.0 g, preferably 1.0-1.5 g, based on 200mL of the liquid culture medium; wherein the preservation number of the stenotrophomonas sp3 is CGMCC No. 18394.

2. The immobilization method according to claim 1, wherein the method comprises carrying out adsorption immobilization at 30 ℃ and at a rotation speed of 140r/min, wherein the initial pH of immobilization is 8.0 to 8.1, the adsorption time is 37.4 to 37.5 hours, the inoculation amount of the cells is 5.0 to 6.0% and the addition amount of the carrier is 0.98 to 1.0g based on 200mL of the liquid medium.

3. The immobilization process according to claim 1 or 2, wherein the support is a zeolite, preferably artificial zeolite particles.

4. The immobilization method according to any one of claims 1 to 3, wherein the initial pH of immobilization is 8.1, the adsorption time is 37.4 hours, the inoculum size is 6.0%, and the artificial zeolite particles are 0.98 g.

5. The immobilization method according to any one of claims 1 to 4, wherein the multivariate regression equation of the immobilization method is:

Y＝82.23+1.54A+0.51B+1.65C+0.096D-3.14AB-3.06AC-1.93AD-2.73BC-2.31BD-1.66CD-4.62A²-4.05B²-5.78C²-4.75D²

wherein Y represents S^2-The oxidation rate, A is the initial pH value of immobilization, B is adsorption time in unit hours, C is the inoculum size in unit percent, and D is the carrier addition amount in unit grams.

6. The immobilization method according to any one of claims 1 to 5, wherein the liquid medium contains: 20g/L glucose, 10g/L peptone, 10g/L yeast extract powder, pH7.0.

7. A composition produced by the immobilization method according to any one of claims 1 to 6.

8. The composition of claim 7, wherein the composition is a microbial inoculum comprising a vector and stenotrophomonas sp3.

Technical Field

The invention belongs to the field of environmental management and relates to the immobilization of stenotrophomonas.

Background

The black and odorous phenomenon of the water body has complex causes, and relates to a plurality of processes such as physics, chemistry, biology and the like, and has more influence factors. Studies have shown S in overlying water^2-Is a key factor causing the water body to turn black. It is mixed with Fe in water²⁺、Mn²⁺、Cu²⁺The plasma metal ions are combined to accumulate and generate metal sulfides, so that the water body is blackened. Thus, S in the black and odorous water body^2-Oxidation to SO of stable valence state₄ ^2-Can avoid the phenomenon of blackening of the water body. S^2-The oxidation process of (A) is very slow in natural environment and mainly depends on indigenous Sulfur Oxidizing Bacteria (SOB) in natural water body, which makes S input from external source^2-Further enrichment in black and odorous water results in deterioration of water quality. S^2-The method for removing pollutants in black and odorous water mainly comprises aeration reoxygenation, sediment dredging, reinforced coagulation, microbiological method and the like. The microbiological method has the advantages of strong pertinence, no secondary pollution to the environment and the like, and has been successfully applied to the treatment of medium and small river channels. At present, some research works have been carried out on the development of microbial agents aiming at the treatment of black and odorous water, mainly including Acinetobacter (Acinetobacter), Lactobacillus (Lactobacillus), Bacillus (Bacillus) and Pseudomonas (Pseudomonas). However, these microbial agentsTakes non-sulfur oxidizing bacteria as main materials and concentrates on COD and NH in water body by microbial inoculum₃Research on removal effect of pollutants such as-N and TP, and rarely relates to a key pollutant S for blackening in black and odorous water^2-The removal of (3) affected the study.

Disclosure of Invention

The inventor of the present application uses inorganic sulfur (S)²)^-Screening out a strain capable of efficiently oxidizing S as a target pollutant^2-The indigenous sulfur-oxidizing bacteria are beneficial to the purification of the black and odorous water body. Meanwhile, the inventors have further searched for a method for immobilizing the bacterium in order to ensure the effect of the bacterium.

In a first aspect, the invention provides a strain of Stenotrophomonas sp3 with a accession number of CGMCC No. 18394.

In a second aspect, there is provided a method of immobilizing stenotrophomonas sp3 of the present invention, comprising:

inoculating stenotrophomonas sp3 into a liquid culture medium, carrying out adsorption fixation at the temperature of 28-32 ℃ and the rotating speed of 130-150 r/min, wherein the initial immobilized pH value is 7.0-9.0, the adsorption time is 37.5-40 h, the inoculation amount of the strain is 5.0% -10.0% and the addition amount of the carrier is 0.5-2.0 g, preferably 1.0-1.5 g, based on 200ml of the liquid culture medium.

Preferably, the method comprises the steps of carrying out adsorption fixation at the rotation speed of 140r/min at the temperature of 30 ℃, wherein the initial pH of the immobilization is 8.0-8.1, the adsorption time is 37.4-37.5 h, the inoculation amount of thalli is 5.0-6.0% and the addition amount of a carrier is 0.98-1.0 g in terms of 200ml of liquid culture medium; preferably, the support is a zeolite; more preferably artificial zeolite particles.

Most preferably, the initial pH of immobilization is 8.1, the adsorption time is 37.4h, the inoculum size is 6.0%, and the artificial zeolite particles are 0.98 g.

The artificial zeolite particles may be commercially available artificial zeolite particles.

Preferably, the multiple regression equation of the immobilization method is:

Y＝82.23+1.54A+0.51B+1.65C+0.096D-3.14AB-3.06AC-1.93AD-2.73 BC-2.31BD-1.66CD-4.62A²-4.05B²-5.78C²-4.75D²

wherein Y represents S^2-The oxidation rate, A is the initial pH of immobilization, B is the adsorption time in hours, C is the inoculum size in grams, and D is the carrier addition.

Preferably, the liquid medium contains: 20g/L glucose, 10g/L peptone, 10g/L yeast extract powder, pH7.0.

In a third aspect, there is provided a composition prepared using the immobilization method described above. Preferably, the composition is a bacterial agent comprising a vector and stenotrophomonas sp3.

In the present invention, "%" used in reference to the glucose concentration and the amount of inoculation represents mass%, unless otherwise specified.

The stenotrophomonas can efficiently oxidize S^2-Effectively reduce S in the water body^2-The content of the active carbon in the black and odorous water can be improved, and the black and odorous water has obvious treatment and purification effects.

By utilizing the immobilization method, stenotrophomonas sp3 can be efficiently immobilized, and the immobilized microbial inoculum can obviously improve the S in the stenotrophomonas black-smelly water body oxidized by the stenotrophomonas^2-。

The stenotrophomonas sp3 is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No.1 of Beijing republic of south China, the preservation number is CGMCC No.18394, and the preservation time is 2019, 8 and 19 days.

Drawings

FIG. 1: strain sp3 phylogenetic tree;

FIG. 2: temperature (A), initial pH (B), initial glucose concentration (C) and initial bacterial concentration (D) for growth of strain sp3 and S^2-The effect of oxidation rate;

FIG. 3: growth Curve and S of Strain sp3^2-An oxidation profile;

FIG. 4: change in inorganic sulfur concentration in each presence, a: experimental groups; b: a control group;

FIG. 5: strain sp3 vs S^2-The major pathway of biological oxidation of (a);

FIG. 6: the removal effect of the strain sp3 on pollutants in a water sample of Beijing east Shahe is achieved;

FIG. 7: influence of immobilized initial pH value on effect of microbial inoculum on black and odorous water body treatment (A: S)^2-The oxidation rate; b: COD and NH₃-removal rate of N and TP);

FIG. 8: influence of immobilization adsorption time on effect of microbial inoculum on treatment of black and odorous water body (A: S)^2-The oxidation rate; b: COD and NH₃-removal rate of N and TP);

FIG. 9: influence of inoculum size on black and odorous water treatment effect of microbial inoculum (A: S)^2-The oxidation rate; b: COD and NH₃-removal rate of N and TP);

FIG. 10: influence of the addition amount of the carrier on the effect of the microbial inoculum on treating black and odorous water (A: S)^2-The oxidation rate; b: COD and NH₃-removal rate of N and TP);

FIG. 11: a contour map and a response surface map; wherein the content of the first and second substances,

a: interaction of two factors of pH and time on S^2-A contour plot of oxidation rate effects;

b: interaction of two factors of pH and time on S^2-A response surface map influenced by oxidation rate;

c: interaction of two factors of pH and inoculum size on S^2-A contour plot of oxidation rate effects;

d: interaction of two factors of pH and inoculum size on S^2-A response surface map influenced by oxidation rate;

e: interaction of two factors of pH and carrier addition amount on S^2-A contour plot of oxidation rate effects;

f: interaction of two factors of pH and carrier addition amount on S^2-A response surface map influenced by oxidation rate;

g: time and inoculum size pairwise factor interaction pair S^2-A contour plot of oxidation rate effects;

h: time and inoculum size pairwise factor interaction pair S^2-A response surface map influenced by oxidation rate;

i: interaction of two factors of time and carrier addition amount on S^2-A contour plot of oxidation rate effects;

j: interaction of two factors of time and carrier addition amount on S^2-A response surface map influenced by oxidation rate;

k: interaction of two factors of inoculation amount and carrier addition amount on S^2-A contour plot of oxidation rate effects;

l: interaction of two factors of inoculation amount and carrier addition amount on S^2-A response surface map influenced by oxidation rate;

FIG. 12: scanning electron micrographs of zeolite particles before and after immobilization of the microbial inoculum, A: before fixing the adsorption microbial inoculum; b: after the adsorbed microbial inoculum is fixed.

Detailed Description

The present invention will be further illustrated by the following examples, but the present invention is not limited thereto.

Unless otherwise specified, the reagents and apparatus used in the following examples are conventional in the art and are commercially available. The methods used are conventional methods, and the person skilled in the art can clearly know how to carry out the described protocol and obtain the corresponding results according to the implementation details.