Application of Bacillus subtilis and its microbial flocculant in gelatin wastewater

Abstract

The present invention belongs to the field of sewage treatment. The present invention provides a Bacillus subtilis MJ-1, which is deposited in the China Center for Type Culture Collection, with a deposit date of October 24, 2022, a deposit number of CCTCC M NO: 20221639, and an address of Wuhan University, Wuhan, China. The beneficial effects of the present invention are as follows: 1. The present invention screened a strain MJ-1 for efficient degradation of gelatin sewage from a natural environment. The Latin scientific name of the efficient degradation strain is Bacillus subtilis, and the strain has certain effects in the laboratory test and pilot stage of gelatin sewage treatment. The strain may be applied to the bioremediation project of gelatin sewage to promote the degradation of gelatin sewage. 2. In the laboratory test experiment of gelatin sewage degradation, the strain MJ-1 can achieve a COD degradation rate of 51.5% for gelatin sewage. At the same time, the strain also has a certain effect in the pilot experiment of gelatin sewage treatment, and the COD degradation rate can reach 47.1% within 4 days.

Description

Technical Field

[0001] The invention belongs to the field of sewage treatment, and specifically relates to application of Bacillus subtilis and a microbial flocculant thereof in gelatin sewage. Background Art

[0002] With the development of economy and science and technology, the gelatin industry has developed into one of the supporting industries for my country's economic development in recent years. The application range of gelatin is very wide, and its main products include edible medical gelatin, color roll gelatin, industrial gelatin, etc. The production process of gelatin includes several major processes such as degreasing, pickling, water washing and neutralization, and gelatin extraction. Each production process will produce a large amount of organic wastewater, and the discharged wastewater is very complex. High calcium, high acid, high chlorine, and high protein are the characteristics of gelatin wastewater. It is difficult to treat and the treatment cost is high. While enterprises produce gelatin in large quantities, they also produce a lot of gelatin wastewater. Improper treatment of gelatin wastewater will cause toxic organic matter and metal ions to be discharged into the natural environment, which will cause serious damage to the natural ecosystem, and will be enriched in animals, plants and humans to a large extent, seriously affecting biological health.

[0003] In response to the pollution problem of gelatin wastewater, researchers at home and abroad have used physical, chemical and biological remediation methods to treat gelatin wastewater and have made some progress, but gelatin wastewater treatment still needs to be studied continuously. The physical treatment method of gelatin wastewater is to use grids, screens and other equipment to filter and precipitate large particle pollutants in gelatin wastewater. Chemical treatment mainly relies on adding chemical reagents to gelatin wastewater, and through the adsorption, flocculation function or chemical reaction of chemical reagents, the pollutants in the wastewater are aggregated, settled or converted into other harmless substances. Common chemical reagents include polyaluminum chloride (PAC) and polyaluminum sulfide chloride (PAC-s). Bioremediation methods include biostimulation and bioenhancement. Bioenhancement is to add efficient degradation strains to gelatin wastewater, and utilize the organic matter in gelatin wastewater through the strain's own metabolic function, so as to achieve the purpose of degrading wastewater; biostimulation is to add nutrients or other supplementary ingredients to gelatin wastewater to stimulate the original microbial strains in the water body to utilize pollutants in wastewater, thereby degrading gelatin wastewater. Physical methods can treat suspended solids in gelatin wastewater, but due to the high cost of treatment equipment and process and the inability to remove metal ions and soluble organic matter in gelatin wastewater, it is generally used as a pretreatment method for gelatin wastewater treatment; chemical methods can only treat colloids and a small number of soluble pollutants in gelatin wastewater by adding chemical reagents, and are generally used as a pretreatment method for biochemical treatment; bioremediation methods are the most popular method for gelatin wastewater treatment due to their high efficiency in wastewater degradation, environmental protection, low cost, and no secondary pollutants. Whether it is bioaugmentation or biostimulation in bioremediation methods, microbial strains are the core focus in their degradation process.

[0004] At present, scholars at home and abroad have isolated some microorganisms that can degrade gelatin wastewater from the natural environment. The main bacterial genera are Pseudomonas, Actinomycetes, photosynthetic bacteria, etc. However, there are still few engineering cases of treating gelatin wastewater by microbial strains. The main reason is that the resources of microbial strains that can degrade gelatin wastewater are not very sufficient, the types of organic matter in gelatin wastewater that can be degraded by microbial strains are limited, and the degradation efficiency is not high enough. Therefore, it is necessary to find or screen more microbial strains that have a degrading effect on gelatin wastewater, and construct a microbial flora with higher and better degradation efficiency for gelatin wastewater through a combination of multiple strains. At the same time, it also accumulates sufficient microbial resources for bioremediation technology to treat gelatin wastewater and provides genetic resources for the construction of efficient engineering bacteria. Summary of the invention

[0005] In order to solve the above problems, in view of the current situation that the microbial strain resources for treating gelatin wastewater with bioremediation technology are insufficient and the degradation efficiency of gelatin wastewater microbial strains is low, the present invention provides an application of Bacillus subtilis and its microbial flocculant in gelatin wastewater.

[0006] The present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a Bacillus subtilis MJ-1, which is deposited in the China Center for Type Culture Collection, with a deposit date of October 24, 2022, a deposit number of CCTCC M NO: 20221639, and a deposit address of Wuhan University, Wuhan, China, with a postal code of 430072.

[0008] The strain was isolated from the sludge of the oxidation ditch of the third-phase sewage treatment of Shaoxing Water Treatment Development Co., Ltd. on Jiangbin Road, Ma'an Town, Keqiao District, Shaoxing City, Zhejiang Province; the bacterium is a Gram-positive bacterium, milky white on the LB plate, the colonies are oval, the colonies are large, the surface is dull and opaque, and it can grow on an 11% high-salt LB plate; the strain MJ-1 is short rod-shaped under a phase contrast microscope, with a size of (0.5-1.2)um×(2-6)um, motile, and the spores are columnar and mesophytic.

[0009] The second aspect of the present invention provides a method for preparing a microbial flocculant, comprising the following steps:

[0010] S1. Inoculate the Bacillus subtilis MJ-1 described in claim 1 into a beef extract peptone medium to obtain a seed solution;

[0011] S2, inoculating the seed solution of step S1 into LB liquid culture medium for fermentation to obtain a fermentation solution with flocculation activity, which is the microbial flocculant.

[0012] Preferably, the components of the LB liquid culture medium include: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, 2.5 g / L FeSO4·7H2O, 0.3 g / L MnSO4·H2O, 0.5 g / L (NH4)6Mo7O 24 ·4H2O, 1g / L ZnSO4·7H2O and deionized water.

[0013] Preferably, the fermentation culture conditions are: 37°C constant temperature shaking culture for 1 day, with a rotation speed of 180 rpm.

[0014] The third aspect of the present invention provides a microbial flocculant, which is obtained by the above method.

[0015] The fourth aspect of the present invention provides an application of a microbial flocculant in gelatin production wastewater, comprising the following steps:

[0016] Add 10L of microbial flocculant to every ton of gelatin production wastewater, treat it aerobically for 3-5 days, control the dissolved oxygen at 2-4mg / L, and keep the temperature at 37℃.

[0017] The beneficial effects of the present invention are:

[0018] 1. The present invention screened a highly efficient gelatin wastewater degradation strain MJ-1 from the natural environment. The highly efficient degradation strain has the Latin name Bacillus subtilis, and the strain has certain effects in the laboratory test and pilot stage of gelatin wastewater treatment. The strain has the possibility of being applied to the bioremediation project of gelatin wastewater to promote the degradation of gelatin wastewater.

[0019] 2. In a laboratory test of gelatin wastewater degradation, the strain MJ-1 has a COD degradation rate of 51.5%. The strain also has a certain effect in a pilot test of gelatin wastewater treatment, with a COD degradation rate of 47.1% within 4 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the growth of Bacillus subtilis MJ-1 on LB medium agar plates;

[0021] Figure 2 This is the growth status of Bacillus subtilis MJ-1 under a phase contrast microscope

[0022] Figure 3 is the growth curve of Bacillus subtilis MJ-1;

[0023] Figure 4 This is a graph showing the growth of Bacillus subtilis MJ-1 on an 11% high salt LB plate. DETAILED DESCRIPTION

[0024] The present invention is described in detail below with reference to specific embodiments.

[0025] The culture medium used in the present invention is as follows:

[0026] The components of the inorganic salt culture medium include: 1g / L K2HPO4, 1g / L KH2PO4, 1g / L (NH4)2SO4, 0.2g / LMgSO4·7H2O, 0.02g / L CaCl2, 1mL / L trace element solution, 1g / L glucose, 1g / L yeast extract, and gelatin wastewater.

[0027] The components of the solid selection culture medium include: 1g / L K2HPO4, 1g / L KH2PO4, 1g / L (NH4)2SO4, 0.2g / LMgSO4·7H2O, 0.02g / L CaCl2, 1mL / L trace element solution, 20g / L agar, and gelatin wastewater.

[0028] The LB medium components include: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, and trace element solution of 2.5 g / L FeSO4·7H2O, 0.3 g / L MnSO4·H2O, 0.5 g / L (NH4)6Mo7O 24 ·4H2O, 1g / L ZnSO4·7H2O, deionized water.

[0029] Example 1: Screening of highly efficient bacterial strains for degrading gelatin wastewater

[0030] The present invention obtains the highly efficient gelatin wastewater degradation strain MJ-1 by sampling, enriching and taming, primary screening and secondary screening of activated sludge from a sewage plant and surrounding contaminated soil. The specific steps are as follows:

[0031] Sample collection: The activated sludge and surrounding contaminated soil from the oxidation ditch process section of the third phase of sewage treatment of Shaoxing Water Treatment Development Co., Ltd., Jiangbin Road, Ma'an Town, Keqiao District, Shaoxing City, Zhejiang Province were collected and placed in sealed bags for storage at an appropriate temperature.

[0032] Activated sludge and soil enrichment and domestication. Weigh 5g of sludge and 5g of soil samples and mix them in an inorganic salt medium with organic pollutants in gelatin wastewater as the main carbon and nitrogen sources. Culture and domesticate them under 37℃ shaking (150rpm) and microaerobic (static) conditions. Continuously transfer the mixed culture to fresh inorganic salt medium for enrichment and domestication for 3 times. When the culture solution is observed to become turbid and the OD value is significantly increased, it is used as the basis for transfer. Take the mixed culture of the last domestication, dilute it into six gradient bacterial suspensions of 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6, and spread it on a solid selective medium agar plate, and place it in a constant temperature incubator at 25℃, 30℃, and 37℃ for 1-7 days. Screen colonies with different morphologies and continuously streak pure culture on LB medium agar plates, and obtain 3 primary screening strains under 37℃ shaking (150rpm) conditions.

[0033] The above three strains were transferred to LB liquid medium, cultured in a constant temperature shaker at 180 rpm and 37 °C for 1 day, and centrifuged (8000 rpm, 5 min) to collect the bacteria, and washed with sterile water for 3 times. After washing, it was diluted with sterile water to OD 600 =1.0. The gelatin wastewater was treated at a dosage of 5% (volume ratio), and the COD of the wastewater was regularly tested by potassium dichromate method. The strain with the best gelatin wastewater degradation effect, MJ-1, was obtained by rescreening according to the COD degradation rate of gelatin wastewater. Table 1 shows the degradation effect of the strain on gelatin wastewater. Under the laboratory conditions of 180rpm and 37℃, the final degradation rate of MJ-1 on gelatin wastewater can reach 51.5%.

[0034] Table 1 Degradation effect of MJ-1 strain on gelatin wastewater in the laboratory

[0035]

[0036] Strain identification. The strain MJ-1 was sent to Hangzhou Qingke Biological Company for sequencing, and then the 16SrDNA sequence was submitted to the NCBI database for sequence comparison analysis. The results showed that the strain MJ-1 had the highest homology with Bacillus subtilis, so the strain MJ-1 was determined to be Bacillus subtilis. The 16S rDNA was amplified using 16s bacterial universal primers 27F and 1492R, and the sequence length was 1378bp. The sequence has been uploaded to the Genbank database with the accession number OL672211. Figure 1 As shown, the strain is milky white on LB medium agar plate, the colonies are oval, the colonies are large, and the surface is dull and opaque; Figure 2 As shown, strain MJ-1 is short rod-shaped under phase contrast microscopy, with a size of (0.5-1.2)um×(2-6)um, motile, columnar spores, and mesophyll.

[0037] The sequence table of strain MJ-1 in this example is as follows:

[0038]

[0039]

[0040] Example 2: Growth curve determination of Bacillus subtilis MJ-1

[0041] Bacillus subtilis MJ-1 was transferred to LB liquid medium and cultured in a constant temperature shaker at 37°C and 180 rpm. Samples were taken every 1 hour. 600 Detect the absorbance at nm and draw a growth curve. Figure 3 ,The experimental results showed that strain MJ-1 was in a slow growth phase before 3 hours, then entered the logarithmic growth phase, 7-12 hours was the end of logarithmic growth, and 12-16 hours was the stable growth phase.

[0042] Example 3: Salt tolerance test of Bacillus subtilis MJ-1

[0043] In order to explore the resistance of Bacillus subtilis MJ-1 under high salinity conditions, its growth in LB culture medium with different salinities was investigated. By adjusting the amount of sodium chloride in the LB culture medium, culture medium with different salt concentrations was prepared, and then different dilution gradient bacterial suspensions of Bacillus subtilis MJ-1 were spread on various high-salt plates, cultured in a constant temperature incubator at 37°C for 24 hours, and the growth of the strain in culture medium with different salt concentrations was detected. The results showed that Bacillus subtilis MJ-1 is salt-tolerant and can grow well in salinity gradients of 1%, 3%, 5%, 7%, 9%, and 11%. Figure 4 The figure shows the growth status of the strain on 11% high salt LB plate.

[0044] Example 4: Gelatin wastewater treatment pilot test

[0045] In order to study the effect of Bacillus subtilis MJ-1 on gelatin wastewater degradation, the test scale was further expanded. 1 ton of wastewater from a gelatin company in Hangzhou, Zhejiang was taken, 10 L of MJ-1 bacterial solution was added at a 1% dosage, and aerobic treatment was carried out for about 4 days. The dissolved oxygen was controlled at about 2 mg / L, the temperature was 37°C, and COD was tested every 24 hours. Finally, the degradation effect of MJ-1 on gelatin wastewater reached 47.1%, and the specific test results are shown in Table 2.

[0046] Table 2 Degradation effect of MJ-1 strain on gelatin wastewater under pilot conditions

[0047]

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis MJ-1, deposited in China Center for Type Culture Collection, deposit date: October 24, 2022, deposit number: CCTCC M NO: 20221639, address: Wuhan University, Wuhan, China.

2. A method for preparing a microbial flocculant, characterized in that: The steps include: S1. Inoculate the Bacillus subtilis MJ-1 described in claim 1 into a beef extract peptone medium to obtain a seed solution; S2, inoculating the seed solution of step S1 into LB liquid culture medium for fermentation to obtain a fermentation solution with flocculation activity, which is the microbial flocculant.

3. The method for preparing a microbial flocculant according to claim 2, characterized in that: The components of the LB liquid culture medium include: 10 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl, 2.5 g / L FeSO4•7H2O, 0.3 g / L MnSO4•H2O, 0.5 g / L (NH4)6Mo7O 24 •4H2O, 1g / L ZnSO4•7H2O and deionized water.

4. The method for preparing a microbial flocculant according to claim 2, characterized in that The fermentation culture conditions are: 37°C constant temperature shaking culture for 1 day, the rotation speed is 180rpm.

5. A microbial flocculant, characterized in that: The microbial flocculant is obtained by the preparation method according to any one of claims 2 to 4.

6. The use of a microbial flocculant in gelatin production wastewater according to claim 5, characterized in that: The steps include: Add 10L of microbial flocculant to every ton of gelatin production wastewater, treat it aerobically for 3-5 days, control the dissolved oxygen at 2-4mg / L, and keep the temperature at 37℃.

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