CN113173647A

CN113173647A - Bacillus amyloliquefaciens MN-13 and application thereof

Info

Publication number: CN113173647A
Application number: CN202110450872.8A
Authority: CN
Inventors: 李红亚; 李术娜; 王树香
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2021-04-26
Filing date: 2021-04-26
Publication date: 2021-07-27
Anticipated expiration: 2041-04-26
Also published as: CN113173647B

Abstract

The invention discloses a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 with the preservation number of CGMCC No. 21504. The strain has good degradation effect on dyes such as active blue, chrome black T, Congo red and the like.

Description

Bacillus amyloliquefaciens MN-13 and application thereof

Technical Field

The invention relates to a strain, in particular to a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 and application thereof.

Background

With the wide application of synthetic dyes in the industries of textile, leather, food, paper and the like, the problem of environmental pollution caused by the production and use of dyes is becoming more serious. Most synthetic dyes are stable in property and extremely difficult to degrade under natural conditions, and some dyes have strong carcinogenic, teratogenic, mutagenic and other toxicity to human beings and animals. When the printing and dyeing wastewater enters the environment, the water system is seriously damaged, and meanwhile, the printing and dyeing wastewater also causes great harm to ecology and human beings along with water circulation. Therefore, the degradation of dyes in wastewater becomes an important issue in environmental regulations.

Among the various dye degradation methods, the microbiological treatment method is receiving much attention due to the advantages of low energy consumption, mild conditions, no secondary pollution, and the like. The dye-degrading microorganisms present a wide variety, mainly including fungi, bacteria, actinomycetes, algae, and the like. The dye degradation fungi mostly generate extracellular lignin degrading enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) through metabolism to realize degradation and detoxification of dyes. In 1999, Kim et al first discovered that the fungus Geotrichum candidum produces a new heme peroxidase, dye decolorization peroxidase (DyP). The peroxidase has broad-spectrum dye decolorizing effect, can decolorize 9 dyes, and especially has the most remarkable decolorizing effect on anthraquinone dyes. Subsequently, researchers successively found the presence of DyPs in bacteria. In addition, research shows that the bacterial laccase produced by bacillus, namely spore exoprotein CotA, has a remarkable decolorizing effect on various synthetic dyes. The Chinese patent application CN112522148A discloses a method for culturing high-performance composite flora by using printing and dyeing wastewater, wherein the composite flora comprises bacillus amyloliquefaciens, pseudomonas putida, shewanella decolorationis and enterococcus faecalis, and the degradation efficiency of different dyes can be improved. International patent application WO2021041603A1 discloses a microbial composition for treating wastewater, water or soil or farm animals, comprising Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus megaterium, etc., and a method for removing pollutants from waste or soil using these strains. However, these are directed to a combination of strains, and do not provide a single strain having a good effect on degradation of the dye.

Disclosure of Invention

The invention aims to provide a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 which has good degradation effect on dyes such as active blue, chrome black T, Congo red and the like.

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 provided by the invention is preserved in China general microbiological culture Collection center (CGMCC) at 12-18 months in 2020, and the preservation number is CGMCC No. 21504. The strain is separated from fresh cow dung and dung of a Simmental beef cattle breeding base of a second branch of an experimental farm of the North river agriculture university.

The invention has the beneficial effects that the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 has obvious decoloring effect on azo dyes and anthraquinone dyes in an inorganic salt culture medium, and the highest degradation rate on chrome black T and Congo red can reach 66.96 percent and 51.72 percent; the degradation rate of active blue can reach 81.77%, and the degradation and decoloration effects of triarylmethane dye crystal violet, aniline blue and malachite green are also achieved to a certain extent, and the degradation rate of the strain to aniline blue in a nutrient broth culture medium is obviously improved and can reach 66.3%. The full-wavelength scanning of an ultraviolet spectrometer and the analysis of the degraded crystal violet solution by an HPLC-MS/MS technology show that the bacterial strain MN-13 can destroy a conjugated system of triarylmethane by removing N-methyl, and degrade the crystal violet into micromolecular aromatic compounds such as mikkonene and the like. The bacillus has wide biological activity and strong stress resistance, can still ensure the biological activity under severe natural conditions, and is easy for industrial production. Compared with the defects of weak growth potential, long growth period, harsh enzyme production conditions and the like of fungi, the bacillus amyloliquefaciens provided by the invention has wider application prospect in the practical application of microbial degradation of dyes.

Drawings

FIG. 1 is an aniline blue decolorizing ring;

FIG. 2 shows the decolorizing and degrading effects of Bacillus amyloliquefaciens MN-13 on 10% dye wastewater (left: control group; right: test group);

FIG. 3 is a UV-visible full-wavelength scan of the dye wastewater after inoculation with strain MN-13;

FIG. 4 construction of phylogenetic tree of MN-13 strain based on the 16S rDNA sequence.

Detailed Description

The present invention will be further described below with reference to specific embodiments.

The materials involved in the test were as follows:

inorganic salt medium (g/L): CaCl₂·2H₂O 0.1，NaCl 1.0，MgSO₄·7H₂O 0.5,NH₄NO₃1.2，K₂HPO₄1.0,MnSO₄0.2(pH adjusted to 7.0).

NB medium (g/L): 10.0 parts of peptone, 3.0 parts of beef extract powder and 5.0 parts of sodium chloride (the pH value is adjusted to 7.2-7.6).

NA Medium (g/L): agar 20.0g was added to NB medium.

Aniline blue culture medium: NA medium containing 0.1g/L aniline blue.

Fermentation medium (g/L): soybean peptone 20.0, corn flour 20.0, MgSO₄0.3,CaCl₂·2H₂O0.2,Na₂HPO₄2.0，NaH₂PO₄1.0, pH 7.0. The above culture media are all sterilized at 121 deg.C under 100kpa for 15 min.

The bacillus amyloliquefaciens MN-13 is separated from fresh cow dung of a second division Simmental beef cattle breeding base of an experimental farm of the northern Hebei agricultural university, is stored in the agricultural waste resource utilization engineering research center of the northern Hebei agricultural university and is stored in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 12 months and 18 days in 2020, and the storage number is CGMCC No. 21504.

Example 1 Primary measurement of decolorizing Effect of Strain MN-13 on dye Aniline blue by decolorizing Ring method

The strain is inoculated into 50mL NB medium and cultured for 18h at 37 ℃ and 180 r/min. 5mL of seed liquid is taken and transferred into a fermentation medium (50mL/250mL) for fermentation for 60 hours, and a proper amount of fermentation liquid is absorbed and filtered by a 0.22 mu m filter membrane. The filtrate will be measured for its decolorizing effect on aniline blue plates using the tube-disc method.

The decolorizing effect of the strain MN-13 fermentation liquid on aniline blue plates is shown in figure 1: the diameter of the decolorizing ring is 33.7 cm.

Example 2 determination of enzyme Activity in Strain MN-13 fermentation broth

Taking a proper amount of fermentation liquor in a sterilized centrifugal tube, centrifuging at 4 ℃ and 6000r/min for 10min to remove thalli, and measuring the enzyme activities of peroxidase and CotA laccase in supernate. Respectively with MnSO₄And veratryl alcohol is taken as a substrate to measure the activity of the decolorizing peroxidase of the extracellular dye of the strain. The CotA enzyme activity was measured using 2,2' -azino-bis (3-ethylotha-6-sulfonic Acid) (ABTS) as a substrate.

At present, a great deal of research reports that exosporium protein CotA derived from bacillus, dye-decolorized peroxidase BsDyP and the like have obvious degradation effects on various dyes. Therefore, the activity of peroxidase and CotA enzyme in the fermentation liquor of the strain MN-13 is measured, and the result is shown in Table 1. From the enzyme activity measurement of the fermentation liquor of the strain MN-13, the strain can produce CotA laccase and extracellular enzymeA peroxidase. Wherein the peroxidase can simultaneously oxidize veratryl alcohol and MnSO₄The catalytic activity of the dye decolorization peroxidase is basically consistent with the property of dye decolorization peroxidase generated by bacillus subtilis reported by Min.

TABLE 1 determination of enzyme Activity in Strain MN-13 fermentation broths

EXAMPLE 3 Effect of Strain MN-13 on degradation of Single dyes

The strain is inoculated into 50mL NB medium and cultured for 18h at 37 ℃ and 180 r/min. 8mL of seed liquid is taken out and put in a sterilized centrifuge tube, centrifuged for 10min at 6000r/min at 4 ℃, the thalli are washed for 3 times by using sterile water, and the components of the culture medium are completely removed by centrifugation. The cleaned thallus is inoculated into an inorganic salt culture medium with the dye concentration of 3.5g/L and cultured at 37 ℃ and 180 r/min. 5mL of fermentation liquor is taken at intervals of 12h, 24h, 48h and 72h for detecting the decolorization rate. Meanwhile, blank control groups without inoculation are set, and each group is provided with 3 parallels.

Determination of decolorization ratio: 5mL of inorganic salt culture medium fermentation liquor containing the dye is centrifuged for 10min at 8000r/min, and supernatant is taken. The medium without the dye was zeroed and the absorbance of each fermentation broth was measured at the maximum absorption wavelength of each dye.

Decolorization rate eta ═ A0-At)/A0X 100%

(At is the absorbance of the dye after degradation, A0 is the absorbance of the control).

The degradation effect of the strain MN-13 on azo dyes and anthraquinone dyes in an inorganic salt culture medium is obviously better than that of triarylmethane crystal violet and malachite green (Table 2). In a dye inorganic salt culture medium with the pH value of 7, the strain has the best decolorization and degradation effects on anthraquinone dye active blue, and the decolorization rate can reach 81.77 percent; the azo dye shows excellent degradation effect, and the highest degradation rate of the azo dye on chrome black T and Congo red can reach 66.96 percent and 51.72 percent; however, the degradation effect on triarylmethane dyes is general, and the degradation rates of crystal violet and malachite green are 23.05% and 14.42%.

TABLE 2 decolorization ratio of MN-13 to dye at different times (% mineral salts medium)

By taking aniline blue as an object, the difference of the degradation effect of the strain MN-13 on triarylmethane dyes under different culture medium conditions is further researched, and the strain is found to have poor decolorization effect on the aniline blue in an inorganic salt culture medium, wherein the degradation rate is 16.8%. If the inorganic salt culture medium is replaced by the NB culture medium with rich nutrition, the degradation rate of the strain MN-13 to aniline blue can reach 66.3 percent. It is shown that the degradation of such dyes by the strains can be further improved by optimizing the culture conditions.

Example 4 decolorization and degradation effects of Strain MN-13 on dye wastewater

The dye wastewater is obtained from laboratories of the liquid crystal dye factories around the baoding, precipitated, filtered, diluted with inorganic salt culture medium containing 2% glucose to 10% dye wastewater content, and sterilized by moist heat at 121 ℃ for 15min for standby. Seed liquid of the strain MN-13 is sucked and inoculated into dye waste liquid with different dilution concentrations, and the dye waste liquid is cultured for 120 hours at 37 ℃ at 180 r/min. Meanwhile, blank control groups without inoculation are set, and each group is provided with 3 parallels. After the fermentation is finished, centrifuging at 6000r/min for 10min to obtain supernatant.

Observing the color change of the supernatants of the experimental group and the control group; the chemical oxygen demand (COD value) of the dye wastewater of the control group and the test group is respectively measured, and the removal rate of the COD value is calculated.

COD removal rate (COD0-CODt)/COD 0X 100%

(CODt is the COD value of the test group; COD0 is the COD value of the control group)

And (3) carrying out full-wavelength scanning (200-800 nm) on the supernatants of the experimental group and the control group by an ultraviolet-visible spectrometer, and inspecting the degradation and decoloration effects.

The effect is as follows: after 120h of culture, the color of the dye wastewater liquid of the experimental group is obviously lighter than that of the control group, and the decolorizing effect is obvious (figure 2). And the absorption intensity of an absorption peak at 500nm-650nm can be seen to be obviously reduced in the spectrum of the ultraviolet-visible full-wavelength scanning result (figure 3), which also indicates that the bacterial strain MN-13 has obvious decoloration and degradation effects on various dyes in the dye wastewater. The COD value removal rate was 42.7%.

EXAMPLE 4 isolation and screening of strains

Weighing 1g of cow dung sample, putting the cow dung sample into a 18X 180mm test tube filled with 9mL of sterile water, uniformly mixing, taking 1mL of cow dung sample into the next same test tube, and sequentially diluting to obtain bacterial suspensions with various concentrations. Respectively take 10^-4、10^-50.1mL of 2 dilution-gradient dilutions were plated on NA medium plates, and then inverted and incubated in a 30 ℃ incubator for 24 h. Picking single colonies with different shapes, transferring to the slant of NA culture medium, culturing at constant temperature of 30 deg.C for 24h, and storing in refrigerator at-4 deg.C for use. The colonies picked were streaked on NA medium plates to check for purity. The separated bacteria were spotted on aniline blue plates, incubated at 24 ℃ for 48 hours, and the size of the destaining circle was observed.

Transferring the strain with the largest decolorizing ring to NA slant, culturing at 37 deg.C for 24 hr, collecting, and storing in 4 deg.C refrigerator.

As a result: obtaining a strain with obvious p-aniline blue decoloration, and naming the strain as MN-13.

Example 5 species identification of Strain MN-13

The physiological and biochemical experiments are carried out by observing the shapes of thalli and colonies of the strains according to the relevant steps of microbiological experimental technology and common bacteria system identification manual.

Extracting strain DNA and amplifying 16S rDNA: extracting total DNA of the strain, and performing PCR amplification by adopting a universal primer 27f/1492 r. And purifying the PCR product by using a kit, and then sending the PCR product to Shanghai Biotech company Limited for sequencing. Similarity analysis and multiple comparison are carried out on the measured gene sequence and the corresponding sequence in the GenBank database, and a phylogenetic tree is constructed by adopting a Neighbor-Joining method.

As a result: colony characteristics of the 24h cultured strain MN-13: near-circular opaque colonies, irregular edges, surface wrinkles, central protrusions. The thalli are rod-shaped, gram-positive, and the size of the thalli is about 3.1 mu m multiplied by 0.9 mu m; the spore size is about 2.0 μm × 0.6 μm, oval, neutral, and non-swelling.

Physiological and biochemical characteristics: a standard strain ATCC23842 of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is selected as a reference strain, and the results of physiological and biochemical tests of the strain MN-13 are shown in Table 3.

TABLE 3 physiological and biochemical assay results for Strain MN-13

Note: "+" indicates positive, the greater the number, the more obvious the phenomenon; "-" indicates negative; "/" No test was performed

According to the colony, the thallus morphology and the physiological and biochemical characteristics, the strain MN-13 is identified to belong to the Bacillus (Bacillus sp.).

16S rDNA sequence analysis and phylogenetic tree of strain MN-13: the sequence length of the 16S rDNA is 1401 bp. The sequences were aligned with the corresponding sequences in the NCBI database by BLAST program, respectively, and phylogenetic trees were constructed using the standard strains with higher similarity (fig. 4). 15 standard strains with homology similarity of more than 98 percent with the 16S rDNA sequence of the strain MN-13 belong to the genus Bacillus. The homology with the 16S rDNA of the standard strain B.amyloliquefaciens DSM 7 can reach 99.95%. And (3) identifying the MN-13 strain as bacillus amyloliquefaciens (B) by combining the determination results of the colony, the thallus morphology and the physiological and biochemical characteristics of the strain MN-13.

Claims

1. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 with the preservation number of CGMCC No. 21504.

2. The use of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 according to claim 1 for degrading dyes.

3. Use according to claim 2, wherein the degradation dye is degradation of azo dyes, anthraquinone dyes or triarylmethane dyes.

4. Use according to claim 2, wherein the dye is one or more of chrome black T, Congo Red, reactive blue, Crystal Violet, Aniline blue, Malachite Green.

5. Use of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) MN-13 according to claim 1 for purifying dye-contaminated wastewater.

6. The use according to claim 5, wherein the dye-contaminated wastewater is a liquid crystal plant dye-contaminated wastewater.