CN112094775A - Bacillus belgii and screening culture method and application thereof - Google Patents

Abstract

The invention provides a Bacillus belgii strain and a screening culture method and application thereof. The Bacillus velezensis is classified and named as Bacillus velezensis B3-4, and the strain is preserved in China center for type culture Collection in 29 months and 5 years 2020, and the preservation number is CCTCC NO: m2020160. The screening culture method comprises the following steps: collecting a sample and storing at low temperature; coating the diluent of the sample on a beef extract peptone culture medium by adopting a gradient dilution method, and culturing at the constant temperature of 15-50 ℃; after the culture medium grows out of the colonies, selecting the colonies with different forms for streak culture; subsequently, a loop of colonies was picked for repeated streaking with microscopic observation until a purified strain was obtained. The Bacillus belgii can effectively degrade and remove organic matters and COD in human excrement and domestic sewage.

Description

Bacillus belgii and screening culture method and application thereof

Technical Field

The invention relates to the technical field of environmental microorganisms, in particular to a bacillus beiLeisi and a screening culture method and application thereof.

Background

As early as 2000, the nation provided a strategic target for toilet modification, and toilet modification was used as an important project for environment modification and healthy human habitation.

As a product of 'toilet revolution', through continuous development, the currently applied environment-friendly toilets have two types of water flush and water-free flush, and due to the shortage of resources in most areas in China, in recent years, a great deal of attention is paid to the water-free biodegradable water flush-free toilet which can realize the functions of flushing-free, excrement recycling and the like. However, the technical emphasis is placed on the structural design of the equipment, and the resources of microbial strains in biological treatment are less involved. For example, patent application publication No. CN101658398A entitled box-type ecological toilet, which was published on 03/2010, describes that enterococcus faecalis, staphylococcus xylosus, bacillus cereus, corynebacterium ammoniagenes, bacillus licheniformis, and motile carnobacterium as degrading bacteria. Patent application publication No. CN103735211A, entitled Water-free ecological toilet, published in 04/23/2014, describes enterococcus faecalis, Staphylococcus xylosus, Cellulosiella, Fusarium, Bacillus licheniformis, and Bacillus mobilis as degrading bacteria. A microbial treatment microbial inoculum for treating human excrement and a preparation method and application thereof are disclosed in 2019, 06, 21 and the patent application publication with the publication number of CN109913389A describes that bacillus licheniformis, pichia pastoris and lactobacillus casei are degradation bacteria. The degrading bacteria provided by the patents are all composite strains, and the types of the degrading bacteria are single and fixed.

Therefore, in order to further enrich the strain resources treated by the water-free biodegradable ecological toilet feces, it is necessary to develop the domestication and screening of the organic matters and COD high-efficiency degrading strains of human feces and domestic wastewater.

Disclosure of Invention

The present invention aims to address at least one of the above-mentioned deficiencies of the prior art. For example, one of the purposes of the invention is to provide bacillus beijerinckii for efficiently degrading and removing organic matters and COD in human excrement and domestic sewage, and a screening culture method and application thereof.

In order to achieve the above object, one aspect of the present invention provides a strain of Bacillus belgii, which is classified and named as Bacillus belgii Bacillus velezensis B3-4, and is deposited in the chinese type culture collection center at 29 months and 5 months in 2020, with the deposit number being CCTCC NO: m2020160.

In an exemplary embodiment of a strain of bacillus belgii of the present invention, the bacillus belgii is a gram-positive bacterium, has spores, is rod-shaped, is motile, and is obligatorily aerobic.

In an exemplary embodiment of a strain of bacillus belgii of the invention, the 16S rDNA sequence of the bacillus belgii is shown in SEQ ID No. 1.

In an exemplary embodiment of the bacillus belgii strain, a colony formed after the bacillus belgii is cultured on a beef extract peptone medium for 24 hours is circular or irregular, and a colony after 48 hours is circular, white, 0.5-1 mm in diameter, irregular in edge and flat and moist.

In an exemplary embodiment of a strain of bacillus belgii of the invention, the bacillus belgii has stress tolerance properties and is capable of growing in a medium having a salt concentration of 0.5-4%.

In another aspect of the present invention, a screening culture method of bacillus belgii is provided, which comprises the following steps: collecting and storing samples at low temperature, wherein the samples comprise human excrement, livestock and poultry excrement, soil, water, sludge and organic fertilizer; coating the diluent of the sample on a beef extract peptone culture medium by adopting a gradient dilution method, and culturing at a constant temperature of 15-50 ℃; after the culture medium grows out of the bacteria, selecting bacterial colonies with different forms from a beef extract peptone flat plate for streak culture; subsequently, a loop of colonies was picked for repeated streaking with microscopic observation until a purified strain was obtained.

In still another aspect, the invention provides an application of the Bacillus belgii or its bacterial suspension or its culture solution or its fermentation product in degradation and removal of organic matter and COD in human excrement and domestic wastewater.

In a further aspect of the present invention, there is provided a biological agent which may comprise the above-mentioned Bacillus belgii or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof.

In still another aspect of the present invention, there is provided an application of the Bacillus belgii or the biological agent in treating organic matter and COD of human excrement and domestic wastewater.

In a further aspect of the invention there is provided the use of the Bacillus belgii or the biological agent in a biodegradable toilet.

Compared with the prior art, the invention has the advantages and beneficial effects that: the Bacillus velezensis B3-4 can effectively degrade and remove organic matters and COD in human excrement and domestic sewage; under pure culture conditions, after inoculation culture for 72 hours, the organic matter degradation rate of the Bacillus belgii on human excrement reaches more than 72.89%, the COD removal rate reaches more than 85.61%, and the treatment effect is obvious; the Bacillus velezensis B3-4 has strong stress resistance, can be used as an excellent treating bacterium for treating human excrement and domestic sewage, and has a good application prospect.

Drawings

The Bacillus belgii of the invention is preserved, and the preservation unit is as follows: china Center for Type Culture Collection (CCTCC), preservation address: in the Wuhan university school of Wuhan 299 in the Wuchang area of Wuhan city, Hubei province, the preservation date is as follows: 29 months 5 in 2020, the preservation number is CCTCC NO: m2020160.

The above and other objects and features of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings, in which:

FIG. 1A shows the microscopic form of Bacillus belgii according to an exemplary embodiment of the present invention; FIG. 1B shows the colony morphology of Bacillus beleisi on beef extract peptone medium;

FIG. 2 shows a phylogenetic diagram of the 16S rDNA sequence of Bacillus belgii in an exemplary embodiment of the invention.

Detailed Description

Hereinafter, Bacillus belgii of the present invention and its sieving culture method and application will be described in detail with reference to the accompanying drawings and exemplary embodiments.

The invention provides a Bacillus belgii strain, which is prepared by adopting the screening culture method. The strain is classified and named as Bacillus velezensis B3-4, is preserved in China Center for Type Culture Collection (CCTCC) in 5-29 th of 2020, and has the preservation number of CCTCC NO: m2020160.

In this example, Bacillus velezensis B3-4 is a gram-positive bacterium, has spores, and the bacterium is rod-shaped, motile, and obligately aerobic.

In this example, the 16S rDNA fragment was amplified by extracting the total DNA of strain B3-4, and the 16S sequence was shown in SEQ ID NO. 1. The measured 16S sequence was aligned at the National Center for Biotechnology Information (NCBI) database of the United states, and B3-4 showed the highest similarity (99.6%) to the model strain Bacillus velezensis CR-502T in GenBank, thereby confirming that B3-4 is classified as Bacillus velezensis B3-4.

In the embodiment, a bacterial colony formed after Bacillus belief (Bacillus velezensis) B3-4 is cultured on a beef extract peptone medium for 24 hours is circular or irregular, and a bacterial colony formed after 48 hours is circular, white (milky white), 0.5-1 mm in diameter, irregular in edge, flat and wet. In the embodiment, Bacillus velezensis B3-4 has stress resistance and can grow in a culture medium with a salt concentration of 0.5-4%.

In another aspect of the present invention, a screening culture method of bacillus belgii is provided, which comprises the following steps:

1) samples (e.g., human feces) are collected and cryopreserved, including human feces, livestock feces, soil, water, sludge, and organic fertilizers.

2) A predetermined amount (for example, 3 to 8g) of the dilution of the sample is applied to a beef extract peptone medium by a gradient dilution method, and incubated at a constant temperature of 15 to 50 ℃. Here, the sample is suitably used in an appropriate amount, and the temperature for cultivation may be adjusted depending on the temperature range of the environment to which the target strain is applied, for example, 5g of the sample may be used, 50 ℃ if the target strain is applied to a high temperature environment, 15 ℃ if the target strain is applied to a low temperature environment, and 30 ℃ if the target strain is applied to a normal temperature environment.

3) After the culture medium grows out of the bacteria, selecting bacterial colonies with different forms from a beef extract peptone flat plate for streak culture.

4) Subsequently (for example, after culturing for 16-18 h), a ring of colonies is picked for repeated streaking, and combined with microscopic observation until a purified strain is obtained, and the purified strain is inoculated into a test tube slant culture medium for preservation. The Bacillus velezensis B3-4 has strong degradation capability and can degrade and remove organic matters and COD (chemical oxygen demand) in human excrement and domestic wastewater.

Preferably, the bacterial suspension of Bacillus velezensis B3-4, or the culture solution thereof, or the fermentation product thereof also has strong degradation capability, and can effectively degrade and remove organic matters and COD in human excrement and domestic wastewater.

In yet another aspect of the invention, a biological agent is provided. In an exemplary embodiment of the biological agent of the present invention, the biological agent may comprise Bacillus belgii (Bacillus velezensis) B3-4 of the present invention or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof.

The biological agent of the invention also has strong degradation capability and can effectively degrade and treat organic matters and COD in human excrement and domestic wastewater.

The Bacillus velezensis B3-4 or the biological agent has good degradation application potential and can be applied to biodegradable toilets.

In order that the above-described exemplary embodiments of the invention may be better understood, further description thereof with reference to specific examples is provided below.

Example 1

(1) Separation, purification and preservation of Bacillus velezensis B3-4 (referred to as "strain B3-4")

Collected human fecal samples are stored at low temperature and taken back to the laboratory. Taking 5g of a fecal sample, coating the fecal sample on a beef extract peptone culture medium by adopting a gradient dilution method, and culturing at constant temperature of 30 ℃. After the colonies grow out, colonies with different forms are selected from the plate and streaked on a beef extract peptone plate. And (5) after culturing for 16h, picking one ring for repeated streak culture, and combining microscopic observation until purification. And inoculating the purified strain into a test tube slant culture medium of a beef extract peptone culture medium for storage.

As shown in FIG. 1, the separated and purified strain B3-4 of the present example was cultured on a beef extract peptone medium, and the colony was circular, white, 0.5-1 mm in diameter, irregular in edge, and flat and wet after 48 hours of culture. Wherein FIG. 1A shows the colony morphology and FIG. 1B is a schematic representation of colonies after streaking on beef extract peptone plates.

(2) Amplification and phylogenetic analysis of 16S rDNA of strain B3-4

Extracting total DNA of the strain, using the total DNA as a template, using 27F and 1492R as primers to amplify a 16S fragment, and using a Bio-RADMCyclerTM instrument for PCR reaction.

Reaction system (50 μ l): mu.l of 2 XPCRMix 25. mu.l each of primers 27F and 1492R (10. mu.M), 1. mu.l of DNA template, and made up to 50. mu.l with ultrapure water; the nucleotide sequences of primers 27F and 1492R are shown in SEQ ID No.2 and SEQ ID No. 3.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 1min, annealing at 54 deg.C for 1min, extension at 72 deg.C for 2min, and circulation for 30 times; final extension at 72 ℃ for 8 min.

The PCR amplification product was detected on 1.0% agarose gel electrophoresis and then sent to Shanghai Bioengineering Co., Ltd for sequence determination. The software DNA man6.0 was used to perform the calculation of gene sequence similarity. The sequencing result is shown in SEQ ID No.1 (sequence table).

The obtained sequence results were compared in the National Center for Biotechnology Information (NCBI) database, and it was found that the 16S rDNA gene sequence of B3-4 had the highest similarity of 99.6% to B3-4 in the database and the model strain Bacillus velezensis CR-502T in GenBank. Based on the NCBI comparison results, the model strain with the highest similarity was selected as the reference strain, and phylogenetic trees (as shown in FIG. 2) and self-developed values (bootstraps) were constructed using the Neighbor-joining method of MEGA 6.0 software (Neighbor-joining).

Based on the above characteristics, the strain B3-4 was identified as Bacillus belgii (Bacillus velezensis). The strain is stored in China center for type culture Collection in 29 th 5 th 2020, and the preservation number is CCTCC NO: m2020160.

(3) Experiment on capability of bacterial strain B3-4 in degrading organic matters and COD in human excrement

B3-4 is inoculated on LB liquid culture medium for activation, when the OD600 of the bacterial suspension for activation culture is 1.0, 5 percent of the bacterial suspension is inoculated into sterilized 100mL MS liquid culture medium with human excrement concentration of 30 percent (w: v), the MS liquid culture medium is subjected to shaking culture at 30 ℃ and 140rpm, and the culture is repeated for 3 times by taking no inoculation as a control.

And (3) measuring the capability of the strain B3-4 for degrading organic matters in excrement: drying filter paper at 80 deg.C to constant weight, weighing and recording (M1), filtering culture solution after culturing for 72 hr, drying filter paper and solid residue at 80 deg.C to constant weight, weighing and recording (M2); the control residue was weighed and recorded in the same way (M3). Human feces organic matter degradation rate (%) (M2-M1) × 100/(M3-M1).

The strain B3-4 has the capability of degrading the COD of human excrement: the culture solution after 72 hours of culture in the above test was collected, centrifuged at 1000rpm for 5min, the supernatant was collected, and the COD content thereof was measured and recorded (T1), and the initial COD content of the treated solution was measured and recorded in the same manner (T2). Human feces COD removal (%) (T2-T1) 100/T1.

The determination result shows that after 72 hours of culture, the degradation rate of organic matters in human excrement treated by inoculating the B3-4 strain reaches 72.89%, and the removal rate of COD reaches 85.61%.

(4) Resistance test of Strain B3-4

And (3) determining the salt tolerance of the strain B3-4: preparing a beef extract peptone culture medium, and adjusting the pH value to 7; adding NaC1 into basic beef extract peptone medium, and preparing culture medium with salt concentration of 0.5%, 1%, 2%, 4%, 8% and 10%. After sterilizing at 121 ℃ for 30min, adding the bacterial suspension to a newly configured culture medium according to 1% (v: v), repeating three times, placing the culture medium in a 30 ℃ constant temperature incubator, performing shake culture at the rotating speed of 150r/min for 48h, and measuring the number of bacteria by using an ultraviolet spectrophotometer under the wavelength lambda of 600.

Growth temperature Range determination of Strain B3-4: preparing a beef extract peptone culture medium, adjusting the pH value to 7, sterilizing at 121 ℃ for 30min, adding bacterial suspension to the newly-prepared culture medium according to 1% (v: v), placing the inoculated culture medium in a constant-temperature incubator with the temperature of 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃ respectively, carrying out shake culture at the rotating speed of 150r/min for 48h, setting 3 times at each temperature, and measuring the quantity of bacteria by using an ultraviolet spectrophotometer under the wavelength lambda of 600.

The result shows that the Bacillus beleisi B3-4 has good stress resistance and stronger salt resistance, and can grow in a beef extract peptone culture medium containing 4% NaCl; the growth temperature range is wide, and the growth can be carried out within the temperature range of 15-50 ℃.

(5) Application of strain B3-4 in biodegradation type toilet experiment

Inoculating B3-4 strain in LB liquid culture medium, performing shaking culture at 28 deg.C, culturing for 24h, inoculating 5% into biodegradable ecological toilet with bamboo residue as filling matrix, adding appropriate amount of water, stirring, and adding their excrement into 2 students every day. Weekly faecal additions were recorded. The total weight was measured weekly. The experimental period was 17 weeks with no inoculation treatment as a control.

The dry matter reduction rate can be calculated by the following formula: r ═ S + F + S0)/(S + F) × 100%; in the formula, S is the mass of the added bamboo slag; f is the total dry matter amount of the added excrement; s0 is the dry matter quantity of the final mixture of the excrement and the bamboo slag.

The results show that: after the inoculation treatment is carried out for 17 weeks, 243.54kg of excrement is added, 231.35kg of excrement is reduced, the human excrement reduction rate is 95%, compared with the human excrement reduction rate of a control treatment, the human excrement reduction rate is increased by 87.5%, and good degradation application potential is shown.

In conclusion, the Bacillus velezensis B3-4 can effectively degrade and remove organic matters and COD in human excrement and domestic sewage; under pure culture conditions, after inoculation culture for 72 hours, the organic matter degradation rate of the Bacillus belgii on human excrement reaches more than 72.89%, the COD removal rate reaches more than 85.61%, and the treatment effect is obvious; the Bacillus velezensis B3-4 has strong stress resistance, can be used as an excellent treating bacterium for treating human excrement and domestic sewage, and has a good application prospect.

Although the present invention has been described above in connection with the exemplary embodiments and the accompanying drawings, it will be apparent to those of ordinary skill in the art that various modifications may be made to the above-described embodiments without departing from the spirit and scope of the claims.

Sequence listing

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<120> Bacillus belgii strain and screening culture method and application thereof

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ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcagaca taaaaggtgg 180

cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240

caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300

acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360

acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420

agaacaagtg ccgttcaaat agggcggcac cttgacggta cctaaccaga aagccacggc 480

taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540

gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600

gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660

gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt 720

aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780

cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac 840

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gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

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Claims (10)

1. The Bacillus belgii is characterized in that the Bacillus belgii is classified and named as Bacillus velezensis B3-4, is preserved in China Center for Type Culture Collection (CCTCC) in 29 months of 2020, and has a preservation number of CCTCC NO: m2020160.

2. The Bacillus belgii of claim 1, wherein the Bacillus belgii is a gram-positive bacterium, has spores, is rod-shaped, is motile, and is obligatorily aerobic.

3. The Bacillus belgii of claim 1, wherein the 16S rDNA sequence of the Bacillus belgii is set forth in SEQ ID No. 1.

4. The Bacillus belgii of claim 1, wherein colonies formed by culturing the Bacillus belgii on a beef extract peptone medium for 24 hours are round or irregular, and colonies formed after 48 hours are round, white, 0.5-1 mm in diameter, irregular in edge and flat and moist.

5. The Bacillus belgii of claim 1, wherein the Bacillus belgii has stress tolerance properties and is capable of growth in a medium having a salt concentration of 0.5-4%.

6. The method for screening and culturing Bacillus belgii according to claim 1, wherein the screening and culturing method comprises the following steps:

collecting and storing samples at low temperature, wherein the samples comprise human excrement, livestock and poultry excrement, soil, water, sludge and organic fertilizer;

coating the diluent of the sample on a beef extract peptone culture medium by adopting a gradient dilution method, and culturing at a constant temperature of 15-50 ℃;

after the culture medium grows out of the bacteria, selecting bacterial colonies with different forms from a beef extract peptone flat plate for streak culture;

subsequently, a loop of colonies was picked for repeated streaking with microscopic observation until a purified strain was obtained.

7. Use of Bacillus belgii or its bacterial suspension or its culture solution or its fermentation product according to claim 1 for degrading and removing organic matter and COD in human feces and domestic wastewater.

8. A biological agent comprising Bacillus belgii or a bacterial suspension thereof or a culture solution thereof or a fermentation product thereof according to claim 1.

9. Use of the bacillus belgii of claim 1 or the biological agent of claim 8 for the treatment of organic matter and COD in human feces and domestic wastewater.

10. Use of the bacillus belgii of claim 1 or the biological agent of claim 8 in a biodegradable toilet.

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