CN108977398B - Bacillus megaterium and application thereof - Google Patents
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Abstract
The invention belongs to the field of microbial application, and particularly relates to bacillus megaterium and application thereof. The specific technical scheme is as follows: a bacillus megaterium strain is preserved in China general microbiological culture Collection center in 2018, 6 months and 11 days, and the preservation addresses are as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the strain name is Bacillus megaterium DFN1, and the preservation number is CGMCC No. 15925. The bacillus megaterium provided by the invention has an excellent treatment effect on common malodor source substances, particularly ammonia gas, hydrogen sulfide, VOC and the like. The invention also provides a deodorizing liquid prepared by utilizing the bacillus megaterium, the deodorizing effect aiming at VOC can reach more than 95%, the deodorizing duration is long, the deodorizing liquid is obviously superior to other similar products sold in the existing market, and the deodorizing liquid can be practically popularized and applied.
Description
Technical Field
The invention belongs to the field of microbial application, and particularly relates to bacillus megaterium and application thereof.
Background
With the development of socio-economy, the requirements of people on the environment in which life is located are increased. As an important environmental pollutant, the malodorous gas has various substances and large influence range, and the treatment of the malodorous gas is gradually paid attention to by people. A large amount of odor-causing microorganisms exist in the livestock manure and the domestic garbage, and a large amount of ammonia gas and hydrogen sulfide are generated in the garbage landfill and composting process, so that the method not only causes serious harm to human bodies, but also pollutes the environment, underground water sources and the like.
The current conventional deodorization methods are: physical deodorization, chemical deodorization, biological deodorization. The physical deodorization method eliminates the odor by means of conversion among solid, liquid and gas phases, only reduces the perception degree of smell by smell, but does not change the chemical properties of the odor fundamentally. Chemical deodorization is the addition of certain chemical agents that change their chemical structure to destroy their odoriferous groups, converting them into odorless or less odoriferous substances. The biological method is a novel malodor treatment method developed in recent years, and a mainstream method of deodorization is being developed gradually because of advantages such as low investment and running costs, high treatment efficiency, no secondary pollution, and the like.
In the deodorizing method for compost plants and refuse landfills, the problems caused by chemical sterilization and deodorization can be solved by adopting biological bacteriostasis and deodorization, and no peculiar smell, no secondary pollution and the like are realized. The biological antibacterial deodorization utilizes the strains to have rapid propagation speed and long survival time, can also produce other beneficial bacterins in the growth process, has no harm to people and livestock, does not pollute the environment, has continuous and strong antibacterial deodorization effect, is a better choice for environmental purification such as compost deodorization, public toilet deodorization, refuse landfill and the like, and can be widely popularized and applied.
House decoration and the like generate a large amount of VOC (volatile organic compounds), which also belongs to a source of malodor and has great influence on human health, and can damage liver, kidney, brain, nervous system and the like, thereby causing serious harm. However, the research on microorganisms for VOC treatment is still in the primary stage at present, and has less applicable microorganisms and undesirable treatment effect.
Disclosure of Invention
The invention aims to provide a bacillus megaterium and application thereof.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a bacillus megaterium strain is preserved in China general microbiological culture Collection center in 2018, 6 months and 11 days, and the preservation addresses are as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the strain name is Bacillus megaterium DFN1, and the preservation number is CGMCC No. 15925.
Correspondingly, the sequencing result of 16Sr DNA of the bacillus megaterium is shown as SEQ ID NO 1.
Accordingly, the application of the bacillus megaterium in treating malodorous substances.
Preferably, the bacillus megaterium is used for treating ammonia gas or hydrogen sulfide.
Preferably, the bacillus megaterium is prepared into a deodorant, and the number of viable bacteria in the deodorant is 1 × 109CFU/ml。
Preferably, the amount of deodorant is 50ml/m2。
Preferably, the use of Bacillus megaterium for the treatment of VOCs.
Preferably, the bacillus megaterium is prepared into a deodorant, and the number of viable bacteria in the deodorant is 1 × 109CFU/ml。
Preferably, the amount of deodorant is 100ml/m2。
The invention has the following beneficial effects:
1. the bacillus megaterium provided by the invention has an excellent treatment effect on common malodor source substances, particularly ammonia gas, hydrogen sulfide, VOC and the like.
2. The invention also provides a deodorizing liquid prepared by utilizing the bacillus megaterium, the deodorizing effect aiming at VOC can reach more than 95%, the deodorizing duration is long, the deodorizing liquid is obviously superior to other similar products sold in the existing market, and the deodorizing liquid can be practically popularized and applied.
Detailed Description
The media referred to herein are as follows:
1. preparation of Vickers salt solution (g/L): k2HPO4 5.0，MgSO4·7H2O 2.5，NaCl 2.5，FeSO4·7H2O 0.05g，MnSO4 0.05g，pH＝7.0。
2. Enrichment medium (g/L): (NH)4)2SO42.0, sodium succinate 13.85, sodium acetate 5.0, glucose 5.0, vickers' salt solution 50mL, pH 7.0.
3. Isolation medium (g/L): (NH)4)2SO40.47, 5.62 parts of sodium succinate and 50mL of Vickers' salt solution, wherein the pH value is 7.0.
4. Separating a solid culture medium: adding 2.0 agar based on the separated culture medium, and sterilizing at 121 deg.C for 30 min. Is used for the research of the separation and purification and the deamination characteristics of heterotrophic nitrifying bacteria.
5. Beef extract peptone medium: 5g of beef extract, 10g of peptone and 5g of NaCl, adding water to 1L, adjusting the pH to 7, sterilizing at 121 ℃ for 30 min.
6. Beef extract solid medium: adding agar of 2.0% on the basis of beef extract peptone culture medium, and using for morphological observation, preservation and activation of the strain.
7. Liquid expanded fermentation medium: 5g of beef extract, 10g of glucose, 5g of peptone and 5g of sodium chloride, and adding water to 1000ml, wherein the pH value is 7.0-7.4.
The first embodiment is as follows: collection, isolation and identification of strains
1. Collecting and separating strains:
(1) the enrichment sample is collected from activated sludge of a secondary sedimentation tank of a sewage treatment plant in Sichuan. Mixing the collected activated sludge and compost sample, acclimatizing with enrichment medium under aerobic condition for 5 days per generation and 4 generations for 20 days, and gradient diluting from 10-2、10-3、10-4、10-5、10-6、10-7、10-8Sucking 0.25mL under dilution, respectively coating on a separation culture medium plate, culturing at constant temperature of 30 ℃, and separating for multiple times to obtain multiple colonies.
(2) Selecting 14 strains with different forms and vigorous growth, carrying out repeated streak culture on the 14 strains for more than three times at the temperature of 30 ℃ and the rotating speed of 160r/min to obtain pure strains, wherein the pure strains are respectively numbered as follows: DNF1-14 strain, cultured on beef extract solid medium for 24h and stored at 4 ℃.
(3) Respectively culturing 14 strains by using a beef extract solid culture medium, respectively inoculating 1ml of the cultured strains into 1L beakers filled with 100g of excrement, acting for 3d at the temperature of 30 ℃, and respectively measuring the odor intensity, the ammonia gas concentration and the hydrogen sulfide concentration in the 14 beakers. Wherein, the odor concentration in the beaker inoculated with the strain No.1 is reduced by 46.5 percent, the ammonia gas concentration is reduced by 55.6 percent, the hydrogen sulfide concentration is reduced by 52.4 percent and is far higher than that of other 13 strains, and the strain is initially judged to have the strongest deodorization capacity and is named as DNF 1.
2. Identification of strains
(1) Morphological characteristics: the observation and detection show that the cell is a long rod-shaped, gram-positive bacterium, has the body length of 1.2-4.0 mu m, can move, is cultured at 37 ℃ for 2-3 days to form spores, has the size of 0.6-1.5 mu m, and is oval. The colony surface is smooth and opaque, and is dirty white. Specific identification features are shown in table 1.
TABLE 1 results of physiological and biochemical tests of DNF1 strain
| Detecting items | The result of the detection | Detecting items | The result of the detection |
| Gram stain | G+ | Milk peptone | + |
| Shape of cell | Long rod shape | Gel liquefaction | + |
| Catalase test | + | Starch hydrolysis | + |
| Movement property | + | Oxidase test | + |
| Nitrate reduction test | - | Good/anaerobic property | Aerobic |
| Glucose | + | Mannitol | + |
| L-arabinose | + | 4％NaCl | + |
| Xylose | + | 50℃ | - |
Attached: "G +" indicates that the bacterium is gram positive, "+" indicates that it is available or can grow, "-" indicates that it is unavailable or cannot grow.
(2) Molecular biological identification: extracting the genome DNA of the strain, and carrying out PCR amplification on a 16S rDNA fragment. The obtained 16SrDNA sequence product is sequenced by Chengdu Ke bioengineering technology service company Limited.
Primers for amplification of 16 SrDNA:
F-primerF27：5’-AGA GTT TGA TCC TGG CTC AG-3’；
R-primerR1492：5’-GGC TAC CTT GTT ACG ACT T-3’。
the PCR reaction system is as follows: 2 × mix: 12.5 μ L, F-PrimerF 27: 1.0 μ L, R-primer R1942: 1.0 μ L, DNA genome: 1.0 μ L of μ L, dd H2O：9.5μL。
The PCR reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 100s, 29 cycles, final extension at 72 deg.C for 5min, and storage at 4 deg.C. mu.L of the PCR reaction product was subjected to 1% agarose gel electrophoresis.
The sequence determined is shown in SEQ ID NO 1. And logging in an NCBI website, and comparing and analyzing nucleic acid data in Genbank by a Blast program, wherein the comparison result shows that the strain is Bacillus megaterium.
The strain is preserved in China General Microbiological Culture Collection Center (China General Microbiological Culture Collection Center) in 6.11.2018 at the preservation addresses of: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the strain name is Bacillus megaterium (Bacillus megaterium) DFN1, and the preservation number is CGMCC No. 15925.
Example two: the deodorizing effect of the strain on the malodorous substances is shown
Inoculating the separated and purified strain into a beef extract peptone culture medium according to the inoculation amount as an inoculating loop, and culturing at 35 ℃ for 24 hours to obtain a seed solution.
1. Feces deodorization:
(1) inoculating the seed solution into the expanded fermentation medium according to the inoculum size of 1% of the volume of the expanded fermentation medium, and performing shake culture at 35 ℃ and 160rmp for 24h to obtain an inoculation solution.
(2) Inoculating 5ml, 10ml, 25ml and 50ml of the inoculation liquid into a 1L beaker filled with 500g of excrement (the inoculation amount is 1.0%, 2.0%, 5.0% and 10.0% V/W respectively), and sealing the beaker by using a preservative film. The same treatment without adding the bacterial liquid and with the same amount of clear water was performed at the same time to serve as a control group. Three replicates per set were set up, maintaining a temperature of 30 ℃. After 5d, the odor intensity, ammonia gas concentration and hydrogen sulfide concentration were measured, and the measurement results are shown in table 2.
TABLE 2 demonstration of deodorizing Effect of the strains on feces
(3) As can be seen from Table 2, the deodorizing effect of feces treated with the bacterial solution of Bacillus megaterium DFN1 added in different inoculum sizes for 5 days showed a tendency of increasing first and then decreasing with the increase of the inoculum size. Wherein, when the inoculation amount is 5 percent, the deodorization effect is best, the odor intensity is reduced by 45.9 percent, the ammonia gas concentration is reduced by 65.4 percent, and the hydrogen sulfide concentration is reduced by 63.5 percent.
2. Deodorizing the refuse landfill:
(1) the inoculation liquid in the excrement deodorization is utilized, the environmental temperature is 30-35 ℃ in a certain refuse landfill of a metropolis, and the inoculation liquid prepared in the excrement deodorization is diluted by clear water until the bacterial concentration is about 1 multiplied by 109CFU/ml to obtain deodorizer, and mixing the deodorizer at a ratio of 50ml/m2Spraying uniformly; the control group was sprayed with an equal amount of clear water. The ammonia concentration and the hydrogen sulfide concentration in the composting plant before and after spraying at the 5 th day were measured, and the measurement results are shown in Table 3.
TABLE 3 deodorization Effect of the strains on landfill
(2) As can be seen from Table 3, after the inoculation liquid is sprayed in different positions of the sewage treatment plant for 5 days, the odor intensity, the ammonia concentration and the hydrogen sulfide concentration are obviously reduced compared with those before spraying, wherein the ammonia concentration is reduced by 52.6% and the hydrogen sulfide concentration is reduced by 42.8% in the lifting station; the ammonia concentration of the primary sedimentation tank is reduced by 58.2 percent, and the hydrogen sulfide concentration is reduced by 38.9 percent; the ammonia concentration of the sludge concentration tank is reduced by 55.4 percent, and the hydrogen sulfide concentration is reduced by 48.6 percent.
(3) In addition, the inventor adds common plant deodorization components such as catechol and resorcinol extracted from plants, inorganic salts such as citrate and yeast composite microbial agents into the deodorant respectively, and performs deodorization tests under the same conditions, and finds that when the bacteria concentration is the same, the deodorant can achieve the equivalent deodorization effect by adding other biologically acceptable common auxiliary materials and beneficial compound bacteria.
Example three: bacterial strain for displaying VOC gas treatment effect
(1) Using the inoculum prepared in example two, it was diluted to a viable count of 1X 109CFU/ml, and preparing into deodorant solution. Uniformly spraying deodorant solution as treatment group, clear water of the same amount as blank control group, and commercially available decoration pollutant spraying treatment agent as positive control group to newly-decorated closed room with spraying amount of 100ml/m2And respectively measuring and calculating the VOC removal rate in the room before spraying and after spraying for 0.5h, 2h, 12h and 48 h.
Wherein the removal rate is (VOC concentration before spraying-VOC concentration at the time of measurement)/VOC concentration before spraying × 100%. The results are shown in Table 4.
Table 4 demonstration of VOC treatment effect by strain
| Group of | 0.5h | 2h | 12h | 48h |
| Treatment group | 91％ | 96％ | 85％ | 80％ |
| Positive control group | 67％ | 69％ | 32％ | 28％ |
| Blank control group | 2％ | / | / | / |
(2) As can be seen from Table 4, compared with the common commercially available finishing pollutant spray treatment agent, the deodorizing liquid provided by the invention has long duration of the effect on VOC treatment and obvious treatment effect.
(3) In addition, the inventor adds common plant deodorization components such as catechol and resorcinol extracted from plants, inorganic salts such as citrate, yeast and other composite microbial agents into the deodorant respectively, and performs deodorization tests under the same conditions, and finds that when the bacteria concentration is the same, the deodorant can achieve the equivalent deodorization effect by adding other biologically acceptable common auxiliary materials and beneficial compound bacteria.
Sequence listing
<110> Youdoujia environmental protection equipment engineering Co., Ltd
<120> bacillus megaterium and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1451
<212> DNA
<213> Bacillus megaterium (Bacillus megaterium)
<400> 1
agcatggcgg gtgctataca tgcagtcgag cgaactgatt agaagcttgc ttctatgacg 60
ttagcggcgg acgggtgagt aacacgtggg caacctgcct gtaagactgg gataacttcg 120
ggaaaccgaa gctaataccg gataggatct tctccttcat gggagatgat tgaaagatgg 180
tttcggctat cacttacaga tgggcccgcg gtgcattagc tagttggtga ggtaacggct 240
caccaaggca acgatgcata gccgacctga gagggtgatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360
acggagcaac gccgcgtgag tgatgaaggc tttcgggtcg taaaactctg ttgttaggga 420
agaacaagta cgagagtaac tgcttgtacc ttgacggtac ctaaccagaa agccacggct 480
aactacgtgc cagcagccgc ggtaatacgt aggtggcaag cgttatcccg gaattattgg 540
gcgtaaagcg cgcgcaggcg gtttcttaag tctgatgtga aagcccacgg ctcaaccgtg 600
gagggtcatt ggaaactggg gaacttgagt gcagaagaga aaagcggaat tccacgtgta 660
gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcggctt tttggtctgt 720
aactgacgct gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttaga gggtttccgc cctttagtgc tgcagctaac 840
gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
ggtcttgaca tcctctgaca actctagaga tagagcgttc cccttcgggg gacagagtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttgatcttag ttgccagcat ttagttgggc actctaaggt gactgccggt 1140
gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta 1200
cacacgtgct acaatggatg gtacaaaggg ctgcaagacc gcgaggtcaa gccaatccca 1260
taaaaccatt ctcagttcgg attgtaggct gcaactcgcc tacatgaagc tggaatcgct 1320
agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg 1380
tcacaccacg agagtttgta acacccgaag tcggtggagt aaccgtaagg agctagccgc 1440
taagtgacag t 1451
Claims (4)
1. A bacillus megaterium strain is characterized in that: in 2018, 6 and 11 months, the culture is preserved in the China general microbiological culture Collection center with the preservation addresses as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the strain name is Bacillus megaterium DFN1, and the preservation number is CGMCC No. 15925.
2. Use of bacillus megaterium for the treatment of malodorous substances as claimed in claim 1, wherein: application of bacillus megaterium in treating volatile organic compounds.
3. Use of bacillus megaterium for the treatment of malodorous substances as claimed in claim 2, wherein: preparing the bacillus megaterium into a deodorant, wherein the number of viable bacteria in the deodorant is 1 multiplied by 109CFU/mL。
4. Use of bacillus megaterium for the treatment of malodorous substances as claimed in claim 3, wherein: the dosage of the deodorant in the substance to be treated is 90-110 mL/m2。
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| CN110106120B (en) * | 2019-05-29 | 2021-02-23 | 浙江省农业科学院 | Microbial deodorizing microbial inoculum |
| CN110157644B (en) * | 2019-05-29 | 2021-07-16 | 浙江省农业科学院 | Application of microbial deodorizing bactericide |
| CN111647534A (en) * | 2020-06-16 | 2020-09-11 | 科里思特(福建)生物科技有限公司 | Bacillus megaterium, preparation and application thereof |
| CN114480181B (en) * | 2022-01-04 | 2023-07-28 | 山东蔚蓝生物科技有限公司 | Bacillus megaterium for deodorizing sludge compost and application thereof |
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