CN112625939A - Bacillus methylotrophicus and application thereof - Google Patents
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Abstract
The invention discloses bacillus methylotrophicus and application thereof, and relates to bacillus methylotrophicus and application thereof. The Bacillus methylotrophicus is Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1, is preserved in China center for type culture collection, is preserved at Wuhan university in Wuhan city, China, has the preservation date of 2018, 12 months and 12 days, and has the preservation number of CCTCC No. M2018884. The invention relates to application of bacillus methylotrophicus in monitoring and evaluating river water pollution. The bacillus methylotrophicus cattle 1 is sensitive to BOD expression, is an indicative organism capable of reflecting the organic matter pollution condition of a water body, and can be used as an index for monitoring the water body environment quality. The invention is applied to the technical field of environmental microorganisms.
Description
Technical Field
The invention relates to a bacillus methylotrophicus and application thereof.
Background
The eutrophication of water is a general problem of water pollution, which is caused by excessive inflow of nutrients such as nitrogen, phosphorus and the like required by organisms into the water, and the data of water quality monitoring at the early stage of the economic river show that nitrogen in the river water quality of the area is mainly overproof organic matters and phosphorus has a certain overproof risk, microorganisms as degraders of the organic matters in the water play a critical role in the quality condition of the polluted river water quality, can degrade all natural organic matters and most artificially synthesized organic matters, and play a great role in controlling the water pollution. The microbial community structure, the microbial species, the microbial quantity and other related microbial indexes can change regularly along with the change of the water quality environment. Therefore, the microorganisms can be used as indexes for evaluating the environmental quality of the water body and indicative biological indexes for representing the water quality condition.
Biochemical Oxygen Demand (BOD) is an important indicator in environmental monitoring. It is a comprehensive index for measuring the content of organic pollutants in water. At present, the BOD5 standard dilution determination method is widely adopted at home and abroad, a water sample is filled in a closed dissolved oxygen bottle, the bottle is protected from light and cultured for 5 days at the temperature of 20 ℃, and the mass concentration difference of dissolved oxygen before and after culture is calculated, so that the dissolved oxygen consumed by each liter of sample is obtained and is expressed by BOD 5. The method has the advantages of complex operation, long required time, multiple interference factors, higher technical requirements on operators and incapability of reflecting the water quality condition in time.
Most of the research on water pollutant indicating microorganisms at home and abroad is the research on the indicating microorganisms of the fecal pollution degree of water. However, the index can only be used as a reference index of hygiene, and the research report on the microorganism indication of urban river sediment pollutants of non-drinking water sources is rare.
Disclosure of Invention
The invention aims to solve the problem of lack of water pollution indicative microorganisms, and provides a methylotrophic bacillus and application thereof.
The Bacillus methylotrophicus strain is Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1, is preserved in China center for type culture collection, is preserved at Wuhan university in Wuhan City, China, has the preservation date of 2018, 12 months and 12 days, and has the preservation number of CCTCC No. M2018884.
The invention discloses application of bacillus methylotrophicus in monitoring and evaluating river water pollution.
The method adopts the Changzhou city river channel Tongji river as a sampling point, collects the surface sediment of the Changji river in 7 months in 2014, collects the sediment in a sterilization sampling bag, quickly brings the sediment to a laboratory by using a sample refrigerator, homogenizes the sediment, removes impurities and the like, and separates and purifies the sediment microorganisms:
one, gradient dilution coating method
10g of bottom mud (avoiding muddy water) is weighed, added into a sterilized triangular flask containing 90mL of sterile distilled water, and placed into a shaking incubator for 120r/min to shake the flask for 2 hours. In a clean bench, the bacterial suspension is sequentially diluted into sterilized distilled water containing 9mL by sucking 1mL by using a pipette with the dilution gradient of 10-2、10-3、10-4、10-5、10-6. The test tube with each dilution gradient is covered with a test tube plug and shaken up and down, and then 100uL of the test tube is sucked by a pipette gun and sprayed into an LB solid culture medium for coating. When in coating, a certain amount of alcohol is dipped in a coating rod which is sterilized, secondary combustion sterilization is carried out on the coating rod on the flame of an alcohol lamp, after the temperature of the coating rod is reduced, coating liquid is carried out on an LB solid culture medium in a cross mode, and then coating is carried out in a mode of drawing circles in the same direction until the coating is finished after feeling the dry and unsmooth feeling. Each dilution gradient coated plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed.
Secondly, separation and purification of bacteria
And (5) finishing the separation and purification of bacteria in a clean bench by using the plate which is subjected to the gradient dilution coating after the culture is finished. Separating different strains according to the shape, diameter, color, viscosity and the like of the strains, dipping the strains by utilizing the sterilized inoculating loop to obtain strains, carrying out four-zone streaking on a beef extract peptone flat culture medium, culturing the four-zone flat in a constant temperature incubator for 24 hours, and then picking out single colonies growing from the four zones to carry out one-zone streaking on the beef extract peptone flat culture medium. After the strain grows out from the zone-scribing plate, observing whether the strain is a single type of pure strain under a microscope, and if not, repeating the previous separation and purification process again until a single strain appears. Culturing the strains in culture media containing BOD with different concentrations, and picking out the microorganisms with the colony number changing along with the change of the concentration of the organic matters as target strains for strain identification. The colony morphology is characterized by roundness, viscosity and neat edge; the cell shape is rod-shaped; gram staining positive; the physiological and biochemical characteristics of the method are VP negative, oxidase negative, catalase positive and amylase positive, and nitrate is reduced. A colony PCR method is adopted, single colony lysate is directly taken as a template to carry out PCR, an amplification product is sequenced, the sequencing result is compared with a target sequence and a reference sequence through CLUSTAL W in MEGA7.0 software Alignment, a phylogenetic evolution tree is constructed, the homology between a target strain and the Bacillus methylotrophicus is the highest and reaches 99.93%, and the target strain is finally named as Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1.
The Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 screened by the invention is sensitive to BOD (biochemical oxygen demand) of environmental organic matters, when BOD is more than 50mg/L, the colony number of the cattle 1 is greatly increased, and the increase rate is 0.044. Therefore, the bacillus methylotrophicus cattle 1 is sensitive to BOD expression, is an indicative organism capable of reflecting the organic matter pollution condition of the water body, and can be used as an index for monitoring the water body environment quality. In addition, the strain provided by the invention is an indigenous strain screened from the polluted bottom sediment, but not a purchased degradation functional strain, so that the problem that the functional strain is inadaptable when being reused in a field is solved, and the instability and uncertainty of functions can be avoided. The native functional strain has strong pertinence, strong adaptability, high activity and good reproducibility.
Drawings
FIG. 1 is a phylogenetic tree of bovine strain 1;
FIG. 2 is a graph showing the effect of BOD on the number of bovine 1 strains.
Detailed Description
The first embodiment is as follows: the Bacillus methylotrophicus strain is Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1, is preserved in China center for type culture collection, is preserved at Wuhan university in Wuhan City, China, and has the preservation date of 12 months and 12 days in 2018 and the preservation number of CCTCC No. M2018884.
This embodiment adopts the city river course of Changzhou city to lead to the ji river as the sampling point, gathers leading to the ji river top layer bed mud in 2014 7 months, and the laboratory is brought back rapidly with the sample fridge to sterilization sampling bag collection, and the bed mud is homogenized, is got rid of processing such as debris, separates and purifies the bed mud microorganism:
one, gradient dilution coating method
10g of bottom mud (avoiding muddy water) is weighed, added into a sterilized triangular flask containing 90mL of sterile distilled water, and placed into a shaking incubator for 120r/min to shake the flask for 2 hours. In a clean bench, the bacterial suspension is sequentially diluted into sterilized distilled water containing 9mL by sucking 1mL by using a pipette with the dilution gradient of 10-2、10-3、10-4、10-5、10-6. The test tube with each dilution gradient is covered with a test tube plug and shaken up and down, and then 100uL of the test tube is sucked by a pipette gun and sprayed into an LB solid culture medium for coating. When in coating, a certain amount of alcohol is dipped in a coating rod which is sterilized, secondary combustion sterilization is carried out on the coating rod on the flame of an alcohol lamp, after the temperature of the coating rod is reduced, coating liquid is carried out on an LB solid culture medium in a cross mode, and then coating is carried out in a mode of drawing circles in the same direction until the coating is finished after feeling the dry and unsmooth feeling. Each dilution gradient coated plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed.
Secondly, separation and purification of bacteria
And (5) finishing the separation and purification of bacteria in a clean bench by using the plate which is subjected to the gradient dilution coating after the culture is finished. Separating different strains according to the shape, diameter, color, viscosity and the like of the strains, dipping the strains by utilizing the sterilized inoculating loop to obtain strains, carrying out four-zone streaking on a beef extract peptone flat culture medium, culturing the four-zone flat in a constant temperature incubator for 24 hours, and then picking out single colonies growing from the four zones to carry out one-zone streaking on the beef extract peptone flat culture medium. After the strain grows out from the zone-scribing plate, observing whether the strain is a single type of pure strain under a microscope, and if not, repeating the previous separation and purification process again until a single strain appears. Culturing the strains in culture media containing BOD with different concentrations, and picking out the microorganisms with the colony number changing along with the change of the concentration of the organic matters as target strains for strain identification. The colony morphology is characterized by roundness, viscosity and neat edge; the cell shape is rod-shaped; gram staining positive; the physiological and biochemical characteristics of the method are VP negative, oxidase negative, catalase positive and amylase positive, and nitrate is reduced. A colony PCR method is adopted, single colony lysate is directly taken as a template to carry out PCR, an amplification product is sequenced, the sequencing result is compared with a target sequence and a reference sequence through CLUSTAL W in MEGA7.0 software Alignment, a phylogenetic evolution tree is constructed, the homology between a target strain and the Bacillus methylotrophicus is the highest and reaches 99.93%, and the target strain is finally named as Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1.
The second embodiment is as follows: the embodiment provides application of a bacillus methylotrophicus strain in monitoring and evaluating river water pollution.
The Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 screened by the embodiment is sensitive to BOD (biochemical oxygen demand) of environmental organic matters, when BOD is larger than 50mg/L, the number of colonies of the cattle 1 is greatly increased, and the increase rate is 0.044. Therefore, the bacillus methylotrophicus cattle 1 is sensitive to BOD expression, is an indicative organism capable of reflecting the organic matter pollution condition of the water body, and can be used as an index for monitoring the water body environment quality. In addition, the strain provided by the embodiment is an indigenous strain screened from the polluted bottom sediment, and is not a purchased degradation functional strain, so that the problem that the functional strain is not suitable when being reused in a field is solved, and the instability and uncertainty of functions can be avoided. The native functional strain has strong pertinence, strong adaptability, high activity and good reproducibility.
The third concrete implementation mode: the second embodiment is different from the first embodiment in that: bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 serving as an indicator microorganism of organic pollutants in a water body is applied to monitoring and evaluation of river water pollution. The rest is the same as the second embodiment.
The fourth concrete implementation mode: the second or third embodiment is different from the first or second embodiment in that: the river water is the Changzhou city's Tongji river water. The other embodiments are the same as the second or third embodiment.
The fifth concrete implementation mode: the screening method of Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 in the embodiment comprises the following steps: adopting Changzhou city river channel Tongji river as sampling point, collecting Tongji river surface layer bottom mud in 2014 and 7 months, bagging and collecting sterilization sampling bags, rapidly bringing the sterilization sampling bags back to a laboratory by using a sample refrigerator, homogenizing the bottom mud, removing impurities and the like, and separating and purifying bottom mud microorganisms:
one, gradient dilution coating method
10g of bottom mud (avoiding muddy water) is weighed, added into a sterilized triangular flask containing 90mL of sterile distilled water, and placed into a shaking incubator for 120r/min to shake the flask for 2 hours. In a clean bench, the bacterial suspension is sequentially diluted into sterilized distilled water containing 9mL by sucking 1mL by using a pipette with the dilution gradient of 10-2、10-3、10-4、10-5、10-6. The test tube with each dilution gradient is covered with a test tube plug and shaken up and down, and then 100uL of the test tube is sucked by a pipette gun and sprayed into an LB solid culture medium for coating. When in coating, a certain amount of alcohol is dipped in a coating rod which is sterilized, secondary combustion sterilization is carried out on the coating rod on the flame of an alcohol lamp, after the temperature of the coating rod is reduced, coating liquid is carried out on an LB solid culture medium in a cross mode, and then coating is carried out in a mode of drawing circles in the same direction until the coating is finished after feeling the dry and unsmooth feeling. Each dilution gradient coated plate was incubated in a 37 ℃ incubator for 24 hours, and the results were observed.
Secondly, separation and purification of bacteria
And (5) finishing the separation and purification of bacteria in a clean bench by using the plate which is subjected to the gradient dilution coating after the culture is finished. Separating different strains according to the shape, diameter, color, viscosity and the like of the strains, dipping the strains by utilizing the sterilized inoculating loop to obtain strains, carrying out four-zone streaking on a beef extract peptone flat culture medium, culturing the four-zone flat in a constant temperature incubator for 24 hours, and then picking out single colonies growing from the four zones to carry out one-zone streaking on the beef extract peptone flat culture medium. After the strain grows out from the zone-scribing plate, observing whether the strain is a single type of pure strain under a microscope, and if not, repeating the previous separation and purification process again until a single strain appears. Culturing the strains in culture media containing BOD with different concentrations, and picking out the microorganisms with the colony number changing along with the change of the concentration of the organic matters as target strains for strain identification.
The formula of the beef extract peptone culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of sodium chloride, 15-20 g of agar, 1000mL of distilled water, pH 7.0-7.2, and sterilizing at 121 ℃ for 20 min.
LB enrichment medium formula (liquid): 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 1000mL of distilled water, pH 7.0 and sterilizing at 121 ℃ for 20 min.
LB enrichment medium formulation (solids): 10g of peptone, 5g of yeast extract, 10g of sodium chloride, 15-20 g of agar, 1000mL of distilled water, pH 7.0 and sterilizing at 121 ℃ for 20 min.
The sixth specific implementation mode: identification of Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 of the present embodiment:
1. strain morphology identification
The strain bovine 1 was cultured on an LB plate for two days, and morphological observation and gram staining were performed thereon, with the results shown in Table 1.
TABLE 1 morphological characterization of the target strains
2. Physiological and biochemical identification of strain
The results of physiological and biochemical identification of the strain bovine 1 are shown in table 2.
TABLE 2 physiological and biochemical identification of target strains
3. Strain 16S rDNA identification
A colony PCR method is adopted, and single colony lysate is directly taken as a template to carry out PCR. The 16S rDNA universal primers were amplified by PCR. 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided. The PCR reaction conditions are as follows: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 51 ℃ for 1min for 30s, 72 ℃ for 2min for 30 s; 10min at 72 ℃. The 16S rDNA sequence is shown in SEQ ID NO: 1 is shown. The sequencing result is compared with the target sequence and the reference sequence through CLUSTALW in the Alignment of MEGA7.0 software, and the parameters are default values. The constructed phylogenetic tree is shown in FIG. 1. The result shows that the strain cattle 1 provided by the invention has high homology with Bacillus methylotrophicus (99.93 percent), and is finally named as Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1.
Functional verification of Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 of the present embodiment on microorganisms indicative of organic pollutants:
test 1: determination of the sensitivity of the strains to organic substances:
inoculating the strain cattle 1 into 10mL of sterile water by using a sterile inoculating loop, and diluting 10-4And (4) absorbing 100uL of the thallus diluent by using a pipette gun, inoculating the thallus diluent into BOD diluents with different concentrations, and culturing for 12 hours at 37 ℃. The culture solution was spread on an LB solid medium, and the number of colonies was counted after 24 hours.
When the BOD concentration is 10mg/L, the number of the cattle 1 colony is 0.3X 106CFU/mL; the number of cattle 1 colonies at a BOD concentration of 30 mg/L was 1.09X 106CFU/mL; the number of cattle 1 colonies at a BOD concentration of 50mg/L was 1.36X 106CFU/mL, the amplification is smaller. The number of cattle 1 colonies at a BOD concentration of 70mg/L was 2.1X 106CFU/mL; the number of cattle 1 colonies at a BOD concentration of 90mg/L was 2.78X 106CFU/mL; the number of cattle 1 colonies at a BOD concentration of 110 mg/L was 3.98X 106CFU/mL. When BOD is more than 50mg/L, the colony number of the cattle 1 is greatly increased, and the growth rate is 0.044. In conclusion, in a certain range, the strain cattle 1 is sensitive to BOD of environmental organic matters and has a certain indication effect.
Test 2, reproducibility test
The results of repeated tests on samples with BOD of 50mg/L are shown in Table 3. The result shows that the average value of the results of 5 times of measurement is 0.3mg/L, the maximum relative deviation is 0, and the requirement of the traditional five-day standard dilution method on the maximum deviation is met.
TABLE 3 reproducibility test
The result shows that the indigenous bacteria cattle 1 provided by the embodiment of the invention is sensitive to BOD expression, is an indicative organism capable of reflecting the organic matter pollution condition of the water body, and can be used as an index for monitoring the water body environment quality.
Sequence listing
<110> university of Harbin
<120> bacillus methylotrophicus and application thereof
<160> 3
<210> 1
<211> 1428
<212> DNA
<213> Bacillus methylotrophicus (Bacillus methylotrophicus)
<400> 1
ctaatacatg caagtcgagc ggacagatgg gagcttgctc cctgatgtta gcggcggacg 60
ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct 120
aataccggat ggttgtctga accgcatggt tcagacataa aaggtggctt cggctaccac 180
ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcgacg 240
atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc 300
ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc 360
gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtgccg 420
ttcaaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca 480
gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggctc 540
gcaggcggtt tcttaagtct gatgtgaaag cccccggctc acccggggag ggtcattgga 600
aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg 660
tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag 720
gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat 780
gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca ttaagcactc 840
cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag 900
cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc 960
tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt ggtgcatggt 1020
tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat 1080
cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa 1140
ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat 1200
ggacagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag 1260
ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca 1320
gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt 1380
ttgtaacacc cgaagtcggt gaggtaacct ttaggagcca gccgccga 1428
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> nucleotide sequence of PCR primer 27F.
<400> 2
<210> 3
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> PCR primer 1492R nucleotide sequence.
<400> 3
GGTTACCTTG TTACGACTT 19
Claims (4)
1. A Bacillus methylotrophicus is characterized in that Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 is preserved in China center for type culture collection, the preservation address is Wuhan university in Wuhan City, the preservation date is 12 months and 12 days in 2018, and the preservation number is CCTCC No. M2018884.
2. The use of a strain of bacillus methylotrophicus according to claim 1 in monitoring and assessment of river water pollution.
3. The application of the Bacillus methylotrophicus strain as claimed in claim 2, wherein the Bacillus methylotrophicus (Bacillus methylotrophicus) cattle 1 is used as an indicator microorganism of organic pollutants in a water body in monitoring and evaluation of river water pollution.
4. The use of the bacillus methylotrophicus according to claim 2, wherein the river water is the normal economic river water of Changzhou city.
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2020
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| US20090014384A1 (en) * | 2003-08-27 | 2009-01-15 | Kobelco Eco-Solutions Co., Ltd. | Novel microorganism and process for treatment of organic solid matter using the microorganism |
| CN1902306A (en) * | 2003-11-14 | 2007-01-24 | 联邦科学及工业研究组织 | Selective enrichiment of microorganisms for desired metabolic properties |
| CN1563354A (en) * | 2004-03-24 | 2005-01-12 | 河北科技大学 | Strain in use sensor of measuring oxygen quantity needed by industrial wastewater or seawater biochemistry-method for cultivating bacillus licheniformis |
| WO2006070391A1 (en) * | 2004-12-28 | 2006-07-06 | Council Of Scientific And Industrial Research | Biological process for reducing chemical and biochemical oxygen demand of pulp and paper industrial effluent |
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| 胡锐等: "新型复合功能菌群的构建及其在近海养殖水体BOD微生物传感器中的应用", 《安徽农业科学》 * |
| 高星爱等: "4株降解低温粪污水芽孢杆菌的分离？鉴定及其降解效率研究", 《安徽农业科学》 * |
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