CN115305218A - Plateau bacillus SX-3 and application thereof in degrading industrial sewage - Google Patents
Plateau bacillus SX-3 and application thereof in degrading industrial sewage Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Abstract
The invention discloses a bacillus plateau SX-3 and application thereof in degrading industrial sewage, and provides a strain for efficiently degrading industrial sewage, namely the bacillus plateau SX-3, which is obtained by screening activated sludge, wherein the degradation rate of the strain in 7d to the industrial sewage reaches 56%, the actual industrial sewage treatment scale of the strain is 50t, SX-3 bacterial liquid 5L is added according to 1/10000 dosage, an aerator controls Dissolved Oxygen (DO) to be 2mg/L, and the degradation rate of the strain SX-3 in 4d to the industrial sewage COD of a certain sewage plant of Shaoxing province can reach 28.0% under the treatment condition of room temperature of 30 ℃, NH 3 The degradation rate of-N can reach 90.2%, and the degradation rate of TN can reach 28.1%. Plateau bacillus SX-3 strain applied to bioremediation of industrial sewageThe possibility in the process promotes the degradation of industrial sewage.
Description
(I) the technical field
The invention relates to a plateau bacillus SX-3 and application thereof in degrading industrial sewage.
(II) background of the invention
With the development of economy, industries such as leather, textile, paint, dye and pharmacy are endlessly developed, the discharge amount of industrial sewage is increasing day by day, the composition of COD in industrial sewage becomes more complex, the composition of COD in industrial sewage usually includes pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs), phenols, dyes and organic heavy metals, and the complex composition causes COD in industrial sewage to have high numerical value and difficult to degrade efficiently. In some cases, these organic contaminants are present in a mixed matrix of industrial waste, which poses a serious health risk to humans, aquatic life and the entire ecosystem, since most of these mixed organic contaminants accumulate in water at concentrations exceeding the discharge limits allowed in the environment. Mixed organic pollutants enter the aquatic environment by being directly discharged into water bodies, non-point runoff, domestic sewage and industrial wastewater, and have extremely high toxicity to human beings, aquatic organisms and the whole environment because the mixed organic pollutants have carcinogenicity and are easy to damage important organs. Therefore, it is necessary to develop a practical and efficient remediation technique to solve the environmental pollution caused by industrial wastewater.
Currently, there are physical, chemical and biological methods for the treatment of industrial wastewater. Physical technologies include membrane separation technology, adsorption and the like, and the physical method has the advantages of small equipment size, low energy consumption, low investment cost and the like, but has more room for improvement in the aspects of efficiency, space requirements, energy, permeation quality and technical skill requirements due to the change of the property and complexity of the wastewater; the chemical technology comprises electrochemistry, chemical flocculation, advanced oxidation and the like, and the chemical method has strong capability of treating sewage with poor biodegradability and large relative molecular mass, but has high investment cost and complex operation and maintenance technology; the biological method comprises an activated sludge method, anaerobic biological treatment, a natural treatment method and the like, the biological method has the advantages of convenient application, low cost, environmental protection and the like, and is considered to be an industrial sewage treatment technology with wide application prospect, but because the components of industrial wastewater are extremely complex, the sewage contains a large amount of toxic organic matters and heavy metal ions, and environmental factors such as high temperature, high salt, unstable pH and the like can influence the growth and metabolism of conventional microorganisms, even cause the death of the microorganisms, and influence the operation of a microbial biochemical system. Under the background, the microbial strengthening technology is generated, and the microbial strengthening technology is characterized in that indigenous and external dominant microbial strains are artificially screened and cultured, domestication is carried out to enable the strains to adapt to the complex environment of industrial sewage and enhance the capacity of efficiently degrading the industrial sewage, and finally, the efficiently degrading strains are added into an original ecological system and the proportion of the efficiently degrading strains in microbial populations is pertinently improved. The microorganism strengthening technology has the advantages of small disturbance to the environment, high degradation efficiency, no secondary pollution, low operation cost and the like, and the key of the technology is to obtain excellent strains for efficiently degrading industrial sewage by screening from the environment.
At present, researchers at home and abroad have separated a plurality of strains which can achieve the purpose of degrading industrial sewage from the environment, wherein the strains mainly comprise bacillus, pseudomonas, microzyme, photosynthetic bacteria and the like, but the success cases of treating the industrial sewage through a microorganism strengthening technology are few at present, the main reason is that the strain resources for efficiently degrading the industrial sewage are not rich enough, the types of the organic matters which can be degraded in the industrial sewage by various strains are limited, and the degradation efficiency is not high. For some large molecules and toxic organic matters in industrial sewage, the large molecules and the toxic organic matters cannot be completely degraded only by a few degrading strains. Therefore, it is very important to screen more strains from the environment which have high-efficiency degradation effect on industrial sewage.
There are two ways for the microorganism to degrade industrial sewage: (1) The microorganism treatment of industrial sewage depends on the metabolism of the microorganism and utilizes organic matters in the sewage as a carbon source required by the growth and metabolism of the microorganism, so that the organic matters in the sewage are converted into CO 2 、H 2 Inorganic substances such as O and the like to achieve the purpose of degrading sewage; (2) The microorganisms treat the industrial sewage through co-metabolism, namely, the microorganisms utilize certain organic matters in the sewage as growth substrates of the microorganisms, and the microorganisms degrade the organic matters and convert certain organic matters (non-growth substrates) which cannot be utilized in the sewage into other organic matters. Therefore, microbial strains for efficiently degrading industrial sewage need to be continuously screened from the environment, industrial sewage degradation strain resources are expanded, enough microbial strain resources are provided for the industrial sewage treatment by the microbial strengthening technology, and meanwhile, the microbial strains are also used for subsequent processesThe construction of the microorganism and the development of the microbial agent provide resources.
Disclosure of the invention
The invention aims to provide a strain for efficiently degrading industrial sewage, namely Bacillus altitudinis SX-3, screened from activated sludge of a sewage plant and application thereof in degrading industrial sewage, and solves the problems that the conventional strain for efficiently degrading industrial sewage is difficult to separate from the environment and the microbial resource of the conventional strain for degrading industrial sewage is not rich enough.
The technical scheme adopted by the invention is as follows:
the invention provides a new strain-Bacillus plateau SX-3 (Bacillus altitudinis SX-3), which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date: 2022, 5/19, accession No.: CCTCC NO: M2022669, address: wuhan, wuhan university, zip code: 430072.
the plateau bacillus SX-3 is a gram-positive bacterium, a bacterial colony on an LB culture medium agar plate is milky white, round and wet in surface, and can well grow on a 7% high-salt LB plate, and simultaneously, a bacterial strain SX-3 is in a straight rod shape under a phase contrast microscope, has a size of (0.2-1.2) Mumx (2.5-6) Mum, and is singly, pairwise or in a fence-shaped arrangement.
The invention also provides application of the Bacillus plateau SX-3 in degrading industrial sewage, wherein the COD of the industrial sewage is 200-500mg/L, and the NH is 3 10-20mg/L of-N, 20-30mg/L of TN, preferably 266mg/L of sewage COD and NH 3 -N 16.12mg/L、TN 27.8mg/L。
Further, the application method comprises the following steps: adding the plateau bacillus SX-3 bacterial liquid into industrial sewage, and treating under the aerobic condition of 30-40 ℃ (preferably 30 ℃), so as to realize degradation of the industrial sewage; the bacterial concentration in the bacterial liquid is 1.0 multiplied by 10 10 -2.8×10 11 CFU/L (preferably 2.8X 10) 11 CFU/L), the ratio of the bacterial liquid to the sewage is 1:1000-10000 (preferably 1; the aerobic refers to that the dissolved oxygen is 1-5mg/L, preferably 2mg/L.
Further, the plateau bacillus SX-3 bacterial liquid is prepared by the following method: (1) Inoculating plateau bacillus SX-3 to an LB solid culture medium flat plate, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; LB solid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract, 20g/L of agar and water as a solvent;
(2) Selecting the colony in the step (1) to be inoculated to an LB liquid culture medium, and carrying out shaking culture at the constant temperature of 37 ℃ and 180rpm for 24 hours to obtain a first-stage seed solution; LB liquid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract and water as a solvent;
(3) Respectively inoculating the primary seed liquid in the step (2) into an LB liquid culture medium (the same as the step 2) according to the inoculation amount of 2% of the volume concentration, and carrying out shake cultivation at 37 ℃ and 180rpm for 1d to obtain a plateau bacillus SX-3 bacterial liquid.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a strain capable of efficiently degrading industrial sewage, namely plateau bacillus SX-3, screened from activated sludge, wherein the degradation rate of the strain in 7d to the industrial sewage reaches 56 percent, the actual industrial sewage treatment scale of the strain is 50t, 5L of SX-3 bacterial liquid is added according to 1/10000 dosage, 2mg/L of Dissolved Oxygen (DO) is controlled by an aerator, the COD degradation rate of the strain SX-3 in 4d to the industrial sewage of a Shaoxing certain sewage plant can reach 28.0 percent under the treatment condition of 30 ℃, and NH 3 is added 3 The degradation rate of-N can reach 90.2%, and the degradation rate of TN can reach 28.1%. The plateau bacillus SX-3 strain has the possibility of being applied to the bioremediation engineering of industrial sewage and promotes the degradation of the industrial sewage.
Description of the drawings
FIG. 1 is a colony morphology diagram of strain SX-3.
FIG. 2 is a micrograph of strain SX-3.
FIG. 3 shows the growth of strain SX-3 on 7% high salt LB plates.
FIG. 4 is a graph of growth of strain SX-3.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of protection of the invention is not limited thereto: the room temperature of the invention is 25-30 ℃.
Example 1: screening of high-efficiency degradation bacterial strain for industrial sewage
The invention screens out the high-efficiency degradation bacterial strain of industrial sewage through enrichment domestication, separation and purification of activated sludge and industrial sewage degradation experiments, and the specific implementation method is as follows:
(1) And (4) enriching and domesticating the activated sludge.
The activated sludge used in the invention is taken from the three-stage sewage treatment oxidation ditch process section of the Anhui saddle Zhenjiang Binjiang Binhui Shaoxing water treatment development Limited company in the Konjac area of Shaoxing city, zhejiang province. The industrial sewage is the effluent of a high-efficiency sedimentation tank of a certain Shaoxing sewage plant, and the COD is 500mg/L.
Adding 80mL of inorganic salt culture medium into a 250mL conical flask, taking 20mL of industrial sewage as a growth substrate for microbial growth, taking 100mL of mixed solution as a first domestication culture medium, adding 5g of activated sludge into the mixed solution, and performing shaking culture at constant temperature of 180rpm and 37 ℃ for 7d (taking the mixed culture solution as a passage basis for obviously becoming turbid or detecting the obviously increased OD value of the culture solution).
And adding 10mL of mixed solution obtained after the first domestication culture for 7 days into a conical flask filled with 40mL of industrial sewage and 50mL of fresh inorganic salt culture medium, performing second domestication for 7d under the same culture condition, finally adding 10mL of mixed solution obtained after the second domestication into the conical flask filled with 70mL of industrial sewage and 20mL of fresh inorganic salt culture medium, and performing third domestication for 7d under the same culture condition.
The inorganic salt culture medium comprises the following components in percentage by weight: 1g/L K 2 HPO 4 、1g/L KH 2 PO 4 、1g/L(NH 4 ) 2 SO 4 、0.2g/L MgSO 4 ·7H 2 O、0.02g/L CaCl 2 1mL/L of trace element solution, and deionized water as a solvent; composition of the trace element solution: 2.5g/L FeSO 4 ·7H 2 O、0.3g/L MnSO 4 ·H 2 O、0.5g/L(NH 4 ) 6 Mo 7 O 24 ·4H 2 O、1g/L ZnSO 4 ·7H 2 And O, the solvent is deionized water.
(2) And (5) separating and purifying strains.
Centrifuging the mixed solution after the third acclimation at 8000rpm for 5min, discarding supernatant, collecting thallus, and diluting the thallus with sterile water to 10% -2 、10 -3 、10 -4 、10 -5 、10 -6 Five gradients, howeverAnd then coating the diluent with different gradients on a solid selective medium plate, placing the plate in a constant-temperature incubator at 37 ℃ for culture, observing the growth condition of the strain on the plate every day, picking out the strain capable of growing, streaking and transferring the strain to the solid enrichment medium plate for multiple times until the colony morphology of the strain on the plate is consistent, observing whether the strain is a single strain by a microscope, and screening to obtain 3 single strains which are respectively marked as a strain SX-1, a strain SX-2 and a strain SX-3.
Solid selection medium composition: 1g/L K 2 HPO 4 、1g/L KH 2 PO 4 、1g/L(NH 4 ) 2 SO 4 、0.2g/L MgSO 4 ·7H 2 O、0.02g/L CaCl 2 1mL/L of trace element solution and 20g/L of agar, wherein the solvent is industrial sewage;
the liquid enrichment medium comprises the following components: 1g/L K 2 HPO 4 、1g/L KH 2 PO 4 、1g/L(NH 4 ) 2 SO 4 、0.2g/L MgSO 4 ·7H 2 O、0.02g/L CaCl 2 1mL/L of trace element solution, 10g/L of tryptone and industrial sewage as a solvent. The solid enrichment culture medium is prepared by adding 20g/L agar into a liquid enrichment culture medium.
The trace element solutions were all: 2.5g/L FeSO 4 ·7H 2 O、0.3g/L MnSO 4 ·H 2 O、0.5g/L (NH 4 ) 6 Mo 7 O 24 ·4H 2 O、1g/L ZnSO 4 ·7H 2 And O, the solvent is deionized water.
(3) And (5) re-screening the strains.
Respectively transferring the strains separated and purified in the step (2) to a liquid enrichment medium (the same as the step 2), and carrying out shake cultivation at constant speed of 180rpm and 37 ℃ for 1d; the thalli is collected by centrifugation (8000rpm 5 min), washed by sterile water for 3 times, and reset in an inorganic salt culture medium after washing, and the OD value is adjusted to 1.0 to serve as seed liquid to be stored in a refrigerator at 4 ℃ for later use.
Experimental groups: according to the volume ratio of 5:95 of seed liquid: preparing 100mL of mixed liquor from industrial sewage, then placing the mixed liquor into a constant temperature shaking table at 180rpm and 37 ℃ for 7d, and detecting the COD of each mixed liquor every 2d, wherein the detection method is a potassium dichromate method (GB 11914-89). And judging the superiority and inferiority of the degradation performance of each strain according to the COD removal rate, wherein the result is shown in the table 1, and re-screening to obtain the strain SX-3 with better industrial sewage degradation effect. Under the same condition, the seed solution is sterilized at the high temperature of 121 ℃ for 30min and then replaces the seed solution in the experimental group to be used as a control group.
The ferrous ammonium sulfate calibration method comprises the following steps: adding 10mL of prepared potassium dichromate standard solution into a 500mL conical flask, adding 100mL of deionized water, then adding 30mL of concentrated sulfuric acid (98%), shaking uniformly, standing and cooling to room temperature, then adding 3 drops of ferron indicator, shaking to make the color uniform, finally titrating with ammonium ferrous sulfate standard solution, wherein the color of the solution is yellow-blue-green-red brown, and the end point is obtained. The ferrous ammonium sulfate concentration is calculated according to equation (1).
Potassium dichromate standard solution (1/6K) 2 CrO 7 =0.2500 mol/L): weighing 12.258g of dry potassium dichromate, dissolving in distilled water, transferring into a 100mL volumetric flask, diluting to a marked line, and shaking up.
Standard solution of ammonium ferrous sulfate [ (NH) 4 )2Fe(SO 4 ) 2 ·6H 2 O≈0.1mol/L]: weighing 39.5g of ferrous ammonium sulfate, dissolving in distilled water, slowly adding 20mL of concentrated sulfuric acid while stirring, cooling, transferring into a 1000mL volumetric flask, adding distilled water to dilute to a marked line, and shaking up.
A ferron indicator: weighing 1.485g of o-phenanthroline (C1) 2 H 8 N 2 ·H 2 O), 0.695g ferrous sulfate (FeSO) 4 ·7H 2 O) was dissolved in water, diluted to 100ml and stored in a brown bottle.
C[(NH 4 ) 2 Fe(SO 4 ) 2 ·6H 2 O]= (0.2500 × 10.00)/V equation (1)
C-the concentration (mol/L) of ferrous ammonium sulfate;
v-titration amount (mL) of ferrous ammonium sulfate standard solution.
The COD determination method comprises the following steps: adding 20mL of water sample to be detected into a 250mL conical flask, measuring 10mL of potassium dichromate standard solution, adding the potassium dichromate standard solution into the conical flask, connecting a condensing device, pouring 30mL of sulfuric acid-silver sulfate solution measured in advance from an upper opening of a condensing tube, slowly shaking the conical flask to uniformly mix the solution, heating the conical flask, and timing for 2 hours when the solution in the conical flask begins to boil. After cooling, the walls of the condenser tube were rinsed with 90mL of distilled water and the flask was removed. And after the solution is cooled again, adding 3 drops of a ferroxyl indicator, titrating by using a ferrous ammonium sulfate standard solution, and recording the dosage of the ferrous ammonium sulfate standard solution, wherein the end point is that the solution is from yellow to reddish brown through bluish green. And (3) while measuring the water sample, performing a blank experiment by using distilled water according to the same steps. Calculating COD value according to the formula (2) and calculating COD degradation rate according to the formula (3).
COD(mg/L)＝C(V 0 -V 1 ) 8X 1000/V equation (2)
The concentration (mol/L) of the C-ammonium ferrous sulfate standard solution in the formula (2);
V 0 -amount of ferrous ammonium sulfate standard solution (mL) at titration blank;
V 1 the amount of standard solution of ferrous ammonium sulfate (mL) used in titrating a water sample;
v-volume of water sample (mL).
COD Removal rate of ＝(C Sample strains -C CK )/C CK Formula (3)
In the formula (3), C Sample strains Adding the degrading strain sample COD (mg/L)
C CK COD of sterile control Water sample (mg/L)
The finally screened strain SX-3 has the best effect on the degradation of industrial sewage (Table 1), and the final degradation rate of SX-3 on the industrial sewage can reach 56% under the condition of 180rpm and 37 ℃ in a laboratory.
TABLE 1 degradation effect of each primary strain on industrial wastewater
(4) And (5) identifying the strain.
The strain SX-3 is sent to Hangzhou Ongke biology company for 16S rDNA sequencing, 16S bacterial universal primers 27F and 1492R are adopted for amplification, the sequence length is 1375bp, then a 16SrDNA sequence (SEQ ID NO. 1) is submitted to an NCBI database for sequence comparison analysis, and the result shows that the strain SX-3 has the highest homology with Bacillus altitudinis, so that the strain SX-3 is determined to be plateau Bacillus. The 16S rDNA sequence has been uploaded to the Genbank database under accession number OL664046.
The sequence of the strain SX-3 is as follows:
GTTACCTCACCGACTTCGGGTGTTGCAACTCTCGTGGTGTGACGGGCGGTG TGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGC GATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGAT TTGTGGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAG CACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTT CCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAAC TAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGA GCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCTAT CTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCG AATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTT CAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCAC TAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTAC CAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACA GACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACC GCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAAT GACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGC GAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCG GCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAA GCAGTTACTCTTGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAAC CTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTC CCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCG ATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCA ACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCA TCCTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAG TTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAA CATCCGGGAGCAAGCT.
the strain SX-3 is inoculated on an LB medium plate and cultured for 1d at 37 ℃, as shown in figure 1, on the LB solid medium plate, the colony is circular, milky white, 0.1-0.4mm in diameter, moist and dull in surface and neat in edge. A colony micrograph of the strain SX-3 is shown in FIG. 2, and the strain SX-3 has a straight rod-shaped cell shape with a size (0.2-1.2) mum X (2.5-6) mum arranged singly, in pairs or in a fence-like arrangement under a phase contrast microscope.
LB liquid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract and water as a solvent. The LB solid medium was an LB liquid medium supplemented with 20g/L agar.
Therefore, the strain SX-3 is identified as Bacillus plateau, named as Bacillus plateau SX-3, and is preserved in China Center for Type Culture Collection (CCTCC) at the address: eight-way No. 299 in Wuchang area, wuhan city, hubei province, zip code: 430072, preservation date: 2022, 5/19, CCTCC preservation number: CCTCC M2022669.
Example 2: plateau bacillus SX-3 growth curve determination
Transferring plateau bacillus SX-3 to LB liquid culture medium, shaking culturing at 37 deg.C and 180rpm, sampling once every half hour, and culturing at OD 600 And detecting the light absorption value at nm and drawing a growth curve. As shown in FIG. 4, the experimental result shows that the strain SX-3 enters a logarithmic growth phase within about 2 hours, enters a terminal growth phase within about 5 hours, enters a stable growth phase within about 9.5 hours, and enters a death phase within about 13 hours.
LB liquid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract and water as a solvent.
Example 3: plateau bacillus SX-3 salinity tolerance test
In order to explore the resistance of the Bacillus plateau SX-3 to high salinity conditions, the growth conditions of the Bacillus plateau SX-3 in LB culture media with different salinity are explored, and the specific operation is as follows:
(1) Inoculating plateau bacillus SX-3 to LB liquid culture medium of NaCl 10g/L, and carrying out shaking culture at 37 ℃ and 180rpm for 24h to obtain seed liquid. LB liquid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract and water as a solvent.
(2) And respectively coating the seed liquid on LB solid medium plates with NaCl of 10g/L, culturing for 24 hours in a constant-temperature incubator at 37 ℃, and detecting the growth condition of the strain. LB solid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract, 20g/L of agar and water as a solvent.
(3) The NaCl concentrations in the steps (1) and (2) are respectively changed to 30g/L, 50g/L, 70g/L and 90g/L, and other operations are the same.
The result shows that the plateau Bacillus SX-3 has wide salinity, and can well grow in salinity gradients of 10g/L, 30g/L, 50g/L and 70 g/L. FIG. 3 shows the growth of the strain on 70g/L high-salt LB plates.
Example 4: preparation of SX-3 pilot-plant bacterial liquid
The SX-3 pilot-plant test bacterium solution is prepared in a step-by-step amplification mode and specifically comprises the following steps:
(1) Inoculating plateau bacillus SX-3 to an LB solid culture medium flat plate, and culturing for 24 hours in a constant-temperature incubator at 37 ℃. LB solid Medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract, 20g/L of agar and water as a solvent.
(2) And (3) selecting the colony in the step (1) to be inoculated into 100mL of LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 180rpm for 24 hours to obtain a first-stage seed solution. LB liquid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract and water as a solvent.
(3) Respectively inoculating the first-stage seed solution of the step (2) into 50 LB liquid culture medium (the same as the step 2) with the volume concentration of 2%, shaking-culturing at 37 ℃ and 180rpm for 1d, combining the culture solutions to serve as a pilot-plant bacteria solution, wherein the volume of the pilot-plant bacteria solution is 5L, and the viable count of the bacteria solution is 2.8 multiplied by 10 11 CFU/L。
Example 5: pilot test degradation effect of SX-3 strain on 50t industrial sewage
Taking three-stage industrial sewage (COD is 266.1mg/L and NH) of Shaoxing certain sewage plant 3 -N16.12 mg/L, TN 27.8 mg/L) 50t, adding SX-3 bacterial solution prepared by the method of example 4 according to the adding amount of 1/10000, carrying out aerobic treatment at 30 ℃ for 4d, controlling the dissolved oxygen at 2mg/L, and detecting COD and NH once by adopting a multi-parameter water quality digestion instrument (Lianhua technology COD ammonia nitrogen detector 5B-3C and Lianhua technology total nitrogen detector LH-TN 100) every 6h 3 N, TN, the results are shown in Table 2. The degradation rate of the final strain SX-3 on COD can reach 28.0 percent, and the degradation rate on NH is improved 3 the-N degradation rate reaches 90.2 percent, and the TN degradation rate reaches 28.1 percent.
TABLE 2 Pilot scale degradation effect of strain SX-3 on 50t industrial wastewater
Sequence listing
<110> Zhejiang industrial university
<120> Bacillus plateau SX-3 and application thereof in degradation of industrial sewage
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1375
<212> DNA
<213> Bacillus plateau (Bacillus altitudinis)
<400> 1
gttacctcac cgacttcggg tgttgcaact ctcgtggtgt gacgggcggt gtgtacaagg 60
cccgggaacg tattcaccgc ggcatgctga tccgcgatta ctagcgattc cagcttcacg 120
cagtcgagtt gcagactgcg atccgaactg agaacagatt tgtgggattg gctaaacctt 180
gcggtctcgc agccctttgt tctgtccatt gtagcacgtg tgtagcccag gtcataaggg 240
gcatgatgat ttgacgtcat ccccaccttc ctccggtttg tcaccggcag tcaccttaga 300
gtgcccaact gaatgctggc aactaagatc aagggttgcg ctcgttgcgg gacttaaccc 360
aacatctcac gacacgagct gacgacaacc atgcaccacc tgtcactctg tccccgaagg 420
gaaagcccta tctctagggt tgtcagagga tgtcaagacc tggtaaggtt cttcgcgttg 480
cttcgaatta aaccacatgc tccaccgctt gtgcgggccc ccgtcaattc ctttgagttt 540
cagtcttgcg accgtactcc ccaggcggag tgcttaatgc gttagctgca gcactaaggg 600
gcggaaaccc cctaacactt agcactcatc gtttacggcg tggactacca gggtatctaa 660
tcctgttcgc tccccacgct ttcgctcctc agcgtcagtt acagaccaga gagtcgcctt 720
cgccactggt gttcctccac atctctacgc atttcaccgc tacacgtgga attccactct 780
cctcttctgc actcaagttt cccagtttcc aatgaccctc cccggttgag ccgggggctt 840
tcacatcaga cttaagaaac cgcctgcgag ccctttacgc ccaataattc cggacaacgc 900
ttgccaccta cgtattaccg cggctgctgg cacgtagtta gccgtggctt tctggttagg 960
taccgtcaag gtgcaagcag ttactcttgc acttgttctt ccctaacaac agagctttac 1020
gatccgaaaa ccttcatcac tcacgcggcg ttgctccgtc agactttcgt ccattgcgga 1080
agattcccta ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc agtgtggccg 1140
atcaccctct caggtcggct acgcatcgtc gccttggtga gccgttacct caccaactag 1200
ctaatgcgcc gcgggtccat ctgtaagtga cagccgaaac cgtctttcat ccttgaacca 1260
tgcggttcaa ggaactatcc ggtattagct ccggtttccc ggagttatcc cagtcttaca 1320
ggcaggttac ccacgtgtta ctcacccgtc cgccgctaac atccgggagc aagct 1375
Claims (7)
1. Plateau Bacillus SX-3 (Bacillus altitudinis SX-3) is preserved in China center for type culture Collection with the preservation date: 2022, 5/19, accession No.: CCTCC NO: m2022669, address: wuhan, wuhan university, zip code: 430072.
2. the application of the Bacillus plateau SX-3 in the step 1 in degrading industrial sewage.
3. The application of claim 2, wherein the COD of the industrial wastewater is 200-500mg/L and NH 3 N is 10-20mg/L, TN is 20-30mg/L.
4. The use according to claim 3, wherein said use is: adding the plateau bacillus SX-3 bacterial liquid into industrial sewage, and treating under the aerobic condition of 30-40 ℃ to realize the degradation of the industrial sewage.
5. The use according to claim 4, wherein the bacterial fluid has a bacterial cell concentration of 1.0X 10 10 -2.8×10 11 CFU/L, the ratio of the bacterial liquid to the sewage is 1:1000-10000.
6. The use of claim 4, wherein aerobic means dissolved oxygen of 1-5mg/L.
7. The use of claim 4, wherein the bacterial liquid of bacillus plateau SX-3 is prepared by the following method: (1) Inoculating plateau bacillus SX-3 to an LB solid medium flat plate, and culturing for 24 hours in a constant-temperature incubator at 37 ℃; LB solid Medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract, 20g/L of agar and water as a solvent;
(2) Selecting the colony in the step (1) to be inoculated to an LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 180rpm for 24 hours to obtain a first-stage seed solution; LB liquid medium: 10g/L of NaCl, 10g/L of tryptone, 5g/L of yeast extract and water as a solvent;
(3) Inoculating the primary seed liquid in the step (2) into an LB liquid culture medium according to an inoculation amount with the volume concentration of 2%, and performing shake cultivation at 37 ℃ and 180rpm for 1d to obtain a plateau bacillus SX-3 bacterial liquid; the LB liquid medium is composed in the same step (2).
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| CN114574383A (en) * | 2022-01-18 | 2022-06-03 | 金华康扬环境科技有限公司 | Efficient compound microbial agent for degrading kitchen garbage as well as preparation method and application thereof |
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| CN114574383A (en) * | 2022-01-18 | 2022-06-03 | 金华康扬环境科技有限公司 | Efficient compound microbial agent for degrading kitchen garbage as well as preparation method and application thereof |
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| CN114854616B (en) * | 2022-02-25 | 2024-02-02 | 河南省科学院生物研究所有限责任公司 | Geobacillus altitudinalis KXY5 and application thereof |
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