CN111378592B - Bacillus licheniformis and method for treating malodorous organic wastewater by using same to purify water - Google Patents
Bacillus licheniformis and method for treating malodorous organic wastewater by using same to purify water Download PDFInfo
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- CN111378592B CN111378592B CN201811611610.XA CN201811611610A CN111378592B CN 111378592 B CN111378592 B CN 111378592B CN 201811611610 A CN201811611610 A CN 201811611610A CN 111378592 B CN111378592 B CN 111378592B
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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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- 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
- C12R2001/10—Bacillus licheniformis
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- 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
Abstract
The invention provides bacillus licheniformis which is classified and named as: bacillus licheniformis (Bacillus licheniformis) GRINML8, the preservation unit is: china center for type culture Collection, Address: wuhan university in Wuhan, China, preservation date: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018675. The bacterium is used for treating the malodorous organic wastewater, organic matters in the wastewater can be utilized as a self carbon source and energy substances to degrade the organic matters in the high-COD organic wastewater, the water body is effectively purified, the environment is protected, the efficiency is high, animals and plants in the water body are not damaged, and an operable process can be provided for degrading organic pollutants in the water.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a Bacillus licheniformis (Bacillus) bacterium, a culture method thereof and a method for treating malodorous organic wastewater and purifying water by using the bacterium.
Background
The substandard discharge of chemical plants and domestic sewage into rivers, lakes and rivers can cause the increase of the contents of carbon, nitrogen, phosphorus, sulfur and the like in the water environment, exceed the self-purification load of the water body, cause the eutrophication of the water body, form a 'black and odorous water body' and harm the life health of animals and plants in the water environment. At present, the conventional treatment methods such as physical adsorption, membrane filtration and other physical sewage interception methods have limited sewage treatment capacity, and the adsorption material is easy to reach the adsorption limit and is not easy to desorb; the chemical treatment method is usually to add some chemical agents for flocculation and precipitation, and the method is not easy to control the adding amount of the agents, has certain toxic action on water animals and plants, and is easy to generate secondary pollution. How to reduce COD in the water body while not damaging aquatic organisms is a key problem for solving the problem of purifying the water body by the organic wastewater. The newly separated bacillus licheniformis can use organic matters in the wastewater as a carbon source and nutrient substances of the bacillus licheniformis to achieve the purpose of purifying the water environment.
Disclosure of Invention
In order to solve the above problems, a first object of the present invention is to provide a highly efficient bacillus licheniformis strain, which can effectively utilize organic matters in wastewater as its own carbon source and energy source substances, and degrade the organic matters in wastewater to achieve the purpose of purifying water environment.
The second purpose of the invention is to provide a culture medium which can enrich, separate and culture the bacteria.
The third purpose of the invention is to provide a method for purifying water body by using the bacterium to treat the malodorous organic wastewater.
In order to achieve the above object, the present invention provides a bacillus licheniformis, which is classified and named as: bacillus licheniformis (Bacillus licheniformis) GRINML8, the preservation unit is: china center for type culture Collection, Address: wuhan university in Wuhan, China, preservation date: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018675.
And (4) observing the morphological characteristics of colonies: the bacterial colony is round, the edge is neat, the surface is smooth and moist, and the bacterial colony has no viscosity and bulges; the bacillus subtilis is in a short rod shape under a microscope, and gram staining shows that the bacillus subtilis is gram-positive bacteria, can generate spores with special resistance under adverse conditions, has good growth vigor in a malodorous organic wastewater environment, and can degrade organic matters in wastewater within 30 days to ensure that COD (chemical oxygen demand) reaches the national V-type water discharge standard.
The bacillus licheniformis can effectively utilize organic matters in the wastewater as self carbon sources and energy substances, and degrade the organic matters in the wastewater to achieve the purpose of purifying the water environment.
The invention also provides a culture medium for isolated culture of the bacillus licheniformis, which comprises the following components in percentage by weight: NH (NH)4Cl 0.1-1g，NaCl 0.2-0.8g，KH2PO4 0.1-0.5g，MgSO4·7H2O 0.1-0.5g，CaCl2 0.01-0.1g，0.01％FeSO41-5mL of the agar gel, 1-2g of sodium succinate, 0.1-1mL/L of a microelement mother solution, 1000mL of distilled water and 20-30g of agar powder, and adjusting the pH value to 7; wherein, the microelement mother liquor comprises the following components: h3BO3 3g/L，CoCl2·6H2O 2g/L，ZnSO4·7H2O 1g/L，MnCl2·4H2O 0.3g/L，Na2MoO4·7H2O 0.3g/L，NiCl2·6H2O 0.2g/L，CuCl2·2H2O 0.1g/L。
The preparation method comprises mixing the above materials, adding agar powder, adjusting pH to 7, sterilizing at 121 deg.C for 30 min, and pouring into flat plate.
The invention also provides a culture medium for enrichment culture of the bacillus licheniformis, which comprises the following components in percentage by weight: yeast extract 0.8g, NH4Cl 1g，(NH4)2SO4 2g，NaAc 3g，MgCl2 0.1g，CaCl2 0.1g，KH2PO4 0.5g，K2HPO40.3g, and 1000mL of distilled water.
The preparation method comprises mixing the above materials, adjusting pH to 7, and sterilizing at 121 deg.C for 30 min.
The enrichment culture method of the bacillus licheniformis comprises the steps of inoculating the bacillus licheniformis strain into an enrichment culture medium, culturing at 35 ℃ under anaerobic condition, enabling the bacillus licheniformis strain to grow and propagate fast, enabling the illumination intensity to reach 3000Lx, and standing and culturing until the concentration of the bacillus licheniformis strain reaches 108one/mL.
The invention also provides a method for treating the malodorous organic wastewater and purifying the water body, which comprises the following steps:
1) adding 5L of malodorous organic wastewater retrieved from a certain chemical plant into a glass container;
2) carrying out enrichment culture on the bacillus licheniformis by adopting the method, inoculating the bacillus licheniformis into a container, setting the illumination intensity to be 3000Lx, and carrying out anaerobic static culture;
3) and detecting COD and the biomass of the bacillus licheniformis in the organic wastewater every 24 hours until the COD value is less than 40mg/L (national class V water discharge standard).
Further, the bacillus licheniformis in the step 2) is inoculated in the culture medium in an amount of 15% (v/v).
The invention has the beneficial effects that:
the invention provides a bacillus licheniformis strain GRINML8, which is used for treating malodorous organic wastewater, can degrade organic matters in the organic wastewater with high COD by using organic matters in the wastewater as a carbon source and an energy substance, effectively purify water, is green and efficient, does not damage animals and plants in the water, can provide an operable process for degrading organic pollutants in water, and achieves the purpose of purifying the organic wastewater by photosynthetic bacteria.
Drawings
FIG. 1 is a photograph of a single colony of Bacillus licheniformis strain GRINML8 provided by the present invention.
FIG. 2 is a photomicrograph of Bacillus licheniformis strain GRINML8 provided by the present invention.
Detailed Description
The present invention is further illustrated by the following examples.
The bacillus licheniformis related by the invention is preserved and named by classification as follows: bacillus licheniformis (Bacillus licheniformis) GRINML8, the preservation unit is: china center for type culture Collection, Address: wuhan university in Wuhan, China, preservation date: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018675.
The culture medium for separating and culturing the bacillus licheniformis (abbreviated as separation culture medium) has the following formula:
NH4Cl 0.5g，NaCl 0.4g，KH2PO4 0.3g，MgSO4·7H2O 0.1g，CaCl2 0.01g，0.01％FeSO42mL, 1.2g of sodium succinate, 0.2mL/L of microelement mother liquor, 1000mL of distilled water and 20g of agar powder; wherein, the microelement mother liquor comprises the following components: h3BO3 3g/L，CoCl2·6H2O 2g/L，ZnSO4·7H2O 1g/L，MnCl2·4H2O 0.3g/L，Na2MoO4·7H2O 0.3g/L，NiCl2·6H2O 0.2g/L，CuCl2·2H2O 0.1g/L。
The preparation method comprises mixing the above materials, adding agar powder, adjusting pH to 7, and sterilizing at 121 deg.C for 30 min. 100mL of the medium was prepared in the above proportions, and one plate was poured per 20 mL.
The culture medium for enrichment culture of the bacillus licheniformis (abbreviated as enrichment culture medium) has the following formula: yeast extract 0.8g, NH4Cl 1g，(NH4)2SO4 2g，NaAc 3g，MgCl2 0.1g，CaCl2 0.1g，KH2PO4 0.5g，K2HPO40.3g, and 1000mL of distilled water.
The preparation method comprises mixing the above materials, adjusting pH to 7, and sterilizing at 121 deg.C for 30 min.
Example 1 Bacillus licheniformis acquisition and identification
The bacillus licheniformis provided by the invention is separated from organic wastewater near a chemical plant in Beijing.
1) Adding yeast powder into organic wastewater with an addition amount of 0.8g/L, standing and enriching at 35 deg.C under anaerobic condition and illumination intensity of 3000Lx for seven daysThe bacterial morphology was judged by microscopic examination. Measuring the absorbance of the culture at 600nm to obtain OD600If the value of (A) is between 0.6 and 0.8.
2) The bacteria in the above culture broth were filtered through a 0.22 μm filter and washed with 20mL of sterile water. The culture medium was inoculated in 100mL enriched medium in an amount of 15% (v/v) and cultured under anaerobic conditions at 35 ℃ to prepare a non-inoculated Control (CK). The growth of the bacteria was observed after two weeks of static culture at an illumination intensity of 3000 Lx.
3) The enrichment medium liquid is diluted by 1, 2, 3, 4 and 5 (respectively corresponding to 10)-1，10-2，10-3，10-4，10-5) 100. mu.L of each dilution was applied to a plate prepared in a separate medium and cultured at 35 ℃ for three days. And selecting a single colony, carrying out plate three-region scribing, and separating to obtain the single colony. After the single colony is picked, the single colony is transferred to a new solid medium plate, and the separation culture is continued by using a plate streaking separation method until the single colony is obtained.
Drawing lines by using a three-zone purification method to obtain the single colony morphology of the bacillus licheniformis as shown in figure 1; the cell morphology was observed under a microscope and is shown in FIG. 2.
The 16S rDNA clone library technology is used for analyzing and identifying the strains. Centrifuging 1mL of bacterial liquid to obtain bacterial sludge, extracting total DNA, and amplifying a 16S rDNA fragment by using a PCR technology and a prokaryotic general primer 27f and a 1492 r. The PCR product was purified and ligated with a T-easy vector of Promega to transform E.coli DH 5. alpha. The white colonies picked were confirmed to be positive by colony PCR, and 4 clones were sequenced by enzyme digestion and typing. The obtained sequence is shown by Blast comparison, and the strain is a bacterium of the genus Bacillus licheniformis (Bacillus) and is named as Bacillus licheniformis GRINML 8.
Example 2 treatment of organic wastewater to purify Water
1) Taking a proper amount of organic wastewater near a certain chemical plant in Beijing to perform a laboratory exploration experiment in a 5L sampling bottle;
the wastewater has the following quality: pH 7.5; COD: 320 mg/L.
2) Inoculating the identified strain of Bacillus licheniformis GRINML8 in a rich mediumCulturing in culture medium under 35 deg.C anaerobic condition, wherein the strain grows and propagates rapidly, the illumination intensity reaches 3000Lx, and the strain concentration reaches 108Per mL; then inoculating the bacterial liquid into a glass container, and setting a control group without inoculation according to the inoculation amount of 15% (v/v) of a culture medium;
3) and detecting COD (chemical oxygen demand) and the biomass of the bacillus licheniformis in the organic wastewater every 24 hours, and continuously operating the whole process for 30 days, wherein the COD value of the organic wastewater is reduced to the national V-type water discharge standard.
The COD is reduced to 19mg/L before and after detection and restoration, (the COD of a control group is up to 300mg/L) and reaches the national V-type water discharge standard, the removal rate of the COD is 94 percent, and the biomass is still 10 percent8The above.
According to the method for treating the organic wastewater and purifying the water body, under the degradation action of the bacillus licheniformis, the organic matters in the wastewater are used as the carbon source and energy source substances of the bacillus licheniformis to degrade the organic matters, so that the water body is effectively purified, the environment-friendly and efficient effects are achieved, animals and plants in the water body are not damaged, an operable process is provided for degrading organic pollutants in the water, and the purpose of purifying the organic wastewater by photosynthetic bacteria is achieved.
Claims (4)
1. A Bacillus licheniformis, characterized in that the classification name is: bacillus licheniformis (Bacillus licheniformis) GRINML8, deposited as: china center for type culture Collection, Address: wuhan university in Wuhan, China, preservation date: year 2018, 10 month 15, deposit accession number: CCTCC NO: m2018675.
2. The method for enrichment culture of Bacillus licheniformis according to claim 1, characterized in that the Bacillus licheniformis according to claim 1 is inoculated into a culture medium, anaerobic static culture is carried out at a culture temperature of 25-35 ℃, the illumination intensity reaches 3000Lx, and the culture is carried out until the bacterial concentration reaches 108Per mL;
the formula of the culture medium is as follows: yeast extract 0.1-1g, NH4Cl 1-3g，(NH4)2SO4 1-5g，NaAc 3-6g，MgCl20.1-0.5g，CaCl2 0.1-0.5g，KH2PO4 0.5-0.9g，K2HPO40.3-0.8g, 1000mL of distilled water, and adjusting the pH to 7.
3. A method for treating malodorous organic wastewater to purify water is characterized by comprising the following steps:
1) adding 5L of malodorous organic wastewater retrieved from a certain chemical plant into a glass container;
2) after the bacillus licheniformis of claim 1 is subjected to enrichment culture by the method of claim 2, the bacillus licheniformis is inoculated into a container, the illumination intensity is set to 3000Lx, and anaerobic static culture is carried out;
3) and detecting COD and the biomass of the bacillus licheniformis in the organic wastewater every 24 hours until the COD value is less than 40 mg/L.
4. The method for treating the malodorous organic wastewater as claimed in claim 3, wherein the Bacillus licheniformis in the step 2) is inoculated in the culture medium at 15 v/v%.
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