CN113337436A - Urea decomposing bacteria and breeding method and application thereof - Google Patents
Urea decomposing bacteria and breeding method and application thereof Download PDFInfo
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- CN113337436A CN113337436A CN202110643795.8A CN202110643795A CN113337436A CN 113337436 A CN113337436 A CN 113337436A CN 202110643795 A CN202110643795 A CN 202110643795A CN 113337436 A CN113337436 A CN 113337436A
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- bacteria
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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
- C02F2101/20—Heavy metals or heavy metal compounds
Abstract
The invention provides a urea decomposing bacterium and a breeding method and application thereof. The urea decomposing bacteria is Staphylococcus succinogenes. The invention also provides a breeding method of the urea decomposing bacteria and application of the urea decomposing bacteria in solidifying mercury and arsenic in high-salt wastewater. The urea decomposing bacteria provided by the invention has good tolerance to heavy metal toxicity, and can adapt to a higher salinity environment. The removal rate of toxic heavy metals Hg and As can reach more than 90 percent, and the material is a good microbial water purification material which can be widely used in arid regions.
Description
Technical Field
The invention relates to the field of sewage treatment, in particular to a urea decomposing bacterium, and a breeding method and application thereof.
Background
Urea decomposing bacteria are an important class of microorganisms in the natural environmentThey are ubiquitous in the environment and are capable of synthesizing urease, specifically catalyzing the hydrolysis of urea to produce CO in and out of cells3 2-And NH4 +The formation of a micron-scale alkaline environment around the cell, resulting in Ca2+With CO3 2-Supersaturation of the solution, and calcium carbonate precipitation is induced to generate. At present, there is a need to provide a good microbial water purification method and material that can be widely used in arid areas.
Disclosure of Invention
The invention provides a urea decomposing bacterium and a breeding method and application thereof. The urea decomposing bacteria provided by the invention has good tolerance to heavy metal toxicity, and can adapt to a higher salinity environment. The removal rate of toxic heavy metals Hg and As can reach more than 90 percent, and the material is a good microbial water purification material which can be widely used in arid regions.
The invention provides a urea decomposing bacterium, which is staphylococcus succinogenes.
The invention also provides application of the urea decomposing bacteria, and the urea decomposing bacteria are applied to solidifying mercury and arsenic in high-salinity wastewater.
The invention also provides a breeding method of the urea decomposition bacteria, which comprises the following steps:
1) collecting a sample of high-salinity environmental sewage;
2) putting the sample into an NB culture medium containing 1-3% NaCl, and carrying out enrichment culture on strains to obtain an enrichment culture solution;
3) putting the enrichment culture solution into an NB culture medium containing 1-3% of urea and 2-4% of NaCl for salt-tolerant bacteria liquid culture, and then diluting to obtain a diluent;
4) culturing the diluent in a culture medium to obtain a bacterial colony;
5) and (4) carrying out purification and separation on the colonies.
According to the breeding method of the ureolytic bacteria provided by the invention, in the step 1), the sample is stored at the temperature of below 4 ℃.
According to the breeding method of the ureolytic bacteria provided by the invention, in the step 2), the sample is placed into an NB culture medium containing 2% NaCl, and the volume ratio of the sample to the NB culture medium is 1: 120-180, preferably 1: 150.
According to the breeding method of the urea decomposition bacteria provided by the invention, in the step 2), the enrichment culture is carried out at the conditions that the rotating speed is 100-150 rpm, preferably 130rpm, and the temperature is 25-30 ℃ and preferably 28 ℃; the time of enrichment culture is 18-25 h, preferably 24 h.
According to the breeding method of the urea decomposition bacteria, provided by the invention, in the step 3), 1-5 mL of suspension of the enrichment culture solution is absorbed, added into 50-200 mL of NB culture medium containing 2% urea and 3% NaCl, cultured for 15-30 h to obtain halotolerant bacteria liquid, 8-15 mL of sterilized deionized water is added into 0.5-2 mL of the halotolerant bacteria liquid for dilution, and 10 times of dilution is respectively adopted by a ten-fold dilution method4、105、106Double dilution.
In the invention, the breeding effect is better by adopting the optimized parameters, the concentration and the proportions.
According to the breeding method of the ureolytic bacteria provided by the invention, in the step 4), the diluent is coated on the surface of a culture medium filled with nutrient agar, and the culture is carried out for 45-50 h at 25-30 ℃.
According to the breeding method of the urea decomposition bacteria provided by the invention, in the step 5), the obtained bacterial colonies are purified and separated by adopting a plate marking method, and the marking purification and separation are carried out for 2-3 times.
According to the breeding method of the ureolytic bacteria provided by the invention, the NB culture medium comprises the following components: 8-15 g/L of peptone, 2-4 g/L of beef powder and 3-7 g/L of sodium chloride; the pH value of the NB culture medium is 7.2-7.8.
The invention has the beneficial effects that: the screened ureolytic bacteria have good tolerance to heavy metal toxicity, and can adapt to higher salinity environment. The removal rate of toxic heavy metals Hg and As can reach more than 90 percent, and the material is a good microbial water purification material which can be widely used in arid regions.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 shows Hg by ureolytic bacteria provided by an embodiment of the present invention2+Schematic diagram of removal of (a);
FIG. 2 shows the pair of As by ureolytic bacteria provided in the embodiment of the present invention3+Schematic diagram of removal of (1).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The instruments and the like are conventional products which are purchased by normal distributors and are not indicated by manufacturers. The process is conventional unless otherwise specified, and the starting materials are commercially available from the open literature. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications.
In the examples of the present invention, the NB medium components: peptone, 10 g/L; 3g/L of beef powder; sodium chloride, 5 g/L; pH, 7.4.
Example 1
The embodiment provides a urea decomposing bacterium, and the method for breeding and solidifying mercury and arsenic comprises the following specific steps: in outdoor high-salt environment sewage, a sewage sample is collected and stored below 4 ℃. In a laboratory1ml of the sample is placed in 150ml of NB medium containing 2% NaCl and placed in a shaker at a rotation speed of 130rpm and a temperature of 28 ℃ for enrichment culture for 24 h. Carrying out enrichment culture on the strains for 24h to form nutrient bacteria liquid. Absorbing 2mL of suspension of the enrichment culture solution, adding the suspension into 100mL of NB culture medium containing 2% urea and 3% NaCl, culturing for 24h, absorbing 1mL of halotolerant bacteria liquid, adding 10mL of sterilized deionized water for dilution, and respectively diluting 10mL of halotolerant bacteria liquid by adopting a tenfold dilution method4、105、106The diluent (2). 0.1ml of each of the above dilutions was added to a petri dish containing a nutrient agar medium, the surface of the medium was uniformly coated with a glass rod at each dilution of 3 parallel samples, and cultured in a constant temperature incubator at 28 ℃ for 48 hours. Each obtained colony is purified and separated by a plate marking method, and after 2-3 times of marking, a single bacterial colony of J3 is obtained.
2% Urea was added to the J3 strain obtained by separation and purification, and pH and OD were observed600The strain J3 can be proved to have the capability of decomposing urea and stronger urease activity.
The gene sequencing, alignment and analysis prove that J3 is Staphylococcus succincus (Staphylococcus succinogenes), which is abbreviated as S. Belongs to staphylococcus, and is moderate halophilic bacteria, and can adapt to water environment with high salt content in Xinjiang.
And (3) adding the purified S.succininus into an NB culture medium, and fermenting for 24 hours in a fermentation tank to obtain the S.succininus urea decomposition microbial inoculum capable of being used in large quantity.
As shown in the attached figure 1-2, the microbial inoculum is added into high-salt waste water (Hg) containing mercury and arsenic2+And As3+The concentrations of (A) were 10. mu.M and 100. mu.M, respectively. No. 0 is blank control without adding humic acid solution (DOM), No. 1 is added with 200 mul of 0.75g/L humic acid solution, No. 2 is added with 1ml of 0.75g/L humic acid solution, No. 3 is added with 5ml of 0.75g/L humic acid solution, No. 4 is added with 10ml of 0.75g/L humic acid solution, so that the final concentrations of the humic acid solutions are 0, 1, 5, 25 and 50mg/L respectively), and Hg is added within 48h2+The total removal rate of the catalyst reaches 90 percent; within 48h, As3+The removal rate of (2) is also 90% or more. In this process, Hg2+Although existence of (2) inhibitsThe method has the advantages that the method is safe and reliable, and has high mercury and arsenic removal effects.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A urea-decomposing bacterium, wherein the urea-decomposing bacterium is Staphylococcus succinogenes.
2. The use of the ureolytic bacteria of claim 1, wherein the ureolytic bacteria is used to solidify mercury and arsenic in high salinity wastewater.
3. A method for selectively breeding a ureolytic bacterium according to claim 1, comprising:
1) collecting a sample of high-salinity environmental sewage;
2) putting the sample into an NB culture medium containing 1-3% NaCl, and carrying out enrichment culture on strains to obtain an enrichment culture solution;
3) putting the enrichment culture solution into an NB culture medium containing 1-3% of urea and 2-4% of NaCl for salt-tolerant bacteria liquid culture, and then diluting to obtain a diluent;
4) culturing the diluent in a culture medium to obtain a bacterial colony;
5) and (4) carrying out purification and separation on the colonies.
4. The method for selectively breeding ureolytic bacteria according to claim 3, wherein the sample is stored at 4 ℃ or lower in step 1).
5. The selective breeding method of ureolytic bacteria of claim 3, wherein in step 2), the sample is placed in NB culture medium containing 2% NaCl, and the volume ratio of the sample to the NB culture medium is 1: 120-180, preferably 1: 150.
6. The selective breeding method of urea decomposition bacteria according to claim 5, wherein in step 2), the enrichment culture is performed at a rotation speed of 100-150 rpm, preferably 130rpm, and a temperature of 25-30 ℃, preferably 28 ℃; the time of enrichment culture is 18-25 h, preferably 24 h.
7. The selective breeding method of urea decomposition bacteria according to claim 3, wherein in step 3), 1-5 mL of the enriched culture solution suspension is sucked, added into 50-200 mL of NB culture medium containing 2% urea and 3% NaCl, cultured for 15-30 h to obtain halotolerant bacteria solution, 8-15 mL of sterilized deionized water is added into 0.5-2 mL of halotolerant bacteria solution for dilution, and 10-fold dilution method is adopted to respectively dilute the halotolerant bacteria solution by ten times4、105、106Double dilution.
8. The method for breeding the ureolytic bacteria of claim 3, wherein in step 4), the diluent is coated on the surface of the nutrient agar medium, and the culture is carried out at 25-30 ℃ for 45-50 h.
9. The breeding method of urea decomposition bacteria according to claim 3, wherein in step 5), the obtained bacterial colonies are purified and separated by a plate-streaking method, and streaking is performed for 2-3 times.
10. The method for breeding the ureolytic bacteria according to any one of claims 3 to 9, wherein the NB medium comprises: 8-15 g/L of peptone, 2-4 g/L of beef powder and 3-7 g/L of sodium chloride; the pH value of the NB culture medium is 7.2-7.8.
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