CN112553113A - Clavus new sphingosine bacillus GBW-HB1906 with broad-salt resistance and application thereof - Google Patents
Clavus new sphingosine bacillus GBW-HB1906 with broad-salt resistance and application thereof Download PDFInfo
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- CN112553113A CN112553113A CN202011515426.2A CN202011515426A CN112553113A CN 112553113 A CN112553113 A CN 112553113A CN 202011515426 A CN202011515426 A CN 202011515426A CN 112553113 A CN112553113 A CN 112553113A
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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
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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
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
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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
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The invention provides a sphingosine bacterium strain GBW-HB1906 with broad salt tolerance and application thereof. The Clavurican sphingosine bacillus GBW-HB1906 is classified and named as the Clavurican sphingosine bacillus Novosphingobium clavifovum, the preservation number is CGMCC No.20365, the bacterial colony of the bacterium is round, yellow, 1-2 μm in diameter, smooth and wet in surface, slightly convex in middle, opaque, neat in edge and free of halo. The sphingosine bacillus clarfenii GBW-HB1906 has broad salt resistance, can normally grow under the salt concentration of 15-35 per mill, has the capacity of resisting and efficiently degrading high-concentration phenol and aniline, and has the degradation rates respectively higher than 65% and 78%. The microbial agent prepared by the sphingosine bacillus clarfurin GBW-HB1906 can effectively improve the treatment effect of the waste water containing phenol and aniline.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a novel sphingosine bacillus with broad salt tolerance GBW-HB1906 and application thereof.
Background
Aniline is one of important organic chemical raw materials in daily life production, has important and wide application value, is applied to the production of more than 300 chemical products and the preparation of intermediates at present, and has wide application in the industries of agriculture, medicine and the like. However, aniline itself has severe toxicity and has serious threat to environment and human health, and wastewater containing the substance belongs to one of the wastewater which is difficult to treat, and can invade human body through channels such as contact between respiratory tract and skin in life, so that hypoxia is caused to organism tissues, so that clinical symptoms such as headache, dizziness, unsmooth breathing and the like are generated, and serious diseases such as teratogenesis, carcinogenesis and the like can be caused when the aniline is seriously contacted with the wastewater for a long time. In addition, often need use a large amount of water to dilute when handling high concentration aniline waste water to reduce aniline concentration, promote the treatment effeciency, lead to the waste of a large amount of water resources. In addition, phenol is also an important chemical raw material, and is applied to a plurality of chemical production, such as raw materials and intermediates in the production process of industries of paper making, oil refining, coking, pesticides, plastics, medicine synthesis and the like. Because of the difficult degradability of the waste water, the waste water is difficult to treat. And the harm to human is very serious, so the research and exploration of the wastewater containing the above two substances have important value and significance.
At present, in the process of wastewater treatment, a biological treatment method is widely applied to the treatment of various wastewater as a component of a key wastewater treatment process. But the wastewater containing high salt, high concentration phenol and aniline has stronger inhibiting effect on microorganisms. Therefore, the key point of breakthrough in the technical field is to find microorganisms capable of resisting salt, efficiently degrading and resisting high-concentration phenol and aniline.
Disclosure of Invention
The invention aims to provide a sphingosine bacterium GBW-HB1906 strain with broad-salt tolerance and application thereof. The sphingosine bacillus clausii GBW-HB1906 has good salt-tolerant growth characteristics, has the characteristic of high-concentration phenol and aniline resistance, and also has the capability of efficiently degrading the high-concentration phenol and aniline.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme:
the invention provides a Clavus new sphingosine bacillus GBW-HB1906 with broad salt tolerance, which is classified and named as Novosphingobium clarifavanum with the preservation number of CGMCC No. 20365.
Furthermore, the colony of the sphingosine bacillus Clarfurin GBW-HB1906 is round, yellow, 1-2 μm in diameter, smooth and wet in surface, slightly convex in middle, opaque, neat in edge and free of halo.
Furthermore, the sphingosine bacterium GBW-HB1906 has broad salt tolerance and can grow well within 15-35 per mill of salinity.
Further, the suitable growth temperature of the sphingosine bacterium Clarfurin GBW-HB1906 is 10-40 ℃.
Further, the optimum growth temperature of the sphingosine bacterium GBW-HB1906 is 25-30 ℃.
Further, the suitable growth pH value of the sphingosine bacterium GBW-HB1906 is 6-9.
Further, the optimum growth pH of the sphingosine bacterium GBW-HB1906 is 7.0-8.0.
Further, the Clarithromycin sphingobacterium GBW-HB1906 is a facultative anaerobe.
Further, the sphingosine bacterium GBW-HB1906 enters the logarithmic growth phase after being cultured for 12h, enters the end stage of logarithmic growth at 28-30h, and the bacterial count reaches 5.0 multiplied by 109CFU/mL。
Further, the Clavuilleumilus GBW-HB1906 is capable of tolerating high concentrations of phenol and aniline.
Further, the sphingosine bacterium GBW-HB1906 can efficiently degrade phenol and aniline in wastewater.
The invention also provides application of the sphingosine bacillus Clarfurin GBW-HB1906 in preparation of a microbial agent for degrading phenol and aniline in wastewater.
Further, when in use, the microbial agent comprises the bacteria with the content of 2.0 multiplied by 109～3.0×109CFU/mL of a Sphingobacterium krafft GBW-HB1906 strain.
Further, the fermentation conditions of the sphingosine bacillus clausii GBW-HB1906 zymocyte liquid are as follows: the tank pressure is 0.2-0.3 MPa, the temperature is 28-30 ℃, the dissolved oxygen is not less than 20%, the stirring speed is 150-180 rpm, and the fermentation time is 32-36 h.
Furthermore, the addition concentration of the sphingosine bacterium clever GBW-HB1906 zymocyte liquid is 70-100 ppm.
Further, the degradation rate of the sphingosine bacillus clausii GBW-HB1906 zymocyte liquid to phenol is higher than 65%.
Further, the degradation rate of the sphingosine bacteria GBW-HB1906 fermentation bacteria liquid to aniline is higher than 78%.
Compared with the prior art, the invention has the following advantages and technical effects:
the sphingosine bacillus clarkii GBW-HB1906 disclosed by the invention is separated from a high-salinity water environment, has a salt-tolerant growth characteristic, and can grow well within a salinity range of 15-35 per mill. The sphingosine bacillus clausii GBW-HB1906 has the characteristic of tolerating high-concentration phenol and aniline, has good degradation capacity for the two substances under the high-concentration condition, and has the degradation rates of 66% and 79% for 96h for the phenol and aniline with the initial concentration of 2000mg/L respectively, so that the sphingosine bacillus clausii GBW-HB1906 is cultured and fermented, and then is prepared into a microbial agent by itself or together with a carrier for degrading the phenol and aniline in waste water, the treatment effect of the waste water containing the phenol and aniline can be effectively improved, and the method has wide application prospect and value in the aspect of microbial treatment of the waste water containing the substances.
Drawings
FIG. 1 is a colony morphology of the Clavus new Sphingobacterium GBW-HB1906 on modified Nutrient Broth (NB) solid medium plates.
FIG. 2 is a growth curve of the novel bacterium Sphingobacterium krafft GBW-HB 1906.
FIG. 3 shows the results of the phenol removal application of said Clarfurin strain GBW-HB 1906.
FIG. 4 shows the results of the aniline removal application test of said Clavus new Sphingobacterium GBW-HB 1906.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples.
In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.
The formulations of the media required in the following examples are as follows:
1. enrichment screening of liquid culture medium for salt-tolerant growth microorganisms: glucose 15g, peptone 3g, K2HPO41.0g，MgSO4·7H2O 0.1g，FeSO4·7H20.5g of O, 20g of NaCl, pH 7.0-8.0 and 1L of distilled water.
2. Separating and purifying a solid culture medium by the salt-tolerant growth microorganisms: glucose 15g, peptone 3g, K2HPO4 1.0g，MgSO4·7H2O 0.1g，FeSO4·7H20.5g of O, 20g of NaCl, 15g of agar powder, 7.0-8.0 of pH and 1L of distilled water.
3. Modified Nutrient Broth (NB) medium: 10g of beef extract, 5g of peptone, 5g of yeast powder, 5g of sodium chloride and FeSO4·7H2O 0.15g/L、MgSO4·7H20.1g/L of O and 1L, pH 7.0.0-7.5 of water.
4. Modified Nutrient Broth (NB) solid medium: 10g of beef extract, 5g of peptone, 5g of yeast powder, 5g of sodium chloride and FeSO4·7H2O 0.15g/L、MgSO4·7H20.1g/L of O, 15g of agar powder and 1L, pH 7.0.0-7.5 of water.
5. Fermentation medium: 100g/L glucose, 30g/L corn steep liquor, 10g/L, NaCl 5g/L, KH peptone2PO40.5g/L、FeSO4·7H2O 0.15g/L、Na2HPO4 3g/L、MgSO4·7H20.1g/L of O, the balance of water, and the pH value of the liquid culture medium is 7.0-7.5.
The above culture medium is sterilized at 121 deg.C for 20min before use, and then stored at room temperature.
Example 1: screening, isolation and identification of sphingosine bacterium clevugh GBW-HB1906
1. Screening and purification of sphingosine bacillus Clarfurin GBW-HB1906
Taking 10mL of mixed wastewater of each section of a biochemical system of a sewage plant, adding the mixed wastewater into 200mL of PBS buffer solution, oscillating for 20min to fully and uniformly mix the samples, centrifuging for 1min at 800rpm, and collecting the sample supernatant for later use. And (3) taking 10mL of the sample supernatant, respectively adding the sample supernatant into conical flasks filled with 200mL of the salt-tolerant growth microorganism enrichment screening liquid culture medium, and carrying out shake culture at 30 ℃ and 180rpm for 3d for enrichment 3 times. After the cultured bacterial suspension is subjected to gradient dilution, 100 mu L of the suspension is coated on a solid culture medium for separating and purifying the salt-tolerant growth microorganisms and is cultured in an incubator at 30 ℃. After 48h, single colonies of different morphologies were picked and streaked on modified Nutrient Broth (NB) solid medium, and isolation and purification were repeated 3 times to obtain a single colony, which was named as GBW-HB 1906.
As shown in FIG. 1, the bacterial colony of the strain GBW-HB1906 on a modified Nutrient Broth (NB) solid medium plate is round, yellow, 1-2 μm in diameter, smooth and wet in surface, slightly convex in middle, opaque, neat in edge and free of halo.
2. Identification of Clavus new Sphingobacterium GBW-HB1906
The DNA of the strain GBW-HB1906 is used as a template, 16S rRNA universal primers are used for amplification, amplified fragments are subjected to sequence determination, the 16S rDNA sequencing result of the obtained strain GBW-HB1906 is compared with the sequence in GenBank for analysis, and the result shows that the strain GBW-HB1906 has the highest homology with Novosphingobium clavifomum, so that the strain GBW-190HB 6 is determined to be the sphingosine bacillus Clarfenii.
The strain GBW-HB1906 is selected and the strain preservation unit of the Clavus new Sphingobacterium GBW-HB1906 is as follows: china general microbiological culture Collection center (CGMCC); address: western road No. 1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: year 2020, month 07, 15; the preservation number of the sphingosine bacterium Novosphingobium clarfavanum is as follows: CGMCC No. 20365.
Example 2: growth determination and physiological and biochemical characteristics of sphingosine bacillus Clarfurin GBW-HB1906
1. Growth assay for Sphingobacterium clarfenii GBW-HB1906
Inoculating the slant cultured sphingosine bacterium GBW-HB1906 into an improved NB culture medium, performing shake culture at constant temperature of 28 ℃ for 40h at pH 7.0-7.5 to obtain GBW-HB1906 bacterial liquid, sampling every 1.0h, measuring absorbance at OD600nm, and drawing a growth curve. As shown in FIG. 2, the experimental results show that GBW-HB1906 is in the growth retardation phase in the first 12h of culture, then enters the logarithmic growth phase, and enters the terminal logarithmic growth phase when being cultured for 28-30h, and the bacterial count reaches 5.0 × 109CFU/mL, 30-36h for the growth stationary phase, followed by the decline phase, completing the entire growth cycle.
2. Physiological and biochemical characteristics of sphingosine bacillus Clarfurin GBW-HB1906
The prepared GBW-HB1906 bacterial liquid is further cultured in NB medium, and then the sphingosine bacterium GBW-HB1906 is detected according to the strain physiological and biochemical detection method of Bergey's Manual of identification of bacteria. As shown in Table 1, the sphingosine bacterium GBW-HB1906 grew normally at 15-40 ℃ and was most suitable for growthThe long temperature is 25-30 ℃; can grow in the pH value range of 6-9, and the optimum growth pH value is 7.0-8.0; it belongs to facultative anaerobe, can grow under aerobic and anaerobic condition; the strain can produce urease, beta-glucosidase, catalase and glycerol, and can degrade power-nitrate, simon citrate and semi-solid agar. In addition, the formula of a medium suitable for growth of Sphingobacterium krafft GBW-HB1906 is as follows: 100g/L glucose, 30g/L corn steep liquor, 10g/L, NaCl 5g/L, KH peptone2PO4 0.5g/L、FeSO4·7H2O 0.15g/L、Na2HPO4 3g/L、MgSO4·7H20.1g/L of O, the balance of water, and the pH value of the liquid culture medium is 7.0-7.5.
TABLE 1 physio-biochemical characteristics of Clavus new Sphingobacterium GBW-HB1906
Note: < + > represents positive, and < - > represents negative.
Example 3: salinity tolerance test of sphingosine bacterium Clarfurin GBW-HB1906
In order to test the salt-tolerant growth characteristics of the sphingosine bacterium GBW-HB1906, the growth conditions of the sphingosine bacterium GBW-HB1906 are detected and analyzed under different salinity conditions. In the test, the culture media with different salt concentrations are prepared by adjusting the amount of sodium chloride in the NB medium, then the slant culture of the Clavus neosphingosine GBW-HB1906 is inoculated into the NB medium with different salt concentrations, and the culture solution of the Clavus neosphingosine GBW-HB1906 is obtained by culturing the slant culture of the Clavus neosphingosine GBW-HB1906 in a constant temperature shaking table with the pH of 7.0-7.5 and the temperature of 30 ℃ and the growth condition of the Clavus neosphingosine in the culture media with different salt concentrations is detected. As shown in Table 2, GBW-HB1906 has salt-tolerant growth characteristics, and can grow well within a salinity range of 15 to 35 ‰.
TABLE 2 growth of Sphingobacterium clarkii GBW-HB1906 at different salt concentrations
Example 4: test of Effect of Sphingobacterium krafft GBW-HB1906 on phenol removal Rate
1. Inoculating the slant cultured sphingosine bacillus Clarfenii GBW-HB1906 into the improved NB culture medium, and culturing in a constant temperature shaking table with pH 7.5 and temperature of 28 deg.C for 28h to obtain sphingosine bacillus Clarfenii GBW-HB1906 seed solution; inoculating the sphingosine bacillus clarfungi GBW-HB1906 seed liquid into a fermentation culture medium according to the volume ratio of 20%, adjusting the tank pressure to 0.2MPa, the temperature to 28 ℃, the dissolved oxygen to be not less than 20%, stirring speed to 160rpm, and fermenting for 34h to obtain the sphingosine bacillus clarfungi GBW-HB1906 zymocyte liquid.
2. In the test process, the content of phenol is detected by adopting a 4-aminoantipyrine spectrophotometry (HJ 503-. The initial concentration of phenol in the water is controlled to 2000mg/L at the beginning of the test, wherein CK is a blank control group without adding sphingosine bacillus Clarfrom GBW-HB1906 zymocyte liquid, F is a test group with adding sphingosine bacillus Clarfrom GBW-HB1906 zymocyte liquid, each group is provided with 3 repeated experiments, the adding concentration of the sphingosine bacillus Clarfrom GBW-HB1906 zymocyte liquid is 100ppm, and the bacterial liquid content is adjusted to 2.0 x 10 before use9～3.0×109CFU/mL; after the treatment is finished, the groups are placed in a shaking table to start the test, sampling is carried out every 4h to detect the change of phenol, the phenol degradation rate is calculated, and the test period is 96 h. In the test process, the temperature is controlled to be 25 ℃, the rotating speed of the shaking table is 180rpm, and the pH value is 7.0-8.0.
As shown in FIG. 3, 96-hour phenol degradation test shows that the removal rate of the sphingosine bacterium GBW-HB1906 zymocyte liquid to phenol with the initial concentration of 2000mg/L is 66%, which indicates that the strain has the capability of efficiently and significantly degrading high-concentration phenol, and has great application value and significance in removing phenol-containing environmental pollution.
Example 5: effect test of sphingosine bacterium Clarfurin GBW-HB1906 on aniline removal Rate
The aniline content is detected by adopting a gas chromatography-mass spectrometry method (HJ 822-2017) in the test process. The initial concentration of aniline in the water is controlled to 2000mg/L at the beginning of the test, wherein CK is a blank control group without adding sphingosine bacillus Clarfenii GBW-HB1906 zymocyte liquid, A is a test group with zymocyte liquid, each group is provided with 3 repeated experiments, the adding concentration of the sphingosine bacillus GBW-HB1906 zymocyte liquid is 100ppm, and the bacterial content of the used bacteria liquid is adjusted to 2.0 x 10 before use9～3.0×109CFU/mL; after the treatment is finished, the groups are placed in a shaking table to start the test, the change of the aniline is sampled and detected every 4 hours, the degradation rate of the aniline is calculated, and the test period is 96 hours. In the test process, the temperature is controlled to be 25 ℃, the rotating speed of the shaking table is 180rpm, and the pH value is 7.0-8.0. As shown in the figure 4, 96-hour aniline degradation test shows that the removal rate of the sphingosine bacterium GBW-HB1906 zymocyte liquid to the aniline with the initial concentration of 2000mg/L is 79%, and the strain is also shown to have the capability of efficiently and obviously degrading high-concentration aniline, so that the strain has important value and significance in the aspect of being applied to the aniline-containing environmental pollution treatment.
The sphingosine bacillus novalaceae GBW-HB1906 can be prepared into a microbial agent alone or together with other carriers, is used for removing phenol and aniline in sewage, wastewater or other environments, has mature preparation process and simple use method, only needs to inoculate seed liquid of the sphingosine bacillus novalaceae GBW-HB1906 into a fermentation medium according to the volume ratio of 20 percent, adjusts the tank pressure to 0.2MPa, the temperature to 28 ℃, the dissolved oxygen to be more than or equal to 20 percent, the stirring speed is 160rpm, and ferments for 32h to prepare the required bacterial liquid; when the using concentration is 100ppm, the phenol/aniline composite material has the capability of efficiently degrading high-concentration phenol and aniline, so that the phenol/aniline composite material has important value and significance in the aspect of treating and repairing the environmental pollution containing phenol or aniline.
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 apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A cladosporium cucurbitacearum GBW-HB1906 with broad salt tolerance is characterized in that the cladosporium cucurbitacearum is classified and named as the cladosporium cucurbitacearum with the preservation number of CGMCC No. 20365.
2. The sphingosine bacterium GBW-HB1906 as claimed in claim 1, wherein the colony of sphingosine bacterium GBW-HB1906 is round, yellow, 1-2 μm in diameter, smooth and moist in surface, slightly convex in the middle, opaque, neat in edge, and halo-free.
3. The sphingosine bacterium GBW-HB1906 as claimed in claim 1, wherein said sphingosine bacterium GBW-HB1906 has broad salt tolerance and can grow well in salinity of 15 to 35 ‰.
4. The sphingosine bacterium GBW-HB1906 as claimed in claim 1, wherein the optimum growth temperature of the sphingosine bacterium GBW-HB1906 is 25-30 ℃.
5. The sphingosine bacterium GBW-HB1906 as claimed in claim 1, wherein the optimum growth pH of said sphingosine bacterium GBW-HB1906 is 7.0-8.0.
6. The sphingosine bacterium GBW-HB1906 as claimed in claim 1, wherein said sphingosine bacterium GBW-HB1906 is capable of efficiently degrading phenol and aniline in wastewater.
7. Use of the bacterium sphingosine bacterium of any of claims 1-6, GBW-HB1906 for the preparation of a microbial agent for the degradation of phenol and aniline in wastewater.
8. The use of claim 7, wherein, when applied, the microbial agent comprises a bacteria content of 2.0 x 109～3.0×109CFU/mL of a Sphingobacterium krafft GBW-HB1906 strain.
9. The use according to claim 8, wherein the fermentation conditions of the sphingosine bacterium GBW-HB1906 zymocyte fluid are as follows: the tank pressure is 0.2-0.3 MPa, the temperature is 28-30 ℃, the dissolved oxygen is not less than 20%, the stirring speed is 150-180 rpm, and the fermentation time is 32-36 h.
10. The use according to claim 8, wherein the sphingosine bacterium GBW-HB1906 fermentation broth is added at a concentration of 70-100 ppm.
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| CN102277312A (en) * | 2011-01-10 | 2011-12-14 | 辽宁石油化工大学 | Strain for degrading polycyclic aromatic hydrocarbons at low temperature and application in bioremediation of groundwater in petroleum hydrocarbon polluted places thereof |
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| CN113444661A (en) * | 2021-06-21 | 2021-09-28 | 温州大学 | Sphingobacterium neoformans and application thereof in wastewater dephosphorization |
| CN113444661B (en) * | 2021-06-21 | 2022-03-04 | 温州大学 | Sphingobacterium neoformans and application thereof in wastewater dephosphorization |
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