CN107129954A - A kind of staphylococcus of degradation of phenol - Google Patents

A kind of staphylococcus of degradation of phenol Download PDF

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CN107129954A
CN107129954A CN201710488862.7A CN201710488862A CN107129954A CN 107129954 A CN107129954 A CN 107129954A CN 201710488862 A CN201710488862 A CN 201710488862A CN 107129954 A CN107129954 A CN 107129954A
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staphylococcus
phenol
bacterial strain
degradation
application
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CN107129954B (en
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许继飞
赵吉
宋晓雪
武琳慧
刘崎峰
王川
包智华
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Inner Mongolia University
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    • 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
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • 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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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/28—Organic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
    • 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

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Abstract

The invention provides one plant of staphylococcus (Staphylococcus caprae) and its application.The Strain Designation is CL, and deposit number is CCTCC NO:M2016305.The bacterium can be in 36h by fluid nutrient medium (the 10g MgSO of 200mg/L concentration phenols4·7H2O、0.2g CaCl2·2H2O, 2 5g KCl, 2.5g tryptones, 10g yeast extracts, NaCl 5%, distilled water are added to 1000ml.Adjust pH be 7.2 or so, 121 DEG C sterilizing 15min) in phenol degrading more than 90%.The staphylococcus CL of the present invention has stronger degradation of phenol ability, the biological prosthetic of environment is polluted available for phenol, with preferable economic value and application prospect.

Description

A kind of staphylococcus of degradation of phenol
Technical field
The present invention relates to microorganism and biodegradation technique field, in particular it relates to a kind of staphylococcus and its in degraded Application in phenol.
Background technology
Phenol and its derivatives are very strong to the toxicity of people and animals and plants, have been put into Environment Priority control pollutant list. Phenol wastewater is mainly derived from the industries such as papermaking, oil refining, synthetic fibers, synthetic rubber, agricultural chemicals, is main in industrial discharge waste water Pollutant.In these phenol wastewater in addition to pollutant containing phenol, contain a large amount of salinities toward contact and make High salt phenol wastewater, this kind of discharge of wastewater can cause severe contamination into environment to soil, surface water and groundwater.Due to high salt Environment is to microorganism growth with suppression and toxic action so that conventional biodegradation technique exists when handling this kind of waste water Bottleneck problem.Halophiles can be directly used in remove high salt industrial wastewater in organic pollution without first reducing waste water salinity, Thus gradually get more and more people's extensive concerning.
Staphylococcus (Staphylococcus caprae) is spherical or slightly oval, and diameter 1.0um or so is arranged in Botryoidalis.Staphylococcus atrichia, it is impossible to move.Without brood cell, pod membrane is generally not formed in addition to a small number of bacterial strains.Easily by conventional alkali Property dyestuff coloring, Gram's staining for the positive.It is its aging, dead or by after leukocytes phagocytic, and some bacterial strains of resistance can It is dyed to Gram-negative.Most staphylococcus energy decomposition glucoses, maltose and sucrose, production acid do not produce gas.Pathogenic bacterium Strain can decompose mannitol.
Staphylococcus is distributed very wide, the skin of healthy birds, feather, eyelid, mucous membrane, enteron aisle Deng Douyou Portugals in nature Grape coccus is present, while normal in microorganism in the bacterium or poultry hatching, raising, processing environment.For staphylococcus to dirt Dye thing degraded research be rarely reported, have research in non-salt environment staphylococcus in 36h, under identical condition, Pyrogentisinic Acid Degraded can reach more than 80%, but the research of Pyrogentisinic Acid there are no the report of correlation in hypersaline environment.
The content of the invention
It is an object of the invention to provide the staphylococcus that one plant is capable of degradation of phenol.
The present invention is separated to one plant of bacterial strain for being capable of degradation of phenol from the bed mud of salt lake.It is raw by strain morphology feature The bacterial strain is accredited as staphylococcus by reason biochemical character and 16S rDNA sequence analyses, is named as CL, the bacterial strain is in June, 2016 It is preserved within 2nd China typical culture collection center (referred to as, address：Wuhan, China Wuhan University, postcode 430072), preservation is compiled Number it is CCTCC NO.M2016305, classify entitled staphylococcus Staphylococcus caprae.
The invention provides the microbial inoculum containing staphylococcus CL.
The invention provides the biological cleanser containing staphylococcus CL.
Microbial inoculum the invention provides staphylococcus CL or containing it or the biological cleanser containing it are in cleaning ambient Application.
Further, the invention provides staphylococcus CL or containing it microbial inoculum or the biological cleanser containing it are in degraded Application in phenol.
Microbial inoculum the invention provides staphylococcus CL or containing it or the biological cleanser containing it are in processing Industry Waste Application in water.
Microbial inoculum the invention provides staphylococcus CL or containing it or the biological cleanser containing it are in processing Medical waste Application in water.
The invention provides applications of the staphylococcus CL in biological cleanser is prepared.
The invention provides applications of the staphylococcus CL in biodegradation agent is prepared.
The biodegradation agent is phenol degrading agent.
The staphylococcus CL of the present invention can be in 36h, and the concentration contained during salinity 0-5% in degraded culture medium is 200mg/L phenol reaches more than 90%, and when salinity is in the case of 10% kind, the degradation rate of Pyrogentisinic Acid is more than 80%.This The staphylococcus CL provided is provided and its microbial inoculum is pollution-free in use, it is nuisanceless, phenol can be applied to and pollute environment It is biological prosthetic, can be widely applied to clean environment field, industrial wastewater or medical field of waste water treatment containing phenol, with compared with Good economic value and application prospect.
Brief description of the drawings
Fig. 1 is bacterial strain CL and close kind of 16S rDNA sequence evolution tree.
Fig. 2 is bacterial strain CL growth and the degradation characteristic figure of phenol.
Fig. 3 is the degraded situation map of bacterial strain CL Pyrogentisinic Acids under different NaCl concentrations.
Fig. 4 is the standard curve (OD of phenol concentration270) figure.
Fig. 5 is degraded situation maps of the bacterial strain CL to different initial phenol concentrations.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.
Unless otherwise specified, chemical reagent used in embodiment is skill used in conventional commercial reagent, embodiment The conventional meanses that art means are well known to those skilled in the art.
The staphylococcus CL of embodiment 1 separation and identification
1st, staphylococcus CL separation
A, by the solid sample collected (salt lake silt, bottom silt, high-salt wastewater handle sludge etc.) 1-5g or 500ml Fluid sample (salt lake saline, seawater, high-salt wastewater etc.) filtrate, be dissolved in 10-50ml fluid nutrient mediums (10g MgSO4· 7H2O、0.2g CaCl2·2H2O, 2-5g KCl, 2.5g tryptone, 10g yeast extracts, NaCl15%, distilled water addition To 1000ml.It is 7.2 or so, 121 DEG C of sterilizing 15min to adjust pH), at room temperature, rotating speed 80-160rpm vibrates 30-120 minutes, Take out and stand 20-40 minutes, draw 100 μ L with pipettor, be uniformly coated on the culture plate of solid medium (aforesaid liquid Culture medium adds 2% agar to obtain), it is placed in 28 DEG C of constant incubators and carries out quiescent culture.
B, until training grow visible colonies when, with oese picking single bacterium colony, connect on Solid media for plates surface Continuous line；
C, so line culture repeatedly, until obtaining pure culture Halophiles bacterial strain.
D, the single bacterium colony of picking pure culture, transfer in the fluid nutrient medium containing phenol, in 28 DEG C, 150rpm perseverance Cultivated in warm shaking table, bacterium solution to muddiness；
Fluid nutrient medium 1L formulas in this step are NaCl 100g, Tris 2g, KCl 2g, KNO3 1g、NH4Cl 5g、 MgSO4·7H2O 24g、MnSO4·H2O 0.688g、CaCl20.12g, glucose 1g, distilled water are added to 1000ml.100ml Sterilized 0.05mol/L Na are added in aforesaid liquid culture medium2HPO4 2ml、0.01mol/L FeSO4 2ml、10mg/mL Yeast extract 2ml, 10mg/mL tryptone 2ml, 10mg/mL phenol 2ml (being 200mg/L phenol solutions after conversion).
E, draw 200 μ L bacterium solutions with pipettor and transfer in the fluid nutrient medium containing phenol described in step d, it is so anti- It is multiple 5-8 times, the Halophiles of the degradation of phenol of one plant of stability and high efficiency is obtained, CL is named as.
2nd, the morphological feature of bacterial strain
Bacterial strain CL dilution spreads observe bacterium colony in golden yellow in solid medium, after 60h, and circular, protuberance, surface is wet Moisten and smooth, colony edge is neat, be in purple after Gram's staining, be gram-positive cocci, and spherical bacterium is with more than two Form be linked to be string-like.
3rd, 16S rDNA are identified
The 16S a length of 1058bp of rDNA partial sequences of the bacterial strain, by the sequence in NCBI websites (http:// Www.ncbi.nlm.nih.gov/ Blast is carried out on) and compares analysis, select has with bacterial strain CL homologous sequence similitude highests Representational bacterial strain, using ortho position phase connection (Neighbor-Joining) and the utilization software building phyletic evolutions of MEGA 6.0 Tree, sees Fig. 1, inspection (bootstrap) 1000 times of bootstrapping.It is staphylococcus to understand bacterial strain CL, with the grape ball delivered at present Bacterium (Staphylococcus caprae ATCC 35538) sequence similarity is up to 99%.
Comprehensive thalli morphology, physio-biochemical characteristics and 16S rDNA gene orders, bacterial strain CL were protected on June 2nd, 2016 It is hidden in China typical culture collection center (referred to as, address：Wuhan, China Wuhan University, postcode 430072), deposit number is CCTCC NO.M2016305, classify entitled staphylococcus Staphylococcus caprae.
The staphylococcus CL of embodiment 2 degradation of phenol performance test
Degradation properties of the staphylococcus CL to phenol in the culture medium containing phenol is detected using high performance liquid chromatography.(training It is 10g MgSO to support base4·7H2O、0.2g CaCl2·2H2O, 2-5g KCl, 2.5g tryptone, 10g yeast extracts, NaCl 5%, distilled water are added to 1000ml.It is 7.2 or so, 121 DEG C of sterilizing 15min to adjust pH, and phenol will according to following experiment Ask and be added)
1st, the degradation characteristic of bacterial strain CL growth and phenol
Under the most suitable growth degradation condition, bacterial strain CL growth and the change curve of phenol concentration are as shown in Figure 2.Bacterial strain CL Enter exponential phase after the deadtime by 12h or so, during this period, bacterial strain is with time almost linear growth, close to 40h And bacterial strain is in and stablized growth period later, now its biomass OD600Maximum is reached, is worth for 1.6.Can be with from phenol concentration curve Find out, with biomass OD600Constantly increase, phenol concentration constantly declines, and final phenol degrading rate is 83%.
2nd, the degraded of bacterial strain CL Pyrogentisinic Acids under different NaCl concentrations
By analysis, the change of NaCl concentration has a significant impact to bacterial strain CL growth and the ability of degradation of phenol (P<0.01).From the figure 3, it may be seen that with the increase of NaCl concentration, bacterial strain CL growth and the degraded of Pyrogentisinic Acid have after first rise The trend of reduction.When NaCl concentration is 5%, bacterial strain CL growth and the degraded of Pyrogentisinic Acid reach maximum, Degradation of Phenol Rate reaches 90%.By Multiple range test, when NaCl concentration by 5% to 10% when, bacterial strain CL biomass (OD600) and it is right The degraded of phenol is without significant changes (P<0.01).
The 3rd, bacterial strain is inoculated into phenol concentration respectively for 0mg/L, 100mg/L, 200mg/L, 400mg/L, 600mg/L, In the culture medium of 800mg/L liquid (formula of the culture medium of liquid is ibid), regulation pH is maintained at 7.0, with 165r/min's Rotating speed is cultivated in temperature is 30 DEG C of constant-temperature table, and other conditions are constant.Survey initial biomass and phenol concentration and Biomass (the OD after 72h is cultivated on constant-temperature table600) and phenol concentration.
Assay method：The nutrient solution 1mL after culture 0h and 60h is drawn, is added in 5mL centrifuge tube, then to centrifuge tube Middle addition 3mL aseptic culture fluid, after mixing, its biomass OD600 is detected using ultraviolet-uisible spectrophotometer.Meanwhile, inhale The nutrient solution 1mL after culture 0h and 60h is taken, is added in 1.5mL centrifuge tube, it is centrifuged under the conditions of 6000r/min 10min, takes supernatant 0.8mL to be added in 5mL centrifuge tube, then adds 2.4mL aseptic culture fluids to centrifuge tube, will after mixing Ultraviolet-uisible spectrophotometer wavelength is set as 270nm, the measure of phenol absorbance is carried out, according to phenol Standard curve (see figure 4) initial phenol concentration and 60h concentration are calculated, so as to calculate phenol degrading rate.
Interpretation of result：By data analysis, the change of phenol concentration is to bacterial strain CL growth and the energy of degradation of phenol Power has a significant impact (P<0.01).As shown in Figure 5, the growth of bacterial strain and the degraded of Pyrogentisinic Acid have elder generation with the increase of phenol concentration The trend reduced after rise.When phenol concentration is 200mg/L, bacterial strain CL growth and the degraded of Pyrogentisinic Acid reach maximum, The degradation rate of wherein Pyrogentisinic Acid reaches 85% or so.Analyzed by Multiple range test, when phenol concentration is more than or less than 200mg/L, the degradation bacteria of growth and Pyrogentisinic Acid to bacterial strain CL has a significant impact (P<0.01).
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (10)

1. a kind of staphylococcus (Staphylococcus caprae) CL, its deposit number is CCTCC NO:M2016305.
2. the microbial inoculum containing staphylococcus CL described in claim 1.
3. the biological cleanser containing staphylococcus CL described in claim 1.
4. the microbial inoculum described in staphylococcus CL or claim 2 described in claim 1 or the Biological clean described in claim 3 Application of the agent in cleaning ambient.
5. the microbial inoculum described in staphylococcus CL or claim 2 described in claim 1 or the Biological clean described in claim 3 Application of the agent in degradation of phenol.
6. the microbial inoculum described in staphylococcus CL or claim 2 described in claim 1 or the Biological clean described in claim 3 Application of the agent in processing industrial wastewater.
7. the microbial inoculum described in staphylococcus CL or claim 2 described in claim 1 or the Biological clean described in claim 3 Application of the agent in medical waste water is handled.
8. applications of the staphylococcus CL in biological cleanser is prepared described in claim 1.
9. applications of the staphylococcus CL in biodegradation agent is prepared described in claim 1.
10. application as claimed in claim 9, it is characterised in that the biodegradation agent is phenol degrading agent.
CN201710488862.7A 2016-06-27 2017-06-23 Staphylococcus for degrading phenol Active CN107129954B (en)

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Publication number Priority date Publication date Assignee Title
CN118460416A (en) * 2024-05-17 2024-08-09 浙江耕盛堂生态农业有限公司 Staphylococcus strain with odor reducing function and application thereof

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CN108048385B (en) * 2018-01-19 2021-07-27 中国农业大学 Strain domestication method for improving degradation efficiency of mycotoxin degrading bacteria
CN108531423B (en) * 2018-04-08 2021-01-26 上海海洋大学 Deep sea halomonas and application thereof in inducing juvenile mytilus coruscus to attach
CN114644993B (en) * 2020-12-21 2023-06-02 有研资源环境技术研究院(北京)有限公司 High-salt-tolerance halomonas and microbial in-situ ecological restoration method for promoting plant growth in saline-alkali soil

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CN104560798B (en) * 2014-12-23 2017-05-31 清华大学 A kind of preparation method of the Halophiles microbial inoculum containing phenol of degrading
CN105087425A (en) * 2015-06-11 2015-11-25 华东理工大学 Halomonas sp. capable of degrading phenols and high-throughput screening method and application thereof

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CN118460416A (en) * 2024-05-17 2024-08-09 浙江耕盛堂生态农业有限公司 Staphylococcus strain with odor reducing function and application thereof

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