CN110079472A - A kind of culture medium of separation of methylbenzene degradation bacteria - Google Patents
A kind of culture medium of separation of methylbenzene degradation bacteria Download PDFInfo
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- CN110079472A CN110079472A CN201910204525.XA CN201910204525A CN110079472A CN 110079472 A CN110079472 A CN 110079472A CN 201910204525 A CN201910204525 A CN 201910204525A CN 110079472 A CN110079472 A CN 110079472A
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- toluene
- culture medium
- degradation
- separation
- methylbenzene
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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
- C12N1/02—Separating microorganisms from their culture media
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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 present invention provides a kind of culture medium of separation of methylbenzene degradation bacteria, the culture medium of the separation of methylbenzene degradation bacteria of 1 L contains NH4NO3 4~6 g、KNO3 2.5~3.5 g、MgCl2·6H2O0.03~0.05 g、(NH4)2HPO4 0.05~0.20 g、ZnCl2 0.05~0.20 g、FeSO4·7H2O 0.003 ~ 0.006 g, Tween 80 1 ~ 2 mL, 1 ~ 5 mL of toluene, pH7.0 ~ 7.5.The degradation of toluene bacterium degradation toluene of the culture medium of separation of methylbenzene degradation bacteria of the present invention, energy high-efficient culture methyl degradation bacteria, culture is high-efficient.Culture method of the present invention is simple, at low cost, pollutes small.
Description
Technical field
The invention belongs to bioengineering fields, and in particular to a kind of culture medium of separation of methylbenzene degradation bacteria.
Background technique
Toluene is a kind of clarified colorless liquid, there is the smell of similar benzene, and relative density 0.866 is volatile, does not dissolve in
Water is soluble in the organic solvents such as ethyl alcohol, benzene, ether, chloroform.Toluene also has inflammability, and steam can be formed with air to explode
Property mixture, is a kind of dangerous material.People suck in a short time higher concentration toluene can acute poisoning, it is acute caused by toluene
Poisoning is mainly shown as the anesthetic effect and vegetative nerve functional disturbance symptom and mucosal irritation symptom of central nervous system,
Severe one even twitches, is clouded in mind, and some may occur in which hysteria sample symptom.Often there is neurasthenic syndrome in toluene slow poisoning,
Also encephalopathy and hepatorenal damage can be caused, it is also possible to cause feminine menstrual abnormal.Toluene can also endanger environment simultaneously, pollute air and water
Source.Phenyl ring has very strong hydrophobicity and stability in toluene chemical structure, is not easy to be degraded.Therefore, the pollution of toluene is
Urgent problem to be solved.
Currently, people mainly pass through the methods of hydrolysis, oxidation, biological adsorption, microbial degradation removal toluene concentration, wherein
Microbial degradation method is the removing relatively effective method of formaldehyde, and low in cost, without secondary pollution.The pass of microbial degradation method
Key relates to separation and purifying to bacterium for degrading, this just needs the culture medium of energy high frequency zone separation.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of culture mediums of energy high frequency zone separation of methylbenzene degradation bacteria.This hair
Bright technical solution are as follows: the culture medium of the separation of methylbenzene degradation bacteria of 1 L, NH is contained in pH7.0 ~ 7.54NO3 4~6 g、KNO3 2.5~
3.5 g、MgCl2·6H2O0.03~0.05 g、ZnCl2 0.05~0.20 g、(NH4)2HPO4 0.05~0.20 g、FeSO4·7H2O
0.003 ~ 0.006 g, Tween 80 1 ~ 2 mL, 1 ~ 5 mL of toluene.
Preferably, the culture medium of the separation of methylbenzene degradation bacteria of 1 L, pH 7.0 ~ 7.5 contain NH4NO3 5 g、KNO3 3 g、
MgCl2·6H2O0.05 g、ZnCl2 0.1 g、(NH4)2HPO4 0.1 g、FeSO4.7H2O 0.005 g、Tween 80 1 mL、
3 mL of toluene.
Preferably, pH is adjusted to 7.4.
Another object of the present invention additionally provides a kind of preparation method of the culture medium of separation of methylbenzene degradation bacteria: weighing
NH4NO3 4~6 g、KNO3 2.5~3.5 g、MgCl2·6H2O0.03~0.05 g、ZnCl2 0.05~0.20 g、(NH4)2HPO4
0.05~0.20 g、FeSO4·7H2O 0.003 ~ 0.006 g, Tween 80 1 ~ 2 mL into beaker, be added 200mL distilled water use
Glass bar stirring and dissolving is added 1 ~ 5 mL of toluene, distilled water is added to be settled to 1 L, adjusts pH to 7.0 ~ 7.5 with pH meter, 121 DEG C go out
Bacterium 30min.
Culture medium of the present invention can provide carbon source, the nitrogen source, nothing that growth needs for degradation of toluene bacterium for degradation of toluene bacterium
Machine element, water and growth substance are able to satisfy the growth demand of degradation of toluene bacterium, have specificity to degradation of toluene bacterium, promote first
The formation of benzene catabolite substantially increases the degradation efficiency of microbiological treatment toluene.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below with reference to embodiment, still
It should be appreciated that these descriptions are only further explanation the features and advantages of the present invention, rather than to the claims in the present invention
Limitation, reagent of the present invention is conventional reagent unless otherwise instructed, can be commercially available by market.
Embodiment one
The culture medium of the separation of methylbenzene degradation bacteria of 1L, contains NH4NO3 4 g、KNO3 2.5 g、MgCl2·6H2O0.03 g、ZnCl2
0.05 g、(NH4)2HPO4 0.05 g、FeSO4·7H2O 0.003 g, Tween 80 1 mL, 1 mL of toluene.
Configuration process: NH is weighed4NO3 4 g、KNO3 2.5 g、MgCl2·6H2O0.03 g、ZnCl2 0.05 g、(NH4)2HPO4 0.05 g、FeSO4·7H2O 0.003 g, Tween 80 1 mL to beaker, 200mL distilled water is added and is stirred with glass bar
Dissolution is added 1 mL of toluene, distilled water is added to be settled to 1 L, adjusts pH to 7.0,121 DEG C of sterilizing 30min with pH meter.
Embodiment two
The culture medium of the separation of methylbenzene degradation bacteria of 1L, contains NH4NO3 5 g、KNO3 3 g、MgCl2·6H2O0.05 g、ZnCl2
0.1 g、(NH4)2HPO4 0.1 g、FeSO4·7H2O 0.005 g, Tween 80 1 mL, 3 mL of toluene.
Configuration process: NH is weighed4NO3 5 g、KNO3 3 g、MgCl2·6H2O0.05 g、ZnCl2 0.1 g、(NH4)2HPO4
0.1 g、FeSO4·7H2O 0.005 g, Tween 80 1 mL to beaker, 200 mL distilled water glass bar stirring and dissolvings are added,
3 mL of toluene is added, distilled water is added to be settled to 1 L, adjusts pH to 7.4,121 DEG C of 30 min of sterilizing with pH meter.
Embodiment three
The culture medium of the separation of methylbenzene degradation bacteria of 1L, contains NH4NO3 6 g、KNO3 3.5 g、MgCl2·6H2O0.05 g、
ZnCl2 0.2 g、(NH4)2HPO4 0.2 g、FeSO4·7H2O 0.006 g, Tween 80 2 mL, 5 mL of toluene.
Configuration process: NH is weighed4NO3 6g、KNO3 3.5g、MgCl2·6H2O0.05g、ZnCl2 0.2g、(NH4)2HPO4
0.20 g、FeSO4·7H2O 0.006g, 80 Tween 2mL to beaker are added 200mL distilled water glass bar stirring and dissolving, add
Enter 5 mL of toluene, distilled water is added to be settled to 1L, adjusts pH to 7.5,121 DEG C of 30 min of sterilizing with pH meter.
Experimental example 1
By sludge sample and enriched medium (1L culture medium contains 10.0 g of tryptone, NaCl5.0 g, 3.0 g of beef extract,
Surplus is distilled water, medium pH 7.4) it is mixed according to the ratio of 8% w/v, culture medium is 5 mL, in 37 DEG C,
100rpm shaking table culture 1 day, from 200 μ L are drawn in above-mentioned culture medium into 5 mL new enriched medium, continuously repeat three
It is secondary, the 3rd cultured products are transferred to toluene isolation medium culture described in embodiment one to embodiment three.According to connecing for 1:50
Kind amount by strain inoculated, to degrading containing the separation of methylbenzene that toluene concentration is 0.866 g/L, (degrade bacterium culture medium by the separation of methylbenzene of 1L
The culture medium of bacterium, contains NH4NO3 4 g、KNO3 2.5 g、MgCl2·6H2O0.03 g、ZnCl2 0.05 g、(NH4)2HPO4
0.05 g、FeSO4·7H2O 0.003 g, Tween 80 1 mL, toluene 1 mL, pH7.0) in, using toluene as strain growth carbon
Source amounts to 6 bottles, while being arranged 1 bottle and be not inoculated with bacterium and doing blank control, and 37 DEG C are put into shaking table and cultivate, by isolation medium shake to
Muddiness applies plate after drawing bacterium solution gradient dilution.Bacterium colony is grown on observation plate, and picking single colonie saves bacterium after enrichment culture
Strain.
Experimental example 2
The strain saved in experimental example 1 is added in the toluene isolation medium of 50mL 0.866 g/L containing toluene, is arranged simultaneously
Control is not inoculated with strain as blank, studies the effect (training of the separation of methylbenzene degradation bacteria of 1L of the decomposition toluene of degradation of toluene bacterium
Base is supported, NH is contained4NO3 4 g、KNO3 2.5 g、MgCl2·6H2O0.03 g、ZnCl2 0.05 g、(NH4)2HPO4 0.05 g、
FeSO4·7H2O 0.003 g, Tween 80 1 mL, toluene 1 mL, pH7.0).The content of toluene uses gas phase color in culture medium
Spectrometry is measured.
The measuring method of toluene:
Reagent: the standard solution of toluene, acetone, chloroform,
Instrument: gas chromatograph (fid detector, packed column are PEG 6000), centrifuge, supersonic wave cleaning machine.
Chromatographic parameter: carrier gas and make-up gas: nitrogen；150 DEG C of injector temperature, column temperature: 150 DEG C；Gasification temperature: 150 DEG C；
Hydrogen flame temperature: 150 DEG C, protection temperature: 130 DEG C；Hydrogen Vapor Pressure: 0.05MPa；Air pressure 0.15MPa, column pressure: 0.12MPa.
Experimental method: it weighs toluene titer of 1 mL containing 1 mol/L NaCl and has in plug plastic bottle in 2 mL, quickly add
Enter 40 μ L chloroforms and 0.16 mL acetone, 3 min of sonic oscillation, then 10000rpm and be centrifuged 1 min, is taken with microsyringe
Bottom organic phase is measured according to above-mentioned chromatographic parameter.
According to above-mentioned toluene measuring method, after measured, the degradation of toluene bacterium of this degradation of toluene bacterium culture medium culture in 7 days drops
Solving toluene efficiency is 90%.
Experimental example 3
Experimental method and process are with experimental example 1, experimental example 2, and difference is that degradation of toluene bacterium culture medium used is pH7.4,1 L's
The culture medium of separation of methylbenzene degradation bacteria, contains NH4NO3 5 g、KNO3 3 g、MgCl2·6H2O0.05 g、ZnCl2 0.1 g、
(NH4)2HPO4 0.1 g、FeSO4·7H2O 0.005 g, Tween 80 1 mL, 3 mL of toluene.
After measured, the degradation of toluene bacterium degradation toluene efficiency of this degradation of toluene bacterium culture medium culture is 100% in 7 days.
Experimental example 4
With experimental example 1, experimental example 2, difference is that separation of methylbenzene degradation bacterium culture medium used is pH7.5 for experimental method and process, 1
The culture medium of the separation of methylbenzene degradation bacteria of L, contains NH4NO3 6 g、KNO3 3.5 g、MgCl2·6H2O0.05 g、ZnCl2 0.2
g、(NH4)2HPO4 0.2 g、FeSO4·7H2O 0.006 g, Tween 80 2 mL, 5 mL of toluene.
After measured, the degradation of toluene bacterium degradation toluene efficiency of this degradation of toluene bacterium culture medium culture is 95% in 7 days.
Experimental example 5
Influence of the pH to degradation toluene efficiency
Strain is inoculated into the culture medium for the content of material as described in example 2 that toluene concentration is 2.6 g/L, respectively in difference
It is cultivated under pH value condition, measures the concentration of toluene daily.As the result is shown when pH is acid or when alkalinity is partially strong, degradation of toluene effect
Fruit is undesirable, can be complete by degradation of toluene in 5 days when pH is 7.0 ~ 7.5, when wherein pH is 7.4, the degradation effect of toluene
Fruit is best.
Experimental example 6
Control: 1L isolation medium contains Na2PO4.7H2O 12.8 g、KH2PO4 3 g、NaCl 0.5 g、NH4Cl 1.0 g、
1M MgSO4 2 mL、CaCl20.1 mL Toluene 20 mL, pH7.0
The present invention: culture medium prepared by embodiment two
It compared prior art culture medium and culture medium prepared by the embodiment of the present invention two, different culture medium culture of isolated goes out toluene
The quantity of degradation bacteria, the results are shown in Table 1.
The different isolation medium isolated strains quantitative comparisons of table 1
From the point of view of contrast and experiment, culture medium culture of the present invention filters out that degradation of toluene bacteria strain is more, and effect is more preferable.
Claims (4)
1. a kind of culture medium of separation of methylbenzene degradation bacteria, it is characterised in that: the separation of methylbenzene degradation bacteria of 7.0 ~ 7.5,1 L of pH
Culture medium contains NH4NO3 4~6 g、KNO3 2.5~3.5 g、MgCl2·6H2O0.03~0.05 g、(NH4)2HPO4 0.05~
0.20 g、ZnCl2 0.05~0.20 g、FeSO4·7H2O 0.003 ~ 0.006 g, Tween 80 1 ~ 2 mL, 1 ~ 5 mL of toluene.
2. the culture medium of separation of methylbenzene degradation bacteria as described in claim 1, it is characterised in that: the toluene of 7.0 ~ 7.5,1 L of pH
The culture medium of degradation bacteria contains NH4NO3 5 g、KNO3 3 g、MgCl2·6H2O0.05 g、(NH4)2HPO4 0.1 g 、ZnCl2
0.1 g、FeSO4.7H2O 0.005g, 80 Tween, 1 mL, 3 mL of toluene.
3. the culture medium of separation of methylbenzene degradation bacteria as claimed in claim 1 or 2, it is characterised in that: pH 7.4.
4. a kind of method for the culture medium for preparing the described in any item separation of methylbenzene degradation bacterias of claim 1 ~ 3, it is characterised in that:
Weigh NH4NO3 4~6 g、KNO3 2.5~3.5 g、MgCl2·6H2O0.03~0.05 g、ZnCl2 0.05~0.20 g、(NH4)2HPO4 0.05~0.20 g、FeSO4·7H2O 0.003 ~ 0.006 g, Tween 80 1 ~ 2 mL into beaker, be added 200 mL it is bis-
Water glass bar stirring and dissolving is steamed, 1 ~ 5 mL of toluene is added, distilled water is added to be settled to 1 L, adjusts pH to 7.0 ~ 7.5 with pH meter,
121 DEG C of 30 min of sterilizing.
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