CN105861375A - Aniline-degrading bacterium and application thereof - Google Patents
Aniline-degrading bacterium and application thereof Download PDFInfo
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- CN105861375A CN105861375A CN201610278249.8A CN201610278249A CN105861375A CN 105861375 A CN105861375 A CN 105861375A CN 201610278249 A CN201610278249 A CN 201610278249A CN 105861375 A CN105861375 A CN 105861375A
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- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
The invention belongs to the technical field of biodegradation and provides Pigmentip haga daeguensis having the aniline degrading capability. The bacterium is a gram-negative bacterium, the bacterial colony is in white, round, smooth and opaque and protrudes slightly, and the rim is neat. The bacterium is preserved in Microbial Culture Preservation Center, Guangdong Province on March, 10<th>, 2016, and the preservation number is GDMCC NO: 60017. The bacterium is applied to deep treatment of low-concentration aniline waste water, can grow well in the low-concentration aniline waste water, is high in aniline degradation speed, free of secondary pollution and safe to use and cannot influence a microbic system of an original treatment system.
Description
Technical field
The invention belongs to biodegradation technique field, be specifically related to a kind of microphenomenon of degradation of aniline and application thereof, especially
Its degradation bacteria relating to a kind of low concentration aniline and application thereof.
Background technology
Aromatic organic compounds has become the most serious of facing mankind to the pollution of surface water and groundwater
One of environmental problem.Aniline, as simplest one-level aromatic amine, is the industrial chemicals of a kind of large usage quantity,
It is widely used in the industries such as printing and dyeing, national defence, pesticide, rubber.Meanwhile, aniline is also that some contain aniline
Intermediate product after compounds biodegradation, its toxicity is extremely strong, is that a kind of relatively conventional water body is dirty
Dye thing.Along with industrial expansion, produce or use that the enterprise of aniline discharged containing aniline industrial waste gas meeting
Atmosphere pollution.Aniline can enter environment by the waste water in industrial undertaking and residential area.For people, benzene
Amine is the formation body of typical metahemoglobin, makes cell lose function of carrying oxygen, has haemolysis, it
Mainly entering human body by the mode such as sucking or eat, is then combined with hemoglobin, reduction is even deprived
Itself and the combination probability of oxygen, ultimately result in the generation of intoxicating phenomenon；And it can damage liver function,
Start an inflammation of the liver, even cause certain cancers.Therefore Aniline categories is listed in master by the state such as the U.S., Japan
Want the pollutant blacklist of monitoring project or priority monitoring.Within 1989, amino benzenes compounds is classified as by China
One of " Environment Priority control pollutant " material.
The method processing aniline waste water at present mainly has Physical, chemical method and bioanalysis.
Physical includes absorption method and extraction, is primarily adapted for use in High Concentration Aniline Wastewater, can realize aniline
Recycling.Absorption method is the method using adsorbing material to process aniline waste water, grasps in engineering application
Making simple, operation is simple, but adsorbent acquisition cost is high, regeneration difficulty, and absorption method often can cause two
Secondary pollution.Extraction is to use energy dissolved contaminants but water-fast extractant, abundant with waste water at it
After mixing contact, utilize pollutant in water and in solvent, different distribution ratios realizes separated from contaminants and carries
A kind of purification method for effluent taken.Processing equipment is required simple by extraction, and technique is simple, but needs equally
Want Regeneration Treatment, and organic solvent is likely to result in secondary pollution.The physics of extraction simply pollutant turns
Move past journey, rather than degraded truly.
Chemical method includes photocatalytic oxidation, supercritical water oxidation method and sonication etc..Photocatalysis
Oxidation technology needs light, catalyst and air, have that energy consumption is low, easy and simple to handle, reaction condition is gentle,
Reaction range is wide, can reduce the outstanding advantages such as secondary pollution.And photocatalytic oxidation is processing high concentration work
Industry waste water exists the problem that nano material is easily assembled, and processing cost is high.Supercritical Water Oxidation Technology
(SCWO) with supercritical water as reaction medium, air, oxygen or hydrogen peroxide etc. are oxidant, by height
Temperature high pressure under radical reaction, the oxidation operations such as aniline are decomposed into carbon dioxide, water, nitrogen with
And the nontoxic micromolecular compound such as inorganic salt, there is the advantage that reaction rate is fast and secondary pollution is little.But
Its weak point is that reaction need to be carried out at high temperature under high pressure, and equipment requirements is high, and cost is high.Ultrasonic degradation
Method is to utilize acoustic cavitation energy to accelerate and control chemical reaction to improve a kind of new technique of reaction rate, has
The advantages such as degradation efficiency is high, the response time is short, facility is simple, floor space is little, but it is huge to consume energy, and makes an uproar
Sound is big, and economy is not ideal enough.
Utilizing amino benzenes compounds in microbial method degrading waste water is a kind of economical and effective and non-secondary pollution
Method, is particularly suitable for the Sewage advanced treatment containing Low Concentration of Benzene aminated compounds.At present, both at home and abroad
Scholar amino benzenes compounds degradation bacteria has been carried out substantial amounts of research work, screened many strains efficient
Degradation bacteria, but it is both for bacterial strain toleration and the research and development to high concentration aniline degradability, still can not
Meet low concentration aniline Sewage advanced treatment and the demand of zero-emission.Therefore, can be quick by screening
The bacterial strain of degradable low concentration aniline, abundant microbial resources, illustrate the quick and complete fall of microorganism
The researchs such as the characteristic of solution aniline are particularly important.
Summary of the invention
In view of this, it is an object of the invention to provide a kind of microphenomenon of degradation of aniline and application thereof.
A kind of microphenomenon of degradation of aniline, is deposited in Guangdong Province's Culture Collection, and deposit number is
GDMCC No:60017.
Microphenomenon of degradation of aniline of the present invention is to screen the printing and dyeing sludge from middle mountain treatment of dyeing wastewater factory, warp
The strain bacterium that aniline domestication, separation and purification obtain.Method particularly includes: carry out washing Fender by printing and dyeing sludge
Decontamination, takes 20g mud and is placed on 200mL with inorganic salt liquid that aniline (50mg/L) is unique carbon nitrogen source
After cultivating 7 days in body culture medium, the inoculum concentration with 10% is transferred to the concentration of aniline inorganic salt as 100mg/L
Fluid medium carries out domestication cultivate 7 days, continue to be transferred to concentration of aniline with the inoculum concentration of 10% the most again
Cultivate 7 days for domestication in the inorganic salt liquid culture medium of 150mg/L.Take appropriate culture fluid dilute through ten times of series
Release, and be coated cultivating with solid medium (100mg/L) surface that aniline is unique carbon nitrogen source,
After cultivating 7 days, picking list bacterium colony carries out purification of ruling, until being pure bacterium for single bacterium colony in culture medium,
Measure the degradation effect of each bacterium respectively, choose dominant strain therein, obtain degradation bacteria AN-4a of aniline.
Degradation bacteria AN-4a of aniline of the present invention is Gram-negative aerobic bacteria, shaft-like, bacteria colony white,
Circle, smooth opaque, slightly projection, neat in edge.Oxidase positive, catalase is positive, lysine
Decarboxylase is negative, and ODC Ornithine decarboxylase is negative, and arginine dihydrolase is negative.Growth temperature 15～45 DEG C,
Growth pH is 5～10.Through 16S rDNA order-checking and tetraploid rice, obtain and aniline of the present invention
Degradation bacteria AN-4a the most close be Pigmentiphaga daeguensis sp., homology is 99%.Knot
Symphysis reason biochemical identification result, determines that degradation bacteria AN-4a of aniline of the present invention belongs to
Pigmentiphaga daeguensis。
Degradation bacteria AN-4a of aniline of the present invention on March 10th, 2016 in Guangdong Province's microorganism
DSMZ (GDMCC) has carried out preservation, and preservation centre address is martyr Road, GuangZhou, China city
Guangdong Microbes Inst, No. 100 5th floors, No. 59 buildings of compound, preserving number is GDMCC No:60017.
Present invention also offers degradation bacteria AN-4a of the described aniline purposes in processing amino benzene analog waste water.
The especially biological reinforced process of low concentration amino benzene analog waste water.
Present invention also offers the processing method of a kind of amino benzene analog waste water, by the degraded of aniline of the present invention
Bacterium 4a is suspended in the sewage containing aniline cultivation.
Wherein, the concentration of Aniline described in described processing method is 10mg/L.
In some embodiments, the inoculum concentration of the degradation bacteria of aniline described in processing method of the present invention
It is more than 20%, preferably 20%.
In some embodiments, cultivating pH value described in processing method of the present invention is 7～9.It is preferably,
PH value is 7.
Cultivation temperature described in processing method of the present invention is 30～40 DEG C.It is preferably 30 DEG C
Incubation time described in processing method of the present invention is more than 12 hours.It is preferably 12 hours.
As shown from the above technical solution, the invention provides a kind of bacterium with degrading aniline ability
Pigmentiphaga daeguensis.This bacterium is gram negative bacteria, bacteria colony white, circular, smooth impermeable
Bright, slightly projection, neat in edge.This bacterium on March 10th, 2016 at Guangdong Province's Microbiological Culture Collection
Center preservation, preserving number is: GDMCC No:60017.This bacterium is applied to low concentration aniline waste water and processes,
Can in the aniline waste water of low concentration well-grown, aniline degradation speed is fast, non-secondary pollution, uses peace
Entirely, the microflora of original processing system is not interfered with.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to reality
Execute the required accompanying drawing used in example or description of the prior art to be briefly described.
Fig. 1 shows the PCR primer sepharose electrophoresis figure of microphenomenon of degradation of aniline AN-4a, and marking 1 and 2 in figure is aniline
The 16S rDNA band of degradation bacteria 4a；
Fig. 2 shows the microphenomenon of degradation of aniline AN-4a degradation effect to aniline under aniline initial concentration different condition
Figure；
Fig. 3 shows the microphenomenon of degradation of aniline AN-4a degradation effect figure to aniline under inoculum concentration different condition；
Fig. 4 shows the microphenomenon of degradation of aniline AN-4a degradation effect to aniline under medium pH value different condition
Figure；
Fig. 5 shows the microphenomenon of degradation of aniline AN-4a degradation effect figure to aniline under cultivation temperature different condition；
Fig. 6 shows microphenomenon of degradation of aniline AN-4a degradation effect figure of aniline under the different response time.
Biological deposits explanation
Classification And Nomenclature: Pigmentiphaga daeguensis is deposited in Guangdong Province on March 10th, 2016
Culture Collection, address is that 5th floor, No. 59 building of compound, martyr Road, GuangZhou, China city 100 is wide
East institute of microbiology of province, deposit number is GDMCC No:60017.
Detailed description of the invention
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Describe, it is clear that described embodiment is only a part of embodiment of the present invention rather than all wholely
Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creativeness
The every other embodiment obtained under work premise, broadly falls into the scope of protection of the invention.
In order to be further appreciated by the present invention, below in conjunction with specific embodiment, the present invention is elaborated.
If no special instructions, the method applied in the present invention is the method that this area is conventional.
Embodiment 1, the separation of strain, purification
Carry out washing mud by printing and dyeing sludge, take 20g mud and be placed on 200mL with aniline (50mg/L) for only
After the inorganic salt liquid culture medium of one carbon nitrogen source is cultivated 7 days, it is transferred to aniline with the inoculum concentration of 10% dense
The inorganic salt liquid culture medium that degree is 100mg/L is tamed, after being further cultured for 7 days, with 10% connect
The amount of kind is transferred in the inorganic salt liquid culture medium that concentration of aniline is 150mg/L tame.Take culture fluid
With dilution spread in solid medium that aniline is unique carbon nitrogen source, after cultivating 7 days, picking list bacterium colony
Line purification, until being pure bacterium for single bacterium colony on flat board, measures the degradation effect of each bacterium respectively, choosing
Take dominant strain AN-4a therein.
Wherein, the formula (1L) of inorganic salt liquid culture medium is: NaCl 0.2g, K2HPO40.2g,
KH2PO40.2g, trace element solution 2mL.
The formula (1L) of trace element solution: FeSO4300mg, CuSO4·5H2O 38mg,
ZnSO4·7H2O 115mg, MnSO4169mg, H3BO3116mg, CoCl 6H2O 24mg,
NaMo·2H2O 17mg。
Solid culture based formulas (1L) is to add 20g agar in inorganic salt liquid culture medium.
Embodiment 2, the physiological and biochemical test of bacterial strain are identified
Strains A N-4a of screening is carried out preliminary Physiology and biochemistry qualification, including colonial morphology detection, leather orchid
The experiment of family name's mensuration, experiment of growth factor, oxidase, catalase reaction, E.C. 4.1.1.18 test, bird
The test of propylhomoserin decarboxylase, arginine dihydrolase experiment etc., described test method uses the mensuration of this area
The conventional methods of this kind of physiological and biochemical index.
Result display strains A N-4a is Gram-negative aerobic bacteria, shaft-like, and bacteria colony white is circular, light
Sliding opaque, slightly projection, neat in edge.Oxidase positive, catalase is positive, and E.C. 4.1.1.18 is cloudy
Property, ODC Ornithine decarboxylase is negative, and arginine dihydrolase is negative.Growth temperature 15～45 DEG C, growth pH
It is 5～10.
Embodiment 3, the 16S rDNA of bacterial strain identify
Identified by 16S rRNA sequence analysis and Biolog microbial identification system, determine strains A N-4a
Belong to Pigmentiphaga daeguensis sp..Specifically comprise the following steps that
Use conventional phenol method to extract the DNA of strains A N-4a, expand 16S rDNA sequence with universal primer.
PCR reaction system (50 μ L) is: 10X PCR buffer 5 μ L, dNTP 4 μ L, primer each 1
μ L, DNA profiling 2.5 μ L, TakaraTaq enzyme 0.25 μ L, ultra-pure water 36 μ L.PCR amplification program is
94℃3min；94 DEG C of 1min, 52 DEG C of 1min, 72 DEG C of 1.5min, 30 circulations；72℃10min.
Sepharose electrophoresis detection amplified production, result is shown in Fig. 1.Amplified production serves the raw limited public affairs of work biotechnology in sea
Department checks order.Sequencing result is as shown in SEQ ID NO:1.
The 16S rDNA sequence inputting GenBank that will record, application BLAST software carries out homology and searches
Rope.Choose the 16S rDNA sequence of different strains and known 16S rDNA carries out homology with GenBank
Property compares.This pcr amplification product carries out in GenBank blast comparison result show, with its phase
That near is Pigmentiphaga daeguensis sp., and homology is 99%.
Bacterial strain physiological and biochemical index deducibility AN-4a shown in 2 belongs to the most in conjunction with the embodiments
Pigmentiphaga daeguensis sp.。
Embodiment 4, microphenomenon of degradation of aniline AN-4a degradation effect to aniline under aniline initial concentration different condition
Experiment
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for is inoculated in LB fluid medium, in 30 DEG C,
Shaking table activated spawn under the conditions of 160r/min, makes the OD600 value of bacterium solution reach 0.5, with 100 μ L activation
Bacterium solution is inoculated in the inorganic salt liquid culture medium that concentration of aniline is 10mg/L, 50mg/L and 100mg/L,
Respectively by N-(1-naphthyl) second two behind 0 hour, 12 hours, 24 hours, 36 hours and 48 hours
The concentration of amine azo spectrphotometric method for measuring aniline, result is shown in Fig. 2.
As seen from Figure 2, under conditions of concentration of aniline is 10mg/L, degradation bacteria after 12h
AN-4a reaches more than 95% to the degradation rate of aniline, and under the conditions of 50mg/L and 100mg/L,
After 12 hours, degradation bacteria AN-4a only reaches 17% and 44% respectively to the degradation rate of aniline.Show degradation bacteria
The efficient degradation of AN-4a suitable low concentration aniline.
The degradation effect of aniline is tested under inoculum concentration different condition by embodiment 5, microphenomenon of degradation of aniline AN-4a,
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for is inoculated in LB fluid medium, in 30 DEG C,
Shaking table activated spawn under the conditions of 160r/min, makes the OD600 value of bacterium solution reach 0.5, with 0.1mL, 0.5
After mL, 1mL, 2mL, 4mL, 8mL activation bacterium solution is centrifugal, being inoculated into 20mL concentration of aniline is
In the inorganic salt solution of 10mg/L, respectively with N-(1-naphthyl) ethylenediamine behind 0 hour and 24 hours
The concentration of azo spectrphotometric method for measuring aniline, result is shown in Fig. 3.
As seen from Figure 3, along with the increase of inoculum concentration, degradation bacteria AN-4a to the degradation rate of aniline by
Step improves.When inoculum concentration reaches more than 4mL, the degradation rate of aniline reaches 100%.Consider that reality should
With, the preferably optimum inoculation amount of degradation bacteria AN-4a is 20%.
Embodiment 6, microphenomenon of degradation of aniline AN-4a degradation effect to aniline under medium pH value different condition
Experiment
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for is inoculated in LB fluid medium, in 30 DEG C,
Shaking table activated spawn under the conditions of 160r/min, makes the OD600 value of bacterium solution reach 0.5, activates bacterium with 4mL
After liquid is centrifugal, being inoculated into 20mL pH value and be respectively 5,6,7,8,9,10, concentration of aniline is 10mg/L
Inorganic salt solution in, respectively with N-(1-naphthyl) ethylenediamine azo luminosity behind 0 hour and 24 hours
Method measures the concentration of aniline, and result is shown in Fig. 4.
As seen from Figure 4, when pH value is 5, the clearance of aniline is minimum, and only 22.5%, along with
When pH value increases to 7, the clearance of aniline reaches 100%, and when pH value is 10, clearance declines
To 74.8%.Showing, degradation bacteria AN-4a the most suitable growth, degraded pH value are between 7～9, it is considered to real
Border situation, it is relatively reasonable that degradation bacteria cultivation pH value is chosen as 7.
The degradation effect of aniline is tested under temperature different condition by embodiment 7, microphenomenon of degradation of aniline AN-4a
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for is inoculated in LB fluid medium, in 30 DEG C,
Shaking table activated spawn under the conditions of 160r/min, makes the OD600 value of bacterium solution reach 0.5, activates bacterium with 4mL
After liquid centrifuging and taking supernatant, it is inoculated in the inorganic salt solution that 20mL concentration of aniline is 10mg/L, point
Be not placed in 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C of shaking tables are cultivated, at 0 hour and 24
By the concentration of N-(1-naphthyl) ethylenediamine azo spectrphotometric method for measuring aniline after hour, result is shown in Fig. 5.
As seen from Figure 5, when 15 DEG C, after 24 hours, aniline clearance is minimum, only reaches 3.3%；20℃
Slightly higher with clearance when 25 DEG C, respectively reach 6.7% and 24.3%；When 30～40 DEG C, clearance reaches 100%.
Show that degradation temperature is in degradation bacteria 4a the most suitable growth between 30～40 DEG C.Consider the practical situation of application,
The cultivation temperature of degradation bacteria AN-4a is chosen to be 30 DEG C.
Embodiment 8, degrading aniline effect after the response time different for microphenomenon of degradation of aniline AN-4a
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for is inoculated in LB fluid medium, in 30 DEG C,
Shaking table activated spawn under the conditions of 160r/min, makes the OD600 value of bacterium solution reach 0.5, activates bacterium with 4mL
After liquid is centrifugal, be inoculated into 20mL pH value be 7, concentration of aniline be 10mg/L inorganic salt solution in,
Be placed in 30 DEG C of shaking tables cultivation, 0 hour, 6 hours, 12 hours, 15 hours, 18 hours, 21
Hour, measure OD600 value after 24 hours and with N-(1-naphthyl) ethylenediamine azo spectrphotometric method for measuring aniline
Concentration.Result is shown in Fig. 6.
As seen from Figure 6, degradation bacteria AN-4a inoculum concentration be 4mL, medium pH value be 7,
Under conditions of cultivation temperature is 30 DEG C, after 12 hours, the degradation rate to aniline reaches 100%, it is achieved completely
Degraded.Bacteria concentration increases over, and shows that degradation bacteria AN-4a utilizes aniline as unique carbon nitrogen source, carries
For the energy needed for growth.After aniline is degradable, bacteria concentration continues to increase, and shows degradation bacteria
AN-4a can utilize the catabolite of aniline, and as nutrient substance, the growth for bacterium provides energy.
Claims (8)
1. a microphenomenon of degradation of aniline, is deposited in Guangdong Province's Culture Collection, and deposit number is
GDMCC No:60017.
2. the purposes in processing low concentration aniline waste water of the microphenomenon of degradation of aniline described in claim 1.
3. a processing method for low concentration aniline waste water, suspends microphenomenon of degradation of aniline described in claim 1
Cultivate in the waste water containing low concentration aniline.
Processing method the most according to claim 3, the concentration of described Aniline is 10mg/L.
Processing method the most according to claim 3, the inoculum concentration of described microphenomenon of degradation of aniline is 20%.
Processing method the most according to claim 3, described cultivation pH value is 7～9.
Processing method the most according to claim 3, described cultivation temperature is 30～40 DEG C.
Processing method the most according to claim 3, described incubation time is more than 12 hours.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109258575A (en) * | 2018-12-10 | 2019-01-25 | 广东工业大学 | A kind of incubation method of frog embryo |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101831391A (en) * | 2009-03-10 | 2010-09-15 | 中国科学院成都生物研究所 | Effective phenylamine degrading strain as well as use and use method thereof |
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2016
- 2016-04-29 CN CN201610278249.8A patent/CN105861375B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831391A (en) * | 2009-03-10 | 2010-09-15 | 中国科学院成都生物研究所 | Effective phenylamine degrading strain as well as use and use method thereof |
Non-Patent Citations (2)
| Title |
|---|
| JUNG-HOON YOON: "pigmentiphaga daeguensis sp.nov., isolated from wastewater of a dye works,and emended description of the genus pigmentiphaga", 《INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY》 * |
| 吕晶华 等: "高效苯胺降解菌的分离鉴定及其降解特性", 《城市环境与城市生态》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109258575A (en) * | 2018-12-10 | 2019-01-25 | 广东工业大学 | A kind of incubation method of frog embryo |
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