CN105861375B - A kind of microphenomenon of degradation of aniline and application thereof - Google Patents
A kind of microphenomenon of degradation of aniline and application thereof Download PDFInfo
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- CN105861375B CN105861375B CN201610278249.8A CN201610278249A CN105861375B CN 105861375 B CN105861375 B CN 105861375B CN 201610278249 A CN201610278249 A CN 201610278249A CN 105861375 B CN105861375 B CN 105861375B
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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
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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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
- 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 belongs to biodegradation technique fields, and the present invention provides a kind of Pigmentiphaga daeguensis with degrading aniline ability.The bacterium is Gram-negative bacteria, and bacteria colony white is round, smooth opaque, slightly protrusion, neat in edge.The bacterium is on March 10th, 2016 in Guangdong Province's Culture Collection preservation, deposit number are as follows: GDMCC No:60017.The bacterium be applied to low concentration aniline waste water advanced treating, can in the aniline waste water of low concentration well-grown, aniline degradation speed is fast, and without secondary pollution, use is safe, will not influence the microflora of original processing system.
Description
Technical field
The invention belongs to biodegradation technique fields, and in particular to a kind of microphenomenon of degradation of aniline and application thereof, more particularly, to
A kind of degradation bacteria and application thereof of low concentration aniline.
Background technique
Aromatic organic compounds ask the environment for the most serious that the pollution of surface water and groundwater has become facing mankind
One of topic.Aniline is a kind of biggish industrial chemicals of dosage as simplest level-one aromatic amine, be widely used in printing and dyeing,
The industries such as national defence, pesticide, rubber.Meanwhile aniline is also the intermediate product after some biodegrades containing amino benzenes compounds, it
Toxicity is extremely strong, is a kind of relatively conventional water pollutant.With the development of industry, it produces or is discharged using the enterprise of aniline
Industrial waste gas containing aniline can pollute atmosphere.Aniline can enter environment by the waste water in industrial enterprise and residential area.To people
Speech, aniline is the formation body of typical ferrihemoglobin, and cell is made to lose function of carrying oxygen, has haemocylolysis, it mainly passes through
It the modes such as sucks or eats and enter human body, then in conjunction with hemoglobin, reduce the combination probability for even having deprived itself and oxygen, most
Lead to the generation of intoxicating phenomenon eventually；And it can damage liver function, start an inflammation of the liver, or even cause certain cancers.Therefore phenyl amines
Pollutant has been included in the pollutant blacklist of main monitoring project or priority monitoring by states such as the U.S., Japan.Phenyl amines in 1989
Compound is classified as one of " Environment Priority controls pollutant " substance by China.
The method of processing aniline waste water mainly has physical method, chemical method and bioanalysis at present.
Physical method includes absorption method and extraction, is primarily adapted for use in High Concentration Aniline Wastewater, it can be achieved that the recycling of aniline is sharp
With.Absorption method is operated in engineering application simply using the method for adsorbent material processing aniline waste water, and operation is simple, but inhales
Attached dose of acquisition cost height, regeneration is difficult, and absorption method often will cause secondary pollution.Extraction is using can dissolved contaminants but not
It is dissolved in the extractant of water, after it is sufficiently mixed contact with waste water, utilizes the pollutant distribution ratio different in solvent in water
Realize a kind of purification method for effluent of separated from contaminants and extraction.Extraction requires simply processing equipment, and technique is simple, but same
Sample needs regeneration treatment, and organic solvent may cause secondary pollution.Extraction is the physical transfer process of a pollutant, and
Degradation in non-real meaning.
Chemical method includes photocatalytic oxidation, supercritical water oxidation method and sonication etc..Photocatalysis oxidation technique
Light, catalyst and air are needed, with low energy consumption, easy to operate, reaction condition is mild, reaction range is wide, can reduce secondary dirt
Outstanding advantages of dye.And photocatalytic oxidation has that nano material is easy aggregation in processing high-concentration industrial-water,
And processing cost is high.Supercritical Water Oxidation Technology (SCWO) is using supercritical water as reaction medium, air, oxygen or hydrogen peroxide etc.
The oxidation operations such as aniline are decomposed by carbon dioxide, water, nitrogen by the radical reaction under high temperature and pressure for oxidant
And the small molecule compound that inorganic salts etc. are nontoxic, have the advantages that reaction rate is fast and secondary pollution is small.But its shortcoming
It is that reaction need to carry out at high temperature under high pressure, equipment requirement is high, and cost is high.Sonication be using acoustic cavitation energy accelerate and
Control chemical reaction improves a kind of new technology of reaction rate, with degradation efficiency is high, the reaction time is short, facility is simple, land occupation
The advantages that area is small, but huge energy consumption, noise is big, and economy is not ideal enough.
It is a kind of economical and effective and method without secondary pollution using amino benzenes compounds in microbial method degrading waste water, especially
It is suitable for the Sewage advanced treatment containing Low Concentration of Benzene aminated compounds.Currently, scholar both domestic and external is to amino benzenes compounds
Degradation bacteria has carried out a large amount of research work, has screened more plants of efficient degradation bacterias, but both for bacterial strain tolerance and right
The research and development of high concentration aniline degradability are not able to satisfy the demand to low concentration aniline Sewage advanced treatment and zero-emission still.
Therefore, the bacterial strain of quick and complete degradation low concentration aniline is capable of by screening, microbial resources is enriched, it is fast to illustrate microorganism
The researchs such as the characteristic of the degradable aniline of speed are particularly important.
Summary of the invention
In view of this, the purpose of the present invention is 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 the printing and dyeing sludge screened from middle mountain treatment of dyeing wastewater factory, is tamed and dociled through aniline
Change, one plant of bacterium that separation and purifying obtain.Method particularly includes: printing and dyeing sludge is carried out to wash Fender decontamination, 20g sludge is taken to be placed on
After 200mL in inorganic salt liquid culture medium that aniline (50mg/L) is unique carbon nitrogen source cultivate 7 days, with 10% inoculum concentration turn
It is connected in the inorganic salt liquid culture medium that concentration of aniline is 100mg/L and carries out domestication culture 7 days, later again with 10% inoculum concentration
Continue to be transferred to domestication culture 7 days in the inorganic salt liquid culture medium that concentration of aniline is 150mg/L.Take appropriate culture solution through ten times
It is serially diluted, and culture is being coated with surface solid medium (100mg/L) that aniline is unique carbon nitrogen source, cultivate 7 days
Afterwards, picking single colonie carries out scribing line purifying, is pure bacterium up to being single bacterium colony on culture medium, measures the degradation effect of each bacterium respectively
Fruit chooses dominant strain therein, obtains the degradation bacteria AN-4a of aniline.
The degradation bacteria AN-4a of aniline of the present invention is Gram-negative aerobic bacteria, and rod-shaped, bacteria colony white is round, smooth
It is opaque, slightly protrusion, neat in edge.Oxidase positive contacts enzyme positive, and lysine decarboxylase is negative, ornithine decarboxylase yin
Property, arginine dihydrolase is negative.15~45 DEG C of growth temperature, growth pH are 5~10.By 16S rDNA sequencing and homology
Compare, obtain most similar with the degradation bacteria AN-4a of aniline of the present invention being Pigmentiphaga daeguensis sp.,
Homology is 99%.In conjunction with Physiology and biochemistry qualification result, determine that the degradation bacteria AN-4a of aniline of the present invention belongs to
Pigmentiphaga daeguensis。
The degradation bacteria AN-4a of aniline of the present invention is on March 10th, 2016 in Guangdong Province's Microbiological Culture Collection
The heart (GDMCC) has carried out preservation, and collection address is the compound the 59th of GuangZhou, China city martyr Road 100 5 building, building Guangdong Province
Institute of microbiology, deposit number are GDMCC No:60017.
The present invention also provides purposes of the degradation bacteria AN-4a of the aniline in processing amino benzene analog waste water.It is especially low
The biological reinforced processing of concentration amino benzene analog waste water.
The present invention also provides a kind of processing methods of amino benzene analog waste water, and the degradation bacteria 4a of aniline of the present invention is suspended
It is cultivated in the sewage containing aniline.
Wherein, the concentration of Aniline described in the 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 be 20% with
On, preferably 20%.
In some embodiments, culture pH value described in processing method of the present invention is 7~9.Preferably, pH value is
7。
Cultivation temperature described in processing method of the present invention is 30~40 DEG C.Preferably 30 DEG C
Incubation time described in processing method of the present invention is 12 hours or more.Preferably 12 hours.
As shown from the above technical solution, the present invention provides a kind of bacterium Pigmentiphaga with degrading aniline ability
daeguensis.The bacterium is Gram-negative bacteria, and bacteria colony white is round, smooth opaque, slightly protrusion, neat in edge.The bacterium in
On March 10th, 2016 is in Guangdong Province's Culture Collection preservation, deposit number are as follows: GDMCC No:60017.Bacterium application
In low concentration aniline wastewater treatment, can in the aniline waste water of low concentration well-grown, aniline degradation speed is fast, without secondary dirt
Dye, use is safe, will not influence the microflora of original processing system.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows the PCR primer agarose electrophoresis figure of microphenomenon of degradation of aniline AN-4a, and mark 1 and 2 is microphenomenon of degradation of aniline 4a in figure
16S rDNA band；
Fig. 2 shows microphenomenon of degradation of aniline AN-4a under aniline initial concentration different condition to the degradation effect figure of aniline；
Fig. 3 shows microphenomenon of degradation of aniline AN-4a under inoculum concentration different condition to the degradation effect figure of aniline；
Fig. 4 shows microphenomenon of degradation of aniline AN-4a under medium pH value different condition to the degradation effect figure of aniline；
Fig. 5 shows microphenomenon of degradation of aniline AN-4a under cultivation temperature different condition to the degradation effect figure of aniline；
Fig. 6 shows the degradation effect figure of microphenomenon of degradation of aniline AN-4a aniline under the different reaction time.
Biological deposits explanation
Classification naming: Pigmentiphaga daeguensis is deposited in Guangdong Province's microbial bacteria on March 10th, 2016
Kind collection, address are the compound the 59th of GuangZhou, China city martyr Road 100 5 building, building Guangdong Microbes Inst, preservation
Number is GDMCC No:60017.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
For a further understanding of the present invention with reference to specific embodiments elaborated to the present invention.Such as without special theory
Bright, the method applied in the present invention is the method for this field routine.
Embodiment 1, the separation of strain, purifying
Printing and dyeing sludge is carried out to wash mud, taking 20g sludge to be placed on 200mL with aniline (50mg/L) is the inorganic of unique carbon nitrogen source
After cultivating 7 days in salt fluid nutrient medium, concentration of aniline is transferred to as the inorganic salt liquid culture of 100mg/L with 10% inoculum concentration
It is tamed in base, after being further cultured for 7 days, concentration of aniline is transferred to 10% inoculum concentration and is trained for the inorganic salt liquid of 150mg/L
It supports and is tamed in base.Take culture solution with aniline be unique carbon nitrogen source solid medium in dilution spread, culture 7 days after,
The scribing line purifying of picking single colonie, is pure bacterium until being single bacterium colony on plate, measures the degradation effect of each bacterium respectively, choose it
In dominant strain AN-4a.
Wherein, the formula (1L) of inorganic salt liquid culture medium are as follows: NaCl 0.2g, K2HPO40.2g, KH2PO40.2g, it is micro
Element Solution 2mL.
The formula (1L) of trace element solution: FeSO4300mg, CuSO4·5H2O 38mg, ZnSO4·7H2O 115mg,
MnSO4169mg, H3BO3116mg, CoCl6H2O 24mg, NaMo2H2O 17mg。
Solid culture based formulas (1L) is to add 20g agar in inorganic salt liquid culture medium.
The physiological and biochemical test identification of embodiment 2, bacterial strain
Preliminary Physiology and biochemistry identification is carried out to the strains A N-4a of screening, including colonial morphology detects, gram measures,
Experiment of growth factor, oxidizing ferment experiment, contact enzyme reaction, lysine decarboxylase test, ornithine decarboxylase test, arginine are double
Hydrolase experiment etc., the test method use the conventional methods of this kind of physiological and biochemical index of measurement of this field.
Strains A N-4a is Gram-negative aerobic bacteria as the result is shown, and rod-shaped, bacteria colony white is round, smooth opaque, slightly
Protrusion, neat in edge.Oxidase positive contacts enzyme positive, and lysine decarboxylase is negative, and ornithine decarboxylase is negative, arginine
Double hydrolases are negative.15~45 DEG C of growth temperature, growth pH are 5~10.
The 16S rDNA identification of embodiment 3, bacterial strain
By the analysis of 16S rRNA sequence and the identification of Biolog microbial identification system, determine that strains A N-4a belongs to
Pigmentiphaga daeguensis sp..Specific step is as follows:
The DNA that strains A N-4a is extracted using conventional phenol method expands 16S rDNA sequence with universal primer.
PCR reaction system (50 μ L) are as follows: 5 μ L of 10X PCR buffer, 4 dNTP μ L, each 1 μ L of primer, 2.5 μ L of DNA profiling,
0.25 μ L of TakaraTaq enzyme, 36 μ L of ultrapure water.PCR amplification program is 94 DEG C of 3min；94 DEG C of 1min, 52 DEG C of 1min, 72 DEG C
1.5min, 30 circulations；72℃10min.Agarose electrophoresis detects amplified production, the result is shown in Figure 1.Amplified production serves Hai Shenggong
Biotechnology Co., Ltd is sequenced.Sequencing result is as shown in SEQ ID NO:1.
The 16S rDNA sequence inputting GenBank that will be measured carries out homology search using BLAST software.It chooses different
The 16S rDNA sequence of bacterial strain simultaneously carries out tetraploid rice with 16S rDNA known in GenBank.The pcr amplification product is existed
Blast comparison result is carried out in GenBank and shows that most similar with it is Pigmentiphaga daeguensis sp., together
Source property is 99%.
Thus bacterial strain physiological and biochemical index deducibility AN-4a shown in 2 belongs to Pigmentiphaga in conjunction with the embodiments
daeguensis sp.。
Embodiment 4, microphenomenon of degradation of aniline AN-4a test the degradation effect of aniline under aniline initial concentration different condition
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for are inoculated into LB liquid medium, in 30 DEG C, 160r/min item
Shaking table activated spawn under part makes the OD600 value of bacterium solution reach 0.5, is inoculated into concentration of aniline with 100 μ L activation bacterium solution as 10mg/
L, small 0 hour, 12 hours, 24 hours, 36 hours and 48 respectively in the inorganic salt liquid culture medium of 50mg/L and 100mg/L
Shi Houyong N- (1- naphthalene) ethylenediamine azo photometry measures the concentration of aniline, as a result sees Fig. 2.
As seen from Figure 2, under conditions of concentration of aniline is 10mg/L, degradation bacteria AN-4a is to aniline after 12h
Degradation rate reach 95% or more, and under the conditions of 50mg/L and 100mg/L, degradation bacteria AN-4a is to aniline after 12h
Degradation rate only reaches 17% and 44% respectively.Show that degradation bacteria AN-4a is suitable for the efficient degradation of low concentration aniline.
Embodiment 5, microphenomenon of degradation of aniline AN-4a test the degradation effect of aniline under inoculum concentration different condition,
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for are inoculated into LB liquid medium, in 30 DEG C, 160r/min item
Shaking table activated spawn under part makes the OD600 value of bacterium solution reach 0.5, activates bacterium solution with 0.1mL, 0.5mL, 1mL, 2mL, 4mL, 8mL
After centrifugation, it is inoculated into the inorganic salt solution that 20mL concentration of aniline is 10mg/L, uses N- (1- behind 0 hour and 24 hours respectively
Naphthalene) ethylenediamine azo photometry measurement aniline concentration, as a result see Fig. 3.
As seen from Figure 3, with the increase of inoculum concentration, degradation bacteria AN-4a steps up the degradation rate of aniline.When connecing
When kind amount reaches 4mL or more, the degradation rate of aniline reaches 100%.Consider practical application, preferably degradation bacteria AN-4a's most preferably connects
Kind amount is 20%.
Embodiment 6, microphenomenon of degradation of aniline AN-4a test the degradation effect of aniline under medium pH value different condition
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for are inoculated into LB liquid medium, in 30 DEG C, 160r/min item
Shaking table activated spawn under part makes the OD600 value of bacterium solution reach 0.5, after 4mL activation bacterium solution centrifugation, is inoculated into 20mL pH value point
Not Wei 5,6,7,8,9,10, concentration of aniline is in the inorganic salt solution of 10mg/L, respectively behind 0 hour and 24 hours with N- (1-
Naphthalene) ethylenediamine azo photometry measurement aniline concentration, as a result see Fig. 4.
As seen from Figure 4, when pH value is 5, the removal rate of aniline is minimum, and only 22.5%, as pH value increases to 7
When, the removal rate of aniline reaches 100%, and when pH value is 10, removal rate drops to 74.8%.Show that degradation bacteria AN-4a is most suitable
Growth, degradation pH value are between 7~9, consider actual conditions, it is relatively reasonable that degradation bacteria culture pH value is selected as 7.
Embodiment 7, microphenomenon of degradation of aniline AN-4a test the degradation effect of aniline under temperature different condition
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for are inoculated into LB liquid medium, in 30 DEG C, 160r/min item
Shaking table activated spawn under part makes the OD600 value of bacterium solution reach 0.5, after activating bacterium solution centrifuging and taking supernatant with 4mL, is inoculated into
20mL concentration of aniline is to be respectively placed in 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C of shaking tables in the inorganic salt solution of 10mg/L
As a result middle culture is shown in Fig. 5 with the concentration of N- (1- naphthalene) ethylenediamine azo photometry measurement aniline behind 0 hour and 24 hours.
As seen from Figure 5,15 DEG C when 24 hours after aniline removal rate it is minimum, only reach 3.3%；It is gone at 20 DEG C and 25 DEG C
Except rate is slightly higher, 6.7% and 24.3% are respectively reached；At 30~40 DEG C, removal rate reaches 100%.Show that degradation temperature is in 30
Degradation bacteria 4a the most suitable growth between~40 DEG C.Consider that the actual conditions of application, the cultivation temperature of degradation bacteria AN-4a are chosen to be 30
℃。
Degrading aniline effect after embodiment 8, the microphenomenon of degradation of aniline AN-4a different reaction time
The microphenomenon of degradation of aniline AN-4a slant strains deposited of going bail for are inoculated into LB liquid medium, in 30 DEG C, 160r/min item
Shaking table activated spawn under part makes the OD600 value of bacterium solution reach 0.5, and after 4mL activation bacterium solution centrifugation, being inoculated into 20mL pH value is
7, concentration of aniline is to be placed in 30 DEG C of shaking tables and cultivate in the inorganic salt solution of 10mg/L, 0 hour, 6 hours, 12 hours, it is 15 small
When, 18 hours, 21 hours, measure OD600 value after 24 hours and measure aniline with N- (1- naphthalene) ethylenediamine azo photometry
Concentration.As a result see Fig. 6.
As seen from Figure 6, degradation bacteria AN-4a inoculum concentration be 4mL, medium pH value 7, cultivation temperature is 30 DEG C
Under the conditions of, 100% is reached to the degradation rate of aniline after 12 hours, is realized degradable.Bacteria concentration increases over time, and shows to degrade
Bacterium AN-4a is using aniline as unique carbon nitrogen source, energy needed for providing growth.After aniline is degradable, bacteria concentration is still
Continue to increase, shows that degradation bacteria AN-4a can provide energy as nutriment using the catabolite of aniline for the growth of bacterium.
Claims (8)
1. one plant of microphenomenon of degradation of aniline AN-4a, the Pseudomonas inPigmentiphaga daeguensis, it is deposited in Guangdong Province microorganism
Culture Collection Center, deposit number are GDMCC No:60017.
2. purposes of the microphenomenon of degradation of aniline AN-4a described in claim 1 in processing low concentration aniline waste water.
3. a kind of processing method of low concentration aniline waste water, microphenomenon of degradation of aniline AN-4a described in claim 1 is suspended in containing low dense
It spends in the waste water of aniline and cultivates.
4. processing method according to claim 3, the concentration of the Aniline is 10mg/L.
5. processing method according to claim 3, the inoculum concentration of the microphenomenon of degradation of aniline AN-4a is 20%.
6. processing method according to claim 3, the culture pH value is 7~9.
7. processing method according to claim 3, the cultivation temperature is 30~40 DEG C.
8. processing method according to claim 3, the incubation time is 12 hours or more.
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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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| 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 |
|---|
| pigmentiphaga daeguensis sp.nov., isolated from wastewater of a dye works,and emended description of the genus pigmentiphaga;jung-hoon yoon;《international journal of systematic and evolutionary microbiology》;20070601;第57卷;1188-1191 * |
| 高效苯胺降解菌的分离鉴定及其降解特性;吕晶华 等;《城市环境与城市生态》;20160220;第57卷;32-34 * |
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