CN103937697B - Bacterial strain for degrading dye with high efficiency - Google Patents
Bacterial strain for degrading dye with high efficiency Download PDFInfo
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- CN103937697B CN103937697B CN201410015741.7A CN201410015741A CN103937697B CN 103937697 B CN103937697 B CN 103937697B CN 201410015741 A CN201410015741 A CN 201410015741A CN 103937697 B CN103937697 B CN 103937697B
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Abstract
The invention discloses a bacterial strain for degrading dye with high efficiency. The bacterial strain is Lysinibacillus sp ((i) Lysinibacillus sp.(/i))FS1, and is preserved in the China Center for Type Culture Collection with the preservation number is CCTCC NO: M 2013561 on Nov, 10th, 2013. The bacterial strain has the advantage of rapid growth and strong environment adaptability, has dye degradation capability with wide spectrum, and has good decolouring effect to a plurality of dyes, maximum decolourization percentage can be reached by culturing for 10 hours, the percent of decolourization can reach more than 90%, and the maximum decolouring concentration can reach 5000mg/L. The bacterial strain can be used for decolouring the printing and dyeing waste water and correlated dye waste water, can effectively avoid the dye molecules which are difficulty performed with natural degradation to directly enter in the water body for environment pollution, no secondary pollution is generated, and the bacterial strain has good ecology efficiency and application prospect. The Lysinibacillus sp can be better used for degrading the dye, fills the blank of domestic technology, and provides a powerful technical base for solving the dye sewage treatment, especially the decolouring problem.
Description
Technical field
The present invention relates to biological technical field, more particularly, to the bacterial strain of a high-efficiency degradation dyestuff.
Background technology
Artificial synthesized dyestuff is widely used in the industries such as printing and dyeing, leather, food, cosmetics, paper products, thus produces a large amount of
Waste water from dyestuff.Colourity height, complicated components are had due to waste water from dyestuff, the features such as chemical stability is strong, biodegradability is poor,
Substantial amounts of dyestuff being discharged in environment with industrial wastewater, most azo dyes have potential three and cause effect (teratogenesis, cause
Cancer, mutagenesis), to environment and human health, there is serious harm.China Pearl River Delta area dyestuff year usage amount is more than 2 at present
×104Ton, wherein has 10～20% dyestuffs to be directly discharged in environment, azo dyes account for the 50% about of total release.
At present the processing method of waste water from dyestuff is had a lot, chemical method such as flocculence, oxidizing process, electrochemical process, Physical
As Bubble-floating Method, supercritical ultrasonics technology, membrane separation process, absorption and extraction etc., although these methods effectively, high treating effect, be processed into
This is higher, easily causes secondary pollution.Although the biological treatment low cost such as traditional activated sludge process, membrane biological process, no two
Secondary pollution, but because the degradability difference of dyestuff is it is impossible to thoroughly be degraded by common microorganism, its application is subject to certain limiting
System.
Separate from environment and filter out the microorganism fungus kind being capable of efficient degradation dyestuff, be applied to waste water from dyestuff
Process, be effective biological treatment, have a good application prospect and ecological benefits.At present to existing lysine bud
Spore bacillus (lysinibacillus sp.) research be concentrated mainly on the ability of its reducing heavy metal or to persistent organic
The degradation capability of pollutant, such as patent cn 102363756a announce one plant of lysine bacilluslysinibacillus sp. gy32Degraded to polychlorinated diphenyl ether bde209, patent cn103395893a discloses one plant and can reduce cr6+Lysine
Bacillus.And domestic at present yet there are no lysine bacillus for dye decolored Research Literature and patent report.
Content of the invention
The technical problem to be solved in the present invention is to fill up the art blank, provides one plant to be capable of efficient degradation dyestuff
Lysine bacillus strain.
The invention solves the problems that another technical problem be that separation method and the cultural method of described bacterial strain are provided.
The purpose of the present invention is achieved by the following technical programs:
There is provided a high-efficiency degradation dyestuff lysine Bacillus strain (lysinibacillus sp.) fs1, in
On November 10th, 2013 is preserved in China typical culture collection center, and (address is that Wuhan City of Hubei China province Luo Jia Shan Wuhan is big
Learn), deposit number is: cctcc no:m 2013561.It is preserved in China typical culture collection center, deposit number is
Cctcc no:m 2013561.
Described bacterial strain is bacillus, Gram-positive, and bacterium colony is circular, smooth surface is flat, neat in edge, faint yellow, impermeable
Bright, have pearly luster, colony diameter 2mm～6mm, the sequence of this bacterial strain 16s rdna is as shown in seq id no.1.Withlysinibacillus fusiformisNbrc 15717(ncbi accession number ab271743) have 99% homology.
The bacterial strain fs1 of efficient degradation dyestuff of the present invention obtains good application effect in terms of dyestuff degraded, especially
It is in terms of decoloring dye waste water.
Invention also provides the separation method of the bacterial strain fs1 of described efficient degradation dyestuff, it is will be dirty for the activity of collection
, through bacterial classification enriched medium enrichment culture to having after obvious decolouring phenomenon, gradient dilution, by the bacterium solution after gradient dilution for mud sample
It is applied on screening and culturing medium flat board, cultivates 24～48h, picking substantially decolours circle, continuously rule, choose single bacterium colony and train in screening for 37 DEG C
Up to after no miscellaneous bacteria, picking single bacterium colony, in decolouring culture medium, obtains final product in 37 DEG C of quiescent culture for foster base culture.
The described separation method of present invention offer separates the bacterial strain fs1 of the efficient degradation dyestuff obtaining in terms of dyestuff degraded
Application, especially has good application effect in terms of decoloring dye waste water.
The consisting of of described enriched medium: beef extract 1g, peptone 2g, glucose 3g, k2hpo42g,
nah2po40.5g, mgso47h2o 0.2g, mnso47h2o 0.02g, fecl3·7h2O 0.01g, cacl20.02g,
Sterilized water 1l, ph 7.2；
The consisting of of described screening and culturing medium: dusty yeast 5g, sodium chloride 5g, peptone 10g, agar 20g, sterilized water 1l,
ph7.2；
Described decolouring the consisting of of culture medium: carbon source 2g, nitrogen source 1g, nah2po40.5g, k2hpo42g, feso4·7h2o
0.01g, mgso4·7h2O 0.2g, cacl20.02g, sterilized water 1l, ph 7.2.
Above culture medium all can be in 121 DEG C of 15～30min that sterilize.
Bacterial strain of the present invention is initially to divide from the activated sludge of Guangzhou, Guangdong industry park wastewater treatment aeration tank
From obtaining.
The cultural method of the bacterial strain fs1 of efficient degradation dyestuff of the present invention, is that described bacterial classification is inoculated in Liquid Culture
In base, at 30～37 DEG C, shaking table culture 10～24h obtains final product the nutrient solution of described bacterial strain；
The consisting of of described fluid nutrient medium: glucose 2g, ammonium chloride 1g, nah2po40.5g, k2hpo42g, feso4·
7h2O 0.01g, mgso4·7h2O 0.2g, cacl20.02g, water 1l, ph7.2.
The strain cultured solution that cultural method culture of the present invention obtains has good application in terms of dyestuff degraded.Especially
It is that have good application effect in terms of decoloring dye waste water.
Bacterial strain of the present invention and its nutrient solution are related to multiple dyestuffs in the application of dye decolored aspect, especially azo are contaminated
Material wastewater degradation is better.
It is further preferable that there is excellent fall to multiple dyestuffs such as methyl orange, acid red b, dimethyl diaminophenazine chloride and/or methylene blues
Solution effect.
Preferably, the concentration range of the most suitably used dyestuff of described application is 80～5000mg/l, can obtain excellent decolouring
Effect.
Preferably, bacterial strainlysinibacillus sp.Fs1 is not higher than 4000mg/l to the decolouring concentration of methylene blue.
Preferably, bacterial strainlysinibacillus sp.The red decolouring concentration of fs1 centering is not higher than 2000mg/l.
Preferably, bacterial strainlysinibacillus sp.Fs1 is not higher than 500mg/l to the decolouring concentration of methyl orange.
Preferably, bacterial strainlysinibacillus sp.Fs1 is not higher than 5000mg/l to the decolouring concentration of acid red b.
Preferably, in described application, it is to cultivate the bacterial strain fs1 of described efficient degradation dyestuff to exponential phase, takes training
Nutrient solution, by volume in the inoculum concentration access decolouring basal medium for 1～5%, adds described dyestuff to carry out normal temperature culture, real
Now decolour.Preferably, the temperature of described normal temperature culture is 10～40 DEG C, ph6～9, incubation time 10～24h.
Preferably, in described application, it is by the bacterial strain of efficient degradation dyestufffs1Strain cultured solution by volume be 1～
5% inoculum concentration accesses in decolouring basal medium, adds described dyestuff to carry out normal temperature culture, realizes decolouring.
Preferably, the temperature of described normal temperature culture is 10～40 DEG C, ph6～9, incubation time 10～24h.
The invention has the advantages that:
The invention provides one plant of new bacterial strain, this strain isolation from the activated sludge of industry park wastewater treatment aeration tank,
Separating step is simple, and preparation cost is relatively low.Described strain growth is rapid, and environmental suitability is strong, has the dyestuff degraded energy of wide spectrum
Multiple dyestuffs are all had a good decolorizing effect by power, and culture 10h can reach maximum percent of decolourization, percent of decolourization up to 90% with
On, maximum decolouring concentration reaches 5000mg/l.
Described bacterial strain is applied to the present invention dyeing waste water and the decoloring dye waste water of correlation is processed, it is possible to resolve existing skill
In art lack efficient degradation effect, adaptive capacity to environment difference the features such as, can be prevented effectively from be difficult to natural degradation dye molecule straight
Tap into the environmental pollution leading to into water body, and non-secondary pollution, there is good ecological efficiency and application prospect.
The present invention first by lysine bacillus (lysinibacillus sp.) it is applied to dyestuff degraded, fill up state
Interior technological gap, processes, for solving dye wastewater, the strong technical foundation of problem offer of especially decolouring.
Brief description
Fig. 1:lysinibacillus sp.The scanning electron microscope (SEM) photograph of fs1, scale=1 m.
Fig. 2:lysinibacillus sp.The phylogenetic tree of fs1.
Fig. 3:lysinibacillus sp.The decolorizing effect to variable concentrations methylene blue for the fs1.
Fig. 4:lysinibacillus sp.The decolorizing effect to variable concentrations dimethyl diaminophenazine chloride for the fs1.
Fig. 5:lysinibacillus sp.The decolorizing effect to variable concentrations methyl orange for the fs1.
Fig. 6:lysinibacillus sp.The decolorizing effect to variable concentrations acid red b for the fs1.
Fig. 7:lysinibacillusSp. fs1 is to the absorption of acid red b and degraded.
Fig. 8:lysinibacillusSp. the spectral scan figure of fs1 degraded acid red b.
Specific embodiment
Further illustrate the present invention with specific embodiment below in conjunction with the accompanying drawings.Unless stated otherwise, what the present invention adopted is former
Reason and method, equipment are principle commonly used in the art, method and apparatus.
Embodiment 1lysinibacillus sp.fs1The separation of bacterial strain obtains
1. bacterial strain activation and enrichment
Take 50ml activated sludge sample (taking from Guangzhou, Guangdong industry park wastewater treatment aeration tank), put into and fill 50ml
(inside there are several beades) in the conical flask of sterilized water, fully break up mud sample in shaking table vibration 30min.Take the suspension mud after breaing up
Sample 5ml, is seeded in the enriched medium filling 100ml, adds the acid red b dyestuff of 30mg/l, 37 DEG C, 200rpm vibration training
Support 16～24h.
The consisting of of described enriched medium: beef extract 1g, peptone 2g, glucose 3g, k2hpo42g,
nah2po40.5g, mgso47h2o 0.2g, mnso47h2o 0.02g, fecl3·7h2O 0.01g,
cacl20.02g, sterilized water 1l, ph 7.2；Culture medium is obtained final product in 121 DEG C of sterilizing 15～30min.
2. bacterial strain screening and purifying
To there is the nutrient solution after the enrichment of obvious decolorizing effect, with SPSS test tube stepwise dilution to 10-1、10-2、10-3、10-4、10-5、10-6、10-7Totally 7 gradients.Each gradient takes the dilution of 0.1ml to coat the acid containing 30mg/l
Property red b screening and culturing medium flat board on, 37 DEG C culture 24～48h.Observe and around single bacterial strain, whether have decolouring to iris out now.Picking
There is the bacterial strain of decoloring ability, 37 DEG C of continuous streak inoculations, on screening and culturing medium flat board, are further purified and confirm to obtain one
High-efficiency degradation dyestuff lysine Bacillus strain (lysinibacillus sp.)fs1, described bacterial strain is bacillus, and leather is blue
Family name is positive, bacterium colony is circular, smooth surface is flat, neat in edge, faint yellow, opaque, have pearly luster, colony diameter 2mm～
6mm, accompanying drawing 1 is the electron-microscope scanning figure of bacterial strain.The sequence of this bacterial strain 16s rdna withlysinibacillus fusiformis
Nbrc 15717(ncbi accession number ab271743) have 99% homology.The phylogenetic tree of this bacterial strain is shown in accompanying drawing 2.
Described bacterial strain is preserved in China typical culture collection center on November 10th, 2013, and (address is Hubei China
Luo Jia Shan Wuhan University of Wuhan City of province), deposit number is: cctcc no:m 2013561.It is preserved in Chinese Typical Representative culture to protect
Tibetan center, deposit number is cctcc no:m 2013561.
Embodiment 2lysinibacillus sp.Fs1 tests to the decolorizing effect of variable concentrations methylene blue
The bacterial strain fs1 of efficient degradation dyestuff of the present invention is inoculated in fluid nutrient medium, in 30～37 DEG C, 150rpm
Lower shaking table culture 10～24h obtains final product the nutrient solution of described bacterial strain；The consisting of of described fluid nutrient medium: glucose 2g, ammonium chloride
1g, nah2po40.5g, k2hpo42g, feso4·7h2O 0.01g, mgso4·7h2O 0.2g, cacl20.02g, water 1l,
ph7.2.
Take the logarithm growth period (od600=1)lysinibacillus sp.Fs1 nutrient solution, by 1%(v/v) access 100ml
Decolouring basal medium in, be separately added into variable concentrations gradient (80mg/l, 500mg/l, 2000mg/l, 3000mg/l,
4000mg/l, 5000mg/l) methylene blue dye.Three parallel, and normal temperature is cultivated, and measures the suction of nutrient solution every 5～24 h
Light value.
Investigate the decoloring ability of bacterial strain fs1 with percent of decolourization.In 200～800(nm) scan dye solution in wave-length coverage
Light absorption value, select the mensure wavelength as dyestuff light absorption value for the wavelength of maximum absorption band m.By nutrient solution to be measured in 5000rpm
Under the conditions of be centrifuged 10min.Supernatant liquor is taken to measure light absorption value.According to formula calculating percent of decolourization:
a0: the initial light absorption value of nutrient solution；a1: the light absorption value after culture a period of time
As shown in Figure 3, methylene blue concentration is that during 80～500mg/l, percent of decolourization just can reach 80% in 10h to result
More than.Although when methylene blue concentration is higher than 500mg/l, bacterial strain fs1 raises with dye strength to its percent of decolourization and drops
Low, but still have certain decoloring ability under 4000mg/l concentration, percent of decolourization is 40%.Bacterial strainlysinibacillus sp.fs1
The highest of methylene blue is decoloured at concentrations up to 4000mg/l.
Embodiment 3lysinibacillus sp.Fs1 tests to the decolorizing effect of variable concentrations dimethyl diaminophenazine chloride
With reference to embodiment 2, cultivate described bacterial strain, take the logarithm growth period (od600=1) bacterial strain fs1 nutrient solution, by 1%(v/
V) access 100ml decolouring basal medium in, be separately added into variable concentrations gradient (80mg/l, 300mg/l, 500mg/l,
1000mg/l, 2000mg/l) neutral red.Other are with embodiment 2.
As shown in Figure 4, when dimethyl diaminophenazine chloride concentration is 300～1000mg/l, percent of decolourization basically reaches result of the test in 10h
Maximum percent of decolourization 90% about, when concentration raises, bacterial strain bleaching time increases about 48h.With the raising of dimethyl diaminophenazine chloride concentration, bacterium
Strain is gradually reduced to its decoloring ability, drops to 30% by 99%.Bacterial strainlysinibacillus sp. fs1Centering is red
Highest is decoloured at concentrations up to 2000mg/l.
Embodiment 4lysinibacillus sp.Fs1 tests to the decolorizing effect of variable concentrations methyl orange
With reference to embodiment 2, cultivate described bacterial strain, take the logarithm growth period (od600=1) bacterial strain fs1 nutrient solution, by 1%(v/
V) access 100ml decolouring basal medium in, be separately added into variable concentrations gradient (80mg/l, 200mg/l, 300mg/l,
400mg/l, 500mg/l) methyl orange dye.Other are with embodiment 2.
As shown in Figure 5, when methyl orange concentration is 80～300mg/l, 10h percent of decolourization is 90% about to result.But work as concentration
When raising further when (400～500mg/l), although percent of decolourization declines, also can remove 60% about.Bacterial strainlysinibacillus sp. fs1Optimal decolouring concentration to methyl orange is 500mg/l.
Embodiment 5lysinibacillus sp. fs1Decolorizing effect checking to variable concentrations acid red b
Take the logarithm growth period (od600=1) bacterial strain fs1 nutrient solution, by 1%(v/v) access 100ml decolouring basis culture
In base, be separately added into variable concentrations gradient (80mg/l, 300mg/l, 500mg/l, 1000mg/l, 2000mg/l, 3000mg/l,
4000mg/l, 5000mg/l) acid red b dyestuff.Other are with embodiment 2.
As shown in Figure 6, when acid red b concentration is 80～1000mg/l, percent of decolourization basically reaches result in 10～24h
Maximum 90% about.Although bringing up to 5000mg/l with acid red b concentration by 80mg/l, bacterial strain is to its decoloring ability by 93%
It is gradually reduced, but minimum also have 28%.There is obvious degradation and decolorization treatment effect.Bacterial strain fs1 decolours to the highest of acid red b
At concentrations up to 5000mg/l.
Embodiment 6lysinibacillusSp. the confirmatory experiment to acid red b absorption and degradation effect for the fs1
Take the logarithm growth period (od600nm=1) bacterial strain fs1 nutrient solution, one of which carries out autoclaving as inactivation group,
Another group does not inactivate as not inactivateing group.Two groups of bacterium solution are fixed with sodium alginate respectively, make diameter 5mm about fixation
Change bead, be inoculated in the decolouring culture medium containing 80mg/l acid red b by 1% inoculum concentration.Other are with embodiment 2.
Result as shown in Figure 7, does not inactivate group percent of decolourization in 20h and just can reach 93% about, and inactivation group percent of decolourization
Within maintaining 0～7% always.The percent of decolourization of inactivation group declines and is because thalline and bead absorption leads to, and does not inactivate group
It is absorption that percent of decolourization declines and bacterial degradation collective effect leads to.
Additionally, scan in 200～800nm wave-length coverage every 3h in incubation not inactivateing group nutrient solution spectrum
Light absorption value.Result as shown in Figure 8, sweep by the spectrum that the curve of spectrum is followed successively by 0h, 3h, 6h, 9h, 12h, 15h, 18h from top to bottom
Retouch curve.It can be seen that, in 0h, the light absorption value of maximum absorption band 515nm is 2.2, cultivates when 3h, 6h, 9h, 12h, 15h, 18h
The light absorption value of big absworption peak is down to 2.0,1.9,1.7,1.5,0.3 respectively, and percent of decolourization is respectively 10%, 14%, 23%, 32%, 86%；
From in figure it can be found that growth with incubation time, the light absorption value of maximum absorption band is gradually reduced, and has not had in 18h
Big absworption peak, curve is substantially smooth, shows acid red b quilt reallylysinibacillusSp. fs1 is degraded.Therefore, this
Bright describedlysinibacillusSp. the biodegradation that fs1 mainly passes through bacterial strain to the decolorization of dyestuff is real
Existing.
sequence listing
<110>Ji'nan University
The bacterial strain of<120>one high-efficiency degradation dyestuffs
<130>
<160> 1
<170> patentin version 3.3
<210> 1
<211> 1413
<212> dna
<213>sequence of bacterial strain 16s rdna
<400> 1
ggctggctcc aaaaggttac ctcaccgact tcgggtgtta caaactctcg tggtgtgacg 60
ggcggtgtgt acaaggcccg ggaacgtatt caccgcggca tgctgatccg cgattactag 120
cgattccggc ttcatgtagg cgagttgcag cctacaatcc gaactgagaa cgactttatc 180
ggattagctc cctctcgcga gttggcaacc gtttgtatcg tccattgtag cacgtgtgta 240
gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttgtcac 300
cggcagtcac cttagagtgc ccaactaaat gatggcaact aagatcaagg gttgcgctcg 360
ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc 420
accgttgccc ccgaagggga aaccatatct ctacagtggt caacgggatg tcaagacctg 480
gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt gcgggccccc 540
gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt 600
tagctgcagc actaaggggc ggaaaccccc taacacttag cactcatcgt ttacggcgtg 660
gactaccagg gtatctaatc ctgtttgctc cccacgcttt cgcgcctcag tgtcagttac 720
agaccagata gtcgccttcg ccactggtgt tcctccaaat ctctacgcat ttcaccgcta 780
cacttggaat tccactatcc tcttctgcac tcaagtctcc cagtttccaa tgaccctcca 840
cggttgagcc gtgggctttc acatcagact taagaaacca cctgcgcgcg ctttacgccc 900
aataattccg gacaacgctt gccacctacg tattaccgcg gctgctggca cgtagttagc 960
cgtggctttc taataaggta ccgtcaaggt acagccagtt actactgtac ttgttcttcc1020
cttacaacag agttttacga accgaaatcc ttcttcactc acgcggcgtt gctccatcag1080
gctttcgccc attgtggaag attccctact gctgcctccc gtaggagtct gggccgtgtc1140
tcagtcccag tgtggccgat caccctctca ggtcggctac gcatcgtcgc cttggtgagc1200
cgttacctca ccaactagct aatgcgccgc gggcccatcc tatagcgaca gccgaaaccg1260
tctttcaata tttcaccatg aggtgaaaca gattattcgg tattagcccc ggtttcccgg1320
agttatccca aactataagg taggttgccc acgtgttact cacccgtccg ccgctaacgt1380
caaaggagca agctccttct ctgttcgctc gac 1413
Claims (2)
1. a high-efficiency degradation dyestuff lysine bacillus (lysinibacillus sp.) fs1, it is preserved in Chinese Typical Representative
Culture collection, deposit number is cctcc no:m 2013561；The 16s rdna of described lysine bacillus fs1
Sequence as shown in seq id no.1；Described lysine bacillus fs1 is bacillus, Gram-positive, and bacterium colony is circular, surface
Smooth flat, neat in edge, faint yellow, opaque, have pearly luster, colony diameter 2mm～6mm.
2. the cultural method of the lysine bacillus fs1 of efficient degradation dyestuff described in claim 1 will be it is characterised in that will rely
Propylhomoserin bacillus fs1 is inoculated in fluid nutrient medium, and at 30～37 DEG C, shaking table culture 10～24h obtains final product described lysine bud
The nutrient solution of spore bacillus fs1, the consisting of of described fluid nutrient medium: glucose 2g, ammonium chloride 1g, nah2po40.5g, k2hpo4
2g, feso4·7h2O 0.01g, mgso4·7h2O 0.2g, cacl20.02g, water 1l, ph7.2.
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