CN102268388B

CN102268388B - Method for screening bacteria capable of decoloring multiple reactive dyes

Info

Publication number: CN102268388B
Application number: CN201110170426.8A
Authority: CN
Inventors: 柳建设; 于文娟; 谢学辉; 王兆慧; 许贺; 洪武林; 张武刚
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2011-06-22
Filing date: 2011-06-22
Publication date: 2014-04-16
Anticipated expiration: 2031-06-22
Also published as: CN102268388A

Abstract

The invention relates to a method for screening bacteria capable of decoloring multiple reactive dyes, which comprises the following steps: (1) dispersing reactive dye sludge with aseptic glass beads to obtain a bacterium suspension, inoculating the bacterium suspension into an enrichment culture medium, and acclimating in the enrichment culture medium for 4 weeks by using dyes with different concentration gradients; (2) taking the acclimated bacterium liquid of which the OD (optical density) is 1, carrying out gradient dilution, uniformly spreading each dilution of bacterium liquid onto a solid culture medium, carrying out anaerobic culture at 35 DEG C for 48 hours, and repeating the step until the bacterial colonies growing on the solid culture medium have a uniform size and a clear edge; scribing the separated single bacterial colony onto a dye-containing solid culture medium, observing the growth conditions of the bacterial colony, inoculating the bacterial colony into a dye-containing liquid culture medium, observing the decoloring effect of the strain, and preserving the strain with good decoloring effect. The separation and purification method provided by the invention is simple and quick, and has the advantages of low cost, environmental friendliness and wide application prospects.

Description

The screening method of a kind of bacterium of the reactive black 5 of decolouring

Technical field

The invention belongs to bacteria screening field, particularly the screening method of a kind of bacterium of the various active dyestuff that decolours.

Background technology

Along with the fast development of dyestuffs industries, the discharge of printing and dyeing industrial waste water and waste water in dye production has caused serious environmental problem.Dyestuff is divided into matching stain, dispersed dye, reactive dyestuffs etc. according to its application.Reactive dyestuffs have become the topmost dye class of cotton fiber dyeing, much larger than other dyestuffs.Azoic dyestuff, because its production method is simple, has very wide spectral range, and has certain fastness, thereby becomes the synthetic dyestuff that commercial applications is maximum.The natural azoic dyestuff that nature exists is little, and industrial azoic dyestuff is almost all artificial sintetics, and its complex structure is various in style, and stability is high, has very strong anti-light, oxidation resistant ability, therefore conventionally more difficult containing the waste water of azoic dyestuff.And azoic dyestuff and its metabolite can work the mischief to HUMAN HEALTH, there is carcinogenic, teratogenesis, mutagenic effect.

Research about reactive dyestuffs decolourings bacterium is existing a lot, but it is relatively less to have the bacterium of broad spectrum decolorizing effect.

Summary of the invention

Technical problem to be solved by this invention is to provide the screening method of a kind of bacterium of the various active dyestuff that decolours, and the method method is simple and efficient, and cost is low, environmentally friendly, has a extensive future.

The screening method of the bacterium of a kind of various active dyestuff that decolours of the present invention, comprising:

(1) bacterial suspension inoculation of making after reactive dyestuffs mud is broken up by sterile glass beads, in enrichment medium, is used the dyestuff that contains different concns gradient in enrichment medium, to tame 4 weeks;

(2) by the bacterium liquid after domestication, get OD value and be 1 bacterium liquid, do gradient dilution, each dilution bacterium liquid is evenly applied on solid medium and cultivates 48h in 35 ℃ of anaerobism, repeat this step until the bacterium colony size growing on solid medium is even, edge clear; The single bacterium colony line being separated to, to containing on the solid medium of dyestuff, is observed to colony growth situation, be inoculated in the liquid nutrient medium containing dyestuff, observe bacterial strain decolorizing effect, the bacterial strain of good decolorizing effect is carried out to preservation, obtain.

Enrichment medium in described step (1) is beef-protein medium, fills a prescription as follows: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L, distilled water 1000mL.

The dyestuff that contains different concns gradient in described step (1) is through the reactive black 5 of bacteriological filtration, Reactive Red 2 39, Reactive Blue 19 100 or reactive brilliant orange x-an, concentration gradient is respectively 50mg/L, 100mg/L, 150mg/L and 200mg/L, and the timed interval that changes concentration is 7d.

Domestication condition in described step (1) is in 35 ℃ of anaerobic acclimations.

Gradient dilution in described step (2) is 10 ^-1～10 ^-7gradient dilution.

Solid medium in described step (2) is the agar powder that adds 17g/L in the beef-protein medium in step (1).

The solid culture based formulas containing dyestuff in described step (2) is extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, distilled water 1000mL, dyestuff 50mg/L.

The liquid culture based formulas containing dyestuff in described step (2) is extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L, distilled water 1000mL, dyestuff 50mg/L.

Described preservation is for doing inclined-plane cryopreservation and Freezing Glycerine preservation, inclined-plane cryopreservation used medium is by after solid medium autoclave sterilization, pour 5mL liquid in clean tube, by test tube lie in domatic on, make to manage interior substratum and form inclined-plane, and on substratum, edge is no more than 2/3 of test tube; Freezing Glycerine preservation glycerine used is the mixed solution that pure glycerin and sterilized water volume ratio are 1: 1, added in the aseptic EP pipe containing equal-volume overnight growth bacterium liquid, and-20 ℃ of preservations, above operation should be carried out on aseptic operating platform.

beneficial effect

Separation purification method of the present invention is simple and efficient, and cost is low, environmentally friendly, has a extensive future; The bacterium that screening obtains can be applicable to the reactive dyestuffs that decolour, and can tolerate percent of decolourization in strong basicity waste water from dyestuff 48h and can reach more than 80%.

Accompanying drawing explanation

Fig. 1 is the molecular structural formula of pattern dye activity black 5.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.

Embodiment 1

(1) bacterial suspension inoculation of active sludge being made, in enrichment medium, is used the enrichment medium that contains different gradient concentration dyestuffs to tame 4 weeks; Wherein enrichment medium is extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L, distilled water 1000mL, pH=7.0-8.0; Adding the bacteriological filtration dyestuff of gradient concentration simultaneously, the concentration of dyestuff in substratum is all raise successively, is reactive black 5 0mg/L, 100mg/L, 150mg/L, 200mg/L, and every 7d improves a concentration gradient, in 30～35 ℃, tames and cultivate 4 weeks under anaerobic condition;

(2) get the sample of domestication after 4 weeks, standing 30 minutes, get upper strata water sample 1mL, do 10 ^-1～10 ^-7gradient dilution.Every kind of weaker concn is got respectively 1mL bacterium liquid in culture dish, be suitable for aseptic spatula and be uniformly coated on solid culture primary surface, culture dish is inverted in 35 ℃ of anaerobic culture boxes and cultivates 48h, picking form is even, sharp-edged single bacterium colony, numbering, on solid medium, rule, culture dish is inverted in 35 ℃ of anaerobic culture boxes and cultivates 48h, observations again, repeatedly several times, and under opticmicroscope, use oily lens head to observe, choose multiple field range observation, guarantee that more than 95% bacterial strain is after pure single bacterial strain, isolated bacterial classification is inoculated on slant medium, at 4 ℃, be stored in and in refrigerator, treat the follow-up Decolorant Test of carrying out.Separately get the bacterium of 600 μ l liquid culture, add 600 μ l50% glycerine, pack in sterilized EP pipe and mix, carry out and be stored in-20 degree after mark.Wherein solid medium is the agar powder that adds 17g/L in enrichment medium, the substratum of cooling formation after autoclave sterilization, slant medium is by after solid medium autoclave sterilization, pour while hot 5mL liquid in clean tube, by test tube lie in domatic on, make to manage interior substratum and form inclined-plane, and on substratum, edge is no more than 2/3 of test tube, more than operates on aseptic operating platform and carries out.

(3) the single bacterium colony of difference picking after separation and purification is in liquid enrichment medium (the extractum carnis 5g/L containing 50mg/L dyestuff, peptone 10g/L, sodium-chlor 5g/L, distilled water 1000mL) middle cultivation, in 35 ℃, 250mL Erlenmeyer flask, under the condition of liquid amount 100mL substratum, cultivate, incubation time is 48h, cultivation is rear by the residual concentration of dyestuff in microscopic examination bacterial growth situation and mensuration solution, thereby determines the bacterial strain with decolouring reactive azo dyes characteristic.

This bacterial strain is 7.0-8.0 in initial pH value, and temperature is 35 ℃, under the condition that the liquid amount of 250mL Erlenmeyer flask is 100mL, inoculation amount is 1%, and dyestuff starting point concentration is 50mg/L, incubation time 48h, then utilize ultraviolet spectrophotometry to measure dye strength, mensuration wavelength is 498nm; The percent of decolourization of dyestuff can reach 91.4%.

Claims

1. a screening method for the bacterium of the reactive black 5 of decolouring, comprising:

(1) bacterial suspension inoculation of making after reactive dyestuffs mud is broken up by sterile glass beads, in enrichment medium, is used the dyestuff that contains different concns gradient in enrichment medium, to tame 4 weeks; Wherein, enrichment medium is beef-protein medium, fills a prescription as follows: extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L, distilled water 1000mL; The dyestuff that contains different concns gradient is the reactive black 5 through bacteriological filtration, and concentration gradient is respectively 50mg/L, 100mg/L, 150mg/L and 200mg/L, and the timed interval that changes concentration is 7d;

(2) by the bacterium liquid after domestication, get OD value and be 1 bacterium liquid, do gradient dilution, each dilution bacterium liquid is evenly applied on solid medium and cultivates 48h in 35 ℃ of anaerobism, repeats this step until the bacterium colony size growing on solid medium is even, edge clear; The single bacterium colony line being separated to, to containing on the solid medium of dyestuff, is observed to colony growth situation, be inoculated in the liquid nutrient medium containing dyestuff, observe bacterial strain decolorizing effect, the bacterial strain of good decolorizing effect is carried out to preservation, obtain; Wherein solid medium is the agar powder that adds 17g/L in the beef-protein medium in step (1); Solid culture based formulas containing dyestuff is extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L, distilled water 1000mL, dyestuff 50mg/L; Liquid culture based formulas containing dyestuff is extractum carnis 5g/L, peptone 10g/L, sodium-chlor 5g/L, distilled water 1000mL, dyestuff 50mg/L.

2. the screening method of the bacterium of a kind of reactive black 5 of decolouring according to claim 1, is characterized in that: the domestication condition in described step (1) is in 35 ℃ of anaerobic acclimations.

3. the screening method of the bacterium of a kind of reactive black 5 of decolouring according to claim 1, is characterized in that: the gradient dilution in described step (2) is 10 ^-1～10 ^-7gradient dilution.