CN103031262A - Paenibacillus sp. strain and microbial exopolysaccharide flocculant generated by same as well as application of flocculant - Google Patents
Paenibacillus sp. strain and microbial exopolysaccharide flocculant generated by same as well as application of flocculant Download PDFInfo
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Abstract
The invention provides paenibacillus sp. strain ZD01 and exopolysaccharide flocculant PE-1 generated by the same, and further provides an application of the flocculant to the aspects of depositing suspended solids in wastewater treatment, decolorizing dye pigments, removing heavy metal ions and the like. The strain is collected in China center for type culture collection (CCTCC) and provided with the collection date of November 20th, 2012 and the collection number of CCTCC No: M 2012463. When simulated wastewater or practical industrial wastewater is treated with a fermentation solution of the unpurified paenibacillus sp. strain ZD01 or the purified PE-1 polysaccharide, both the paenibacillus sp. strain ZD01 and the PE-1 polysaccharide have higher activity of flocculating the turbid suspended particles, decolorizing the dye pigments and removing the heavy metal ions. The high-efficiency microbial flocculant has a potential application to treatment of wastewater such as life wastewater, industrial wastewater and the like, and especially plays an important role in the wastewater pre-treatment process, so that the subsequent treatment steps and costs are reduced.
Description
(1) technical field
The present invention relates to a strain series bacillus (Paenibacillus sp.) ZD01, and by its Microbial exopolysaccharides flocculation agent and application thereof of fermenting and producing.
(2) background technology
Along with China's industry fast development, the trade effluent and the sanitary sewage that contain the pollutents such as heavy metal ion, pigment and other suspended particles discharge in a large number, and pollution on the environment is day by day serious.Flocculence is one of important method of wastewater treatment, and oneself is widely used in a plurality of fields such as plumbing, wastewater treatment and sludge dewatering this technology.Its objective is the cohesion such as the pollutent that makes suspended particle in the sewage, pigment, heavy metal ion for large flocs unit, in order to separate from water, so the quality of flocculation agent directly affects the effect of purification of water quality.Composition according to flocculation agent is divided into it: inorganic flocculating agent such as aluminium salt, molysite and inorganic polymer flocculant; The macromolecule organic flocculating agent of synthetic such as polyacrylamide and polymine etc.; Natural biological flocculant such as sodium alginate and other microbial flocculants etc.
The composition of the microbial flocculant of having reported so far has polysaccharide, protein, glycoprotein and nucleic acid etc., and is wherein in the majority with polysaccharide.Microbial exopolysaccharides (exopolysaccharide, EPS) refers to that microorganism cells is synthetic and be secreted into the long-chain high molecular polymer that the class outside the born of the same parents is coupled together by a plurality of monose dehydration condensations in process of growth.Wherein the part polysaccharide can have the impurity aggegations such as solid suspended particle, bacterial chip and other colloidal particles that make difficult degradation in the waste water, the flocculating properties of precipitation, can become novel microbial flocculant.This class flocculation agent is compared with traditional inorganic flocculating agent and the polymeric flocculant of synthetic, have the mankind and environment nontoxic, safety, efficient, the distinct advantages such as biodegradable non-secondary pollution have larger application potential aspect city domestic sewage, the trade effluent.
(3) summary of the invention
The exocellular polysaccharide flocculation agent PE-1 that the purpose of this invention is to provide a strain series bacillus (Paenibacillus sp.) ZD01 and produce, and the application of the aspects such as removal of the decolouring of precipitation, coloring matter of this flocculation agent suspended solid in wastewater treatment and heavy metal ion further is provided.
The technical solution used in the present invention is:
One strain series bacillus (Paenibacillus sp.) ZD01, be preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, the Classification And Nomenclature of proposal are Paenibacillus sp. ZD01, preservation date: on November 20th, 2012, deposit number CCTCC No:M 2012463.
How from many habitats gather soil sample to bacterial strain class bud pole bacterium provided by the invention (Paenibacillus sp.) ZD01, adopt plate dilution method to separate, and have the performance that produces the thing polysaccharide flocculant through evaluation.
Bacterial strain provided by the invention bacterium colony after the nutrient broth flat board is cultivated 24h is rounded, neat in edge, and smooth surface is moistening, and is colourless opaque, cultivates 5-7 days rear surface projection folds.
Bacterial strain gramstaining provided by the invention is variable, aerobic, shaft-like, and long 2. 8 ~ 4.8 μ m are wide by 0.3 ~ 1. 11 m, have peritrichous, can produce and hold the oval brood cell of life; Oxydase, catalase activity are arranged, and the nitrate reductase reaction is positive.Do not produce hydrogen sulfide, can be hydrolyzed casein food grade, gelatin.
The application of described series bacillus ZD01 in preparation Microbial exopolysaccharides flocculation agent.
The invention still further relates to the Microbial exopolysaccharides flocculation agent PE-1 that is produced by described series bacillus ZD01 fermentation, it is a kind of microbial polysaccharide that outside aerobic fermentation is secreted into born of the same parents, is produced by bacterial strain, its skeleton is comprised of glucose, glucuronic acid, seminose, wood sugar, rhamnosyl, its mol ratio is 1:1:2:2:0.1, molecular weight 7 * 10
6Dalton.
Described Microbial exopolysaccharides flocculation agent PE-1 makes by the following method: series bacillus CCTCC No:M 2012463 is seeded to fermention medium, 28～32 ℃, 100 ~ 200rpm vortex shaking culture 3 ~ 4 days obtains containing the fermented liquid of described Microbial exopolysaccharides flocculation agent PE-1; Described fermention medium is composed as follows: sucrose 30 ~ 60g/L; Yeast extract 0.1 ~ 1.0g/ L; Peptone 5 ~ 8g/ L; Potassium primary phosphate 1 ~ 5g/ L; Sal epsom 0.1 ~ 1.0g/ L; Iron(ic) chloride 0.01 ~ 0.05g/ L; Manganous chloride tetrahydrate 0.01 ~ 0.05g/ L, solvent are distilled water, pH7.0 ~ 7.5.
Usually also needed to carry out the seed enlarged culturing before fermentation culture, seed culture medium can be conventional nutrient broth medium, and concrete grammar can be as follows:
(1) seed culture: make conventional nutrient broth medium, inoculating strain (Paenibacillus sp.) ZD01,30 ℃, 200rpm cultivates 24h, obtains seed liquor;
(2) fermentation: seed liquor is forwarded to fermention medium with 5～10%(v/v) inoculum size, and 28～32 ℃, 100 ~ 200rpm vortex shaking culture 3 ~ 4 days obtains containing the fermented liquid of flocculation agent.The composition of fermention medium: sucrose 50g/ L; Yeast extract 0.5g/ L; Peptone 6.5g/ L; Potassium primary phosphate 2g/ L; Sal epsom 0.5g/ L; Iron(ic) chloride 0.02g/ L; Manganous chloride tetrahydrate 0.02g/ L, solvent are distilled water, pH7.2.
By above-mentioned steps, flocculation agent output of the present invention is more than 25g/L.
This flocculation agent can directly be used for sewage disposal with the fermented liquid form, also can after purifying, be used for sewage disposal, concrete method of purification was as follows: with fermented liquid in 8000rpm centrifugal 10 ~ 25 minutes, high fermentation liquid is to add N,O-Diacetylmuramidase (200,000 U/g, Pang Bo is biological, and add-on is every liter of fermented liquid 0.5g) and Sumizyme MP (20,000 U/g, Pang Bo is biological, add-on is every liter of fermented liquid 1g), 40 ℃ of insulation 2h; Sevag reagent (chloroform-propyl carbinol volume ratio is 5: 1 mixed solutions) remove residual albumen until aqueous phase without the uv-absorbing of 280nm; The ethanol that adds 2 ~ 5 times of volumes stirs after 30 minutes 4 ℃ of refrigerators placements and spends the night, and 6000 ~ 12000rpm got precipitation in centrifugal 10 ~ 25 minutes, and vacuum-drying gets Microbial exopolysaccharides flocculation agent PE-1.
The invention still further relates to the application of described Microbial exopolysaccharides flocculation agent PE-1 in sewage disposal, specifically can be used for the precipitation of suspended solid in the waste water, the decolouring of coloring matter and the aspects such as removal of heavy metal ion.
Beneficial effect of the present invention is mainly reflected in: utilize the PE-1 polysaccharide behind unpurified (Paenibacillus sp.) ZD01 fermented liquid provided by the invention or the purifying, when treatment of simulated waste water or actual trade effluent, all have higher outstanding turbid suspended particle flocculation activity, coloring matter decolouring activity and removal of heavy metal ions activity.The present invention prepares the high-effective microorganism flocculation agent, has potential use in wastewater treatments such as life, industry, and especially performance acts on greatly in the preprocessing process of sewage, reduces step and the cost of subsequent disposal.
(4) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: the zymotechnique of polysaccharide flocculant PE-1
1, series bacillus CCTCC No:M 2012463 is inoculated on the nutrient agar inclined-plane, cultivates 72h for 30 ℃; Nutrient agar is prepared by following composition: peptone 10g/L, and beef powder 3g/L, sodium-chlor 5g/L, agar 15g/L, solvent are distilled water, pH7.2.
2, seed culture: slant strains is accessed the nutrient broth seed culture medium (being contained in the 250mL triangular flask) of 50mL routine, in 30 ℃, 200rpm shaking culture 24h, be seed liquor; The nutrient broth seed culture medium is prepared by following composition: peptone 10g/L, and beef powder 3g/L, sodium-chlor 5g/L, solvent are distilled water, pH7.2.
3, tank top fermentation: with seed liquor with in the inoculum size of the 10% volume ratio access fermention medium, under 30 ℃, pH 6.8 ~ 7.2 conditions, 1. 0vvm that ventilate, rotating speed 300rpm cultivated 96 hours, got fermented liquid; Fermention medium forms: sucrose 50g/L; Yeast extract 0.5g/ L; Peptone 6.5g/ L; Potassium primary phosphate 2g/ L; Sal epsom 0.5g/ L; Iron(ic) chloride 0.02g/ L; Manganous chloride tetrahydrate 0.02g/ L, solvent are distilled water, pH7.2.
4, Crude polysaccharides yield determination: behind 10 times of the fermented liquid distilled water dilutings, 10000rpm removed bacterial sediment in centrifugal 20 minutes, added 3 times of ethanol, and 10000rpm is after centrifugal 20 minutes, and the survey dry weight is also calculated the flocculation agent yield more than 25g/L.
Embodiment 2: the extraction purification technique of exocellular polysaccharide flocculation agent PE-1
1, fermentation liquor pretreatment: fermented liquid 100L, centrifugal 20 minutes of 8000rpm gets supernatant;
2, protease treatment: supernatant constant temperature to 40 ℃, transfer pH7.0 with 100g/L NaOH, add the N,O-Diacetylmuramidase (200,000 U/g, Pang Bo is biological) of 50g and the Sumizyme MP (20,000 U/g, Pang Bo is biological) of 100g, insulation 2h;
3, the Sevag method is removed a small amount of residual foreign protein: the polysaccharide soln after the protease treatment mixes with the volume ratio of 5:1 with Sevag reagent (chloroform-propyl carbinol volume ratio is 5: 1 mixed solutions), jolting 30 minutes, centrifugal 30 minutes of 8000rpm, keep the Sevag reagent that water adds water volume 1/ 5 again, repeat said process, until water is without OD
280Absorption value;
4, precipitation, separate: add the 300L dehydrated alcohol in the fermented liquid after the above-mentioned processing (step 3 water), stir after 30 minutes, 4 ℃ of constant temperature spend the night;
5, centrifugal drying: 8000rpm got precipitation in centrifugal 20 minutes, vacuum-drying, the exocellular polysaccharide flocculation agent PE-1(that gets behind the purifying is designated hereinafter simply as PE-1).
Embodiment 3: the flocculation agent compositional analysis
1, flocculation agent hydrolysis: the PE-1 behind the 20mg purifying is dissolved in 2 M trifluoroacetic acid solution, and 105 ℃ are hydrolyzed 8 hours, and rotary evaporation is removed residual trifluoroacetic acid;
2, monose 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) derivatize: various monose standard substance and PE-1 hydrolysate are dissolved in respectively 200 μ L 0.3M NaOH solution, each adds 200 μ L 0.5M PMP methanol solutions, 70 ℃ were reacted 70 minutes, add the neutralization of 240 μ L 0.3M HCl solution after the cooling, add water to 2mL, each adds the residual PMP of 2mL chloroform extraction, re-extract 3 times;
3, derivatize monose HPLC separation determination: chromatographic column: common C18,250 mm * 4.6 mm, 5 μ m; Moving phase: 0.1M phosphate buffered saline buffer (pH 6.7)-acetonitrile (V: V is 83: 17); Column temperature: 25 ℃; Detect wavelength: 245nm; Flow velocity: 1 mL/ minute; Sampling volume: 10 μ L;
What 4, PE-1 formed determines: according to the measurement result of HPLC, determine that PE-1 is comprised of glucose, glucuronic acid, seminose, wood sugar, rhamnosyl, its ratio is 1:1:2:2:0.1.
Embodiment 4: Crude polysaccharides fermented liquid and PE-1 kaolin flocculating property are measured
1, kaolin suspension preparation: add 10mL kaolin suspension (4g/L) and 0.5mL 10g/L CaCl in the 25mL colorimetric cylinder
2Solution mixes;
2, flocculation treatment: add 0.25mL fermented liquid to be measured or the PE-1(25g/L of purifying), vigorous stirring (500rpm) 2 minutes, 50rpm stirred 5 minutes, static 5 minutes.
3, determination of activity: get supernatant liquor is determined at the 550nm place with spectrophotometer absorbancy, with inoculation medium or distilled water do not compare.
4, active calculating: the flocculating rate calculation formula is:
Flocculating rate=(B-A)/A * 100%
Wherein, A be testing sample in the absorbancy at 550nm place, B is that control sample is in the absorbancy at 550nm place.
5, through measuring, the flocculating rate of fermented liquid and PE-1 is respectively 85% and 87%.
Embodiment 5: Crude polysaccharides fermented liquid and PE-1 textile industry coloring matter removal capacity are measured
1, the preparation of textile industry coloring matter waste water: the pigment methylene blue (methylene blue) of commonly using in the textile industry and Cationic Red X-GRL (cationic Red X-GRL) are as pigment to be measured.Add respectively 10mg methylene blue or Cationic Red X-GRL in the 100ml deionized water;
2, flocculation treatment: add 0.1mL fermented liquid to be measured or the PE-1(25g/L of purifying), vigorous stirring under the mixed solution magnetic stirring apparatus (200rpm) 12 hours, centrifugal 5 minutes of 4000rpm.
3, determination of activity: supernatant spectrophotometric determination optical density value, its Methylene Blue is measured wavelength 370nm; Cationic Red X-GRL is measured wavelength 530nm.
4, active calculating: the pigment removal calculation formula is:
Decolouring activity=(C
0-C
e)/C
0* 100%
C wherein
0Be the starting point concentration of pigment in the waste water, C
eProcess the residual concentration of pigment in the rear sample for decolouring.
5, through measuring, fermented liquid and PE-1 pigment clearance rate see the following form:
| Pigment | Fermented liquid decolouring active (%) | PE-1 decolouring active (%) |
| Methylene blue | 65 | 65 |
| Positive red X-GRL | 68 | 70 |
Embodiment 6: Crude polysaccharides fermented liquid and the performance measurement of PE-1 removal of heavy metal ions
1, the preparation of effluent containing heavy metal ions: representative heavy metal Ion reagent CuSO
4, CoCl
2, AlCl
3, PbCl
2Respectively be made into 2mM solution with deionized water;
2, flocculation treatment: add respectively 0.4mL fermented liquid to be measured (25g/L) or PE-1 polysaccharide soln (25g/L) in the 100ml ionic liquid, 30 ℃, 200rpm stirred 24 hours, centrifugal 20 minutes of 10000rpm;
3, determination of activity: supernatant corresponding residual ion content in residual each heavy metal wastewater thereby of Atomic Absorption Spectrometry;
4, active calculating: removal of heavy metal ions rate calculation formula is:
Removal of heavy metal ions rate=(C
0-C
e)/C
0* 100%
C wherein
0Be the starting point concentration of corresponding ion in the waste water, C
eFor processing the residual concentration of rear sample intermediate ion.
5, through measuring, fermented liquid and PE-1 removal of heavy metal ions rate see the following form:
| Ion | Fermented liquid ion remaval rate (%) | PE-1 ion remaval rate (%) |
| Cu 2+ | 51 | 49 |
| Co 2+ | 42 | 40 |
| Pb 2+ | 25 | 58 |
| Al 3+ | 70 | 69 |
Embodiment 7: Crude polysaccharides fermented liquid and PE-1 actual industrial purification of waste water performance measurement
1, flocculation treatment: add 1mL CaCl in the 100ml trade effluent
2(10g/L), add respectively again the PE-1(25g/L of 3mL fermented liquid to be measured (25g/L) or 3mL purifying), 200rpm stirred 30 minutes, and 40rpm continues to stir 5 minutes, leaves standstill 10 minutes.
2, determination of activity: method is routinely measured the variation of turbidity, pigment and the chemical oxygen demand (COD) (COD) of supernatant;
3, active calculating: the clearance calculation formula is:
Clearance=(C
0-C
e)/C
0* 100%
C wherein
0Be the initial value of corresponding entry in the waste water, C
eFor processing residual value in the rear sample.
5, through measuring, fermented liquid and PE-1 pollutants removal rate see the following form:
| Ion | Fermented liquid clearance (%) | PE-1 clearance (%) |
| COD | 70 | 67 |
| Colourity | 80 | 85 |
| Turbidity | 88 | 90 |
Conclusion: the series bacillus of the present invention unpurified fermented liquid of (Paenibacillus sp.) ZD01 bacterial strain or its fermentation are by the exocellular polysaccharide flocculation agent PE-1 of purifying, when treatment of simulated waste water or actual trade effluent, all have higher outstanding turbid suspended particle flocculation activity, coloring matter decolouring activity and removal of heavy metal ions active, have the important application prospect in field of waste water treatment such as life, industry.
Claims (5)
1. a strain series bacillus (Paenibacillus sp.) ZD01 is preserved in Chinese Typical Representative culture collection center, address: China, Wuhan, Wuhan University, postcode 430072, preservation date: on November 20th, 2012, deposit number CCTCC No:M 2012463.
2. the application of series bacillus ZD01 as claimed in claim 1 in preparation Microbial exopolysaccharides flocculation agent.
3. the Microbial exopolysaccharides flocculation agent PE-1 that is produced by the described series bacillus ZD01 fermentation of claim 1, its skeleton is comprised of glucose, glucuronic acid, seminose, wood sugar, rhamnosyl, and its mol ratio is 1:1:2:2:0.1, molecular weight 7 * 10
6Dalton.
4. Microbial exopolysaccharides flocculation agent PE-1 as claimed in claim 3, it is characterized in that described Microbial exopolysaccharides flocculation agent PE-1 makes by the following method: series bacillus CCTCC No:M 2012463 is seeded to fermention medium, 28～32 ℃, 100 ~ 200rpm vortex shaking culture 3 ~ 4 days obtains containing the fermented liquid of described Microbial exopolysaccharides flocculation agent PE-1; Described fermention medium is composed as follows: sucrose 30 ~ 60g/L; Yeast extract 0.1 ~ 1.0g/ L; Peptone 5 ~ 8g/ L; Potassium primary phosphate 1 ~ 5g/ L; Sal epsom 0.1 ~ 1.0g/ L; Iron(ic) chloride 0.01 ~ 0.05g/ L; Manganous chloride tetrahydrate 0.01 ~ 0.05g/ L, solvent are distilled water, pH7.0 ~ 7.5.
5. the application of Microbial exopolysaccharides flocculation agent PE-1 claimed in claim 3 in sewage disposal.
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