CN104560819B - A kind of acinetobacter calcoaceticus and the microbial flocculant made from its fermentation - Google Patents
A kind of acinetobacter calcoaceticus and the microbial flocculant made from its fermentation Download PDFInfo
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Abstract
The present invention provides a kind of acinetobacter calcoaceticus FL 2 and its application.The deposit number for the acinetobacter calcoaceticus that the present invention is provided is CGMCC No.9380, with fast growth, stability is strong, flocculant yield is high, it is efficient the features such as, it can be used for the microbial flocculant of fermenting and producing microbial flocculant, particularly fermenting and producing for the coating cloud waste water that flocculates.
Description
Technical field
The invention belongs to microbial flocculant field.Specifically, being capable of fermenting and producing microorganism the present invention relates to one kind
The new acinetobacter calcoaceticus of flocculant, and by the obtained microbial flocculant of its fermentation.
Background technology
Paint spray booth is the paint booth for specializing in spraying liquid coating, is one of the key equipment in industrial coating workshop.With existing
For the development of coating process, spraying consumption increasingly increases, and thus also occurring spray painting in spraying process can not be coated on workpiece
And the problem of turn into " cross spray " coating cloud.
" cross and spray " coating cloud contains a large amount of volatile organic solvents for being unfavorable for health, severe contamination surrounding environment and sky
Gas.Coating is bonded to be difficult to clear up on the ground, can also be entered in recirculated water and be caused environmental pollution, and coating resource is wasted, and pair is set
Standby, pipeline also results in the harm such as clog.
Mode for indoor circulation water process of painting mainly includes mechanical curettage method, adsorption-flocculation partition method etc..Flocculation
Agent is that a class can make the floccule body precipitation into larger with flocculate suspended particles of the colloid in liquid or the material floated.Flocculation
Agent is broadly divided into three major types：Inorganic flocculating agent, organic flocculant and microbial flocculant.Inorganic flocculating agent mainly include aluminium salt and
Molysite.But the sludge that aluminium salt is produced is widely used in agricultural, Aluminium in Soil content is caused to raise, aluminium evil occurs in plant, so that shadow
Plant normal growth is rung, in addition it is dead；Enter the health that food chain has influence on human body with these crops simultaneously.Molysite is to gold
Category has corrosiveness, can also cause to carry color in processing water, and the iron of high concentration has not to health and ecological environment
Profit influence.And it is polyacrylamide to be obtained in organic flocculant most widely used, and monomer is even more to have strong nerve
Toxicity and carcinogenicity.
Microbial flocculant is the macromolecule metabolite for having flocculation activity that a class is produced by microorganism, and composition has sugared egg
In vain, polysaccharide, protein, cellulose and DNA etc..It is to utilize microbial technique, by microbial fermentations such as bacterium, fungies, is carried
Take, it is refined obtained from, new, efficient, nontoxic, non-secondary pollution the water treatment agent with Biodegradable and security.
Microbial flocculant is the focus studied both at home and abroad at present.
The content of the invention
In view of the above-mentioned problems, it is an object of the invention to provide a kind of new acinetobacter calcoaceticus, the acinetobacter calcoaceticus has growth speed
The characteristics of degree is fast, stability is strong, and the new microbial flocculant for being made and being capable of efficient flocculating coating cloud waste water that can ferment, because
This acinetobacter calcoaceticus of the invention has a good application prospect.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
On the one hand, the present invention provides a kind of acinetobacter calcoaceticus (Acinetobacter sp.) FL-2, the acinetobacter calcoaceticus kind
Deposit number is CGMCC No.9380.
Bacterial strain provided by the present invention is Acinetobacter lwoffii FL-2, in being the waste water for molding Co., Ltd from Jiangsu Korea Spro one
Isolated, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 25th, 2014
(abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica), preserving number is
CGMCC No.9380.It has following biological characteristics：On beef extract-peptone agar medium, 24h is cultivated at 37 DEG C,
Bacterium colony is produced, and is rendered as circle, milky, Edge divider is smooth, toughness.
Shown in the following sequence of 16S rDNA gene sequencing results of the bacterial strain：
SEQ ID NO:1
GGCTTACACATGCAGTCGAGCGGGGAAAAGTAGCTTGCTACTTTACCTAGCGGCGGACGGGTGAGTAAT
GCTTAGGAATCTGCCTATTAGTGGGGGACAACATCTCGAAAGGGATGCTAATACCGCATACGTCCTACGGGAGAAAG
CAGGGGACCTTCGGGCCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAG
GCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC
AGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTTTGGTTGT
AAAGCACTTTAAGCGAGGAGGAGGCTACCGAGATTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCG
GCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGCGCGT
AGGTGGCCAATTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTGGCTAGAGTAT
GGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCC
ATCTGGCCTAATACTGACACTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGT
AAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAG
TACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGC
AACGCGAAGAACCTTACCTGGTCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTTACATA
CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCT
TATTTGCCAGCGGGTTAAGCCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAA
GTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACCACGCGAGTGGA
TGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAAT
CGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCA
CCAGAAGTAGGTAGTCTAACCTTAGGGAGGACGCTACCACGTGCAGTTGC
It can cultivate or preserve in the following manner the acinetobacter calcoaceticus of the present invention：
(1) Nostoc commune Vanch is preserved using beef extract-peptone agar medium inclined-plane, and culture medium prescription is beef extract 3g, egg
White peptone 10g, NaCl 5g, agar 15-20g, distilled water 1000mL.
(2) laboratory fluids culture uses beef extract-peptone fluid nutrient medium, and culture medium prescription is beef extract 3g, albumen
Peptone 10g, NaCl 5g, distilled water 1000mL.
(3) bulk fermentation culture uses bulk fermentation culture medium, and culture medium prescription is glucose 20g, KH2PO42g,
K2HPO45g, (NH4)2SO40.2g, NaCl 0.1g, MgSO4﹒ 7H2O 0.5g, urea 0.2g, yeast extract 0.5g, distilled water
1000mL。
It is demonstrated experimentally that the acinetobacter calcoaceticus that the present invention is provided, which can ferment, produces the microbial flocculation of efficient flocculating coating cloud waste water
Agent, therefore, another aspect, the present invention provide described acinetobacter calcoaceticus and are preparing the microbial flocculant for the coating cloud waste water that flocculates
In purposes.The coating cloud waste water mainly includes a large amount of coating cloud particles, depending on paint vehicle and solvent, cosolvent used etc., bag
Containing such as polyacrylic resin, polyurethane, alkyd resin, aromatic solvent.In addition, it is demonstrated experimentally that in addition to flocculation,
The microbial flocculant that the present invention is provided can also have the effect of difficult degradation compound in degrading waste water, can effectively reduce useless
Water COD.Therefore, although the present invention is using term " flocculant ", it should be understood that the term also has the implication of " degradation agent ".
Another aspect, the present invention provide it is a kind of be used for flocculate and/or coating cloud waste water of degrading microbial flocculant, it is described micro-
Biological flocculant is fermented by acinetobacter calcoaceticus of the present invention to be made.
Preferably, the microbial flocculant is the zymotic fluid of acinetobacter calcoaceticus of the present invention, or the zymotic fluid
Centrifuged supernatant, or the material extracted from the centrifuged supernatant.For example, by the thing extracted from supernatant
Matter is dissolved into certain density solution in water, you can used directly as flocculant.
Preferably, the microbial flocculant is prepared by the method comprised the following steps：
In bulk fermentation culture medium, at 25-35 DEG C, with 120-200r/min rotating speed, the culture present invention's is motionless
Bacillus 60-80h, obtains zymotic fluid.
It is further preferred that the microbial flocculant is prepared by the method comprised the following steps：
(1) beef extract-peptone agar medium is used, the inclined-plane of the acinetobacter calcoaceticus is prepared；
(2) the inclined-plane picking colony prepared from step (1) is inoculated into constant temperature at beef extract-peptone fluid nutrient medium, 37 DEG C
Culture 24h obtains seed liquor, wherein the bacteria concentration in the seed liquor is 1.5 × 106Individual/mL；
(3) seed liquor for preparing step (2) is seeded to bulk fermentation culture medium according to 3% amount, in 30 DEG C, 160r/
72h is cultivated under conditions of min；
Optionally, methods described also includes：
(4) zymotic fluid for preparing step (3) obtains supernatant at 4 DEG C with 8000r/min centrifugations 20min；
(5) optionally, the absolute ethyl alcohol of precooling at 4 DEG C of two volumes, 4 are added in the supernatant prepared to step (4)
Stand overnight, then centrifuged under the same conditions with step (4), incline supernatant at DEG C, will be deposited in bag filter and dialyse
48h, is then freeze-dried.
The formula of beef extract-peptone agar medium is beef extract 3g, peptone 10g, NaCl in the step (1)
5g, agar 15-20g, distilled water 1000mL；The formula of beef extract-peptone fluid nutrient medium is beef extract in the step (2)
3g, peptone 10g, NaCl 5g, distilled water 1000mL；The formula of bulk fermentation culture medium is glucose in the step (3)
20g, KH2PO42g, K2HPO45g, (NH4)2SO40.2g, NaCl 0.1g, MgSO4﹒ 7H2O 0.5g, urea 0.2g, yeast extract
0.5g, distilled water 1000mL.The various culture mediums that the present invention is used are carrying out conventional sterilant using preceding.
Another aspect, the present invention provide it is a kind of be used for flocculate and/or coating cloud waste water of degrading microbial flocculation composition, institute
State microbial flocculation composition and include microbial flocculant of the present invention；
Preferably, the microbial flocculation composition also includes cation, and the cation is selected from Na+、K+、Mg2+、Ca2+、
Cu2+、Fe3+、Al3+In one or more.Cation can further enhance flocculation as auxiliary flocculant.It is preferred that
Ground, the cation is selected from Mg2+、Ca2+、Cu2+、Fe3+、Al3+In one or more, more preferably Ca2+。
Another further aspect, the present invention provides a kind of method flocculated and/or degrade coating cloud waste water, and methods described includes sending out this
Bright described acinetobacter calcoaceticus, which is seeded in coating cloud waste water, to be cultivated, or by microbial flocculant of the present invention and coating cloud waste water
Mixing, or microbial flocculation composition of the present invention is mixed with coating cloud waste water.
Further aspect, the present invention also provides the acinetobacter calcoaceticus or microbial flocculant or microbial flocculation composition
Purposes in flocculating and/or degrading coating cloud waste water.
Coating cloud waste water suspension concentration is high and contains the high molecular polymer of substantial amounts of difficult degradation, and its comparison of ingredients is complicated.
The solid waste that domestic and international paint line flocculation is produced at present is easily caused secondary dirt mainly using the method processing such as landfill, burning
Dye；Also there are indivedual producers using coagulating sedimentation-chemical oxidization method to cut down solid waste.Due to the use of hydrogen peroxide in the above method
Costly, this method is suitable only for the factory that waste water output is small, concentration is low；And the strong oxidizing property and corrosivity of hydrogen peroxide, deposit
In potential safety hazard.Although it can make organic matter degradation in waste water to discharge standard, on the whole, the place for coating cloud waste water
Reason still has many deficiencies：Processing equipment is more complicated, and floor space is wider, and one-time investment is very high.Divided using microbial method
The macromolecule organic in coating cloud waste water is solved, relevant document report is not yet seen, mainly due to most of high molecular polymers
Biodegradability is poor, strand is difficult to be broken in the presence of microorganism, and therefore, it is difficult to degrade.
In this regard, the invention provides a kind of new acinetobacter calcoaceticus.The acinetobacter calcoaceticus is isolated in waste water, with growth
The characteristics of speed is fast, stability is strong, and the new microbial flocculant for being made and being capable of efficient flocculating coating cloud waste water that can ferment.
It is demonstrated experimentally that for coating cloud waste water, the obtained zymotic fluid of Acinetobacter lwoffii FL-2 fermentations of the invention can realize that 90% is left
Right flocculating effect, flocculating effect is very notable.Also, the thus obtained nontoxic nothing of zymotic fluid of Acinetobacter lwoffii FL-2 fermentations
Evil, the problem of reducing caused by chemical type flocculant that solid waste amount is big, bring secondary pollution, production cost is low, it is ensured that environment
Safety.Moreover, from the point of view of the amplitude of the coating cloud waste water COD reduction before and after degraded, the microorganism has to dissolved organic matter in water
Preferable degradation effect；Also, before and after to degraded in terms of the mass spectrometry results of paint slag, the composition of paint slag there occurs very big change
Become, it is this to change mainly as caused by the microorganism.Because the main component in paint slag is macromolecule filming material, therefore can
To infer that the acinetobacter calcoaceticus has certain degradation capability to polymer substance in paint slag.In summary, Luo Fei of the invention is not
Lever bacterium FL-2 can flocculate coating cloud waste water, and can reduce the load to environment, have a good application prospect.
Brief description of the drawings
Hereinafter, specific embodiments of the present invention are described in detail with reference to accompanying drawing, wherein：
Fig. 1 shows the colonial morphology of acinetobacter calcoaceticus of the present invention.
Fig. 2 shows the infrared spectrogram of obtained dried microorganism flocculant in the embodiment of the present invention 2.
Fig. 3 shows influence of the zymotic fluid dosage to flocculating rate in embodiment 3.
Fig. 4 shows the Contrast on effect of acinetobacter calcoaceticus in embodiment 3 to (left side) before coating cloud waste water flocculating (right side) afterwards.
Fig. 5 shows influence of the temperature to flocculating rate in embodiment 3.
Fig. 6 shows influence of the various cations to flocculating rate in embodiment 3.
7a in Fig. 7 show in embodiment 2 obtained dried microorganism flocculant different amplification (1000 times and
10000 times) under scanning electron microscope (SEM) photograph；7b is shown in embodiment 2 in obtained dried microorganism flocculant (left side) and embodiment 3
Flocculated microbial flocculant (right side) the dried scanning electron microscope (SEM) photograph (2000 times) painted.
Fig. 8 shows the change of coating cloud waste water COD in embodiment 4.
Fig. 9 show in embodiment 4 paint slag degraded before and degraded 30 days after mass spectrographic change.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these are changed and replacement is each fallen within protection scope of the present invention.
Following culture medium is used in embodiment：
(1) beef extract-peptone agar medium, culture medium prescription is beef extract 3g, peptone 10g, NaCl 5g, agar
15-20g, distilled water 1000mL.
(2) beef extract-peptone fluid nutrient medium, culture medium prescription is beef extract 3g, peptone 10g, NaCl 5g, distillation
Water 1000mL.
(3) bulk fermentation culture medium, culture medium prescription is glucose 20g, KH2PO42g, K2HPO45g, (NH4)2SO40.2g, NaCl 0.1g, MgSO4﹒ 7H2O 0.5g, urea 0.2g, yeast extract 0.5g, distilled water 1000mL.
The calculating of coating cloud waste water flocculating method and flocculating rate：
Coating cloud is calculated using the change of absorbance before and after ultraviolet-visible spectrophotometer method measure coating cloud wastewater treatment
Flocculating rate.Ultraviolet spectra full wavelength scanner is carried out to coating cloud waste water first, finds in its characteristic absorption peak, the present invention and is defined as ripple
Long 520nm.This wavelength is set to the setting wavelength on spectrophotometer, the change of 520nm absorbances before and after the processing is determined, from
And calculate flocculating rate.Calculation formula is as follows：
Wherein, S1、S2The 520nm absorbances before and after coating cloud wastewater treatment are represented respectively.
The calculating of kaolin flucculation process and flocculating rate：
4g/L kaolin solution 50mL is taken, pH is to 8 or so for regulation, adds acinetobacter calcoaceticus zymotic fluid 8mL, adds
1%CaCl2Solution 2mL is shaken up as auxiliary flocculant, and flocculating effect is observed after standing 10min.The liquid of 2cm under liquid level is taken,
Its absorbance in 550nm (maximum absorption band) is determined, is changed according to the absorbance for adding flocculant process and blank
To flocculating rate.Calculation formula is as follows：
Kaolin flocculating rate=(A-B)/A × 100%
A and B represents blank with adding the 550nm absorbances after flocculant process respectively.
Embodiment 1
1st, the isolation and purification of acinetobacter calcoaceticus of the invention
The acinetobacter calcoaceticus of the present invention is separated in being the waste water for molding Co., Ltd from Jiangsu Korea Spro one using plate streak
Arrive.
The separation of flocculated bacteria：The waste water fetched is inoculated into beef extract-peptone fluid nutrient medium, 37 DEG C incubated
24h.Pick a small amount of bacterium solution with transfer needle to rule on beef extract-peptone agar plate, timing observation colony growth situation.So
Plate streak is used afterwards, and picking single bacterium colony is transferred on beef extract-peptone agar slant and saved backup respectively.
The screening of the acinetobacter calcoaceticus of producing microbial flocculant：Screened by the flocculating effect to aqueous suspension ofkaolin.
(1) primary dcreening operation：Each single bacterium colony chosen is inoculated into bulk fermentation culture medium, 160r/min is cultivated at 30 DEG C
72h, obtains zymotic fluid.
50mL 4g/L aqueous suspension ofkaolin is added in each 150mL triangular flasks, 8mL zymotic fluids is added, quickly shakes
Swing and 10min is stood after 1min, then observe the flocculation situation of aqueous suspension ofkaolin in each bottle.According to the number and size of flco
To decide whether to retain the bacterium.The bacterial strain for remaining with flocculation activity carries out secondary screening hereafter again, eliminates the bacterial strain without flocculation activity.
(2) secondary screening：The strain with flocculation that primary dcreening operation is obtained is isolated and purified again, and bulk fermentation is inoculated in respectively
In culture medium, 160r/min cultivates 72h at 30 DEG C, obtains zymotic fluid.
Secondary screening is carried out according to kaolin flucculation process, kaolin flocculating rate is calculated, flocculating rate is more than 60% reservation, on the contrary
Give up.Select flocculating rate highest bacterial strain repeatedly to be passed on, screening obtains the stable bacterial strain FL-2 of flocculating property, is used as this hair
Bright aimed strain.
2nd, strain idenfication
(1) Microbiological Characteristics
On beef extract-peptone agar medium flat board, 24h is cultivated at 37 DEG C, bacterium colony is presented circular, raised, milky white
Color, Edge divider, smooth, toughness.Colonial morphology is shown in Fig. 1.
(2) physio-biochemical characteristics
After testing, the bacterial strain Gram's staining is feminine gender, and V-P experiments are positive, and methyl red test is negative, indole test sun
Property, gelatin liquefaction test feminine gender, the Starch Hydrolysis experiment positive, the Nagler's reaction positive, oxidase test feminine gender, urine enzyme test
The positive, catalase test is positive, and nitrate reduction test is positive, and nitrite reduction test is negative, the examination of glucose oxidative fermentation
Test as oxidized form, production acid, sterile film is formed.
(3) molecular biological characteristic
The following SEQ ID NO of 16S rDNA gene sequencing results of the bacterial strain:Shown in 1：
GGCTTACACATGCAGTCGAGCGGGGAAAAGTAGCTTGCTACTTTACCTAGCGGCGGACGGGTGAGTAAT
GCTTAGGAATCTGCCTATTAGTGGGGGACAACATCTCGAAAGGGATGCTAATACCGCATACGTCCTACGGGAGAAAG
CAGGGGACCTTCGGGCCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAG
GCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC
AGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTTTGGTTGT
AAAGCACTTTAAGCGAGGAGGAGGCTACCGAGATTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCG
GCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGCGCGT
AGGTGGCCAATTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTGGCTAGAGTAT
GGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCC
ATCTGGCCTAATACTGACACTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGT
AAACGATGTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAG
TACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGC
AACGCGAAGAACCTTACCTGGTCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTTACATA
CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCT
TATTTGCCAGCGGGTTAAGCCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAA
GTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCTACCACGCGAGTGGA
TGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAAT
CGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCA
CCAGAAGTAGGTAGTCTAACCTTAGGGAGGACGCTACCACGTGCAGTTGC
It is accredited as Acinetobacter lwoffii (Acinetobacter lwoffii) FL-2.In beef extract-peptone agar culture
Obtained Acinetobacter lwoffii is preserved in base inclined-plane, the common micro- life of China Committee for Culture Collection of Microorganisms is preserved in
Thing center, preserving number is CGMCC No.9380.
Embodiment 2
Strain seed liquor culture：Picking is few from Acinetobacter lwoffii FL-2 beef extract-peptone agar medium inclined-plane
Bacterium is measured, is seeded in beef extract-peptone fluid nutrient medium, incubated 24h, as seed liquor at 37 DEG C, wherein the seed
Bacteria concentration in liquid is 1.5 × 106Individual/mL.
A large amount of liquid fermentations：Seed liquor is inoculated into bulk fermentation culture medium to (250mL triangles are bottled in 3% ratio
Enter 100mL fermentation mediums) in, shaking table 160r/min cultivates 72h at 30 DEG C.
By obtained zymotic fluid on high speed freezing centrifuge at 4 DEG C with 8000r/min centrifuge 20min, obtain supernatant
Liquid, adds in supernatant and is stood overnight at the absolute ethyl alcohol of precooling at 4 DEG C of two volumes, 4 DEG C, then in identical centrifugal condition
Lower centrifugation, incline supernatant, will be deposited in bag filter after the 48h that dialyses, is dried to obtain on freeze drier, is that the Lip river is luxuriant and rich with fragrance not
Lever bacterium FL-2 microbial flocculant sterlings, are characterized for infrared analysis and ESEM.Infrared spectrogram is shown in Fig. 2, scanning electricity
Mirror figure is shown in Fig. 7 a.
Embodiment 3
Present embodiments provide flocculating experiment of the microbial flocculant to coating cloud waste water of Acinetobacter lwoffii FL-2 generations.
1st, the preparation of coating cloud waste water is simulated
Daily plastic beverage bottle is taken, running water 100mL is added, pH to 12 is adjusted with 1mol/L NaOH solution, then
6 blob of paint are instilled with a suction pipe, are acutely shaken up, what is obtained is simulation coating cloud waste water.
2nd, the preparation of microbial flocculation agent solution and auxiliary cationic solution
As described in Example 2, the zymotic fluid that shaking table 160r/min at 30 DEG C of the present invention cultivates 72h is prepared, directly as wadding
The medicament of solidifying experiment.
In addition, preparing 1% respectively contains Na+、K+、Mg2+、Ca2+、Cu2+、Fe3+, Al3+NaCl, KCl, MgSO4、
CaCl2、CuCl2、FeCl3、Al2(SO4)3Solution is used as auxiliary cationic solution.
3rd, experimental design and result
First group：Influence of the zymotic fluid dosage to flocculating rate
Zymotic fluid 5mL, 10mL, 15mL, 20mL, 25mL are separately added into the bottle of the coating cloud waste water containing preparation, is shaken up, it is quiet
10min is put, flocculating effect is observed.
As a result show, when the dosage that ferments is 10mL, flocculating rate can reach up to 89.1%.As a result it is shown in Fig. 3.Contain
Waste water situation in the bottle of coating cloud waste water before and after flocculation is shown in Fig. 4.
Second group：Influence of the temperature to flocculating rate
By 10mL zymotic fluids respectively at 20 DEG C, 35 DEG C, 60 DEG C, 80 DEG C, 30min is kept in 100 DEG C of water-baths, then adds and contains
In the bottle of the coating cloud waste water of preparation, shake up, stand 10min, observe flocculating effect.
As a result show, when flocculant is after 35 DEG C are handled, flocculating rate is up to 92.4%, is as a result shown in Fig. 5.
3rd group：Aid in influence of the cation to flocculating rate
Various auxiliary cationic solution 2mL are first separately added into the bottle of the coating cloud waste water containing preparation, zymotic fluid is added
10mL, observes influence of the various cationic solutions additions to flocculating rate.
As a result show, cation Mg2+、Ca2+、Cu2+、Fe3+、Al3+More other cations have the rush flocculation become apparent to make
With, and Ca ought be added2+During solution, flocculating rate reaches highest 90.2%.As a result it is shown in Fig. 6.
It will be dried after microbial flocculant precipitation after flocculation, compared with the drying flocculant before the flocculation of embodiment 2, two
Person's scanning electron microscope (SEM) photograph is shown in Fig. 7 b.
Embodiment 4
Present embodiments provide degradations of the Acinetobacter lwoffii FL-2 to coating cloud waste water.
1st, the measure of coating cloud waste water COD
Using 3 first groups of coating cloud waste water of embodiment, using waste water continuous several days before flocculating and after flocculation as sample, room temperature
Under, the liquid at 2cm below liquid level is taken respectively, the COD in waste water is determined using potassium dichromate method, observes its change.
The change of coating cloud waste water COD is as shown in Figure 8.The Acinetobacter lwoffii FL-2 of a part is still suffered from after flocculation in waste water,
And it can be seen that the COD in waste water is gradually reduced with the passage of time, illustrate flocculated bacteria present in waste water to useless
Soluble organic matter has certain degradation in water, and degradation effect is preferable.
2nd, the composition measurement of slag is painted after flocculating
Paint slag after first group of flocculation in embodiment 3 is picked up, excessive moisture is filtered off, it is standby.
At ambient temperature, with sawdust with painting slag according to mass ratio 1:1 ratio is mixed as compost material, using implementation
Load closed appearance after the seed liquor of example 2, the Acinetobacter lwoffii FL-2 for being inoculated with 5% (according to bacterium solution and the mass ratio of compost material)
In device, place 30 days.
Respectively by closed preceding paint slag mixture and it is closed 30 days after mixture xylene soluble, centrifugation, mistake
Filter 2-3 times, be diluted to after certain proportion, with Finnigan companies of U.S. LCQ advantageI types electrospray mass spectrometer, ESI from
Component, composition transfer of the detection paint slag specimen product before and after degraded.
Mass spectrogram is as shown in Figure 9 before and after degraded.As can be seen from the figure m/z=511.18 peak is disappeared, m/z=
121.08 peak do not change.It is 510 molecule (molecular weight=peak value -1) that 511.18 peak, which represents molecular weight,.In addition, after degraded
Molecule of many molecular weight 176,254,314 etc. is also had more.It may determine that the flocculant of the present invention to paint from the change at peak
Chemical substance in slag generates certain degradation.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deformed, and without departing from the spirit of the present invention, all should belong to the model of appended claims of the present invention
Enclose.
Claims (13)
1. a kind of acinetobacter calcoaceticus (Acinetobacter sp.) FL-2, it is characterised in that the deposit number of the acinetobacter calcoaceticus is
CGMCC No.9380。
2. acinetobacter calcoaceticus described in claim 1 prepare be used to flocculate and/or the microbial flocculant for coating cloud waste water of degrading in
Purposes.
3. it is a kind of be used for flocculate and/or coating cloud waste water of degrading microbial flocculant, it is characterised in that the microbial flocculant
It is made as the acinetobacter calcoaceticus fermentation described in claim 1.
4. microbial flocculant according to claim 3, it is characterised in that the microbial flocculant is claim 1
The zymotic fluid of described acinetobacter calcoaceticus, or the zymotic fluid centrifuged supernatant；
Wherein, the zymotic fluid is prepared by the method comprised the following steps：
In bulk fermentation culture medium, at 25-35 DEG C, with 120-200r/min rotating speed, cultivate described in claim 1 not
Lever bacterium 60-80h, obtains zymotic fluid.
5. microbial flocculant according to claim 4, it is characterised in that the microbial flocculant is by including following step
It is prepared by rapid method：
(1) beef extract-peptone agar medium is used, the inclined-plane of the acinetobacter calcoaceticus is prepared；
(2) the inclined-plane picking colony prepared from step (1) is inoculated into incubated at beef extract-peptone fluid nutrient medium, 37 DEG C
24h obtains seed liquor, wherein the bacteria concentration in the seed liquor is 1.5 × 106Individual/mL；
(3) seed liquor for preparing step (2) is seeded to according to seed liquor and the volume ratio of bulk fermentation culture medium for 3% amount
Bulk fermentation culture medium, 72h is cultivated under conditions of 30 DEG C, 160r/min.
6. microbial flocculant according to claim 5, it is characterised in that methods described also includes：
(4) zymotic fluid for preparing step (3) obtains supernatant at 4 DEG C with 8000r/min centrifugations 20min.
7. microbial flocculant according to claim 6, it is characterised in that methods described also includes：
(5) add and stood at the absolute ethyl alcohol of precooling at 4 DEG C of two volumes, 4 DEG C in the supernatant prepared to step (4)
At night, then centrifuged under the same conditions with step (4), incline supernatant, will be deposited in bag filter the 48h that dialyses, Ran Houleng
It is lyophilized dry.
8. the microbial flocculant according to any one of claim 5 to 7, it is characterised in that beef in the step (1)
The formula of cream peptone agar medium is beef extract 3g, peptone 10g, NaCl 5g, agar 15-20g, distilled water 1000mL；
The formula of beef extract-peptone fluid nutrient medium is beef extract 3g, peptone 10g, NaCl 5g, distilled water in the step (2)
1000mL；The formula of bulk fermentation culture medium is glucose 20g, KH in the step (3)2PO42g, K2HPO45g, (NH4)2SO40.2g, NaCl 0.1g, MgSO4, 7H2O 0.5g, urea 0.2g, yeast extract 0.5g, distilled water 1000mL.
9. it is a kind of be used to flocculate and/or coating cloud waste water of degrading microbial flocculation composition, it is characterised in that microorganism wadding
Solidifying composition includes the microbial flocculant according to any one of claim 3 to 8.
10. microbial flocculation composition according to claim 9, it is characterised in that the microbial flocculation composition is also
Comprising cation, the cation is selected from Na+、K+、Mg2+、Ca2+、Cu2+、Fe3+、Al3+In one or more.
11. microbial flocculation composition according to claim 10, it is characterised in that the cation is Ca2+。
12. a kind of method flocculated and/or degrade coating cloud waste water, it is characterised in that methods described is included described in claim 1
Acinetobacter calcoaceticus be seeded in coating cloud waste water cultivate, or by the microbial flocculant any one of claim 3 to 8 with
Coating cloud waste water is mixed, or the microbial flocculation composition any one of claim 9 to 11 is mixed with coating cloud waste water.
13. microbial flocculant any one of acinetobacter calcoaceticus or claim 3 to 8 described in claim 1 or
Purposes of the microbial flocculation composition in flocculating and/or degrading coating cloud waste water any one of claim 9 to 11.
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