CN103045510A - High-yield cellulase bacillus licheniformis with flocculation and application of same - Google Patents
High-yield cellulase bacillus licheniformis with flocculation and application of same Download PDFInfo
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention relates to high-yield cellulase bacillus licheniformis with flocculation and application of the high-yield cellulase bacillus licheniformis. The invention discloses a strain of Bacilluslicheniformis Bl-2 with a preservation number of CCTCC (China Center For Type Culture Collection) No.M 2012458. A water quality modifier can be separately prepared from the strain or can be prepared by compounding the strain with other water purification microorganism strains according to a certain proportion. An application effect test shows that the water quality modifier can effectively remove waste components in aquaculture water or other polluted water, has an excellent degradation effect on cellulase precipitates, can obviously improve water quality of the polluted water, obviously improves visibility of a water ruler, cannot cause secondary pollution and has good development and application prospects in the field of wastewater treatment.
Description
Technical field
The invention belongs to microbial technique and sewage treatment area, but be specifically related to a strain High Cellulase Production and have the Bacillus licheniformis of throwing out, and the application of this bacterial strain on culture fishery and other polluted-water water quality regulation.
Background technology
A most important link is how effectively to carry out water quality regulation in the culture fishery.In the aquaculture process, because the problems such as body eutrophication, Water quality ammonia nitrogen and nitrite that feed deposition etc. causes exceed standard, green alga hypertrophy, water body is gone from bad to worse, and the aquatic animal disease frequent occurrence has become the bottleneck that hinders China's culture fishery development.
At present, utilize flocculation agent that the aquaculture water is carried out water quality regulation and obtained effective progress.But inorganic salts flocculation agent consumption is large, easily environment is caused secondary pollution; The residue of organic synthesis high score subclass flocculation agent is difficult for degraded, and monomer whose has stronger toxic action; Microbial flocculant is that a class is by the special polymer meta-bolites that is difficult for the coagulation sedimentations such as solid suspended particle, somatic cells and colloidal particle of degraded in the made sewage of microorganisms, these polymer meta-bolitess comprise polysaccharide, protein, ester class, DNA class etc., and certified microbial flocculant major part belongs to polysaccharose substance so far.Microbial flocculant is a kind of water conditioner with efficient, nontoxic, non-secondary pollution of Biodegradable and security, because it is with low cost, promotes rapidly in aquaculture and is widely used.The microorganism that can produce the flocculation composition mainly derives from soil, is more and more paid close attention at present and approves, on the road that replaces inorganic salts and high score subclass flocculation agent, microbial flocculant is being played the part of very important role.
Yet, compare with non-cellulose waste component (comprising carbohydrate, fat and protein etc.), cellulose waste composition in the aquaculture water, especially the microorganism that the Cellulose precipitates after flocculating is difficult to be flocculated in the agent is usually thoroughly degraded, cause the Mierocrystalline cellulose in the water body constantly to be piled up, the flocculation purifying water effect is not good.
Summary of the invention
Defects for existing microbial flocculant exists the invention provides High Cellulase Production Bacillus licheniformis and application thereof that a strain has throwing out.Bacterial strain of the present invention has the ability of good throwing out and High Cellulase Production, it can effectively be removed waste component in aquaculture water and other polluted-waters separately or with the composite water conditioner of making of other other water purification microorganism strains, and purifying water effect is good.
Technical scheme of the present invention is as follows:
The invention provides High Cellulase Production Bacillus licheniformis (Bacilluslicheniformis) BL-2 that a strain has throwing out, deposit number is CCTCC No.M 2012458.
The invention provides described Bacillus licheniformis BL-2, the application of CCTCC No.M 2012458 on aquaculture and polluted-water water quality regulation is that this Bacillus licheniformis BL-2 is independent or mix rear as the water quality improvement of water conditioner for aquaculture and polluted-water with other water purification microorganism strains.
Its further technical scheme is:
Described water purification microorganism strains is selected from subtilis, Rhodopseudomonas palustris, plant lactobacillus, the yeast saccharomyces cerevisiae one or more.
Described water conditioner is the viable bacteria pulvis.
Useful technique effect of the present invention is:
But the invention discloses a strain High Cellulase Production and have Bacillus licheniformis (Bacillus licheniformis) the BL-2(deposit number of throwing out: CCTCC No.M 2012458).This bacterial strain itself has good throwing out (flocculating rate reaches 91%) on the one hand, and its meta-bolites can make inter-adhesive the accumulating in of organic or inorganic thing particulate in the polluted-water generate together zoogloea, finally forms flocks, makes water body purification; On the one hand because this bacterial strain has the ability (cellulase activity reaches 137.2U/mL) of High Cellulase Production, can collaborative above-mentioned flocculating effect thoroughly degraded and remove cellulose waste composition in the water body and the Cellulose precipitates after the flocculation, play splendid flocculation and purifying water effect; With bacterial strain of the present invention separately or with other water purification microorganism strains composite water conditioner that can be made into by a certain percentage, the effect test shows, this water conditioner can significantly improve the water quality of aquaculture water and polluted-water, obviously improve above-mentioned water body scale visibility, can not cause secondary pollution, have preferably development and application prospect at sewage treatment area.
High Cellulase Production Bacillus licheniformis (Bacilluslicheniformis) BL-2 with throwing out provided by the present invention, be preserved in Chinese Typical Representative culture collection center (CCTCC) on November 14th, 2012, deposit number: CCTCC M 2012458, preservation address: China, Wuhan, Wuhan University.This bacterial strain is hereinafter to be referred as Bacillus licheniformis BL-2CCTCC M 2012458.
Description of drawings
Fig. 1 is bacterial strain enlarged culturing process flow sheet in the embodiment of the invention 1.
Embodiment
Below in conjunction with embodiment the specific embodiment of the present invention is further described, following examples are convenient to understand better the present invention, but do not limit the present invention.
Screening, separation and the enlarged culturing of embodiment 1 Bacillus licheniformis BL-2 CCTCC M 2012458
Pollute collection bed mud sample 2g the pond from Jiangsu Province's Yixing City, add the 98mL stroke-physiological saline solution, in 37 ℃ of shaking culture 20min on shaking table, shaking speed 120rpm; (medium component by weight percentage ratio is as follows: CMC 5g/l to draw the substratum of 1mL supernatant liquor adding take Xylo-Mucine CMC as sole carbon source, ammonium sulfate 1g/l, sodium-chlor 5g/l, magnesium sulfate heptahydrate 0.5g/l, magnesium sulfate monohydrate 0.1g/l, all the other compositions are water) in, shaking culture 6h continued; Get cultivate 10000 times of pregnant solution dilutions after, (medium component by weight percentage ratio is as follows: CMC 5g/l to be applied to plate culture medium take carboxymethyl cellulose (CMC) as sole carbon source with spreading rod, ammonium sulfate 1g/l, sodium-chlor 5g/l, magnesium sulfate heptahydrate 0.5g/l, magnesium sulfate monohydrate 0.1g/l, agar 15g/l, all the other compositions are water), cultivate 48h in 37 ℃ in constant incubator, according to the diameter of growth transparent circle that bacterium colony produces on the substratum, filtering out a strain can be than the bacterial strain of good utilisation CMC, be numbered BL-2, picking list bacterium colony also is seeded to the LB slant culture and carries out enlarged culturing after based on 37 ℃ of overnight incubation.
Described enlarged culturing method is as follows:
1. seed culture
Go bail for and have the BL-2 on the LB inclined-plane and place 100 ℃ of water-bath thermal treatment 10min, take out and be cooled to rapidly room temperature, inoculate to the 500mL triangular flask that the 50mL seed culture medium is housed, in 37 ℃ of shaking culture 24h, shaking speed 150rpm obtains viable count 1 * 10
7～3 * 10
10The activated seed liquid of individual/mL; Above-mentioned seed culture based component is as follows: peptone 10g/L, sodium-chlor 10g/L, yeast powder 5g/L, and with the distilled water preparation, until all regulating pH value to 7.5～7.6 after the dissolving, again to the agar powder that wherein adds 15g/L, in 121 ℃ of sterilization 20min.
2. liquid fermentation and culture
Above-mentioned activated seed liquid is seeded to the 200L fermentor tank that liquid fermentation medium is housed, fermentation culture 8h, get again primary fermentation liquid and be seeded to 5000L fermentor tank continuation fermentation culture 12～36h that liquid fermentation medium is housed, keep air flow 1: 2vvm in the fermenting process, mixing speed 200rpm, pressure 1kg/m
2, 37 ℃ of leavening temperatures are 6.0 with NaOH and HCl solution controlled fermentation pH value; Aforesaid liquid fermentation culture based component is as follows: glucose 20g/L, yeast powder 10g/L, peptone 15g/L, potassium primary phosphate 5g/L, sal epsom 1g/L, and with the distilled water preparation, pH 7.0; It is complete to ferment, and obtains gemma and adds up to 2 * 10
11～2 * 10
12The fermented liquid of cfu/mL.
3. the collection of gemma pulvis
With above-mentioned fermented liquid process centrifugal (rotating speed 5000rpm) and Plate Filtration, or use wheat bran, rice bran to adsorb to remove fermented liquid; Collect thalline, the spray-dried gemma pulvis (spore pulvis) that namely gets.
The concrete technology flow process is referring to Fig. 1.
The evaluation of embodiment 2 Bacillus licheniformis BL-2 CCTCC M 2012458
1. bacterium colony morphological features
BL-2 forms irregular, the trichoid circular bacterium colony in edge after overnight incubation on the LB solid medium, lawn is canescence, and drying is opaque, spreads shape; Be Gram-positive with observation by light microscope, shaft-like, size is about 0.7～0.8 μ m * 2～3 μ m, and without pod membrane, amphitrichous can move, and it is shaft-like that nourishing body is shaped as rod, forms gemma and is shaped as ellipse, slightly biased length.
2. physiological and biochemical property
Embodiment 1 separating obtained bacterial strain is carried out Physiology and biochemistry to be identified.Its major physiological biochemical character is referring to table 1.
Table 1
| Test subject | The result |
| The gemma shape | Ellipse, slightly biased length |
| Thalli morphology | Rod is shaft-like |
| Caseinhydrolysate | + |
| Hydrolyzed starch | + |
| The VP reaction | + |
| Gelatin hydrolysate | + |
| D-Glucose produces acid | + |
| L-arabinose produces acid | + |
| Citrate trianion | + |
| The D-wood sugar produces acid | + |
| Propionic salt | + |
| N.F,USP MANNITOL produces acid | + |
| The phenylalanine defatting enzyme | + |
| The glucose aerogenesis | - |
| Tyrosine hydrolysis | - |
| Indoles | + |
| Catalase | + |
| Nitrate reduction | + |
Annotate: "-" expression is negative; "+" expression is positive.
3. genetics characteristics
Adopt 16S rDNA order-checking to carry out the taxonomy Molecular Identification.Utilize the 16SrDNA of round pcr amplification BL-2, wherein, upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 ', downstream primer: 5 '-AAGGAGGTGATCCAGCCGC-3 '.Pcr amplification product has 99% homology between this 16SrDNA base sequence and the Bacillus licheniformis type strain Bacillus licheniformis DSM 13 through sequencing analysis as can be known.
In conjunction with above-mentioned morphological specificity, physiological and biochemical property and heredity feature, can identify that this BL-2 bacterial strain belongs to Bacillus licheniformis (Bacillus licheniformis).
The mensuration of embodiment 3 Bacillus licheniformis BL-2 CCTCC M 2012458 cellulase-producing abilities
The go bail for Bacillus licheniformis BL-2 that has the LB slant medium and be seeded to the liquid culture medium, the liquid culture medium is loaded on the 250mL triangular flask, liquid amount 15mL, in 37 ℃ of fermentation 48h, it is complete to ferment, and gets fermented liquid; The percentage ratio meter is as follows by weight for aforesaid liquid culture medium composition: 4% maltose, 1% peptone, 0.5% sodium-chlor, 0.2% dipotassium hydrogen phosphate, all the other compositions are water); With the cellulase assay method (the CMC method, concrete operations are referring to such as Publication about Document: Mabdels, M.﹠amp; Weber, J.1969.Theproduction of cellulases, Cellulases and their applications.Gould, R.F. (ed.), Amer.Chem.Soc., Washington.p.391.) measure fermented liquid cellulase content, its enzyme work can reach 53.4U/mL after testing.
The Bacillus licheniformis BL-2 that has the LB slant medium and be seeded to the 7.5L liquid fermentation tank that liquid fermentation medium the is housed 32h that ferments goes bail for, keep air flow 1: 1vvm in the fermenting process, mixing speed 700rpm, 37 ℃ of leavening temperatures, it is complete to ferment, and gets fermented liquid; Measure fermented liquid cellulase content with cellulase assay method (the same), its enzyme work can reach 137.2U/mL after testing.
Above-mentioned test-results shows that Bacillus licheniformis BL-2 CCTCC M 2012458 of the present invention has the ability of High Cellulase Production, cellulose components in the water body that can be used for degrading.
The mensuration of embodiment 4 Bacillus licheniformis BL-2 CCTCC M 2012458 flocculation abilities and the application on aquaculture and polluted-water water quality regulation
The flocculation ability measuring method is as follows:
Test group: the kaolin suspension liquid that in the 50mL colorimetric cylinder, adds final concentration 5g/L, 1% calcium chloride solution 1mL, embodiment 1 gained fermented liquid 0.5mL(sample), regulate pH value to 6.0～7.0, be settled to 50mL with distilled water, measure OD in 721 visible spectrophotometers
550nmBlank: distilled water.The flocculating rate calculation formula is: (OD
Contrast-OD
Sample)/OD
Contrast* 100%.After testing, take the kaolin suspension liquid as contrast, test group places that flocculating rate reaches 91% after 4 days.
According to 1000cfu/m
3The gemma quantity of water body is sprayed on Jiangsu Province's Yixing City urban refuse pollution water body with embodiment 1 made gemma pulvis, observes water quality situation and the surveyors' staff visibility (cm) of this water body of 0～4d, and the result is referring to table 2.
Table 2
| Time | 0d | 1d | 2d | 3d | 4d |
| The scale visibility | 5cm | 6.2cm | 12cm | 17.9cm | 19cm |
Gemma pulvis with embodiment 1 made Bacillus licheniformis BL-2, subtilis viable bacteria pulvis (available from Wuhan Kenuo Biology Science ﹠ Technology Co., Ltd.), (former bacterial strain is available from Chinese common micro-organisms culture presevation administrative center for Rhodopseudomonas palustris viable bacteria pulvis, deposit number: CGMCC No.1.2349, can adopt and well known to a person skilled in the art that any method makes the viable bacteria pulvis), (former bacterial strain is available from Chinese common micro-organisms culture presevation administrative center for plant lactobacillus viable bacteria pulvis, deposit number: CGMCC No.1.124, can adopt and well known to a person skilled in the art that any method makes the viable bacteria pulvis) and Active Dry Yeast (available from Angel Yeast Co.,Ltd) according to several 3: 1: 0.8 of viable bacteria (comprising gemma): 0.3: 1 ratio is mixed, be made into the viable bacteria pulvis that viable bacteria adds up to (comprising gemma) 10,000,000,000 cfu/g, be sprayed on Jiangsu Province Yixing City chinese carp according to 20g/ mu/Mi Shuiti and raise together with the pool, observe water quality situation and the surveyors' staff visibility (cm) in this fish pond of 0～4d, the result is referring to table 3.
Table 3
| Time | 0d | 1d | 2d | 3d | 4d |
| The scale visibility | 15cm | 20cm | 35cm | 36cm | 35cm |
By table 2～table 3 data as can be known, Bacillus licheniformis BL-2 CCTCC M 2012458 of the present invention has good flocculation and water purification effect, can effectively remove the waste component in aquaculture water and other polluted-waters, Cellulose precipitates is had splendid degradation effect.
If no special instructions, related each reagent of above embodiment and raw material are the commercial goods.
Claims (4)
1. a strain has High Cellulase Production Bacillus licheniformis (Bacilluslicheniformis) BL-2 of throwing out, and deposit number is CCTCC No.M 2012458.
2. the described Bacillus licheniformis BL-2 of claim 1, the application of CCTCC No.M 2012458 on aquaculture and polluted-water water quality regulation is characterized in that: this Bacillus licheniformis BL-2 is mixed afterwards as the water quality improvement of water conditioner for aquaculture and polluted-water separately or with other water purification microorganism strains.
3. described Bacillus licheniformis BL-2 according to claim 2, the application of CCTCC No.M 2012458 on aquaculture and polluted-water water quality regulation is characterized in that: described water purification microorganism strains is selected from subtilis, Rhodopseudomonas palustris, plant lactobacillus, the yeast saccharomyces cerevisiae one or more.
4. described subtilis BL-2 according to claim 2, the application of CCTCC No.M 2012458 on aquaculture and polluted-water water quality regulation is characterized in that: described water conditioner is the viable bacteria pulvis.
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| CN104150606A (en) * | 2013-05-13 | 2014-11-19 | 无锡中科活力生物技术有限公司 | Aquaculture water in-situ purification composite microbial membrane and using method thereof |
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| EP3095855A4 (en) * | 2014-06-03 | 2017-05-17 | Jiangnan University | Efficient bottom treatment bacillus, composite bottom treatment inoculant prepared using same and applications thereof |
| CN105441358A (en) * | 2015-12-19 | 2016-03-30 | 佛山市艳晖生物科技有限公司 | On-site fermented bacillus licheniformis preparation for aquaculture farm and applications thereof |
| CN105441358B (en) * | 2015-12-19 | 2018-12-21 | 佛山市艳晖生物科技有限公司 | Bacillus licheniformis preparation and its application for the fermentation of aquatic farm scene |
| CN110129230A (en) * | 2019-05-20 | 2019-08-16 | 江南大学 | A kind of microorganism formulation and its application in the sanitary sewage disposal of Expressway Service |
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| CN113897303A (en) * | 2021-08-20 | 2022-01-07 | 中国海洋大学 | Microbial flocculant for aquaculture and preparation method and application thereof |
| CN113897303B (en) * | 2021-08-20 | 2024-02-02 | 中国海洋大学 | Microbial flocculant for aquaculture and preparation method and application thereof |
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