CN102220404A - Preparation method of compound microbial flocculant for cyanobacterial bloom - Google Patents
Preparation method of compound microbial flocculant for cyanobacterial bloom Download PDFInfo
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- CN102220404A CN102220404A CN2011100790641A CN201110079064A CN102220404A CN 102220404 A CN102220404 A CN 102220404A CN 2011100790641 A CN2011100790641 A CN 2011100790641A CN 201110079064 A CN201110079064 A CN 201110079064A CN 102220404 A CN102220404 A CN 102220404A
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Abstract
The invention relates to a preparation method of a compound microbial flocculant for cyanobacterial bloom. The preparation method comprises the following steps: respectively inoculating Serratia and Bacillus into a sterilized nutrient broth medium for culture; respectively taking out 1mL of bacterial liquids of Serratia and Bacillus after culturing for 24h, and inoculating into a sterilized fermentation medium for fermentation culture; treating a broth as a seed liquid after culturing for 72 h and carrying out complex culture for 72h under conditions of a culture temperature of 35 DEG C, a rotation speed of 160rpm, an initial pH of a medium of 8.0, and an inoculation amount of the seed liquid of 10 ml/L; and carrying out centrifugal separation on the broth, and taking out a supernatant liquor. So the compound microbial flocculant is obtained. The preparation method is simple, fast and low cost, the flocculant prepared in the invention has the advantages of small dosage in use, strong stability and easy storage, can effectively flocculate cyanobactreial water samples with a flocculation rate of 92%, and has an excellent application prospect.
Description
Technical field
The invention belongs to the preparation field of compound microbial flocculation agent, particularly a kind of preparation method who is used for the compound microbial flocculation agent of blue-green alga bloom.
Background technology
The flocculation agent widespread use with in industrial circles such as sewage disposal, foodstuff production and biological fermentations; but at present widely used inorganic and residue organic polymer coargulator can cause problem of environmental pollution usually; even the mankind had toxic actions such as carcinogenic, teratogenesis; forbidden by many countries or the use of limiting the quantity of, thus need that exploitation is efficient, safety non-toxic, novel flocculant free from environmental pollution.
Biological flocculant mainly is the high-molecular biologic polymkeric substance with flocculation activity by microorganism secreted generation under Incubation Condition, has safety non-toxic, advantage such as environmentally friendly.At present, the U.S., Britain, Japan and other countries are studied biological flocculant, filter out multiple bacterium for producing flocculant, and have carried out serial work in the Gene Handling of flocculating conditions, mechanism, produce flocculant, purifying, character and the application facet of flocculation agent.
Microbial flocculant has different throwing outs to different suspended substances, and microbial flocculant is used for the research of body eutrophication less at present.
Summary of the invention
It is simple and efficient that technical problem to be solved by this invention provides this method of preparation method of a kind of compound microbial flocculation agent that is used for blue-green alga bloom, cost is low, husky thunder bacterium (Serratia) and genus bacillus (Bacillus) are to the equal non-resistant of general microbiotic, be easy to deactivation, guaranteed the ecological security that uses; The flocculation agent consumption that makes is few, and stability is strong, be easy to preserve, and the blue-green algae water sample that can effectively flocculate, flocculating rate can reach 92%, has a good application prospect.
A kind of preparation method who is used for the compound microbial flocculation agent of blue-green alga bloom of the present invention comprises:
Husky thunder bacterium Serratia and bacillus (commercially available) are inserted respectively in the nutrient broth medium of sterilization and cultivate, the bacterium liquid of getting husky thunder bacterium of 1mL and genus bacillus after 24 hours respectively inserts in the fermention medium of sterilizing and carries out fermentation culture, cultivate behind the 72h this fermented liquid as seed liquor, in culture temperature is after 35 ℃, shaking speed are to carry out compound cultivation 72h under the condition that 160rpm, the initial pH of substratum are 8.0, the seed liquor inoculum size is 10ml/L, fermented liquid is carried out centrifugation, get supernatant liquor and promptly get the compound microbial flocculation agent.
Described nutrient broth medium consist of peptone 10g, extracted beef powder 4.0g, NaCl 5.0g, yeast extract powder 3.0g, distilled water 1L; PH is 7.0～7.2.
Described nutrient broth medium is in 115 ℃ of sterilization 30min.
Described fermention medium consist of glucose 20g, K
2HPO
45g, KH
2PO
43g, NaCl 0.3g, urea 0.3g, yeast extract paste 0.6g, MgSO
47H
2O 0.5g, distilled water 1L; PH is 7.5～8.5.
Glucose is in 115 ℃ of sterilization 30min in the described fermention medium, and other composition is in 121 ℃ of sterilization 20min.
Husky thunder bacterium Serratia, bacteria colony white, circle, convex surface, colony edge is neat, peritrichous, motion, cell is shaft-like, Gram-reaction is negative.Bacillus bacterium colony yellow, circle, convex surface, colony edge is neat.Find that with electron microscope and scanning electron microscopic observation bacterial strain is shaft-like behind the gramstaining, Gram-reaction is positive, the adnation flagellum.
Beneficial effect
(1) the present invention is simple and efficient, and cost is low, and husky thunder bacterium (Serratia) and genus bacillus (Bacillus) are easy to deactivation to the equal non-resistant of general microbiotic, has guaranteed the ecological security that uses;
(2) the flocculation agent output height that makes, consumption is few, and stability is strong, is easy to preserve, and can effectively be used in actual water sample, the blue-green algae water sample that can effectively flocculate, flocculating rate can reach 92%, has a good application prospect.
Description of drawings
Fig. 1 (1) is the scanning electron microscope of husky thunder bacterium (Serratia), and (2) are the scanning electron microscope of genus bacillus (Bacillus);
Fig. 2 is husky thunder bacterium (Serratia) and the growth curve of genus bacillus (Bacillus) under different time;
Fig. 3 (1) is the influences of different culture temperature to compound microbial flocculation agent flocculence;
(2) be of the influences of different shaking speed to compound microbial flocculation agent flocculence;
(3) be the influence of the initial pH of different substratum to compound microbial flocculation agent flocculence;
(4) be of the influences of different bacterium liquid consumptions to compound microbial flocculation agent flocculence;
(5) be of the influence of different vaccination amount to compound microbial flocculation agent flocculence;
(6) be different 1%CaCl
2The dosage of solution is to the influence of compound microbial flocculation agent flocculence;
(7) be the influences of different pH to compound microbial flocculation agent flocculence;
Fig. 4 is compound microbial flocculation agent flocculating effect figure to the blue-green algae water sample under different action times.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The flocculence measuring method: add 0.4g kaolin in the 150mL beaker, add water 80ml distilled water, regulating pH is 4.5, and the massfraction that adds 4mL is 1%CaCl
2Solution and 1.5mL composite flora fermented liquid, adding distil water is to 100mL then.Normal temperature stirs 2min with constant temperature blender with magnetic force down under the condition of 200r/min, leave standstill 5min under the normal temperature.Draw the supernatant liquor at 50mL place, under the wavelength of 550nm, measure optical density value ODi with ultraviolet-visible pectrophotometer (752 type), replace the composite flora fermented liquid with the nonvaccinated fresh fermention medium of 2mL simultaneously, the identical experiment of other operational condition records optical density value OD in contrast
0
The flocculating rate calculation formula is:
Screening of efficient flocculating bacterium and evaluation:
(1) bacterial strain screens from active sludge, insert respectively in the Erlenmeyer flask (250mL) that fermention medium (50mL) is housed separating each bacterial strain that obtains, place shaking table to cultivate 64h in 30 ℃, 150r/min, get supernatant liquor and carry out the flocculation activity test, the inoculation bacterium colony carries out in Bechtop;
(2) primary dcreening operation of bacterium for producing flocculant of microbe: in the 100mL small beaker, add 0.2g kaolin, 40ml distilled water, 2ml1%CaCl
2The aqueous solution, 2ml thalline fermented supernatant fluid add water to 50mL then, all shake the static 15min in back, serve as contrast with the kaolin suspension liquid of the substratum that do not add bacterium simultaneously or are contrast with the kaolin suspension liquid, with the absorbancy of spectrophotometer at 550nm place survey supernatant liquor;
(3) the multiple sieve of bacterium for producing flocculant of microbe: multiple sieve is more accurate than primary dcreening operation, and detailed process is as follows: inoculation is in the 250mL Erlenmeyer flask that fills the 50mL substratum preferably with flocculation activity, and 150r/min surveys flocculation activity behind 30 ℃ of cultivation 64h.
Groping of optimal culture condition:
(1) add the 50ml fermention medium respectively in 6 250ml Erlenmeyer flasks, regulating pH is 7.0, adds the seed liquor of husky thunder bacterium Serratia of 1mL and bacillus respectively.Respectively at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, shaking speed is in the vertical double shaking table of 150rpm carries out compound cultivation, composite flora XL1.Behind the 72h, get 2ml fermented liquid supernatant liquid respectively, measure its flocculating rate;
(2) getting shaking speed respectively is 120rpm, 140rpm, 160rpm, 180rpm, 200rpm, 220rpm, at 35 ℃ of following constant temperature culture 72h, measures flocculating rate respectively;
(3) regulate the initial pH of substratum and be respectively 5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0, insert composite flora XL1, under 35 ℃, 160rpm, cultivate 72h, survey the flocculating rate that produces flocculation agent;
(4) the initial pH of substratum is adjusted to 8.0, inserts composite flora XL1 and under 35 ℃, 160rpm, cultivate 72h.In the 100ml Kaolin clay suspension, add the supernatant liquor of 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL composite flora XL1 fermented liquid respectively, measure flocculating rate separately;
(5) the initial pH of substratum is adjusted to 8.0, the seed liquor that in 5 250mL Erlenmeyer flasks that the 50mL fermention medium is housed, adds 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL composite flora XL1 respectively, after under 35 ℃, 160rpm, cultivating 72h, the 1.5mL fermented liquid supernatant of respectively asking for liquid is measured its flocculating rate;
(6) composite flora XL1 is 8.0 at the initial pH of substratum, culture temperature is that 35 ℃, shaking speed are that 160rpm, inoculum size are after carrying out fermentation culture 72h under the condition of 1ml, get the 1.5mL supernatant liquor respectively, add 0mL, 1mL, 2mL, 3mL, 4mL, 5mL, 6mL1%CaCl respectively
2In the 100ml Kaolin clay suspension of solution, survey flocculating rate separately;
(7) with composite flora XL1 by after carrying out fermentation culture 72h under the obtained top condition of above test, get 1.5ml supernatant liquor and 4ml1%CaCl
2It is in 3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0 the kaolin standard suspension that solution adds pH respectively, and measures flocculating rate separately;
(8) composite flora XL1 is cultivated according to the optimal culture condition of above test after, to add pH be 4.5, add 4.0ml1%CaCl to get the 1.5mL supernatant liquor
2In the Kaolin clay suspension of solution, stir 2min with 200rpm, get time of repose then respectively and be 3min, 6min, 9min, 12min, 15min, 18min, 21min is some detection time, measures the absorbancy of this moment with constant temperature blender with magnetic force.And contrast with the absorbancy of the Kaolin clay suspension that adds fresh culture, determine the flocculating rate of each time point.
Preferred embodiment is defined as:
Husky thunder bacterium Serratia and bacillus are inserted respectively in the nutrient broth medium of sterilization and cultivate, the bacterium liquid of getting husky thunder bacterium of 1mL and genus bacillus after 24 hours respectively inserts in the fermention medium of sterilizing and carries out fermentation culture, cultivate behind the 72h this fermented liquid as seed liquor, in culture temperature is after 35 ℃, shaking speed are to carry out compound cultivation 72h under the condition that 160rpm, the initial pH of substratum are 8.0, the seed liquor inoculum size is 10ml/L, fermented liquid is carried out centrifugation, get supernatant liquor and promptly get the compound microbial flocculation agent.
Enrichment medium is glucose 10g, K
2HPO
45.0g, urea 0.5g, yeast extract paste 0.5g, water 1.0L, pH7.0～7.2.Its fermention medium is glucose 20g, K
2HPO
45g, KH
2PO
43g, NaCl 0.3g, urea 0.3g, yeast extract paste 0.6g, MgSO
47H
2O 0.5g, distilled water 1.0L, pH7.5～8.5.
Solid medium is peptone 10g, extractum carnis 5.0g, NaCl 5.0g, agar 18g, water 1.0L, pH7.0～7.2.
The practical application test:
The blue-green algae water sample of getting the 44.5ml logarithmic phase is in the 100mL beaker, and the massfraction that adds 2mL is 1%CaCl
2The supernatant liquor of solution and 0.75mL composite flora fermented liquid, normal temperature stirs 2min with constant temperature blender with magnetic force down under the condition of 200r/min, then the blue-green algae water sample is put into the blue-green algae illumination box and carry out illumination cultivation, measure the flocculating rate of this flocculation agent behind the 24h the blue-green algae water sample.Calculate flocculating rate with blood cell counting.
The flocculating rate calculation formula is:
Flocculating rate/(%)=[(to the cell density of the cell density-supernatant liquor of algae liquid in the same old way)/to the cell density of algae liquid in the same old way] * 100
Composite flora XL1 institute produce flocculant can reach 92% to the flocculating rate of the blue-green algae water sample of logarithmic phase after measured.
The result shows, uses the compound microbial flocculation agent flocculation blue-green algae water body that this composite flora is cultivated, the control blue-green alga bloom, and the eutrophication of alleviating water body is feasible.
Claims (5)
1. preparation method who is used for the compound microbial flocculation agent of blue-green alga bloom comprises:
Husky thunder bacterium (Serratia) and genus bacillus (Bacillus) are inserted respectively in the nutrient broth medium of sterilization and cultivated, the bacterium liquid of getting husky thunder bacterium of 1mL and genus bacillus after 24 hours respectively inserts in the fermention medium of sterilizing and carries out fermentation culture, cultivate behind the 72h this fermented liquid as seed liquor, in culture temperature is after 35 ℃, shaking speed are to carry out compound cultivation 72h under the condition that 160rpm, the initial pH of substratum are 8.0, the seed liquor inoculum size is 10ml/L, fermented liquid is carried out centrifugation, get supernatant liquor and promptly get the compound microbial flocculation agent.
2. a kind of preparation method who is used for the compound microbial flocculation agent of blue-green alga bloom according to claim 1, it is characterized in that: described nutrient broth medium consist of peptone 10g, extracted beef powder 4.0g, NaCl 5.0g, yeast extract powder 3.0g, distilled water 1L; PH is 7.0～7.2.
3. a kind of preparation method who is used for the compound microbial flocculation agent of blue-green alga bloom according to claim 2 is characterized in that: described nutrient broth medium is in 115 ℃ of sterilization 30min.
4. a kind of preparation method who is used for the compound microbial flocculation agent of blue-green alga bloom according to claim 1 is characterized in that: described fermention medium consist of glucose 20g, K
2HPO
45g, KH
2PO
43g, NaCl 0.3g, urea 0.3g, yeast extract paste 0.6g, MgSO
47H
2O 0.5g, distilled water 1L; PH is 7.5～8.5.
5. a kind of preparation method who is used for the compound microbial flocculation agent of blue-green alga bloom according to claim 4 is characterized in that: glucose is in 115 ℃ of sterilization 30min in the described fermention medium, and other composition is in 121 ℃ of sterilization 20min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676422A (en) * | 2012-01-17 | 2012-09-19 | 大连理工大学 | Bacillus for producing microbial flocculant and use of bacillus in microalgae recovery |
| CN103936122A (en) * | 2014-04-18 | 2014-07-23 | 芜湖凯奥尔环保科技有限公司 | Deodorizing powdery blue algae treating agent and production method thereof |
| CN113604407A (en) * | 2021-09-03 | 2021-11-05 | 中冶华天南京工程技术有限公司 | Composite microbial algaecide and preparation method and application thereof |
| CN114891673A (en) * | 2022-05-07 | 2022-08-12 | 华南理工大学 | Biological flocculant, preparation method thereof and application thereof in blue algae treatment |
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| CN101219828A (en) * | 2008-01-24 | 2008-07-16 | 云南大学 | Blue algae water bloom bioflocculation agent and production |
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2011
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| CN101219828A (en) * | 2008-01-24 | 2008-07-16 | 云南大学 | Blue algae water bloom bioflocculation agent and production |
Non-Patent Citations (1)
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676422A (en) * | 2012-01-17 | 2012-09-19 | 大连理工大学 | Bacillus for producing microbial flocculant and use of bacillus in microalgae recovery |
| CN102676422B (en) * | 2012-01-17 | 2013-05-08 | 大连理工大学 | Bacillus for producing microbial flocculant and use of bacillus in microalgae recovery |
| CN103936122A (en) * | 2014-04-18 | 2014-07-23 | 芜湖凯奥尔环保科技有限公司 | Deodorizing powdery blue algae treating agent and production method thereof |
| CN103936122B (en) * | 2014-04-18 | 2015-12-02 | 芜湖凯奥尔环保科技有限公司 | A kind of deodorizing powdery blue algae treatment agent and preparation method thereof |
| CN113604407A (en) * | 2021-09-03 | 2021-11-05 | 中冶华天南京工程技术有限公司 | Composite microbial algaecide and preparation method and application thereof |
| CN113604407B (en) * | 2021-09-03 | 2024-02-06 | 中冶华天南京工程技术有限公司 | Composite microbial algicidal fungicide and preparation method and application thereof |
| CN114891673A (en) * | 2022-05-07 | 2022-08-12 | 华南理工大学 | Biological flocculant, preparation method thereof and application thereof in blue algae treatment |
| CN114891673B (en) * | 2022-05-07 | 2023-11-17 | 华南理工大学 | Biological flocculant, preparation method thereof and application thereof in blue algae treatment |
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Application publication date: 20111019 |