CN103173378A - Method for fast screening microbial flocculant producing bacteria - Google Patents
Method for fast screening microbial flocculant producing bacteria Download PDFInfo
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- CN103173378A CN103173378A CN2012105670496A CN201210567049A CN103173378A CN 103173378 A CN103173378 A CN 103173378A CN 2012105670496 A CN2012105670496 A CN 2012105670496A CN 201210567049 A CN201210567049 A CN 201210567049A CN 103173378 A CN103173378 A CN 103173378A
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Abstract
The invention provides a method for fast screening microbial flocculant producing bacteria. The preparation method comprises the steps of: (1) microbial enrichment culture: putting activated sludge into a flask, carrying out static settlement for 10 min, taking 10 ml of supernate, adding the supernate into a 250 ml triangular flask containing 100 ml of sterilized LB liquid culture medium or PDA liquid culture medium or Gause-1 liquid culture medium, and culturing in a shaking table in a speed of 200 revolutions per minute at 28-37 DEG C, wherein the concentration of enriched bacteria liquid is 1.0 CFU/ml; and (2) preparation of optimized fermented culture liquid: sequentially putting 28-32 g of soluble starch, 4-6 g of K2HPO4, 1.5-2.5 g of KH2PO4, 3-5 g of yeast extracts, 0.8-1.2 g of KNO3, 0.4-0.6 g of NaCl, 0.4-0.6 g of MgSO4.7H2O and 0.008-0.013 g of FeSO4 into a container containing 950-1050 ml of distilled water, regulating the pH to 7.5-8.5 by utilizing NaOH, and carrying out sterilization for 25-30 min at 110-150 DEG C, so as to obtain the optimized fermented culture liquid. The preparation method further comprises the steps of (3) microbial fermentation culture; (4) separation and purification of strains; and (5) testing of a flocculation rate.
Description
Technical field
The present invention relates to the screening of bacterium for producing flocculant of microbe, the method for be particularly useful for fast, high-effective microorganism being cultivated the generation microbial flocculant.
Background technology
Along with water pollution problems is day by day serious, it is more and more severeer that water treatment problems also becomes, the method of water treatment has the several different methods such as absorption, chemical oxidation, electrodialysis, biochemistry, ion-exchange, wherein flocculence is a kind of comparatively effective and lower-cost pretreatment process, has been widely used in the numerous areas such as sewage disposal, foodstuff production and industrial fermentation.Flocculation agent divides two large classes: the one, take aluminium salt, molysite and multipolymer thereof as main inorganic flocculating agent; The 2nd, take polyacrylamide as main synthetic polymeric flocculant, this traditional flocculation agent has certain corrodibility and toxicity, easily forms secondary pollution, and HUMAN HEALTH is had very large harm, and flocculating effect affected by the water quality water temperature condition larger.Biological flocculant can be described as third generation flocculation agent, a kind of microbial metabolites or chemical modification natural organic high-molecular flocculant with efficient flocculating activity, it is to utilize biotechnology, fermentation culture by microorganism, extraction from microbial metabolites, purifying and the New Type Water Treatment Chemicals that obtains, its main component has protein, polysaccharide, lipid, Mierocrystalline cellulose, DNA and the thalline of flocculation activity is arranged.Not only use range is wide, consumption is few for biological flocculant, cost is lower, can degrade voluntarily, environment is not produced secondary pollution, and treatment effect is high, and some unmanageable high-concentration waste waters are flocculated.
Literature search discloses: 1. a kind of composite flora 1(BAFRT4+CYGS1 of filtering out of Zhang Zhiqiang), under the Optimal Medium condition, COD clearance and the percent of decolourization of indigo dyeing waste water reached respectively 79.2% and 87.6%; 2. the strain gas bacillus that filters out from soil of Wang Shuguang can produce flocculating effect better and the microbial flocculant of stable performance; 3. the meta-bolites that separates the bacterial strain that obtains from active sludge such as Chen Yuehua can be applied to treatment of dyeing and printing; 4. the patent of invention CN 101503709A of China provides a kind of method of utilizing Bacillus licheniformis to prepare microbial flocculant; 5. patent of invention CN1616358A provide a kind of with the stalk bacterium as the flocculation agent bacterial classification, produce the method for flocculation agent through fermentation culture, but these methods that prepare flocculation agent all will be through the primary dcreening operation of bacterial strain, multiple sieve, the isolation identification of bacterial classification, the conventional steps such as fermentation culture of bacterial strain, its process is complicated, complex steps.Although the advantage of microbial flocculant makes it have wide distant view, many experts and scholars have also carried out the research to biological flocculant, but so far, microbial flocculant is not widely applied in practice, wherein loaded down with trivial details cultivation screening process, long growth cycle make the high cost of actual production, are the major causes that microbial flocculant is difficult to promote.Good production bacterial classification normally separates from different environmental samples, purifying, more further obtains after screening through flocculating experiment, and this process workload is large, and the cycle is long, and the screening target is uncertain, so screening efficiency is low.
The present invention utilizes the mutual metabolism between microorganism, by enrichment and the fermentation culture of microflora, obtains having the microflora of obvious flocculating effect, separate for this group again and obtain the efficient flocculating bacterial strain, and by the Optimal Medium composition, improve flocculation agent output, the cycle of the bacterium for producing flocculant of microbe that screens is short, efficient is high, with strong points, overcome traditional microbe to screen complicated, the shortcoming of complex steps, can reduce production costs, be easy to realize suitability for industrialized production.
Summary of the invention
The object of the invention is to: the rapid screening method that provides overcomes traditional bacterium for producing flocculant of microbe screening method complexity, cycle length, inefficient deficiency, realizes the quick, efficient of sample screened.
The object of the present invention is achieved like this: a kind of method of rapidly screening microbial bacterium for producing flocculant, implement by step:
The enrichment culture of step 1 microorganism:
Get active sludge water in beaker, staticly settle 10min, get supernatant liquor 10ml, add in the 250ml triangular flask of LB liquid nutrient medium that 100ml sterilization is housed or PDA liquid nutrient medium or Gause I liquid nutrient medium, at 28-37 ℃, cultivate in the 200rpm shaking table, the enrichment bacterial concentration is 1.0CFU/ml;
PDA liquid nutrient medium wherein: get fresh potato decortication, be cut into approximately 2cm
2Fritter, the beaker of putting into 1500ml boils 30min, then uses 4 layers of filtered through gauze, gets its filtrate and adds glucose 20g, is complementing to 1000ml with distilled water, 121 ℃ the sterilization 25min, get finished product;
Gause I liquid nutrient medium wherein: get Zulkovsky starch 20g, KNO
31g, NaCl 0.5g, K
2HPO
43H
2O 0.5g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g puts into 1000ml distilled water, regulates pH 7.6 with NaOH, and temperature is below 121 ℃, and sterilization 25min obtains;
LB liquid nutrient medium wherein: get peptone 10g, yeast soaks powder 5g, NaCl 5g, puts into 1000ml distilled water, regulate with NaOH the 25min that sterilizes under 7.0,121 ℃ of pH, get finished product;
Step 2 is optimized the preparation of fermentation culture:
With Zulkovsky starch 28-32g, K
2HPO
44-6g, KH
2PO
41.5-2.5g, yeast extract paste 3-5g, KNO
30.8-1.2g, NaCl 0.4-0.6g, MgSO
47H
2O 0.4-0.6g, FeSO
40.008-0.013g put into successively the container of 950-1050ml distilled water, regulating pH with NaOH is the 25-30min that sterilizes under 7.5-8.5, temperature 110-115 ℃, gets finished product;
The fermentation culture of step 3 microorganism:
Step 1 enrichment culture liquid is optimized in fermentation culture by the inoculum size access step 2 of 1-2:100 cultivated, temperature 25-30 ℃, time 30-40h, shaking speed 100-200rpm; Get supernatant 2ml after medium centrifugal, add 100ml waste water, measure flocculating rate; Its flocculation agent main component is polysaccharide, is the metabolic secretion thing in the growth process;
The separation and purification of step 4 bacterial classification:
Select the fermented liquid of high flocculating effect to dilute with sterilized water, make the dilution bacterium liquid of different gradients, the dilution bacterium liquid of getting each gradient of 0.1ml is coated on LB solid medium, PDA solid medium and Gause I solid medium, 28-37 ℃ of standing cultivation, after growing single bacterium colony, picking list bacterium colony is again through 28-37 ℃, the cultivation of 200rpm shaking table, concentration reaches after 1.0CFU/ml the inoculum size access fermentation culture by 1.5:100,25-30 ℃, the 150rpm shaking table is cultivated 30-40h and is measured its flocculating rate, result must produce the bacterial strain of flocculation agent, and flocculating rate reaches 80-90%;
The measuring method of step 5 flocculating rate: take 4g kaolin and be dissolved in the Kaolin clay suspension that is configured to 4g/L in 1000ml distilled water, take 1gCaC l and be dissolved in and be configured to 1% solution in 100ml distilled water; Get the 100ml Kaolin clay suspension in the 250ml beaker, regulate pH7.0 with NaOH, add the CaCl solution of 2ml 1% and the fermentation culture of 2ml, first rapid stirring 2min slowly stirs 3min again, get supernatant liquor after standing 10min, measure its absorbancy with spectrophotometer at the 550nm place, do controlled trial with the Kaolin clay suspension that adds fermention medium, the flocculating rate calculation formula: flocculating rate=(A-B)/A * 100%; Wherein A is the absorbancy of contrast supernatant liquor at the 550nm place; B adds the sample supernatant liquor of fermented liquid in the absorbancy at 550nm place.
mechanism of the present invention and effect: the flocculation agent main component of microorganisms is glycoprotein, polysaccharide, protein, Mierocrystalline cellulose and DNA etc., it is a kind of meta-bolites that produces in microbial cultivation process, can be obtained by one or more microorganism culturing, its output and flocculating effect not only are subjected to the impact of bacterium for producing flocculant culture condition, also relevant with the method for flocculation agent separating-purifying, the present invention utilizes the mutual metabolism between microorganism, enrichment and fermentation culture by microflora, obtain having the microflora of obvious flocculating effect, separate for this group again and obtain the efficient flocculating bacterial strain, and by the Optimal Medium composition, initial pH value, temperature, incubation time and rotating speed provide optimal culture condition and the method for purification of bacterium for producing flocculant, shorten culture cycle, improve flocculation agent output.
Beneficial effect of the present invention: temperature, time, rotating speed and fermentation culture based component to microorganism culturing are optimized, and the bacterial strain flocculating effect that obtains is better, and flocculating rate reaches more than 80%; The flocculation agent composition of microorganisms is through being accredited as polysaccharide, and is nontoxic, can degrade voluntarily under field conditions (factors); The method is simple to operate, quick, can realize the screening of a large amount of samples, and applied range shows technical progress.
Embodiment
The present invention will be further described below in conjunction with embodiment
Embodiment 1
The screening of flocculation activity bacterium: get sludge sewage, getting after precipitation supernatant 10ml adds in the 250ml triangular flask that 100ml LB liquid nutrient medium is housed, 37 ℃, the 200rpm shaking table cultivate 48h to bacterial concentration be 1.0CFU/ml, optimize in fermentation culture 30 ℃ according to the inoculum size access of 1.5:100, the 150rpm shaking table is cultivated 30h, get supernatant 2ml after medium centrifugal, add in the 100ml petrochemical wastewater, measure flocculating rate; Through identifying, the flocculation agent composition is mainly polysaccharide, is the metabolic secretion thing in the growth process.
The fermented liquid with high flocculating effect that records dilutes with sterilized water, make the dilution bacterium liquid of different gradients, the diluent of getting each gradient of 0.1ml is applied on the LB solid medium, after 37 ℃ of standing cultivation 24h, picking list bacterium colony is rule and is separated until colonial morphology is consistent, single bacterium colony is again through 37 ℃, the 200rpm shaking table is cultivated 48h, concentration reaches after 1.0CFU/ml the inoculum size access fermentation culture by 1.5:100,30 ℃, the 150rpm shaking table is cultivated 30h and is measured its flocculating rate, result obtains the bacterial strain that 2 strains produce flocculation agent, and flocculating rate reaches 81.2%.
LB liquid nutrient medium used is by peptone 10g, and yeast soaks powder 5g, and NaCl 5g puts into 1000ml distilled water, regulates with NaOH that under 7.0,121 ℃ of pH, sterilization 25min obtains, and optimization fermented liquid substratum used is by Zulkovsky starch 30g, K
2HPO
45g, KH
2PO
42g, yeast extract paste 4g, KNO
31g, NaCl 0.5g, MgSO
47H
2O 0.5g, FeSO
40.01g put into 1000ml distilled water, regulate pH 7.5 with NaOH, at 115 ℃ of temperature, sterilization 30min obtains.
The measuring method of flocculating rate: take 4g kaolin and be dissolved in the Kaolin clay suspension that is configured to 4g/L in 1000ml distilled water, take 1g CaCl and be dissolved in and be configured to 1% solution in 100ml distilled water; Get the 100ml Kaolin clay suspension in the 250ml beaker, regulate pH7.0 with NaOH, add the CaCl solution of 2ml 1% and the fermentation culture of 2ml, first rapid stirring 2min slowly stirs 3min again, get supernatant liquor after standing 10min, measure its absorbancy with spectrophotometer at the 550nm place, do controlled trial with the Kaolin clay suspension that adds fermention medium, the flocculating rate calculation formula:
Flocculating rate=(A-B)/A * 100%
A is the absorbancy of contrast supernatant liquor at the 550nm place
B adds the sample supernatant liquor of fermented liquid in the absorbancy at 550nm place
Embodiment 2
The saccharomycetic screening of flocculation activity: get fresh potato decortication, be cut into approximately 2cm
2Fritter, the beaker of putting into 1500ml boils 30min, then uses 4 layers of filtered through gauze, gets its filtrate and adds glucose 20g, is complementing to 1000ml with distilled water, natural pH, 121 ℃ the sterilization 25min obtain the PDA liquid nutrient medium.
Get sludge sewage, getting after precipitation supernatant 10ml adds in the 250ml triangular flask that 100ml PDA liquid nutrient medium is housed, 28 ℃, the 200rpm shaking table cultivate 72h to bacterial concentration be 1.0CFU/ml, inoculum size access according to 1.5:100 is optimized in fermentation culture, 26 ℃, the 150rpm shaking table is cultivated 35h, get supernatant 2ml after medium centrifugal, add in 100ml crow petrochemical wastewater, measure flocculating rate, through identifying, the flocculation agent composition is mainly polysaccharide, is the metabolic secretion thing in the growth process.
The fermented liquid with high flocculating effect that records dilutes with sterilized water, make the dilution bacterium liquid of different gradients, the diluent of getting each gradient of 0.1ml is applied on the PDA solid medium, after 28 ℃ of standing cultivation 48h, picking list bacterium colony is rule and is separated until colonial morphology is consistent, single bacterium colony is again through 28 ℃, the 200rpm shaking table is cultivated 72h, concentration reaches after 1.0CFU/ml the inoculum size access fermentation culture by 1.5:100,26 ℃, the 150rpm shaking table is cultivated 35h and is measured its flocculating rate, result obtains the bacterial strain that 1 strain produces flocculation agent, and flocculating rate reaches 89%.
Embodiment 3
The actinomycetic screening of flocculation activity: get sludge sewage, getting after precipitation supernatant 10ml adds in the 250ml triangular flask that 100ml Gause I liquid nutrient medium is housed, 28 ℃, the 200rpm shaking table cultivate 6d to bacterial concentration be 1.0CFU/ml, optimize in fermentation culture 28 ℃ according to the inoculum size access of 1.5:100, the 150rpm shaking table is cultivated 40h, get supernatant 2ml after medium centrifugal, add in 100ml crow petrochemical wastewater, measure flocculating rate; Through identifying, the flocculation agent composition is mainly polysaccharide, is the metabolic secretion thing in the growth process.
The fermented liquid with high flocculating effect that records dilutes with sterilized water, make the dilution bacterium liquid of different gradients, the diluent of getting each gradient of 0.1ml is applied on the PDA solid medium, after 28 ℃ of standing cultivation 6d, picking list bacterium colony is rule and is separated until colonial morphology is consistent, single bacterium colony is again through 28 ℃, the 200rpm shaking table is cultivated 5d, concentration reaches after 1.0CFU/ml the inoculum size access fermentation culture by 1.5:100,28 ℃, the 150rpm shaking table is cultivated 40h and is measured its flocculating rate, result obtains the bacterial strain that 1 strain produces flocculation agent, and flocculating rate reaches 87.9%.
Gause I liquid nutrient medium used is by Zulkovsky starch 20g, KNO
31g, NaCl 0.5g, K
2HPO
43H
2O 0.5g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g puts into 1000ml distilled water, regulates pH 7.6 with NaOH, and temperature is below 121 ℃, and sterilization 25min obtains.
The raw material that present method is selected: Zulkovsky starch producer is Tianjin chemical reagent three factories; K
2HPO
4Producer is Tianjin Hedong District red rock chemical reagent work; KH
2PO
4Producer is Tianjin bright Fine Chemical Co., Ltd forever; Yeast extract paste producer is Beijing extensive and profound in meaning star biotechnology limited liability company; K
2HPO
43H
2O producer is Guangzhou profit exhibition chemical industry company limited; MgSO
47H
2O producer is Tianjin Fengchuan Chemical Reagent Science ﹠ Technology Co., Ltd.; FeSO
47H
2O producer is the Xi'an chemical reagent factory; Kaolinic producer is gold source, Datong kaolin limited liability company; Shaking table model used is HZQ-Z.
Claims (1)
1. the method for a rapidly screening microbial bacterium for producing flocculant is characterized in that: implement by step:
The enrichment culture of step 1 microorganism:
Get active sludge water in beaker, staticly settle 10min, get supernatant liquor 10ml, add in the 250ml triangular flask of LB liquid nutrient medium that 100ml sterilization is housed or PDA liquid nutrient medium or Gause I liquid nutrient medium, at 28-37 ℃, cultivate in the 200rpm shaking table, the enrichment bacterial concentration is 1.0CFU/ml;
PDA liquid nutrient medium wherein: get fresh potato decortication, be cut into approximately 2cm
2Fritter, the beaker of putting into 1500ml boils 30min, then uses 4 layers of filtered through gauze, gets its filtrate and adds glucose 20g, is complementing to 1000ml with distilled water, 121 ℃ the sterilization 25min, get finished product;
Gause I liquid nutrient medium wherein: get Zulkovsky starch 20g, KNO
31g, NaCl 0.5g, K
2HPO
43H
2O 0.5g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g puts into 1000ml distilled water, regulates pH 7.6 with NaOH, and temperature is below 121 ℃, and sterilization 25min obtains;
LB liquid nutrient medium wherein: get peptone 10g, yeast soaks powder 5g, NaCl 5g, puts into 1000ml distilled water, regulate with NaOH the 25min that sterilizes under 7.0,121 ℃ of pH, get finished product;
Step 2 is optimized the preparation of fermentation culture:
With Zulkovsky starch 28-32g, K
2HPO
44-6 g, KH
2PO
41.5-2.5g, yeast extract paste 3-5g, KNO
30.8-1.2g, NaCl 0.4-0.6g, MgSO
47H
2O 0.4-0.6g, FeSO
40.008-0.013g put into successively the container of 950-1050ml distilled water, regulating pH with NaOH is the 25-30min that sterilizes under 7.5-8.5, temperature 110-115 ℃, gets finished product;
The fermentation culture of step 3 microorganism:
Step 1 enrichment culture liquid is optimized in fermentation culture by the inoculum size access step 2 of 1-2:100 cultivated, temperature 25-30 ℃, time 30-40h, shaking speed 100-200rpm; Get supernatant 2ml after medium centrifugal, add 100ml waste water, measure flocculating rate; Its flocculation agent main component is polysaccharide, is the metabolic secretion thing in the growth process;
The separation and purification of step 4 bacterial classification:
Select the fermented liquid of high flocculating effect to dilute with sterilized water, make the dilution bacterium liquid of different gradients, the dilution bacterium liquid of getting each gradient of 0.1ml is coated on LB solid medium, PDA solid medium and Gause I solid medium, 28-37 ℃ of standing cultivation, after growing single bacterium colony, picking list bacterium colony is again through 28-37 ℃, the cultivation of 200rpm shaking table, concentration reaches after 1.0 CFU/ml the inoculum size access fermentation culture by 1.5:100,25-30 ℃, the 150rpm shaking table is cultivated 30-40h and is measured its flocculating rate, result must produce the bacterial strain of flocculation agent, and flocculating rate reaches 80-90%;
The measuring method of step 5 flocculating rate: take 4g kaolin and be dissolved in the Kaolin clay suspension that is configured to 4g/L in 1000ml distilled water, take 1g CaCl and be dissolved in and be configured to 1% solution in 100ml distilled water; Get the 100ml Kaolin clay suspension in the 250ml beaker, regulate pH7.0 with NaOH, add the CaCl solution of 2ml 1% and the fermentation culture of 2ml, first rapid stirring 2min slowly stirs 3min again, get supernatant liquor after standing 10min, measure its absorbancy with spectrophotometer at the 550nm place, do controlled trial with the Kaolin clay suspension that adds fermention medium, the flocculating rate calculation formula: flocculating rate=(A-B)/A * 100%; Wherein A is the absorbancy of contrast supernatant liquor at the 550nm place; B adds the sample supernatant liquor of fermented liquid in the absorbancy at 550nm place.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088107A (en) * | 2013-01-25 | 2013-05-08 | 北京大北农科技集团股份有限公司 | Screening method and detecting method of flocculating agent producing strain for aquatic products |
| CN104478091A (en) * | 2014-11-27 | 2015-04-01 | 新疆德蓝股份有限公司 | High-efficiency ammonia nitrogen degradation composite strain culture method |
| CN112391320A (en) * | 2020-11-27 | 2021-02-23 | 江苏南资环保科技有限公司 | Strain capable of remarkably improving sedimentation performance of high-density sedimentation tank and application thereof |
| CN112481139A (en) * | 2020-12-22 | 2021-03-12 | 华东理工大学 | Culture medium for producing emodin by using marine fungus aspergillus flavus HN4-13 and preparation method thereof |
| CN112710617A (en) * | 2020-07-02 | 2021-04-27 | 科之杰新材料集团(广东)有限公司 | Flocculation performance test evaluation method of flocculant for washing sand |
-
2012
- 2012-12-24 CN CN2012105670496A patent/CN103173378A/en active Pending
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088107A (en) * | 2013-01-25 | 2013-05-08 | 北京大北农科技集团股份有限公司 | Screening method and detecting method of flocculating agent producing strain for aquatic products |
| CN104478091A (en) * | 2014-11-27 | 2015-04-01 | 新疆德蓝股份有限公司 | High-efficiency ammonia nitrogen degradation composite strain culture method |
| CN104478091B (en) * | 2014-11-27 | 2016-02-03 | 新疆德蓝股份有限公司 | A kind of cultural method of high-efficiency ammonia nitrogen degradation composite bacteria |
| CN112710617A (en) * | 2020-07-02 | 2021-04-27 | 科之杰新材料集团(广东)有限公司 | Flocculation performance test evaluation method of flocculant for washing sand |
| CN112391320A (en) * | 2020-11-27 | 2021-02-23 | 江苏南资环保科技有限公司 | Strain capable of remarkably improving sedimentation performance of high-density sedimentation tank and application thereof |
| CN112391320B (en) * | 2020-11-27 | 2022-07-08 | 江苏南资环保科技有限公司 | Strain capable of remarkably improving sedimentation performance of high-density sedimentation tank and application thereof |
| CN112481139A (en) * | 2020-12-22 | 2021-03-12 | 华东理工大学 | Culture medium for producing emodin by using marine fungus aspergillus flavus HN4-13 and preparation method thereof |
| CN112481139B (en) * | 2020-12-22 | 2022-08-05 | 华东理工大学 | Culture medium for producing emodin by using marine fungus aspergillus flavus HN4-13 and preparation method thereof |
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Application publication date: 20130626 |