CN105462955A - Efficient selective breeding method of biological flocculant producing strain - Google Patents
Efficient selective breeding method of biological flocculant producing strain Download PDFInfo
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Abstract
The invention relates to an efficient selective breeding method of a biological flocculant producing strain. The method comprises the steps that 1, a method of combining ultraviolet mutagenesis with chemical mutagenesis is adopted by a flocculation strain to selectively breed a mutant strain, the genetic stability is good, and the flocculation rate reaches 96.8 percent and is increased by 25.1 percent compared with an original strain; 2, the selectively bred strain is good in flocculation activity and low in dosing quantity for treating wastewater, discharged precipitates are few compared with poly aluminum chloride and biodegradable or can serve as additives of animal feed to be utilized, and secondary pollution to the environment can be avoided. Various indexes of a treated water sample are all superior to those of the poly aluminum chloride which is widely applied at present.
Description
Technical field
The present invention relates to a kind of selection of high-performance bio bacterium for producing flocculant.
Background technology
Existing bacterium for producing flocculant is much that separation, purifying filter out from the active sludge of sewage work.But existing bacterial strain flocculating rate is lower, need to improve flocculating effect further by artificially breeding.
Summary of the invention
The present invention is directed to above-mentioned defect, object is to provide a kind of selection that can increase substantially a kind of high-performance bio bacterium for producing flocculant of producing strains flocculating rate.
The technical solution used in the present invention is for this reason: the present invention carries out according to following steps:
1) from cryopreserved seed inclined-plane, get a ring subtilis, access is equipped with in the triangular flask of fermention medium, in 28-30 DEG C, under 180-200r/min concussion cultivate 30-32h, centrifugation thalline, makes 10 with physiological saline
6individual/mL archespore suspension is for subsequent use;
2) ultraviolet mutagenesis:
Aseptically, get above-mentioned cell suspending liquid for subsequent use, be spread evenly across the culture dish surface that screening culture medium is housed, locate lucifuge under culture dish being placed in ultraviolet lamp and irradiate, culture dish is good with black paper bag, is placed in 28-30 DEG C of constant temperature, lucifuge is cultivated; Single bacterium colony that after 3-4d, the raw state of picking is good, is inoculated in fermention medium, in 28-30 DEG C, 180-200r/min concussion cultivation 30-32h;
Described fermention medium: glucose 8-10g, K
2hP0
41-3g, KH
2p0
41-2g, NaCl0.1-0.3g, urea 0.4-0.6g, yeast extract paste 0.5-0.7g, distilled water 1000mL, pH7.0-7.5, each material can zoom in or out in proportion;
Described screening culture medium:
Glucose 6-8g, dimixo-octyl phthalate 8-11, KH
2p0
41-2g, KH
2p0
40.5-1g, NaCl0.1-0.3g, 0.5g, yeast extract paste 0.6-0.8g, agar 18-20g, manganous sulfate 0.001-0.003g, distilled water 980-1000mL, pH7.0-7.5, each material can zoom in or out in proportion.
Preferred:
1) from cryopreserved seed inclined-plane, get a ring subtilis, access 200-210mL is equipped with in the triangular flask of fermention medium, in 28-30 DEG C, under 180-200r/min concussion cultivate 30-32h, centrifugation thalline, makes 10 with physiological saline
6individual/mL archespore suspension is for subsequent use.
2) ultraviolet mutagenesis:
Aseptically, get above-mentioned cell suspending liquid 0.1-0.2mL for subsequent use, be spread evenly across the culture dish surface that screening culture medium is housed, under culture dish being placed in 20w ultraviolet lamp, 28-30cm place lucifuge irradiates 40,80,120 seconds, culture dish is good with black paper bag, is placed in 28-30 DEG C of constant temperature, lucifuge is cultivated, and often processes do 3 parallel tests.Single bacterium colony that after 3-4d, the raw state of picking is good, is inoculated in fermention medium, and in 28-30 DEG C, 180-200r/min concussion cultivation 30-32h, the flocculating rate of fermented liquid is surveyed in sampling respectively.And compare with the fermentation culture inoculated without the subtilis of uv irradiating.
2) following seed selection is carried out after step:
Chemomorphosis method:
Get ultraviolet mutagenesis bacterial strain one ring, be inoculated in fermention medium, in 30-31 DEG C, under 120-140r/min concussion cultivate 30-32h, centrifugal collecting cell, after respectively washing 1-2 time with stroke-physiological saline solution and buffer solution of potassium phosphate, by washed cell suspension in the potassium phosphate solution containing 10-11g/L5-bromouracil (BU), 28-30 DEG C, 120-140r/min concussion cultivation 30-32min; Get the dilution of the suspension 0.16-0.18mol/L hypo solution after 10-12mL process, centrifugal collecting cell, after respectively washing 1 time with buffer solution of potassium phosphate and sterilized water, evenly be coated on screening culture medium flat board, 28-30 DEG C of constant temperature culture, from flat board, the larger single bacterium colony of picking, is inoculated in fermention medium, in 28-30 DEG C, 120-140r/min concussion cultivation 30-32h.
Advantage of the present invention is: 1) the present invention is flocculated the method that bacterial strain adopts ultraviolet mutagenesis and chemomorphosis to combine, and select mutant strain, genetic stability is good, and its flocculating rate reaches 96.8%, improves 25.1% than former bacterial strain.2) the bacterial strain flocculation activity selected is excellent, disposes of sewage adding less, and discharge throw out is few compared with polymerize aluminum chloride, and biodegradable, or is used as the additive of animal-feed, can avoid causing secondary pollution to environment.And the indices of process water sample is all better than the polymerize aluminum chloride of current widespread use.
Accompanying drawing explanation
Fig. 1 is ultraviolet induction secondary fermentation liquid flocculation schematic diagram.
Fig. 2 is that fermented liquid input amount is to flocculating effect effect diagram.
Embodiment
Specific description is made to the present invention below:
1 test method:
1.1 substratum:
1.1.1 fermention medium:
Glucose 8-10g, K
2hP0
41-3g, KH
2p0
41-2g, NaCl0.1-0.3g, urea 0.4-0.6g, yeast extract paste 0.5-0.7g, distilled water 1000mL, pH7.0-7.5.
1.1.2 screening culture medium:
Glucose 6-8g, dimixo-octyl phthalate 8-11, KH
2p0
41-2g, KH
2p0
40.5-1g, NaCl0.1-0.3g, 0.5g, yeast extract paste 0.6-0.8g, agar 18-20g, manganous sulfate 0.001-0.003g, distilled water 980-1000mL, pH7.0-7.5.
1.2 seed culture
From cryopreserved seed inclined-plane, get a ring subtilis, access 200-210mL is equipped with in the triangular flask of fermention medium, in 28-30 DEG C, under 180-200r/min concussion cultivate 30-32h, centrifugation thalline, makes 10 with physiological saline
6individual/mL archespore suspension is for subsequent use.
1.3 ultraviolet mutagenesis
Aseptically, get above-mentioned cell suspending liquid 0.1-0.2mL for subsequent use, be spread evenly across the culture dish surface that screening culture medium is housed, under culture dish being placed in 20w ultraviolet lamp, 28-30cm place lucifuge irradiates 40,80,120 seconds, culture dish is good with black paper bag, is placed in 28-30 DEG C of constant temperature, lucifuge is cultivated, and often processes do 3 parallel tests.Single bacterium colony that after 3-4d, the raw state of picking is good, is inoculated in fermention medium, and in 28-30 DEG C, 180-200r/min concussion cultivation 30-32h, the flocculating rate of fermented liquid is surveyed in sampling respectively.And compare with the fermentation culture of the inoculation without uv irradiating.
Flocculating rate measuring method:
In 150mL graduated cylinder, add 100-110mL kaolin (4g/L) suspension liquid, add 1-2%CaCl
2solution 4-4.3mL and 44-4.2mL nutrient solution, 200-220r/min rapid stirring 2-3min, 100-110r/min stir 5-6min, after leaving standstill 15-17min, get supernatant liquor and measure 550nm place absorbancy.Replace nutrient solution to compare with the liquid nutrient medium not connecing bacterial classification simultaneously, calculate flocculating rate.
Flocculating rate=(A-B)/A × 100%
A contrasts the absorbancy of supernatant liquor, the absorbancy of B sample supernatant.
1.4 chemomorphosis methods
Get ultraviolet mutagenesis bacterial strain one ring, be inoculated in the triangular flask that 100-110mL fermention medium is housed, in 30-31 DEG C, under 120-140r/min concussion cultivate 30-32h, centrifugal collecting cell, after respectively washing 1-2 time with stroke-physiological saline solution and 0.1mol/L buffer solution of potassium phosphate (pH7.0), by washed cell suspension in the potassium phosphate solution (pH7.0) containing 10-11g/L5-bromouracil (BU), 28-30 DEG C, 120-140r/min concussion cultivation 30-32min; Get the suspension 0.16-0.18mol/L hypo solution 100mL after 10-12mL process to dilute, centrifugal collecting cell, after respectively washing 1 time with 0.1mol/L buffer solution of potassium phosphate and sterilized water, get that 0.1-0.2mL is even to be coated on screening culture medium flat board, 28-30 DEG C of constant temperature culture, the larger single bacterium colony of picking from flat board, be inoculated in fermention medium, in 28-30 DEG C, 120-140r/min concussion cultivation 30-32h, the flocculating rate of fermented liquid is surveyed in sampling, and result filters out aimed strain by experiment.
1.5 wastewater treatment efficiency tests
The strain X N-3 filtered out, be inoculated in the triangular flask that 350mL fermention medium is housed, in 30-31 DEG C, under 120-140r/min concussion cultivate 30-32h, collect fermented liquid and be used for sewage disposal experiment.In order to prove the verity of experiment effect, this effect test entrusts sewage disposal key lab of Hebei province---the application experiment that Hebei Sang Wote Water Company LLC carries out.
Get city domestic sewage 1000-1100mL, about holding temperature 23-26 DEG C, adjust ph is 7-7.2, the fermented liquid of biological flocculant XN-3 is dropped into by 2mL/kg, rapid stirring 5-7min under 200-220r/min rotating speed, then adjusting rotary speed is 80-90r/min, low rate mixing 10-12min, stops stirring.Treatment solution is squeezed in settling bowl, standing sedimentation 40-45min, get clear liquor and measure the indexs such as COD, BOD, SS, colourity, pH value respectively.Release clear liquor, the throw out of discharging bottom settling bowl after centrifugation, measure weight of precipitate (weight in wet base).Often process in triplicate, average.According to aforesaid method, drop into polymeric aluminum chlorides solution (30%) by 3mL/kg, and measure the indices after disposing of sewage, contrast as a comparison.
The analytical procedure of each Testing index:
COD, dichromate titration GB11914-89; BOD, dilution and inoculation method GB7488-87; SS, weighting method GB11901-89; Colourity, extension rate method GB11903-89; PH value, glass electrode method GB6920-86.
2 results and analysis
2.1 ultraviolet induction
UV treatment has stronger lethal effect to B. subtilis cell.When irradiation time is 40S, induction generation 3 plant mutant bacterial strain; When irradiation time is 80S, induction generation 2 plant mutant bacterial strain.When irradiation time is 120S, produce without mutant strain.The 5 strain dissociants that ultraviolet induction produces, cultivate through purifying, are labeled as UH-1 ~ UH-5 respectively.Through 301 DEG C, 120r/min concussion cultivates after 30h, the fermented liquid flocculating rate of sampling and measuring, is shown in Fig. 1 respectively.
As seen from Figure 1: except UH-1, the flocculating rate of other 4 strain dissociants all increases than former bacterial strain ring subtilis, especially UH-2 and UH-4 flocculating rate improves 13.7%, 10.5% respectively, reaches 91.2% and 90.7%, and all has good genetic stability.Based on this, using the starting strain of UH-2 and UH-4 as chemomorphosis.
2.2 chemomorphosis
In order to improve the flocculating effect of UH-2 and UH-4 further, 5-bromouracil (BU) is utilized to carry out chemomorphosis to these two bacterial strains.Through primary dcreening operation and the multiple sieve of dissociant, 8 strains are filtered out.Through being inoculated into fermention medium, concussion measures the flocculating rate of each bacterial strain as table 1 after cultivating 30h.
The flocculating effect of table 1 chemomorphosis bacterial strain
| Strain number | Flocculating rate % | Strain number | Flocculating rate % |
| UH-2-1 | 93.6 | UH-4-1 | 94.3 |
| UH-2-2 | 95.7 | UH-4-2 | 65.4 |
| UH-2-3 | 89.8 | UH-4-3 | 97.5 |
| UH-2-4 | 96.8 | UH-4-4 | 92.5 |
| UH-2 | 91.2 | UH-4 | 90.7 |
From table 1: after second time chemomorphosis, except UH-2-3, the flocculating effect of other dissociants all has raising in various degree, especially UH-2-4 and UH-4-3, flocculating rate reaches 96.8% and 97.5%, improves 5.9% than UH-2, improves 7.5% than UH-4.Finally selected UH-4-3 is as last bacterial strain, for abridged table is stated, UH-4-3 called after XN-3.
2.3 dosages affect flocculating effect
At 100mL kaolin (4g/L) suspension liquid, add 1%CaCl
2solution 4mL, subtilis and dissociant XN-3 fermented liquid input amount are respectively: 0.5,1.0,1.5,2.0,3.0mL, investigate fermented liquid dosage on the impact of flocculating effect as shown in Figure 2.
As seen from Figure 2: along with dosage increase subtilis and XN-3 flocculating rate increase sharply, when dosage is 4mL, subtilis and XN-3 occur maximum value simultaneously, and then along with dosage, decline phenomenon all appears in flocculating rate.This may be because too much drop into fermented liquid, and system viscosity increases, and is unfavorable for bridge linking effect on the contrary and reduces flocculation activity; Meanwhile, under identical dosage, dissociant XN-3 flocculating rate is all higher than former Strains B. subtilis.Showing that XN-3 bacterial strain flocculation activity is excellent, reaching in identical flocculating rate situation, significantly can reduce addition, reduce biological flocculant use cost.
2.4 sewage disposal results
By simultaneous test, show biological flocculant XN-3 process city domestic sewage, treated water sample indices is all better than flocculation agent---the polymerize aluminum chloride of current widespread use.After XN-3 disposes of sewage, precipitate particles is comparatively large, is easy to filter dehydration, can shortens the settlement treatment time, and have good decolorizing effect, can improve the water quality after sewage treating efficiency and process.
Claims (3)
1. a selection for high-performance bio bacterium for producing flocculant, is characterized in that, carries out according to following steps:
1) from cryopreserved seed inclined-plane, get a ring subtilis, access is equipped with in the container of fermention medium, in 28-30 DEG C, under 180-200r/min concussion cultivate 30-32h, centrifugation thalline, makes 10 with physiological saline
6individual/mL archespore suspension is for subsequent use;
2) ultraviolet mutagenesis:
Aseptically, get above-mentioned cell suspending liquid for subsequent use, be spread evenly across the culture dish surface that screening culture medium is housed, locate lucifuge under culture dish being placed in ultraviolet lamp and irradiate, culture dish is good with black paper bag, is placed in 28-30 DEG C of constant temperature, lucifuge is cultivated; Single bacterium colony that after 3-4d, picking growth conditions is good, is inoculated in fermention medium, in 28-30 DEG C, 180-200r/min concussion cultivation 30-32h;
Described fermention medium: glucose 8-10g, K
2hP0
41-3g, KH
2p0
41-2g, NaCl0.1-0.3g, urea 0.4-0.6g, yeast extract paste 0.5-0.7g, distilled water 1000mL, pH7.0-7.5, each material can zoom in or out in proportion;
Described screening culture medium:
Glucose 6-8g, dimixo-octyl phthalate 8-11, KH
2p0
41-2g, KH
2p0
40.5-1g, NaCl0.1-0.3g, 0.5g, yeast extract paste 0.6-0.8g, agar 18-20g, manganous sulfate 0.001-0.003g, distilled water 980-1000mL, pH7.0-7.5, each material can zoom in or out in proportion.
2. the selection of a kind of high-performance bio bacterium for producing flocculant according to claim 1, is characterized in that,
1) from cryopreserved seed inclined-plane, get a ring subtilis, access is equipped with in the triangular flask of 200-210mL fermention medium, in 28-30 DEG C, under 180-200r/min concussion cultivate 30-32h, centrifugation thalline, makes 10 with physiological saline
6individual/mL archespore suspension is for subsequent use;
2) ultraviolet mutagenesis:
Aseptically, get above-mentioned cell suspending liquid 0.1-0.2mL for subsequent use, be spread evenly across the culture dish surface that screening culture medium is housed, under culture dish being placed in 20w ultraviolet lamp, 28-30cm place lucifuge irradiates 40,80,120 seconds, culture dish is good with black paper bag, is placed in 28-30 DEG C of constant temperature, lucifuge is cultivated, and often processes do 3 parallel tests; Single bacterium colony that after 3-4d, the raw state of picking is good, be inoculated in fermention medium, in 28-30 DEG C, 180-200r/min concussion cultivation 30-32h, the flocculating rate of fermented liquid is surveyed in sampling respectively, and compares with the fermentation culture inoculated without the subtilis of uv irradiating.
3. the selection of a kind of high-performance bio bacterium for producing flocculant according to claim 1, is characterized in that, 2) carry out following seed selection after step:
Chemomorphosis method:
Get ultraviolet mutagenesis bacterial strain one ring, be inoculated in fermention medium, in 30-31 DEG C, under 120-140r/min concussion cultivate 30-32h, centrifugal collecting cell, after respectively washing 1-2 time with stroke-physiological saline solution and buffer solution of potassium phosphate, by washed cell suspension in the potassium phosphate solution containing 10-11g/L5-bromouracil (BU), 28-30 DEG C, 120-140r/min concussion cultivation 30-32min; Get the dilution of the suspension 0.16-0.18mol/L hypo solution after 10-12mL process, centrifugal collecting cell, after respectively washing 1 time with buffer solution of potassium phosphate and sterilized water, evenly be coated on screening culture medium flat board, 28-30 DEG C of constant temperature culture, from flat board, the larger single bacterium colony of picking, is inoculated in fermention medium, in 28-30 DEG C, 120-140r/min concussion cultivation 30-32h.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117925475A (en) * | 2024-01-30 | 2024-04-26 | 重庆港力环保股份有限公司 | Bacillus clathratus capable of efficiently reducing chromaticity, breeding method thereof and compound microbial inoculum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154168A (en) * | 2011-01-07 | 2011-08-17 | 石家庄市兴柏生物工程有限公司 | Abamectin producing bacterium and preparation method thereof |
| CN102628066A (en) * | 2012-04-17 | 2012-08-08 | 山西大学 | Preparation method and application of microbial flocculant |
| CN103214101A (en) * | 2012-01-19 | 2013-07-24 | 中国中化股份有限公司 | Microbial flocculating agent, and preparation and use thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154168A (en) * | 2011-01-07 | 2011-08-17 | 石家庄市兴柏生物工程有限公司 | Abamectin producing bacterium and preparation method thereof |
| CN103214101A (en) * | 2012-01-19 | 2013-07-24 | 中国中化股份有限公司 | Microbial flocculating agent, and preparation and use thereof |
| CN102628066A (en) * | 2012-04-17 | 2012-08-08 | 山西大学 | Preparation method and application of microbial flocculant |
Non-Patent Citations (1)
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| 杨森等: "枯草芽孢杆菌诱变育种研究进展", 《农产品加工：学刊》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117925475A (en) * | 2024-01-30 | 2024-04-26 | 重庆港力环保股份有限公司 | Bacillus clathratus capable of efficiently reducing chromaticity, breeding method thereof and compound microbial inoculum |
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Application publication date: 20160406 |