CN102127513A - Method for culturing Bacillus subtilis by using potato starch wastewater - Google Patents
Method for culturing Bacillus subtilis by using potato starch wastewater Download PDFInfo
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- CN102127513A CN102127513A CN2010100337214A CN201010033721A CN102127513A CN 102127513 A CN102127513 A CN 102127513A CN 2010100337214 A CN2010100337214 A CN 2010100337214A CN 201010033721 A CN201010033721 A CN 201010033721A CN 102127513 A CN102127513 A CN 102127513A
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Abstract
The invention relates to a method for culturing Bacillus subtilis by using potato starch wastewater, and belongs to the field of biotechnology. The method is characterized in that: a fermentation medium takes the potato starch wastewater as a main nutrient source; and the method comprises the following culture steps of: preparing the medium; activating strains; preparing liquid seeds; performing liquid state fermentation; and preparing a bacterial agent. The Bacillus subtillis produced by the technical scheme can efficiently degrade organic phosphorus pesticide residue on leaves of vegetables in situ, promote the growth of the vegetables, reduce the content of nitrate and nitrite in the vegetables and guarantee food safety; and the technical scheme also can effectively recycle the potato starch wastewater, reduce emission of high-concentration wastewater and protect ecological environment.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method of utilizing high concentrated organic wastewater to cultivate subtilis.
Technical background
In recent years, China's Potato Industry development has all formed the industrialization base in many areas rapidly.The annual yam starch total amount of producing reaches more than 40 ten thousand tons, nearly 3,000,000 tons of processing potato, more than 800 ten thousand tons of waste discharges.These processing wastewater major parts are thrown in river and the reservoir, cause severe contamination.In the yam starch waste water, what pollute mainly is protein liquid, accounts for about 25% of waste water total amount.Main nutrient composition is protein, polysaccharide, mineral substance of potato itself etc. in the protein liquid, and total content is 4%-5.5%, and wherein protein content is 0.9%～2.1%, and chemical oxygen demand (COD) (COD) content is at 39000-45000mg/L.Its pollution mechanism and harm is, protein liquid is emitted on and discharges gases such as hydrogen sulfide, ammonia in the river reservoir after the spontaneous fermentation, and wafing has one foul smell in air; In water, because organic concentration is too high, various microorganism growth breedings are very fast, raised growth breeding comprising harmful microorganism or pathogenic bacterium, not only can directly encroach on hydrocoles, and because microbial growth and organic oxidizing reaction, the dissolved oxygen in the water is consumed totally, make hydrocoles because of anoxic dead (drawing certainly: " feed wide-angle " the 13rd phase in 2007,32-34 page or leaf).
(application number: 200810063868.0) method that discloses a kind of air supporting-UASB-SBR that offers medicine is handled starch wastewater to Chinese invention patent.This method mainly is by the suspended substance in the air flotation pool removal water, the air flotation pool water outlet flows into the UASB anaerobic reactor, most organism is decomposed into inorganic molecules material and methane, excess sludge enters sludge thickener, water outlet flows into the preaeration settling tank, and the water outlet gravity flow of preaeration settling tank enters SBR and carries out the aerobe processing.China's utility model patent (application number: 200820131168.6) disclose protein embrane method retrieving arrangement in a kind of waste water produced in potato starch manufacturing, be applicable to recovery of protein in the starch production wastewater, concentrate, the rate of recovery is more than 50%.China's utility model patent (application number: 200720126184.1) disclose a kind of nanofiltration and reclaimed oligose device in the waste water produced in potato starch manufacturing, be applicable to oligose in the waste water recovery, concentrate, the rate of recovery is more than 80%.China's utility model patent (application number: 200620005277.4) disclose and a kind of the proteinic waste water that contains that produces in the yam starch production process is carried out heat treated and extracts proteinic Equipment for Heating Processing.Do not find at present the method for bibliographical information as yet with yam starch waste water cultivation subtilis.
The strain that subtilis (Bacillus subtilis) N14 bacterial strain is our laboratory separation screening comes out from the blade face of rape has the degrading organic phosphor pesticides characteristic, and can promote the wild strain of plant-growth.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preservation date is on November 2nd, 2009, classification called after subtilis (Bacillus subtilis), and deposit number is: CGMCC No.3374.The 16S rDNA gene order of this bacterial strain has been delivered to the GenBank database, and accession number is GU086422.On http://www.ncbi.nlm.nih.gov/ website, compare with blast program and the 16S rDNA gene order of logining bacterial isolates, the result shows with the similarity of subtilis (Bacillus subtilis) the highest, can reach more than 99%.This bacterial strain can utilize the organic matter in the yam starch waste water to grow fast for nutrition.
Summary of the invention
The purpose of this invention is to provide a kind of yam starch waste water that utilizes and be main raw material, cultivate the method for subtilis (Bacillus subtilis) N14 bacterial strain, and be mixed with microbial inoculum, help preserving and agricultural application.
The production method of microbial inoculum of the present invention is as follows:
1, substratum preparation:
1) culture presevation substratum (solid): ammonium sulfate 0～3g, dipotassium hydrogen phosphate 0～2g, sal epsom 0～0.1g, agar 20g adds yam starch waste water to 1L, pH7.0-7.5;
2) strain activation and culture base (liquid): ammonium sulfate 0～3g, dipotassium hydrogen phosphate 0～2g, sal epsom 0～0.1g adds yam starch waste water to 1L, pH7.0-7.5;
3) seed culture medium (liquid): ammonium sulfate 0～30g, dipotassium hydrogen phosphate 0～20g, sal epsom 0～1.0g adds yam starch waste water to 10L, pH7.0-7.5;
4) fermention medium (liquid): ammonium sulfate 0～0.5kg, dipotassium hydrogen phosphate 0～0.4kg, sal epsom 0～20g adds yam starch waste water to 200L, pH7.0-7.5;
Above substratum is all at 121 ℃ of sterilization 15-30min.
2, bacterial classification switching and activation: the subtilis (Bacillus subtilis) that the picking inclined-plane is preserved is to the culture presevation culture medium flat plate, 35 ℃ rule, choose single bacterium colony continuously and cultivate twice after, picking list bacterium colony is in the strain activation and culture base, in 35 ℃, 150-200r/min shaking table shaking culture 12-24h;
3, liquid seeds preparation: in the fermentor tank of the seed culture medium that high-temperature sterilization is housed, according to the inoculum size inoculation step 2 activatory organophosphorus pesticide degradation bacteriums of 5%-20%, in 25-37 ℃, blowing air is cultivated 12-24h, obtains liquid seeds.
4, liquid state fermentation: in the fermentor tank of the fermention medium that high-temperature sterilization is housed, organophosphorus pesticide degradation bacterium liquid seeds according to inoculum size inoculation step 3 preparation of 5%-20%, in 25-37 ℃, blowing air is cultivated 18-60h, obtain viable bacteria body culture, cell concentration reaches more than 6,000,000,000 CFU/mL.
5, fungicide preparation: cultured fermented liquid adds glycine (massfraction 0.2%-2%) and glycerine (massfraction 0.2%-5%), the can preservation after bacterial count.Dilute with water 500-1000 doubly during use.
The subtilis (Bacillus subtilis) that is produced by the technical solution of the present invention residual organophosphorus pesticide of crop surface of can effectively degrading promotes plant growth, and low production cost, has also realized the resource utilization of high concentrated organic wastewater.Below describe enforcement of the present invention in detail by specific embodiment, purpose is to help the reader to understand spirit of the present invention better, but not as to the qualification of the scope of the present invention.
Embodiment 1: the cultural method of subtilis (Bacillus subtilis) N14 bacterial strain
1, substratum preparation:
1) culture presevation substratum (solid): ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, sal epsom 0.1g, agar 20g adds yam starch waste water to 1L, pH7.0-7.5;
2) strain activation and culture base (liquid): ammonium sulfate 2g, dipotassium hydrogen phosphate 2g, sal epsom 0.1g adds yam starch waste water to 1L, pH7.0-7.5;
3) seed culture medium (liquid): ammonium sulfate 20g, dipotassium hydrogen phosphate 20g, sal epsom 1.0g adds yam starch waste water to 10L, pH7.0-7.5;
4) fermention medium (liquid): ammonium sulfate 0.5kg, sal epsom 20g, dipotassium hydrogen phosphate 0.4kg adds yam starch waste water to 200L, pH7.0-7.5;
Above substratum is all at 121 ℃ of sterilization 15-30min.
2, bacterial classification switching and activation: the organophosphorus pesticide degradation bacterium that the picking inclined-plane is preserved is to the culture presevation culture medium flat plate, 35 ℃ rule, choose single bacterium colony continuously and cultivate twice after, picking list bacterium colony in the strain activation and culture base, in 35 ℃, 150r/min shaking table shaking culture 20h;
3, liquid seeds preparation: in the fermentor tank of the seed culture medium that high-temperature sterilization is housed, according to 5% inoculum size inoculation organophosphorus pesticide degradation bacterium, in 37 ℃, blowing air is cultivated 24h, obtains liquid seeds.
4, liquid state fermentation: in the fermentor tank of the fermention medium that high-temperature sterilization is housed, according to 10% inoculum size inoculation organophosphorus pesticide degradation bacterium liquid seeds, in 37 ℃, blowing air is cultivated 36h, obtains viable bacteria body culture, and cell concentration reaches 5,000,000,000 CFU/mL.
5, fungicide preparation: cultured fermented liquid adds glycine (massfraction 1%) and glycerine (massfraction 2%), the can preservation after bacterial count.Dilute with water 500-1000 doubly during use.
Embodiment 2: subtilis (Bacillus subtilis) N14 is to the degradation effect of rape blade face SD-1750 pesticide residue
1) chooses two identical rape plot, each 30m
2, evenly spray 80% DDT EC agricultural chemicals of 1000 times of dilutions;
2) behind the sprinkling SD-1750 12h, insect is killed, and evenly spraying pesticide degradation bacterial agent (with 600 times of clear water dilutions) compares to spray clear water;
3) behind the sprinkling microbial inoculum 24h, it is residual that five point samplings detect rape blade face SD-1750, the treatment group of discovery sprinkling microbial inoculum is compared with the control group that does not spray microbial inoculum, and the SD-1750 residual quantity significantly reduces, and is lower than the residual national limit standard of SD-1750 (0.1mg/kg) in the vegetables.
Embodiment 3: subtilis (Bacillus subtilis) N14 promotes growth of rape, improves rapeseed quality
1) chooses two identical rape plot, each 30m
2, every vegetable plot is all used urea according to 30 kilograms/mu rate of fertilizer application in the seedling phase, and wherein while applies the fermented liquid (consumption is according to 80 liters/mu) of subtilis (Bacillus subtilis) N14 bacterial strain again.
2) after 2 weeks of fertilising, pluck 30 in rape sample at random in every vegetable plot, weigh, and detect the content of nitrate and nitrite in the rape; The result shows, uses the rape of subtilis N14 bacterial strain fermentation liquor and compares with the contrast that does not apply fermented liquid, and strain heavily increases by 15%, and nitrate content reduces by 30%, and nitrite content reduces by 70%.
Subtilis used in the present invention (Bacillus subtilis) N14 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date is on November 2nd, 2009, classification called after subtilis (Bacillus subtilis).Deposit number is: CGMCC No.3374.
Claims (1)
1. utilize yam starch waste water to cultivate the method for subtilis (Bacillus subtilis), it is characterized in that: the fermentation training
Supporting base is main nutrient matter source with yam starch waste water, and culturing step comprises:
(1) substratum preparation:
1) culture presevation substratum (solid): ammonium sulfate 0～3g, dipotassium hydrogen phosphate 0～2g, sal epsom 0～0.1g, agar 20g adds yam starch waste water to 1L, pH 7.0-7.5;
2) strain activation and culture base (liquid): ammonium sulfate 0～3g, dipotassium hydrogen phosphate 0～2g, sal epsom 0～0.1g adds yam starch waste water to 1L, pH 7.0-7.5;
3) seed culture medium (liquid): ammonium sulfate 0～30g, dipotassium hydrogen phosphate 0～20g, sal epsom 0～1.0g adds yam starch waste water to 10L, pH 7.0-7.5;
4) fermention medium (liquid): ammonium sulfate 0～0.5kg, dipotassium hydrogen phosphate 0～0.4kg, sal epsom 0～20g adds yam starch waste water to 200L, pH 7.0-7.5;
Above substratum is all at 121 ℃ of sterilization 15-30min;
(2) bacterial classification switching and activation: the subtilis (Bacillus subtilis) that the picking inclined-plane is preserved is to the culture presevation culture medium flat plate, 35 ℃ rule, choose single bacterium colony continuously and cultivate twice after, picking list bacterium colony is in the strain activation and culture base, in 35 ℃, 150-200r/min shaking table shaking culture 12-24h;
(3) liquid seeds preparation: in the fermentor tank of the seed culture medium that high-temperature sterilization is housed, according to the inoculum size inoculation step 2 activatory organophosphorus pesticide degradation bacteriums of 5%-20%, in 25-37 ℃, blowing air is cultivated 12-24h, obtains liquid seeds;
(4) liquid state fermentation: in the fermentor tank of the fermention medium that high-temperature sterilization is housed, organophosphorus pesticide degradation bacterium liquid seeds according to inoculum size inoculation step 3 preparation of 5%-20%, in 25-37 ℃, blowing air is cultivated 18-60h, obtain viable bacteria body culture, cell concentration reaches more than 6,000,000,000 CFU/mL;
(5) fungicide preparation: cultured fermented liquid adds glycine (massfraction 0.2%-2%) and glycerine (massfraction 0.2%-5%), the can preservation after bacterial count.Dilute with water 500-1000 doubly during use.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965293A (en) * | 2011-09-01 | 2013-03-13 | 中国科学院生态环境研究中心 | Production of organophosphorus pesticide degrading bacterial agent by citric acid waste water |
| CN103931660A (en) * | 2013-01-17 | 2014-07-23 | 无锡中科活力生物技术有限公司 | Method utilizing critic acid wastewater to produce bacillus amyloliquefaciens bio-control inoculant |
| CN104263783A (en) * | 2014-10-09 | 2015-01-07 | 哈尔滨艾克尔食品科技有限公司 | Method for preparing protein peptide through waste water produced from potato starch producing |
| CN105779335A (en) * | 2016-03-15 | 2016-07-20 | 甘肃省农业科学院生物技术研究所 | Formula and preparation method of micro-ecological deodorant |
| CN106167777A (en) * | 2016-08-05 | 2016-11-30 | 四川农业大学 | A kind of cultural method of the bacillus amyloliquefaciens producing Substance |
| CN106434418A (en) * | 2016-08-05 | 2017-02-22 | 四川农业大学 | Culture medium for bacillus amyloliquefaciens culture medium for producing bacteriostatic active substances |
| CN106867937A (en) * | 2017-03-11 | 2017-06-20 | 鲁东大学 | With the bacterial strain of pea protein wastewater liquid fermenting and producing Bacillus subtilis natto microbial inoculum, method and application |
| CN108277183A (en) * | 2018-03-16 | 2018-07-13 | 中科美大（北京）生态环境工程有限公司 | A method of preparing animal probiotics using potato starch wastewater |
| CN110643654A (en) * | 2019-10-17 | 2020-01-03 | 浙江大学 | Method for producing polysaccharide by fermenting starch wastewater |
| CN111057667A (en) * | 2019-12-24 | 2020-04-24 | 天津科技大学 | Complex microbial inoculant and preparation method and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965293A (en) * | 2011-09-01 | 2013-03-13 | 中国科学院生态环境研究中心 | Production of organophosphorus pesticide degrading bacterial agent by citric acid waste water |
| CN103931660A (en) * | 2013-01-17 | 2014-07-23 | 无锡中科活力生物技术有限公司 | Method utilizing critic acid wastewater to produce bacillus amyloliquefaciens bio-control inoculant |
| CN103931660B (en) * | 2013-01-17 | 2018-08-24 | 无锡中科活力生物技术有限公司 | Bacillus amyloliquefaciens biocontrol agent is produced using citric acid wastewater |
| CN104263783A (en) * | 2014-10-09 | 2015-01-07 | 哈尔滨艾克尔食品科技有限公司 | Method for preparing protein peptide through waste water produced from potato starch producing |
| CN105779335A (en) * | 2016-03-15 | 2016-07-20 | 甘肃省农业科学院生物技术研究所 | Formula and preparation method of micro-ecological deodorant |
| CN106167777A (en) * | 2016-08-05 | 2016-11-30 | 四川农业大学 | A kind of cultural method of the bacillus amyloliquefaciens producing Substance |
| CN106434418A (en) * | 2016-08-05 | 2017-02-22 | 四川农业大学 | Culture medium for bacillus amyloliquefaciens culture medium for producing bacteriostatic active substances |
| CN106867937A (en) * | 2017-03-11 | 2017-06-20 | 鲁东大学 | With the bacterial strain of pea protein wastewater liquid fermenting and producing Bacillus subtilis natto microbial inoculum, method and application |
| CN108277183A (en) * | 2018-03-16 | 2018-07-13 | 中科美大（北京）生态环境工程有限公司 | A method of preparing animal probiotics using potato starch wastewater |
| CN110643654A (en) * | 2019-10-17 | 2020-01-03 | 浙江大学 | Method for producing polysaccharide by fermenting starch wastewater |
| CN111057667A (en) * | 2019-12-24 | 2020-04-24 | 天津科技大学 | Complex microbial inoculant and preparation method and application thereof |
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Application publication date: 20110720 |