CN1186275C - Long-acting microbial water purifying agent and its producing method - Google Patents
Long-acting microbial water purifying agent and its producing method Download PDFInfo
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- CN1186275C CN1186275C CNB021349983A CN02134998A CN1186275C CN 1186275 C CN1186275 C CN 1186275C CN B021349983 A CNB021349983 A CN B021349983A CN 02134998 A CN02134998 A CN 02134998A CN 1186275 C CN1186275 C CN 1186275C
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Abstract
The present invention provides a long-acting microbial water purifying agent and a producing method thereof, wherein the long-acting microbial water purifying agent is a kind of microbial fermentation liquid, wherein the long-acting microbial water purifying agent contains bacillus licheniformis AS1.813, bacillus megatherium ACCC10008, pseudomonas fluorescens AS1.867, pseudomonas putida AS1.1003, streptobacillus AB93179, and geotrichum candidum AS2.1175, and the total count of the six kinds of bacteria can reach more than 5.0*10<9>cfu/ml. The present invention has the producing method that the six kinds of the bacteria are used as strains, after the slant activating culture and the first level liquid activating culture for the six kinds of the strains are respectively carried out, the mixing activating culture is carried out in a seed tank, the strains enter a large fermentation tank to carry out the mixed fermentation culture, nutrient solution is properly supplied during the fermentation period, and the total count of the six strains reach more than 5.0*10<9>cfu/ml. The product can be used for water quality purification of an aquatic culture water body and can be used for the overall process of freshwater culture and marine culture.
Description
Technical field
The present invention relates to a kind of microbial water-purifying agent and production method thereof that is used for aquaculture, environmental protection.
Background technology
Microbial water-purifying agent of the present invention belongs to a type of EM.EM is the english abbreviation of Effective Microorganisms, i.e. efficacious microbial colony, and to be Japan at first teach in innovation and creation at the beginning of the eighties than good according to the husband for it, at that time by 10 genus, more than 80 kind of a kind of complex microorganism preparations that microorganism is formed.Because this product is the beneficial microorganism complex body, thus it does not contain any chemical objectionable impurities, have no side effect, free from environmental pollution.The EM technology can be used for improving the throughput of agricultural, animal husbandry, forestry, water industry; administering aspects such as environment, protection environment and human health care; huge, irreplaceable effect and potentiality are all arranged; be described as present range of influence maximum, Application Areas the strongest the widest, comprehensive high-new biotechnology, have remarkable economic efficiency, ecological benefits, social benefit and application prospects.At present, the EM technology not only in Japan's wide popularization and application, is also drive on boldly to field depths such as farming, forestry, husbandary and fishing, environmental protection, medical treatment, industry in more than 90 countries and regions such as the U.S., Thailand, Brazil, France.China introduced since 1991, at present in Guangdong, provinces such as Jiangxi, Sichuan, Fujian have carried out a lot of production applications.The EM technology possesses following function on aquaculture: 1. obviously improve day weight gain; 2. eqpidemic disease there is peculiar prevention effect; 3. obviously improve breeding potential; 4. eliminate the feces of livestock and poultry stench.EM can make the ight soil recycle, with farming, woods, herd, fishing etc. interweaves, and forms the efficient synthesized environmental protection chain that the oneself purifies, makes full use of.The aspect of environmental protection simultaneously also has powerful effect: purify water, make water circulation use, decomposing residual agricultural chemicals. the water that purifies rural well water or pond pollutes, so that drink more healthy water, guarantee human health, reduce pathogenicity bo derivative and bad algae, increase biophase, reduce the generation of mud, reduce stench.
When specifically carrying out EM production, concrete purposes according to product, carry out the optimization of microorganism species, simultaneously concrete production method is also varied, total target all is in order to reduce production costs, production method is simple as far as possible, produces the efficacious microbial colony of high density at short production cycle as far as possible.In the existing microorganism water purification agent producing process, easy for what cultivate, produce, usually adopt single or two to three bacterial classifications, ferment under certain condition, because the substratum versatility that adopts is relatively poor, make different strain extent of growth differ greatly, often have a certain bacterial classification that to a certain degree growth is arranged with different growth characteristics, and the difficult growth of other bacterial classification; Even single culture, because substratum collocation and zymotechnique is unreasonable, its bacteria concentration is not high yet, fermentation period is also long relatively partially, thereby make that the result of use of microbial water-purifying agent is not too remarkable, the time of guaranteeing the quality simultaneously is also shorter, thereby prior art has greatly limited the production of the significant product of this class function of viable bacteria microbial water-purifying agent and applies.
The purpose of this invention is to provide a kind of long-acting microbial water purifying agent and production method thereof, to overcome deficiency and the problem that prior art exists with high density efficacious microbial colony.
Long-acting microbial water purifying agent provided by the present invention is the microbial fermentation solution that is made by the inoculation of macrospecies amount, fermentation by six kinds of different microorganisms, contain Bacillus licheniformis AS1.813, bacillus megaterium ACCC10008, the false pseudomonas bacillus AS1.867 of fluorescent, pseudomonas putida AS1.1003, streptobacillus AB93179 and geotrichum candidum AS2.1175 in this fermented liquid, the total count of six kinds of bacterium reaches 5.0 * 10
9More than the cfu/ml.
The physical resource of described six kinds of bacterium is: 1, Bacillus licheniformis AS1.813 (Bacillus licheniformis), be preserved in CGMCC, Chinese common micro-organisms DSMZ; Bacillus megaterium ACCC10008 (Bacillusmegatherium de Bary) is preserved in CGMCC, Chinese common micro-organisms DSMZ; Geotrichum candidum AS2.1175 (Geotrichum candidum Link) is preserved in CGMCC, Chinese common micro-organisms DSMZ; Pseudomonas putida AS1.1003 (Pseudomonas putida) is preserved in CGMCC, Chinese common micro-organisms DSMZ; Streptobacillus AB93179 (Streptobacillus sp)., be preserved in CCTCC, Chinese typical culture collection center; The false pseudomonas bacillus AS1.867 (Pseudomonas fluorescens Migula) of fluorescent is preserved in CGMCC, Chinese common micro-organisms DSMZ.
(write by China Committee for Culture Collection of Microorganisms in " Chinese bacterial classification catalogue " for above-mentioned bacterial classification, nineteen eighty-three, light industry press publishes) and " Chinese typical culture collection center culture catalogue " (in September, 1998 Wuhan University, the chief editor: open record Tao Tianshen), wherein: Bacillus licheniformis AS1.813 (Bacillus licheniformis), bacillus megaterium ACCC10008 (Bacillus megatherium de Bary), geotrichum candidum AS2.1175 (Geotrichum candidumLink), pseudomonas putida AS1.1003 (Pseudomonas putida), the false pseudomonas bacillus AS1.867 of fluorescent (Pseudomonasfluorescens Migula) is documented in the P55 of " Chinese bacterial classification catalogue " respectively, P57, P293, P166, the pages such as P165; (Streptobacillus sp. is documented in the P115 page of " Chinese typical culture collection center culture catalogue " to streptobacillus AB93179.
Long-acting microbial water purifying agent of the present invention adopts following method to produce preparation: with Bacillus licheniformis AS1.813, bacillus megaterium ACCC10008, the false pseudomonas bacillus AS1.867 of fluorescent, pseudomonas putida AS1.1003, streptobacillus AB93179 and geotrichum candidum AS2.1175 is bacterial classification, with described six kinds of bacterial classifications respectively after inclined-plane, level liquid activation culture, carrying out admixture activation at seeding tank cultivates, insert again and carry out mixing fermentation culture in the big fermentor tank, make the total count of six kinds of bacterium reach 5.0 * 10
9More than the cfu/ml.
Described six kinds of bacterial classifications are in the technology of seeding tank activation culture, and every kind of bacterium is with identical inoculum size inoculation.
Carry out in the technology of fermentation culture at big fermentor tank, preferably adopt the following substratum of composition (calculating): glucose 15～25, urea 1～3 by every liter of contained grammes per square metre g/l of substratum, yeast powder 2～5, corn steep liquor 4～8, peptone 8～12, yeast extract paste 8～12, pH7.0; And adopt Continuous Flow to add the nutritional supplementation substratum of high density during the fermentation, and its component is (weight percent): glucose 35～45, and yeast extract paste 15～25, all the other are water, pH7.0.
Concrete fermentation technology process of the present invention is:
A) open the freeze-drying pipe of Bacillus licheniformis AS1.813, bacillus megaterium ACCC10008, geotrichum candidum AS2.1175, pseudomonas putida AS1.1003, streptobacillus AB93179, six kinds of bacterial classifications of the false pseudomonas bacillus AS1.867 of fluorescent respectively, select single bacterium colony that growth is the fastest, bacterium colony is maximum with ordinary method, be inoculated on the preservation inclined-plane with inoculating needle and cultivate;
B) slant strains activation: cultured preservation inclined-plane kind is lined respectively on the above-mentioned different productions inclined-plane of above new preparation and activate;
C) level liquid of bacterial classification activation: above-mentioned cultured activated inclined plane is inoculated respectively with transfering loop encircle in different level liquid activation mediums and cultivate, make the bacterium number of liquid strain reach 2.0 * 10
9More than the cfu/ml;
D) activation of seeding tank secondary liquid is planted and is cultivated: above-mentioned cultured respectively six kinds of level liquids activation is planted shake up back balanced mix under aseptic condition, then by 1～5% inoculum size, insert in the seeding tank secondary liquid activation medium, aerated culture 8～10 hours treats that the bacterium number reaches 1.0 * 10
9More than the cfu/ml, stop to cultivate;
E) cultivation in the big fermentor tank: will above-mentioned cultured secondary liquid activation kind be equipped with in the fermentor tank that disinfects substratum by 10～20% inoculum size access and cultivate, 30～36 ℃, rotating speed 180～250r/m, air quantity are 20～30M
3Air/M
3Substratum hour carried out aerated culture 6～8 hours, began stream after fermenting simultaneously 1～2 hour and added, and flow acceleration is by 0.01～0.03M
3Nutritional supplementation substratum/M
3Fermented liquid hour, fermenting stops stream after 4～5 hours and adds.Treat that the bacterium number reaches 5.0 * 10
9More than the cfu/ml, stop fermentation culture.
The present invention adds the bacterium of having gone out, dissolving high concentration glucose and yeast extract paste solution well by proper flow in the inoculation of six kinds of equal macrospecies amounts of different microorganisms, the fermenting process, make in the fermented liquid carbon source relative constant with the former nutrition of nitrogen, simultaneously, the fermentation whole process adopts constant temperature, big air quantity technology, thereby make that six kinds of bacterium all can balanced, growth fast, this production method zymotechnique is simple, fermentation period short, fermentation finishes, and main Nutrious fermented composition runs out of substantially, the concentration bacterium of every kind of bacterium reaches 10 after the fermentation ends
9More than the cpu/ml.The microbial water-purifying agent product that this technology is produced because have that above characteristics are all higher as the concentration of six kinds of bacterium, residual main Nutrious fermented composition is few etc. in the product, thereby have long quality-guarantee period, result of use and significantly reach advantages such as long-lasting.This technology has overcome the deficiencies in the prior art effectively.This product is used for the purification of water quality of aquaculture water in aquatic products, light, sea farming whole process all can be used.Every mu (1 meter of the depth of water) discharges 1 kilogram first, later weight according to the water pollution degree, every 20-30 days dispensing 0.5-1 kilograms.Can keep water quality condition good for a long time.The environmental protection aspect according to the weight of water pollution degree, applies different consumptions.
Embodiment
Use 6M
3Fermentor tank is produced long-acting microbial water purifying agent, and technological process is as follows:
1. activated inclined plane bacterial classification: with inoculating needle with 1. Bacillus licheniformis AS1.813 (Bacilluslicheniformis) of the bacterial classification of preservation, 2. bacillus megaterium ACCC10008 (Bacillus megatherium de Bary), 3. the false pseudomonas bacillus AS1.867 (Pseudomonas fluorescens Migula) of fluorescent, 4. pseudomonas putida AS1.1003 (Pseudomonas putida), 5. streptobacillus AB93179 (Streptobacillus sp.) inoculate respectively one the ring line on the above-mentioned production inclined-plane of above new preparation, the chemical constitution on production inclined-plane following (g/L): glucose 20, urea 4.0, corn steep liquor 6, peptone 10, yeast extract paste 10, agar 20, pH7.0.Each kind respectively connects 3, cultivates 18 hours in 32 ℃; With inoculating needle the bacterial classification geotrichum candidum AS2.1175 of preservation being inoculated a ring respectively lines on the above-mentioned production inclined-plane of above new preparation, connect 3 production inclined-planes, the chemical constitution on production inclined-plane following (g/L): 6Bx ° of wort 1000ml, agar 20, the pH nature was cultivated 24 hours in 28 ℃.
2. the level liquid of bacterial classification activation: cultured activated inclined plane is inoculated respectively with transfering loop encircle in above-mentioned different level liquid activation mediums, wherein 1.～5. number kind level liquid activation medium chemical constitution following (g/L): glucose 20, urea 4.0, corn steep liquor 6, peptone 10, yeast extract paste 10, pH7.0.Substratum is loaded in the 1000ml triangular flask, and the triangular flask loading amount is 40ml, and each kind respectively connects 18 bottles, and in 33 ℃ of reciprocating type shaking table shaking culture 15 hours, shaking speed was 110r/m, and stroke is 75mm, makes the bacterium number of liquid strain reach 2.0 * 10
9More than the cfu/ml; 6. number kind geotrichum candidum AS2.1175 encircles in corresponding level liquid activation medium with inoculation articulating two, the liquid activation medium is loaded in the 1000ml triangular flask, the triangular flask loading amount is 100ml, connect 7 bottles, in 28 ℃ of reciprocating type shaking table shaking culture 24 hours, shaking speed is 120r/m, and stroke is 75mm, makes the bacteria concentration of liquid strain reach 2.0 * 10
9More than the cfu/ml.
3. the cultivation of secondary liquid activation kind of (600L seeding tank): above-mentioned cultured respectively six kinds of level liquids activation kind is shaken up back each kind under aseptic condition get the 670ml balanced mix, insert then in the secondary liquid activation medium (seeding tank), the chemical constitution following (g/L) of secondary liquid activation kind of substratum: glucose 20, urea 2, yeast powder 5, corn steep liquor 10, peptone 10, yeast extract paste 10, pH7.0.The canned 400L substratum of 600L seed, 33 ℃, rotating speed 200r/m, air quantity are 2M
3/ hour, carry out aerated culture, keep tank pressure 0.1Mpa; Since six hours, detect bacteria concentration by microscopy in conjunction with optical density(OD) (OD), treat that the bacterium number reaches 1.0 * 10
9More than the cfu/ml, stop to cultivate.
4. the cultivation of complex microorganism preparations in big fermentor tank: 4M is equipped with in above-mentioned cultured respectively secondary liquid activation kind of a 400L access
3Disinfected in the fermentor tank of substratum and cultivated fermention medium component (g/L): glucose 20, urea 2, yeast powder 3, corn steep liquor 6, peptone 10, yeast extract paste 10, pH7.0.34 ℃, rotating speed 220r/m, air quantity are 140M
3Air/hour, carried out aerated culture 6-8 hour, began stream after fermenting simultaneously 1.5 hours and add, flow acceleration is pressed 0.041M
3The nutritional supplementation substratum/hour, fermenting stops stream after 5 hours and adds, and keeps tank pressure 0.1Mpa.Since 4 hours, treat that bacteria concentration reaches 5.0 * 10
9More than the cfu/ml, stop fermentation culture.
5. make finished product: after stopping fermentation culture, at first turn down ventilation, keep tank pressure 0.02Mpa, rotating speed is reduced to 100r/m from 220r/m, begins to put jar, adopts aseptic technique simultaneously, puts into the jar of packing of different volumes with 4 flexible pipes directly, fast.
Claims (5)
1. long-acting microbial water purifying agent, be a kind of microbial fermentation solution, it is characterized in that wherein containing Bacillus licheniformis AS1.813, bacillus megaterium ACCC10008, the false pseudomonas bacillus AS1.867 of fluorescent, pseudomonas putida AS1.1003, streptobacillus AB93179 and geotrichum candidum AS2.1175, the total count of six kinds of bacterium reaches 5.0 * 10
9More than the cfu/ml.
2. the preparation method of the described long-acting microbial water purifying agent of claim 1, it is characterized in that with Bacillus licheniformis AS1.813, bacillus megaterium ACCC10008, the false pseudomonas bacillus AS1.867 of fluorescent, pseudomonas putida AS1.1003, streptobacillus AB93179 and geotrichum candidum AS2.1175 be bacterial classification, with described six kinds of bacterial classifications respectively after inclined-plane, level liquid activation culture, carrying out admixture activation at seeding tank cultivates, insert again and carry out mixing fermentation culture in the big fermentor tank, make the total count of six kinds of bacterium reach 5.0 * 10
9More than the cfu/ml.
3. the preparation method of long-acting microbial water purifying agent according to claim 2 is characterized in that described six kinds of bacterial classifications in the technology of seeding tank activation culture, and the inoculum size of every kind of bacterium is identical.
4. the preparation method of long-acting microbial water purifying agent according to claim 2, it is characterized in that carrying out in the technology of fermentation culture at big fermentor tank, the substratum composition is calculated as follows by every liter of contained grammes per square metre g/l of substratum: glucose 15～25, urea 1～3, yeast powder 2～5, corn steep liquor 4～8, peptone 8～12, yeast extract paste 8～12, pH7.0; And adopt Continuous Flow to add the nutritional supplementation substratum of high density during the fermentation, and the weight percent of its component is: glucose 35～45, and yeast extract paste 15～25, all the other are water, pH7.0.
5. according to the preparation method of claim 2,3 or 4 described long-acting microbial water purifying agents, it is characterized in that concrete fermentation technology process is:
A) open the freeze-drying pipe of Bacillus licheniformis AS1.813, bacillus megaterium ACCC10008, geotrichum candidum AS2.1175, pseudomonas putida AS1.1003, streptobacillus AB93179, six kinds of bacterial classifications of the false pseudomonas bacillus AS1.867 of fluorescent respectively, select single bacterium colony that growth is the fastest, bacterium colony is maximum with ordinary method, be inoculated on the preservation inclined-plane with inoculating needle and cultivate;
B) slant strains activation: cultured preservation inclined-plane kind is lined respectively on the above-mentioned different productions inclined-plane of above new preparation and activate;
C) level liquid of bacterial classification activation: above-mentioned cultured activated inclined plane is inoculated respectively with transfering loop encircle in different level liquid activation mediums and cultivate, make the bacterium number of liquid strain reach 2.0 * 10
9More than the cfu/ml;
D) activation of seeding tank secondary liquid is planted and is cultivated: above-mentioned cultured respectively six kinds of level liquids activation is planted shake up back balanced mix under aseptic condition, then by 1～5% inoculum size, insert in the seeding tank secondary liquid activation medium, aerated culture 8～10 hours treats that the bacterium number reaches 1.0 * 10
9More than the cfu/ml, stop to cultivate;
E) cultivation in the big fermentor tank: will above-mentioned cultured secondary liquid activation kind be equipped with in the fermentor tank that disinfects substratum by 10～20% inoculum size access and cultivate, 30～36 ℃, rotating speed 180～250r/m, air quantity are 20～30M
3Air/M
3Substratum hour carried out aerated culture 6～8 hours, began stream after fermenting simultaneously 1～2 hour and added, and flow acceleration is by 0.01～0.03M
3Nutritional supplementation substratum/M
3Fermented liquid hour, fermenting stops stream after 4～5 hours and adds, and treats that the bacterium number reaches 5.0 * 10
9More than the cfu/ml, stop fermentation culture.
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Cited By (1)
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| CN102168032A (en) * | 2010-10-29 | 2011-08-31 | 中国水产科学研究院南海水产研究所 | Staphylococcus bacterial strain HGMC 412 for degrading marine organic matters and application thereof |
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| CN102168031B (en) * | 2010-10-29 | 2012-09-12 | 中国水产科学研究院南海水产研究所 | Marine organic matter degradation Halomonas sp. strain GHMC414 and application thereof |
| CN102219310B (en) * | 2011-04-11 | 2013-06-19 | 武汉中傲天乐生物科技有限公司 | Environment-friendly composite microbial preparation, preparation thereof, and application thereof to aquaculture |
| CN103484413A (en) * | 2013-10-14 | 2014-01-01 | 青岛蔚蓝生物集团有限公司 | Pseudomonas putida strain and application thereof |
| CN103964582A (en) * | 2014-01-22 | 2014-08-06 | 刘佳 | Water quality modifier |
| CN104232527A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus megaterium preparation |
| CN104388348A (en) * | 2014-11-21 | 2015-03-04 | 上海寄绿生物环保科技有限公司 | Microbial preparation for purifying sewage and deodorizing garbage and preparation method thereof |
| CN106676021A (en) * | 2015-11-06 | 2017-05-17 | 丹阳市尚德生物科技有限公司 | Microbial preparation for river course water quality restoration, and preparation method thereof |
| US10174283B2 (en) * | 2017-01-12 | 2019-01-08 | Cisbay Global Inc. | Method for cleaning water dispensers via use of selectively bred and cultivated generations of microbes |
| CN107251859A (en) * | 2017-05-25 | 2017-10-17 | 江苏沃纳生物科技有限公司 | A kind of preparation of multi-functional microbial inoculum compound of promotion shrimp crab shell and application process |
| CN116711808B (en) * | 2023-05-29 | 2024-10-15 | 广东鼎烨生态环保产业有限公司 | Microbial feed additive and preparation method thereof |
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2002
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102168032A (en) * | 2010-10-29 | 2011-08-31 | 中国水产科学研究院南海水产研究所 | Staphylococcus bacterial strain HGMC 412 for degrading marine organic matters and application thereof |
| CN102168032B (en) * | 2010-10-29 | 2012-08-29 | 中国水产科学研究院南海水产研究所 | Staphylococcus bacterial strain HGMC 412 for degrading marine organic matters and application thereof |
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