CN105602995A - Method for preparing Enteromorpha bio-fertilizer through liquid deep quick fermentation - Google Patents
Method for preparing Enteromorpha bio-fertilizer through liquid deep quick fermentation Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a method for preparing Enteromorpha bio-fertilizer through liquid deep quick fermentation. The method mainly includes steps activation of culture, cultivation of seed liquid, liquid deep quick fermentation and preparation of the Enteromorpha bio-fertilizer. In the aspect of fermentation preparation, liquid deep fermentation technology is combined with special fermentation equipment like a bio-reactor, so that realtime monitoring of the fermentation process is realized and yield of a target product is increased while the thallus metabolism process is regulated and controlled effectively. By using the fermentation preparation method, solid enteromorpha powder can be directly used as a main raw material for fermentation, so that fermentation period is shortened remarkably, production cost is lowered, and needs of industrialized production are met.
Description
Technical field
The invention belongs to technical field of biological fermentation, relate to the microbial fermentation production method of Enteromorpha bio-fertilizer, relate in particular to the method that deep liquid Rapid Fermentation is prepared Enteromorpha bio-fertilizer.
Background technology
Liquid deep layer fermenting typically refer to bioreactors such as utilizing fermentation tank to fluid nutrient medium and material continuously ferment, the operation such as batch fermentation or fed-batch fermentation, in Real-Time Monitoring sweat, biological metabolism is adjusted in time in changing and is synthetic controlling product, thereby effectively improves substrate utilization ratio and product yield. Than traditional shake flask fermentation, the feature such as liquid deep layer fermenting technology has with short production cycle, and productive rate is high, and production process is easily controlled, constant product quality. Therefore prepare aspect object product and have a good application prospect in microorganism fermentation scale.
Along with the exploitation of marine resources, marine alga processing and higher value application thereof embody great commercial value gradually. Seaweed organism fertilizer is the novel sea biologic product taking marine algae extract or fermentation of seaweed thing as core substance. Wherein contain a large amount of organic matters, vitamin, the peculiar algal polysaccharides of mineral matter element and marine alga self, alga oligosaccharides, alginic acid and multiple natural plants growth regulator, can play beneficial functional to the improvement of plant growth and soil quality. Along with the appearance in succession of different seaweed bio fertilizer products, its rapid scale manufacture mode becomes study hotspot gradually.
Li Guoting etc. are taking natural seaweed as primary raw material, be equipped with a small amount of potassium hydroxide and under condition of high voltage, carry out alkaline hydrolysis to destroy the string in marine alga, thereby discharge the natural plants such as seaweeds, gibberellin growth regulatory substance, by the composite seaweed manure of preparing. Although simple to operate, this Mechanical Method energy consumption is higher and product yield is lower, be not suitable for realizing the scale preparation of product; Wang Mingpeng etc. mention chemical methodes such as adopting acid, alkali and organic solvent and process alginic cell in the research of seaweed organism fertilizer, cell are cleared up or endogenous substance solubilising can effectively extract the in-house active component of marine alga, for the preparation of seaweed bio fertilizer. But this type of chemical method reagent is residual serious, and biological safety is poor, and environment is existed to huge potential hazard; By contrast, microbe fermentation method because of its biological safety is good, its lytic activity component content is high and rapidly and efficiently single-minded feature become gradually the main way of producing seaweed organic fertilizer.
Enteromorpha marine stock biomass is huge, and traditional shake flask fermentation technique is loaded down with trivial details, and productive rate is lower, and scale can not be satisfied with the needs of actual production far away. The present invention applies to liquid deep layer fermenting technology the preparation of Enteromorpha bio-fertilizer; and the main component using solid waterside lichenin as fermentation medium of novelty; not only simplify greatly the configuration of fermentation medium; and contribute to the large-scale production of Enteromorpha bio-fertilizer, for its later stage suitability for industrialized production has been made good technology place mat.
Do not prepare at present report and the patent of Enteromorpha bio-fertilizer about the deep liquid Rapid Fermentation relevant to the application.
Summary of the invention
The present invention aims to provide a kind of method that deep liquid Rapid Fermentation is prepared Enteromorpha bio-fertilizer; first fermentation process of the present invention activates bacterial strain; and by bioreactor real-time monitoring fermentation condition; taking solid waterside lichenin as raw material, realize the scale preparation of Enteromorpha bio-fertilizer through liquid fermentation, Separation of Solid and Liquid and complex process.
For achieving the above object, the present invention is achieved by the following technical solutions:
Deep liquid Rapid Fermentation is prepared a method for Enteromorpha bio-fertilizer, and described method comprises following concrete steps:
(1) actication of culture: inoculation is carried out in activation medium to activation culture, initial pH6.8-7.2, cultivation temperature 30-32 DEG C, shaking speed 160-200rpm, incubation time 36-48h;
(2) preparation of seed liquor: the culture medium after actication of culture in step (1) is inoculated in seed culture medium and cultivates and obtain seed liquor with 5-8% inoculum concentration, initial pH6.5-7.2, cultivation temperature is 28-30 DEG C, shaking speed 160-200rpm, incubation time 30-36h;
(3) deep liquid Rapid Fermentation: seed liquor described in step (2) is inoculated in to the fermentation of carrying out Enteromorpha bio-fertilizer in the fermentation medium of bioreactor with 8-10% inoculum concentration and prepares zymotic fluid, liquid amount is the 50-70% of biological reactor volume, pH6.8-7.5, cultivation temperature 24-28 DEG C, rotating speed of agitator 200-600rpm, throughput 0.5-2.5vvm, dissolved oxygen 30-70%, incubation time 18-24h;
(4) preparation of Enteromorpha bio-fertilizer: the zymotic fluid in step (3) is carried out to Separation of Solid and Liquid, liquid part is collected and make described Enteromorpha bio-fertilizer by a great number of elements, trace element and nutriment composite.
Further improvement to technique scheme: described bacterial strain is that deposit number is the alternately monad A321 of CCTCCNO:M2012132.
Further improvement to technique scheme: described activation medium consists of: by percentage to the quality, sea grass polysaccharide 0.5-2.0%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, surplus is water.
Further improvement to technique scheme: described seed culture medium consists of: by percentage to the quality, sea grass polysaccharide 0.5-2.0%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, surplus is water.
Further improvement to technique scheme: described fermentation medium consists of: by percentage to the quality, particle diameter is 40-200 object solid waterside lichenin 1.0-8.0%, organic nitrogen source 0.01-0.05%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, surplus is water.
Further improvement to technique scheme: described sea grass polysaccharide obtains by hot-water process's lixiviate or autoclaving lixiviate.
Further improvement to technique scheme: in described step (1), the liquid amount of activation medium is 25mL/100mL.
Be one or more in beef extract, yeast extract and peptone to the described organic nitrogen source of further improvement of technique scheme.
Further improvement to technique scheme: the soluble polysaccharide content in described step (3) in zymotic fluid is 0.2-1.2%, content of reducing sugar is 0.02-0.3%, and protein content is 0.1-0.6%, and the content of organic matter is 0.5-3.0%, solid content is 1.0-5.0%, by percentage to the quality.
Further improvement to technique scheme: in described step (4), zymotic fluid is realized Separation of Solid and Liquid by plate-frame filtering in conjunction with the method for low-speed centrifugal.
Method provided by the invention has following characteristics:
(1) taking large-scale green damp algae Enteromorpha solid as primary raw material, wide material sources, marine stock biomass is huge; (2) pass through deep liquid fast fermentation in fixed in conjunction with bioreactor, improved greatly substrate utilization ratio and product yield; (3) in zymotic fluid, contain the Plant growth-promoting effect factors such as numerous active polysaccharides, oligosaccharides and organic matter, the Enteromorpha bio-fertilizer of preparing in conjunction with process for formulating has good environment friendly and biological safety.
Detailed description of the invention
Below in conjunction with specific embodiment, technical scheme of the present invention is further described in detail.
Embodiment 1
In the present invention, bacterial strain uses therefor is the alternately monad A321(separating in the seawater of Qingdao sea areaAlteromonascolwellianaA321), alternately monad A321 was deposited in the Chinese Typical Representative culture collection center (CCTCC) that is positioned at Wuhan, China Wuhan University on April 23rd, 2012, and deposit number is CCTCCNO:M2012132.
The described alternately morphological feature of monad A321 and the application for a patent for invention that physiological and biochemical property is CN103451119A referring to publication number.
1. actication of culture: described alternately monad A321 inoculation is carried out to activation culture in activation medium, culture medium liquid amount 25mL/100mL, initial pH6.8,30 DEG C of cultivation temperature, shaking speed 180rpm, incubation time 48h;
Described activation medium consists of: sea grass polysaccharide 0.5-2.0%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, all the other compositions are water. Sea grass polysaccharide of the present invention obtains by hot-water process's lixiviate (extracting in boiling water 1-4 hour) or autoclaving lixiviate (115 DEG C are extracted 30min).
2. the preparation of seed liquor: the culture medium after actication of culture in step 1 is inoculated in seed culture medium and cultivates and obtain seed liquor with 6% inoculum concentration, initial pH7.0, cultivation temperature is 28 DEG C, shaking speed 180rpm, incubation time 36h;
Described seed culture medium consists of: sea grass polysaccharide 0.5-2.0%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, all the other compositions are water.
Seed culture medium specifically can be divided into first order seed culture medium and secondary seed medium according to the size of fermentation scale.
3. deep liquid Rapid Fermentation: seed liquor described in step 2 is inoculated in to the fermentation preparation of carrying out Enteromorpha bio-fertilizer in the fermentation medium of bioreactor with 8% inoculum concentration, liquid amount is 50% of biological reactor volume, pH6.8,26 DEG C of cultivation temperature, rotating speed of agitator 200rpm, throughput 1.5vvm, dissolved oxygen 60%, incubation time 24h;
Described fermentation medium consists of: particle diameter is 40-200 object solid waterside lichenin 1.0-8.0%, organic nitrogen source 0.01-0.05%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, all the other compositions are water.
In the present invention, deep liquid Rapid Fermentation is batch fermentation, one or more in fed-batch fermentation and continuous ferment process. Bioreactor is pilot scale, industrially scalable fermentation tank and related microorganisms Fermentation Engineering equipment thereof.
4. the preparation of Enteromorpha bio-fertilizer: the zymotic fluid in step 3 is realized to Separation of Solid and Liquid by plate-frame filtering in conjunction with the method for low-speed centrifugal, by the collection of liquid part and by the composite Enteromorpha bio-fertilizer of preparing of a great number of elements, trace element and relevant nutriment.
Embodiment 2
1. actication of culture: described alternately monad A321 inoculation is carried out to activation culture in activation medium (formula is with embodiment 1), culture medium liquid amount 25mL/100mL, initial pH7.0,32 DEG C of cultivation temperature, shaking speed 160rpm, incubation time 48h;
2. the preparation of seed liquor: the culture medium after actication of culture in step 1 is inoculated in seed culture medium (formula is with embodiment 1) and is cultivated with 8% inoculum concentration, initial pH6.8, cultivation temperature is 28 DEG C, shaking speed 160rpm, incubation time 36h;
3. deep liquid Rapid Fermentation: seed liquor in step 2 is inoculated in to the fermentation preparation of carrying out Enteromorpha bio-fertilizer in the fermentation medium (formula with embodiment 1) of bioreactor with 10% inoculum concentration, liquid amount is 60% of biological reactor volume, pH7.2,24 DEG C of cultivation temperature, rotating speed of agitator 200rpm, throughput 1.0vvm, dissolved oxygen 50%, incubation time 20h;
4. the preparation of Enteromorpha bio-fertilizer: the zymotic fluid in step 3 is realized to Separation of Solid and Liquid by plate-frame filtering in conjunction with the method for low-speed centrifugal, by the collection of liquid part and by the composite Enteromorpha bio-fertilizer of preparing of a great number of elements, trace element and relevant nutriment.
Above embodiment is only in order to technical scheme of the present invention to be described, but not is limited; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, the technical scheme that still can record previous embodiment is modified, or part technical characterictic is wherein equal to replacement; And these amendments or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (10)
1. deep liquid Rapid Fermentation is prepared a method for Enteromorpha bio-fertilizer, it is characterized in that described method comprises following concrete steps:
(1) actication of culture: inoculation is carried out in activation medium to activation culture, initial pH6.8-7.2, cultivation temperature 30-32 DEG C, shaking speed 160-200rpm, incubation time 36-48h;
(2) preparation of seed liquor: the culture medium after actication of culture in step (1) is inoculated in seed culture medium and cultivates and obtain seed liquor with 5-8% inoculum concentration, initial pH6.5-7.2, cultivation temperature is 28-30 DEG C, shaking speed 160-200rpm, incubation time 30-36h;
(3) deep liquid Rapid Fermentation: seed liquor described in step (2) is inoculated in to the fermentation of carrying out Enteromorpha bio-fertilizer in the fermentation medium of bioreactor with 8-10% inoculum concentration and prepares zymotic fluid, liquid amount is the 50-70% of biological reactor volume, pH6.8-7.5, cultivation temperature 24-28 DEG C, rotating speed of agitator 200-600rpm, throughput 0.5-2.5vvm, dissolved oxygen 30-70%, incubation time 18-24h;
(4) preparation of Enteromorpha bio-fertilizer: the zymotic fluid in step (3) is carried out to Separation of Solid and Liquid, liquid part is collected and make described Enteromorpha bio-fertilizer by a great number of elements, trace element and nutriment composite.
2. deep liquid Rapid Fermentation according to claim 1 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: described bacterial strain is that deposit number is the alternately monad A321 of CCTCCNO:M2012132.
3. deep liquid Rapid Fermentation according to claim 2 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: described activation medium consists of: by percentage to the quality, sea grass polysaccharide 0.5-2.0%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, surplus is water.
4. deep liquid Rapid Fermentation according to claim 2 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: described seed culture medium consists of: by percentage to the quality, sea grass polysaccharide 0.5-2.0%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, surplus is water.
5. deep liquid Rapid Fermentation according to claim 2 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: described fermentation medium consists of: by percentage to the quality, particle diameter is 40-200 object solid waterside lichenin 1.0-8.0%, organic nitrogen source 0.01-0.05%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate 0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, surplus is water.
6. the method for preparing Enteromorpha bio-fertilizer according to the deep liquid Rapid Fermentation described in claim 3 or 4, is characterized in that: described sea grass polysaccharide obtains by hot-water process's lixiviate or autoclaving lixiviate.
7. deep liquid Rapid Fermentation according to claim 1 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: in described step (1), the liquid amount of activation medium is 25mL/100mL.
8. deep liquid Rapid Fermentation according to claim 5 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: described organic nitrogen source is one or more in beef extract, yeast extract and peptone.
9. deep liquid Rapid Fermentation according to claim 1 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: the soluble polysaccharide content in described step (3) in zymotic fluid is 0.2-1.2%, content of reducing sugar is 0.02-0.3%, protein content is 0.1-0.6%, the content of organic matter is 0.5-3.0%, solid content is 1.0-5.0%, by percentage to the quality.
10. deep liquid Rapid Fermentation according to claim 1 is prepared the method for Enteromorpha bio-fertilizer, it is characterized in that: in described step (4), zymotic fluid is realized Separation of Solid and Liquid by plate-frame filtering in conjunction with the method for low-speed centrifugal.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106578379A (en) * | 2016-12-09 | 2017-04-26 | 中国海洋大学 | Method for preparing functional feed additive through biological glycolysis of enteromorpha |
| CN110655424A (en) * | 2019-11-06 | 2020-01-07 | 中国农业科学院烟草研究所 | Preparation method and application of biofertilizer taking enteromorpha and burkholderia pyrrocinia as raw materials |
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| CN103352016A (en) * | 2013-06-27 | 2013-10-16 | 中国海洋大学 | Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha |
| CN103451119A (en) * | 2012-06-03 | 2013-12-18 | 中国海洋大学 | Alteromonas and method thereby for producing gel-type enteromorpha polysaccharide degrading enzyme by using Alteromonas |
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Patent Citations (3)
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| CN103451119A (en) * | 2012-06-03 | 2013-12-18 | 中国海洋大学 | Alteromonas and method thereby for producing gel-type enteromorpha polysaccharide degrading enzyme by using Alteromonas |
| CN103352016A (en) * | 2013-06-27 | 2013-10-16 | 中国海洋大学 | Method for preparing biological fertilizer by utilizing Alteromonas colwelliana A321 to ferment enteromorpha |
| CN104262008A (en) * | 2014-09-30 | 2015-01-07 | 青岛嘉瑞生物技术有限公司 | Process for preparing liquid seaweed fertilizer through enzymolysis and fermentation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106578379A (en) * | 2016-12-09 | 2017-04-26 | 中国海洋大学 | Method for preparing functional feed additive through biological glycolysis of enteromorpha |
| CN110655424A (en) * | 2019-11-06 | 2020-01-07 | 中国农业科学院烟草研究所 | Preparation method and application of biofertilizer taking enteromorpha and burkholderia pyrrocinia as raw materials |
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