CN105602995B - A kind of method that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer - Google Patents
A kind of method that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer Download PDFInfo
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- CN105602995B CN105602995B CN201610038944.7A CN201610038944A CN105602995B CN 105602995 B CN105602995 B CN 105602995B CN 201610038944 A CN201610038944 A CN 201610038944A CN 105602995 B CN105602995 B CN 105602995B
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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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- C05—FERTILISERS; MANUFACTURE THEREOF
- 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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Abstract
The invention discloses a kind of methods that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer.This method step mainly includes four part of preparation of actication of culture, the culture of seed liquor, deep liquid Rapid Fermentation and Enteromorpha bio-fertilizer.With the professional Zymolysis Equipment such as liquid deep layer fermenting technology combination bioreactor in terms of preparation of fermenting, the real time monitoring of fermentation process is realized, the yield of purpose product is improved while Effective Regulation bacterial metabolism process.Fermentation preparation of the invention can directly ferment using solid enteromorpha powder as primary raw material, while significantly shortening fermentation period, reduce production cost, meet the demand of industrialization production.
Description
Technical field
The invention belongs to technical field of biological fermentation, are related to the microbial fermentation production method of Enteromorpha bio-fertilizer, especially relate to
And the method that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer.
Background technique
Liquid deep layer fermenting typically refers to carry out fluid nutrient medium and material using bioreactors such as fermentors continuous
The operation such as fermentation, batch fermentation or fed-batch fermentation, it is timely while biological metabolism changes in real-time monitoring fermentation process
Adjustment and control Product formation, to effectively improve substrate utilization ratio and product yield.Compared to traditional shake flask fermentation, liquid
Body submerged fermentation technology has the features such as with short production cycle, yield is high, and production process is easy to control, stable product quality.Therefore
It has a good application prospect in terms of microbial fermentation prepare with scale purpose product.
With the exploitation of marine resources, seaweed processing and its higher value application gradually embody great commercial value.Sea
Algae bio-fertilizer is using marine algae extract or fermentation of seaweed object as the novel sea biological agent of core substance.Wherein containing a large amount of
Algal polysaccharides specific to organic matter, vitamin, mineral matter element and seaweed itself, alga oligosaccharides, alginic acid and a variety of days
Right plant growth regulator can play beneficial functional to the improvement of plant growth and soil quality.As different seaweed are raw
The successive appearance of object fertilizer product, rapid scale production preparation method are increasingly becoming research hotspot.
Li Guoting etc. using natural seaweed as primary raw material, be equipped with a small amount of potassium hydroxide carry out under high pressure alkaline hydrolysis with
The plant fiber in seaweed is destroyed, to release the natural plants growth regulatory substance such as seaweeds, gibberellin, is made by compounding
Standby seaweed manure.Although easy to operate, the Mechanical Method energy consumption is higher and product yield is lower, is not suitable for realizing product
Prepare with scale；Wang Mingpeng etc. is mentioned in the research of seaweed organism fertilizer using at the chemical methodes such as acid, alkali and organic solvent
Alginic cell is managed, makes cell resolution or endogenous substance solubilising that can effectively extract the active constituent in seaweed tissue, to be used for seaweed
The preparation of biological organic fertilizer.But such chemical method reagent residual is seriously, biological safety is poor, and to environment, there are huge latent
It is endangering；In contrast, microbe fermentation method is because its biological safety is good, lytic activity component content is high and rapidly and efficiently
Single-minded feature is increasingly becoming the main way of production seaweed organic fertilizer.
Enteromorpha marine stock biomass is huge, and traditional shake flask fermentation technique is cumbersome, and yield is lower, and scale far can not expire
It is enough to the needs of actual production.The present invention by liquid deep layer fermenting Technology application in the preparation of Enteromorpha bio-fertilizer, and it is innovative
Using solid enteromorpha powder as the main component of fermentation medium, the configuration of fermentation medium, Er Qieyou are not only greatly simplified
Help the large-scale production of Enteromorpha bio-fertilizer, makees good technology place mat for its later period industrialized production.
Currently without the report and patent for preparing Enteromorpha bio-fertilizer about deep liquid Rapid Fermentation relevant to the application.
Summary of the invention
The present invention is intended to provide a kind of method that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer, fermentation of the present invention
Method first activates bacterial strain, and by bioreactor real-time monitoring fermentation condition, using solid enteromorpha powder as raw material, warp
Liquid fermentation, separation of solid and liquid and complex process realize the prepare with scale of Enteromorpha bio-fertilizer.
For achieving the above object, the present invention is achieved by the following scheme:
A kind of method that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer, the method includes steps in detail below:
(1) strain inoculated actication of culture: is subjected to activation culture, initial pH6.8-7.2, culture temperature in activation medium
30-32 DEG C, shaking speed 160-200rpm, incubation time 36-48h of degree；
(2) culture medium after actication of culture in step (1) preparation of seed liquor: is inoculated in seed training with 5-8% inoculum concentration
It supporting and carries out culture acquisition seed liquor in base, initial pH6.5-7.2, cultivation temperature is 28-30 DEG C, shaking speed 160-200rpm,
Incubation time 30-36h；
(3) seed liquor described in step (2) deep liquid Rapid Fermentation: is inoculated in biological respinse with 8-10% inoculum concentration
Fermentation liquid is prepared in the fermentation that Enteromorpha bio-fertilizer is carried out in the fermentation medium of device, and liquid amount is biological reactor volume
50-70%, pH6.8-7.5,24-28 DEG C of cultivation temperature, rotating speed of agitator 200-600rpm, ventilatory capacity 0.5-2.5vvm, dissolved oxygen
30-70%, incubation time 18-24h；
(4) preparation of Enteromorpha bio-fertilizer: the fermentation liquid in step (3) is separated by solid-liquid separation, and liquid portion is collected simultaneously
The Enteromorpha bio-fertilizer is made by the compounding of a great number of elements, microelement and nutriment.
Further improvement to above-mentioned technical proposal: the bacterial strain is the friendship that deposit number is CCTCC NO: M2012132
For monad A321.
Further improvement to above-mentioned technical proposal: the activation medium composition are as follows: by percentage to the quality, Enteromorpha is more
Sugared 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 above-mentioned technical proposal: the seed culture medium composition are as follows: by percentage to the quality, Enteromorpha is more
Sugared 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 above-mentioned technical proposal: the fermentation medium composition are as follows: by percentage to the quality, partial size is
Solid the enteromorpha powder 1.0-8.0%, organic nitrogen source 0.01-0.05%, dipotassium hydrogen phosphate 0.05-0.5%, potassium sulfate of 40-200 mesh
0.2-2.0%, potassium nitrate 0.05-0.6%, magnesium sulfate 0.02-0.1%, surplus are water.
Further improvement to above-mentioned technical proposal: the sea grass polysaccharide is extracted by hot-water process or autoclaving extracts
It obtains.
Further improvement to above-mentioned technical proposal: the liquid amount of activation medium is 25mL/ in the step (1)
100mL。
Organic nitrogen source described in further improvement to above-mentioned technical proposal is one of beef extract, yeast extract and peptone
Or it is a variety of.
Further improvement to above-mentioned technical proposal: the soluble polysaccharide content in the step (3) in fermentation liquid is
0.2-1.2%, content of reducing sugar 0.02-0.3%, protein content 0.1-0.6%, content of organic matter 0.5-3.0%, solid content
Content is 1.0-5.0%, by percentage to the quality.
Further improvement to above-mentioned technical proposal: in the step (4) fermentation liquid by plate-frame filtering combination low speed from
The method of the heart, which is realized, to be separated by solid-liquid separation.
Method provided by the invention has the following characteristics that
(1) using large-scale green tide algae Enteromorpha solid as primary raw material, from a wealth of sources, marine stock biomass is huge；(2) pass through liquid
Deep layer fast fermentation in fixed combination bioreactor, greatly improves substrate utilization ratio and product yield；(3) in fermentation liquid
Containing the Plant growth-promoting effects factor such as numerous active polysaccharides, oligosaccharides and organic matter, the Enteromorpha bio-fertilizer prepared in conjunction with process for formulating
With good environment friendly and biological safety.
Specific embodiment
Technical solution of the present invention is further described in detail combined with specific embodiments below.
Embodiment 1
Bacterial strain uses therefor is the Alteromonad A321(separated in the seawater of Qingdao sea area in the present inventionAlteromonas colwellianaA321), Alteromonad A321 is deposited on April 23rd, 2012 positioned at Wuhan, China Wuhan University
China typical culture collection center (CCTCC), deposit number are CCTCC NO: M2012132.
The morphological feature and physiological and biochemical property of the Alteromonad A321 is referring to 103451119 A of Publication No. CN
Application for a patent for invention.
1. actication of culture: the Alteromonad A321 strain inoculated being carried out activation culture in activation medium, is trained
Feeding base liquid amount 25mL/100mL, initial pH6.8,30 DEG C of cultivation temperature, shaking speed 180rpm, incubation time 48h；
The activation medium composition are as follows: 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%, remaining ingredient are water.Sea grass polysaccharide of the present invention passes through hot water
Method extracts (extracting in boiling water 1-4 hours) or autoclaving extraction (115 DEG C of extraction 30min) obtains.
2. the preparation of seed liquor: the culture medium after actication of culture in step 1 is inoculated in seed culture medium with 6% inoculum concentration
In carry out culture obtain seed liquor, initial pH7.0, cultivation temperature be 28 DEG C, shaking speed 180rpm, incubation time 36h；
The seed culture medium composition are as follows: 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%, remaining ingredient are water.
Seed culture medium particularly may be divided into primary-seed 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 the hair of bioreactor with 8% inoculum concentration
The fermentation preparation of Enteromorpha bio-fertilizer is carried out in ferment culture medium, liquid amount is 50%, pH6.8 of biological reactor volume, cultivation temperature
26 DEG C, rotating speed of agitator 200rpm, ventilatory capacity 1.5vvm, dissolved oxygen 60%, incubation time is for 24 hours；
The fermentation medium composition are as follows: partial size is solid the enteromorpha powder 1.0-8.0%, organic nitrogen source 0.01- of 40-200 mesh
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%, remaining
Ingredient is water.
Deep liquid Rapid Fermentation is batch fermentation in the present invention, one in fed-batch fermentation and continuous ferment process
Kind is a variety of.Bioreactor is pilot scale, industrially scalable fermentor and its related microorganisms Fermentation Engineering equipment.
4. the preparation of Enteromorpha bio-fertilizer: the fermentation liquid in step 3 is real by the method for plate-frame filtering combination low-speed centrifugal
It is now separated by solid-liquid separation, the compounding that liquid portion is collected to and passed through a great number of elements, microelement and related nutritional substance prepares Enteromorpha
Bio-fertilizer.
Embodiment 2
1. actication of culture: by the Alteromonad A321 strain inoculated in activation medium (formula is with embodiment 1)
Activation culture is carried out, culture medium liquid amount 25mL/100mL, initial pH7.0, are trained by 32 DEG C of cultivation temperature, shaking speed 160rpm
Support time 48h；
2. the preparation of seed liquor: the culture medium after actication of culture in step 1 is inoculated in seed culture medium with 8% inoculum concentration
It is cultivated in (formula is with embodiment 1), 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 trained with the fermentation that 10% inoculum concentration is inoculated in bioreactor
The fermentation preparation that Enteromorpha bio-fertilizer is carried out in base (formula is with embodiment 1) is supported, liquid amount is the 60% of biological reactor volume,
PH7.2,24 DEG C of cultivation temperature, rotating speed of agitator 200rpm, ventilatory capacity 1.0vvm, dissolved oxygen 50%, incubation time 20h；
4. the preparation of Enteromorpha bio-fertilizer: the fermentation liquid in step 3 is real by the method for plate-frame filtering combination low-speed centrifugal
It is now separated by solid-liquid separation, the compounding that liquid portion is collected to and passed through a great number of elements, microelement and related nutritional substance prepares Enteromorpha
Bio-fertilizer.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than is limited；Although referring to aforementioned reality
Applying example, invention is explained in detail, for those of ordinary skill in the art, still can be to aforementioned implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these are modified or replace
It changes, the spirit and scope for claimed technical solution of the invention that it does not separate the essence of the corresponding technical solution.
Claims (6)
1. a kind of method that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer, it is characterised in that the method includes in detail below
Step:
(1) strain inoculated actication of culture: is subjected to activation culture, initial pH6.8-7.2, cultivation temperature in activation medium
30-32 DEG C, shaking speed 160-200rpm, incubation time 36-48h；The bacterial strain is that deposit number is CCTCC NO:
The Alteromonad A321 of M2012132；
(2) it the preparation of seed liquor: is trained being inoculated in seed culture medium after actication of culture in step (1) with 5-8% inoculum concentration
It supports and obtains seed liquor, initial pH6.5-7.2, cultivation temperature is 28-30 DEG C, shaking speed 160-200rpm, incubation time 30-
36h；
(3) seed liquor described in step (2) deep liquid Rapid Fermentation: is inoculated in bioreactor with 8-10% inoculum concentration
Fermentation liquid is prepared in the fermentation that Enteromorpha bio-fertilizer is carried out in fermentation medium, and liquid amount is the 50- of biological reactor volume
70%, pH6.8-7.5,24-28 DEG C of cultivation temperature, rotating speed of agitator 200-600rpm, ventilatory capacity 0.5-2.5vvm, dissolved oxygen 30-
70%, incubation time 18-24h；
(4) preparation of Enteromorpha bio-fertilizer: the fermentation liquid in step (3) is separated by solid-liquid separation, liquid portion is collected and passed through
The Enteromorpha bio-fertilizer is made in the compounding of a great number of elements, microelement and nutriment.
2. the method that deep liquid Rapid Fermentation according to claim 1 prepares Enteromorpha bio-fertilizer, it is characterised in that: described
The composition of fermentation medium are as follows: by percentage to the quality, partial size is the solid enteromorpha powder 1.0-8.0% of 40-200 mesh, 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.
3. the method that deep liquid Rapid Fermentation according to claim 1 prepares Enteromorpha bio-fertilizer, it is characterised in that: described
The liquid amount of activation medium is 25mL/100mL in step (1).
4. the method that deep liquid Rapid Fermentation according to claim 2 prepares Enteromorpha bio-fertilizer, it is characterised in that: described
Organic nitrogen source is one of beef extract, yeast extract and peptone or a variety of.
5. the method that deep liquid Rapid Fermentation according to claim 1 prepares Enteromorpha bio-fertilizer, it is characterised in that: described
Soluble polysaccharide content in step (3) in fermentation liquid is 0.2-1.2%, content of reducing sugar 0.02-0.3%, and protein content is
0.1-0.6%, content of organic matter 0.5-3.0%, solid content 1.0-5.0%, by percentage to the quality.
6. the method that deep liquid Rapid Fermentation according to claim 1 prepares Enteromorpha bio-fertilizer, it is characterised in that: described
Fermentation liquid is realized by the method for plate-frame filtering combination low-speed centrifugal and is separated by solid-liquid separation in step (4).
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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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 海藻生物肥研究进展与展望;王明鹏等;《生物技术进展》;20150331;第5卷(第3期);第158-163页 * |
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