CN105602995B - A kind of method of liquid submerged rapid fermentation to prepare prolifera biological fertilizer - Google Patents

A kind of method of liquid submerged rapid fermentation to prepare prolifera biological fertilizer Download PDF

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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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王鹏
江晓路
乔乐克
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Qingdao Marine Biomedical Research Institute Co Ltd
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    • 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
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    • C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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Abstract

本发明公开了一种液体深层快速发酵制备浒苔生物肥的方法。该方法步骤主要包括菌种活化、种子液的培养、液体深层快速发酵和浒苔生物肥的制备四部分。在发酵制备方面运用液体深层发酵技术结合生物反应器等专业发酵设备，实现了发酵过程的实时监控，在有效调控菌体代谢过程的同时提高了目的产物的产量。本发明的发酵制备方法可直接将固体浒苔粉作为主要原料进行发酵，在显著缩短发酵周期的同时，降低了生产成本，符合产业化生产的需求。The invention discloses a method for preparing prolifera bio-fertilizer by liquid deep rapid fermentation. The steps of the method mainly include four parts: activation of bacterial species, cultivation of seed liquid, rapid liquid submerged fermentation and preparation of prolifera biological fertilizer. In terms of fermentation preparation, liquid submerged fermentation technology combined with professional fermentation equipment such as bioreactors realizes real-time monitoring of the fermentation process, effectively regulating the metabolic process of bacteria and improving the yield of target products. The fermentation preparation method of the present invention can directly use the solid prolifera powder as the main raw material for fermentation, which not only significantly shortens the fermentation period, but also reduces the production cost, and meets the needs of industrialized production.

Description

A kind of method that deep liquid Rapid Fermentation prepares Enteromorpha bio-fertilizer
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)

1.一种液体深层快速发酵制备浒苔生物肥的方法，其特征在于所述方法包括以下具体步骤：1. a method for preparing prolifera bio-fertilizer by liquid deep fermentation rapidly, it is characterized in that described method comprises the following concrete steps: （1）菌种活化：将菌株接种于活化培养基中进行活化培养，初始pH6.8-7.2，培养温度30-32℃，摇床转速160-200rpm，培养时间36-48h；所述菌株为保藏编号为CCTCC NO ：M2012132的交替单胞菌A321；(1) Activation of strains: The strains are inoculated into the activation medium for activation and culture, the initial pH is 6.8-7.2, the culture temperature is 30-32°C, the shaking speed is 160-200rpm, and the culture time is 36-48h; the strains are: The preservation number is the Altomonas A321 of CCTCC NO: M2012132; （2）种子液的制备：将步骤（1）中菌种活化后以5-8%接种量接种于种子培养基中进行培养获得种子液，初始pH6.5-7.2，培养温度为28-30℃，摇床转速160-200rpm，培养时间30-36h；(2) Preparation of seed liquid: inoculate the activated bacteria in step (1) with 5-8% inoculum in the seed medium for cultivation to obtain seed liquid, the initial pH is 6.5-7.2, and the cultivation temperature is 28-30 ℃, shaker speed 160-200rpm, culture time 30-36h; （3）液体深层快速发酵：将步骤（2）中所述种子液以8-10%接种量接种于生物反应器的发酵培养基中进行浒苔生物肥的发酵制备得到发酵液，装液量为生物反应器体积的50-70%，pH6.8-7.5，培养温度24-28℃，搅拌桨转速200-600rpm，通气量0.5-2.5vvm，溶解氧30-70%，培养时间18-24h；(3) Liquid submerged rapid fermentation: inoculate the seed liquid described in the step (2) with 8-10% inoculum in the fermentation medium of the bioreactor, and carry out fermentation of the prolifera bio-fertilizer to prepare a fermentation liquid. It is 50-70% of the volume of the bioreactor, pH 6.8-7.5, culture temperature 24-28°C, stirring paddle speed 200-600rpm, ventilation 0.5-2.5vvm, dissolved oxygen 30-70%, culture time 18-24h ; （4）浒苔生物肥的制备：将步骤（3）中的发酵液进行固液分离，将液体部分收集并通过大量元素、微量元素及营养物质的复配制得所述浒苔生物肥。(4) Preparation of prolifera biological fertilizer: the fermentation broth in step (3) is subjected to solid-liquid separation, the liquid part is collected, and the prolifera biological fertilizer is prepared by compounding macroelements, trace elements and nutrients. 2.根据权利要求1所述的液体深层快速发酵制备浒苔生物肥的方法，其特征在于：所述发酵培养基的组成为：以质量百分比计，粒径为40-200目的固体浒苔粉1.0-8.0%，有机氮源0.01-0.05%，磷酸氢二钾0.05-0.5%、硫酸钾0.2-2.0%、硝酸钾0.05-0.6%、硫酸镁0.02-0.1%，余量为水。2. the method that liquid submerged rapid fermentation according to claim 1 prepares prolifera biological fertilizer, it is characterized in that: the composition of described fermentation medium is: in mass percent, particle diameter is 40-200 object solid prolifera powder 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%, and the balance is water. 3.根据权利要求1所述的液体深层快速发酵制备浒苔生物肥的方法，其特征在于：所述步骤（1）中活化培养基的装液量为25mL/100mL。3 . The method for preparing prolifera bio-fertilizer by liquid submerged rapid fermentation according to claim 1 , characterized in that: in the step (1), the liquid filling amount of the activation medium is 25 mL/100 mL. 4 . 4.根据权利要求2所述的液体深层快速发酵制备浒苔生物肥的方法，其特征在于：所述有机氮源为牛肉膏、酵母膏和蛋白胨中的一种或多种。4 . The method for preparing prolifera bio-fertilizer by liquid submerged rapid fermentation according to claim 2 , wherein the organic nitrogen source is one or more of beef extract, yeast extract and peptone. 5 . 5.根据权利要求1所述的液体深层快速发酵制备浒苔生物肥的方法，其特征在于：所述步骤（3）中发酵液中的可溶性多糖含量为0.2-1.2%，还原糖含量为0.02-0.3%，蛋白含量为0.1-0.6%，有机质含量为0.5-3.0%，固形物含量为1.0-5.0%，以质量百分比计。5. The method for preparing prolifera biofertilizer by liquid submerged rapid fermentation according to claim 1, characterized in that: in the step (3), the soluble polysaccharide content in the fermentation liquid is 0.2-1.2%, and the reducing sugar content is 0.02% -0.3%, protein content is 0.1-0.6%, organic matter content is 0.5-3.0%, solid content is 1.0-5.0%, in mass percentage. 6.根据权利要求1所述的液体深层快速发酵制备浒苔生物肥的方法，其特征在于：所述步骤（4）中发酵液通过板框过滤结合低速离心的方法实现固液分离。6 . The method according to claim 1 , characterized in that: in the step (4), the fermented liquid is separated from the solid-liquid by the method of plate-and-frame filtration combined with low-speed centrifugation. 7 .
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CN110655424A (en) * 2019-11-06 2020-01-07 中国农业科学院烟草研究所 A kind of preparation method and application of biological fertilizer using prolifera and Burkholderia pyrrole as raw materials

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