CN103739322B - A kind of preparation method of composite bio-fertilizer - Google Patents

Abstract

The invention discloses a preparation method of biological compound fertilizer, which belongs to the technical field of agricultural fertilizers. The preparation method of the biological compound fertilizer comprises setting the parts by weight as follows: 5-18 parts of Bacillus colloidus, 2-6 parts of phosphorus solubilizing bacteria, 5-10 parts of potassium solubilizing bacteria, and 18-30 parts of Bacillus subtilis culture , 5-10 parts of Aspergillus awamori culture and 3-8 parts of Lactobacillus plantarum agent are mixed or granulated after mixing. The fertilizer of the present invention can enhance the drought resistance of crops. The colloidal bacillus in the compound strain of the present invention is a silicate bacterium with strong potassium-decomposing ability screened from the roots of crops. Developed by industrial fermentation; the bacteria can secrete auxin substances and gibberellin substances and other growth-promoting substances to increase roots and seedlings; decompose silicon in the soil for plants to use, thicken the waxy layer of plants, and improve water retention capacity of plants ; Can promote the formation of soil aggregate structure, prevent soil compaction; destroy soil capillarity, prevent soil moisture from evaporating.

Description

A kind of preparation method of biological compound fertilizer

technical field

The invention relates to a biological fertilizer processing method, which belongs to the technical field of agricultural fertilizers, in particular to a preparation method of biological compound fertilizer.

Background technique

Fertilizer pollution has become a major public hazard in the world today. Agricultural experts have unanimously called for reducing the amount of chemical fertilizers to control the environment. An industry expert and academician Chen Wenxin of China Agricultural University believes that the excessive application of chemical fertilizers in my country has reached its limit. Taking nitrogen fertilizer as an example, my country's average application rate is nearly three times that of the United States and more than eight times that of Australia. This has caused a series of serious problems such as the destruction of soil structure, the reduction of natural biodiversity and ecosystem stability. Dr. Li Jun, director of the Agricultural Microbiology Professional Committee of the Chinese Society for Microbiology, pointed out that vigorously promoting the use of microbial fertilizers combined with chemical fertilizers is one of the ways to solve this problem.

Microbial fertilizer is a microbial living product that uses microbial life activities and its products to cause crops to obtain specific fertilizer effects. It can improve soil fertility, improve fertilizer utilization, inhibit crops from absorbing harmful substances such as heavy metals and pesticides, and purify and restore soil. It plays an irreplaceable role in promoting the utilization of crop stalks and urban waste, and improving the quality of agricultural products. The efficacy of microbial fertilizers is mainly achieved through the synergistic effect on traditional chemical fertilizers and organic fertilizers, the improvement and activation of soil, and the physiological effects of microorganisms.

Therefore, microbial fertilizers have a variety of fertility and efficacy of chemical fertilizers, organic fertilizers, and beneficial biological bacteria. They are ideal fertilizers for activating fertilizer nutrients, improving fertilizer effects, improving crop quality, and promoting agricultural production and efficiency. Microbial fertilizer is a living fertilizer, and its function is mainly completed by the life activities of a large number of beneficial microorganisms it contains. Only when these beneficial microorganisms are vigorously multiplying and metabolizing, can material transformation and beneficial metabolites be continuously formed. Therefore, the types of beneficial microorganisms in microbial fertilizers and whether their life activities are vigorous are the basis of their effectiveness, unlike other fertilizers that are based on the form and amount of major elements such as nitrogen, phosphorus, and potassium. Because microbial fertilizers are living preparations, their fertilizer efficiency is closely related to the number, strength and surrounding environmental conditions of viable bacteria, including temperature, moisture, pH, nutritional conditions and the repulsion of indigenous microorganisms that originally lived in the soil.

For many years, most of the processing methods of microbial fertilizers only use finished bacterial agents mixed with fillers, lacking fermentation metabolites, which seriously affect the fertilizer efficiency of microbial fertilizer products. For example, the patent application with publication number CN103396252A discloses a compound microorganism containing amino acids The fertilizer, wherein the finished microbial agent added is composed of Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium mixed in a certain proportion.

Moreover, the agricultural microbial agents currently on the market are mainly single microbial strains, which are difficult to restore the micro-ecological structure. At the same time, the synergy between common strains and crops is weak, which is unfavorable such as improving plant stress resistance and root nutrient absorption. For example, the publication number is The patent application of CN102888356A discloses a method for preparing microbial fertilizer by using Bacillus subtilis and its application. Although some composite microbial agents have appeared on the market, their functions are single, or their use methods are limited, and the effect of increasing crop production after use is still unsatisfactory. A compound microbial agent or fertilizer with comprehensive functions and good yield-increasing effects.

Due to the long-term lack of investment in research on microbial fertilizers in my country, my country's microbial fertilizer industry still has problems such as low overall level, insufficient technological innovation, and unstable product quality and application effects.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a preparation method of biological compound fertilizer.

The preparation method of the biological compound fertilizer is as follows: according to the following parts by weight, the bacterial agents are mixed, or granulated after mixing:

5-18 parts of Bacillus colloidus, 2-6 parts of phosphate solubilizing bacteria, 5-10 parts of potassium solubilizing bacteria, 18-30 parts of Bacillus subtilis culture, 5-10 parts of Aspergillus awamori culture, 3- 8 copies;

The preparation of the Bacillus subtilis culture: transfer and cultivate the Bacillus subtilis from the inclined plane, transfer the seed liquid after step-by-step expansion into the fermentation tank, and after the fermentation is completed, the fermentation liquid is filtered and dried to obtain the Bacillus subtilis culture .

The preparation method of Lactobacillus plantarum agent: transfer and cultivate Lactobacillus plantarum from an inclined plane, transfer the seed liquid after step-by-step expansion into a fermenter, and after fermentation, the fermented liquid is concentrated to 45% of the original volume by low-temperature negative pressure vacuum to obtain Bacterial Concentrate. Add carrier: Add the mixed carrier to the concentrated solution and mix evenly; the weight ratio of the concentrated solution to the carrier is 0.5-0.6:1, and the carrier composition is: 20-30 parts of CaCO3, 10-15 parts of dextrin. Fluidized bed drying, drying temperature 50 ℃.

Preparation of Aspergillus awamori culture: strain culture, solid fermentation culture: inoculate spore liquid into Aspergillus oryzae solid-state fermentation culture material, cultivate at 26-33°C until mycelium is covered with culture material, dry in a low-temperature fluidized bed, and pulverize the dry matter.

The bacterial classification that the present invention adopts is as follows:

Bacillus subtilis CGMCC7926

Lactobacillus plantarum CGMCC7928

Aspergillus awamori CGMCC3.6484

The preservation unit of the above three strains is the China Common Microorganism Culture Collection and Management Center. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China; Zip code: 100101.

The phosphate solubilizing bacteria is Bacillus megaterium, and the phosphate solubilizing bacteria agent is provided by Cangzhou Wangfa Biotechnology Research Institute, address: 807-812, Area 1, Building A, Yihe International Business Center, Jiefang West Road, Yunhe District, Cangzhou City, Hebei, China.

Potassium-dissolving bacteria were provided by Guangzhou Weiyuan Biotechnology Co., Ltd., address: 506, No. 101, Science Avenue, Luogang District, Guangzhou.

Hebei Baoding Ruigu Biotechnology Co., Ltd. provides Bacillus colioids powder.

Beneficial effect

The fertilizer of the invention can enhance the drought resistance ability of crops. The colloidal bacillus in the composite strain of the invention is a silicate bacterium with strong potassium decomposing ability, which is developed through industrial fermentation using a specific culture medium. The bacteria can secrete growth-promoting substances such as auxin substances and gibberellin substances to increase roots and seedlings; decompose silicon in the soil for plant utilization, thicken the waxy layer of plants, and improve water retention capacity of plants; can promote soil aggregate structure Formation, prevent soil compaction; destroy soil capillarity, prevent soil moisture from evaporating.

The fertilizer of the present invention can improve soil. Beneficial microorganisms in fertilizers can produce sugar substances and combine with plant mucus, mineral embryos and organic colloids, which can improve the structure of soil aggregates, enhance the physical properties of soil and reduce the loss of soil particles. Under certain conditions, they can also participate in Humus formation. Therefore, the application of microbial fertilizers can improve the physical properties of soil and help to improve soil fertility.

The method of using this product is simple, the amount of use per mu is 0.3-1 kg, and the cost is only 80-100 yuan/mu, and the surface is sprayed before seed dressing or irrigation during the growth period.

The transformation of soil biological enzymes can play a significant role in reducing the cost of agricultural production, reducing the use of chemical fertilizers, restoring soil ecological fertility and effectively increasing crop yields, giving full play to the effectiveness of soil organic matter.

detailed description

Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be considered as illustrative rather than limiting the scope of the invention, the spirit and scope of which is defined only by the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, various changes or modifications to the reaction conditions and separation and extraction conditions in these embodiments also fall within the protection scope of the present invention.

The following examples can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way. Example 1

Bacillus subtilis (Bacillus subtilis) Li-2013-02, the strain has been preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short) on July 15, 2013, the address is: Beichen West Road 1, Chaoyang District, Beijing, China No. 3 Courtyard), the preservation number is CGMCCNo.7926.

The characteristic of the strain is that it has high enzyme activity for producing high temperature-resistant α-amylase, and strong heat resistance and acid resistance.

The high temperature-resistant α-amylase enzyme activity prepared by the strain is 30000-35000u/ml; the applicable temperature range is 105-115°C, the optimum reaction temperature is 110°C, and the enzyme activity is completely stable at 110°C; the applicable reaction pH range is 3.0-7.0, when the pH value is 3.0, the enzyme activity is completely stable, and the optimum reaction pH value is 4.2.

Described bacterial strain characteristic is as follows:

The colony color of the strain on the solid plate is milky white, the surface is dry and opaque, and the edges are neat, and it is an aerobic bacterium with motility. Microscopic examination was long rod-shaped, Gram stain was positive. The bacteria can use citrate, nitric acid reduction, and V-P test to be positive.

The Bacillus subtilis (Bacillus subtilis) Li-2013-02 is obtained from a strain of Bacillus subtilis Li-2013 producing high-temperature resistant α-amylase through ultraviolet-lithium chloride-diethyl sulfate compound mutagenesis screening, and the specific screening steps as follows:

(1) Preparation of bacterial suspension

Inoculate the Li-2013 single colony grown after streaking on the plate into the seed medium, culture at 40°C for 12 hours at 100 r/min, take 1 mL of the culture medium, centrifuge, wash twice with normal saline, and resuspend with 9 mL of physiological in salt water.

(2) Ultraviolet-Lithium Chloride-Diethyl Sulfate Compound Mutagenesis

The bacterial suspension was placed on a sterile plate, stirred and irradiated for 100s under a UV lamp with a distance of 30cm and a power of 15w. The irradiated bacteria solution was diluted and spread on the lithium chloride plate after gradient dilution, and the bacteria solution without ultraviolet irradiation was diluted and spread on the plate as a control. Wrap the uniformly coated plate above with black cloth or newspaper, incubate at 40°C for 48 hours, screen out the plate with the largest ratio of hydrolysis circle to colony diameter on the plate that grows colonies, pick it up and store it on a slant, and prepare bacteria after purification. Suspension, after gradient dilution, was fully mixed with diethyl sulfate stock solution, and treated with shaking at 40°C for 40 minutes, and the treated bacterial solution was spread on lithium chloride plate after gradient dilution.

(3) Preliminary screening of high-yield strains

Put the uniformly coated plates above and culture them at 40°C for 48 hours. On the plates where the colonies grew, the ones with a larger ratio of the hydrolysis circle to the colony diameter were selected and stored on the slope. After purification, three strains of Li-2013-01 were obtained. Li-2013-02, Li-2013-03.

(4) Shake flask fermentation re-screening

The obtained three strains Li-2013-01, Li-2013-02, and Li-2013-03 were carried out in a 250mL shake flask containing 30mL fermentation medium, and the seed inoculum was 10% (V/V). Cultivate at 40°C and 100r/min for 72 hours, and centrifuge to collect the fermentation supernatant to obtain crude enzyme liquid.

(5) Determination of enzyme activity

The definition of enzyme activity unit: 1mL of crude enzyme solution, under the conditions of 105°C and pH4.2, liquefy 1mg of soluble starch in 1min,

That is, one enzyme activity unit, expressed in U/mL.

It was determined that the strain Li-2013-02 was the most stable strain with the highest yield, and the enzyme activity reached 30000U/mL.

The lithium chloride plate: starch 1%, peptone 1%, (NH) ₂ SO ₄ 0.4%, K ₂ HPO ₄ 0.8%, CaCl ₂ 0.2%, lithium chloride 0.9%, agar 2%.

The seed medium: 0.5% of yeast powder, 1% of peptone, 1% of soluble starch, and 1% of NaCl.

The fermentation medium: corn flour 5%-15%, bean cake flour 4%-10%, (NH) ₂ SO ₄ 0.4%, K ₂ HPO ₄ 0.8%, CaCl ₂ 0.2%.

The shake flask culture conditions: the bacterium is cultured in a 250 mL shake flask containing 30 mL of fermentation medium, the inoculum size is 10% (V/V), 100 r/min, 40° C. for 72 hours.

The high temperature resistant α-amylase has the following enzymatic properties:

(1) The temperature range of the enzyme is wide, and the optimum action temperature is between 100-110°C, and the temperature stability is better if it is stored below 110°C, but the temperature stability is poor if it is stored above 110°C for a long time.

(2) The optimum reaction pH value of the enzyme is 4.2. There is high enzyme activity between pH 3.0-7.0, and the enzyme activity is completely stable at pH 3.0.

(3) Enzyme activity: the enzyme activity of the high temperature-resistant α-amylase prepared from the mutant strain Li-2013-02 provided by the present invention is 30000-35000 U/ml.

The Lactobacillus plantarum (Lactobacillus plantarum) Li-2013-01 provided by the present invention is preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, the preservation number is CGMCCNo.7928, and the preservation address is: 1 Beichen West Road, Chaoyang District, Beijing No. 3 Courtyard, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101. The deposit date is July 15, 2013. The characteristics of the strain are as follows: observed under a microscope, the strain is short rod-shaped, Gram staining is positive, without bristles, and does not produce spores; on solid medium, the bacterial colony is white, smooth, dense, and the shape is Round with neat edges. The physical and chemical characteristics are: catalase (-), gelatin liquefaction (-), indole test (+), motility (-), fermentation gas production (-), nitrite reduction (-), fermentation gas production ( -), producing hydrogen sulfide gas (-), growing in MRS medium at pH 4.5 (+).

Plantarum lactobacillus of the present invention adopts following process to carry out breeding:

Original starting strain → test tube activation → diethyl sulfate (DES) mutagenesis → plate primary screening → nitrosoguanidine (NTG) mutagenesis → plate primary screening → shake flask secondary screening → passage stability test.

The original starting strain was CICC20242, which was purchased from China Industrial Microbiology Culture Collection Management Center.

The original bacterial strain adopted in the present invention has a lactic acid output of 12.5 g/L in a xylan medium. In order to increase its lactic acid production, the strain was mutagenized sequentially with DES and NTG. The mutagenesis was first screened on a MRS calcium carbonate plate, and then fermented in a 500mL shake flask. Excellent Lactobacillus plantarum strains, and then conduct subculture experiments to evaluate their genetic stability.

The results of the genetic stability of the strain CGMCCNo.7928 showed that after ten consecutive subcultures, all performance indicators were relatively stable, heredity was good, and the traits did not recover. Therefore, the strain CGMCCNo.7928 was selected as the target strain obtained by selection.

The target strain CGMCCNo.7928 was tested in a 10L fermenter. The results showed that after 72 hours of fermentation, with xylan as the carbon source, the lactic acid concentration of Lactobacillus plantarum CGMCCNo.7928 could reach 57g/L, which was 356g/L higher than that of the starting strain. %.

The target strain CGMCCNo.7928 was tested in a 10L fermenter, and the results showed that after 72 hours of fermentation, the lactic acid concentration of Lactobacillus plantarum CGMCCNo.7928 could reach 68g/L with glucose as the carbon source.

The specific process is as follows:

Medium:

Liquid MRS xylan medium (beef extract 2g, peptone 10g, yeast extract 5g, xylan 20g, sodium acetate 5g, ammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.05g, after dissolving one by one, tap water to volume 1000mL, adjust pH7.0-7.2); MRS xylan screening solid medium (beef extract 2g, peptone 10g, yeast extract 5g, xylan 90g, sodium acetate 5g, lemon Ammonium phosphate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, manganese sulfate heptahydrate 0.05g, dissolve one by one, tap water to volume 1000mL, adjust pH 7.0-7.2, add 20g agar).

1. Diethyl Sulfate (DES) Mutation Breeding

1) Take a ring of Lactobacillus plantarum on the inclined surface of the test tube on the ultra-clean bench, insert it into a 250mL Erlenmeyer flask filled with 50mL liquid MRS xylan medium, and culture it at 200rpm at 40°C for about 12h, so that the bacteria are in logarithmic Early growth period.

2) Take 5 mL of the bacterial liquid, centrifuge at 5000 rpm for 10 minutes to collect the bacterial cells, and wash twice with normal saline.

3) Dilute with pH7.0 phosphate buffer to 107/mL bacterial suspension.

4) Take 32mL of pH7.0 potassium phosphate buffer, 8mL of bacterial suspension, and 0.4mL of DES in a 150mL Erlenmeyer flask pre-placed in the rotor and mix thoroughly so that the final concentration of DES is 1% (v/v).

5) React in a shaker at 30° C. at 150 rpm for 30 min, take 1 mL of the mixed solution, and add 0.5 mL of 25% Na ₂ S ₂ O ₃ solution to stop the reaction.

6) Dilute and spread on the MRS xylan screening solid medium plate containing 90g/L xylan. After culturing at 40°C for 2 to 3 days, pick the strain with the largest transparent circle/colony diameter, and label it as DES bacteria.

2. Nitrosoguanidine Mutagenesis

1) Take the DES ring of Lactobacillus plantarum on the inclined surface of the test tube on the ultra-clean bench, insert it into a 250mL Erlenmeyer flask containing 50mL liquid MRS xylan medium, and cultivate it at 200rpm at 40°C for about 12h, so that the bacteria are in the right condition. Early growth period.

2) Take 5 mL of the bacterial solution and centrifuge at 5000 rpm for 10 minutes to collect the bacterial cells, and wash twice with normal saline.

3) Dilute with pH 6.0 phosphate buffer to 107/mL bacterial suspension.

4) Transfer 10 mL of bacterial suspension to a 100 mL Erlenmeyer flask, add 10 mg of NTG to prepare an NTG solution with a final concentration of 10 mg/mL, and add 4-5 drops of acetone to facilitate the dissolution of NTG.

5) Shake and react at 200 rpm for 30 min at 30° C., centrifuge at 5000 rpm for 10 min to collect the bacterial cells, wash with sterile saline several times, and terminate the reaction.

6) Appropriately dilute, take 0.2mL of the final dilution of the bacterial solution, dilute and spread it on the MRS xylan screening solid medium plate containing 90g/L xylan. After culturing at 40° C. for 2 to 3 days, pick 150 strains with larger transparent circle/colony diameter.

3. Shake flask re-screening

1) Take a ring of Lactobacillus plantarum on the slant of each test tube on the ultra-clean bench, insert it into a 250mL Erlenmeyer flask filled with 50mL liquid MRS xylan medium, culture at 200rpm, 40°C for 3-4 days, and test every day Glucose concentration and L-lactic acid concentration changes. After the fermentation, the xylan consumption rate, lactic acid production rate, lactic acid conversion rate and heteroacid content of 150 strains were compared.

2) Select the strain with fast xylan metabolism rate, high lactic acid concentration, high conversion rate and low heteroacid content as the final strain, named Li bacteria.

4. Genetic Stability Test

The Li-2013-01 strain was continuously subcultured on the slant for ten times, and the fermentation status after each subculture was detected by re-screening in shake flasks. The experiment found that after ten consecutive passages on the slant, the traits of the strain did not change significantly, and all performance indicators were normal, indicating that the strain had strong genetic stability.

The preparation method of Awamori Aspergillus agent:

Activation culture of slant strains: transfer Aspergillus awamori slant strains to the slant medium, and culture at 27°C for 3 days.

Solid first-level seed culture: pick the Aspergillus awamori slant strain and insert it into a 500 ml Erlenmeyer flask containing 100 grams of medium for seed culture, and cultivate it at 30°C for 3 days.

Solid secondary seed culture: Stir the above-mentioned cultivated solid primary seeds into pieces and add them to a 5000 ml Erlenmeyer flask containing 1000 grams of medium for seed cultivation. Culture conditions: 3 days at 30°C.

Solid fermentation culture: crush the seeds of the secondary shaker flask, add them to the fermentation tank or tray with sterilized medium, mix them evenly, and then cultivate them. The material is turned over once an hour, and the cultivation time is 5-7 days; the cultivation of solid koji material adopts the common koji material culture technology; the culture can be completed when the culture material is covered with hyphae, and the medium is pre-treated by high-temperature cooking and sterilization, and the sterilization conditions are controlled The temperature is 121°C, and the time is 1 hour.

Drying and crushing: After fermentation, the compost is dried on a fluidized bed or other drying equipment, the drying temperature is controlled at 60°C, and the moisture content is below 10%, then the solid compost is crushed, and the pore size of the crushed material is above 60 mesh .

Medium composition: solid raw materials: bran 80%, bean cake powder 10%, cornstarch 10%, add the same amount of tap water; the initial pH is natural.

Example 2

A kind of biological compound fertilizer, the number of parts by weight is: 12 parts of colloid bacillus, 5 parts of phosphorus solubilizing bacteria, 8 parts of potassium solubilizing bacteria, 25 parts of bacillus subtilis culture, 7 parts of aspergillus awamori culture, 6 parts of plant lactobacillus agent share.

Preparation method of Bacillus subtilis culture:

Obtaining of fermented liquid: Obtaining Bacillus subtilis fermented liquid by step-by-step expansion culture of slanted strains;

(1) First-level seed cultivation: insert the slant strain of Bacillus subtilis into a 500-milliliter shake flask, with a medium loading capacity of 100 milliliters, a rotary shaker at 180 rpm, a cultivation temperature of 30° C., and a cultivation time of 24 hours;

(2) secondary seed cultivation: the primary seed is inserted into a 500 milliliter secondary seed shake flask according to the inoculum size of 10%, and the cultivation condition is the same as that of the primary seed;

(3) Tertiary seed cultivation: insert the secondary seed into a 5000 milliliter three-stage seed shaking flask with 10% inoculum size, 1000 milliliters of culture medium, 100 rpm of rotary shaker, 30° C. of cultivation temperature, and cultivate Time 24 hours;

(4) First-level seed tank cultivation: connect the third-level seeds with 10% inoculation amount into a first-level seed tank with a total volume of 150L, the fermentation medium loading capacity is 100L, the cultivation temperature is 28°C, the stirring speed is 100 rpm, and the ventilation Volume (V/V) 1:0.5, tank pressure 0.05MPa, culture time 24 hours;

(5) Fermentation culture: the first-level seed tank strains are inserted into the total volume of 1.5 tons of second-level seed tanks with 10% inoculum, and the fermentation medium loading is 1 ton. The culture conditions are 28°C of culture temperature and 100 rpm of stirring speed. Min, ventilation volume (V/V) 1:0.5, tank pressure 0.05MPa, culture time 24 hours. After the fermentation is completed, the fermented liquid is filtered and dried to obtain the Bacillus subtilis culture containing the Bacillus subtilis agent.

Medium composition: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO ₃ 1%, pH 6.8.

The preparation method of plant lactobacillus agent:

(1) Primary seed cultivation: insert Lactobacillus plantarum into a 500-ml shaker flask, the culture medium is 100 ml, the culture temperature is 30°C, and the culture time is 24 hours;

(2) secondary seed cultivation: the primary seed is inserted into a 500 milliliter secondary seed shake flask according to the inoculum size of 10%, and the cultivation condition is the same as that of the primary seed;

(3) Tertiary seed cultivation: insert the secondary seeds with 10% inoculum size into 5000 milliliters of the tertiary seed shake flasks, the medium loading capacity is 1000 milliliters, the cultivation temperature is 30° C., and the cultivation time is 24 hours;

(4) First-level seed tank cultivation: put the third-level seeds into the first-level seed tank with a total volume of 150L with 5% inoculation amount, the fermentation medium loading capacity is 100L, the culture temperature is 30°C, the tank pressure is 0.05MPa, and the culture time is 18 Hour;

(5) Fermentation tank cultivation: the strains of the first-level seed tank are inserted into the second-level seed tank with a total volume of 3 tons with 5% inoculation amount, the fermentation medium loading capacity is 2 tons, and the culture conditions are culture temperature 30 ° C, tank pressure 0.05 MPa , The incubation time was 22 hours. After the fermentation is completed, the fermented liquid is vacuum-concentrated to 45% of the original volume by low-temperature negative pressure to obtain a concentrated bacterial liquid. Add carrier: add the mixed carrier to the concentrated solution and mix evenly; the weight ratio of the concentrated solution to the carrier is 0.5:1, the carrier composition is: 25 parts of CaCO ₃ , 12 parts of dextrin, fluidized bed drying, drying temperature 50 ℃.

The medium composition is: casein peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 0.5%, sodium acetate 0.5%, diamine citrate 0.2%, Tween800.1%, K ₂ HPO ₄ 0.2%, MgSO ₄ .7H ₂ O0.02%, MnSO ₄ .H ₂ O0.005%, CaCO ₃ 2%, agar 1.5%, pH6.8.

Example 3

A biological compound fertilizer, the number of parts by weight is: 10 parts of colloidal bacillus, 6 parts of phosphorus solubilizing bacteria, 7 parts of potassium solubilizing bacteria, 27 parts of Bacillus subtilis culture, 9 parts of Aspergillus awamori culture, 7 parts of plant lactobacillus agent share.

The preparation of the Bacillus subtilis culture: transfer and cultivate the Bacillus subtilis from the inclined plane, transfer the seed liquid after step-by-step expansion into the fermentation tank, and after the fermentation is completed, the fermentation liquid is filtered and dried to obtain the Bacillus subtilis culture .

The preparation method of Lactobacillus plantarum agent: transfer and cultivate Lactobacillus plantarum from an inclined plane, transfer the seed liquid after step-by-step expansion into a fermenter, and after fermentation, the fermented liquid is concentrated to 45% of the original volume by low-temperature negative pressure vacuum to obtain Bacterial Concentrate. Add carrier: add the mixed carrier to the concentrated solution, and mix evenly; the weight ratio of the concentrated solution to the carrier is 0.5:1, and the carrier composition is: 25 parts of CaCO ₃ and 12 parts of dextrin. Fluidized bed drying, drying temperature 50 ℃.

Preparation of Aspergillus awamori culture: strain culture, solid fermentation culture: spore liquid inoculated into Aspergillus oryzae solid-state fermentation culture material, cultured at 27-33°C until mycelia covered with culture material, dried in a low-temperature fluidized bed, and crushed to dry matter.

Example 4

Product effect experiment

Selection of test site and test design: The test was carried out in Babao Village, Huamachi Town, Yanchi County, Ningxia, from April 27th to September 30th, 2009.

10 mu of corn was planted in the experimental field, and 0.5 kg of the product of the present invention per mu was used when planting, and 0.5 kg of the product of the invention was used by hoeing and loosening the soil for about 1 month after emergence, and the control group used conventional fertilizers.

The corn yield of the corn field using the invented product reached 650 kg, and that of the control group reached 500 kg; the use of the invented product increased the yield of corn per mu by 30%. Spring wheat was planted in the plot in the second year, and the yield of spring wheat reached 400 kg, which was 20% higher than that of the control group. And the soil structure of the test field is good, without large lumps and compaction.

Claims (5)

1. a preparation method for composite bio-fertilizer, the microbial inoculum formed according to following parts by weight mixes, or granulates after mixing:

Colloid bacillus cereus 5-18 part, phosphate solubilizing bacteria 2-6 part, potassium solubilizing bacteria 5-10 part, subtilis culture 18-30 part, Aspergillus awamori culture 5-10 part, plant lactobacillus agent 3-8 part; Described subtilis preserving number is CGMCCNo.7926, and described plant lactobacillus deposit number is CGMCCNo.7928;

The preparation method of described subtilis culture is as follows: the acquisition of fermented liquid: cultivate subtilis from inclined-plane switching, seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermented liquid that ferments obtains subtilis culture after Plate Filtration, drying;

The preparation method of described plant lactobacillus agent is as follows: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% of original volume, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5-0.6:1, and vehicle group becomes: CaCO ₃20-30 part, dextrin 10-15 part; Fluidised bed drying, drying temperature 50 DEG C;

The preparation method of described Aspergillus awamori culture is as follows: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 26-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing.

2. the preparation method of a kind of composite bio-fertilizer according to claim 1, is characterized in that, composite bio-fertilizer parts by weight consist of: colloid bacillus cereus 12 parts, phosphate solubilizing bacteria 5 parts, potassium solubilizing bacteria 8 parts, subtilis culture 25 parts, Aspergillus awamori culture 7 parts, plant lactobacillus agent 6 parts.

3. the preparation method of a kind of composite bio-fertilizer according to claim 2, is characterized in that, the preparation method of subtilis culture is as follows:

The acquisition of fermented liquid: adopt slant strains to spread cultivation step by step and obtain fermentation of bacillus subtilis liquid;

(1) first order seed is cultivated: subtilis slant strains accessed in 500 ml shake flasks, substratum loading amount 100 milliliters, rotary shaker 180 revs/min, culture temperature 30 DEG C, incubation time 24 hours;

(2) secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;

(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, rotary shaker 100 revs/min, culture temperature 30 DEG C, incubation time 24 hours;

(4) first class seed pot is cultivated: the first class seed pot being 150L with 10% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours;

(5) fermentation culture: it is 1.5 tons of secondary seed tanks that first class seed pot bacterial classification is accessed cubic capacity with 10% inoculum size, fermention medium loading amount 1 ton, culture condition culture temperature 28 DEG C, stirring velocity 100 revs/min, ventilation (V/V) 1:0.5, tank pressure 0.05MPa, incubation time 24 hours; The complete fermented liquid that ferments obtains containing bacillus subtilis microbial inoculum at interior subtilis culture after Plate Filtration, drying;

Substratum forms: glucose 6%, yeast extract 1%, peptone 0.2%, CaCO ₃1%, pH6.8;

The preparation method of plant lactobacillus agent is as follows:

(1) first order seed is cultivated: accessed by plant lactobacillus bacterial classification in 500 ml shake flasks, substratum loading amount 100 milliliters, culture temperature 30 DEG C, incubation time 24 hours;

(2) secondary seed cultivate: by first order seed according to 10% inoculum size access in 500 milliliters of secondary seed shaking flasks, culture condition is identical with first order seed;

(3) three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum size, substratum loading amount 1000 milliliters, culture temperature 30 DEG C, incubation time 24 hours;

(4) first class seed pot is cultivated: the first class seed pot being 150L with 5% inoculum size access cubic capacity by three grades of seeds, fermention medium loading amount 100L, culture temperature 30 DEG C, tank pressure 0.05MPa, incubation time 18 hours;

(5) fermentor cultivation: it is 3 tons of secondary seed tanks that first class seed pot bacterial classification is accessed cubic capacity with 5% inoculum size, fermention medium loading amount 2 tons, culture condition culture temperature 30 DEG C, tank pressure 0.05MPa, incubation time 22 hours; The complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% of original volume, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO ₃25 parts, 12 parts, dextrin, fluidised bed drying, drying temperature 50 DEG C;

Substratum consists of: casein peptone 1%, beef extract 1%, yeast extract 0.5%, glucose 0.5%, sodium acetate 0.5%, dibasic ammonium citrate 0.2%, Tween800.1%, K ₂hPO ₄0.2%, MgSO ₄.7H ₂o0.02%, MnSO ₄.H ₂o0.005%, CaCO ₃2%, agar 1.5%, pH6.8.

4. the preparation method of a kind of composite bio-fertilizer according to claim 1, is characterized in that, composite bio-fertilizer parts by weight consist of: colloid bacillus cereus 10 parts, phosphate solubilizing bacteria 6 parts, potassium solubilizing bacteria 7 parts, subtilis culture 27 parts, Aspergillus awamori culture 9 parts, plant lactobacillus agent 7 parts.

5. the preparation method of a kind of composite bio-fertilizer according to claim 4, it is characterized in that, described subtilis culture preparation: cultivate subtilis from inclined-plane switching, seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermented liquid that ferments obtains subtilis culture after Plate Filtration, drying;

The preparation method of plant lactobacillus agent: from inclined-plane switching culturing plants Bacterium lacticum, the seed liquor after spreading cultivation step by step is transferred in fermentor tank, and the complete fermentation liquor low-temperature negative-pressure that ferments is concentrated in vacuo to 45% of original volume, obtains bacterium concentrated solution; Add carrier: in concentrated solution, add the carrier mixed, mix; The weight ratio of concentrated solution and carrier is 0.5:1, and vehicle group becomes: CaCO ₃25 parts, 12 parts, dextrin; Fluidised bed drying, drying temperature 50 DEG C;

Prepared by Aspergillus awamori culture: spawn culture, and solid fermentation is cultivated: spore liquid is inoculated in aspergillus oryzae solid state fermentation culture material, and 27-33 DEG C is cultured to mycelia and covers with culture material, and low temperature fluidized bed dry, pulverize dry thing.