CN105802889A - Multiple species inoculant for enhancing sediment nutrient and preparation method thereof - Google Patents
Multiple species inoculant for enhancing sediment nutrient and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a multiple species inoculant for enhancing sediment nutrient. The multiple species inoculant comprises azotobacter chroococcum, bacillus megaterium and paenibacillus mucilaginosus; a volume ratio of bacteria solution of the azotobacter chroococcum to bacteria solution of the bacillus megaterium to bacteria solution of the paenibacillus mucilaginosus is 3:1:1. According to the invention, a segmented aerated culture method is adopted to carry out culture expansion, and the temperature is also changed with time of bacteria solution culture. The multiple species inoculant disclosed by the invention has the beneficial effects that the bacterial proportion of the multiple species inoculant can effectively enhance the sediment nutrient of an organic fertilizer, and the organic fertilizer can be decomposed to a great degree so as to enable crops to adsorb the organic fertilizer; compared with a multiple species inoculant which does not adopt segmented aerated culture, the multiple species inoculant adopting segmented aerated culture is more beneficial to bacterium breeding, enables a living bacteria count to be improved, and is beneficial for forming bacillus stearothermophilus spores; according to the multiple species inoculant disclosed by the invention, in different periods, different temperatures are adopted to culture bacillus, which enables the living bacteria count to be improved and is beneficial for forming spores.
Description
Technical field
The present invention relates to microorganism field, more particularly to a kind of composite bacteria agent capable promoting matrix nutrition and preparation method thereof.
Background technology
Nitrogen fixing bacteria is one of highly important functional group in soil ecosystem, plays irreplaceable effect in soil nitrogen circulates.It can be used as the Inoculant of microbial manure to compare chemical fertilizer and have that cost is low, use safety, lasting effect is good, volume increase is stable, non-renewable energy consumption less, to features such as environment and food safety, economic benefit height.Nitrogen-fixing bacteria are utilized extensively to carry out all over the world as the research of Field inoculation agent and achieve some gratifying achievements.The growth of plant is had facilitation and with certain yield increasing effect by many results of study display Azotobacter.But owing to the specificity of bacterial strain Yu host plant is stronger, different niches is different from the kind of plant nitrogen-fixing bacteria and characteristic, therefore, separate from specific habitat with plant rhizosphere obtain high-efficiency nitrogen-fixing bacterial strain with develop applicable different niches and plant special bio bacterial manure have be of great significance.
Colloid bacillus cereus (Bacillusmucilaginous) has another name called bacillusmusilaginosiengineering, domestic is normally referred to as silicate bacteria, is a kind of multi-functional bacterium.Agriculturally, colloid bacillus cereus has potassium decomposing, molten phosphorus effect and is easily formed root system sociales characteristic, is one comparatively important in current China microbial manure product.Industrial aspect, colloid bacillus cereus is easily generated significant extracellular polysaccharide pod membrane in the C/N culture medium of nitrogen-free agar or bigger, utilizes the obvious microbial flocculant of the flocculation production advantage of this polysaccharide to have a extensive future.In metallurgical industry, colloid bacillus cereus can be applicable to antibacterial leaching and improves the characteristic of some mineral material, especially to the exploitation of low-grade mineral and recycle some useful metal and have good prospect.Aquaculture aspect, the protein of colloid bacillus cereus growth course generation, organic acid etc., can be used as feed supplement, improve the value of feedstuff.Therefore improve the viable bacteria of colloid bacillus cereus or Number of spores and quality has important real world applications meaning.
Bacillus megaterium, for producing spore bacillus, and is gram positive bacteria and aerobic bacteria, is also saprophytic bacteria in common oil.Industrially it is used for producing glucose isomerase, is also the decomposer of organophosphor simultaneously, therefore, manufacture phosphorous bacterial fertilizer agriculturally can be used for.Bacillus megaterium has effect of organophosphor in soil of well degrading, and is the conventional strain producing biological organic fertilizer, is also the conventional strain making water body inorganic agent, is administered on Nicotiana tabacum L. by it unique to improving tobacco fermentation flavouring effect.
Existing culture technique, cultivate nitrogen-fixing bacteria negligible amounts, and most bacterium pod membrane is relatively thin or is formed without pod membrane, cause that antibacterial is in preservation process, lack nutrition and mortality, affect shelf-life and product quality.
The function of pod membrane: 1. antiphagocytosis: pod membrane, because of its hydrophilic and space occupy-place, barrier action, is effective against the cytophagous phagocytosis of host.2. adhesion: capsular polysaccharide can make antibacterial adhesion to each other, it is possible to attach to histiocyte or xenobiotic surface；Bacterial capsule in biological wastewater treatment has biological adsorption effect, the Organic substance in waste water, inorganic matter and colloid is adsorbed on bacterial body surface.3. the damaging action of anti-harmful substance: be in bacterial cell outermost layer, pod membrane just as the armor can effectively protect thalline from or less by various sterilization, antibacterial substance damage, such as lysozyme, complement etc..4. resist drying effect: capsular polysaccharide is high degree of hydration molecule, water content, more than 95%, can help the dry threat to existence of antibacterial opposing.5., when lacking nutrition, pod membrane can be utilized makes carbon source and the energy, and some pod membranes also can make nitrogenous source.
The existing culture technique also majority of bacillus cereus rests on the yeast culture stage, and spore forming rate is low, and overall quantity is few, and the microbial inoculum not forming spore is easily affected by environment in storage, causes mortality.Spore is the life entity that in biosphere, resistance is the strongest, heat resistanceheat resistant, shoulder chemicals and radioprotective etc. in very prominent, the hypopus that spore is antibacterial can be changed into vegetative state cell under optimum conditions again.The trophocyte of antibacterial is just death in 10 minutes when 70～80 DEG C, and spore can also be survived several hours at 120～140 DEG C, trophocyte is dead soon in 5% phenol solution, spore but can be survived 15 days, most of enzymes of spore are inactive, metabolic activity is extremely low, so, spore is resistant to the hypopus of extraneous poor environment.
Therefore the number of spore directly affects the survival rate of bacillus cereus.
Existing antibacterial spreads cultivation technology, and bacterial reproduction speed is relatively slow, and survival rate is low, and the spore of bacillus cereus becomes rate not enough, and the time-to-live of bacillus cereus is short, has had a strong impact on product quality and the shelf-life of composite bacteria agent capable.
Summary of the invention
Instant invention overcomes the deficiencies in the prior art, a kind of composite bacteria agent capable promoting matrix nutrition and preparation method thereof is provided, solve existing microbial inoculum matrix nutrition enhancement dynamics is strong not, in microbial inoculum, bacterial content is not enough, the spore of bacillus cereus becomes rate not enough, time-to-live is shorter, the problem having a strong impact on product quality and shelf-life.
For solving above-mentioned technical problem, the present invention by the following technical solutions:
A kind of composite bacteria agent capable promoting matrix nutrition, described composite bacteria agent capable includes azotobacter chroococcum, bacillus megaterium and gel-shaped series bacillus, the bacterium solution of described azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus bacterium solution volume ratio be 2-4:1:1；The bacterium solution bacteria containing amount of described azotobacter chroococcum is 80-100 hundred million/ml, and the bacterium solution bacteria containing amount of described bacillus megaterium is 4.5-5 hundred million/ml, and the bacterium solution bacteria containing amount of described colloid bacillus cereus is 4.5-5 hundred million/ml.
A kind of preparation method of the composite bacteria agent capable promoting matrix nutrition, it includes three steps:
(1) amplification culture of azotobacter chroococcum bacterial strain: load azotobacter chroococcum culture fluid in shaking flask, is then seeded in shaking flask by azotobacter chroococcum one-level isogenic stocks and carries out spreading cultivation for the first time, and the temperature that spreads cultivation is 29-31 DEG C, and cultivation cycle is 36-40h；Then the azotobacter chroococcum bacterium solution after first time spreads cultivation being seeded to the one-level tank equipped with azotobacter chroococcum culture fluid and carry out aerobic culture, cultivation temperature is 29.5-30.5 DEG C, and cultivation cycle is 14-16h；Then azotobacter chroococcum bacterium solution after one-level tank spreads cultivation being seeded to two grades of tanks equipped with azotobacter chroococcum culture fluid and carries out aerobic culture, cultivation temperature is 28-32 DEG C, and cultivation cycle is 20-24h；
(2) amplification culture of gel-shaped series bacillus bacterial strain: load gel-shaped series bacillus culture fluid in shaking flask, then gel-shaped series bacillus one-level isogenic stocks is seeded in shaking flask and carries out spreading cultivation for the first time, the temperature that spreads cultivation is 31-33 DEG C, and cultivation cycle is 36-40h；Then the gel-shaped series bacillus bacterium solution after first time spreads cultivation being seeded to the one-level tank equipped with gel-shaped series bacillus culture fluid and carry out aerobic culture, cultivation temperature is 28-32 DEG C, and cultivation cycle is 20-24h；Then the gel-shaped series bacillus bacterium solution after one-level tank spreads cultivation being seeded to two grades of tanks equipped with gel-shaped series bacillus culture fluid and carry out aerobic culture, cultivation temperature is 28-34 DEG C, and cultivation cycle is 20-24h；
(3) amplification culture of bacillus megaterium bacterial strain: load bacillus megaterium culture fluid in shaking flask, then bacillus megaterium one-level isogenic stocks is seeded in shaking flask and carries out spreading cultivation for the first time, the temperature that spreads cultivation is 32.5-33.5 DEG C, and cultivation cycle is 36-40h；Then the bacillus megaterium bacterium solution after first time spreads cultivation being seeded to the one-level tank equipped with bacillus megaterium culture fluid and carry out aerobic culture, cultivation temperature is 32.5-33.5 DEG C, and cultivation cycle is 14-16h；Then bacillus megaterium bacterium solution after one-level tank spreads cultivation being seeded to two grades of tanks equipped with bacillus megaterium culture fluid and carries out aerobic culture, cultivation temperature is 28-33.5 DEG C, and cultivation cycle is 20-24h；
(4) bacterium solution that step (1)-(3) are finally obtained according to the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus the ratio mix homogeneously that bacterium solution volume ratio is 3:1:1, gnotobasis carries out fill.
Further, in described step (1), azotobacter chroococcum is seeded to one-level tank and carries out aerobic culture, and the ventilation before 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/h；In described step (1), azotobacter chroococcum is seeded in two grades of tanks and carries out aerobic culture, and the ventilation before 3h is 12m3After/h, 3h to ventilation before 6h be 18m3After/h, 6h to the ventilation before 9h be 24m3The later ventilation of/h, 9h is 42-48m3/h。
Further, in described step (2), gel-shaped series bacillus bacterium solution is seeded to one-level tank and carries out aerobic culture, and the ventilation before 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/h；In described step (2), gel-shaped series bacillus bacterium solution is seeded in two grades of tanks and carries out aerobic culture, and the ventilation before 3h is 12m3After/h, 3h to ventilation before 6h be 18m3After/h, 6h to the ventilation before 9h be 24m3The later ventilation of/h, 9h is 42-48m3/ h, is 28-30 DEG C in two grades of tank cultivation temperature 12h, and 12h-24h cultivation temperature is 32-33 DEG C, and after 24h, temperature rises to 33-34 DEG C.
Further, in described step (3), bacillus megaterium bacterium solution is seeded to one-level tank and carries out aerobic culture, and cultivation cycle is the ventilation before 14-16h, 3h is 1m3After/h, 3h to ventilation before 6h be 2m3After/h, 6h to ventilation before 9h be 3m3/h；In described step (3), bacillus megaterium bacterium solution is seeded to one-level tank and carries out aerobic culture, and cultivation cycle is the ventilation before 20-24h, 3h is 20m3After/h, 3h to ventilation before 6h be 25m3/ h, 6h rear venting amount is 35m3Being 28-30 DEG C in/h, cultivation temperature 12h, 12h-24h cultivation temperature is 32-33 DEG C, and after 24h, temperature rises to 33.5 DEG C, and tank pressure is 0.05M.
Further, the preparation method of the inoculum of described azotobacter chroococcum: take sucrose 10-11g, ammonium sulfate 0.5-0.6g, potassium dihydrogen phosphate 0.2-0.3g, yeast extract 0.5-0.6g, sodium citrate 0.5-0.6g, magnesium sulfate 0.2-0.3g, dipotassium hydrogen phosphate 0.7-0.8g, tap water 1000ml are configured to the solution of pH7.0-7.2, and sterilizing is standby.
Further, the preparation method of the inoculum of described gel-shaped series bacillus: take soluble starch 5.0-6.0g, sucrose 2.0-3.0g, ammonium sulfate 0.8-1.0g, soybean cake powder 0.8-1.0g, potassium dihydrogen phosphate 2.5-3.0g, magnesium sulfate 1.5-2.0g, calcium carbonate 1.5-2.0g, ferric chloride 0.2-0.3g, yeast extract 1.0-1.5g, tap water 1000ml are configured to the solution of PH7.2-7.5, and sterilizing is standby.
Further, the preparation method of the inoculum of described bacillus megaterium: glucose 2.0-3.0g, calcium carbonate 5.0-6.0g, magnesium sulfate 0.4-0.6g, potassium dihydrogen phosphate 0.2-0.4g, ammonium sulfate 0.8-1.0g, yeast extract 0.8-1.0g, sodium chloride 1.0-1.5g, soybean cake powder 3.0-3.5g, starch 5.0-6.0g, tap water 1000ml are configured to the solution of PH7.0-7.5, and sterilizing is standby.
Compared with prior art, the invention has the beneficial effects as follows:
(1) the strain ratio of the present invention can effectively promote the matrix nutrition of fertilizer, can decompose fertilizer significantly and enable crop to absorb.
(2) present invention adopts segmentation aerobic culture to be more beneficial for breeding of antibacterial than unsegmented aerobic culture, and living bacteria count promotes, and is conducive to the formation of bacillus spore；Under non-optimal ventilation, thalline will be unable to reach maximum output, and affects spore forming rate.Constant temperature culture causes that a large amount of thalline is dead in the sporulation phase, it is impossible to form spore, and the period that the present invention is different adopts different temperature that bacillus cereus is cultivated, and living bacteria count promotes, and is conducive to the formation of spore.
(3) culture fluid of conventional azotobacter chroococcum is that mannitol 10g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.2g, sodium chloride 0.2g, calcium carbonate 1g add water formulated, nitrogen-fixing bacteria reproductive number in nitrogenous culture medium is more, the culture fluid of azotobacter chroococcum of the present invention selects yeast extract as nitrogenous source, can promoting bacterial concentration in a large number, ammonium sulfate, yeast extract provide the breeding of multiple nitrogenous source (organic nitrogen source is with inorganic nitrogen-sourced composite) nitrogen-fixing bacteria preferably.Sodium citrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate play the effect of pH buffer agent in the medium, make the ph growing environment that nitrogen-fixing bacteria keep relative stability in incubation.The culture fluid that the present invention adopts considerably increases the quantity of azotobacter chroococcum viable bacteria, rises to the viable count of at least 80 hundred million/ml from the viable count of about the 2000000000/ml of conventional formulation.
(4) culture fluid of conventional colloid bacillus cereus is that potassium nitrate 1g, dipotassium hydrogen phosphate 0.5g, sodium chloride 0.5g, ferrous sulfate 0.01g, soluble starch 20g, magnesium sulfate 0.5g add water formulated, the culture fluid of colloid bacillus cereus of the present invention adopts soluble starch, ferric chloride to be conducive to colloid bacillus to form pod membrane and spore, soybean cake powder and ammonium sulfate, yeast extract are multiple types nitrogenous source, provide the nitrogenous source of different demand for each growth period of antibacterial.Sucrose can provide early stage exponential phase carbon source, carbon source needed for soluble starch offer formation spore.Calcium carbonate is the key factor that colloid bacillus cereus forms spore.The culture fluid that the present invention adopts considerably increases the quantity of colloid bacillus cereus viable bacteria, rises to the viable count of about 500,000,000/ml from the viable count of about the 300000000/ml of conventional formulation, and spore rate also rises at least 90% from 30%.
(5) culture fluid of conventional bacillus megaterium is that Carnis Bovis seu Bubali cream 20.0g, peptone 10.0g, sodium chloride 5.0g add water formulated, the culture fluid of bacillus megaterium of the present invention adopts soybean cake powder and ammonium sulfate, yeast extract to be multiple types nitrogenous source, provides the nitrogenous source of different demand for each growth period of antibacterial.Glucose can provide early stage exponential phase carbon source, and early stage bacillus megaterium is higher for quick carbon source demand, carbon source needed for starch offer formation spore.Calcium carbonate is the key factor that colloid bacillus cereus forms spore.The culture fluid that the present invention adopts considerably increases the quantity of bacillus megaterium viable bacteria, rises to the viable count of about 500,000,000/ml from the viable count of about the 300000000/ml of conventional formulation, and spore rate also rises at least 90% from 30%.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.Embodiments of the present invention include but not limited to the following example.
[embodiment 1]
A kind of composite bacteria agent capable promoting matrix nutrition, described composite bacteria agent capable includes azotobacter chroococcum, bacillus megaterium and gel-shaped series bacillus, the bacterium solution of described azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus bacterium solution volume ratio be 2:1:1；The bacterium solution bacteria containing amount of described azotobacter chroococcum is 98.6 hundred million/ml, and the bacterium solution bacteria containing amount of described bacillus megaterium is 4.7 hundred million/ml, and the bacterium solution bacteria containing amount of described colloid bacillus cereus is 5.0 hundred million/ml.
Wherein, the amplification culture method of described azotobacter chroococcum bacterial strain is: load azotobacter chroococcum inoculum 120mlml in the triangle shaking flask of 500ml, inoculation azotobacter chroococcum one-level isogenic stocks 7.5ml, then shaking flask, rotary shaker setting speed 90-100rpm, cultivation 36h, cultivation temperature is 29 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 75% of described one-level tank azotobacter chroococcum inoculum, described inoculum concentration is the 1% of the loading amount of one-level tank azotobacter chroococcum inoculum, then carries out aerobic culture, cultivation cycle is the ventilation before 14h, 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/ h, one-level tank speed setting rotating speed 90-100rpm, one-level tank cultivation temperature is 29.5 DEG C, and tank pressure is 0.05Mpa.Then the bacterium solution transferred species after being fermented by one-level tank is to two pole tanks, loading amount is tank body the 75% of two grades of tank azotobacter chroococcum inoculums, described two grades of tank inoculum concentrations are the 1%-5% of the loading amount of azotobacter chroococcum inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 24h, 3h is 15m3After/h, 3h to ventilation before 6h be 25m3After/h, 6h to the ventilation before 9h be 30m3The later ventilation of/h, 9h is 42m3/ h, two grades of tank speed setting rotating speed 90-100rpm, two grades of tank cultivation temperature are 28 DEG C, and tank pressure is 0.05Mpa.The preparation method of the inoculum of described azotobacter chroococcum: take sucrose 10g, ammonium sulfate 0.5g, potassium dihydrogen phosphate 0.2g, yeast extract 0.5g, sodium citrate 0.5g, magnesium sulfate 0.2g, dipotassium hydrogen phosphate 0.7g, tap water 1000ml are configured to the solution of pH7.0-7.2, and sterilizing is standby.
The amplification culture method of described gel-shaped series bacillus bacterial strain is: load gel-shaped series bacillus inoculum 60ml in the triangle shaking flask of 500ml, inoculation gel-shaped series bacillus one-level isogenic stocks 3.0ml, then shaking flask, rotary shaker setting speed 190-200rpm, cultivation 36h, cultivation temperature is 32.5 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 75% of described one-level tank gel-shaped series bacillus inoculum, described bacterium solution inoculum concentration is the 1% of the loading amount of one-level tank gel-shaped series bacillus inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 14h, 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/ h, one-level tank speed setting rotating speed 90-100rpm, cultivation temperature is 29.5 DEG C, tank pressure is that the bacterium solution transferred species after then one-level tank is fermented by 0.05Mpa. is to two pole tanks, loading amount is tank body the 75% of two grades of tank gel-shaped series bacillus inoculums, described two grades of tank bacterium solution inoculum concentrations are the 1% of the loading amount of gel-shaped series bacillus inoculum, then carry out aerobic culture, cultivation cycle is the ventilation before 24h, 3h is 12m3After/h, 3h to ventilation before 6h be 18m3After/h, 6h to the ventilation before 9h be 24m3The later ventilation of/h, 9h is 42-48m3/ h, two grades of tank speed setting rotating speed 90-100rpm, are 28 DEG C in two grades of tank cultivation temperature 12h, and 12h-24h cultivation temperature is 32 DEG C, and after 24h, temperature rises to 33 DEG C, and tank pressure is 0.05Mpa.The preparation method of the inoculum of described gel-shaped series bacillus: take soluble starch 5.0g, sucrose 2.0g, ammonium sulfate 0.8g, soybean cake powder 0.8g, potassium dihydrogen phosphate 2.5g, magnesium sulfate 1.5g, calcium carbonate 1.5g, ferric chloride 0.2g, yeast extract 1.0g, tap water 1000ml are configured to the solution of PH7.2-7.5, and sterilizing is standby.
The amplification culture method of described bacillus megaterium bacterial strain is: load bacillus megaterium inoculum 60ml in the triangle shaking flask of 500ml, inoculation bacillus megaterium one-level isogenic stocks 3.0ml, then shaking flask, rotary shaker setting speed 190-200rpm, cultivation 12, cultivation temperature is 32.5 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 75% of described one-level tank bacillus megaterium inoculum, described bacterium solution inoculum concentration is the 1% of the loading amount of one-level tank bacillus megaterium inoculum, then carries out aerobic culture, cultivation cycle is the ventilation before 14h, 3h is 1m3After/h, 3h to ventilation before 6h be 2m3After/h, 6h to ventilation before 9h be 3m3Ventilation after/h, 9h is 4m3/ h, one-level tank speed setting rotating speed 190-200rpm, cultivation temperature is 32.5 DEG C, and one-level tank tank pressure is 0.05Mpa；Then the bacterium solution transferred species after being fermented by one-level tank is to two pole tanks, loading amount is tank body the 75% of two grades of tank bacillus megaterium inoculums, described two grades of tank bacterium solution inoculum concentrations are the 1% of the loading amount of bacillus megaterium inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 24h, 3h is 20m3After/h, 3h to ventilation before 6h be 25m3/ h, 6h rear venting amount is 35m3/ h, two grades of tank speed setting rotating speed 190-200rpm, in cultivation temperature 12h be 28 DEG C, and 12h-24h cultivation temperature is 32 DEG C, and after 24h, temperature rises to 33.5 DEG C, and two grades of tank tank pressures are 0.05Mpa.The preparation method of the inoculum of described bacillus megaterium: glucose 2.0g, calcium carbonate 5.0g, magnesium sulfate 0.4g, potassium dihydrogen phosphate 0.2g, ammonium sulfate 0.8g, yeast extract 0.8g, sodium chloride 1.0g, soybean cake powder 3.0g, starch 5.0g, tap water 1000ml are configured to the solution of PH7.0-7.5, and sterilizing is standby.
By above-mentioned through amplification culture, the antibacterial bacterium solution finally obtained according to the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus the ratio mix homogeneously that bacterium solution volume ratio is 2:1:1, sterile filling can obtain promote matrix nutrition composite bacteria agent capable.
[embodiment 2]
A kind of composite bacteria agent capable promoting matrix nutrition, described composite bacteria agent capable includes azotobacter chroococcum, bacillus megaterium and gel-shaped series bacillus, the bacterium solution of described azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus bacterium solution volume ratio be 4:1:1；The bacterium solution bacteria containing amount of described azotobacter chroococcum is 8,000,000,000/ml, and the bacterium solution bacteria containing amount 4.5 hundred million/ml of described bacillus megaterium, the bacterium solution bacteria containing amount of described colloid bacillus cereus is 4.5 hundred million/ml.
Wherein, the amplification culture method of described azotobacter chroococcum bacterial strain is: load azotobacter chroococcum inoculum 150ml in the triangle shaking flask of 500ml, inoculation azotobacter chroococcum one-level isogenic stocks 7.5ml, then shaking flask, rotary shaker setting speed 90-100rpm, cultivation 40h, cultivation temperature is 31 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 80% of described one-level tank azotobacter chroococcum inoculum, described inoculum concentration is the 5% of the loading amount of one-level tank azotobacter chroococcum inoculum, then carries out aerobic culture, cultivation cycle is the ventilation before 16h, 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/ h, one-level tank speed setting rotating speed 90-100rpm, one-level tank cultivation temperature is 30.5 DEG C, and tank pressure is 0.05Mpa.Then the bacterium solution transferred species after being fermented by one-level tank is to two pole tanks, loading amount is tank body the 80% of two grades of tank azotobacter chroococcum inoculums, described two grades of tank inoculum concentrations are the 5% of the loading amount of azotobacter chroococcum inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 28h, 3h is 15m3After/h, 3h to ventilation before 6h be 25m3After/h, 6h to the ventilation before 9h be 30m3The later ventilation of/h, 9h is 42-45m3/ h, two grades of tank speed setting rotating speed 90-100rpm, two grades of tank cultivation temperature are 32 DEG C, and tank pressure is 0.05Mpa.The preparation method of the inoculum of described azotobacter chroococcum: take sucrose 11g, ammonium sulfate 0.7g, potassium dihydrogen phosphate 0.4g, yeast extract 0.7g, sodium citrate 0.7g, magnesium sulfate 0.4g, dipotassium hydrogen phosphate 0.9g, tap water 1000ml are configured to the solution of pH7.0-7.2, and sterilizing is standby.
The amplification culture method of described gel-shaped series bacillus bacterial strain is: load gel-shaped series bacillus inoculum 80ml in the triangle shaking flask of 500ml, inoculation gel-shaped series bacillus one-level isogenic stocks 3.0ml, then shaking flask, rotary shaker setting speed 190-200rpm, cultivation 48h, cultivation temperature is 33.5 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 80% of described one-level tank gel-shaped series bacillus inoculum, described bacterium solution inoculum concentration is the 5% of the loading amount of one-level tank gel-shaped series bacillus inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 16h, 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/ h, one-level tank speed setting rotating speed 90-100rpm, cultivation temperature is 30.5 DEG C, tank pressure is that the bacterium solution transferred species after then one-level tank is fermented by 0.05Mpa. is to two pole tanks, loading amount is tank body the 80% of two grades of tank gel-shaped series bacillus inoculums, described two grades of tank bacterium solution inoculum concentrations are the 5% of the loading amount of gel-shaped series bacillus inoculum, then carry out aerobic culture, cultivation cycle is the ventilation before 30h, 3h is 12m3After/h, 3h to ventilation before 6h be 18m3After/h, 6h to the ventilation before 9h be 24m3The later ventilation of/h, 9h is 42-48m3/ h, two grades of tank speed setting rotating speed 90-100rpm, are 30 DEG C in two grades of tank cultivation temperature 12h, and 24h cultivation temperature is 35 DEG C, and after 24h, temperature rises to 34 DEG C, and tank pressure is 0.05Mpa.The preparation method of the inoculum of described gel-shaped series bacillus: take soluble starch 6.0g, sucrose 3.0g, ammonium sulfate 1.0g, soybean cake powder 1.0g, potassium dihydrogen phosphate 3.0g, magnesium sulfate 2.0g, calcium carbonate 2.0g, ferric chloride 0.4g, yeast extract 1.5g, tap water 1000ml are configured to the solution of PH7.2-7.5, and sterilizing is standby.
The amplification culture method of described bacillus megaterium bacterial strain is: load bacillus megaterium inoculum 80ml in the triangle shaking flask of 500ml, inoculation bacillus megaterium one-level isogenic stocks 3.0ml, then shaking flask, rotary shaker setting speed 190-200rpm, cultivation 16h, cultivation temperature is 33.5 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 80% of described one-level tank bacillus megaterium inoculum, described bacterium solution inoculum concentration is the 5% of the loading amount of one-level tank bacillus megaterium inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 14-16h, 3h is 1m3After/h, 3h to ventilation before 6h be 2m3After/h, 6h to ventilation before 9h be 3m3Ventilation after/h, 9h is 4m3/ h, one-level tank speed setting rotating speed 190-200rpm, cultivation temperature is 33.5 DEG C, and one-level tank tank pressure is 0.05Mpa；Then the bacterium solution transferred species after being fermented by one-level tank is to two pole tanks, loading amount is tank body the 80% of two grades of tank bacillus megaterium inoculums, described two grades of tank bacterium solution inoculum concentrations are the 5% of the loading amount of bacillus megaterium inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 36h, 3h is 20m3After/h, 3h to ventilation before 6h be 25m3/ h, 6h rear venting amount is 35m3/ h, two grades of tank speed setting rotating speed 190-200rpm, in cultivation temperature 12h be 30 DEG C, and 12h-24h cultivation temperature is 33 DEG C, and after 24h, temperature rises to 33.5 DEG C, and two grades of tank tank pressures are 0.05Mpa.The preparation method of the inoculum of described bacillus megaterium: glucose 3.0g, calcium carbonate 6.0g, magnesium sulfate 0.6g, potassium dihydrogen phosphate 0.4g, ammonium sulfate 1.0g, yeast extract 1.0g, sodium chloride 1.2g, soybean cake powder 3.5g, starch 6.0g, tap water 1000ml are configured to the solution of PH7.0-7.5, and sterilizing is standby.
By above-mentioned through amplification culture, the antibacterial bacterium solution finally obtained according to the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus the ratio mix homogeneously that bacterium solution volume ratio is 4:1:1, sterile filling can obtain promote matrix nutrition composite bacteria agent capable.
[embodiment 3]
A kind of composite bacteria agent capable promoting matrix nutrition, described composite bacteria agent capable includes azotobacter chroococcum, bacillus megaterium and gel-shaped series bacillus, the bacterium solution of described azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus bacterium solution volume ratio be 3:1:1；The bacterium solution bacteria containing amount of described azotobacter chroococcum is 10,000,000,000/ml, and the bacterium solution bacteria containing amount of described bacillus megaterium is 500,000,000/ml, and the bacterium solution bacteria containing amount of described colloid bacillus cereus is 500,000,000/ml.
Wherein, the amplification culture method of described azotobacter chroococcum bacterial strain is: load azotobacter chroococcum inoculum 135ml in the triangle shaking flask of 500ml, inoculation azotobacter chroococcum one-level isogenic stocks 4.5ml, then shaking flask, rotary shaker setting speed 90-100rpm, cultivation 38h, cultivation temperature is 30 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 77.5% of described one-level tank azotobacter chroococcum inoculum, described inoculum concentration is the 3% of the loading amount of one-level tank azotobacter chroococcum inoculum, then carries out aerobic culture, cultivation cycle is the ventilation before 15h, 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/ h, one-level tank speed setting rotating speed 90-100rpm, one-level tank cultivation temperature is 30 DEG C, and tank pressure is 0.05Mpa.Then the bacterium solution transferred species after being fermented by one-level tank is to two pole tanks, loading amount is tank body the 77.5% of two grades of tank azotobacter chroococcum inoculums, described two grades of tank inoculum concentrations are the 3% of the loading amount of azotobacter chroococcum inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 26h, 3h is 15m3After/h, 3h to ventilation before 6h be 25m3After/h, 6h to the ventilation before 9h be 30m3The later ventilation of/h, 9h is 42-45m3/ h, two grades of tank speed setting rotating speed 90-100rpm, two grades of tank cultivation temperature are 30 DEG C, and tank pressure is 0.05Mpa.The preparation method of the inoculum of described azotobacter chroococcum: take sucrose 10.5g, ammonium sulfate 0.6g, potassium dihydrogen phosphate 0.3g, yeast extract 0.6g, sodium citrate 0.6g, magnesium sulfate 0.3g, dipotassium hydrogen phosphate 0.8g, tap water 1000ml are configured to the solution of pH7.0-7.2, and sterilizing is standby.
The amplification culture method of described gel-shaped series bacillus bacterial strain is: load gel-shaped series bacillus inoculum 70ml in the triangle shaking flask of 500ml, inoculation gel-shaped series bacillus one-level isogenic stocks 1.8ml, then shaking flask, rotary shaker setting speed 190-200rpm, cultivation 42h, cultivation temperature is 32.5-33.5 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 77.5% of described one-level tank gel-shaped series bacillus inoculum, described bacterium solution inoculum concentration is the 1%-5% of the loading amount of one-level tank gel-shaped series bacillus inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 15h, 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/ h, one-level tank speed setting rotating speed 90-100rpm, cultivation temperature is 30 DEG C, tank pressure is that the bacterium solution transferred species after then one-level tank is fermented by 0.05Mpa. is to two pole tanks, loading amount is tank body the 77.5% of two grades of tank gel-shaped series bacillus inoculums, described two grades of tank bacterium solution inoculum concentrations are the 3% of the loading amount of gel-shaped series bacillus inoculum, then carry out aerobic culture, cultivation cycle is the ventilation before 27h, 3h is 12m3After/h, 3h to ventilation before 6h be 18m3After/h, 6h to the ventilation before 9h be 24m3The later ventilation of/h, 9h is 42-48m3/ h, two grades of tank speed setting rotating speed 90-100rpm, are 29 DEG C in two grades of tank cultivation temperature 12h, and 12h-24h cultivation temperature is 33.5 DEG C, and after 24h, temperature rises to 33.5 DEG C, and tank pressure is 0.05Mpa.The preparation method of the inoculum of described gel-shaped series bacillus: take soluble starch 5.5g, sucrose 2.5g, ammonium sulfate 0.9g, soybean cake powder 0.9g, potassium dihydrogen phosphate 2.75g, magnesium sulfate 1.75g, calcium carbonate 1.75g, ferric chloride 0.3g, yeast extract 1.25g, tap water 1000ml are configured to the solution of PH7.2-7.5, and sterilizing is standby.
The amplification culture method of described bacillus megaterium bacterial strain is: load bacillus megaterium inoculum 70ml in the triangle shaking flask of 500ml, inoculation bacillus megaterium one-level isogenic stocks 1.8ml, then shaking flask, rotary shaker setting speed 190-200rpm, cultivation 14h, cultivation temperature is 33 DEG C；Then it is seeded to one-level tank to spread cultivation, loading amount is tank body the 77.5% of described one-level tank bacillus megaterium inoculum, described bacterium solution inoculum concentration is the 3% of the loading amount of one-level tank bacillus megaterium inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 15h, 3h is 1m3After/h, 3h to ventilation before 6h be 2m3After/h, 6h to ventilation before 9h be 3m3Ventilation after/h, 9h is 4m3/ h, one-level tank speed setting rotating speed 190-200rpm, cultivation temperature is 33 DEG C, and one-level tank tank pressure is 0.05Mpa；Then the bacterium solution transferred species after being fermented by one-level tank is to two pole tanks, loading amount is tank body the 77.5% of two grades of tank bacillus megaterium inoculums, described two grades of tank bacterium solution inoculum concentrations are the 1%-5% of the loading amount of bacillus megaterium inoculum, then aerobic culture is carried out, cultivation cycle is the ventilation before 30h, 3h is 20m3After/h, 3h to ventilation before 6h be 25m3/ h, 6h rear venting amount is 35m3/ h, two grades of tank speed setting rotating speed 190-200rpm, in cultivation temperature 12h be 29 DEG C, and 12h-24h cultivation temperature is 32.5 DEG C, and after 24h, temperature rises to 33.5 DEG C, and two grades of tank tank pressures are 0.05Mpa.The preparation method of the inoculum of described bacillus megaterium: glucose 2.5g, calcium carbonate 5.5g, magnesium sulfate 0.5g, potassium dihydrogen phosphate 0.3g, ammonium sulfate 0.9g, yeast extract 0.9g, sodium chloride 1.1g, soybean cake powder 3.25g, starch 5.5g, tap water 1000ml are configured to the solution of PH7.0-7.5, and sterilizing is standby.
By above-mentioned through amplification culture, the antibacterial bacterium solution finally obtained according to the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus the ratio mix homogeneously that bacterium solution volume ratio is 3:1:1, sterile filling can obtain promote matrix nutrition composite bacteria agent capable.
[ventilation contrast experiment 1]
Change the ventilation of the one-level tank aerobic culture of the amplification culture method of azotobacter chroococcum bacterial strain in embodiment 1 into 8m3/ h, the ventilation of two grades of tank aerobic culture changes 50m into3/ h, other steps keep consistent with the amplification culture method of the azotobacter chroococcum bacterial strain in embodiment 1.
Change the ventilation of the one-level tank aerobic culture of the amplification culture method of gel-shaped series bacillus bacterial strain in embodiment 1 into 4m3/ h, the ventilation of two grades of tank aerobic culture changes 40m into3/ h, other steps keep consistent with the amplification culture method of the gel-shaped series bacillus bacterial strain in embodiment 1.
Change the ventilation of the one-level tank aerobic culture of the amplification culture method of bacillus megaterium bacterial strain in embodiment 1 into 4m3/ h, the ventilation of two grades of tank aerobic culture changes 30m into3/ h, other steps keep consistent with the amplification culture method of the bacillus megaterium bacterial strain in embodiment 1.
Table 1
Conclusion: the data of table 1 show, segmentation aerobic culture is more beneficial for breeding of antibacterial than unsegmented aerobic culture, and living bacteria count promotes, and is conducive to the formation of bacillus spore.Under non-optimal ventilation, thalline will be unable to reach maximum output, and affects spore forming rate.
[temperature comparisons tests 1]
Changing the temperature of two grades of tank aerobic culture of the amplification culture method of gel-shaped series bacillus bacterial strain in embodiment 1 into 30 DEG C, other steps keep consistent with the amplification culture method of the gel-shaped series bacillus bacterial strain in embodiment 1.
Changing the temperature of two grades of tank aerobic culture of the amplification culture method of bacillus megaterium bacterial strain in embodiment 1 into 30 DEG C, other steps keep consistent with the amplification culture method of the bacillus megaterium bacterial strain in embodiment 1.
Table 2
Conclusion: table 2 data show, adopts different temperature that bacillus cereus is cultivated different periods, and living bacteria count promotes, and is conducive to the formation of spore, and constant temperature culture causes that a large amount of thalline is dead in the sporulation phase, it is impossible to form spore.
[application example 1]
Raw material: the composite bacteria agent capable 1 kilogram promoting matrix nutrition that will produce in chicken manure 2 tons, powder of straw or leaf powder 0.5 ton, Semen Maydis flour 2.5 kilograms, embodiment 1.
Chicken manure hot fermentation: be deposited in fermentation vat by chicken manure, generally the time of fermentation is 5-7 days；Chicken manure after the degree of depth is fermented, with powder of straw or leaf powder, Semen Maydis flour, the composite bacteria agent capable promoting matrix nutrition mixes, and the dispensing being stirred piles wide 1.5-2 rice, and the strip of high 0.3-0.4 rice, upper cover straw screen or mat or the piece of sack carry out aerobic fermentation composting.In generally banking up 24 hours, temperature rises to 50 DEG C, and in 48 hours, temperature can rise to 60-70 DEG C, and during fermentation so high temperature can kill all source of disease bacterium and worm's ovum, grass seed etc..Testing according to composting, spring and summer compost in autumn composting generally needs 6 days, generally needs winter 7 days, treats that fertilizer covers with white hypha up and down, namely all becomes thoroughly decomposed, and dry in the sun is sieved a little.
[application example 2]
Raw material in application example 1 is changed into: the composite bacteria agent capable 1 kilogram promoting matrix nutrition that will produce in chicken manure 2 tons, powder of straw or leaf powder 0.5 ton, Semen Maydis flour 2.5 kilograms, embodiment 2.
Compost step is according to the compost step of application example 1.
[application example 3]
Raw material in application example 1 is changed into: the composite bacteria agent capable 1 kilogram promoting matrix nutrition that will produce in chicken manure 2 tons, powder of straw or leaf powder 0.5 ton, Semen Maydis flour 2.5 kilograms, embodiment 3.
Compost step is according to the compost step of application example 1.
[comparison example 1]
Raw material in application example 1 is changed into: by chicken manure 2 tons, powder of straw or leaf powder 0.5 ton, Semen Maydis flour 2.5 kilograms, humic acid 1 kilogram.
Compost step is according to the compost step of application example 1.
[comparison example 2]
Antibacterial is cultivated according to the training method in embodiment 1, then according to the ratio mix homogeneously that bacterium solution volume ratio is 1:1:1 of the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus, makes composite bacteria agent capable.
Raw material by application example 1: the composite bacteria agent capable 1 kilogram promoting matrix nutrition produced in embodiment 1 changes the composite bacteria agent capable 1 kilogram that this comparison example produces into, and compost step is according to the compost step of application example 1.
[comparison example 3]
Antibacterial is cultivated according to the training method of any one embodiment in embodiment 1-3, then according to the ratio mix homogeneously that bacterium solution volume ratio is 5:1:1 of the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus, make composite bacteria agent capable.
Raw material by application example 1: the composite bacteria agent capable 1 kilogram promoting matrix nutrition produced in embodiment 1 changes the composite bacteria agent capable 1 kilogram that this comparison example produces into, and compost step is according to the compost step of application example 1.
[comparison example 4]
Antibacterial is cultivated according to the training method of any one embodiment in embodiment 1-3, then according to the ratio mix homogeneously that bacterium solution volume ratio is 3:2:1 of the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus, make composite bacteria agent capable.
Raw material by application example 1: the composite bacteria agent capable 1 kilogram promoting matrix nutrition produced in embodiment 1 changes the composite bacteria agent capable 1 kilogram that this comparison example produces into, and compost step is according to the compost step of application example 1.
[field test]
Test site: experimental plot, Modern Agricultural Park, Jintan, Jin Long township, Luzhou City Longmatan District.
Test period: in July ,-2015 in March, 2015.
Experimental field basic condition: be experimental field positioned at moistening monsoon climatic region, subtropical zone, for the yellow sandy loam that examination soil is medium fertility, soil fertility is more uniform, and illumination is better, and irrigation and drainage are convenient, and front late autumn in season is as Brassica campestris L, test area 2 mu.
For studying thing: beautiful No. 2 Semen Maydiss in river.
EXPERIMENTAL DESIGN: this test is for arranging four unit, i.e. seven unit of T1, T2, T3, T4, T5, T6, T7, each cellar area is 0.5 mu, T1 uses the fertilizer that application example 1 produces, and T2 uses the fertilizer that application example 2 produces, and T3 uses the fertilizer that application example 3 produces, T4 uses the fertilizer that comparison example 1 produces, T5 uses the fertilizer that comparison example 2 produces, and T6 uses the fertilizer that comparison example 3 produces, and T7 uses the fertilizer that comparison example 4 produces.
After corn planting, carrying out emergence rate observation, survey result is, T1:90.8%, T2:89.8%, T3:91.2%, T4:84.5%, T5:87.2%, T6:86.9%, T7:87.5%.
The Semen Maydis jointing stage, each unit Semen Maydis plant height, single-strain fresh weight, side radical, root fresh weight, leaf area per plant being investigated, survey result is in Table 3:
Table 3
Volume analysis: after harvest corn, singly receives singles, and the yield of each unit is, T1:316.5kg, T2:314.3kg, T3:321.1kg, T4:301.7kg, T5:308.2kg, T6:307.5kg, T7:306.9kg.
Data above show effect of the fertilizer with the composite bacteria agent capable that with the addition of the present invention be better than contrast test produce fertilizer, illustrate simultaneously the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus the composite bacteria agent capable that bacterium solution volume ratio is 2-4:1:1 the enhancement effect of fertilizer substrate is better than the composite bacteria agent capable of other bacterium solution volume ratios.
It is embodiments of the invention as mentioned above.The present invention is not limited to above-mentioned embodiment, and anyone should learn the structure change made under the enlightenment of the present invention, and every have same or like technical scheme with the present invention, each falls within protection scope of the present invention.
Claims (8)
1. the composite bacteria agent capable promoting matrix nutrition, it is characterized in that: described composite bacteria agent capable includes azotobacter chroococcum, bacillus megaterium and gel-shaped series bacillus, the bacterium solution of described azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus bacterium solution volume ratio be 2-4:1:1；The bacterium solution bacteria containing amount of described azotobacter chroococcum is 80-100 hundred million/ml, and the bacterium solution bacteria containing amount of described bacillus megaterium is 4.5-5 hundred million/ml, and the bacterium solution bacteria containing amount of described colloid bacillus cereus is 4.5-5 hundred million/ml.
2. the preparation method of a kind of composite bacteria agent capable promoting matrix nutrition as claimed in claim 1, it is characterised in that: it includes three steps:
(1) amplification culture of azotobacter chroococcum bacterial strain: load azotobacter chroococcum culture fluid in shaking flask, is then seeded in shaking flask by azotobacter chroococcum one-level isogenic stocks and carries out spreading cultivation for the first time, and the temperature that spreads cultivation is 29-31 DEG C, and cultivation cycle is 36-40h；Then the azotobacter chroococcum bacterium solution after first time spreads cultivation being seeded to the one-level tank equipped with azotobacter chroococcum culture fluid and carry out aerobic culture, cultivation temperature is 29.5-30.5 DEG C, and cultivation cycle is 14-16h；Then azotobacter chroococcum bacterium solution after one-level tank spreads cultivation being seeded to two grades of tanks equipped with azotobacter chroococcum culture fluid and carries out aerobic culture, cultivation temperature is 28-32 DEG C, and cultivation cycle is 20-24h；
(2) amplification culture of gel-shaped series bacillus bacterial strain: load gel-shaped series bacillus culture fluid in shaking flask, then gel-shaped series bacillus one-level isogenic stocks is seeded in shaking flask and carries out spreading cultivation for the first time, the temperature that spreads cultivation is 31-33 DEG C, and cultivation cycle is 36-40h；Then the gel-shaped series bacillus bacterium solution after first time spreads cultivation being seeded to the one-level tank equipped with gel-shaped series bacillus culture fluid and carry out aerobic culture, cultivation temperature is 28-32 DEG C, and cultivation cycle is 20-24h；Then the gel-shaped series bacillus bacterium solution after one-level tank spreads cultivation being seeded to two grades of tanks equipped with gel-shaped series bacillus culture fluid and carry out aerobic culture, cultivation temperature is 28-34 DEG C, and cultivation cycle is 20-24h；
(3) amplification culture of bacillus megaterium bacterial strain: load bacillus megaterium culture fluid in shaking flask, is then seeded in shaking flask by bacillus megaterium one-level isogenic stocks and carries out spreading cultivation for the first time, and the temperature that spreads cultivation is 32.5-33.5 DEG C, and cultivation cycle is 36-40h;Then the bacillus megaterium bacterium solution after first time spreads cultivation being seeded to the one-level tank equipped with bacillus megaterium culture fluid and carry out aerobic culture, cultivation temperature is 32.5-33.5 DEG C, and cultivation cycle is 14-16h；Then bacillus megaterium bacterium solution after one-level tank spreads cultivation being seeded to two grades of tanks equipped with bacillus megaterium culture fluid and carries out aerobic culture, cultivation temperature is 28-33.5 DEG C, and cultivation cycle is 20-24h；
(4) bacterium solution that step (1)-(3) are finally obtained according to the bacterium solution of azotobacter chroococcum, the bacterium solution of bacillus megaterium, gel-shaped series bacillus the ratio mix homogeneously that bacterium solution volume ratio is 3:1:1, gnotobasis carries out fill.
3. the preparation method of a kind of composite bacteria agent capable promoting matrix nutrition according to claim 2, it is characterised in that: in described step (1), azotobacter chroococcum is seeded to one-level tank and carries out aerobic culture, and the ventilation before 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/h；In described step (1), azotobacter chroococcum is seeded in two grades of tanks and carries out aerobic culture, and the ventilation before 3h is 12m3After/h, 3h to ventilation before 6h be 18m3After/h, 6h to the ventilation before 9h be 24m3The later ventilation of/h, 9h is 42-48m3/h。
4. the preparation method of a kind of composite bacteria agent capable promoting matrix nutrition according to claim 2, it is characterised in that: in described step (2), gel-shaped series bacillus bacterium solution is seeded to one-level tank and carries out aerobic culture, and the ventilation before 6h is 2m3/ h, 6-9h ventilation is 4m3Ventilation after/h, 9h is 6m3/h；In described step (2), gel-shaped series bacillus bacterium solution is seeded in two grades of tanks and carries out aerobic culture, and the ventilation before 3h is 12m3After/h, 3h to ventilation before 6h be 18m3After/h, 6h to the ventilation before 9h be 24m3The later ventilation of/h, 9h is 42-48m3/ h, is 28-30 DEG C in two grades of tank cultivation temperature 12h, and 12h-24h cultivation temperature is 32-33 DEG C, and after 24h, temperature rises to 33-34 DEG C.
5. the preparation method of a kind of composite bacteria agent capable promoting matrix nutrition according to claim 1, it is characterized in that: in described step (3), bacillus megaterium bacterium solution is seeded to one-level tank and carries out aerobic culture, cultivation cycle is the ventilation before 14-16h, 3h is 1m3After/h, 3h to ventilation before 6h be 2m3After/h, 6h to ventilation before 9h be 3m3/h；In described step (3), bacillus megaterium bacterium solution is seeded to one-level tank and carries out aerobic culture, and cultivation cycle is the ventilation before 20-24h, 3h is 20m3After/h, 3h to ventilation before 6h be 25m3/ h, 6h rear venting amount is 35m3Being 28-30 DEG C in/h, cultivation temperature 12h, 12h-24h cultivation temperature is 32-33 DEG C, and after 24h, temperature rises to 33.5 DEG C, and tank pressure is 0.05M.
6. the preparation method of a kind of composite bacteria agent capable promoting matrix nutrition according to claim 2, it is characterized in that: the preparation method of the inoculum of described azotobacter chroococcum: take sucrose 10-11g, ammonium sulfate 0.5-0.6g, potassium dihydrogen phosphate 0.2-0.3g, yeast extract 0.5-0.6g, sodium citrate 0.5-0.6g, magnesium sulfate 0.2-0.3g, dipotassium hydrogen phosphate 0.7-0.8g, tap water 1000ml are configured to the solution of pH7.0-7.2, and sterilizing is standby.
7. the preparation method of a kind of composite bacteria agent capable promoting matrix nutrition according to claim 2, it is characterized in that: the preparation method of the inoculum of described gel-shaped series bacillus: take soluble starch 5.0-6.0g, sucrose 2.0-3.0g, ammonium sulfate 0.8-1.0g, soybean cake powder 0.8-1.0g, potassium dihydrogen phosphate 2.5-3.0g, magnesium sulfate 1.5-2.0g, calcium carbonate 1.5-2.0g, ferric chloride 0.2-0.3g, yeast extract 1.0-1.5g, tap water 1000ml are configured to the solution of PH7.2-7.5, and sterilizing is standby.
8. the preparation method of a kind of composite bacteria agent capable promoting matrix nutrition according to claim 2, it is characterized in that: the preparation method of the inoculum of described bacillus megaterium: glucose 2.0-3.0g, calcium carbonate 5.0-6.0g, magnesium sulfate 0.4-0.6g, potassium dihydrogen phosphate 0.2-0.4g, ammonium sulfate 0.8-1.0g, yeast extract 0.8-1.0g, sodium chloride 1.0-1.5g, soybean cake powder 3.0-3.5g, starch 5.0-6.0g, tap water 1000ml are configured to the solution of PH7.0-7.5, and sterilizing is standby.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106717908A (en) * | 2016-12-14 | 2017-05-31 | 渠县金穗农业科技有限公司 | A kind of propagation method of bletilla |
| CN109797118A (en) * | 2019-03-11 | 2019-05-24 | 大连大学 | A kind of composite bacteria agent for the cow concentrated feed that ferments |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106717908A (en) * | 2016-12-14 | 2017-05-31 | 渠县金穗农业科技有限公司 | A kind of propagation method of bletilla |
| CN109797118A (en) * | 2019-03-11 | 2019-05-24 | 大连大学 | A kind of composite bacteria agent for the cow concentrated feed that ferments |
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