CN106085922A - Small-sized microbial ecosystem method prepares multifunctional composite microbe microbial inoculum and application - Google Patents

Small-sized microbial ecosystem method prepares multifunctional composite microbe microbial inoculum and application Download PDF

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CN106085922A
CN106085922A CN201610657751.XA CN201610657751A CN106085922A CN 106085922 A CN106085922 A CN 106085922A CN 201610657751 A CN201610657751 A CN 201610657751A CN 106085922 A CN106085922 A CN 106085922A
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何圣平
凌仕信
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Zhongshan Moist Biological Technology Co Ltd
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Abstract

The present invention discloses small-sized microbial ecosystem method and prepares multifunctional composite microbe microbial inoculum and application.The present invention initially sets up small-sized microbial ecosystem, the microorganism seed of more than three kinds mutual the most not antagonisms is inoculated into small-sized microbial ecosystem mixed culture, obtain the multiple composite flora seed with microbial compound enzyme, then the fungus decomposing different vegetable material enzyme is cultivated according to a conventional method, obtain fungus shake-flask seed, again multiple composite flora seed and fungus shake-flask seed are inoculated into Solid nutritional base solid fermentation in proportion, after fermentation ends, are prepared as multifunctional composite microbe microbial inoculum.The present invention can solve the existing microbial bacterial agent function singleness being applied to agricultural and environment, bad adaptability, between strain, synergism ability is weak, new, powerful compound enzyme system cannot be produced, a difficult problem that production cost is high, it is achieved microbial bacterial agent low cost, high-efficient cleaning clean process and recycling agricultural and environmental pollution garbage.

Description

Small-sized microbial ecosystem method prepares multifunctional composite microbe microbial inoculum and application
Technical field
The invention belongs to biological manufacture and agriculture and field of environmental technology, relate to complex micro organism fungicide and application, specifically Relate to small-sized microbial ecosystem method and prepare multifunctional composite microbe microbial inoculum and application.
Background technology
Currently, the solid waste environmental pollution that animal excrements, human production activity produce is on the rise, by this A little pollutant harmless treatments, recycling face huge challenge.
At agricultural and environmental area, for realizing dye thing harmless treatment, all kinds of microniological proudcts of recycling, life Thing microbial inoculum continues to bring out, and has a characteristic that (1) major part microniological proudcts are with low content of technology, product identical, and preparation is simple thick Put；(2) excavation and selection-breeding are applied to agriculture fewer with the excellent novel bacterial of environment naturally；(3) engineering bacteria technology requires height, and Its function has specificity, is discharged in environment the existence because of indigenous microorganism, and its stability takes long enough with safety Assessment, it is impossible to extensive for agricultural and environment in the short time.
Microorganism major part in existing microbial inoculum product be all carry out mixing according to various straightforward procedures proportioning repeatedly, liquid Fermentation, carrier adsorption, be dried etc. technique be prepared.According to prior art, in strain culturing with microbial inoculum preparation process, multiple It is fewer that microorganism mixed fungus fermentation disposably obtains product, and with high costs.Most list strain sterile culture that use, finally Each single strain is obtained product according to the ratio mixing arbitrarily determined, in these products application to agricultural and environment, it is impossible to real Alternate between existing multiple-microorganism, symbiosis, Co metabolism, the microorganism in product and the group's relation between former indigenous microorganism are unstable Fixed, bad adaptability, the synergism ability between microorganism is weak, it is impossible to produce new, powerful compound enzyme system, product function Property target cannot realize.When being especially applicable to environmental pollution treatment, these products carry out biology to changeable environmental contaminants Harmless treatment is not thorough, and recycling difficulty strengthens.
Therefore, exploring new method and the technology path of invention microorganism Hybrid NC machine tool, developing low-cost, purposes are wide, novel Multifunctional composite microbe microbial inoculum becomes current necessity.
Summary of the invention
In order to overcome the defect of prior art, the present invention provides one to utilize small-sized microbial ecosystem method to prepare many merits The method of energy complex micro organism fungicide, provides the multifunctional composite microbe microbial inoculum prepared by said method simultaneously and answers With；The existing microbial bacterial agent function singleness being applied to agricultural and environment, bad adaptability, microbial bacteria interspecific coordination can be solved Ability to function is weak, it is impossible to produce new, powerful compound enzyme system, a difficult problem that production cost is high, it is achieved microbial bacterial agent is low Cost, high-efficient cleaning clean process agricultural and environmental pollution garbage, and recycling.
For achieving the above object, the present invention adopts the following technical scheme that the preparation method of multifunctional composite microbe microbial inoculum, Realized by small-sized microbial ecosystem method, comprise the steps:
Step one: set up small-sized microbial ecosystem, is inoculated into small-sized micro-by the microorganism seed of more than three kinds mutual the most not antagonisms Bioecosystem is mixed, cultivate can alternate, symbiosis, micropopulation that Co metabolism is good, jointly increase in same environment Grow, it is thus achieved that the composite thallus of multiple-microorganism group, by composite thallus shake-flask culture, it is thus achieved that composite flora seed；Described micro-life Thing can be antibacterial, fungus, actinomycetes etc.；Described microorganism seed can be natural separation and selection-breeding bacterial strain, it is also possible in state Family's Organism Depositary is bought and is obtained；
Step 2: synchronize to use conventional method to produce the fungus shake-flask seed decomposing different vegetable material enzymes；
Step 3: composite flora seed and fungus shake-flask seed are inoculated into Solid nutritional base, the Solid nutritional base through having inoculated Carry out solid fermentation, after fermentation ends, be prepared as multifunctional composite microbe microbial inoculum.
The preparation method of multifunctional bio microbial inoculum of the present invention is to use the preparation of microbial ecosystem method to realize, and specifically wraps Include following steps:
Step one: set up small-sized microbial ecosystem: include that prepared by nature culture, prepared by initial incubation liquid, initial incubation Liquid sterilizing, sterility test, small-sized microecosystem are formed；
(1) prepared by natural culture:
The most in percentage by weight, 1000g nature culture contains material and ratio be: animal fresh excreta 30%-50%, Activated sludge 5%-10%, the 40-60 mesh natural soils 5%-10% that sieves, powder of straw 10%-15% of fineness 80 mesh, sieve 40-60 mesh Organic waste 5%-10%, sugar slag 15%-20%, ordinary beer 5L；
2. by animal fresh excreta, activated sludge, the 40-60 mesh natural soils that sieve, fineness 80 mesh powder of straw, sieve 40-60 mesh Organic waste, sugar slag are mixed into uniform initial former base, then mix homogeneously preliminary with 5L medicated beer for initial former base, will mix Initial former base containing medicated beer is placed in agitator stirring mixing 5-10 minute, uniformly after be prepared as nature culture, be placed in container In standby；
(2) prepared by initial incubation liquid: by above-mentioned standby natural culture heated and boiled 30-40 minute, stands cooling 2-5 hour, Treat that proportion is more than the species precipitate of water, when upper liquid is in sub-translucent, small particle half suspension in vessel, takes liquid after filtration and turn Entering volume is in 1 L triangular flask, and each triangular flask liquid amount is 500ml, standby as initial incubation liquid；
(3) initial incubation liquid sterilizing: the triangular flask that will be equipped with initial incubation liquid puts into high-pressure steam sterilizing pan, at steam pressure 1.05kg/cm2, temperature 121 DEG C, sterilizing 40～50min, sterilizing terminate after the most standby；
(4) sterility test: aseptically, takes the initial incubation liquid 2-5ml after standby sterilizing, accesses in sterilized petri dishes, puts Enter constant incubator 35 DEG C-37 DEG C degree constant temperature culture 18-24 hour, grow without microorganism, then initial by after step (3) sterilizing Culture fluid uses as aseptic initial incubation liquid；If any growth of microorganism, then again prepare by step (1)-(4)；
(5) small-sized microecosystem is formed
1., in 5 triangular flasks after aseptic initial incubation liquid being sub-packed in sterilizing under cleaning condition, the volume of triangular flask is 500ml, the liquid amount of each triangular flask is 250ml, under the conditions of natural pH, forms independent small-sized life in each triangular flask State system；The quantity of described triangular flask can also determine according to microbe species to be seeded；
2. under cleaning condition, each triangular flask is inoculated the microorganism of 1-5 kind the most not antagonism, put under sterile board room temperature natural Grow 18-24 hour；Described microorganism is the one in bacillus cereus or fungus；
3., after 18-24 hour, take out in sterile board the triangular flask 2. processed through step, under cleaning condition according to equal-volume, etc. Ratio mixes, and then material in triangular flask is all proceeded to sterile chamber；Sterile chamber is airtight, often it is positioned in clean environment Temperature is cultivated 10-16 hour naturally, obtains multi-cultur es and mixes bacterium solution, standby；Or by de-to the bacillus cereus in sterile chamber or fungus warp Water is dried, it is thus achieved that the dormancy thalline of complex microorganism；
Step 2: synchronize to use conventional method to produce the fungus shake-flask seed decomposing different vegetable material enzymes, the specifically side of preparation Method is,
(1) make PDA slant medium, seed culture medium, culture medium respectively, fungus strain is seeded to the training of PDA inclined-plane Support base to cultivate, it is thus achieved that fungus inclined-plane viable bacteria kind, then fungus inclined-plane viable bacteria kind be forwarded to seed culture medium and carry out liquid cultivation, Obtain liquid fungus seed, then liquid fungus seed is transferred in culture medium cultivation, it is thus achieved that produce and decompose different plant material Expect the fungus liquid seed of specific enzyme, preserve, standby；
(2) seed activation is cultivated: be inoculated in activation culture shaking flask by fungus liquid seed, and activation culture obtains liquid fermentation kind Son, i.e. prepares fungus shake-flask seed；
Step 3: multi-cultur es mixes bacterium cultivation
(1) prepared by compound bacteria seed: the multi-cultur es that step one room temperature is cultivated naturally is mixed bacterium solution, is divided in and goes out under cleaning condition In triangular flask after bacterium, shaking table shaken cultivation 24-72 hour, shaking speed 100-200r/min, cultivation initial temperature is room temperature, Every intensification in 3 hours 1-2 DEG C, after cultivating 24-72 hour, as micro organism quantity >=2x10 in shaking flask7During cfu/ml, lived The shaking flask complex microorganism seed changed；During described shaking table shaken cultivation, when microbe inoculation is bacillus cereus, cultivation temperature 38 When ± 1 DEG C, stopping heating up, when microbe inoculation is fungus, cultivation temperature controls at 23-35 DEG C；
(2) prepared by complex micro organism fungicide: obtain in fungus shake-flask seed step 2 obtained and step 3 (1) is compound micro- Biological seed is respectively according to 8-10%(v/w, ml/g) inoculum concentration be inoculated in solid medium, solid medium can be by Wheat bran 50%-70%, soybean cake 10%-15%, sugar slag 5%-10%, powder of straw 5%-10%, food processing leftover bits and pieces 5%-10% are according to weight Percentage ratio is prepared from；Described food processing leftover bits and pieces can be the one in postfermented tea powder, cane powder, dehydrated potato powder, tapioca starch Or it is several；After complex microorganism seed is inoculated into solid medium, fermenting cellar solid fermentation 48-96 hour, it is thus achieved that solid is multiple Close microorganism seed；Continue to be inoculated into expansion fermentation culture in solid medium with the solid composite microbe seed obtained, connect Kind of amount is weight percentage 5%-6%, sends into fermenting cellar solid fermentation 48-96 hour, and sweat temperature controls at 32-37 DEG C, Moisture Control is about 65%, and pH is natural, after expanding fermentation ends, and micro organism quantity >=2x108During cfu/g, 55-60 DEG C is dried, Moisture Control is at 15%-20%；Size-reduced, be screened to 60-80 mesh, it is thus achieved that multifunctional composite microbe microbial inoculum, room temperature preserve.
Wherein, when carrying out the preparation of fungus shake-flask seed according to above-mentioned steps two, conventional method can be used for difference Fungus select different culture medium prescriptions and activation culture based formulas.In PDA culture medium, most of funguses all can well give birth to Long, PDA culture medium is mainly used in for fungus inclined-plane viable bacteria kind of transferring；Seed culture medium, for fungus inclined-plane viable bacteria kind again Switching liquid is cultivated, and makes culture medium nutrition more horn of plenty with comprehensively, and it is more preferable that fungal mycelia can grow, and becomes liquid fungus kind Son；Culture medium is cultivated in culture medium for again being transferred by liquid fungus seed.Preferably culture medium prescription Can be as follows:
1. slant medium (PDA): potato dextrose agar (PDA) culture medium, its way is to weigh 200g Rhizoma Solani tuber osi, cleans Peeling chopping, the 1000ml that adds water boils half an hour, filtered through gauze, then adds 15g glucose and 18g agar, takes advantage of after fully dissolving Hot filtered through gauze, subpackage test tube, every test tube about 10ml, about the 20 minutes rear test tubes that take out of 121 DEG C of sterilizings are put inclined-plane, are store after cooling Deposit standby；
2. seed culture medium: soluble starch 2-3%, glucose 1-1.5%, KH2PO40.1-0.25%, MgSO40.1- 0.3%, yeast extract 0.5-0.75%, NaNO30.2-0.35%, natural pH, 121 DEG C of autoclaving 20min；
3. culture medium: Testa Tritici 4-6%, (NH4)2SO40.1-0.2%, KH2PO40.2-0.35%, sodium acetate 0.1- 0.2%, ascorbic acid 0.1-0.2%, MgSO40.15-0.25%, carbamide 0.25-0.5%, natural pH, 121 DEG C of autoclavings 20min；
4. fungus seed activation cultivation formula and method: peptone 5.0-5.5 g/L, glucose 10.0-15 g/L, magnesium sulfate 0.5-0.8 g/L, yeast extract powder 2.0-3.0g/L, dipotassium hydrogen phosphate 1-3g/L, pH7.0-7.2 nutritional solution, shaking flask rotating speed For 100-160rpm, at a temperature of 23-35 DEG C, cultivate 25h-28h.
The small-sized microbial ecosystem that the inventive method is formed is mainly used in the Hybrid NC machine tool of various bacillus cereus, also Can be used for the Hybrid NC machine tool of fungus.Small-sized microbial ecosystem principle of the present invention is as follows: this system is according to various spore The alternate of bacillus ecotone in its natural state, symbiosis, Co metabolism principle, the most do not use chemical reagent to make with biochemical preparation For nutrient source；Intrasystem source of nutrition is the residue nutrient of garbage and pollutant, and in system, natural pH and temperature constantly become Changing, nutritional condition, close to polluting wild strain animation in environment, makes multiple-microorganism under imitative natural state, jointly gives birth to Exist in same environment；This system can cultivate required plurality of target flora, substitutes existing high cost mixed culture liquid state fermentation Method and apparatus.
Preferably technical scheme can be to be achieved by the steps of:
Step one: set up small-sized microbial ecosystem, prepares including nature culture, prepared by initial incubation liquid, initial incubation Liquid sterilizing, sterility test, small-sized microecosystem are formed；
(1) prepared by natural culture
1. preparing nature culture by following percentage by weight, material and ratio that 1000g nature culture contains be: animal is new Fresh feces 30%-50%, activated sludge 5%-10%, the 40-60 mesh natural soils 5%-10% that sieves, fineness 80 mesh powder of straw 10%-15%, Sieve 40-60 mesh organic waste 5%-10%, sugar slag 15%-20%, ordinary beer 5L；
2. the preparation method of natural culture: by animal fresh excreta, activated sludge, sieve 40-60 mesh natural soils, fineness 80 Mesh powder of straw, the 40-60 mesh organic waste that sieves, sugar slag are mixed into uniform initial former base, then will be the most preliminary with 5L medicated beer Mix homogeneously, was placed in agitator stirring mixing 5-10 minute by mixing the initial former base containing medicated beer, uniformly after be prepared as from So culture, is placed in containers for future use；
(2) prepared by beginning culture fluid: by above-mentioned standby natural culture heated and boiled 30-40 minute, stands cooling 2-5 hour, treats Proportion, more than the species precipitate of water, when upper liquid is in sub-translucent, small particle half suspension in vessel, takes top separatory after filtration Body, proceeds in 3-5 1 L triangular flask standby as initial incubation liquid；
(3) initial incubation liquid sterilizing: initial incubation liquid is put into high-pressure steam sterilizing pan, in steam pressure 1.05kg/cm2, temperature Under the conditions of spending 121 DEG C, sterilizing 40～50min, after sterilizing terminates, standby；
(4) sterility test: take the initial incubation liquid 0.5-1ml after the sterilizing of step (3), directly cultivate at ready aseptic LB In base plate, 35 DEG C of-37 DEG C of constant temperature culture 18-24 hour, grow without microorganism, it is thus achieved that aseptic initial incubation liquid；If any micro- Biological growth, is prepared by step (1)-(4) again；
(5) small-sized microecosystem is formed
1. step (1)-(4) are prepared in the triangular flask after aseptic initial incubation liquid is sub-packed in 8 sterilizings under cleaning condition, Strain pH is natural, forms independent small-sized ecosystem in each triangular flask；
2. under cleaning condition, respectively by bacillus subtilis, bacillus polymyxa, bacillus megaterium, stearothermophilus spore Bacillus is inoculated in triangular flask, every 2 triangular flasks inoculation same strain, inoculates complete putting into and naturally grows under sterile board room temperature 18-24 hour；
3., after 18-24 hour, take out the triangular flask in sterile board, mix successively than 1:1:1:1 according to equal-volume under cleaning condition Close, then proceed to sterile chamber, formed in sterile chamber and there is the small-sized micro-of multiple-microorganism kind and multiple-microorganism group Bioecosystem；Sterile chamber is positioned over room temperature in clean environment become multi-cultur es after naturally cultivating 10-16 hour and mix bacterium Liquid, takes out, is placed in 4 DEG C of refrigerator and saves backup；Or by preserving after sub-for mixed strain dehydrate, obtain complex microorganism dormancy bacterium Body；
Step 2: synchronization employing conventional method production aspergillus niger shake-flask seed, Eurotium cristatum shake-flask seed:
(1) make PDA slant medium, seed culture medium, culture medium respectively, aspergillus niger, Eurotium cristatum strain are divided It is not seeded to PDA slant medium cultivate, it is thus achieved that inclined-plane viable bacteria kind, then inclined-plane viable bacteria kind is forwarded to seed culture medium and carries out Liquid is cultivated, it is thus achieved that liquid fungus seed, then is transferred by liquid fungus seed in culture medium cultivation, obtains aspergillus niger respectively Liquid seed and Eurotium cristatum liquid seed, preserve, standby；
1. prepared by PDA slant medium: potato dextrose agar (PDA) culture medium, and its way is to weigh 200g Rhizoma Solani tuber osi, washes Only removing the peel chopping, the 1000ml that adds water boils half an hour, filtered through gauze, then adds 15g glucose and 18g agar, after fully dissolving Filtered through gauze while hot, subpackage test tube, every test tube about 10ml, 121 DEG C of sterilizings are taken out test tube pendulum inclined-plane, are stored after cooling after 20 minutes Standby；
2. prepared by seed culture medium: soluble starch 2-3%, glucose 1-1.5%, KH2PO4 0.1-0.25%, MgSO4 0.1-0.3%, yeast extract 0.5-0.75%, NaNO30.2-0.35%, natural pH, 121 DEG C of autoclaving 20min；
3. prepared by culture medium: Testa Tritici 4-6%, (NH4)2SO40.1-0.2%, KH2PO40.2-0.35%, sodium acetate 0.1- 0.2%, ascorbic acid 0.1-0.2%, MgSO40.15-0.25%, carbamide 0.25-0.5%, natural pH, 121 DEG C of autoclavings 20min；
(2) seed activation is cultivated: being inoculated in activation culture shaking flask by aspergillus niger liquid seed, activating recipe is: peptone 5.0-5.5 g/L, glucose 10.0-15 g/L, magnesium sulfate 0.5-0.8 g/L, yeast extract powder 2.0-3.0g/L, phosphoric acid Hydrogen dipotassium 1-3g/L, in pH7.0-7.2 nutritional solution, shaking flask rotating speed is 100-160rpm, cultivates 25h-at a temperature of 23-35 DEG C 28h, it is thus achieved that liquid fermentation seed, obtains the fungus shake-flask seed of aspergillus niger；Eurotium cristatum liquid seed is inoculated into activation Cultivating activation culture in shaking flask, shaking flask Middle nutrition formula of liquid is: sucrose 60-80g/L, 100 mesh fineness postfermented tea powder 10-20g/L or Person dark tea slurry fluid 0.8-1%, dipotassium hydrogen phosphate 3-5g/L, pH5.5-6.5, shaking flask rotating speed is 100-130rpm, at 26-32 DEG C Lower cultivation 90-100 hour, it is thus achieved that the fungus shake-flask seed of liquid fermentation seed, i.e. Eurotium cristatum；
Step 3: multi-cultur es mixes bacterium cultivation
(1) composite flora bacillus cereus cultivating seeds: the multi-cultur es that 3. step one (5th) item walks acquisition is mixed bacterium solution and is divided in In 5-10 the triangular flask containing initial incubation liquid, triangular flask volume is 1L, and liquid amount is 500ml, vibrates on activation shaking table Fluctuation temperature culture 40-72h, vibration Fluctuation temperature culture condition is as follows: shaking speed 160-200r/min, initial temperature is 20 DEG C, every 3 Hour raise 1-2 DEG C, when being adjusted to 38 ± 1 DEG C in temperature, stop raise；Vibration Fluctuation temperature culture terminates, it is thus achieved that liquid fermentation is multiple Close seed liquor；Then liquid fermentation seed liquor is inoculated into 10kg by wheat bran, rice according to the inoculative proportion (v/w, ml/g) of 10% In the solid medium that bran, soybean cake, Semen Maydis powder are mixed with according to mass ratio 10:1:3:2, fully mix, at 37 DEG C, cultivate 3 My god, obtain main composite flora bacillus cereus solid seed；
(2) fungi flora aspergillus niger and coronoid process dissipate capsule bacterium cultivating seeds:
1. by the fungus shake-flask seed of aspergillus niger according to the inoculative proportion (v/w, ml/g) of 10% be inoculated into 5kg by wheat bran, Testa oryzae, In the solid medium that soybean cake, dehydrated potato powder mix according to mass ratio 7:1:1:1, fully mix, at 37 DEG C, cultivate cultivation 3 My god, obtain aspergillus niger solid seed；
2. by the fungus shake-flask seed of Eurotium cristatum according to the inoculative proportion (v/w, ml/g) of 5% be inoculated into 5kg by postfermented tea powder, In the solid medium that cane powder mixes according to mass ratio 1:1, fully mix, initial stage temperature 25 DEG C, after 48 hours, temperature is adjusted Joint is to 28 DEG C-32 DEG C, and humidity is 70%, and pH value regulates to 5-5.5, cultivates 5 days continuously, obtains Eurotium cristatum solid seed；
3. solid amplification culture: the main composite flora bacillus cereus solid seed for preparing above-mentioned, auxiliary flora black fermented preparation Mould solid seed, Eurotium cristatum solid seed uniformly mix according to weight ratio 7:2:1, add to according to the inoculative proportion of 6-8% In the solid medium that 100kg is mixed to prepare according to mass ratio 8:1:0.5:0.5 by wheat bran, soybean cake, Semen Maydis powder, postfermented tea powder, fill Divide mixing, temperature 32 DEG C in the early stage, wait after 24 hours and temperature is adjusted to 35 DEG C-37 DEG C, cultivate continuously under the conditions of pH is natural 5-7 days；After cultivation terminates, it is dried at 55-65 DEG C, it is thus achieved that product effective active bacterium 2x109Cfu/g, water content 15%-20%, Pulverize through chain type grinder, cross 60-100 mesh sieve, prepare multifunctional composite microbe microbial inoculum.
Microbial population of the present invention can be antibacterial, fungus, actinomycetes etc., and described microorganism seed can be Natural separation and selection-breeding bacterial strain, it is also possible to buy at country's Organism Depositary and obtain.
The multifunctional bio microbial inoculum using the inventive method to prepare can be applicable to organic matter decomposing inoculant and processes solid Organic waste, it is also possible to as ecological feed additive, adds in feedstuff, it is also possible to as bio-feritlizer.
Relative to prior art, beneficial effects of the present invention is as follows:
(1) bacillus cereus obtained after mixed bacterium is cultivated or fungus mixing liquid seeds, the most directly amplification culture, can be direct Become composite bacteria agent capable product；
(2) bacillus cereus obtained after mixed bacterium is cultivated or fungus mixing liquid seeds, can be with many after dehydrate preserves Plant microorganism and be mixed with various complex micro organism fungicide；
(3) achieving the Hybrid NC machine tool of multi-cultur es, investment reduction, technique is simple, easy to operate, it is possible to decrease microbial bacterial agent produces Cost 40%-60%, economic benefit is fairly obvious；
(4) breeding in common ecosystem because of microorganism, define good alternate, symbiosis, Co metabolism relation, new answers Synthase system self-assembling formation, provides new method and technology path to exploitation Multifunction microbial bacterial agent.
Detailed description of the invention
Being described in further details the present invention below by embodiment, these embodiments are only used for the present invention is described, and Do not limit the scope of the invention.
Embodiment 1 specifically prepares complex micro organism fungicide product: it is micro-that strain used by the present embodiment derives from the Chinese Academy of Sciences DSMZ of biological study institute, strain and numbered: bacillus subtilis CGMCC1.1086, bacillus polymyxa CGMCC1.0548, bacillus megaterium CGMCC1.0217, bacstearothermophilus CGMCC1.3380, aspergillus niger CGMCC3.0316, Eurotium cristatum CGMCC3.0448.
Employing following steps realize:
Step one: set up small-sized microbial ecosystem, prepares including nature culture, prepared by initial incubation liquid, initial incubation Liquid sterilizing, sterility test, small-sized microecosystem are formed and mix the production of strain；
(1) prepared by natural culture:
The most in percentage by weight, 1000g nature culture contains material and ratio be: animal fresh excreta 40%, activity Mud 10%, the 40-60 mesh natural soils 10% that sieve, the powder of straw 10% of fineness 80 mesh, the 40-60 mesh organic waste 5% that sieves, sugar slag 20%, ordinary beer 5L；
2. by animal fresh excreta, activated sludge, the 40-60 mesh natural soils that sieve, fineness 80 mesh powder of straw, sieve 40-60 mesh Organic waste, sugar slag are mixed into uniform initial former base, then mix homogeneously preliminary with 5L medicated beer for initial former base, will mix Initial former base containing medicated beer is placed in agitator stirring mixing 10 minutes, uniformly after be prepared as nature culture, be placed in container Standby；
(2) prepared by initial incubation liquid: by above-mentioned standby natural culture heated and boiled 30 minutes, stands cooling 4 hours, treats proportion More than the species precipitate of water, when upper liquid is in sub-translucent, small particle half suspension in vessel, takes liquid after filtration and proceed to volume Being in 1 L triangular flask, each triangular flask liquid amount is 500ml, standby as initial incubation liquid；
(3) initial incubation liquid sterilizing: the triangular flask that will be equipped with initial incubation liquid puts into high-pressure steam sterilizing pan, at steam pressure 1.05kg/cm2, temperature 121 DEG C, sterilizing 50min, sterilizing terminate after the most standby；
(4) sterility test: aseptically, takes the initial incubation liquid 2-5ml after standby sterilizing, accesses in sterilized petri dishes, puts Enter constant incubator 35 DEG C-37 DEG C degree constant temperature culture 18-24 hour, grow without microorganism, then initial by after step (3) sterilizing Culture fluid uses as aseptic initial incubation liquid；If any growth of microorganism, then again prepare by step (1)-(4)；
(5) small-sized microecosystem is formed and mixes the production of strain
1. step (1)-(4) are prepared in the triangular flask after aseptic initial incubation liquid is sub-packed in 8 sterilizings under cleaning condition, Strain pH is natural, forms independent small-sized ecosystem in each triangular flask；
2. under cleaning condition, respectively by bacillus subtilis, bacillus polymyxa, bacillus megaterium, stearothermophilus spore Bacillus is inoculated in triangular flask, every 2 triangular flasks inoculation same strain, inoculates complete putting into and naturally grows under sterile board room temperature 18-24 hour；
3., after 18-24 hour, take out the triangular flask in sterile board, mix successively than 1:1:1:1 according to equal-volume under cleaning condition Close, then proceed to sterile chamber, formed in sterile chamber and there is the small-sized micro-of multiple-microorganism kind and multiple-microorganism group Bioecosystem；Sterile chamber is positioned over room temperature in clean environment become mixed strain after naturally cultivating 10-16 hour and take Go out, be placed in 4 DEG C of refrigerator and save backup (or after dehydrate, preserving complex microorganism dormancy thalline)；
Step 2: synchronize to use conventional method to produce the fungus shake-flask seed decomposing different vegetable material enzymes, select fungus bacterium Kind for Aspergillus niger, Eurotium cristatum, make PDA slant medium, seed culture medium, culture medium respectively, by aspergillus niger, Eurotium cristatum strain is seeded to PDA slant medium respectively and cultivates, it is thus achieved that inclined-plane viable bacteria kind, strain transfer of then being lived on inclined-plane Carry out liquid cultivation to seed culture medium, it is thus achieved that liquid fungus seed, then liquid fungus seed is transferred in culture medium training Supporting, obtain aspergillus niger liquid seed and Eurotium cristatum liquid seed respectively, preserve, standby, concrete preparation method is as follows:
(1) conventional method is used to prepare PDA slant medium, seed culture medium, the cultivation of product enzyme respectively by following culture medium prescription Base:
1. PDA slant medium: potato dextrose agar (PDA) culture medium, its way is to weigh 200g Rhizoma Solani tuber osi, cleans and goes Skin shreds, and the 1000ml that adds water boils half an hour, filtered through gauze, then adds 15g glucose and 18g agar, after fully dissolving while hot Filtered through gauze, subpackage test tube, every test tube about 10ml, 121 DEG C of sterilizings are taken out test tube pendulum inclined-plane, are stored standby after cooling after 20 minutes With；
2. seed culture medium: soluble starch 2%, glucose 1%, KH2PO4 0.2%, MgSO4 0.2%, yeast extract 0.5- 0.75%, NaNO30.3%, natural pH, 121 DEG C of autoclaving 20min；
3. culture medium: Testa Tritici 4-6%, (NH4)2SO40.2%, KH2PO40.3%, sodium acetate 0.1%, ascorbic acid 0.1%, MgSO40.2%, carbamide 0.3%, natural pH, 121 DEG C of autoclaving 20min；
(2) seed activation is cultivated: being inoculated in activation culture shaking flask by aspergillus niger liquid seed, activating recipe is: peptone 5.2 g/L, glucose 12g/L, magnesium sulfate 0.5 g/L, yeast extract powder 2.0g/L, dipotassium hydrogen phosphate 2g/L, pH7.0- 7.2 nutritional solutions, shaking flask rotating speed is 100-160rpm, cultivates 25h-28h, it is thus achieved that liquid fermentation seed, i.e. at a temperature of 23-35 DEG C Obtain Aspergillus niger fungus shake-flask seed；Eurotium cristatum liquid seed is inoculated into activation culture in activation culture shaking flask, in shaking flask Nutrient solution prescription is: sucrose 70g/L, dark tea slurry fluid 0.9%, dipotassium hydrogen phosphate 4g/L, pH5.5-6.5, and shaking flask rotating speed is 120rpm, cultivates 90-100 hour, it is thus achieved that the fungus shake-flask seed of liquid fermentation seed, i.e. Eurotium cristatum at 26-32 DEG C；
Step 3: multi-cultur es mixes bacterium cultivation
(1) composite flora bacillus cereus cultivating seeds: will be placed in what 4 DEG C of refrigerator saved backup in step one (5th) item the 3. step Bacillus subtilis, bacillus polymyxa, bacillus megaterium, bacstearothermophilus mix strain and are divided in containing initial In 5-10 triangular flask of culture fluid, triangular flask volume is 1L, and liquid amount is 500ml.Vibrate on activation shaking table Fluctuation temperature culture 40-72h, vibration Fluctuation temperature culture condition is as follows: shaking speed 160-200r/min, and initial temperature is 20 DEG C, raises every 3 hours 1-2 DEG C, when being adjusted to 38 ± 1 DEG C in temperature, stop raising；Vibration Fluctuation temperature culture terminates, it is thus achieved that liquid fermentation compound seed liquid； Then liquid fermentation seed liquor is inoculated into 10kg by wheat bran, Testa oryzae, soybean cake, jade according to the inoculative proportion (v/w, ml/g) of 10% Rice flour is mixed with in solid medium according to mass ratio 10:1:3:2, fully mixes, and cultivates 3 days at 37 DEG C, obtains main Composite flora bacillus cereus solid seed；
(2) fungi flora aspergillus niger solid seed and Eurotium cristatum solid cultivating seeds:
1. by the fungus shake-flask seed of aspergillus niger according to the inoculative proportion (v/w, ml/g) of 10% be inoculated into 5kg by wheat bran, Testa oryzae, In the solid medium that soybean cake, dehydrated potato powder mix according to mass ratio 7:1:1:1, fully mix, at 37 DEG C, cultivate cultivation 3 My god, obtain aspergillus niger solid seed；
2. by the fungus shake-flask seed of Eurotium cristatum according to the inoculative proportion (v/w, ml/g) of 5% be inoculated into 5kg by postfermented tea powder, In the solid medium that cane powder mixes according to mass ratio 1:1, fully mix, initial stage temperature 25 DEG C, after 48 hours, temperature is adjusted Joint is to 28 DEG C-32 DEG C, and humidity is 70%, and pH value regulates to 5-5.5, cultivates 5 days continuously, obtains Eurotium cristatum solid seed；
3. solid amplification culture: the main composite flora bacillus cereus solid seed for preparing above-mentioned, auxiliary flora black fermented preparation Mould solid seed, Eurotium cristatum solid seed uniformly mix according to weight ratio 7:2:1, add to according to the inoculative proportion of 6-8% In the solid medium that 100kg is mixed to prepare according to mass ratio 8:1:0.5:0.5 by wheat bran, soybean cake, Semen Maydis powder, postfermented tea powder, fill Divide mixing, temperature 32 DEG C in the early stage, wait after 24 hours and temperature is adjusted to 35 DEG C-37 DEG C, cultivate continuously under the conditions of pH is natural 5-7 days；After cultivation terminates, it is dried at 55-65 DEG C, it is thus achieved that product effective active bacterium 2x109Cfu/g, water content 15%-20%, Pulverize through chain type grinder, cross 60-100 mesh sieve, prepare multifunctional composite microbe microbial inoculum.
Embodiment 2 multifunctional composite microbe microbial inoculum application in agricultural with environmental area: the present invention prepares Multifunctional composite microbe microbial inoculum has good bioenzyme activity, after measured, cellulase therein, saccharifying enzyme, amylase The enzymatic activity such as property, urease, protease, pectase, xylanase, superoxide dismutase (SOD), lipase, phytase is very Height, various enzyme work are between 280-350U/g；Biotoxin such as aflatoxin (B1, B2, G1, G2), ochratoxin, mycete Toxin etc. do not detect；Bio-feritlizer that other countries specify and the relevant index of bio-fermentation agent are the most qualified.Prepared by the present invention The main application mode of the multifunctional composite microbe microbial inoculum that obtains may is that (1) processes solid as organic matter decomposing inoculant Organic waste, it is achieved the recycling of solid organic castoff；(2) use as ecological feed additive；(3) as raw These several application modes are described as follows by thing Fertilizer application.
One, solid organic castoff is processed as organic matter decomposing inoculant
1, ultimate principle: feces of livestock and poultry+straw stalk+organic waste+multifunctional bio microbial inoculum=clean+resource
(1) feces of livestock and poultry+straw stalk+organic waste is by nitrogenous Organic substance (protein-based aminoacid, nonprotein Class uric acid, carbamide, hippuric acid), carbohydrate (polysaccharide starch, cellulose, hemicellulose, lignin, pectin Matter, disaccharidase class maltose, sucrose, lactose, monosaccharide glucose, galactose, fructose, xylose, mannose)；Lipid (fats glycerol, fatty acid), Phosphorous species (phospholipid, nucleic acid, phytic acid), sulfur material (cysteine, leucine, vitamin B, sulfur peace element, biotin, thioctic acid element) etc. composition；
(2) decomposition of protein: protein resolves into polypeptide, dipeptides (aminoacid) under the effect of protease.Microorganism is being given birth to Producing specific enzyme in long and metabolic process, each microorganism produces different enzymes, be enzyme in action.Enzyme similarly is catalysis Agent, as reduced fat in the gallbladder matter of human body.The enzyme of the most secretions of microbial biomass is the most, acts on the strongest.Aminoacid is at deaminizating Ammonia, carboxylic acid, keto acid is produced under the effect of enzyme；Under the effect of decarboxylase, produce carbon dioxide, amine substance；In ammoxidation Aldehyde is become under the effect of enzyme；Aldehyde generates organic acid under conditions of aerobic.Aminoacid under anaerobic decomposes (referred to as corrupt work With) produce strength stench, because to form a large amount of intermediate product, such as mercaptan, butyric acid, indole, formaldehyde indole, hydrogen sulfide and ammonia nitrogen Deng.Aminoacid decomposes (referred to as putrefaction) methane, CO under aerobic conditions2, ammonia, water and hydrogen, this just requires constantly turning Oxygenation；
(3) decomposition of nonprotein, non-proteinaceous, under aerobic conditions, decomposes ammonification and CO2, then at the work of nitrobacteria Under with, become nitre state ammonia (inorganic nitrogen) and be available for plant and directly absorb (streptomycin, promise Ka Shi, penicillin etc.).Nitrobacteria is all Typical aerobic type autotrophic microbe, can utilize the energy that chemical reaction is released by CO2Synthesis of organic substance.Simultaneously at the bar of anaerobism Under part, also a bacterioid carries out Denitrification to nitrate nitrogen, i.e. nitrate reduction become nitrogen or intermediate product nitrite, The processes such as ammonia, its result can cause the nitrogen discharge in fecaluria；
(3) decomposition of carbohydrate: starch, cellulose, hemicellulose, lignin, pectic substance are at different microbial enzymes Under effect, polysaccharide hydrolysis becoming disaccharidase, be hydrolyzed into monosaccharide, be oxidized to glucose, glucose, under the conditions of having oxidation, generates CO2、 Water, ATP, big energy (generates heat), under anaerobic, produces organic acid, alcohols, methane, CO2, hydrogen (bacillus, mycete, Actinomycetes etc.)；
(4) fat is under aerobic conditions, produces glycerol under the effect of lipase, ultimately produces CO by repeatedly decomposing2, water, Energy (ATP, heating)；Fatty acid finally also produces CO under oxidative conditions2, water, energy；
(5) lipid (lecithin, the cephalin) effect by various enzymes, becomes glycerol, fatty acid, phosphoric acid, choline, gallbladder ammonia enter again Enter above circulation；
(6) Phosphorous species decomposes, and phospholipid nucleic acid, under the effect of nuclease, becomes nucleotide, hydrolysis Phos (nucleoside, phosphoric acid) It is absorbed by plants.Phytic acid, under the effect of phytase, becomes phosphoric acid, inositol；
(7) sulphur-containing substance, sulphur-containing substance, under conditions of aerobic, finally produce sulfate radical and the reaction of some heavy metal produces salt Class, it is possible to by microbial assimilation, is incorporated in Biomass；Produce hydrogen sulfide (rotten-egg odour) under anaerobic.
2, the method that the multifunctional microbial microbial inoculum of the present invention processes solid organic castoff is used: by multi-functional for the present invention compound Microbial bacterial agent joins in solid waste, mix homogeneously, carries out becoming thoroughly decomposed preparing fertilizer by solid organic castoff.Example As: according to percentage by weight preparation need fermentation process pollute environment solid organic castoff, as feces of livestock and poultry 55-70%, Straw stalk 15-20%, organic waste 5-20%, be subsequently adding the multifunctional microbial microbial inoculum 0.01-0.05% of the present invention, mix homogeneously, C/N controls in 20-25:1 scope, and moisture controls below 60%, builds stack height 1.5 meters-1.8 meters, width 2-5 rice, length Degree sets according to site condition, it is ensured that oxygen supply, heap temperature reaches about 60 DEG C, and in 24-48 hour, turning is once.General 72 little Time interior deodorize, within 10-15 days, become thoroughly decomposed, directly as fertilizer use.
This application mode had both solved problem of environmental pollution, achieved again the utilization of resources of garbage.
Two, use as ecological feed additive: the multifunctional composite microbe microbial inoculum of the present invention is added in feedstuff, Addition 0.1%-0.5%(mass percent), the pestilence phase, stress, disease treatment, reload period addition suitably increase to 1%。
This kind of application mode it is achieved that
1. suppress the breeding of harmful bacteria, make intestinal flora keep balance；Suppression and the breeding of prevention intestinal toxic bacterium, make probiotics Quantity occupies advantage；The breeding of cause of disease coliform, clostruidium, Salmonella, β hemolytic bacterioid etc. can be suppressed；
2. produce digestive enzyme, synthetic vitamin, the digestive enzyme such as amylase and protease and vitamin B group can be produced, additionally The synthesis of vitamin A also has been found to；
3. strengthen immunization, by siberian crabapple cell in stimulation intestinal, increase the formation of local antibody, thus increase huge biting carefully Cytoactive；Additive for microbe feedstuff can make a large amount of accumulations in liver have the vitamin A strengthening immunization；
4. producing hydrogen peroxide, hydrogen peroxide all has detrimental effect to several potential pathogenic microorganisms, and it is special by some Material formed in some substrate；
5. optimization of ecological environment: probiotics, enzyme preparation, in animal intestinal metabolic process, have been decomposed and have been difficult to be absorbed by animal Thick protein, phytase and antinutritional factor, hence it is evident that prevented and treated growing of fly larvae, effectively cut off the source of ammonia, foul smell, Make the concentration of harmful gas in animal wastes obtain effective reduction, improve feeding environment, reduce ammonia and human body is invaded Evil, prevents the generation of livestock and poultry respiratory and intestinal tract disease.
Three, use as bio-feritlizer: the multifunctional composite microbe microbial inoculum of the present invention can be used as bio-feritlizer, it is adaptable to Various crops and industrial crops, every mu uses 2-3 kilogram, can make base manure and topdress；Coordinate organic Fertilizer application better, Every mu of crop belts compounding application organic fertilizer 200-250 kilogram.Complex micro organism fungicide uses as bio-feritlizer, has following Advantage and effect.
1. improvement soil
(1) by the amount reproduction of probiotics, a large amount of probioticss at the root system of plant formed around dominant population, it is suppressed that its The vital movement of his harmful bacteria；
(2) fast decoupled soil organic substance, promotes the formation of soil granular, and can loose soil by the activity of probiotics Earth, the fertilizer conservation of soil, fertilizer, water conservation, water supply and breathability are the most well regulated；
(3) pesticide residues in soil are decomposed, it is to avoid residual pesticide produces poisoning to crop of lower season.Also growing process is led to The harmful substance crossing root system discharge decomposes.
2. fixed nitrogen, phosphorus decomposing, potassium decomposing function.Can partly utilize the nitrogen in air, produce phase by probiotics growth metabolism The enzyme answered and acid, can be carried out the phosphorus of slightly solubility, potash fertilizer (in soil, difficultly soluble phosphatic fertilizer accounts for 95%, and slightly solubility potassium accounts for 98%) in soil Decompose, thus become the absorbent phosphorus potash fertilizer of plant.Therefore, it is possible to be greatly improved the crop utilization rate to fertilizer, thus reduce Using of fertilizer.
3. improve crop quality.While phosphorus decomposing, potassium decomposing, it is possible to promote the release of Trace Elements in Soil, by crop institute Utilizing, the material needed for beneficial bacterial metabolic produces various plants simultaneously, such as little molecule aminoacid, growth-promoting substance, vitamin Deng.
4. reaching the effect of Biological control disease, pouring root can suppress the pathogenic bacteria in soil, is sprayed onto blade face, can prevent disease Invasion.
5. promote early ripening of the crops and extend collection period.Owing to soil physico-chemical property is improved, soil nutrient is abundant and flat Weighing apparatus, in soil, fertilizer can preferably well be absorbed by crop, therefore, it is possible to promote early ripening of the crops and extend collection period.
6. with fertilizer with the use of, it is possible to continual improvement soil, 2-3 just can make soil be fully achieved production The standard of organic crops, and because probiotics can fast decoupled organic substance be Crop, so overcoming organic fertilizer The feature that fertilizer efficiency is slow, the feature that single organic fertilizer yields poorly.
7. bio-bacterial manure is unlike chemical fertilizer, disposably brings the solubility nutrient of excess into soil, bio-bacterial manure Can avoid environmental pollution, and metabolism is constantly constantly bred in probiotics around root system of plant, constantly, non-excess to work Thing provides nutrition.
8. use test through field, crop quality tool is improved significantly.Use the vegetables of multifunctional bio microbial inoculum Dish nitrate content reduces 38.9～67.1mg/kg, reduces about 30% than traditional fertilization；Vitamin C content adds 139.5mg/ Kg, sugar content averagely adds 8.3mg/kg.
9. effect of increasing production, is combined with chemical fertilizer under application conditions in crop belts, than traditional fertilizer method of application volume increase 8%～ 10%, account for the 27.9.% of volume increase sum, volume increase 10%～15% account for 24.8%, volume increase 15%～20% account for 23.2%, increase production 20% Above accounts for 24.1%.
The multifunctional composite microbe microbial inoculum of the present invention, except above-mentioned several frequently seen application mode, it is also possible to further Develop its other application in agricultural with field of environmental technology.

Claims (6)

  1. The method that the most small-sized microbial ecosystem method prepares multifunctional composite microbe microbial inoculum, it is characterised in that: use small-sized Microbial ecosystem method realizes, and described small-sized microbial ecosystem method comprises the steps:
    Step one: set up small-sized microbial ecosystem, is inoculated into small-sized micro-by the microorganism seed of more than three kinds mutual the most not antagonisms Bioecosystem is mixed, cultivate can alternate, symbiosis, micropopulation that Co metabolism is good, jointly increase in same environment Grow, it is thus achieved that the composite thallus of multiple-microorganism group, by composite thallus shake-flask culture, it is thus achieved that composite flora seed；
    Step 2: use conventional method to produce the fungus shake-flask seed decomposing different vegetable material enzymes；
    Step 3: composite flora seed and fungus shake-flask seed are inoculated into Solid nutritional base, the Solid nutritional base through having inoculated Carry out solid fermentation, after fermentation ends, be prepared as multifunctional composite microbe microbial inoculum.
  2. Method the most according to claim 1, it is characterised in that: comprise the following specific steps that:
    Step one: set up small-sized microbial ecosystem, prepares including nature culture, prepared by initial incubation liquid, initial incubation Liquid sterilizing, sterility test, small-sized microecosystem are formed:
    (1) prepared by natural culture:
    The most in percentage by weight, 1000g nature culture contains material and ratio be: animal fresh excreta 30%-50%, Activated sludge 5%-10%, the 40-60 mesh natural soils 5%-10% that sieves, powder of straw 10%-15% of fineness 80 mesh, sieve 40-60 mesh Organic waste 5%-10%, sugar slag 15%-20%, ordinary beer 5L；
    2. by animal fresh excreta, activated sludge, the 40-60 mesh natural soils that sieve, fineness 80 mesh powder of straw, sieve 40-60 mesh Organic waste, sugar slag are mixed into uniform initial former base, then mix homogeneously preliminary with 5L medicated beer for initial former base, will mix Initial former base containing medicated beer is placed in agitator stirring mixing 5-10 minute, uniformly after be prepared as nature culture, be placed in container In standby；
    (2) prepared by initial incubation liquid: by above-mentioned standby natural culture heated and boiled 30-40 minute, stands cooling 2-5 hour, Treat that proportion is more than the species precipitate of water, when upper liquid is in sub-translucent, small particle half suspension in vessel, takes liquid after filtration and turn Entering volume is in 1 L triangular flask, and each triangular flask liquid amount is 500ml, standby as initial incubation liquid；
    (3) initial incubation liquid sterilizing: the triangular flask that will be equipped with initial incubation liquid puts into high-pressure steam sterilizing pan, at steam pressure 1.05kg/cm2, temperature 121 DEG C, sterilizing 40～50min, sterilizing terminate after the most standby；
    (4) sterility test: aseptically, takes the initial incubation liquid 2-5ml after standby sterilizing, accesses in sterilized petri dishes, puts Enter constant incubator 35 DEG C-37 DEG C degree constant temperature culture 18-24 hour, grow without microorganism, then initial by after step (3) sterilizing Culture fluid uses as aseptic initial incubation liquid；If any growth of microorganism, then again prepare by step (1)-(4)；
    (5) small-sized microecosystem is formed
    1., in 5 triangular flasks after aseptic initial incubation liquid being sub-packed in sterilizing under cleaning condition, the volume of triangular flask is 500ml, the liquid amount of each triangular flask is 250ml；Under the conditions of natural pH, in each triangular flask, form independent small-sized life State system；
    2. under cleaning condition, each triangular flask is inoculated the microorganism of 1-5 kind the most not antagonism, put under sterile board room temperature natural Grow 18-24 hour；Described microorganism is the one in bacillus cereus or fungus；
    3., after 18-24 hour, take out in sterile board the triangular flask 2. processed through step, under cleaning condition according to equal-volume, etc. Ratio mixes, and then material in triangular flask is all proceeded to sterile chamber；Sterile chamber is airtight, often it is positioned in clean environment Temperature is cultivated 10-16 hour naturally, obtains multi-cultur es and mixes bacterium solution, standby；Or by de-to the bacillus cereus in sterile chamber or fungus warp Water is dried, it is thus achieved that the dormancy thalline of complex microorganism；
    Step 2: the fungus shake-flask seed of the employing conventional method different vegetable material enzymes of production decomposition:
    (1) make PDA slant medium, seed culture medium, culture medium respectively, fungus strain is seeded to the training of PDA inclined-plane Support base to cultivate, it is thus achieved that fungus inclined-plane viable bacteria kind, then fungus inclined-plane viable bacteria kind be forwarded to seed culture medium and carry out liquid cultivation, Obtain liquid fungus seed, then liquid fungus seed is transferred in culture medium cultivation, it is thus achieved that produce and decompose different plant material Expect the fungus liquid seed of specific enzyme, preserve, standby；
    (2) seed activation is cultivated: be inoculated in activation culture shaking flask by fungus liquid seed, and activation culture obtains liquid fermentation kind Son, i.e. prepares fungus shake-flask seed；
    Step 3: multi-cultur es mixes bacterium cultivation
    (1) prepared by compound bacteria seed: the multi-cultur es that step one room temperature is cultivated naturally is mixed bacterium solution, is divided in and goes out under cleaning condition In triangular flask after bacterium, shaking table shaken cultivation 24-72 hour, shaking speed 100-200r/min, cultivation initial temperature is room temperature, Every intensification in 3 hours 1-2 DEG C, after cultivating 24-72 hour, as micro organism quantity >=2x10 in shaking flask7During cfu/ml, lived The shaking flask complex microorganism seed changed；During described shaking table shaken cultivation, when microbe inoculation is bacillus cereus, cultivation temperature 38 When ± 1 DEG C, stopping heating up, when microbe inoculation is fungus, cultivation temperature controls at 23-35 DEG C；
    (2) prepared by complex micro organism fungicide: obtain in fungus shake-flask seed step 2 obtained and step 3 (1) is compound micro- Biological seed is according to 8-10%(v/w, ml/g) inoculum concentration be inoculated in solid medium, solid medium is by wheat bran 50%- 70%, soybean cake 10%-15%, sugar slag 5%-10%, powder of straw 5%-10%, food processing leftover bits and pieces 5%-10% are according to percentage by weight system For forming；Described food processing leftover bits and pieces is one or more in postfermented tea powder, cane powder, dehydrated potato powder, tapioca starch；Compound micro- After biological seed is inoculated into solid medium, fermenting cellar solid fermentation 48-96 hour, it is thus achieved that solid composite microbe seed； Continuing to be inoculated into expansion fermentation culture in solid medium with the solid composite microbe seed obtained, inoculum concentration is solid culture The 5%-6% of basic weight amount, sends into fermenting cellar solid fermentation 48-96 hour, and sweat temperature controls at 32-37 DEG C, moisture Control Natural at 60-65%, pH；After expanding fermentation ends, micro organism quantity >=2x108During cfu/g, 55-60 DEG C is dried, moisture Control At 15%-20%；Size-reduced, be screened to 60-80 mesh, it is thus achieved that multifunctional composite microbe microbial inoculum, room temperature preserve.
  3. 3. the method for claim 1, it is characterised in that: use the method to prepare the concrete of multifunctional composite microbe bacterium Step is as follows:
    Step one: set up small-sized microbial ecosystem, prepares including nature culture, prepared by initial incubation liquid, initial incubation Liquid sterilizing, sterility test, small-sized microecosystem are formed；
    (1) prepared by natural culture
    1. preparing nature culture by following percentage by weight, material and ratio that 1000g nature culture contains be: animal is new Fresh feces 30%-50%, activated sludge 5%-10%, the 40-60 mesh natural soils 5%-10% that sieves, fineness 80 mesh powder of straw 10%-15%, Sieve 40-60 mesh organic waste 5%-10%, sugar slag 15%-20%, ordinary beer 5L；
    2. the preparation method of natural culture: by animal fresh excreta, activated sludge, sieve 40-60 mesh natural soils, fineness 80 Mesh powder of straw, the 40-60 mesh organic waste that sieves, sugar slag are mixed into uniform initial former base, then will be the most preliminary with 5L medicated beer Mix homogeneously, was placed in agitator stirring mixing 5-10 minute by mixing the initial former base containing medicated beer, uniformly after be prepared as from So culture, is placed in containers for future use；
    (2) prepared by beginning culture fluid: by above-mentioned standby natural culture heated and boiled 30-40 minute, stands cooling 2-5 hour, treats Proportion, more than the species precipitate of water, when upper liquid is in sub-translucent, small particle half suspension in vessel, takes top separatory after filtration Body, proceeds in 3-5 1 L triangular flask standby as initial incubation liquid；
    (3) initial incubation liquid sterilizing: initial incubation liquid is put into high-pressure steam sterilizing pan, in steam pressure 1.05kg/cm2, temperature Under the conditions of spending 121 DEG C, sterilizing 40～50min, after sterilizing terminates, standby；
    (4) sterility test: take the initial incubation liquid 0.5-1ml after the sterilizing of step (3), directly cultivate at ready aseptic LB In base plate, 35 DEG C of-37 DEG C of constant temperature culture 18-24 hour, grow without microorganism, it is thus achieved that aseptic initial incubation liquid；If any micro- Biological growth, is prepared by step (1)-(4) again；
    (5) small-sized microecosystem is formed
    1. step (1)-(4) are prepared in the triangular flask after aseptic initial incubation liquid is sub-packed in 8 sterilizings under cleaning condition, Strain pH is natural, forms independent small-sized ecosystem in each triangular flask；
    2. under cleaning condition, respectively by bacillus subtilis, bacillus polymyxa, bacillus megaterium, stearothermophilus spore Bacillus is inoculated in triangular flask, every 2 triangular flasks inoculation same strain, inoculates complete putting into and naturally grows under sterile board room temperature 18-24 hour；
    3., after 18-24 hour, take out the triangular flask in sterile board, mix successively than 1:1:1:1 according to equal-volume under cleaning condition Close, then proceed to sterile chamber, formed in sterile chamber and there is the small-sized micro-of multiple-microorganism kind and multiple-microorganism group Bioecosystem；Sterile chamber is positioned over room temperature in clean environment become multi-cultur es after naturally cultivating 10-16 hour and mix bacterium Liquid, takes out, is placed in 4 DEG C of refrigerator and saves backup；Or by preserving after sub-for mixed strain dehydrate, obtain complex microorganism dormancy bacterium Body；
    Step 2: synchronization employing conventional method production aspergillus niger shake-flask seed, Eurotium cristatum shake-flask seed:
    (1) make PDA slant medium, seed culture medium, culture medium respectively, aspergillus niger, Eurotium cristatum strain are divided It is not seeded to PDA slant medium cultivate, it is thus achieved that inclined-plane viable bacteria kind, then inclined-plane viable bacteria kind is forwarded to seed culture medium and carries out Liquid is cultivated, it is thus achieved that liquid fungus seed, then is transferred by liquid fungus seed in culture medium cultivation, obtains aspergillus niger respectively Liquid seed and Eurotium cristatum liquid seed, preserve, standby；
    1. prepared by PDA slant medium: potato dextrose agar, and its way is to weigh 200g Rhizoma Solani tuber osi, cleans and goes Skin shreds, and the 1000ml that adds water boils half an hour, filtered through gauze, then adds 15g glucose and 18g agar, after fully dissolving while hot Filtered through gauze, subpackage test tube, every test tube about 10ml, 121 DEG C of sterilizings are taken out test tube pendulum inclined-plane, are stored standby after cooling after 20 minutes With；
    2. prepared by seed culture medium: soluble starch 2-3%, glucose 1-1.5%, KH2PO4 0.1-0.25%, MgSO4 0.1- 0.3%, yeast extract 0.5-0.75%, NaNO3 0.2-0.35%, natural pH, 121 DEG C of autoclaving 20min；
    3. prepared by culture medium: Testa Tritici 4-6%, (NH4)2SO40.1-0.2%, KH2PO40.2-0.35%, sodium acetate 0.1- 0.2%, ascorbic acid 0.1-0.2%, MgSO4 0.15-0.25%, carbamide 0.25-0.5%, natural pH, 121 DEG C of autoclavings 20min；
    (2) seed activation is cultivated: being inoculated in activation culture shaking flask by aspergillus niger liquid seed, activating recipe is: peptone 5.0-5.5 g/L, glucose 10.0-15 g/L, magnesium sulfate 0.5-0.8 g/L, yeast extract powder 2.0-3.0g/L, phosphoric acid Hydrogen dipotassium 1-3g/L, in pH7.0-7.2 nutritional solution, shaking flask rotating speed is 100-160rpm, cultivates 25h-at a temperature of 23-35 DEG C 28h, it is thus achieved that liquid fermentation seed, obtains aspergillus niger shake-flask seed；Eurotium cristatum liquid seed is inoculated into activation culture shake Activation culture in Ping, shaking flask Middle nutrition formula of liquid is: sucrose 60-80g/L, 100 mesh fineness postfermented tea powder 10-20g/L or postfermented tea Lixiviating solution 0.8-1%, dipotassium hydrogen phosphate 3-5g/L, pH5.5-6.5, shaking flask rotating speed is 100-130rpm, cultivates at 26-32 DEG C 90-100 hour, it is thus achieved that the shake-flask seed of liquid fermentation seed, i.e. Eurotium cristatum；
    Step 3: multi-cultur es mixes bacterium cultivation
    (1) composite flora bacillus cereus cultivating seeds: the multi-cultur es that 3. step one (5th) item walks acquisition is mixed bacterium solution and is divided in In 5-10 the triangular flask containing initial incubation liquid, triangular flask volume is 1L, and liquid amount is 500ml, vibrates on activation shaking table Fluctuation temperature culture 40-72h, vibration Fluctuation temperature culture condition is as follows: shaking speed 160-200r/min, initial temperature is 20 DEG C, every 3 Hour raise 1-2 DEG C, when being adjusted to 38 ± 1 DEG C in temperature, stop raise；Vibration Fluctuation temperature culture terminates, it is thus achieved that liquid fermentation is multiple Close seed liquor；Then liquid fermentation seed liquor is inoculated into 10kg by wheat bran, rice according to the inoculative proportion (v/w, ml/g) of 10% In the solid medium that bran, soybean cake, Semen Maydis powder are mixed with according to mass ratio 10:1:3:2, fully mix, at 37 DEG C, cultivate 3 My god, obtain main composite flora bacillus cereus solid seed；
    (2) fungi flora aspergillus niger and coronoid process dissipate capsule bacterium cultivating seeds:
    1. by the shake-flask seed of aspergillus niger according to the inoculative proportion (v/w, ml/g) of 10% be inoculated into 5kg by wheat bran, Testa oryzae, soybean cake, In the solid medium that dehydrated potato powder mixes according to mass ratio 7:1:1:1, fully mix, cultivate 3 days at 37 DEG C, obtain Aspergillus niger solid seed；
    2. the shake-flask seed of Eurotium cristatum is inoculated into 5kg by postfermented tea powder, Caulis Sacchari sinensis according to the inoculative proportion (v/w, ml/g) of 5% In the solid medium that powder mixes according to mass ratio 1:1, fully mix, initial stage temperature 25 DEG C, after 48 hours, temperature is adjusted to 28 DEG C-32 DEG C, humidity is 70%, and pH value regulates to 5-5.5, cultivates 5 days continuously, obtains Eurotium cristatum solid seed；
    3. solid amplification culture: the main composite flora bacillus cereus solid seed for preparing above-mentioned, aspergillus niger solid kind Son, Eurotium cristatum solid seed uniformly mix according to weight ratio 7:2:1, according to the inoculative proportion of 6-8% add to 100kg by In the solid medium that wheat bran, soybean cake, Semen Maydis powder, postfermented tea powder are mixed to prepare according to mass ratio 8:1:0.5:0.5, fully mix, Temperature 32 DEG C in the early stage, wait after 24 hours and temperature are adjusted to 35 DEG C-37 DEG C, cultivate 5-7 days continuously under the conditions of pH is natural；Training Support after terminating, be dried at 55-65 DEG C, it is thus achieved that product effective active bacterium 2x109Cfu/g, water content 15%-20%, through chain-type Pulverizer is pulverized, and crosses 60-100 mesh sieve, prepares multifunctional composite microbe microbial inoculum.
  4. 4. the method for claim 1, it is characterised in that: use the multifunctional composite microbe bacterium that the method prepares Agent processes solid organic castoff as organic matter decomposing inoculant.
  5. 5. the method for claim 1, it is characterised in that: use the multifunctional composite microbe bacterium that the method prepares Ecological feed additive it is used as described in agent.
  6. 6. the method for claim 1, it is characterised in that: use the multifunctional composite microbe bacterium that the method prepares Agent is used as bio-feritlizer.
CN201610657751.XA 2016-08-12 2016-08-12 Small-sized microbial ecosystem method prepares multifunctional composite microbe microbial inoculum and application Pending CN106085922A (en)

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CN108148779A (en) * 2018-01-16 2018-06-12 天津生机集团股份有限公司 A kind of organic matter decomposing inoculant and preparation method thereof
CN108265018A (en) * 2018-04-04 2018-07-10 北京航天恒丰科技股份有限公司 A kind of composite bacteria agent that fertilizer is prepared using stalk fermentation and preparation method thereof

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