CN103981138A - Microbial inoculant, and preparation method and application thereof - Google Patents

Microbial inoculant, and preparation method and application thereof Download PDF

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CN103981138A
CN103981138A CN201410234581.5A CN201410234581A CN103981138A CN 103981138 A CN103981138 A CN 103981138A CN 201410234581 A CN201410234581 A CN 201410234581A CN 103981138 A CN103981138 A CN 103981138A
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microbiobacterial agent
preparation
sodium
chlor
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CN103981138B (en
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周礼红
廖瀚峰
丁旭
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周礼红
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    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00Technologies for solid waste management
    • Y02W30/40Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention relates to the field of biological products, and particularly relates to a microbial inoculant, and a preparation method and application thereof. The preparation method of the microbial inoculant comprises the following steps: respectively activating Brevibacillus brevis 50L-3a, Brevibacillus agri and Bacillus amyloliquefaciens strains to prepare bacterium suspensions; culturing the three bacterium suspensions to obtain primary seed bacterium suspensions; mixing the three primary seed bacterium suspensions, and culturing to obtain a secondary seed mixed bacterium suspension; and fermenting and culturing until the spore concentration is (2-5)*10<12>CFU/ml and the spores account for more than 85% of the total count in the fermentative bacterium solution, thereby obtaining the microbial inoculant. The preparation method is simple and is low in cost. The microbial inoculant can be used for deodorizing municipal domestic waste, kitchen waste and livestock/poultry manure and preparing organic fertilizers by fermentative rotting, has the advantages of favorable deodorizing effect and short deodorizing time, has the function of preparing organic fertilizers by fermentative rotting, changes wastes into the organic fertilizers, and has important meanings for environmental protection.

Description

A kind of preparation method of microbiobacterial agent and the microbiobacterial agent obtaining and application
Technical field
The present invention relates to field of biological product, in particular to a kind of preparation method and the microbiobacterial agent obtaining and application of microbiobacterial agent.
Background technology
Along with the enhancing of people's environmental consciousness, also more and more higher to the requirement of environmental quality, also more responsive to the pollution bringing because of stench.The material that produces stench not only can make people produce unhappy and detest and feel, and many repugnant substances health life even of also endangering people, thereby abroad some country has just started the research of this aspect earlier, stench is carried out to special legislation, odor pollution is separated separately from topsoil to a kind of as public hazards.
Removing of foul gas is a very important technology, generally has chemical method, physics and biological process.Wherein, microbial deodorant technology in biological process is to utilize efficient adsorption, absorption and the Degradation of the peculiar microorganism of the repugnant substance that can transform or degrade to purify foul gas, change stench is odorless, not containing any pharmaceutical chemicals, also not containing transgenic product composition, can not cause secondary pollution, represent the direction of biological environmental production industry development.And the existing microbial deodorant technology deodorizing time is long, microbial inoculum complicated process of preparation, cost is higher.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of microbiobacterial agent and the microbiobacterial agent obtaining and application, to solve the above problems.
The preparation method that a kind of microbiobacterial agent is provided in an embodiment of the present invention, comprises the following steps:
(a) by after Brevibacillus brevis 50L-3a, soil bacillus brevis and Bacillus amyloliquefaciens strain difference solid activation culture, make concentration and be 1-3 * 10 8three kinds of bacteria suspensions of CFU/ml;
(b) three kinds of described bacteria suspensions being take respectively to the inoculum size that percent by volume is 3-7% is seeded to first order seed substratum, cultivates to obtain concentration and be 1-4 * 10 10three kinds of first order seed bacteria suspensions of CFU/ml;
(c) three kinds of described first order seed bacteria suspensions are mixed with equal-volume, the inoculum size that the percent by volume of then take is 2-3% is forwarded to secondary seed medium, and cultivating and obtaining mixed bacterium concentration is 2-5 * 10 11the secondary seed plastc ring of CFU/ml;
(d) the described secondary seed plastc ring inoculum size that by volume percentage ratio is 2-6% is forwarded to bacteria fermentation substratum, ferment to gemma concentration be 2-5 * 10 12cFU/ml, gemma number account for the more than 85% of described zymocyte liquid total count, obtain microbiobacterial agent.
Preferably, in described step (a), adopt the aseptic water washing thalline that contains tween 80, the percent by volume that described tween 80 accounts for described sterilized water is 0.04-0.06%, mixes and obtains described bacteria suspension.
Preferably, in described step (b), by weight, described first order seed medium component comprises: 1000 parts of Semen Maydis powder 10-14 part, yeast extract 4-6 part, sodium-chlor 6-10 part and distilled water, the pH of described first order seed substratum is 7.1-7.3.
Preferably, in described step (c), by weight, described secondary seed medium composition comprises: 1000 parts of Semen Maydis powder 10-14 part, vinasse powder 8-12 part, peanut meal powder 4-6 part, sodium-chlor 5-7 part and distilled water, the pH of described secondary seed medium is 7.1-7.3.
Preferably, in described step (d), by weight, described bacteria fermentation medium component comprises: Semen Maydis powder 13-15 part, vinasse powder 14-16 part, peanut meal powder 6-8 part, wheat bran 14-16 part, sodium-chlor 2-4 part, K 2hPO 43-5 part, MnSO 41000 parts of 0.03-0.05 part and distilled water, the pH of described bacteria fermentation substratum is 7.1-7.3.
Preferably, in described step (b), described step (c) and described step (d), culture condition is 35-38 ℃, 210-240rpm/min shaking culture.
Preferably, the pH value of described microbiobacterial agent is adjusted to 5-5.5, adds sodium-chlor, extremely the mass percent of described sodium-chlor and described microbiobacterial agent is 8-10%, makes liquid microbial microbial inoculum.
Preferably, the pH value of described microbiobacterial agent is adjusted to 5.5-6, adds successively sodium-chlor and Sodium Propionate, the weight of described sodium-chlor and described Sodium Propionate is respectively 4-6% and the 1.5-3.0% of the weight of described microbiobacterial agent, obtains prefabricated solution;
In described prefabricated solution, add carrier, the mass volume ratio of described carrier and described prefabricated solution is 1-1.5:1-1.5, dries to water content lower than 10% in 45-60 ℃, makes solid-state microorganism microbial inoculum;
Wherein said carrier is the mixture that the mass ratio of vinasse powder and wheat bran is 1-1.5:1-1.5.
The embodiment of the present invention also provides the microbiobacterial agent of being prepared by aforesaid method.
The embodiment of the present invention also provides this microbiobacterial agent deodorizing and fermentation maturity in domestic waste, rubbish from cooking and the feces of livestock and poultry to prepare the application of fertilizer.
The preparation method of the microbiobacterial agent that the embodiment of the present invention provides, first Brevibacillus brevis 50L-3a, soil bacillus brevis and bacillus amyloliquefaciens are activated respectively to rear cultivation and obtain respectively primary seed solution, then primary seed solution is fermented after mixed culture according to a certain percentage again, obtaining gemma concentration is 2-5 * 10 12cFU/ml, gemma number account for more than 85% microbiobacterial agent of fermented liquid total count.The method of preparing microbiobacterial agent is simple, and the microbiobacterial agent cost obtaining is low.The microbiobacterial agent of preparation deodorizing and fermentation maturity in domestic waste, rubbish from cooking and feces of livestock and poultry are prepared the application of fertilizer, the microbiobacterial agent obtaining is good deodorization effect not only, the deodorizing time is short, and there is the effect that fermentation maturity is prepared fertilizer, rubbish is become to fertilizer, environmental protection is significant.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
The preparation method that a kind of microbiobacterial agent is provided in an embodiment of the present invention, comprises the following steps:
(a) by after Brevibacillus brevis 50L-3a, soil bacillus brevis and Bacillus amyloliquefaciens strain difference solid activation culture, make concentration and be 1-3 * 10 8three kinds of bacteria suspensions of CFU/ml;
(b) three kinds of described bacteria suspensions being take respectively to the inoculum size that percent by volume is 3-7% is seeded to first order seed substratum, cultivates to obtain concentration and be 1-4 * 10 10three kinds of first order seed bacteria suspensions of CFU/ml;
(c) three kinds of described first order seed bacteria suspensions are mixed with equal-volume, the inoculum size that the percent by volume of then take is 2-3% is forwarded to secondary seed medium, and cultivating and obtaining mixed bacterium concentration is 2-5 * 10 11the secondary seed plastc ring of CFU/ml;
(d) the described secondary seed plastc ring inoculum size that by volume percentage ratio is 2-6% is forwarded to bacteria fermentation substratum, ferment to gemma concentration be 2-5 * 10 12cFU/ml, gemma number account for the more than 85% of described zymocyte liquid total count, obtain microbiobacterial agent.
Wherein, Brevibacillus brevis 50L-3a bacterial strain, the applicant's patent applied for, and be deposited in Chinese Typical Representative culture collection center, preserving number is CCTCC NO:M2013597; Soil bacillus brevis (brevibacillusagri), purchased from Chinese industrial microbial strains preservation administrative center; Preserving number is: CICC23082; Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), purchased from Chinese common micro-organisms culture presevation administrative center; Preserving number is: CGMCC1.10901.Brevibacillus brevis 50L-3a is high temperature bacterium, and optimum growth temperature is 49-51 ℃; Soil bacillus brevis is mesophilic bacteria, and optimum growth temperature is 36-38 ℃; Bacillus amyloliquefaciens is mesophilic bacteria, and optimum growth temperature is 30-35 ℃.The microbiobacterial agent that the present invention adopts the bacterial strain of these three kinds of differing temps scopes to make, growth temperature subject range is wider, and the temperature range that can grow is at 8 ℃-70 ℃; The microbial safety of the microbiobacterial agent forming, level of security is all 4 grades; Can be used for rubbish fast deodorization and the quick fermentation production fertilizer that becomes thoroughly decomposed; And the preparation method of this microbiobacterial agent is fairly simple, and cost is lower.
Preferably, in described step (a), adopt the aseptic water washing thalline that contains tween 80, the percent by volume that described tween 80 accounts for described sterilized water is 0.04-0.06%, mixes and obtains described bacteria suspension.Adopt the tween 80 of this concentration that the thalline on solid medium is washed in a state of excitement, thalline is spread out, and obtains the bacterium liquid of homogeneous, inoculum size is more accurate, and the bacterium liquid of homogeneous is beneficial at postvaccinal substratum and disperses, and is beneficial to the growth of thalline, saves the growth time of thalline.Wherein fermented liquid total count is gemma thalline number and non-gemma thalline number.
Preferably, in described step (b), by weight, described first order seed medium component comprises: 1000 parts of Semen Maydis powder 10-14 part, yeast extract 4-6 part, sodium-chlor 6-10 part and distilled water, the pH of described first order seed substratum is 7.1-7.3.After the composition of first order seed substratum is weighed, mix, regulate pH value, then adopt autoclave sterilization, adopt conventional 121 ℃, sterilizing 20-30min, afterwards, is cooled to the first order seed substratum that room temperature obtains aseptic be directly used in inoculation.Adopt this substratum to cultivate respectively Brevibacillus brevis 50L-3a, soil bacillus brevis and bacillus amyloliquefaciens, growth time is shorter, and the thalli growth obtaining is active.
Preferably, in described step (c), by weight, described secondary seed medium composition comprises: 1000 parts of Semen Maydis powder 10-14 part, vinasse powder 8-12 part, peanut meal powder 4-6 part, sodium-chlor 5-7 part and distilled water, the pH of described secondary seed medium is 7.1-7.3.After the composition of secondary seed medium is weighed, mix, regulate pH value, then adopt autoclave sterilization, adopt conventional 121 ℃, sterilizing 25-35min, afterwards, is cooled to the secondary seed medium that room temperature obtains aseptic be directly used in inoculation.Adopt the mixed bacteria liquid of this culture medium culturing Brevibacillus brevis 50L-3a, soil bacillus brevis and bacillus amyloliquefaciens, every kind of thalli growth speed, the thalli growth obtaining is active.
Preferably, in described step (d), by weight, described bacteria fermentation medium component comprises: Semen Maydis powder 13-15 part, vinasse powder 14-16 part, peanut meal powder 6-8 part, wheat bran 14-16 part, sodium-chlor 2-4 part, K 2hPO 43-5 part, MnSO 41000 parts of 0.03-0.05 part and distilled water, the pH of described bacteria fermentation substratum is 7.1-7.3.After the composition of bacteria fermentation substratum is weighed, mix, regulate pH value, then adopt autoclave sterilization, adopt conventional 121 ℃, sterilizing 25-35min, afterwards, is cooled to the bacteria fermentation substratum that room temperature obtains aseptic be directly used in inoculation.Adopt this fermention medium to carry out fermentation culture to the mixed solution of three kinds of bacterium, every kind of thalli growth speed, in the fermented liquid obtaining bacterium to count concentration high, and gemma number to account in fermented liquid the ratio of total count high.
The granularity of the Semen Maydis powder wherein, relating in substratum in the present invention, vinasse powder, peanut meal powder and wheat bran is 1.5-2mm.In addition the reagent using in the present invention, is common biochemical reagents.
Preferably, in described step (b), described step (c) and described step (d), culture condition is 35-38 ℃, 210-240rpm/min shaking culture.This culture condition, thalli growth temperature is suitable, and shaking culture has guaranteed the homogeneous of bacterium liquid, prevents thalline caking, is beneficial to the single Fast Growth of thalline.
For the ease of preserving and carrying, microbiobacterial agent obtained above is made to liquid microbial microbial inoculum system and solid-state microorganism microbial inoculum system.
Particularly, the pH value of described microbiobacterial agent is adjusted to 5-5.5, adds sodium-chlor, extremely the mass percent of described sodium-chlor and described microbiobacterial agent is 8-10%, makes liquid microbial microbial inoculum.
Particularly, the pH value of described microbiobacterial agent is adjusted to 5.5-6, adds successively sodium-chlor and Sodium Propionate, the weight of described sodium-chlor and described Sodium Propionate is respectively 4-6% and the 1.5-3.0% of the weight of described microbiobacterial agent, obtains prefabricated solution;
In described prefabricated solution, add carrier, the mass volume ratio of described carrier and described prefabricated solution is 1-1.5:1-1.5, dries to water content lower than 10% in 45-60 ℃, makes solid-state microorganism microbial inoculum;
Wherein said carrier is the mixture that the mass ratio of vinasse powder and wheat bran is 1-1.5:1-1.5.
The embodiment of the present invention also provides the microbiobacterial agent of being prepared by aforesaid method.
The embodiment of the present invention also provides this microbiobacterial agent deodorizing and fermentation maturity in domestic waste, rubbish from cooking and the feces of livestock and poultry to prepare the application of fertilizer.
The preparation method of the microbiobacterial agent that the embodiment of the present invention provides, first Brevibacillus brevis 50L-3a, soil bacillus brevis and bacillus amyloliquefaciens are activated respectively to rear cultivation and obtain respectively primary seed solution, then primary seed solution is fermented after mixed culture according to a certain percentage again, obtaining gemma concentration is 2-5 * 10 12cFU/ml, gemma number account for more than 85% microbiobacterial agent of fermented liquid total count.The method of preparing microbiobacterial agent is simple, and the microbiobacterial agent cost obtaining is low.The microbiobacterial agent of preparation deodorizing and fermentation maturity in domestic waste, rubbish from cooking and feces of livestock and poultry are prepared the application of fertilizer, the microbiobacterial agent obtaining is good deodorization effect not only, the deodorizing time is short, and there is the effect that fermentation maturity is prepared fertilizer, rubbish is become to fertilizer, environmental protection is significant.
Embodiment 1
Adopt following steps to prepare microbiobacterial agent:
(a) by Brevibacillus brevis 50L-3a, soil bacillus brevis and Bacillus amyloliquefaciens strain respectively after solid activation culture, adopt and contain the aseptic water washing thalline that percent by volume is 0.04% tween 80, mix to such an extent that concentration is 1 * 10 8three kinds of bacteria suspensions of CFU/ml;
(b) three kinds of bacteria suspensions all being take to the inoculum size that percent by volume is 3% is seeded to first order seed substratum, cultivates to obtain concentration and be 1 * 10 10three kinds of first order seed bacteria suspensions of CFU/ml; Wherein, first order seed substratum makes in the following manner: take respectively 1000 parts of 10 parts of Semen Maydis powder, 4 parts of yeast extracts, 6 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.1, then adopts 121 ℃, and sterilizing 20min, is cooled to room temperature and get final product;
(c) three kinds of first order seed bacteria suspension equal-volumes are mixed, the inoculum size that the percent by volume of then take is 2% is forwarded to secondary seed medium, and cultivating and obtaining mixed bacterium concentration is 2 * 10 11the secondary seed plastc ring of CFU/ml; Wherein, secondary seed medium makes in the following manner: take respectively 1000 parts of 10 parts of Semen Maydis powder, 8 parts, vinasse powder, 4 parts, peanut meal powder, 5 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.1, then adopt 121 ℃, sterilizing 25min, is cooled to room temperature and get final product;
(d) the secondary seed plastc ring inoculum size that by volume percentage ratio is 2% is forwarded to bacteria fermentation substratum, ferment to gemma concentration be 2 * 10 12cFU/ml, gemma number account for 85% of described zymocyte liquid total count, obtain microbiobacterial agent; Wherein, bacteria fermentation substratum makes in the following manner: take respectively 13 parts of Semen Maydis powder, 14 parts, vinasse powder, 6 parts, peanut meal powder, 14 parts, wheat bran, 2 parts, sodium-chlor, K 2hPO 43 parts, MnSO 41000 parts of 0.03 part and distilled water, mix, and regulating pH value is 7.1, then adopts 121 ℃, and sterilizing 25min, is cooled to room temperature and get final product;
In step (b), step (c) and step (d), culture condition is 35 ℃, 240rpm/min shaking culture;
The microbiobacterial agent obtaining is prepared to fertilizer for domestic refuse deodorizing and fermentation maturity, is specially:
Get 2 tons of domestic refuses and pack Shui great Chi into, domestic refuse is piled high 1.8-2 rice; Microbiobacterial agent obtained above is inoculated in domestic refuse heap by the inoculum size of 60-75ml/ ton domestic refuse, and turning mixes, and piles high 1.8-2 rice, plastic covering film on scrap heap, normal temperature fermentation 120-144h.
After domestic refuse microbe inoculation microbial inoculum 5-6h, junk-heap body middle part temperature reaches 57 ℃ ± 2 ℃; When 10-34h is arrived in fermentation, junk-heap body middle part temperature reaches 78 ℃ ± 3 ℃; Scrap heap after fermentation ends stink for to be down to 2-3 level from 5 grades; While continuing fermentation to 120-144h, domestic refuse fermentation reaches becomes thoroughly decomposed.
Embodiment 2
Adopt following steps to prepare microbiobacterial agent:
(a) by Brevibacillus brevis 50L-3a, soil bacillus brevis and Bacillus amyloliquefaciens strain respectively after solid activation culture, adopt and contain the aseptic water washing thalline that percent by volume is 0.05% tween 80, mix to such an extent that concentration is 2 * 10 8three kinds of bacteria suspensions of CFU/ml;
(b) three kinds of bacteria suspensions all being take to the inoculum size that percent by volume is 5% is seeded to first order seed substratum, cultivates to obtain concentration and be 2 * 10 10three kinds of first order seed bacteria suspensions of CFU/ml; Wherein, first order seed substratum makes in the following manner: take respectively 1000 parts of 12 parts of Semen Maydis powder, 5 parts of yeast extracts, 8 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.2, then adopts 121 ℃, and sterilizing 25min, is cooled to room temperature and get final product;
(c) three kinds of first order seed bacteria suspension equal-volumes are mixed, the inoculum size that the percent by volume of then take is 3% is forwarded to secondary seed medium, and cultivating and obtaining mixed bacterium concentration is 4 * 10 11the secondary seed plastc ring of CFU/ml; Wherein, secondary seed medium makes in the following manner: take respectively 1000 parts of 12 parts of Semen Maydis powder, 10 parts, vinasse powder, 5 parts, peanut meal powder, 6 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.2, then adopt 121 ℃, sterilizing 30min, is cooled to room temperature and get final product;
(d) the secondary seed plastc ring inoculum size that by volume percentage ratio is 3% is forwarded to bacteria fermentation substratum, ferment to gemma concentration be 4 * 10 12cFU/ml, gemma number account for 90% of described zymocyte liquid total count, obtain microbiobacterial agent; Wherein, bacteria fermentation substratum makes in the following manner: take respectively 14 parts of Semen Maydis powder, 15 parts, vinasse powder, 7 parts, peanut meal powder, 15 parts, wheat bran, 3 parts, sodium-chlor, K 2hPO 44 parts, MnSO 41000 parts of 0.04 part and distilled water, mix, and regulating pH value is 7.2, then adopts 121 ℃, and sterilizing 30min, is cooled to room temperature and get final product;
In step (b), step (c) and step (d), culture condition is 37 ℃, 220rpm/min shaking culture;
The pH value of microbiobacterial agent obtained above is adjusted to 5, adds sodium-chlor, the mass percent of sodium-chlor and microbiobacterial agent is 8%, makes liquid microbial microbial inoculum.
The liquid microbial microbial inoculum obtaining is prepared to fertilizer for rubbish from cooking deodorizing and fermentation maturity, is specially:
Get 2 tons of rubbishes from cooking and pack great Chi into, pile high 1.8-2 rice; The inoculum size of above-mentioned liquid microbial microbial inoculum 45-55ml/ ton rubbish from cooking is inoculated in rubbish from cooking heap, and turning mixes, and piles high 1.8-2 rice, the upper plastic covering film of rubbish from cooking heap, environment normal temperature fermentation 110-130h.
5-6h after the liquid microbiobacterial agent of rubbish from cooking heap inoculation, rubbish from cooking heap body middle part temperature reaches 57 ℃ ± 2 ℃; When 15-30h is arrived in fermentation, rubbish from cooking heap body middle part temperature reaches 76 ℃ ± 3 ℃; Rubbish from cooking heap after fermentation ends stink for to be down to 2-3 level from 5 grades; While continuing fermentation to 110-130h, rubbish from cooking fermentation reaches becomes thoroughly decomposed.
Embodiment 3
Adopt following steps to prepare microbiobacterial agent:
(a) by Brevibacillus brevis 50L-3a, soil bacillus brevis and Bacillus amyloliquefaciens strain respectively after solid activation culture, adopt and contain the aseptic water washing thalline that percent by volume is 0.06% tween 80, mix to such an extent that concentration is 3 * 10 8three kinds of bacteria suspensions of CFU/ml;
(b) three kinds of bacteria suspensions all being take to the inoculum size that percent by volume is 7% is seeded to first order seed substratum, cultivates to obtain concentration and be 4 * 10 10three kinds of first order seed bacteria suspensions of CFU/ml; Wherein, first order seed substratum makes in the following manner: take respectively 1000 parts of 14 parts of Semen Maydis powder, 6 parts of yeast extracts, 10 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.3, then adopt 121 ℃, sterilizing 30min, is cooled to room temperature and get final product;
(c) three kinds of first order seed bacteria suspension equal-volumes are mixed, the inoculum size that the percent by volume of then take is 2% is forwarded to secondary seed medium, and cultivating and obtaining mixed bacterium concentration is 5 * 10 11the secondary seed plastc ring of CFU/ml; Wherein, secondary seed medium makes in the following manner: take respectively 1000 parts of 14 parts of Semen Maydis powder, 12 parts, vinasse powder, 6 parts, peanut meal powder, 7 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.3, then adopt 121 ℃, sterilizing 35min, is cooled to room temperature and get final product;
(d) the secondary seed plastc ring inoculum size that by volume percentage ratio is 6% is forwarded to bacteria fermentation substratum, ferment to gemma concentration be 5 * 10 12cFU/ml, gemma number account for 95% of described zymocyte liquid total count, obtain microbiobacterial agent; Wherein, bacteria fermentation substratum makes in the following manner: take respectively 15 parts of Semen Maydis powder, 16 parts, vinasse powder, 8 parts, peanut meal powder, 16 parts, wheat bran, 4 parts, sodium-chlor, K 2hPO 45 parts, MnSO 41000 parts of 0.05 part and distilled water, mix, and regulating pH value is 7.3, then adopts 121 ℃, and sterilizing 35min, is cooled to room temperature and get final product;
In step (b), step (c) and step (d), culture condition is 38 ℃, 210rpm/min shaking culture;
The pH value of microbiobacterial agent obtained above is adjusted to 5.5-6, adds successively sodium-chlor and Sodium Propionate, the weight of sodium-chlor and Sodium Propionate is respectively 4-6% and the 1.5-3.0% of microbiobacterial agent weight, obtains prefabricated solution;
In prefabricated solution, add carrier, the mass volume ratio of carrier and prefabricated solution is 1-1.5:1-1.5, dries to water content lower than 10%, makes solid-state microorganism microbial inoculum for 45-60 ℃;
Wherein carrier is the mixture that the mass ratio of vinasse powder and wheat bran is 1-1.5:1-1.5.
The solid-state microorganism microbial inoculum obtaining is prepared to fertilizer for fresh pig manure deodorizing and fermentation maturity, is specially:
Get 2 tons of fresh pig manures and pack great Chi into, pile high 1.8-2 rice; Above-mentioned solid-state microorganism microbial inoculum is inoculated in fresh pig dunghill by the inoculum size of 45-55g/ ton pig manure, and turning mixes, and piles high 1.8-2 rice, plastic covering film on fresh pig dunghill, environment normal temperature fermentation 100-120h.
Fresh pig dunghill inoculation solid-state microorganism microbial inoculum 3-4h, fresh pig dunghill body middle part temperature reaches 57 ℃ ± 2 ℃; When 9-33h is arrived in fermentation, fresh pig dunghill body middle part temperature reaches 76 ℃ ± 3 ℃; Fresh pig dunghill after fermentation ends stink for to be down to 2-3 level from 5 grades.While continuing fermentation to 100-120h, fresh pig manure fermentation reaches and becomes thoroughly decomposed.
Embodiment 4
Adopt following steps to prepare microbiobacterial agent:
(a) by Brevibacillus brevis 50L-3a, soil bacillus brevis and Bacillus amyloliquefaciens strain respectively after solid activation culture, adopt and contain the aseptic water washing thalline that percent by volume is 0.05% tween 80, mix to such an extent that concentration is 2 * 10 8three kinds of bacteria suspensions of CFU/ml;
(b) three kinds of bacteria suspensions all being take to the inoculum size that percent by volume is 4% is seeded to first order seed substratum, cultivates to obtain concentration and be 3 * 10 10three kinds of first order seed bacteria suspensions of CFU/ml; Wherein, first order seed substratum makes in the following manner: take respectively 1000 parts of 12 parts of Semen Maydis powder, 5 parts of yeast extracts, 8 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.2, then adopts 121 ℃, and sterilizing 25min, is cooled to room temperature and get final product;
(c) three kinds of first order seed bacteria suspension equal-volumes are mixed, the inoculum size that the percent by volume of then take is 3% is forwarded to secondary seed medium, and cultivating and obtaining mixed bacterium concentration is 3 * 10 11the secondary seed plastc ring of CFU/ml; Wherein, secondary seed medium makes in the following manner: take respectively 1000 parts of 12 parts of Semen Maydis powder, 10 parts, vinasse powder, 5 parts, peanut meal powder, 6 parts, sodium-chlor and distilled water, mix, regulating pH value is 7.2, then adopt 121 ℃, sterilizing 30min, is cooled to room temperature and get final product;
(d) the secondary seed plastc ring inoculum size that by volume percentage ratio is 4% is forwarded to bacteria fermentation substratum, ferment to gemma concentration be 4 * 10 12cFU/ml, gemma number account for 97% of described zymocyte liquid total count, obtain microbiobacterial agent; Wherein, bacteria fermentation substratum makes in the following manner: take respectively 14 parts of Semen Maydis powder, 15 parts, vinasse powder, 7 parts, peanut meal powder, 15 parts, wheat bran, 3 parts, sodium-chlor, K 2hPO 44 parts, MnSO 41000 parts of 0.04 part and distilled water, mix, and regulating pH value is 7.2, then adopts 121 ℃, and sterilizing 30min, is cooled to room temperature and get final product;
In step (b), step (c) and step (d), culture condition is 37 ℃, 220rpm/min shaking culture;
The pH value of microbiobacterial agent obtained above is adjusted to 5.5, adds sodium-chlor, the mass percent of sodium-chlor and microbiobacterial agent is 10%, makes liquid microbial microbial inoculum.
The liquid microbial microbial inoculum obtaining is prepared to fertilizer for fresh chicken droppings deodorant and fermentation maturity, is specially:
Get 2 tons of fresh chicken manures and pack great Chi into, fresh chicken manure is piled high 1.8-2 rice;
Liquid microbial microbial inoculum obtained above is inoculated in fresh chicken manure heap by the inoculum size of the fresh chicken manure of 35-50ml/ ton, and turning mixes, and piles high 1.8-2 rice, the upper plastic covering film of fresh chicken manure heap, environment normal temperature fermentation 100-120h.
After the liquid microbiobacterial agent 3-4h of fresh chicken manure heap inoculation, fresh chicken manure heap body middle part temperature reaches 57 ℃ ± 2 ℃; When 8-33h is arrived in fermentation, new refuse heap body middle part temperature reaches 77 ℃ ± 3 ℃; New refuse heap stink after fermentation ends is down to 2-3 level from 5 grades.While continuing fermentation to 100-120h, fresh chicken manure fermenting reaches and becomes thoroughly decomposed.
In addition, can also to elect as can be cow dung, horsehit, sheep excrement, duck excrement, goose excrement etc. to feces of livestock and poultry.
Microbiobacterial agent preparation method provided by the invention is simple, and cost is lower, and the microbial safety rank that forms microbiobacterial agent is 4 grades, safe; The temperature of the microbiobacterial agent growth obtaining is at 8 ℃-70 ℃, can be applicable to deodorizing and fermentation maturity in domestic waste, rubbish from cooking and feces of livestock and poultry and prepare fertilizer, microbiobacterial agent good deodorization effect provided by the invention, 1-2d can be down to 2-3 level by 5 grades, and the deodorizing time is short; Fermentation maturity time 3-5d, the efficiency of rubbish being become thoroughly decomposed into fertilizer is high, and environmental protection is significant.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (10)

1. a preparation method for microbiobacterial agent, is characterized in that, comprises the following steps:
(a) by after Brevibacillus brevis 50L-3a, soil bacillus brevis and Bacillus amyloliquefaciens strain difference solid activation culture, make concentration and be 1-3 * 10 8three kinds of bacteria suspensions of CFU/ml;
(b) three kinds of described bacteria suspensions being take respectively to the inoculum size that percent by volume is 3-7% is seeded to first order seed substratum, cultivates to obtain concentration and be 1-4 * 10 10three kinds of first order seed bacteria suspensions of CFU/ml;
(c) three kinds of described first order seed bacteria suspensions are mixed with equal-volume, the inoculum size that the percent by volume of then take is 2-3% is forwarded to secondary seed medium, and cultivating and obtaining mixed bacterium concentration is 2-5 * 10 11the secondary seed plastc ring of CFU/ml;
(d) the described secondary seed plastc ring inoculum size that by volume percentage ratio is 2-6% is forwarded to bacteria fermentation substratum, ferment to gemma concentration be 2-5 * 10 12cFU/ml, gemma number account for the more than 85% of described zymocyte liquid total count, obtain microbiobacterial agent.
2. the preparation method of microbiobacterial agent according to claim 1, it is characterized in that, in described step (a), adopt the aseptic water washing thalline that contains tween 80, the percent by volume that described tween 80 accounts for described sterilized water is 0.04-0.06%, mixes and obtains described bacteria suspension.
3. the preparation method of microbiobacterial agent according to claim 1, it is characterized in that, in described step (b), by weight, described first order seed medium component comprises: 1000 parts of Semen Maydis powder 10-14 part, yeast extract 4-6 part, sodium-chlor 6-10 part and distilled water, the pH of described first order seed substratum is 7.1-7.3.
4. the preparation method of microbiobacterial agent according to claim 1, it is characterized in that, in described step (c), by weight, described secondary seed medium composition comprises: 1000 parts of Semen Maydis powder 10-14 part, vinasse powder 8-12 part, peanut meal powder 4-6 part, sodium-chlor 5-7 part and distilled water, the pH of described secondary seed medium is 7.1-7.3.
5. the preparation method of microbiobacterial agent according to claim 1, it is characterized in that, in described step (d), by weight, described bacteria fermentation medium component comprises: Semen Maydis powder 13-15 part, vinasse powder 14-16 part, peanut meal powder 6-8 part, wheat bran 14-16 part, sodium-chlor 2-4 part, K 2hPO 43-5 part, MnSO 41000 parts of 0.03-0.05 part and distilled water, the pH of described bacteria fermentation substratum is 7.1-7.3.
6. the preparation method of microbiobacterial agent according to claim 1, is characterized in that, in described step (b), described step (c) and described step (d), culture condition is 35-38 ℃, 210-240rpm/min shaking culture.
7. according to the preparation method of the microbiobacterial agent described in claim 1-6 any one, it is characterized in that, the pH value of described microbiobacterial agent is adjusted to 5-5.5, add sodium-chlor, mass percent to described sodium-chlor and described microbiobacterial agent is 8-10%, makes liquid microbial microbial inoculum.
8. according to the preparation method of the microbiobacterial agent described in claim 1-6 any one, it is characterized in that, the pH value of described microbiobacterial agent is adjusted to 5.5-6, add successively sodium-chlor and Sodium Propionate, the weight of described sodium-chlor and described Sodium Propionate is respectively 4-6% and the 1.5-3.0% of the weight of described microbiobacterial agent, obtains prefabricated solution;
In described prefabricated solution, add carrier, the mass volume ratio of described carrier and described prefabricated solution is 1-1.5:1-1.5, dries to water content lower than 10% in 45-60 ℃, makes solid-state microorganism microbial inoculum;
Wherein said carrier is the mixture that the mass ratio of vinasse powder and wheat bran is 1-1.5:1-1.5.
9. the microbiobacterial agent that prepared by the preparation method described in a claim 1-8 any one.
10. a microbiobacterial agent claimed in claim 9 deodorizing and fermentation maturity in domestic waste, rubbish from cooking and feces of livestock and poultry prepared the application of fertilizer.
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CN104609905A (en) * 2015-02-09 2015-05-13 习水县和平有机肥业有限公司 Organic fertilizer fermentation method
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CN111019863A (en) * 2019-12-26 2020-04-17 青岛尚德生物技术有限公司 Deodorizing microbial agent for removing odor in dry latrine and application thereof
CN113186126A (en) * 2021-04-16 2021-07-30 天津益农生物科技有限公司 Microbial agent

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