CN107384823A - One plant of grease degrading strain and its application - Google Patents

One plant of grease degrading strain and its application Download PDF

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Publication number
CN107384823A
CN107384823A CN201710649424.4A CN201710649424A CN107384823A CN 107384823 A CN107384823 A CN 107384823A CN 201710649424 A CN201710649424 A CN 201710649424A CN 107384823 A CN107384823 A CN 107384823A
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bacillus subtilis
compost
microbial
livestock
grease
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Inventor
刘标
刘惠知
贺月林
尹红梅
许隽
杜东霞
王震
许丽娟
吴迎奔
陈薇
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HUNAN PROVINCE MICROBIOLOGY INSTITUTE
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HUNAN PROVINCE MICROBIOLOGY INSTITUTE
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F1/00—Fertilisers made from animal corpses, or parts thereof
    • C05F1/005—Fertilisers made from animal corpses, or parts thereof from meat-wastes or from other wastes of animal origin, e.g. skins, hair, hoofs, feathers, blood
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • Y—GENERAL 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/20—Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
    • Y—GENERAL 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention belongs to microbial technology field, provide one plant of grease degrading strain and its application, present invention separation screening from the soil by grease contaminated areas obtains a bacillus subtilis (Bacillus subtilis) KF 1, the bacterial strain being capable of high temperature resistant, with stronger animal and plant fat degradation capability, yielding lipase vigor is high, and lipase activity reaches 42.31U/mL after shake flask fermentation culture 72h；The microbial inoculum prepared using the bacterial strains of bacillus subtilis KF 1 is applied in livestock and poultry dead volume compost, can accelerate compost maturity, significantly reduces the fat content of composting production, improves seed germination index, lifts the quality of composting production.

Description

One plant of grease degrading strain and its application
Technical field
The invention belongs to microbial technology field, is related to plant height effect grease degrading strain and its application, especially exists Application in livestock and poultry dead volume compost.
Background technology
China is animal husbandry big country, and cultivation scale is at the forefront in the world.By taking live pig as an example, according to statistics, China live pig in 2015 Livestock on hand 4.51 hundred million, deliver 7.08 hundred million for sale.In the case where cultivation scale constantly expands, every year because various diseases cause livestock and poultry The death rate accounts for the 5%~10% of cultivation sum, generates the epidemic-infected animal corpse of enormous amount.Dead livestock and poultry carries substantial amounts of disease Substance, the sound development to the mankind and animal husbandry cause serious threat.How harmless treatment dead livestock and poultry corpse, be current The problem of in the urgent need to address during breeding scale.Promulgated within 2013 according to the Ministry of Agriculture of China《The innoxious place of animals died of illness Manage technical specification》, China's animals died of illness innoxious process for treating mainly includes four classes, i.e., burning method, change preparation method, bury method and hair Ferment method, above technology have application in animals died of illness harmless treatment.Burning method is handled thoroughly spoil, passes through high temperature Pathogenic microorganism can be utterly destroyed, but air pollution can be caused, and needs a large amount of fuel, it is costly；Change preparation method locating During reason, foul gas and waste water are also easy to produce, and it is costly.Though burial method has, processing speed is fast, low cost and other advantages, But underground water and soil pollution can be caused, and this method long processing period.Fermentation method refers to spoil and rice chaff, wood chip etc. Auxiliary material mixes, and biological heat or the specific microorganism of addition using spoil, spoil of fermenting, finally animals died of illness is become For the process of organic fertilizer.Research shows both at home and abroad, residual by can preferably decompose livestock and poultry to dead livestock and poultry progress compost treatment Body, and the high temperature in composting process can effectively kill pathogenic microorganism and realize innoxious, be a kind of inexpensive dead livestock and poultry processing Method.
The compost bacterium developed at present is primarily directed to the composition in feces of livestock and poultry and domestic waste, and livestock and poultry are dead Body compost is different from the compost of other species in various physicochemical properties, in the market dedicated for livestock and poultry dead volume compost microbial inoculum compared with Few, fat content is up to 20.3% in the organic fertilizer for causing to obtain using fermentation method, and floorboard with high oil content is to influence livestock and poultry corpse fertilizer The principal element of materialization product quality, and composting arrangement is also adversely affected.Therefore, searching and exploitation can accelerate compost The microbial strains of middle oils degradation, the quality of composting production can be lifted, contribute to the harmless treatment of livestock and poultry dead volume and resource Change and utilize.
Application No. CN201710203146.X patent《A kind of bacillus subtilis of efficient degradation grease and its should With》The bacillus subtilis 6-loil of one plant of degraded grease is provided, and describes it at home in kitchen garbage aerobic compost Application.Different from the grease composition in kitchen garbage, the grease in livestock and poultry dead volume is animal fat, saturated fat therein Acid content is higher, the more difficult degradation of the grease in compost.Therefore, screening can in efficient degradation livestock and poultry dead volume grease microbial bacteria Strain, help to promote application of the composting process in terms of livestock and poultry dead volume is handled.The bacterial strain KF-1 provided in the present invention is to livestock and poultry dead volume Oil and grease degradation rate in compost is significantly higher than degradeds of the bacillus subtilis 6-loil to grease in kitchen garbage up to 87.3% Rate, have a extensive future.
The content of the invention
In order to solve the above-mentioned technical problem, present invention aims at provide one plant of oils degradation bacterium and its microbial inoculum in livestock and poultry Application in dead volume compost.
To achieve these goals, the technical solution adopted by the present invention is：
A kind of bacillus subtilis (Bacillus subtilis) KF-1, has been preserved in Chinese microorganism strain preservation pipe Reason committee common micro-organisms center, its deposit number are CGMCC No.14120, and preservation date is on May 15th, 2017, are protected Tibetan address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica.
Bacillus subtilis KF-1 of the present invention is used for grease of degrading.Described bacillus subtilis KF-1 can be prepared into Microbial inoculum, degrade animal fat or vegetable fat.Microbial inoculum prepared by bacillus subtilis KF-1 can also be used for livestock and poultry dead volume Compost maturity.The preparation process of bacillus subtilis KF-1 microbial inoculums comprises the following steps：
(1) preparation of seed liquor：Slant strains are inoculated with a ring into beef extract-peptone fluid nutrient medium, 35 DEG C, 180r/min cultivates 10h to logarithmic phase, is transferred to 1% inoculum concentration in the 500mL triangular flasks equipped with 100mL fermentation mediums, 35 DEG C, 180r/min cultivate 24h as seed liquor；
(2) fermented and cultured：Seed liquor is inoculated into fermented and cultured according to 3% inoculum concentration of fermentation medium volume fraction In base, 72h is cultivated under conditions of 35 DEG C, 180r/min, i.e. acquisition bacillus subtilis KF-1 microbial inoculums, withered grass gemma in microbial inoculum Bacillus KF-1 viable count is more than 1.0 × 109CFU/mL；
Fermentation medium components：Corn flour 20g, peptone 10g, MnSO40.2g, NaCl 0.5g, K2HPO40.12g, KH2PO40.25g, distilled water 1000mL, pH 7.0~7.2.
Detailed process when microbial inoculum prepared by bacillus subtilis KF-1 is used for livestock and poultry dead volume compost maturity is as follows：
Composting substrate presses sick dead pig and rice straw 3:1 fresh weight mass ratio is mixed, and composting substrate gross mass is 360kg, it is 25 that C/N is adjusted during compost:1, water content 50%；The addition of microbial bacterial agent is the 1% of composting substrate weight in wet base； Microbial inoculum is uniformly spread in raw material in composting substrate raw material whipping process, and 1.2m × 1.2m × 0.8m heap body is piled after mixing, Terminate compost in compost progress turning in the 7th, 14,21 day, the 30th day.
The bacillus subtilis microbial agent of the present invention is applied in livestock and poultry dead volume compost, can speed up compost maturity, significantly drop The fat content of low composting production, seed germination index is improved, lift the quality of composting production.
The present invention has advantages below：
1. bacillus subtilis KF-1 has stronger oils degradation ability, there is degradation to animal and plant fat, and Resistant against high temperatures.
2. the bacterial strain has higher yielding lipase active, lipase active reaches 42.31U/ after liquid fermentation and culture 72h mL。
3. the strain fermentation process is simple, growth and breeding speed is fast, cost is low, be advantageous to industrialized production.
4. the microbial inoculum prepared by the bacterial strain is applied in livestock and poultry dead volume compost, compost maturity is can speed up, significantly reduces heap The fat content of fertilizer product, seed germination index is improved, lift the quality of composting production.
Brief description of the drawings
Fig. 1 is that 4 plants of bacterial strains that primary dcreening operation obtains compare the degradation rate of grease in nutrient solution；
Fig. 2 is colonial morphology figures of the bacterial strain KF-1 on neutral red culture；
Fig. 3 is bacterial strain KF-1 microscopic morphology structure charts；
Fig. 4 is under the same conditions, bacterial strain KF-1 compares the degradation rate of different greases；
Fig. 5 is bacterial strain KF-1 yielding lipase curve maps；
Fig. 6 is the change that KF-1 microbial inoculums handle livestock and poultry dead volume compost temperature；
Fig. 7 is the change that KF-1 microbial inoculums handle livestock and poultry dead volume compost fat content；
Fig. 8 is the change that KF-1 microbial inoculums handle livestock and poultry dead volume compost seed germination index.
Embodiment
The embodiment carried out below around bacterial strain of the present invention is described in detail, but these embodiment party Formula is merely to illustrate the present invention, rather than limitation the scope of the present invention.
Culture medium involved in the present invention is as follows：
1. enriched medium：NaCl 0.5g, peptone 5.0g, beef extract 0.5g, lard are gradually incrementally added by the cycle, PH 7.0~7.2, the 000mL of distilled water 1.121 DEG C of sterilizing 20min.
2. neutral red culture：1 000mL beef-protein medium is separately added into lard stearin 10mL's and 0.6% Neutral red aqueous solution 1mL.121 DEG C of sterilizing 20min.
3. culture medium of degrading：Grease 10g, (NH4)2SO45g, Na2HPO42g, KH2PO42g, NaCl 2g, add distilled water It is settled to 1L, pH 7.0~7.4.
4. culture medium：Sucrose 0.50g, peptone 2.00g, K2HPO4·3H2O 0.10g, (NH4)2SO40.10g, MgSO4·7H2O 0.05g, 1g lards, 100mL, natural pH, 115 DEG C of sterilizing 15min are settled to distilled water.
5. beef-protein medium：Beef extract 3g, peptone 5g, NaCl 5g, pH 7.0~7.2, distilled water 1000mL；
6. fermentation medium components：Corn flour 20g, peptone 10g, MnSO4 0.2g, NaCl 0.5g, K2HPO4 0.12g, KH2PO40.25g, distilled water 1000mL, pH 7.0~7.2.
Embodiment 1：The acclimating of grease degrading strain and screening
By from for a long time by the soil that grease contaminated areas collects with after 10 times of dilutions of sterilized water, 5mL dilutions are taken to add Into 45mL enrichment culture liquid, 30 DEG C, 180rmin-1Shaken cultivation, it was 1 cycle per 4d, inoculation 10mL bacterium solutions are newly trained to 90mL To promote the growth of bacterial strain in nutrient solution, domestication continues 5 cycles.Initial volume read-out is gradually incremented by with the cycle, respectively 0.8, 1.6、2.4、3.2、4.0mL·L-1。
The bacterium solution 1mL sterile distilled water gradient dilutions for taking last time to tame, take 10 respectively–4、10–5、10–6Three ladders The bacterium solution coating dimethyl diaminophenazine chloride grease flat board of degree, 30 DEG C of incubated 3~5d, observe colony growth situation and whether periphery of bacterial colonies becomes It is red.Show that bacterial strain can cut grease if reddening and produce aliphatic acid.The bacterial strain that primary dcreening operation is selected in is inoculated into 1% inoculum concentration Using lard as in the degraded nutrient solution of sole carbon source, 30 DEG C, 150rmin–1Under the conditions of cultivate 96h, use ultraviolet spectrophotometry Absorbance of the determination sample under 223nm wavelength, while using lard as solute, petroleum ether is solvent, and it is dense to determine different lards The absorbance of degree, standard curve is drawn, lard content and the oil and grease degradation rate of each bacterial strain are calculated according to standard curve.As a result Grease as shown in figure 1,4 plants of bacterial strains that primary dcreening operation obtains can degrade in nutrient solution, wherein bacterial strain KF-1 degradation rate highest, reach To 79.6%.
Embodiment 2：High-efficiency grease degradation bacteria KF-1 identification
After carrying out morphology, micro- sem observation to degradation rate highest bacterial strain KF-1, bacterium is extracted with DNA extraction kit Genomic DNA, PCR amplification use 16S rDNA universal primers 27F：
5'-AGAGTTTGATCCTGGCTCAG-3', 1492R：5'-GGTTACCTTGTTACGACTT-3'.PCR reacts bar Part：95 DEG C of pre-degeneration 3min；94 DEG C of denaturation 1min, 56 DEG C of annealing 1min, 72 DEG C of extension 2min, react and carry out 30 circulations；Most 72 DEG C of extension 10min afterwards, 4 DEG C of terminations.PCR primer is transferred to Shanghai bioengineering Co., Ltd after purification using QIAquick Gel Extraction Kit Its sequence is determined, then submits ncbi database to carry out BLAST sequence and compares analysis.
After bacterial strain KF-1 cultivates 4d on neutral red culture, periphery of bacterial colonies substantially reddens (Fig. 2), and thalline is in shaft-like, shape Into gemma (Fig. 3).Using bacterial strain KF-1 genomic DNA as template, after entering performing PCR amplification using 27F, 1492R universal primer, obtain About 1457bp 16S rDNA sequences are obtained, its 16S rDNA sequence is as shown in SEQ.ID.NO.1.The sequence by BLAST with Report that sequence carries out sequence analysis in NCBI GenBank databases, Blast comparison results are shown：KF-1 and withered grass gemma The homology of bacillus is up to 99%, and combining form feature, it is bacillus subtilis (Bacillus subtilis) to identify KF-1.
Embodiment 3：Degradation capabilities of the KF-1 to different greases
Bacillus subtilis KF-1 is inoculated in containing 1% lard, peanut oil, soybean oil, sesame oil by 1% inoculum concentration Oils degradation culture medium in, in 30 DEG C, 96h is cultivated under the conditions of 180rpm, according to method described in embodiment 1 calculate bacterial strain pair The degradation rate of various greases.The degradation rate of different greases of the KF-1 to being determined is as shown in Figure 4.As a result show, KF-1 is to each oil Fat has degradation capability, and its degradation effect to lard and peanut oil is substantially better than soybean oil and sesame oil.
Embodiment 4：Grease degrading strain KF-1 yielding lipase determinations of activity
Bacterial strain KF-1 is inoculated into beef extract-peptone nutrient solution and cultivates 18h, then with 1% inoculum concentration access producing enzyme training Support base, 30 DEG C, 180rmin–1, per 8h, the 000g of zymotic fluid 8 once, is centrifuged 10min, determines lipase in supernatant by sampling Enzyme activity, so as to draw out strain enzyme-producing curve.Lipase activity uses olive oil titration measuring.Fig. 5 results show, bacterial strain KF-1 is less in 0~32h producing enzymes, and enzyme activity significantly increases in 32h after fermentation liquid, and lipase active is most when cultivating 72h, in zymotic fluid Height, reach 42.31U/mL, higher than most of it has been reported that the yielding lipase bacterial strain crossed, this illustrates the bacterial strain yielding lipase ability It is stronger.
Embodiment 5：The preparation of KF-1 microbial inoculums and its application in livestock and poultry dead volume compost
The preparation of 1.KF-1 microbial bacterial agents
(1) preparation of seed liquor：The bacillus subtilis KF-1 of slant preservation is inoculated with a ring to beef extract-peptone liquid In body culture medium, 30 DEG C, 180r/min culture 10h to logarithmic phase.It is transferred to 1% inoculum concentration equipped with 100mL fermentation mediums 500mL triangular flasks in, 30 DEG C, 180r/min cultivate 24h as seed liquor；(2) fermented and cultured：Seed liquor is trained according to fermentation 3% inoculum concentration for supporting matrix fraction is inoculated into fermentation medium, 72h is cultivated under conditions of 35 DEG C, 180r/min, i.e., Bacillus subtilis KF-1 microbial inoculums can be obtained, bacillus subtilis KF-1 viable count is more than 1.0 × 10 in microbial inoculum9CFU/mL。
2. application of the microbial inoculum in livestock and poultry dead volume compost
Composting test is divulged information indoors, and greenhouse is interior to be carried out, 30 days altogether.Composting substrate presses sick dead pig and rice straw 3:1 (fresh weight mass ratio) is mixed, and heap body gross mass is 360kg, and it is about 25 to adjust C/N during compost:1, water content is about 50%. This experiment sets 2 processing：(1) (CK) is compareed：Do not add microbial inoculum；(2) test group (T1)：Add the micro- of composting substrate weight in wet base 1% Bacteria agent.Microbial inoculum is uniformly spread in raw material in windrow whipping process, and about 1.2m × 1.2m × 0.8m heap is piled after mixing Body, each processing repeat 3 heaps, and each detection project result takes the average value of 3 repetition heap bodies.Carried out within the 7th, 14,21 day in compost Turning, respectively in compost the 0th, 5,10,15,20,25,30d sample about 150g respectively in 3 points of top layer, middle level, bottom, It is well mixed.
Temperature measuring is in daily noon 11:30 determine heap body core temperature and environment temperature with mercurial thermometer respectively. In composting process, compost temperature change is the most directly perceived and most important index whether reflection fermentation is normally carried out.Fig. 6 results show Show, in whole composting process, the central temperature of compost material is above environment temperature, the heap temperature change of control group and treatment group Trend is basically identical.CK groups compost reach a high temperature the phase for the 8th day (50 DEG C of ＞), maximum temperature be 62 DEG C；And it with the addition of KF-1 Microbial inoculum group reaches a high temperature the phase on the 4th day in compost, and maximum temperature is 63 DEG C.The heap body high temperature (50 DEG C of ＞) of 2 different disposals maintains Time equal ＞ 10d, meet the innoxious requirement (GB 7959-2012) of During High-Temperature Composting, but the high temperature number of days (14d) of test group is big In the high temperature number of days (11d) of control group.This explanation adds a certain proportion of KF-1 microbial inoculums, can shorten compost and reach a high temperature the phase Time, extend megathermal period number of days, be advantageous to the harmless treatment of compost.
The measure of fat content uses soxhlet extraction.Fat content in composting process in each group compost as shown in fig. 7, With the progress of compost, the fat content of each group constantly reduces.During compost the 5th day, the fat content degraded of each group is less and right According to organizing, there was no significant difference (P ＞ 0.05) with the fat content of test group；At the end of compost, the oil content of control group is 19.98%, the degradation rate of grease is 24.8%, and the oil content of test group is reduced to 3.35%, and oil and grease degradation rate reaches 87.3%.It was found from oil and grease degradation rate, a certain proportion of KF-1 microbial inoculums are added in livestock and poultry dead volume compost can significantly reduce oil The content of fat.
The measure of seed germination index (GI):Take the fresh samples of 10g to add 100mL distilled water, vibrate 1h.5mL filtrates are drawn, are added Enter to being covered with the 9cm culture dishes of 2 filter paper, each culture dish is uniformly put into 20 radish seeds, light culture 48h at 30 DEG C, surveys Determine germination percentage and root long, while be used as and compareed by the use of distilled water.Its calculation formula is:GI=(percentage of seedgermination of compost treatment × Seed root long)/(percentage of seedgermination of control × seed root long) × 100%.
The seed germination index (GI) of composting production is one of most important index for evaluating compost maturity.Work as germination When index is more than 80%, it is completely decomposed to be considered as compost.From figure 8, it is seen that in compost 15d, the GI of T1 processing reaches 89.1%, and the GI of CK processing is still less than 80%；To compost, the seed germination index of CK, T1 processing is respectively 96.5%th, 114.2%.As a result illustrate, a certain proportion of KF-1 is added in compost can accelerate the decomposed of compost, improve compost The rotten degree of product.
SEQUENCE LISTING
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<120>One plant of grease degrading strain and its application
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Claims (6)

1. bacillus subtilis (Bacillus subtilis) KF-1 that one plant of preserving number is CGMCC No.14120.
2. the application of the bacillus subtilis KF-1 described in claim 1, it is characterised in that for grease of degrading.
3. application according to claim 2, it is characterised in that described bacillus subtilis KF-1 is prepared into microbial inoculum, Degraded animal fat or vegetable fat.
4. application according to claim 3, it is characterised in that the microbial inoculum for preparing bacillus subtilis KF-1 is used for livestock and poultry Dead volume compost maturity.
5. the application according to claim 3 or 4, it is characterised in that the preparation process bag of bacillus subtilis KF-1 microbial inoculums Include following steps：
(1) preparation of seed liquor：Slant strains are inoculated with a ring into beef extract-peptone fluid nutrient medium, 35 DEG C, 180r/ Min cultivates 10h to logarithmic phase, is transferred to 1% inoculum concentration in the 500mL triangular flasks equipped with 100mL fermentation mediums, 35 DEG C, 180r/min cultivates 24h as seed liquor；
(2) fermented and cultured：Seed liquor is inoculated into fermentation medium according to 3% inoculum concentration of fermentation medium volume fraction In, 72h is cultivated under conditions of 35 DEG C, 180r/min, i.e. acquisition bacillus subtilis KF-1 microbial inoculums, bacillus subtilis in microbial inoculum Bacterium KF-1 viable count is more than 1.0 × 109CFU/mL；
Fermentation medium components：Corn flour 20g, peptone 10g, MnSO40.2g, NaCl 0.5g, K2HPO40.12g, KH2PO4 0.25g, distilled water 1000mL, pH 7.0~7.2.
6. application according to claim 5, it is characterised in that the microbial inoculum for preparing bacillus subtilis KF-1 is used for livestock and poultry Detailed process during dead volume compost maturity is as follows：
Composting substrate presses sick dead pig and rice straw 3:1 fresh weight mass ratio is mixed, and composting substrate gross mass is 360kg, heap It is 25 that C/N is adjusted when fertile:1, water content 50%；The addition of microbial bacterial agent is the 1% of composting substrate weight in wet base；Microbial inoculum is in heap Uniformly it is spread in fertilizer ground substance raw material whipping process in raw material, 1.2m × 1.2m × 0.8m heap body is piled after mixing, in compost 7th, carry out turning within 14,21 days, terminate compost at the 30th day.
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CN113308404A (en) * 2021-06-01 2021-08-27 中农新科(苏州)有机循环研究院有限公司 Screening method of grease degrading strain and grease degrading product
CN116987641A (en) * 2023-08-07 2023-11-03 湖南省微生物研究院 Corrosion-promoting nitrogen-preserving composting microbial inoculum and preparation method and application thereof

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