CN110093300A - A kind of method of bacillus composite bacteria agent of degrading maize straws and preparation method thereof, degrading straw - Google Patents
A kind of method of bacillus composite bacteria agent of degrading maize straws and preparation method thereof, degrading straw Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- 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
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Abstract
The method that the present invention specifically discloses bacillus composite bacteria agent of a kind of degrading maize straws and preparation method thereof, degrading straw.Microbial inoculum of the present invention includes microorganism: bacillus amyloliquefaciens, secondary bigcatkin willow bacillus, bacillus licheniformis, bacillus.Microbial inoculum is made by being mixed, taming to microorganism in the present invention；And the synergistic effect by utilizing microorganism, the Utilization ability to complicated organic substrates is promoted, the organic matter of corn stover and the degradation efficiency of cellulosic are improved, treatment effect is stablized；Under normal temperature condition, which significantly improves the fermentation efficiency of corn stover in a short time, and corn stover degradation efficiency reaches 31.5% in five days, and stablizes in the level, the fast degradation processing and energy application suitable for agricultural stalk of the microbial inoculum.
Description
Technical field
The present invention relates to microbial inoculum preparation technical fields, and in particular to a kind of bacillus composite bacteria agent of degrading maize straws
And preparation method thereof, the method for degrading straw.
Background technique
China is exactly large agricultural country from ancient times, and agricultural crop straw annual output is up to 8.7 hundred million tons, since crop material is difficult to corruption
Solution, while modern life consciousness is gradually increased so that peasant is more willing to directly to use chemical fertilizer and is reluctant time-consuming and laborious to utilize stalk
Returning to the field, therefore the stalk of annual up to one third is directly burnt or is discarded, if this part stalk energy is efficiently utilized,
Alternative more than 6,000 ten thousand tons of coals, and become China's big problem urgently to be resolved how to efficiently use stalk resource.
China's stalk comprehensive utilization mainly includes five major class such as " fodder, base-material, turning sludge into fuel, raw material, Fertilizer Transformeds "
Tens kinds of technical methods consider that degradation returning to the field should become the main path of stalk comprehensive utilization from environment, economy etc..And
The above are cellulose, hemicellulose and lignin for the 80% of stalk total solids content, it is difficult to degrade, become and hinder stalk fertilizer
One of obstacle of application.Using the conventional physicals chemical method degrading straw such as photochemical degradating, hydrolysis, thermal degradation, throwing can be faced
Big, at high cost, secondary pollution problems are provided, and biological method degrading straw is compared to the former significant effect, economical and efficient.Stalk is raw
Returning to the field after object compost and degradation, it is possible to provide organic fertilizer abundant improves soil soil property, therefore, carries out crop using bioanalysis
The degradation of stalk and cropland application have very big application potential.
Currently the research about straw degradative microorganism focuses primarily upon fungi, bacterium and actinomyces etc., and bacterium has
Breeding it is fast, the period is short, is easy to the features such as industrial production that the research about Cellulase Producing Bacteria is made increasingly to cause to close extensively
Note.The bacterium with cellulose degradation ability isolated at present mainly includes hot rod Pseudomonas (Caldibacillus), false monospore
Pseudomonas (Pseudomonas), Cytophaga (Cytophaga), Erwinia (Erwinia), the raw thermophilic fiber bacterium of spore
(Sporacytophga), Cellfalcicula (Cellfacicula), bacillus (Bacillus), Cellulomonas
(Cellulomonas) etc..Wherein, bacillus G+ bacteria can produce gemma, to extraneous injurious factor
Resistance is strong, and its is widely distributed, very quickly can sprout and grow at a suitable temperature, is the height of biodegrade and fermentation
Microbe groups are imitated, can produce metabolism product abundant, are suitable for the application of in lignocellulose degradation and soil activation.
Summary of the invention
For current China for the processing mode of stalk mainly to burn or directly discard, to effective benefit of stalk
It is lower with rate, the significant wastage of resource is caused, it is a kind of for promoting corn stover to degrade it is an object of the invention to propose
Microbial inoculum and preparation method thereof, using from feces of livestock and poultry, sludge, soil mixture separation obtain and solution starch through Molecular Identification
Bacillus (Bacillus amyloliquefaciens strain m164b), secondary bigcatkin willow bacillus (Bacillus
Paralicheniformis strain Bac84), bacillus licheniformis (Bacillus licheniformis strain),
Bacillus (Bacillus sp.strain LA134), degrades to stalk using above-mentioned strain, can cropland application, change
Kind soil property, and the wasting of resources, environmental pollution can be avoided.
Because the mode of the processing of traditional stalk is not able to satisfy demand used in connection with, for further expansion use scope
It is restricted with field, is not able to satisfy relevant use demand；The present invention is by developing the gemma of degrading maize straws a kind of
Bacillus composite bacteria agent is not satisfactory to solve the problems, such as to efficiently use shortcoming in a manner of stalk and straw utilization rate now, can also be with
Meet the use demand of related fields.
To realize that above-mentioned target, the present invention are achieved by the following technical programs:
A kind of microbial inoculum for promoting corn stover to degrade, consists of the following compositions based on percentage: solution starch gemma bar
Bacterium 25%, secondary bigcatkin willow bacillus 25%, bacillus licheniformis 25% and bacillus 25%；The bacillus amyloliquefaciens
(Bacillus amyloliquefaciens strain m164b) deposit number ACCC 04304, the pair bigcatkin willow bacillus
(Bacillus paralicheniformis strain Bac84) deposit number ATCC 9945a, the bacillus licheniformis
(Bacillus licheniformis strain) deposit number ACCC 11080, the bacillus (Bacillus
Sp.strain LA134) deposit number ACCC 06386.
Preferably, the preparation method of the microbial inoculum for promoting corn stover to degrade, comprising the following steps:
S1. by bacillus amyloliquefaciens (Bacillus amyloliquefaciens strain m164b), secondary bigcatkin willow bud
Spore bacillus (Bacillus paralicheniformis strain Bac84), bacillus licheniformis (Bacillus
Licheniformis strain), bacillus (Bacillus sp.strain LA134) is seeded to LB Liquid Culture respectively
It is cultivated in base；
S2. exponential phase is grown to it, is combined by various compatibilities, obtain optimization mixed bacterial, and saved.
Preferably, the viable count in the microbial inoculum is 1.0 × 108-1.0×109CFU/mL。
It is highly preferred that described is further, the LB culture medium the preparation method comprises the following steps:
Tryptone 10g, yeast extract 5g, NaCl 5g are settled to 1000ml, adjust pH to 7, then wet at 121 DEG C
Heat sterilization 25 minutes, it should be noted that if solid medium, then add the agar of 15-20g/L.
Preferably, the condition of culture of the culture medium are as follows: under the conditions of 37 DEG C of ± 2 DEG C of temperature, 180 ± 10r/min shaking table
Cultivate 24~36h.
Preferably, the work of the bacillus amyloliquefaciens, secondary bigcatkin willow bacillus, bacillus licheniformis and bacillus
The ratio between bacterium number is 1:1:1:1.
Preferably, described save as will mix by the glycerol that steam sterilizing concentration is 70-90% with bacterium solution, be put in-
It is saved in 20 DEG C of refrigerators.
Preferably, the strain is cultivated comprise the steps of: in the medium
S1. liquid fermentation medium is prepared, composite bacteria agent is inoculated on liquid fermentation medium, obtains fermentation liquid；
S2. a certain proportion of stalk, degrading straw will be added in the fermentation liquid being prepared.
Preferably, the liquid fermentation medium is inoculated with 10% composite bacteria agent, with volume fraction.
Preferably, the additive amount of the stalk in the fermentation liquid is that 2g stalk is added in every 100ml fermentation liquid.
It is highly preferred that the liquid fermentation medium is the preparation method comprises the following steps: by MgSO4·7H2O 0.5g, KNO30.5g,
Na2HPO41.8g, KH2PO40.3g, NaC l 5g add water to be settled to 1000ml, 121 DEG C moist heat sterilization 25 minutes.
Specific embodiment
The present invention is further elaborated combined with specific embodiments below, the embodiment is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.Test method as used in the following examples is routine unless otherwise specified
Method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
Embodiment 1
A kind of microbial inoculum for promoting corn stover to degrade, consists of the following compositions based on percentage: solution starch gemma bar
Bacterium 25%, secondary bigcatkin willow bacillus 25%, bacillus licheniformis 25% and bacillus 25%；The bacillus amyloliquefaciens
(Bacillus amyloliquefaciens strain m164b), the pair bigcatkin willow bacillus (Bacillus
Paralicheniformis strain Bac84), bacillus licheniformis (the Bacillus licheniformis
Strain), the bacillus (Bacillus sp.strain LA134).
A kind of preparation method of the microbial inoculum for promoting corn stover to degrade comprising the steps of:
S1. original mixed flora source is added in stalk powder culture medium according to weight percent 10% and carries out mixing training
After supporting 15 days, sampling is diluted to 10-1To 10-6Different gradients take 0.1ml to be applied on straw powder Selective agar medium in 37 respectively
DEG C ± 2 DEG C of 2~3d of culture, select growing way is good on straw powder Selective agar medium single colonie repeatedly plate streaking culture until obtaining
Obtain pure culture.Obtained strain is respectively bacillus amyloliquefaciens (Bacillus amyloliquefaciens through Molecular Identification
Strain m164b), secondary bigcatkin willow bacillus (Bacillus paralicheniformis strain Bac84), lichens bud
Spore bacillus (Bacillus licheniformis strain), bacillus (Bacillus sp.strain LA134).
S2. use LB culture medium by bacillus amyloliquefaciens (Bacillus amyloliquefaciens strain
M164b), secondary bigcatkin willow bacillus (Bacillus paralicheniformis strain Bac84), bacillus licheniformis
(Bacillus licheniformis strain), bacillus (Bacillus sp.strain LA134) live respectively
Change, expand culture.Wherein, LB culture medium includes tryptone 10g/L, NaCl 10g/L, yeast extract 5g/L, remaining be from
Sub- water.Culture medium passes through 121 DEG C, 20 minutes high-temperature sterilizations.It should be noted that if being solid medium, then 15- is added
The agar of 20g/L.
S3. by bacillus amyloliquefaciens, secondary bigcatkin willow bacillus, bacillus licheniformis and the gemma after expanding culture
The mixing of bacillus bacterium solution obtains the microbial inoculum for promoting corn stover to degrade.Viable count through plate count, in the microbial inoculum
It is 2.2 × 108CFU/mL, wherein the bacillus amyloliquefaciens, secondary bigcatkin willow bacillus, bacillus licheniformis and gemma bar
The ratio between viable count of bacterium is 1:1:1:1.
S4. it will be mixed for 50% glycerol with bacterium solution by steam sterilizing concentration, be put in -20 DEG C of refrigerators and save；It uses
When take bacterium solution 2% to activate rejuvenation in LB culture medium, put into production use.
Embodiment 2
A kind of microbial inoculum for promoting corn stover to degrade, consists of the following compositions based on percentage: solution starch gemma bar
Bacterium 25%, secondary bigcatkin willow bacillus 25%, bacillus licheniformis 25% and bacillus 25%；The bacillus amyloliquefaciens
(Bacillus amyloliquefaciens strain m164b), the pair bigcatkin willow bacillus (Bacillus
Paralicheniformis strain Bac84), bacillus licheniformis (the Bacillus licheniformis
Strain), the bacillus (Bacillus sp.strain LA134).
A kind of preparation method of the microbial inoculum for promoting corn stover to degrade comprising the steps of:
S1. by MgSO4·7H2O 0.5g, KNO30.5g, Na2HPO41.8g, KH2PO40.3g, NaCl 5g add water fixed
Hold to 1000ml, 121 DEG C obtain liquid fermentation medium in moist heat sterilization 25 minutes, and composite bacteria agent is inoculated with according to 10% ratio
Onto liquid fermentation medium, fermentation liquid is obtained.
S2. 2g stalk is added in every 100ml fermentation liquid, the stalk is dry to constant weight.At room temperature in 180 ± 10r/min
Degrading straw under shaking table speed.
S3. it ferments two days later, more apparent muddiness occurs in culture medium, it can be seen that fraction stalk is broken；To third
It when, clearly, stalk has been broken into filiform to culture medium turbidity；When by the 5th day, most straw degradatives
At bits shape, at this point, blank control group remnants stalk dry weight is 1.85g, and composite bacteria agent treated experimental group remnants stalk is dry
Weight is 1.37g, and straw degradative rate is 31.5%, is suitable for corn stover original position returning to the field, and the stalk fermentation liquid ingredient after degradation
It is abundant, it can be used for fertilizer development.
One, straw degradative is tested
Test group: the microbial inoculum of the culture of embodiment 2 is degraded as test group；Control group: solution starch bud is taken respectively
Spore bacillus, secondary bigcatkin willow bacillus, bacillus licheniformis and bacillus individually respectively degrade to stalk.
It is as follows that the single bacterial strain of 4 kinds of microbial inoculums of control group or the combination of other bacterial strains carry out condition of culture:
S1. by MgSO4·7H2O 0.5g, KNO30.5g, Na2HPO41.8g, KH2PO40.3g, NaCl 5g add water fixed
Hold to 1000ml, 121 DEG C of moist heat sterilizations, 25 minutes liquid fermentation mediums, by any four single bacterium (viable count 2.2 ×
108CFU/mL), any four bacterial strain combines (viable count 2.2 × 108CFU/mL) liquid fermentation is inoculated into according to 10% ratio
In culture medium, fermentation liquid is obtained.
S2. 2g stalk is added in every 100ml fermentation liquid, the stalk is dry to constant weight.At room temperature in 180 ± 10r/min
Degrading straw under shaking table speed.(test result is as shown in table 1).
The test of 1 straw degradative of table
The test of 2 straw degradative of table
| Other bacterial strains combine straw degradative rate maximum value | 23.3% |
By the test data in Tables 1 and 2 it is found that the microbial inoculum prepared through the invention for stalk degradation rate highest,
Degradation effect is preferable, and treatment effect is stablized.The microbial inoculum is added in reaction system, and corn stover degradation efficiency reaches
31%, and it is stably maintained at the level, effectively degradation can be carried out to stalk and is utilized, and avoid the waste of stalk resource, and be better than
The exclusive use of this 4 kinds of bacterial strains or other combination straw degradative rates, there is preferable degradation effect.
It can be seen that provided by the present invention for promoting the microbial inoculum of corn stover degradation that can effectively solve crop material hardly possible
The technical issues of to degrade.The microbial inoculum makes reaction system at normal temperature and can reach higher position reason effect.The microbial inoculum is thrown
It is added in reaction system, using the synergistic effect between microorganism, improves the degradation efficiency of corn stover, and degradation rate stablizes maintenance
In higher level, the whole service stage carries out at room temperature.The microbial inoculum is particularly suitable for maize straw resourceization and utilizes field.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected
The limitation of range is protected, although explaining in detail referring to preferred embodiment to the present invention, those skilled in the art are answered
Work as understanding, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the reality of technical solution of the present invention
Matter and range.
Claims (10)
1. a kind of microbial inoculum for promoting corn stover to degrade, which is characterized in that including following component based on percentage: solution starch
Bacillus 25%, secondary bigcatkin willow bacillus 25%, bacillus licheniformis 25% and bacillus 25%；The solution starch gemma
Bacillus (Bacillus amyloliquefaciens strain m164b) deposit number ACCC 04304, the pair bigcatkin willow gemma
Bacillus (Bacillus paralicheniformis strain Bac84) deposit number ATCC 9945a, the lichens gemma bar
Bacterium (Bacillus licheniformis strain) deposit number ACCC 11080, the bacillus (Bacillus
Sp.strain LA134) deposit number ACCC 06386.
2. a kind of preparation method of microbial inoculum for promoting corn stover to degrade described in claim 1, which is characterized in that including
Following steps:
S1. by bacillus amyloliquefaciens (Bacillus amyloliquefaciens strain m164b), secondary bigcatkin willow gemma bar
Bacterium (Bacillus paralicheniformis strain Bac84), bacillus licheniformis (Bacillus
Licheniformis strain), bacillus (Bacillus sp.strain LA134) is seeded to LB Liquid Culture respectively
It is cultivated in base；
S2. exponential phase is grown to it, is combined by various compatibilities, obtain optimization mixed bacterial, and saved.
3. the microbial inoculum according to claim 1 for promoting corn stover to degrade, which is characterized in that the work in the microbial inoculum
Bacterium number is 1.0 × 108-1.0×109CFU/mL。
4. a kind of bacterial preparation process for promoting corn stover to degrade according to claim 2, which is characterized in that institute
State the condition of culture of culture medium are as follows: under the conditions of 37 DEG C of ± 2 DEG C of temperature, 180 ± 10r/min shaking table culture, 24~36h.
5. a kind of bacterial preparation process for promoting corn stover to degrade according to claim 2, which is characterized in that institute
The ratio between viable count of the bacillus amyloliquefaciens, secondary bigcatkin willow bacillus, bacillus licheniformis and bacillus stated is 1:1:1:
1。
6. a kind of bacterial preparation process for promoting corn stover to degrade according to claim 2, which is characterized in that institute
Saving as stating will be mixed by the glycerol that steam sterilizing concentration is 70-90% with bacterium solution, is put in -20 DEG C of refrigerators and is saved.
7. a kind of bacterial preparation process for promoting corn stover to degrade according to claim 2, which is characterized in that institute
The strain stated is cultivated comprise the steps of: in the medium
S1. liquid fermentation medium is prepared, composite bacteria agent is inoculated on liquid fermentation medium, obtains fermentation liquid；
S2. a certain proportion of stalk, degrading straw will be added in the fermentation liquid being prepared.
8. a kind of bacterial preparation process for promoting corn stover to degrade according to claim 7, which is characterized in that institute
The liquid fermentation medium stated is inoculated with 10% microbial inoculum, with volume fraction.
9. a kind of bacterial preparation process for promoting corn stover to degrade according to claim 7, which is characterized in that institute
The additive amount for the stalk in fermentation liquid stated is that 2g stalk is added in every 100ml fermentation liquid.
10. a kind of preparation method of microbial inoculum for promoting corn stover to degrade according to claim 7, includes following step
It is rapid:
S1. by MgSO4·7H2O 0.5g, KNO30.5g, Na2HPO41.8g, KH2PO40.3g, NaCl 5g, add water to be settled to
1000ml, 121 DEG C obtain liquid fermentation medium in moist heat sterilization 25 minutes, and composite bacteria agent is inoculated into liquid according to 10% ratio
On body fermentation medium, fermentation liquid is obtained；
S2. 2g stalk is added in every 100ml fermentation liquid, the stalk is dry to constant weight.At room temperature in 180 ± 10r/min shaking table
Degrading straw under speed；
S3. it ferments 4~6 days, obtains stalk fermentation liquid.
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| CN111676163B (en) * | 2020-06-18 | 2022-04-29 | 浙江工业大学 | Microbial agent for high-temperature biodegradation of kitchen waste and application thereof |
| CN113151098A (en) * | 2021-05-08 | 2021-07-23 | 齐鲁工业大学 | Alkali-resistant composite microbial pretreatment microbial inoculum for wheat straw pulping and application thereof |
| CN113817639A (en) * | 2021-09-26 | 2021-12-21 | 黑龙江省农业科学院耕作栽培研究所 | Efficient corn straw degradation composite microbial inoculum, and preparation method and application thereof |
| CN114045234A (en) * | 2021-11-03 | 2022-02-15 | 哈尔滨工业大学 | Bacterial liquid for promoting aerobic fermentation of corn straw to produce heat, preparation method and application |
| CN115043674A (en) * | 2022-05-24 | 2022-09-13 | 广东省科学院生物与医学工程研究所 | Method for quickly decomposing and drying fresh banana straws |
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