CN108865927A - The bacterium bacterial strain and its fermentation culture method of low temperature glycolysis corn stover and application - Google Patents
The bacterium bacterial strain and its fermentation culture method of low temperature glycolysis corn stover and application Download PDFInfo
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Abstract
The invention discloses the bacterium bacterial strain of low temperature glycolysis corn stover and its fermentation culture method and applications.The present invention discloses the bacillus pumilus bacterial strain of one plant of separation first, and microbial preservation number is：CGMCC No.15177.The related parameter of the fermentation preparation of separated bacillus pumilus bacterial strain is optimized in the present invention.The separated bacillus pumilus of the present invention can all be grown at different temperatures, under 4 DEG C of cryogenic conditions, still have vigorous growing power.The separated bacterial strain of the present invention can efficient glycolysis stalk under cryogenic, glycolysis stalk can be united and applied in individually or with EM bacterium.Glycolysis corn stover, stalk glycolysis effect are in be obviously improved at low temperature after the fermentation liquid of bacterial strain of the present invention is mixed with EM bacterium solution, and glycolysis rate improves 47.4%.The separated bacillus pumilus bacterial strain of present invention application prospect in preparing straw decomposing inoculant, especially corn stover decomposing agent is extensive.
Description
Technical field
The decomposed bacterium bacterial strain of cold ground corn stover separated the present invention relates to one plant, further relates to the cold ground straw decomposing
Application in bacterium bacterial strain glycolysis corn stover efficient at low temperature belongs to separation and the application neck of cold ground straw decomposing bacterium
Domain.
Background technique
The nutrition such as agricultural crop straw cellulose rich in, hemicellulose, lignin, protein and mineral matter element
Substance is a kind of reproducible living resources.But Chinese straw utilization rate is less than 33%, mainly since stalk is in Tanaka's hardly possible
In collection, transport is inconvenient, most of to be incinerated or waste treatment.Heilongjiang Province is the big province of Chinese agriculture, every annual output stalk quantity
Huge, these stalks are since environment temperature is low, returning to the field condition and popularization degree be not high, and many stalks are incinerated or discard, not only
Valuable crop genetic resource is wasted, huge pollution also is caused to environment.Stalk is mainly rich in cellulose, hemicellulose and wood
Quality, many fungies, bacterium and actinomyces etc. can eccrine fiber element enzyme, hemicellulase and lignoenzyme and decomposing straws.By
The content of cellulose highest in stalk, therefore, screening, there is the microorganism of high-cellulose enzyme Enzymatic characteristic to become research stalk corruption
Ripe dose of one of important directions.
EM bacterium is by photosynthetic bacteria group, lactobacillus, yeast flora, the fermentation multiple-microorganisms group such as der Pilz and actinomyces
At a profitable strain, application when have many advantages, such as that at low cost, application method is easy, in agriculture field, animal husbandry
Field, aquatic products field and water pollution field etc. are widely used.Many test results have in verified EM bacterium efficient
The microorganism of decomposing straw cellulose, and these microorganisms also can normally play a role under cryogenic, therefore, benefit
Decomposed stalk bacterium coldly is screened with EM bacterium filter out and be capable of the high efficient strain of decomposed stalk under cryogenic, will tremble with fear for increase
The strain of ground straw decomposing microbial inoculum forms and the application of straw decomposing inoculant provides practical basis and theoretical foundation.
Summary of the invention
First technical problem to be solved by this invention is to provide the efficient cryogenic glycolysis corn stover of one plant of separation
Bacterium bacterial strain；
Second technical problem to be solved by this invention is to provide the cold ground straw decomposing bacterium bacterial strain at low temperature
Application in efficient glycolysis corn stover.
In order to solve the above technical problems, the technical solution adopted by the present invention is that：
The present invention obtains 23 bacterium by carrying out 6 separation and purifying to the microorganism in cold ground straw decomposing object altogether
Strain；Under different temperatures condition of culture, 26 bacterial strains of separation are cultivated, observe its growing state, carry out low temperature stalk
The primary dcreening operation of decomposed bacterium, filter out can under 15 DEG C of cryogenic conditions normal growth bacterium be 7 plants, respectively A3, B7, C1, C4,
D7, E5 and E14.The present invention further to be just sieved to can under cryogenic normal growth 7 plants of bacterium bacterium carry out secondary screening,
Observation screening obtains the ratio between transparent loop diameter and colony diameter (D/d) size in the Congo red solid medium of CMC- of bacterium, knot
Fruit discovery, B7 plants have the ratio between biggish transparent loop diameter and colony diameter (D/d), are 2.68；In view of transparent circle size one
Determine that the ability that bacterial strain generates cellulase can be embodied in degree, illustrates that screened bacterial strain B7 has stronger cellulase
Ability.
The straw decomposing bacterium B7 screened is carried out bacterium colony to the present invention and thalli morphology is observed.As a result, it has been found that this is thin
Bacterium can form single bacterium colony on solid medium.The bacterium colony of bacterial strain B7 is with yellow, translucent, smooth wet, side
Edge is irregular, and the circular colonies slightly swelled, thallus is thin rod shape, and circle end individually or in short chain arranges, is rendered as gram
The positive, by observing above, the preliminary straw decomposing bacterium bacterial strain B7 for inferring screening is bacillus.According to phylogenetic tree
As a result, determine B7 be bacillus pumilus (bacillus pumilus), which is named as bacillus
pumilus-B7。
The Bacillus pumilus-B7 bacterial strain that the present invention obtains screening carries out Identification of Cold Tolerance, as a result, it has been found that the bacterium
Strain can be grown at different temperatures, under the conditions of 4 DEG C, still had vigorous growing power, shown that the decomposed bacterial strain has
Stronger cold resistance.Morita point out feature that psychrotrophs have be 0-5 DEG C can growth and breeding, the most suitable growth temperature
For degree at 15 DEG C or more, maximum growth temperature is higher than 20 DEG C.Therefore, the present invention screens the Bacillus pumilus-B7 bacterium obtained
Strain is psychrotrophs, can be in northern low temp area normal growth.
The mechanism that the Bacillus pumilus-B7 bacterial strain that the present invention obtains separation submits patent to approve carries out preservation,
Its microbial preservation is numbered：CGMCC No.15177；Classification naming is：Bacillus pumilus Bacillus pumilus.It protects
Hide unit：China Committee for Culture Collection of Microorganisms's common micro-organisms center；The preservation time is on January 11st, 2018；It protects
Hide address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The preparation of the fermentation liquid of Bacillus pumilus-B7 bacterial strain of the present invention includes：By the Bacillus
Pumilus-B7 strain inoculated is cultivated in fluid nutrient medium, collect bacterium solution to get.
Wherein, the fluid nutrient medium Ke Yi Wei He Qixunshi minimal medium is as basic culture medium, the nitrogen
Source can be peptone, yeast extract, ammonium sulfate or urea, additive amount 0.2wt%；Preferably, the nitrogen source is albumen
Peptone；By volume percentage, the inoculum concentration of the bacterial strain can be 1-9%, it is preferred that the inoculum concentration of bacterial strain is 5%；It is described
Cultivation temperature can be 10-40 DEG C, preferably 25 DEG C；The initial pH value of fluid nutrient medium can be 4-10, preferably 7；It is described
Time of fermented and cultured can be 1-7d, preferably 3d.
The optimum nitrogen source screening experiment of fermentation medium is found, using peptone and yeast extract as in nitrogen source medium
The activity of cellulase-producing is higher, and the activity of cellulase is generated on addition ammonium sulfate and urea crop nitrogen source medium
It is lower, and difference is very significant.Comprehensive comparison is suitable for cold ground straw decomposing bacterium Bacillus pumilus-B7 bacterium
The culture optimum nitrogen source of strain cellulase-producing is peptone.
The optimum inoculation amount the selection result of fermentation finds that the difference of inoculum concentration will affect strain liquid fermentation and produce cellulose
The activity of enzyme, with the increase of inoculum concentration, the activity of straw decomposing bacteria-produced cellulase, which is presented, first to be risen, and what is declined afterwards becomes
Gesture, the bacterium all have higher fiber element enzymatic activity.When inoculum concentration is 1% and 3%, producing enzyme and enzyme activity are gradually increased.Inoculation
After amount is more than 5%, enzyme activity is begun to decline, it may be possible to which since bacterial content is excessive, nutriment is limited in environment, thalli growth
A large amount of consumption nutriments and enzymatic productivity declines.Comprehensive comparison, optimum inoculation amount 5%.
Fermentation temperature screening experiment as a result, it has been found that, bacterial strain Bacillus pumilus-B7 is the enzyme at 10-30 DEG C of temperature
Vigor gradually increases, enzyme activity highest when to 30 DEG C, but the vigor of producing enzyme, with 25 DEG C of ratios, difference is not significant.With temperature liter
Height, producing enzyme vigor are begun to decline, and at 40 DEG C, the producing enzyme vigor of bacterial strain all drops to minimum.It can be seen from these results
The bacterial strain of this experiment sieving has a very wide inulinase-producing activity section, and low temperature is more advantageous to its producing enzyme.Comprehensive comparison,
The optimum temperature for determining the strain fermentation is 25 DEG C.
The screening experiment of culture medium optimal initial pH value as a result, it has been found that, in peracid, cross straw decomposing bacterium in the environment of alkali
CMC enzyme activity it is very low, or even not producing enzyme.When pH is 5 to 8, decomposed bacterium has inulinase-producing activity, in the initial pH of culture medium
When being 7, there is highest CMC enzyme activity, when pH is increased to 8, conspicuousness decline all occurs in the activity of producing enzyme.These knots
Fruit shows that the low temperature straw decomposing bacterial strain of this experiment sieving is more suitable for the producing enzyme under acid and neutrallty condition, determines best training
Supporting base initial pH value is 7.
The screening experiment of best incubation time as a result, it has been found that, the inulinase-producing activity of bacterial strain is presented with the extension of incubation time
First rise, rear downward trend.When cultivating 3d, all there is best inulinase-producing activity, CMC enzyme activity gradually drops after 3d
It is low, but still there is stronger enzymatic activity.The best incubation time for determining cold ground straw decomposing bacterial fermentation is 3d.
In order to verify the glycolysis effect of screened efficient cryogenic glycolysis stalk bacterium bacterial strain Bacillus pumilus-B7
Fruit, the present invention carry out filter paper glycolysis test using decomposed bacterium.According to filter paper glycolysis test result as it can be seen that the present invention is screened
Straw decomposing bacterium bacterial strain Bacillus pumilus-B7 have stronger filter paper glycolysis effect.
The present invention further carries out efficient cryogenic glycolysis stalk bacterium Bacillus pumilus-B7 and the cooperation of EM bacterium
The test of glycolysis stalk, when 30d, in two kinds of processing, there is weightless and blackening phenomena in stalk, but low being added to
In warm efficiently glycolysis stalk bacterium Bacillus pumilus-B7 combination, the degree of stalk weightlessness and blackening is bigger, and measurement is beautiful
The weight-loss ratio of rice stalk, the weight-loss ratio for being added to efficient cryogenic glycolysis stalk bacterium Bacillus pumilus-B7 combination are
56%, and the processing of EM fermented liquid is only used, the weight-loss ratio of stalk is 38%, and glycolysis rate improves 47.4%, and in stalk
Be added to efficient cryogenic glycolysis stalk bacterium Bacillus pumilus-B7 in digest process, ferment 30d when, what stalk rotted
Degree is preferable, easily broken.Separated Bacillus pumilus-B7 bacterial strain of the invention can at low temperature efficiently as a result,
Glycolysis stalk can be applied to glycolysis crop material, especially corn stover.
The present invention further discloses the Bacillus pumilus-B7 bacterial strains in preparing straw decomposing inoculant
Using.
The invention also discloses a kind of rice straw decomposing agents, including：Bacillus pumilus-B7 of the present invention
The fermentation liquid of bacterial strain.
The fermentation liquid of the separated Bacillus pumilus-B7 bacterial strain of the present invention can be applied individually to any glycolysis stalk,
It can also be matched with EM bacterium bacterium solution applied to glycolysis, stalk glycolysis rate will be significantly improved.
The invention also discloses a kind of corn stover decomposing agents, including：Bacillus pumilus-B7 of the present invention
The fermentation liquid and EM bacterium bacterium solution of bacterial strain.Preferably, the fermentation liquid of Bacillus pumilus-B7 bacterial strain and EM bacterium bacterium solution are pressed
According to 1:1 volume ratio composition.
The present invention is not particularly limited the EM bacterium bacterium solution, and commercially available EM bacterium bacterium solution is suitable for the present invention.
The separated Bacillus pumilus-B7 bacterial strain of the present invention can be grown at different temperatures, in 4 DEG C of items
Under part, still there is stronger cold resistance with vigorous growing power, it being capable of efficient glycolysis stalk under cryogenic；It can be with
Individually or glycolysis stalk is united and applied in EM bacterium；By the fermentation liquid of Bacillus pumilus-B7 bacterial strain with EM fermented liquid
Glycolysis corn stover, stalk glycolysis significant effect improve at low temperature after mixing, and glycolysis rate improves 47.4%.
Detailed description of the invention
Fig. 1 is the transparent circle of decomposed bacterium on the Congo red culture medium of CMC-；
Fig. 2 is the colonial morphology (A) and microscopic morphology (B) of decomposed bacterium；
Fig. 3 is the phylogenetic tree that bacterial strain B7 is constructed based on 16SrDNA sequence homology；
Fig. 4 is influence of the different nitrogen sources to strain enzyme-producing；
Fig. 5 is influence of the different vaccination amount to strain fermentation；
Fig. 6 is influence of the different temperatures to strain fermentation；
Fig. 7 is influence of the initial pH of different culture medium to 3 strain enzyme-producings；
Fig. 8 is the influence of different incubation times fermentation；
Fig. 9 is resolution ratio of the bacterial strain to filter paper.
Specific embodiment
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with describing
And it is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This
Field technical staff should be understood that without departing from the spirit and scope of the invention can be to technical solution of the present invention
Details and form are modified or are replaced, but these modifications or substitutions each fall within protection scope of the present invention.
The separation and identification of the cold ground of embodiment 1 straw decomposing bacterium
1. test method
1.1 test material
1.1.1 strain and stalk
EM bacterium bacterium solution used in this test is provided by Heilongjiang Academy of Agricultural Sciences.Corn stover picks up from Northeast Agricultural University
Experimental plot.
1.2 test method
1.2.1 ground stalk EM mycocriny of trembling with fear is ripe
In December, 2016, in Harbin City, Heilongjiang Province Northeast Agricultural University experimental plot, outdoor temperature at subzero 12 DEG C extremely
Between 25 DEG C, crushing straw and EM bacterium solution mixture are embedded in zanjon, covered using untreated stalk and plastic cloth, it is rotten
Ripe decomposition 60d.The straw decomposing object of taking-up is in state of rotting, accidental strip or the incomplete decomposed stalk fragment of fractionlet shape.
1.2.2 the separation of cold ground straw decomposing bacterium
1.2.2.1 in straw decomposing object bacterium bacterial strain separation, primary dcreening operation and preservation
(1) in decomposed object bacterium bacterial strain separation：It weighs 5g straw decomposing object and 50mL sterile water is added in conical flask,
37 DEG C are cultivated for 24 hours on shaking table.It is crossed, is separated on strain isolation culture medium with connecing collarium and dipping decomposed object suspension supernatant
Out after single colonie, bacterium is connect on LB agar medium, is saved.
(2) under cryogenic conditions in decomposed object bacterium bacterial strain primary dcreening operation：It can be in cryogenic conditions using cold ground straw decomposing bacterium
Lower growth carries out primary dcreening operation using bacterial strain of the different temperatures to concentration and separation.Using 4 DEG C, 10 DEG C, 15 DEG C, 25 DEG C and 37 DEG C isothermals
Degree condition, cultivates separated bacterial strain, observes strain growth situation, filter out can normal growth under cryogenic bacterial strain.
(3) preservation of bacterial strain：The single colonie isolated is classified, bacterium connects 37 DEG C of cultures in LB liquid medium
For 24 hours, in 50% glycerol after taking bacterial solution to be placed in high pressure sterilization, -80 DEG C of preservations.
1.2.2.2 the secondary screening of cold ground straw decomposing bacterium
It is screened using low temperature and obtains whether microorganism has the ability of decomposition of cellulose as foundation, to cold ground straw decomposing
Bacterium carries out secondary screening.The bacterium that can be grown under cryogenic that primary dcreening operation is obtained, is seeded on the Congo red culture medium of CMC-,
It cultivates to strain growth.Transparent circle size on culture medium is observed, transparent circle and bacterial strain diameter ratio (D/d) are calculated.Bacterial strain exists
Transparent circle size is able to reflect the big of bacterial strain decomposition of cellulose ability to a certain extent on the Congo red solid medium of CMC-
Small, transparent circle is bigger, and the ability of bacterial strain decomposition of cellulose is stronger.
1.2.3 the identification of cold ground straw decomposing bacterium
1.2.3.1 the Morphological Identification of cold ground straw decomposing bacterium
By secondary screening to bacterium bacterial strain be inoculated in LB solid and fluid nutrient medium respectively, for 24 hours afterwards observation bacterium colony appearance and
Thalli morphology carries out taxonomic identification to bacterium using Gram's stain.
1.2.3.2 the Molecular Identification of cold ground straw decomposing bacterium
From being inoculated in the LB culture medium that purified single bacterium has fallen on 30mL on LB plating medium, 37 DEG C are cultivated
For 24 hours, rotten as cold ground stalk using the 16SrDNA sequence universal primer 27F/541R of Bacteria Identification using the DNA of SDS bacterium
The Molecular Identification primer of ripe bacterium carries out PCR amplification, and PCR product to Harbin Bo Shi Bioisystech Co., Ltd is surveyed
Sequence.It in the sequence inputting GenBank that sequencing is obtained, is compared using BLAST, carries out sequence homology analysis, and use
MEGA5.0 software building phylogenetic tree carries out Molecular Identification to the kind of bacterium.
1.2.4 the Identification of Cold Tolerance of cold ground straw decomposing bacterium
It will screen and identify that obtain straw decomposing bacterium is inoculated in LB liquid medium and LB solid medium respectively early period
On, upgrowth situation of the record bacterial strain under 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C of condition of culture determines sieve
The cold resistance of the straw decomposing bacterium of choosing.
2. test result
2.1. the primary dcreening operation of cold ground straw decomposing bacterium
By carrying out 6 separation and purifying to the microorganism in cold ground straw decomposing object, 23 bacterial strains are obtained altogether.?
Under different temperatures condition of culture, isolated bacterial strain is cultivated, observes its growing state, carries out low temperature straw decomposing bacterium
Primary dcreening operation.The result shows that filter out can under 15 DEG C of cryogenic conditions normal growth bacterium be 7 plants, respectively A3, B7, C1,
C4, D7, E5 and E14.
The secondary screening of 2.2 cold ground straw decomposing bacteriums
To be just sieved to can under cryogenic normal growth 7 plants of bacterium bacterium carry out secondary screening, mainly by CMC- it is rigid
Transparent circle diameter on arnotto solid medium carries out secondary screening.Observation screening obtains the Congo red solid training of CMC- of bacterium
The ratio between transparent loop diameter and colony diameter (D/d) size in base is supported, wherein B7 has biggish transparent loop diameter and colony diameter
The ratio between (D/d), be 2.68, transparent circle size illustrate to a certain extent bacterial strain generate cellulase ability, illustrate bacterial strain B7
Ability with stronger cellulase (see Fig. 1).
The morphological observation of 2.3 cold ground straw decomposing bacterium
2.3.1 the morphological observation of decomposed bacterium
The straw decomposing bacterium B7 screened is cultivated on LB solid and fluid nutrient medium, observe bacterium colony and
Thalli morphology.The result shows that the bacterium can form single bacterium colony on solid medium.The bacterium colony of bacterial strain B7 is tool
There is yellow, translucent, smooth wet, edge is irregular, and the circular colonies slightly swelled, thallus is thin rod shape, circle end, individually
Or in short chain arrangement (see Fig. 2-A).It is rendered as Gram-positive (see Fig. 2-B), by observing above, preliminary deduction screening
Straw decomposing bacterium bacterial strain is all bacillus.
The Molecular Identification of 2.4 cold ground straw decomposing bacterium
It is analyzed by bacterial 16 S rDNA sequence, molecular biology identification is carried out to the decomposed bacterium B7 that screening obtains, wherein
The acquisition 16SrDNA sequence fragment length of amplification is 1449bp.
BLAST comparison and phylogenetic tree building are carried out to the sequence of acquisition, as a result as shown in Figure 3.According to systematic growth
Tree as a result, determine B7 be bacillus pumilus (bacillus pumilus), this result with front Morphological Identification have
There is consistency, which is named as bacillus pumilus-B7.
The Identification of Cold Tolerance of 2.5 cold ground straw decomposing bacterium
Identification of Cold Tolerance is carried out to the Bacillus pumilus-B7 bacterial strain that screening obtains.The decomposed bacterium of screening is connect
Kind is cultivated, the results showed that the bacterial strain can give birth at different temperatures in the culture medium of LB under different cultivation temperatures
It is long, under the conditions of 4 DEG C, still there is vigorous growing power, show that these decomposed bacterium have stronger cold resistance.Morita refers to
The feature that psychrotrophs have out be 0-5 DEG C can growth and breeding, optimum growth temperature is at 15 DEG C or more, highest growth
Temperature is higher than 20 DEG C.Therefore, this experiment sieving obtain decomposed bacterial strain be psychrotrophs, can northern low temp area just
It is frequently grown.
The straw decomposing bacterium fermentation condition optimization test of the cold ground of test example 1
1. test method
Influence of the different nitrogen source of 1.1 culture mediums to decomposed bacterial fermentation performance
In order to determine that the influence to the fermentation of decomposed bacterium of culture medium different nitrogen sources, this test use the He Qixun without nitrogen source
Family name's minimal medium adds different nitrogen sources, the nitrogen source of addition is peptone, ferment as basic culture medium on this basis
Female cream, ammonium sulfate and urea, additive amount 0.2%, each 4 bottles of processing, if 3 repetitions.Ferment 3d under the conditions of 25 DEG C, takes bacterium
Liquid 0.2mL is measured for cellulase activity, using DNS measuring method.
Influence of the 1.2 strain inoculum concentrations to decomposed bacterial fermentation
In order to observe the influence that different strain inoculum concentration ferments to decomposed bacterium, the different strain inoculum concentration of this experimental design.
1%, 3%, 5%, 7%, 9% 5 inoculum concentration of setting is handled.It is connect in the liquid fermentation medium using peptone as nitrogen source
Kind, 7.0,25 DEG C of initial pH value of medium cultures, each 5 bottles of processing, if 3 repetitions.When cultivating 3d under the conditions of 25 DEG C,
Bacterium solution 0.2mL is taken, is measured for cellulase activity.
Influence of 1.3 cultivation temperatures to decomposed bacterial fermentation
In order to observe the influence that different cultivation temperatures ferment to decomposed bacterium, the different strain culturing temperature of this experimental design.
Provided with 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C of totally 7 cultivation temperature processing.Using peptone as nitrogen source
It is inoculated with respectively with 5% inoculum concentration in liquid fermentation medium, initial pH value of medium 7.0, each 7 bottles of processing, if 3
It repeats.When cultivating 3 d, bacterium solution 0.2mL is taken, is measured for cellulase activity.
Influence of 1.4 initial pH value of medium to decomposed bacterial fermentation
In order to observe the influence that different culture medium initial pH value ferments to decomposed bacterium, the different culture medium of this experimental design
Initial pH value.Provided with 4,5,6,7,8,9,10 totally 7 different culture medium initial pH value processing.Using peptone as the liquid of nitrogen source
Body fermentation medium is basic culture medium, adjusts the initial pH of culture medium, is inoculated with 5% inoculum concentration, 25 DEG C of cultures, each place
7 bottles of reason, if 3 repetitions.When cultivating 3d, bacterium solution 0.2mL is taken, is measured for cellulase activity.
Influence of 1.5 incubation times to decomposed fungin enzymatic activity
In order to observe the influence that different incubation times ferment to decomposed bacterium, the different incubation time of this experimental design.Wherein
1d, 2d, 3d, 4d, 5d, 6d, 7d totally 7 different incubation time processing are arranged in bacterium, train by the liquid fermentation of nitrogen source of peptone
Base is supported, initial pH value of medium 7 is inoculated with 5% inoculum concentration, 25 DEG C of cultures, each 7 bottles of the processing of bacteria test, by experiment
After the incubation time culture of design, bacterium solution 0.2mL is taken respectively, is measured for cellulase activity.
2. experimental result
2.1 optimum nitrogen source the selection results
The optimum nitrogen source of the cold ground straw decomposing bacterial fermentation of screening is screened.As a result as shown in figure 4, by result
As can be seen that higher as the activity of cellulase-producing in nitrogen source medium using peptone and yeast extract, and in addition ammonium sulfate
It is lower with the activity of generation cellulase in urea crop nitrogen source medium, and difference is very significant.Comprehensive comparison is fitted
Culture optimum nitrogen source together in cold ground straw decomposing bacteria-produced cellulase is peptone.
Influence of 2.2 inoculum concentrations to fermentation
The optimum inoculation amount of the cold ground straw decomposing bacterial fermentation of screening is screened, as a result as shown in Fig. 5.By tying
The difference that fruit can be seen that inoculum concentration will affect the activity of strain liquid fermentation cellulase-producing, with the increase of inoculum concentration,
The activity of straw decomposing bacteria-produced cellulase, which is presented, first to be risen, and rear downward trend, the bacterium all has higher fiber element enzyme
Activity.When inoculum concentration is 1% and 3%, producing enzyme and enzyme activity are gradually increased.Inoculum concentration is more than after 5%, and enzyme activity is begun to decline,
It may be since bacterial content is excessive, nutriment is limited in environment, and thalli growth largely consumes nutriment and enzymatic productivity
Decline.Comprehensive comparison, optimum inoculation amount 5%.
Influence of 2.3 temperature to fermentation
As seen from Figure 6, bacterial strain Bacillus pumilus-B7 is that enzyme activity gradually increases at 10-30 DEG C of temperature,
Enzyme activity highest when to 30 DEG C, but the vigor of producing enzyme, with 25 DEG C of ratios, difference is not significant.As temperature increases, producing enzyme vigor starts
Decline, at 40 DEG C, the producing enzyme vigor of bacterial strain all drops to minimum.The bacterial strain of this experiment sieving it can be seen from these results
With a very wide inulinase-producing activity section, low temperature is more advantageous to its producing enzyme.Comprehensive comparison determines the strain fermentation
Optimum temperature is 25 DEG C.
Influence of the initial pH of 2.4 culture mediums to fermentation
As seen from Figure 7, in peracid, the CMC enzyme activity of crossing straw decomposing bacterium in the environment of alkali it is very low, or even do not produce
Enzyme.When pH is 5 to 8, decomposed bacterium has inulinase-producing activity, when the initial pH of culture medium is 7, has highest CMC enzyme activity
Power, when pH is increased to 8, all there is conspicuousness decline in the activity of producing enzyme.These results indicate that the low temperature of this experiment sieving
Straw decomposing bacterial strain is more suitable for the producing enzyme under acid and neutrallty condition, determines that optimal medium initial pH value is 7.
Influence of 2.5 incubation times to producing enzyme
As seen from Figure 8, the inulinase-producing activity of bacterial strain presents and first rises, what is declined afterwards becomes with the extension of incubation time
Gesture.When cultivating 3d, all there is best inulinase-producing activity, CMC enzyme activity gradually decreases after 3d, but still has stronger
Enzymatic activity.The best incubation time for determining cold ground straw decomposing bacterial fermentation is 3d.
The application effect test of the glycolysis stalk of the cold ground of test example 2 straw decomposing bacterium
1. test method
1.1 straw decomposing bacterium filter paper glycolysis test
At the beginning of taking the straw decomposing bacterium Bacillus pumilus-B7 screened to be inoculated in culture medium with 5% inoculum concentration
On the filter paper fluid nutrient medium that beginning pH is 7,15 DEG C of cultures.After inoculation, continuous to measure per the weight-loss ratio for measuring filter paper for 24 hours
8d.Each 8 bottles of processing, if 3 repetitions.The measuring method of filter paper weight-loss ratio is to take filtering fermentation liquor, and residuals are placed in baking
In case 80 DEG C drying to constant weight, and then measure filter paper weight-loss ratio.
1.2 efficient cryogenic glycolysis stalk bacteriums compound the test of glycolysis stalk with EM bacterium
Using the efficient cryogenic glycolysis stalk ferment product and EM fermented liquid 1 after fermentation:1 mixing, in 4 DEG C of conditions
Lower glycolysis corn stover.The processing method of corn stover：For 24 hours with the NaOH solution soaking corn stalk section of 2mol/L, it is washed to
PH=7,80 DEG C of drying box drying, the stalk section after drying crush 100 mesh sieving for standby.The composition of fermentation, according to fermented maize
Amount add 5%EM fermented liquid, 5% efficient cryogenic glycolysis stalk ferment product, 1% brown sugar, humid control is in 35%-
Between 65%, ferment 30d, is control with EM fermented liquid.After glycolysis, the weight-loss ratio of stalk is measured, measuring method is the same as filter
Paper weight-loss ratio method is identical.
2. test result
Glycolysis effect of 2.1 bacteriums to filter paper
In order to verify screening cold ground straw decomposing bacterium Bacillus pumilus-B7 glycolysis effect, use is decomposed
Bacterium carries out filter paper glycolysis test.After bacterial treatment, the resolution ratio of filter paper is as shown in Figure 9.It can be seen from the results that using
1~3d after Bacillus pumilus-B7 processing, with the extension of processing time, the weight-loss ratio of filter paper is significantly risen, after 3d
Slow ascendant trend is presented in the resolution ratio of filter paper, has reached highest in 15d, resolution ratio, can by result above up to 75% or more
To find out, the straw decomposing bacterium Bacillus pumilus-B7 of this experiment sieving has stronger filter paper glycolysis effect.
2.2 efficient cryogenic glycolysis stalk bacteriums and EM bacterium cooperate glycolysis stalk test result
Under cryogenic, using efficient cryogenic glycolysis stalk bacterium Bacillus pumilus-B7 and EM bacterium cooperate into
The glycolysis of row corn stover is tested, and when 30d, in two kinds of processing, weightless and blackening phenomena occurs in stalk, but
It is added in efficient cryogenic glycolysis stalk bacteria combination, the degree of stalk weightlessness and blackening is bigger, measures the weightlessness of corn stover
Rate, the weight-loss ratio for being added to efficient cryogenic glycolysis stalk bacteria combination is 56%, and only uses the processing of EM fermented liquid, stalk
Weight-loss ratio be 38%, glycolysis rate improves 47.4%, and efficient cryogenic glycolysis straw are added to during straw decomposing
Stalk bacterium, ferment 30d when, the rotten degree of stalk is preferable, easily broken.
Claims (10)
1. bacillus pumilus (bacillus pumilus) bacterial strain of one plant of separation, which is characterized in that its microbial preservation is compiled
Number it is：CGMCC No.15177.
2. a kind of method for preparing bacillus pumilus described in claim 1 (bacillus pumilus) bacterial strain fermentation liquor,
It is characterized in that, including：Strain inoculated described in claim 1 is subjected to fermented and cultured in fermentation medium, collects fermentation liquid,
To obtain the final product.
3. according to the method for claim 2, it is characterised in that：The nitrogen source of the fermentation medium be peptone, yeast extract,
Ammonium sulfate or urea, additive amount 0.2wt%；Preferably, the nitrogen source is peptone.
4. according to the method for claim 2, it is characterised in that：It will be by volume percentage, by bacterial strain described in claim 1
It is inoculated into fermentation medium and is cultivated according to the inoculum concentration of 1-9%, it is preferred that by bacterial strain described in claim 1 according to 5%
Inoculum concentration be inoculated into fermentation medium and cultivated.
5. according to the method for claim 2, it is characterised in that：The fermented and cultured temperature is 10-40 DEG C, preferably
25-30 DEG C, most preferably 25 DEG C.
6. according to the method for claim 2, it is characterised in that：The initial pH value of the fermentation medium is 4-10, preferably
For 5-8, most preferably 7.
7. according to the method for claim 2, it is characterised in that：The time of the fermented and cultured is 1-7d, preferably 3d.
8. a kind of decomposing agent of glycolysis stalk, which is characterized in that including：Bacillus pumilus described in claim 1
(bacillus pumilus) microbial inoculum or fermentation liquid and EM bacterium bacterium solution；Preferably, the volume ratio of the two is 1:1.
9. application of bacillus pumilus (bacillus pumilus) bacterial strain in glycolysis stalk described in claim 1.
10. application of the decomposing agent according to any one of claims 8 in glycolysis stalk.
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