CN103255077B - Bacillus subtilis YB-04 and microorganism preparation thereof, and application of bacillus subtilis YB-04 in straw degradation - Google Patents
Bacillus subtilis YB-04 and microorganism preparation thereof, and application of bacillus subtilis YB-04 in straw degradation Download PDFInfo
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- CN103255077B CN103255077B CN201210518981.XA CN201210518981A CN103255077B CN 103255077 B CN103255077 B CN 103255077B CN 201210518981 A CN201210518981 A CN 201210518981A CN 103255077 B CN103255077 B CN 103255077B
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- bacillus subtilis
- stalk
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- straw
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a strain of Bacillus subtilis YB-04 and a preparation method of Bacillus subtilis YB-04 agent, wherein the preservation number of the Bacillus subtilis YB-04 is CGMCC NO.6497. According to the present invention, the Bacillus subtilis YB-04 provides a significant straw degradation effect, provides significant bacterial inhibition effects on wheat root take-all diseases, sheath blight and the like, and presents broad-spectrum bacterial inhibition effects, wherein a degradation rate can be 49% when the Bacillus subtilis YB-04 separately acts on straws, when the Bacillus subtilis YB-04 and fungi mixedly act on straws to achieve moistening and rotten surface of the straw, a degradation rate achieves the maximal value 59%, and a degradation effect of the experiment combination is far superior to a degradation effect of the commercially available bacterial agent; and the Bacillus subtilis YB-04 agent further presents broad-spectrum bacterial inhibition effects, wherein the Bacillus subtilis YB-04 agent provides good bacterial inhibition effects for cotton verticillium dahlia, wheat take-all pathogen, wheat sheath blight and other rhizosphere plant diseases, straws can be degraded, rhizosphere pathogenic bacteria can be inhibited, rhizosphere microorganism community can be regulated, and good development utilization prospects are provided.
Description
Technical field
The present invention relates to a kind of microbial technology field, be specifically related to a kind of subtilis YB-04
,its microbial preparation and the application in straw degradative thereof.
Background technology
Straw-returning more and more comes into one's own in recent years, and on the one hand large-scale agriculture production need to be processed stalk nearby, to save labour; On the other hand, use merely the growth that chemical fertilizer is unfavorable for soil fertility for a long time.In agricultural crop straw, contain the organic nutrients such as abundant nitrogen ﹑ phosphorus ﹑ potassium, after straw-returning, will increase the content of the soil organism, improve native earth knot structure ﹑ culture fertility, have large quantity research to show after straw-returning, soil nutrient and organic content to be all improved.But under field conditions (factors), straw decomposition speed is slow, if field also in a large number, stalk is decomposition fully, can affect to broadcast kind of a quality ﹑ and emerge and seedling growth.Stalk also can exert an influence to second stubble crop for disease and pest provides the place of surviving the winter after covering.Along with the development of mechanize, straw-returning has become the integral part of advanced agriculture production, because stalk nature decomposition speed is excessively slow, straw-returning has brought a series of problem, and further investigation straw degradative, will fundamentally solve soil disease, crop straw burning, the problems such as pollution of agricultural products.Stalk fast degradation is that one realizes resource circulation utilization technology, has the potentiality of exploitation in agriculture Sustainable development.Microbial straw decomposition agent is the more direction of stalk fast degradation technical study, and stalk decomposition agent is mainly made up of the bacterial strain of strongly secreting straw degradative enzyme.Under the comprehensive action of microbial straw decomposition agent, cellulosic molecule is thoroughly disintegrated, after organism mineralization is decomposed, detritus forms organic matter then.Along with continuous researchdevelopment, the composite degradation bacteria strains of people not only can promote the fast degradation of stalk, simultaneously also to the future development that suppresses disease and pest.
Subtilis (
bacillus subtilis), have that growth and breeding is fast, nutrition is simple, can produce the contrary gemma of heat-resistant, good to person poultry harmless, environment compatibility, can suppress plurality of plant diseases, be difficult for making farm crop to develop immunity to drugs.The advantages such as and mass production processes is simple, cost is also lower, uses conveniently, and the shelf lives is long, some bacterial strain plays good effect in degrading straw, therefore, is a kind of desirable degraded preparation.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of subtilis YB-04 with the effect of antagonism take-all, and has provided concrete cultivation application method.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
Isolated a kind of subtilis YB-04, its deposit number is CGMCC NO.6497.
A kind of microbial preparation, is characterized in that, contained activeconstituents is that above-mentioned subtilis YB-04 is or/and its meta-bolites.
Described subtilis YB-04 is applied to straw degradative with liquid formulation, liquid bacterial preparation process: the subtilis YB-04 preserving is moved on TSA test tube slant substratum, cultivate at 28～35 DEG C and within 24～36 hours, obtain thalline; In bacterial classification access LB liquid medium in test tube, at 28～32 DEG C, concussion is cultivated and within 12～16 hours, is made seed liquor; Liquid fermentation medium is: get glucose, Semen Maydis powder, soybean cake powder, dipotassium hydrogen phosphate and add water and be mixed with the nutrient solution containing glucose 1.2%, Semen Maydis powder 1.8%, soybean cake powder 3%, dipotassium hydrogen phosphate 0.2%; After fermentation equipment and medium sterilization, seed liquor is inoculated in to the above nutrient solution preparing by 5% inoculum size, at 27～34 DEG C, rotating speed 180rpm, PH8.0 condition bottom fermentation was cultivated after at least 36～48 hours, collected fermented liquid, obtained liquid bacterial agent.
Described matrix substratum adds water and is mixed with by getting glucose, Semen Maydis powder, soybean cake powder, containing glucose 9～12g/L, Semen Maydis powder 11～16g/L, soybean cake powder 12～16g/L mass ratio make through high pressure moist heat sterilization after mixing.
The present invention has actively useful effect:
Isolated subtilis YB-04 CGMCC NO. 6497, has obvious degradation effect to stalk, and obvious to fungistatic effects such as wheat root gaeumannomyces graminis disease, banded sclerotial blights, finds that it shows the comparatively fungistatic effect of wide spectrum.Independent role is in the time of stalk, and degradation rate can reach 49%, and when during in stalk, making the more moistening corruption in stalk surface with fungi mixing effect, degradation rate has reached maximum value 59%, and the Degradation of experiment combination is far superior to the Degradation of commercially available microbial inoculum.Just the stalk after solution carries out content of cellulose mensuration, the content of cellulose of finding each test group all declines to some extent, in stalk after degrading with 3 kinds of fungi combinations, content of cellulose becomes 44.7%, Mierocrystalline cellulose in visible stalk is degraded in a large number, the one-piece construction of stalk is upset, thereby stalk obtains good degraded.
Subtilis YB-04 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 3rd, 2012, it is referred to as CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, the deposit number of this subtilis YB-04 is CGMCC NO.6497.
Brief description of the drawings
Fig. 1 is the colony characteristics figure of bacterial strain YB-04;
Fig. 2 is the molecular evolution tree graph of bacterial strain YB-04;
Fig. 3 is the design sketch of P3, P4, the independent degrading straw of P5;
Fig. 4 is P3, P4, the P5 design sketch respectively at YB-04, L8 combined degradation stalk;
Fig. 5 is the design sketch of P3, P4, P5 mixed degradation stalk;
Fig. 6 is the design sketch of P3, P4, P5 and YB-04, L8 mixed degradation stalk;
Fig. 7 is the design sketch of commercially available microbial inoculum 1 degrading straw;
Fig. 8 is the design sketch of commercially available microbial inoculum 2 degrading straws.
Embodiment
In order to make the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in the scope of the present invention of placing restrictions on for the present invention is described, NM specific experiment method in the following example, experimental technique carries out routinely conventionally.
Separation, the screening of embodiment 1 subtilis YB-04
Sample collecting: the corn that sample collecting is rotting from multiple fields, base, Yuanyang, academy of agricultural sciences of Henan Province and wheat stalk (acquisition time: 6-2010 October in 2010; Concrete collecting location: base, Yuanyang, academy of agricultural sciences of Henan Province, gathers people: Xie Maofang), take back with freshness protection package.
The isolation and purification of subtilis: get the rotten stalk of adopting, weigh 1g and put into the triangular flask that contains 100mL sterilized water, 2h fully vibrates on 28 DEG C of constant temperature oscillators.With dilution spread flat band method by 10
-1-10
-7sample liquid coat in LB flat board, 37 DEG C cultivate one day, on LB flat board, carry out respectively purifying according to colonial morphology.And substratum is inverted in to 37 DEG C of constant temperature culture spends the night, last picking form, color and luster, size and the similar single bacterium colony of subtilis are transferred on LB plate culture medium, after 37 DEG C of cultivation 2d, record its numbering, then select single colony inoculation and preserve to LB slant medium.
Table 1 has larger transparent circle bacterial strain
Separate through pedotheque microorganism, isolate altogether the similar bacterial isolates of 20 strain, be relatively total to obtain effect thin 3 strains preferably, called after YB-04, L4 and L8 respectively through repeated screening.This 3 strain bacterium all can vigorous growth in its corresponding screening culture medium, as shown in table 1, in starch degradation screening culture medium and proteolytic degradation screening culture medium, produce the hyaluronic circle that is greater than 20mm, show that YB-04 is rich in the enzyme of degraded starch and protein, has the potentiality of good degrading straw.
The qualification of embodiment 2 subtilis YB-04
(1)the qualification of physiology and morphology Biochemical Classification
?adopt colonial morphology to observe and microscopic observation method qualification YB-04 bacterial strain, and searching document is determined its kind.Individual morphological feature, comprises gramstaining anti-Ying ﹑ gemma Guan Cha ﹑ thalline size etc.; Colony's morphologic observation, observe Jun fall Xing Zhuan ﹑ bacterium colony Biao face Te Zheng ﹑ Jun fall Bian Yuan ﹑ bacterium colony color and whether secrete soluble pigment etc.; The biochemical examination of raw reason is tested ﹑ and is crossed oxide compound enzyme and survey and determine ﹑ V.P examination and test ﹑ sugar fermentation examination and test the examination of ﹑ Starch Hydrolysis and test ﹑ gelatin liquefaction ﹑ Citrate trianion profit ﹑ indoles and try to test ﹑ salt tolerance test etc.
(2)molecular Identification: extracting genome DNA is with reference to following steps: 1. cultivate the bacterial cultures of 5mL to state of saturation, get the centrifugal 1min of culture 12000rpm of 1.2mL.2. throw out adds the sterilized water of 500 μ L, repeatedly blows and beats and makes it resuspended with suction pipe.The Proteinase K that adds the 10%SDS of 30 μ L and the 20mg/ μ L of 10 μ L, mixes, in 37 DEG C of insulation 1h.3. the NaCL that adds 100 μ L5mol/L, fully mixes, then adds 80 μ LCTAB/NaCL solution, mixes, and bathes 10min in 60 DEG C of temperature.4. add the chloroform of 750 μ L: primary isoamyl alcohol (24:1), mix, leave standstill 5min, the centrifugal 5min of 12000rpm, gets supernatant.5. in supernatant, add isopyknic chloroform: primary isoamyl alcohol (24:1), extracting once again.6. draw the freezing Virahol that supernatant adds 0.6 times of volume.Mix gently until DNA precipitates 4 DEG C of standing 2-4h.7. the centrifugal supernatant of abandoning, 1mL75% alcohol washing for precipitation, centrifugal 5min, abandons supernatant, makes alcohol volatilization completely, is resuspended in 30 μ L deionized waters.Upstream and downstream primer for the 16Sr DNA sequence dna that increases is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, is respectively 16SF:5 ‵-AGAGTTTGATCATGGCICAG-3 ‵, 16SR: ‵-TAGGGTTACCTTGTTACGACTT-3 ‵.Taking genomic dna as template, utilize primer 16SF and 16SR to carry out pcr amplification.PCR reaction system is 25 μ l:10 × PCR Buffer 2.5 μ l, template 1 μ l, dNTP (10 mM each) 0.5 μ l, primer 16SF (10 mM) 1 μ l, 16SR (10 mM) 1 μ l, (5 U/ μ are 0.25 μ l l), adds deionized water to 25 μ l for archaeal dna polymerase.Pcr amplification condition: 94 DEG C of 5 min; 94 DEG C of 1 min; 55 DEG C of 30 sec; 72 DEG C of 1 min; 33 circulations; 72 DEG C of 10 min.PCR reaction product adopts 1% agarose gel electrophoresis to detect.1400bp size object band adopts DNA to reclaim test kit (AXYGEN company) and reclaims after purifying, be connected with T-carrier (Takara PMD-19T), transformed competence colibacillus cell, then on the flat board that contains Amp, IPTG, X-Gal, carry out blue hickie screening, and carry out bacterium colony PCR and filter out recombinant plasmid.Empirical tests is that the positive colony of object fragment is delivered to the biological company limited of the raw work in Shanghai and checked order.Sequencing result, by retrieval Genbank (http://www.nebi.nlm.nih.gov/Blast/), adopts BLAST to carry out sequence identity comparison.Meanwhile, download Bacillus strain 16SrDNA sequence, and use CLUSTAL X (Version 1.8) software to carry out contraposition arrangement, be aided with artificial check and correction, the sequence obtaining is used MEGA3.1 molecular evolution genetic analysis software analysis.Calculate on genetic distance basis in Kimura 2-parameter method, adopt adjacent method to build NJ phylogenetic tree, bootstrapping method of inspection (bootstrap) inspection for the degree of confidence of the each branch of genealogical tree, carries out 1000 circulations altogether.
(3) bacterial strain YB-04 morphological specificity: the bacterial strain of the better YB-04 of multiple sieve effect is cultivated to 1d in LB culture plate at 28 DEG C, observe its colonial morphology and color.YB-04 is well-grown on LB substratum, and bacterium colony is creamy white, and there are no pigment formation, colonial morphology is irregular, there is protuberance on surface, and forms irregular fold, (see figure 2).Cultivate after 24d and produce slight fishy odor, while provoking, bacterium colony becomes one and have mucus.Gramstaining (the G that is positive
+).
(4) Physiology and biochemistry of bacterial strain YB-04 qualification: with reference to the method for " the outstanding Bacteria Identification handbook of uncle (the 8th edition " and " common bacteria identification handbook ", measure and observe its some biochemical indexes.
From table 2, bacterial strain YB-04 can make gelatine liquefication, Starch Hydrolysis, nitrate reduction; Can make milk peptonize, can produce catalase.Can not make cellulose hydrolysis, glucose aerogenesis.Can utilize glucose, fructose, semi-lactosi, wood sugar, maltose, raffinose and N.F,USP MANNITOL.Bacterial strain YB-04 tolerance NaCl concentration is 7%, and in the time of pH<5 or pH>9, bacterial strain can not be grown, and Citrate trianion utilizes positive, the gelatine liquefication positive.According to " uncle outstanding Bacteria Identification handbook " (the 8th edition) and " common bacteria identification handbook ", the morphological specificity of YB-04 and physiological and biochemical property all meet subtilis (
bacillus subtilis) criteria for classification.
The physiological and biochemical property of table 2 bacterial strain YB-04
Note: "+": positive findings Positive results; “ – ": negative findings Negative results
(5) the Molecular Identification result of bacterial strain YB-04: correlated series in YB-04 sequence and GenBank database is carried out to BLAST analysis, result show 130 with the homologous sequence of this sequences match, choose the 16S rDNA sequence construct phylogenetic tree of the higher and representative bacterial strain of homology, as shown in Fig. 3, bacterial strain YB-04 with
bacillus subtilis(OYKN9W5701S) the same branch in evolutionary tree, homology is 100%.Comprehensive above-mentioned morphology, physiological and biochemical property and YB-04 and relevant bacterial strain 16SrDNA sequence homology analysis result are subtilis by YB-04 identification of strains
(Bacillus subtilis).
Embodiment 3 YB-04 microbiobacterial agent preparation methods
(1) strain culturing is inoculated into subtilis YB-04 on TSA test tube slant substratum with preparation, cultivates at 28～35 DEG C and within 24～36 hours, obtains thalline;
(2) seed liquor is prepared in test tube strains access TSB nutrient solution prepared by step (1), and at 28～32 DEG C, concussion is cultivated and within 12～16 hours, made seed liquor;
(3) fermention medium preparation is got respectively glucose, Semen Maydis powder, soybean cake powder, dipotassium hydrogen phosphate and is added water and be mixed with containing glucose 1.2%, Semen Maydis powder 1.8%, soybean cake powder 3%, dipotassium hydrogen phosphate 0.2% nutrient solution;
(4) liquid fermenting is produced after fermentation equipment and medium sterilization, seed liquor is inoculated in to the nutrient solution that step (3) prepares by 5% inoculum size, at 27～34 DEG C, rotating speed 180rpm, PH8.0 condition bottom fermentation was cultivated after at least 36～48 hours, collected fermented liquid, obtained liquid bacterial agent.
The enzyme activity determination of embodiment 4:YB-04 bacterial strain
The preparation of seed liquor: fungi is inoculated in respectively on PDA test tube slant, 28 DEG C of constant temperature culture to test tube slants cover with spore.Add 10mL sterilized water fully to shake, gained bacteria suspension is seed liquor.Microbionation is in being equipped with the test tube of 8mL LB nutrient solution, and 37 DEG C of constant temperature culture 24h gained bacteria suspensions are bacterium seed liquor.
Seed liquor is inoculated in liquid culture medium, and 37 DEG C, 180r/min ferments.After having fermented, by centrifugal fermented liquid 5000r/min 10min, gained supernatant is crude enzyme liquid.
Diastatic activity measuring method: according to document (Peng Huayuan, Lu Hongmei, Zeng Xiangqin. the mutagenesis screening of high yield zytase bacterial strain and product enzymatic property [J] thereof. brewing science and technology, 2006,10:34-36.) in test tube, add 1mL crude enzyme liquid, 70 DEG C of water-bath 15min add the citrate buffer of 1mL pH5.06 again, add the NaOH of 4mL 0.4moL/L in contrast.Test tube is incubated to 10min in 40 DEG C of water-baths, adds 2mL starch solution to shake up, put into immediately 40 DEG C of water-baths and react 5min, after taking-up, add rapidly the NaOH of 4mL 0.4moL/L with termination reaction.Extract reaction solution 2 mL and add 2 mL3,40 DEG C of water-bath 5min of 5-dinitrosalicylic acid, are settled to 25mL after cooling.Measure its light absorption value at 520nm place, reference standard curve calculation enzyme is lived.
The enzymic activity of the each bacterial strain of table 3
Protease activity measuring method: according to document (Xu Ying, what on National Day, Chen Qi and etc. the impact [J] of Buffer Systems on Elastase Production by Bacillus sp. EL 31410. Journal of Agricultural Biotechnology, 2004,1(6): 709-713.), to the crude enzyme liquid that adds 1mL in test tube, blank in contrast, is placed in 40 DEG C of water-bath preheating 5min, then 2% casein solution that adds 1mL, is accurately incubated 10min.After adding immediately the trichoroacetic acid(TCA) solution 15min of 2mL 0.4M, draw supernatant 1mL, add sodium carbonate solution and the 1mL fehling reagent of 5mL0.4M, 20min develops the color in 40 DEG C of water-baths.Under 680nm, measure light absorption value, reference standard curve calculation enzyme is lived.
The bacterial strain with larger transparent circle is carried out to enzymatic production, and enzyme activity determination the results are shown in Table 3.It is not 113.47 u/mL and 134.95 u/mL that visible YB-04 amylase and protease activity reach best result for second day in fermentation, higher than L4 and the L8 bacterial strain of same batch fermentation, can predict that YB-04 is rich in straw degradative enzyme, has the prospect of exploitation degraded preparation.
Embodiment 5:YB-04 degradation bacteria liquid fermenting stalk condition of enzyme production is optimized
Straw degradative process is mainly the digestibility and utilization process of the enzyme that produces of various bacterium to stalk, and therefore conventional enzymatic productivity is weighed the utilize ability of degradation bacteria strains to stalk, and the size of enzymatic productivity is generally weighed by the unit enzyme work size in fermented liquid.The present invention has studied the fermented stalk enzymatic productivity of bacterial strain YB-04 under liquid condition, has indicated the factor that affects these strain enzyme-producings.
(1) impact of fermentation time on degradation bacteria strains enzymatic productivity: fermentation time is one of factor affecting strain enzyme-producing, the fermentation initial stage is along with the enzyme of the prolongation fermented liquid of time is lived generally in rising trend; Along with exhausting and the Degradation of zymoprotein of nutritive substance, the enzyme of fermented liquid is lived and will be occurred decline.By 1% inoculum size, pH nature, shaking speed 180r/min, 37 DEG C are carried out fermentation culture.Once, METHOD FOR CONTINUOUS DETERMINATION 5d, studies enzyme that degradation bacteria strains produces relation over time alive in sampling in every 24h hour.
(2) impact of pH on degradation bacteria strains enzymatic productivity: different microorganisms has different tolerances to pH, therefore its product enzyme process is subject to the impact of pH larger.Bacterium generally adapts to growth and product enzyme under alkaline environment.The pH value of YB-04 strain enzyme-producing substratum is set to 5 levels (7 ﹑ 7.5 ﹑ 8 ﹑ 8.5 ﹑ 9).According to 1% inoculum size, 37 DEG C of 180r/min of shaking table carry out fermentation culture.The fermented liquid of getting fermentation 48h carries out enzyme activity determination, the enzyme work of research fermented liquid and the variation relation of pH.
(3) impact of temperature on degradation bacteria strains enzymatic productivity: temperature is also one of index affecting occurring in nature production by biological enzyme, the product enzyme temperature of the fungi of speaking more greatly is generally 30 DEG C of left and right, and the product enzyme temperature of bacterium is generally 37 DEG C of left and right.Fungi culture medium is set as respectively (24 ﹑ 26 ﹑ 28 ﹑ 32 ﹑ 34) 5 temperature levels, and bacterium culture medium is set as respectively (30 ﹑ 35 ﹑ 37 ﹑ 42 ﹑ 47) 5 temperature levels.According to 1% inoculum size, pH nature, 180r/min carries out fermentation culture.Fermentation is measured enzyme after 48h and is lived, and the enzyme of research fermented liquid is lived and the relation of leavening temperature.
(4) impact of inoculum size on degradation bacteria strains enzymatic productivity: the size of inoculum size affect bacterial classification colony number, thereby affect production of enzyme, but not inoculum size is the bigger the better.Be subject to the impact of dissolved oxygen and nutritive substance, study suitable inoculum size and just can make fermented liquid there is the highest enzyme work.On pH nature, 37 DEG C, 28 DEG C of bacteriums of fungi, under 180r/min condition, inoculum size is set to (0.5% ﹑ 1% ﹑ 1.5% ﹑ 2% ﹑ 2.5%) 5 levels, the enzyme of measuring after fermentation 48h is lived, and research fermentation broth enzyme is lived and the relation of inoculum size.
(5) impact of nitrogenous source on degradation bacteria strains enzymatic productivity: nitrogenous source is that microorganism growth is necessary, the C/N of stalk is the suitableeest C/N that 50:1 is far longer than microorganism growth, therefore in fermentation culture, will have certain nitrogenous source, different nitrogen sources also has different impacts to enzymatic productivity.Nitrogenous source in this research ferment product is that yeast soaks powder, uses respectively yeast extract paste, peptone, and Tryptones, substitutes.By 1% inoculum size, at pH nature, 37 DEG C, 28 DEG C of bacteriums of fungi, ferment under 180r/min condition, and the enzyme of measuring after fermentation 48h is lived, and research fermentation broth enzyme is lived and the relation of carbon source addition.
(6) interpretation of result: the fermented liquid of YB-04 bacterial strain is carried out to single factors optimization experiment of the aspects such as enzyme work, pH, temperature, inoculum size, nitrogenous source utilization, can find out: 1. YB-04, the fermentation of L1 ﹑ L8 bacterium reached product enzyme maximum value after 2 days, one-way analysis of variance finds that the enzyme after bacterial strain YB-04, L1 ﹑ L8 fermentation 2d is lived and there is notable difference other day, therefore ferments and within two days, is the best fermentation time of YB-04 two strain bacterium; 2. along with the variation of pH, the unit enzyme of fermented liquid is lived and is constantly changed, and visible pH value can have influence on the product enzyme process of bacterial strain.Result shows YB-04, in the time of pH 7.5, producing proteinase activity best result Wei 135.01U/mL, through one-way analysis of variance find YB-04 in the time of pH 7.5 the institute amylase that produces and other level there were significant differences, the optimal pH that proteolytic enzyme is produced in fermentation is 7.5, and it is 8 that diastatic optimal pH is produced in fermentation; 3. under condition of different temperatures, the enzyme work of fermented liquid has obvious variation.Therefore determining of optimum temperuture is also the important factor of fermentation condition optimization.YB-04 Dian Fen Mei ﹑ proteolytic enzyme in the time of 37 DEG C all has the highest enzyme to live, and diastatic activity is respectively: 142.59U/mL, protease activity is respectively: 133.14 U/mL, show that through variance analysis the difference during with 40 DEG C is not remarkable at YB-0437 DEG C.Consider based on economic condition, fermentation condition is defined as 37 DEG C; 4. along with the enzyme of the increase fermented liquid of inoculum size live in rising trend, but when inoculum size reaches a certain amount of, enzyme is lived and is started again to decline.When the inoculum size of YB-04 is 1%, enzyme is alive the highest, and diastatic activity is respectively 137.81 U/mL, and protease activity is respectively 154.09 U/mL.Show that YB-04 has notable difference with other level under 1% inoculum size condition through variance analysis, thus the suitableeest inoculum size be 1%; 5. in the time soaking powder as nitrogenous source taking yeast, YB-04 diastatic activity is up to 151.36U/mL, and when taking trypsinase as nitrogenous source, protease activity is 121.16U/mL.While soaking powder as nitrogenous source through variance analysis is known taking yeast, the enzyme of YB-04 fermented liquid is lived, and there were significant differences with other level, and therefore the suitableeest nitrogenous source of YB-04 is that yeast soaks powder.
The antibacterial general survey of embodiment 6:YB-04 is fixed
Taking 10 kinds of pathogenic bacterias as target bacterium, adopt the method for dull and stereotyped face-off to measure the antagonistic action of YB-04 bacterial strain to these 10 kinds of pathogenic bacterias, measure its antimicrobial spectrum.
Table 4 YB-04 bacterial strain antimicrobial spectrum
Note: " +++ ": antibacterial band is more than 10mm; " ++ ": the antibacterial 5～10mm that is with;
"+": antibacterial band < 5mm; "-": antibacterial band < 3mm
Determine the antibacterial band of YB-04 to 10 kinds of pathogenic bacterias, result is as shown in table 4, finds that it shows the comparatively fungistatic effect of wide spectrum, wherein better to rhizosphere disease fungistatic effects such as verticillium dahliae, gaeumannomyces graminis, wheat hypochnuss.Show that YB-04 not only can degrading straw, can also suppress rhizosphere pathogenic bacteria, regulate rhizospheric microorganism group, there is fine exploitation prospect.
Embodiment 7: strain combination degraded wheat stalk test
(1) co-culture experiments of degradation bacteria: between the microorganism of the ecosystem, relation is very complicated, and interdependence is vied each other again.By by two or more inoculation on same flat board, observe between them whether have antagonistic action, could determine whether multiple bacterial strains can co-cultivation.The bacterial strain that this experiment obtains screening is inoculated on PDA flat board between two, by the observation of colony diameter, analyzes between two kinds of bacterium whether can produce antagonistic action.
(2) bacterial strain uses therefor: YB-04 be in patent of the present invention, protect bacterial strain subtilis (
baciLLus stbtiLis), P4 be black-koji mould (
aspergillus niger), P3 be aspergillus oryzae (
aspergiLLus oryzae) bacterial strain, P5 be Penicillium chrysogenum (
penicillium chrysogenum) bacterial strain, L8 be bacillus amyloliquefaciens (
b. amyLaLiquefaiens) bacterial strain, separate in the stalk that rots by this laboratory.Microbial inoculum 1 and microbial inoculum 2 are respectively purchased from Bai Hui bio tech ltd, Hebi.
(3) degrading straw test: accurately take 2g and put into culture dish at 60 DEG C of fresh wheat stalks that are dried to constant weight, carry out the effect research of strain degradation wheat stalk, the making of bacteria suspension is with reference to aforementioned seed liquor manufacture method.Single culture is degraded to: add 1mL bacteria suspension moisturizing to 10mL to being placed with in the culture dish of 2g stalk; Combined degradation is: be placed with in the plate of stalk and add bacterium liquid, every kind of bacterium liquid is 1mL, and moisturizing is to 10mL.
(4) degradation effect analysis: after Degrading experiment is complete, by appearance features, and the changes in weight situation of stalk is evaluated the degradation effect of stalk.Rotten stalk, can be according to the black Huang of color (Huang ﹑ Wei Huang ﹑ He Huang ﹑) be divided into (1 ﹑ 2 ﹑ 3 ﹑ 4) level Four, according to the smell (Mei Wei ﹑ An Wei ﹑ Jiu Wei ﹑ putrid taste of stalk) be divided into (1 ﹑ 2 ﹑ 3 ﹑ 4) level Four, rot according to feel softening degree (Ying ﹑ Wei Ruan ﹑ Ruan ﹑) be divided into (1 ﹑ 2 ﹑ 3 ﹑ 4) level Four, the progression of three kinds of features is summed up to the rotten degree that is stalk, level is said larger, and rotten degree is larger
[80].Within every 5 days, observe a straw degradative situation, record the progression of straw degradative.
The mycelium on stalk surface is clean with distilled water flushing, and 60 DEG C are dried to the weight of constant weight weighing residue stalk, calculate straw degradative rate, and residue stalk is carried out to content of cellulose mensuration.Content of cellulose measuring method: take 0.1g stalk in test tube, add acetic acid and nitric acid mixed solution (volume ratio is 1/1) 5mL, cover lid heats 30min in boiling water bath, is transferred to adding distil water in large centrifuge tube and is diluted to 45 mL, the cooling rear centrifugal supernatant that goes, 60 DEG C of oven dry.The sample of getting after oven dry adds the (sulfuric acid that massfraction is 10% in mixed solution, concentration is the potassium bichromate of 0.1mol/L) 10 mL, shake up rear boiling water bath 15min, all be transferred to titration in clean triangular flask, adding massfraction is 20% KI solution 5 mL, with the Sulfothiorine of 0.2mol/L be titrated to solution just aobvious blue 2 and half a minute in nondiscoloration, separately make a blank that does not add stalk.
Content of cellulose calculation formula: X=k (a-b)/(n × 24)
In formula: the concentration that k is Sulfothiorine, a is the volume of blank titration sodium sulfate used, b is the volume of solution Sulfothiorine that titration consumes, the quality that n is rice husk.
(5) be total to culture experiment result
Table 5 is culture experiment strain growth speed altogether
By two kinds of different inoculation, on flat board, taking single culture cultivation as contrast, the bacterium colony size of bacterial strain on flat board is in table 5.Speed when growth velocity during by bacterial strain co-cultivation and single culture is carried out difference analysis, analytical results shows that P value is all greater than 0.05, therefore the not property of there are differences of the growth velocity of co-cultivation and single culture, therefore shows not have antagonistic action between bacterial strain, can co-cultivation.
(6) straw degradative interpretation of result
Table 6 strain combinations degrading straw scheme
Note: microbial inoculum 1/ microbial inoculum 2 is purchased from Bai Hui bio tech ltd, Hebi; In every kind of combination, bacterium liquid all adds 1ml.
In previous methods, introducing is the Study on degradation that stalk is carried out in 5 strain bacterium combinations, and assembled scheme is in table 6, and the degraded progression in straw degradative process is as shown in table 7.As seen from the above table, when off-test, the stalk of bacterial classification combination of two degraded and the stalk of single degraded have reached identical rotten progression, but the degradation speed of stalk is greater than the speed that single culture is when strain combination.And fashionable when there being genus bacillus to add, the degradation speed of stalk is faster than not adding the experimental group degradation speed of genus bacillus, rot deep.In test, 3 fungal strains and the combination of 2 strain gemma make the rotten progression of stalk reach 12 grades, and the rotten progression of two kinds of commercially available microbial inoculums reaches respectively 9 grades of 8 ﹑, visible P3+P4+P5+(YB-04+L8) respond well to straw degradative.From Fig. 3-8, in the time only having fungi effect and stalk, although a large amount of mycelium is adhered on stalk surface, stalk surface is drier, when adding after bacterium the more moistening corruption in stalk surface more obvious.The utilization of fungi (P3+P4+P5) mixing effect stalk after stalk is obviously greater than single fungi and acts on stalk, adds the test batch stalk of bacterium also to occur the corruptions such as obvious blackening.Can observe these two kinds of microbial inoculums composite effective not as mixing in research to the degradation effect of stalk.
Table 7 straw degradative progression
Note: the rotten degree of the larger explanation of progression is more obvious
Table 8 straw degradative rate
The variation of table 9 content of cellulose
In the rear stalk of straw degradative rate and degraded, content of cellulose is in table 8 and table 9.Known, fungi and bacterium mixing effect are after stalk, the degradation rate of the degradation rate of stalk apparently higher than fungi independent role in stalk, when 5 kinds of bacterium mixing effects are during in stalk, the degradation rate of stalk has reached maximum value, but being not far longer than the compound action of single fungi and bacterium, is not simple addition when visible bacterial strain mixed degradation stalk.From the degradation rate of experiment combination and commercially available microbial inoculum relatively, the Degradation of experiment combination is far superior to the Degradation of commercially available microbial inoculum.
Embodiment 8 straw degradative simulation field experiments
(1) test method: in flowerpot, in (15cm × 12cm), add the vegetable garden soil that 8cm is thick, above even spreading wheat stalk 100g.With the bacterium liquid sprinkling stalk of different treatment, bacterium liquid and treatment process are shown in embodiment 3, and consumption is 10mL.Then till overlying soil is just in time filled flowerpot on stalk after, put into 25 DEG C of greenhouses and degrade after 30d, measure the degradation rate of stalk, and respectively process Nitrogen In Soils phosphorus potassium and organic content.Taking the stalk that do not add the processing of bacterium liquid as contrast.
(2) interpretation of result
In pot experiment, the degradation rate of stalk is in table 10, and the nitrogen phosphorus potassium after straw degradative in soil and organic content are in table 11.From table 10, degradation rate under natural condition in stalk 30d is 55.55%, adds the degradation rate of various processing of bacterium liquid all higher than the degradation rate under natural condition, and wherein the straw degradative rate of 5 kinds of bacterium combined treatment is 79.61%, reach maximum value, be significantly higher than the degradation rate under natural condition.
Table 10 is simulated field experiment straw degradative rate
Note: lowercase is significance analysis in 0.05 level
From table 11, stalk can make nitrogen phosphorus potassium and the organic content in soil increase after degraded in soil, nitrogen phosphorus potassium and organism increased value in the larger soil of straw degradative rate are larger, the Nitrogen In Soils phosphorus potassium and the organic content that do not add stalk are respectively 31mg/mL ﹑ 42mg/mL ﹑ 240 mg/mL ﹑ 0.1%, Nitrogen In Soils phosphorus potassium content after 5 kinds of bacterium combination degrading straws has reached 58 mg/mL ﹑ 52 mg/mL ﹑ 674 mg/mL, 1.3%, the content of Nitrogen In Soils phosphorus potassium is significantly higher than contrast, also illustrate that 5 kinds of potted plant stalk effects of bacterium combination degraded are best, provide scientific basis for further developing combination degradation bacteria.
Table 11 Nitrogen In Soils phosphorus potassium and organic content
Claims (4)
1. a subtilis YB-04, its deposit number is CGMCC NO.6497.
2. a preparation method for microbial bacteria preparation, comprises the steps:
(1) strain culturing and preparation: subtilis YB-04 described in claim 1 is inoculated on TSA test tube slant substratum, cultivates at 28～35 DEG C and obtain thalline for 24～36 hours;
(2) seed liquor preparation: in test tube strains access TSB nutrient solution prepared by step (1), concussion is cultivated and made seed liquor for 12～16 hours at 28～32 DEG C;
(3) fermention medium preparation: get respectively glucose, Semen Maydis powder, soybean cake powder, dipotassium hydrogen phosphate and add water and be mixed with the nutrient solution containing glucose 1.2%, Semen Maydis powder 1.8%, soybean cake powder 3%, dipotassium hydrogen phosphate 0.2%;
(4) liquid fermenting is produced: after fermentation equipment and medium sterilization, seed liquor is inoculated in to the nutrient solution that step (3) prepares by 5% inoculum size, cultivates after 36～48 hours at 27～34 DEG C of bottom fermentations, collect fermented liquid and obtain microbial inoculum.
3. the application of bacillus subtilis strain YB-04 claimed in claim 1 in crop material degraded.
4. bacillus subtilis strain YB-04 claimed in claim 1 is in control wheat rhizosphere banded sclerotial blight and the application in gaeumannomyces graminis disease.
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