CN1580242A - Laterosporo short bacillus, and its fermentating process and use - Google Patents

Abstract

The invention provides a kind of Brevibacillus laterosporus (STP-B.latCGMCCNo.1119), the character follows this. G+, haulm shape, the shape is (0.4-0.5)*(2-2.5)mu. The spore adnation is from neutrality to sub-neutrality. The spore bursa will enlarge. Most of the spore is vertebra aitch, the remainder is disciform. VP-, ph value of culture fluid is 7.2, amylolysis-, gelatin dispergation-, decomposition casein+, katalase+, oxidase-, cirate -, dextrose aerogenesis-, dextrose acidgenesis+, L-arabinose acidgenesis-, xylose acidgenesis-, mannit acidgenesis+, azotate deocidisation+, methyl red+, pH5.7 growth+.

Description

A kind of side spore bacillus brevis, its fermenting process and application

Technical field

The present invention relates to a kind of new isolating microorganism and application thereof, in particular, relate to a kind of side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat and application thereof.

Background technology

The use of chemical fertilizer has made in the agriculture production a very big step, but along with more and more a large amount of uses, has brought negative effects such as soil compaction, environmental pollution, crop downgrade.Fertilizer becomes ecological fertilizer of new generation likely so bio-feritlizer, especially inorganic, organic, biological three limits are become attached to.But Liu Tong most bio-feritlizers are because effective bacterium of using is functional poor in the market, and most bacterial strains are not full gemma.Cultivate that production technique is coarse, simple and crude, the biological bacteria survival time short, be difficult to the fertilizer quality, the fertilizer efficiency instability that store, make, do not form commodity.Even compound granulation, but because of not resisting the high temperature in the production process, not anti-drying, and mass mortality.The full sporeformer of minority is also because of inorganic fertilizer can not melt altogether, salt resistance is poor, seriously hindered promoting the use of and develop of bio-fertilizer.

In order to solve the problem of above-mentioned bio-feritlizer, side spore bacillus brevis of the present invention (Brevibacilluslaterosporus) STP-B.lat is a bacterial classification outstanding in the function yeast, mensuration and research through science series, proved that its unique function, physiological property are applicable to industrialized production fully, satisfied the ecotope demand on border, process of crop growth soil microorganisms district.

Summary of the invention

The invention provides a kind of side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat CGMCCNo.1119.It is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 23rd, 2004, and it abbreviates CGMCC as, and deposit number is CGMCC No.1119.

Through identifying that side spore bacillus brevis STP-B.lat of the present invention has following mycology feature:

G ⁺, shaft-like, size is (0.4-0.5) * (2-2.5) μ.The gemma adnation is neutral to inferior neutral.Sporangiocyst expands.The gemma great majority are vertebra shape, also have oval-shaped.VP ^-, nutrient solution pH7.2.The starch hydrolysis ^-Gelatine liquefication ⁺Decompose casein ⁺Catalase ⁺Oxydase ^-Citrate trianion utilizes ^-The glucose aerogenesis ^-Glucose produces acid ⁺L-arabinose produces acid ^-Wood sugar produces acid ^-N.F,USP MANNITOL produces acid ⁺Nitrate reduction ⁺Methyl red test ⁺The PH5.7 growth ⁺On anaerobic culture medium, grow, in 7%NaCl, do not grow.

The present invention also provides a kind of strain inclined plane that contains microorganism, and described microorganism is side spore bacillus brevis STP-B.lat of the present invention.

The aforesaid strain inclined plane that contains microorganism, it uses the storage medium of being made by the reagent of following weight parts:

Peptone 5.9 ‰

Glucose 4.8 ‰

NaCl????????3.6‰

KCl?????????0.9‰

MgSO ₄??????0.24‰

MnCl ₂??????0.004‰

Yeast powder 0.5 ‰

Agar 20 ‰

CaCO ₃??????2‰

The pH nature.

The present invention also provides a fermenting process, and the bacterial classification that it uses is the described side spore of claim 1 bacillus brevis (Brevibacillus laterosporus) STP-B.lat.

Aforesaid fermenting process, it uses the substratum of being made by the reagent of following weight parts:

Peptone 5.9 ‰

Glucose 4.8 ‰

NaCl????????3.6‰

KCl?????????0.9‰

MgSO ₄??????0.24‰

MnCl ₂??????0.004‰

Yeast powder 0.5 ‰

CaCO ₃??????2‰

The pH nature;

Perhaps use the substratum of making by the reagent of following weight parts:

Semen Maydis powder 15 ‰

Soybean cake powder 11.45 ‰

K ₂HPO ₄???1‰

Yeast extract paste 0.7 ‰

CaCO ₃??????0.5‰

Corn steep liquor 10 ‰

Trace element 1 ‰

Defoamer is an amount of

The pH nature.

The present invention also provides a kind of tunning, and it contains side spore bacillus brevis STP-B.lat of the present invention.

The present invention also provides a kind of bio-feritlizer, and it contains side spore bacillus brevis STP-B.lat of the present invention, perhaps contains above-mentioned tunning.

The present invention also provides described side spore bacillus brevis STP-B.lat to have application in the bio-feritlizer that promotes plant-growth and/or fixed nitrogen function in preparation.

The present invention also provides described side spore bacillus brevis STP-B.lat to have application in the bio-feritlizer of parallel off fungi, nematode function in preparation.

Description of drawings

Fig. 1 is a side spore bacillus brevis STP-B.lat living spore curve in one embodiment.

Fig. 2 is the artwork of the fermenting process embodiment of side spore bacillus brevis of the present invention (Brevibacillus laterosporus) STP-B.lat.

Embodiment

The following examples will be further explained the present invention, but the present invention is not limited only to these embodiment, the scope that these embodiment do not limit the present invention in any way.

The separation screening of embodiment 1 side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat

The separation screening of side spore bacillus brevis STP-B.lat:

1) 1) separation of side spore bacillus brevis STP-B.lat and the used substratum of purifying:

The separation of side spore bacillus brevis STP-B.lat and purifying are aided with N.F,USP MANNITOL substratum and improvement Ashby substratum based on improvement D ǒ bereiner substratum, and the prescription of each substratum is as follows:

Improvement D ǒ bereiner substratum:

Raw material content of starting materials consumption

Sucrose 10.0g Sucus Mali pumilae 5.0g

K ₂HPO ₄·H ₂O???0.1g??????K ₂HPO ₄·H ₂O?0.4g

MgSO ₄·7H ₂O????0.2g??????NaCl????????????0.1g

CaCl·2H ₂O??????0.02g?????FeCl ₃??????????0.01g

Na ₂MoO ₄·H ₂O??0.002g????H ₂O???????????1000ml

Transferring pH with NaOH is 7.2.

NN: do not add peptone, be nitrogen-free agar;

LN: add peptone 0.2g, be low nitrogen substratum;

HN: add peptone 5g, be high nitrogen substratum.

The N.F,USP MANNITOL substratum:

Raw material content of starting materials consumption

N.F,USP MANNITOL 10.0g K ₂HPO ₄3H ₂O 0.5g

MgSO ₄·7H ₂O????0.2g??????NaCl?????????????0.2g

CaCl·2H ₂O??????0.2g??????FeCl ₃???????????0.01g

Yeast extract paste 0.3g H ₂O 1000ml

Improvement Ashby substratum:

Raw material content of starting materials consumption

Glucose (or N.F,USP MANNITOL) 10.0g CaCO ₃5.0g

K ₂HPO ₄?????????0.2g??????K ₂SO ₄????????0.2g

MgSO ₄·7H ₂O??0.2g??????NaCl???????????0.2g

Agar 18.2g H ₂O 1000ml

2) separation, the purifying of rhizosphere side spore bacillus brevis STP-B.lat

Adopt dilution-plate method separating rice rhizosphere side spore bacillus brevis (Brevibacilluslaterosporus) STP-B.lat from soil.Rhizosphere soil mixing with adopting back takes by weighing 10g root and native mixture, grinds in mortar, adds sterilized water to 100ml, is diluted to 10 by 10 times of dilution methods ^-5, totally three repetitions.Get 10 ^-4With 10 ^-5Each 0.1ml of the soil supension of two concentration evenly is applied in the nitrogen-free agar plate, cultivates after 3 days down for 30 ℃, and different bacterium colony labels are picked out, and surveys its acetyiene reduction activity (ARA) on improvement D ǒShi LN substratum.Then, generally select acetyiene reduction activity to be higher than 20n mol C ₂H ₄The bacterial strain of/hr/ml is eliminated non-activity and active low bacterial strain, separates purification with plate streak, and single bacterium colony of selecting different shape again picks out, and selects the significant bacterium colony of ARA value to continue to separate, up to selecting the higher pure bacterial strain of ARA.

3) acetyiene reduction activity (ARA) of rhizosphere side spore bacillus brevis STP-B.lat is measured

The D ǒShi LN solid medium that in 15 * 150mm test tube, adds the improvement of 4.5ml, 121 ℃ of autoclavings 15 minutes, beveling.The inoculation back is cultivated 18～24hrs down at 30 ℃, and tampon is changed to the soft rubber ball sealing, extracts the air of 1.8ml, injects the C of 1.6ml ₂H ₂, after continuing to cultivate 24～36hrs, get 100 μ l gas samples and on SQ-204 type gas chromatograph, measure ethene peak value, standard gas C ₂H ₂Concentration is 138mgml ^-1The working conditions of gas chromatograph is set to: 100 ℃ of sensing chamber's temperature, and 60 ℃ of column temperatures, 60 ℃ of injector temperatures, column length are 1m, gauge outfit nebulizer gas pressure nitrogen is 0.95kgcm ^-2, hydrogen is 0.8kgcm ^-2

The acetyiene reduction activity method of calculation are:

Through work in a few years, from up to a hundred the samples in all parts of the country, separate the function yeast that using value is arranged.Recording side spore bacillus brevis STP-B.lat activity after the acetylene reduction is 2210～2330n mol C ₂H ₄/ hr/ml.

The evaluation of side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat:

Through identifying that side spore bacillus brevis STP-B.lat of the present invention has following mycology feature:

G ⁺, shaft-like, size is (0.4-0.5) * (2-2.5) μ.The gemma adnation is neutral to inferior neutral.Sporangiocyst expands.The gemma great majority are vertebra shape, also have oval-shaped.VP ^-, nutrient solution pH7.2.The starch hydrolysis ^-Gelatine liquefication ⁺Decompose casein ⁺Catalase ⁺Oxydase ^-Citrate trianion utilizes ^-The glucose aerogenesis ^-Glucose produces acid ⁺L-arabinose produces acid ^-Wood sugar produces acid ^-N.F,USP MANNITOL produces acid ⁺Nitrate reduction ⁺Methyl red test ⁺The PH5.7 growth ⁺On anaerobic culture medium, grow, in 7%NaCl, do not grow.

The short growing nitrogen-fixing function of embodiment 2 side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat bacterial strains:

The acetylene reduction method:

(1) measuring method: in 15 * 150mm test tube, add the low nitrogen solid medium of improvement D ǒShi of 4.5ml, 121 ℃ of autoclavings 15 minutes, beveling.The inoculation back is cultivated 18～24hrs down at 30 ℃, and tampon is changed to the soft rubber ball sealing, extracts the air of 1.8ml, injects the C of 1.6ml ₂H ₂, after continuing to cultivate 24～36hrs, get 100 gas samples and on SQ-204 type gas chromatograph, measure ethene peak value, standard gas C ₂H ₂Concentration is 138mgml ^-1The working conditions of gas chromatograph is set to: 100 ℃ of sensing chamber's temperature, and 60 ℃ of column temperatures, 60 ℃ of injector temperatures, column length are 1m, gauge outfit nebulizer gas pressure nitrogen is 0.95kgcm ^-2, hydrogen is 0.8kgcm ^-2, air is 0.6kgcm ^-2

(2) measurement result: the ethene reducing activity (ARA) of side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat is 2210～2330n mol C ₂H ₄/ hr/ml.

¹⁵ The N labelling method:

(1) measures bacterial strain: side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat.

(2) measuring method:

A) with D ǒShi culture medium prescription, every 1000ml adds 5g agar and 0.2g peptone, is made into the low nitrogen semisolid medium of improvement D ǒShi, gives in the triangular flask of 25ml and injects 10ml, 121 ℃ of autoclavings 20 minutes.Then, vertical percutaneous puncture-inoculation is with soft rubber ball sealing triangular flask.

B) according to the calculating of consumption, add in right amount at Plastic Bottle ( ¹⁵NH ₂) ₄SO ₄, bleed, add argon gas again, bleed 3 times, inject an amount of LiBrO then, generate ¹⁵N ₂Standby.( ¹⁵NH ₂) ₄SO ₄Produce by Shanghai Chemical Research Inst, its ¹⁵The N abundance is 10.12%.

C) extract the air of having inoculated in the triangular flask out, add argon gas, repeat 3 times, add 8% oxygen then, all the other skies add ¹⁵N ₂, cultivate after 2 days in 30 ℃ of incubators, carefully pour culture into nitre and boil in the pipe, 80 ℃ of oven dry down.

D) on the MU-1305 type mass spectrograph of Electronic Speculum mass spectrum testing laboratory of Atomic Energy Utilization Inst. of China Agricultural Sciences Academy, in the test sample product ¹⁵The isotopic abundance of N, mass spectrometric precision are 0.5%.Test conditions is to add vitriol oil 5ml, catalyst mixture (K in the sample ₂SO ₄: Se=500: 1) 2ml, use H after nitrated ₂SO ₄Absorb.

(3) measurement result: side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat's ¹⁵The N atomic percent surpasses 0.311%.

Nif gene identification method:

(1) measures bacterial strain: side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat.

(2) for the examination plasmid:

A) plasmid pSA30 contains the nifHDKE of klepsiella pneumoniae (Klebsiella pneumonia), and 6.2kb, carrier are pCAY18,4.0kb, and tool EcoRI restriction enzyme site, the Tc resistance is provided by Atomic Energy Utilization Inst. of China Agricultural Sciences Academy's biotechnology.

B) plasmid pHU8 contains the nifH of Brasil diazotrophic spirillum (Azospirillum Brasilense), 0.6kb, carrier is pUC19,2.8kb, tool RcoRI and HindIII double enzyme site, the Amp resistance is provided by Agricultural biotechnologies National Key Laboratory of Beijing Agricultural University.

(3) main agents.Tris, agarose, Proteinase K, N,O-Diacetylmuramidase, the RNA enzyme, restriction enzyme EcoRI, HindIII, main biochemical reagents such as single endonuclease digestion λ DNA/HindIII, double digestion λ DNA/HindIII, EcoRI and the big fragment of archaeal dna polymerase Klenow are all available from magnificent company and Promega company; α-32P-dCTP is buied by Beijing Fu Rui company; Other is homemade analytical reagent.The collocation method of reagent is mainly by " molecular cloning test direction " operation.

(4) a large amount of method extraction and purifications of bacteria total DNA:

A) after 1～2 day, get 20ml bacterium liquid with the high nitrogen culture medium culturing of D ǒShi under 30 ℃, behind centrifugal 15 minutes precipitations of the 6000rpm thalline, be suspended among the TE centrifugal again.

B) behind the 5ml TE suspension thalline, add N,O-Diacetylmuramidase 100 μ l (50mg/ml), 37 ℃ of down effects after 1 hour add 10% SDS and the 30 μ l Proteinase Ks (10mg/ml) of 0.5ml successively, 37 ℃ of effects 1 hour down.

C), after adding 5M NaCl and making its final concentration be 0.1M, add 2 times of volume dehydrated alcohols successively with behind the isopyknic phenol, phenol/chloroform/primary isoamyl alcohol, each extracting of chloroform/primary isoamyl alcohol 1～2 time.Spend the night under-20 ℃.

D) with 10000～12000rpm rotating speed centrifugal 15～20 minutes, stay precipitation to abandon liquid, wash precipitation with 70% ethanol, with the TE dissolving, add 37 ℃ of 30 μ lRNA enzymes (10mg/ml) effect 1～2hrs down ,-20 ℃ of preservations are standby.

(5) bacterial plasmid extracts (alkaline lysis):

A) the LB liquid nutrient medium was cultivated thalline after 24 hours, with 8000rpm rotating speed precipitation thalline.

B) add solution I (50mmol/l glucose, 25mmol.LTrisCl, 10mmol/l EDTA) 8ml, placed 30 minutes.

C) add solution II (0.2mmol/L NaOH, 1%SDS) 16ml, place the ice several minutes to mix the back and placed 20 minutes.

D) add solution III (K+3mmol/L, C ₂H ₃COO-5mmol/L) after 12ml is mixed, placed 20 minutes, make its renaturation.

E) 5000～12000rpm is centrifugal 15 minutes, gets supernatant and reinforces propyl alcohol and make final volume be 0.6 times to be mixed, and room temperature is placed after 20 minutes for 30 ℃, with 8500～12000rpm rotating speed centrifugal 18～20 minutes, abandons liquid.

F) add an amount of 70% ethanol, place and washed precipitation in 10 minutes, add 5ml TE dissolution precipitation after, add isopyknic phenol mixing, centrifugal 10 minutes with rotating speed 8000rpm.

G) get supernatant liquor and add equal-volume phenol/chloroform/primary isoamyl alcohol, chloroform/primary isoamyl alcohol mixing after, centrifugal 15～20 minutes respectively.

H) get supernatant liquor 5M NaCl, making its final concentration is 0.1M, adds 2 times of dehydrated alcohols again, and it is centrifugal that mixing is positioned over-20 ℃ of backs of spending the night, and discards liquid, and the ethanol with 70% is cleaned, and control is done, and adds an amount of TE dissolving.

(6) enzyme is cut:

A) the pSA30 plasmid is cut with the EcoRI enzyme; The pHU8 plasmid is cut with EcoRI and HindIII enzyme.

B) preparation electrophoresis check enzyme is cut effect and is taken pictures.

(7) the sepharose freeze thawing is reclaimed:

A) get the 6.2kb band and the pHU8 plasmid 0.6kb band blob of viscose of pSA30 plasmid, add an amount of phenol.

B) after-20 ℃ of refrigerator and cooled are frozen, melt again and smash to pieces, centrifugal to broken back for several times repeatedly, extract upper aqueous phase, abandon bottom phenol.

C) add equal amounts of phenolic/chloroform/primary isoamyl alcohol, behind the mixing centrifugal 3～5 minutes, extract supernatant.

D) add equal amounts of chloroform/primary isoamyl alcohol, behind the mixing centrifugal 3～5 minutes, extract supernatant.

E) add NaCl solution and make final concentration be 0.1M after, add 2 times of dehydrated alcohols ,-20 ℃ are spent the night.

F) with rotating speed 12000rpm centrifugal 15～20 minutes, abandon liquid, get precipitation, add 70% ethanol and wash.

G) after an amount of TE dissolving, detect through agarose gel electrophoresis.

(8) gene probe equipment and blotting hybridization:

A) cut DNA and prepare electrophoresis with the EcoRI enzyme, soaked blob of viscose 15 minutes with 0.25N HCl 500ml.

B) in 1.5M NaCl/0.5M NaOH solution and 3M NaCl/0.5M TrisCl solution, soak 20 minutes twice respectively.

C) give and to add an amount of 20 * SSC in the big culture dish and make and soak, after driving bubble out of, the point sample hole is placed on glue on the filter paper down, drives bubble out of, get large-area slightly nitrocellulose filter, soak with 6 * SSC, be tiled on the glue, total head is sealed film all around, add Xinhua's filter paper number layer on it, add about 2 cun thieving papers then, and press and to add an amount of weight, spend the night.

D) take out nitrocellulose filter, the absorption open fire also dries, and 80 ℃ of oven dry were immersed 6 * SSC immersion after 10～15 minutes with nitrocellulose filter after 2 hours, put into pre-washing lotion 42 ℃ of water-baths, jog 1～2 hour.

E) will immerse in the 50ml prehybridization solution, after jog was washed film in 3～5 hours in 68 ℃ of water-baths, preparation probe: 1. the plasmid DNA that reclaims is got 0.5 μ l and 0.5 μ lTE (pH=8.3) puts into little centrifuge tube, mixing, in boiling water, boiled 2 minutes, and put into frozen water very soon, make its sex change; Add 2. for the little centrifuge tube in the ice bath: H2O, 24 μ l, 5 * buffer, 10 μ l, dgTP 1 μ l, daTP 1 μ l, dtTP 1 μ l, BSA 2 μ l and E1.8 μ l; 3. get 5 μ l[α- ³²P] dCTP injects little centrifuge tube mixing, makes overall solution volume reach 50 μ l; 4. adding isotopic little centrifuge tube at room temperature placed more than 1 hour; 5. little centrifuge tube is put into boiling water after 2 minutes, put into ice bath very soon.6. the probe 50 μ l solution of sex change are clicked and entered in the prehybridization solution that is placed with nitrocellulose filter, 68 ℃ of following water-baths are spent the night, and it is hybridized; 7. at room temperature wash film with 2 * SSC/0.1%SDS solution and 1 * SSC/0.1%SDS solution respectively; 8. suitably dry, under-70 ℃ in magazine with the X-ray film press mold after 3 days, develop, photographic fixing.

(9) qualification result: the total DNA of side spore bacillus brevis STP-B.lat and the nifHDKE gene probe of klepsiella pneumoniae have weak positive hybridization signal at the 20kb place, at 6kb and 4kb place two stronger positive signal are arranged; At 16kb and 6kb place two very strong positive hybridization signals are arranged with the nifH gene probe of Brasil diazotrophic spirillum.

The phosphorus decomposing of embodiment 3 side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat, potassium decomposing, fixed nitrogen are renderd a service and other functional examination

The phosphorus decomposing efficacy determinations

Culture medium prescription is as follows:

Raw material	Amount	Raw material	Amount	Raw material	Amount
						Glucose	10g	(NH 4) 2SO 4	0.5g	NaCl	0.3g

KCl	?0.3g	MgSO 4	0.3g	?FeSO 4	?0.03g
						MnSO 4	?0.03g	Yeast powder	0.5g	?CaCO 3	?0.1g
Yelkin TTS	?0.2g	Distilled water	1000ml	Ground phosphate rock	?0.1％

Annotate: transfer pH=7.0～7.5

The packing triangular flask is sterilized, and bacterium is measured in inoculation, and 30 ℃ of concussions were cultivated 4 days, and filtration, plasma radiation spectrography are surveyed rapid available phosphorus.Phosphorus decomposing 176.58 μ g/ml improve 17.1 times (three repetitions, get its average) than CK.

The potassium decomposing efficacy determinations

Culture medium prescription is as follows:

Raw material	Amount	Raw material	Amount	Raw material	Amount
						Base sugar	5g	Na 2HPO 4	2g	MgSO 4	0.5g
FeCl 3	0.05g	CaCO 3	0.1g	Water	1000ml
						pH	7.0	Feldspar Powder	0.1％	The packing triangular flask	Sterilization

Inoculate and measure 30 ℃ of concussion cultivations of bacterium 4 days, filter, get filtrate, with the potassium of aas determination parsing.Potassium decomposing 13.7 μ g/ml improve 27% (three repetitions, get its average) than CK.

Fixed nitrogen, resolve other element, secretion plant hormone efficacy determinations

1# oxysuccinic acid substratum (water 1000ml, pH7.2)

Raw material	Amount	Raw material	Amount	Raw material	Amount	Raw material	Amount
								Sucrose	10g	K 2HPO 4	0.1g	MgSO 4	0.2g	CaCl 2	0.02g
Oxysuccinic acid	5g	KH 2PO 4	0.4g	NaCl	0.1g	FeCl 3	0.01g
								Sodium orthomolybdate	0.002g	Peptone	0.2g	Agar	20g

2# Sunmorl N 60S substratum (water 1000ml, pH7.4～7.6)

Raw material	Amount	Raw material	Amount	Raw material	Amount
						N.F,USP MANNITOL	5g	K 2HPO 4	0.2g	MgSO 4	0.2g
Sodium Glutamate	0.4g	Agar	20g	NaCl	0.2g
						Sunmorl N 60S	5g	Yeast extract paste	0.4g	Mix little liquid	2ml

Put into the inclined-plane after above-mentioned substratum packing 200 * 200mm Boiling tube sterilization, connect test organisms and cultivated 24 hours for 30 ℃, change anti-mouthful of plug draws air, annotate acetylene gas, continue to cultivate 24 hours, take out and measure.

3# plant hormone secretion substratum

Raw material	Amount	Raw material	Amount	Raw material	Amount	Raw material	Amount
								Glucose	2g	KH 2PO 4	2g	MgSO 4	0.5g	CaCO 3	0.1g
Starch	3g	Yeast powder	0.4g	(NH 4) 2SO 4	0.3g	1％FeCl 3	1.5ml

The packing triangular flask, sterilization back inoculation test bacterium, 30 ℃ of concussions were cultivated 5 days, and filtration or centrifugal clear liquid are surveyed hormone-content.

The gas chromatography determination nitrogenase activity, liquid chromatography for measuring excretory hormone, the potassium that aas determination is resolved, plasma emission spectrometry is measured other element of resolving.

The result:

1) nitrogenase activity:

On the oxysuccinic acid substratum: 934.2232n mol C ₂H ₄//hour;

On the active culture base: 517.2827n mol C ₂H ₄//hour.

2) microbiotic recovery is renderd a service in secreting hormone and the promotion soil

After follow the tracks of using that side spore bacillus brevis STP-B.lat makes, soil investigation is the result show, actinomycetes increase by 8.4 times, and vinelandii increase by 39 times, G ⁺Bacterium and G ^-The bacterium ratio increases by 4.1 times.The mechanism of action is: 1. actinomycetes are actively participated in organic decomposition, particularly decomposing the later stage, can well decompose the plant residue that becomes thoroughly decomposed, for crop provides nutrition, and can in its vital movement, produce meta-bolites---microbiotic, plant hormone etc., can grow by stimulating plant, regulate metabolism, suppress the generation of fungal disease.2. the nitrogen of vinelandii in can fixed air formed individual cells, and after the cytoclasis, nitrogen accumulation is plant utilization in soil.Can produce Plant hormones regulators,gibberellins, cytokinin, indolylacetic acid and pathogeny inhibitor in its vital movement again, so the energy stimulating growth, the regulation and control metabolism alleviates the pathogeny bacterium and infects.3. G ⁺Increasing of bacterium means the reinforcement of protein Decomposition, the increase of soil nitrogen accumulation.

Side spore bacillus brevis STP-B.lat secretes 4 kind of plant hormone (Z, IPA, GA in the vital movement process ₃, IAA), its amount can reach 838.2 μ g/100ml.

The fauna microbial species	The effective bacterium number in STP-B.lat processing field (individual/g)	The effective bacterium number in contrast field (individual/g)	Comparison is according to field increment (%)	Effectively bacterium increases multiple
					Aerobic bacteria	1.5×10 7	1.0×10 7	50	0.5
Anerobe	3.6×10 6	1.5×10 6	140	1.4
					Yeast and mould	1.1×10 4	4.0×10 3	175	1.8
Actinomycetes	2.8×10 5	3.3×10 3	838	8.4
					Pseudomonas	7.3×10 3	3.9×10 4	-81	-0.8
Vinelandii	4.0×10 4	＜1.0×10 3	＞3900	39
					G -Bacterium	1.1×10 5	9.6×10 6	-69	-0.7
G +Bacterium	1.5×10 7	26.7∶1	59	0.6
					G +Bacterium/G -Bacterium	136.4∶1		411	4.1

3) side spore bacillus brevis STP-B.lat analytical capabilities is measured

Substratum number	?1	?2	?3
				Zn	?25.9	?11.25	?6.51
B	?/	?/	?/
				Mo	?4.55	?/	?/
Be	?1.5	?0.55	?/
				Mg	?525	?/	?629

?Mn	?72	?/	?0.4
				?Si	?/	?/	?/
?Ai	?94.15	?/	?6.7
				?Sr	?15.95	?/	?/
?Ba	?15.95	?/	?6.66
				?Co	?2.25	?/	?/
?Ni	?5.85	?/	?0.09
				?Zr	?13.35	?/	?/
?Nb	?22.2	?/	?/
				?Ti	?/	?/	?0.67
?P	?430	?/	?/
				?V	?2.35	?/	?/
?Fe	?155	?/	?/
				?Ca	?21620	?/	?536
?Sn	?3.15	?/	?/
				?Bi	?9.5	?/	?/
?Ga	?46.65	?/	?/
				?In	?26.05	?/	?/
?Cu	?5.35	?/	?/

4) the mechanism effect of side spore bacillus brevis STP-B.lat parallel off fungi, nematode:

Chitin is a line eggs wall shell, also is the main moiety of pathomycete cell wall.Side spore bacillus brevis STP-B.lat produces powerful outside enzyme, and effectively cracking fungi, the outer cell wall of nematode have suffered the elimination of essence when making it form larva.This test adds chitin solution by the side spore bacillus brevis STP-B.lat that grows on flat board, transparent haloing appearred in rear plate upside spore bacillus brevis STP-B.lat periphery of bacterial colonies in 48 hours.Chitin is totally resolved.All farm crop root system nematode tumors of side spore bacillus brevis STP-B.lat that used obviously reduce in the practice.

The influence of embodiment 4 boundary's condition offside spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat

The influence of differing temps offside spore bacillus brevis STP-B.lat:

If 15, seven treatment temps such as 20,30,35,40,45 ℃, each is handled 5 times and repeats, and with the inclined-plane inoculation, cultivates 20 hours in 30 ℃ of incubators earlier, through handling, is placed on respectively under the differing temps and cultivates 24 hours, surveys ARA at SQ-204 type chromatographic instrument.

Conclusion:

Under condition of different temperatures, side spore bacillus brevis STP-B.lat subject range is wide than general utility functions bacterium all.The high reactivity value of bacterium all is 35 ℃ of appearance, and the optimum temperuture that bacterium is described is 35 ℃.Under cold condition bacterium is then played restraining effect, and strong to the restraining effect of general utility functions bacterium, suppressing the active half temperature of general utility functions bacterium is 25 ℃, but offside spore bacillus brevis STP-B.lat then is 20 ℃ (with 30 ℃ time ARA be contrast); The nitrogenase activity that at high temperature also suppresses the general utility functions bacterium as 40 ℃ the time, plays restraining effect to the activity of general utility functions bacterium, and offside spore bacillus brevis STP-B.lat then plays a driving role.

The influence of different pH offside spore bacillus brevis STP-B.lat:

1) different pH are to the active influence of bacterium:

The configuration liquid nutrient medium, with different pH values below the NaOH furnishing: 4.5,5.5,6.5,7.0,7.5,8.0,9.0,10.0,11.5,13.0, totally ten processing, install to respectively in the little triangular flask of 20ml, every bottle adds the 8ml liquid nutrient medium, adds the bacterium liquid that 2ml cultivated 24 hours again, three repetitions, cultivated 24 hours on treated 30 ℃ of following earthquake devices, on the GC-14 type chromatographic instrument of Tianjin, island, survey ARA.

Conclusion:

When hanging down pH (＜4.0), no matter to general function bacterium or side spore bacillus brevis STP-B.lat, all performance suppresses, along with increasing of pH value, its activity value also raises gradually, it is faster than general function bacterium that the ARA value of side spore bacillus brevis STP-B.lat is recovered, and occurs the peak during pH7.5, and the optimal pH that bacterium is described is pH7.5.After peak value, along with the rising of pH value, the activity of general function bacterium is suppressed, the value when being lower than pH7.0, and when pH reached 13.0, its activity inhibited reached 50%, but this illustrates that also the general function bacterium can stand high pH; But offside spore bacillus brevis STP-B.lat, after the pH value continues to raise, though its ARA value slightly descends than peak value, but all compare according to the ARA value of (pH7.0) high, even the ARA value is still compared according to high nearly 50% under the situation of pH13.0, illustrate at alkaline condition downside spore bacillus brevis STP-B.lat still can show high nitrogenase activity.

2) different pH are to the influence of bacteria growing amount:

After having surveyed the ARA value, same processing bacterium liquid poured out mixing after, draw 0.5ml and add the dilution of 2.5ml sterilized water again, under the 560nm condition, with Tianjin, island its OD value of spectrophotometric instrumentation.

Conclusion:

When hanging down pH, bacteria biomass reduces, consistent on this aspect with the bacterium activity value, rising along with pH, the biomass of bacterium increases thereupon, reach a small leak when pH6.5, biomass slightly descends then, and the general function bacterium is consistent with variation and its activity value variation tendency of side spore bacillus brevis STP-B.lat.The biomass variation of side spore bacillus brevis STP-B.lat is consistent basically; And the biomass of general function bacterium variation is not consistent.

Different [NH4 ⁺ ] offside spore bacillus brevis STP-B.lat influence:

1) different [NH ₄ ⁺] offside spore bacillus brevis STP-B.lat activity influence:

With (NH ₄) ₂SO ₄Dispose different concentration, capacity is the little triangular flask of the 10ml bacterium liquid that 4.5ml grown 24 hours of packing into, adds 0.5ml different concns [NH respectively ₄ ⁺] solution, 0,20,30,40,50,80,100,200ppm contrast adds the sterilized water of 0.5ml, makes final concentration be:, totally nine processing repeat for three times.

Conclusion:

[NH ₄ ⁺] inhibited to the nitrogenase activity expression, the someone is reported in 20ppm, also is reported in 30ppm or 40ppm.Under 30ppm, [NH ₄ ⁺] inhibited to the general function bacterium, but (10～20ppm) then have promoter action under lower concentration, and under lower concentration, the hormesis of general function bacterium is better than side spore bacillus brevis STP-B.lat, but side spore bacillus brevis STP-B.lat can stand higher [NH ₄ ⁺], when 50ppm, the general function bacterium loses activity fully, and offside spore bacillus brevis STP-B.lat only suppress its active 50%, when 80ppm, side spore bacillus brevis STP-B.lat still has faint expression, illustrates that side spore bacillus brevis STP-B.lat can stand higher [NH ₄ ⁺], prove by different bacterium and make up, can filter out the bacterial strain of anti-ammonia.

2) different [NH ₄ ⁺] increment is influenced

Treatment process is with " different pH are to the influence of bacteria growing amount ", has surveyed ARA value after, same processing bacterium liquid poured out mixing after, absorption 0.5ml adds the 2.5ml sterilized water and dilutes, under the 560nm condition, usefulness Tianjin, island its OD value of spectrophotometric instrumentation.

Conclusion:

Different [NH ₄ ⁺] to the effect basically identical of general function bacterium and side spore bacillus brevis STP-B.lat, all along with [NH ₄ ⁺] increase, the biomass of bacterium increases thereupon.Compare with the activity value of bacterium, activity of bacterium and the biomass of bacterium are also inconsistent, though at high [NH ₄ ⁺] under, the biomass of bacterium increases, but the activity of bacterium is by high density NH ₄ ⁺-N suppresses.

The influence of different carbon, nitrogenous source offside spore bacillus brevis STP-B.lat:

1) different carbon, nitrogenous source are to active influence:

Prepare the liquid nutrient medium (seeing the 4th～5 page of the application for details) of different carbon, nitrogenous source combination.With capacity is the little triangular flask of 20ml, and every bottle of liquid nutrient medium that adds 8ml adds the bacterium liquid that 2ml cultivated 24 hours again, three repetitions, and measuring method is with " different pH are to the active influence of bacterium ".

Conclusion:

Different carbon, nitrogen make up bacterium activity influence difference, when being only nitrogen source with the peptone, side spore bacillus brevis STP-B.lat nitrogenase activity value in the substratum that with N.F,USP MANNITOL is carbon source is the highest, next is that sucrose, starch are minimum, activity was the highest when the general function bacterium then was carbon source with the oxysuccinic acid, next is minimum in glucose, the starch, and this point is consistent with side spore bacillus brevis STP-B.lat.When being nitrogenous source with the yeast extract paste, side spore bacillus brevis STP-B.lat is consistent when active performance is nitrogenous source with peptone in different carbon sources; The activity performance of general function bacterium also is with minimum in the highest starch of activity in the oxysuccinic acid, and this variation tendency is similar with the variation in peptone.Illustrating that bacterium active performance in different carbon sources is different, is the expression that carbon source is more suitable for the general function bacterium with the oxysuccinic acid, with N.F,USP MANNITOL is being the activity expression that carbon source is more suitable for side spore bacillus brevis STP-B.lat.When being nitrogenous source with the peptone, though to the ARA value of general function bacterium or side spore bacillus brevis STP-B.lat all than the activity value height in yeast extract paste, be more suitable for the nitrogenase of bacterium when illustrating with the peptone, activity expression to nitrogenous source.

2) different carbon, nitrogenous source influence increment

Treatment process is with " influences of different pH offside spore bacillus brevis STP-B.lat ", has surveyed ARA value after, same processing bacterium liquid poured out mixing after, absorption 0.5ml adds the 2.5ml sterilized water and dilutes, under the 560nm condition, usefulness Tianjin, island its OD value of spectrophotometric instrumentation.

Conclusion:

When being nitrogenous source with the peptone, in different carbon sources, the general function bacteria biomass is the highest in the oxysuccinic acid intermediate value, secondly is sucrose, and is in the carbon source at glucose, N.F,USP MANNITOL and starch, and biomass is consistent basically; Side spore bacillus brevis STP-B.lat biomass also is the highest in oxysuccinic acid, is that biomass is minimum in the carbon source with starch.When being nitrogenous source with the yeast extract paste, the biomass of general function bacterium is still the highest in the oxysuccinic acid, and is minimum in N.F,USP MANNITOL, and side spore bacillus brevis STP-B.lat is in these several different carbon sources, and biomass changes relatively more consistent.The biomass of general function bacterium changes consistent with its ARA value variation, and the biomass of side spore bacillus brevis STP-B.lat changes and its ARA value changes difference greatly.

The influence of weedicide offside spore bacillus brevis STP-B.lat:

1) method:

Single weedicide: with the little triangular flask of 10ml, pack into 24 hours bacterium liquid 4.5ml of liquid medium within growth, the weedicide that adds the 0.5ml different concns then respectively, contrast adds sterilized water, 0,10,20,50,100,500,1000,5000,10000ppm make final concentration be at last:, repeat for three times, be placed on after the processing to shake on the homemade KS type Kang Shi oscillator and cultivate 24 hours (30 ℃), on the GC-14 type gas chromatograph of Tianjin, island, survey ARA.

Mixed herbicide: three kinds of weedicides mix in twos, have three kinds of combinations, specifically see result and analysis, and treatment process is the same.

2) effect:

A) influence of single weedicide offside spore bacillus brevis STP-B.lat:

The effect of Londax offside spore bacillus brevis STP-B.lat:

Londax plays restraining effect to the general function bacterium, and along with concentration increases, its restraining effect effect is also obvious, and when concentration reached 1000ppm, ARA had only 50% of contrast; Offside spore bacillus brevis STP-B.lat then plays a driving role, and along with concentration increases, its effect is also obvious, illustrates that Londax offside spore bacillus brevis STP-B.lat has just effect effect.

The effect of Facet offside spore bacillus brevis STP-B.lat:

No matter to general function bacterium or offside spore bacillus brevis STP-B.lat, Facet plays restraining effect basically, the general function bacterium there is promoter action at low-down concentration end, along with concentration increases, Facet is more violent to the effect of general function bacterium, and it changes than very fast, when 100ppm, ARA has only contrast 50%, and when concentration reached 200ppm, the ARA value of general function bacterium was suppressed basically; Its effect of offside spore bacillus brevis STP-B.lat is then relatively more steady, and when 200ppm, the ARA value has only 40% of contrast, slowly reduces thereafter.

The influence of Machete offside spore bacillus brevis STP-B.lat:

Machete is more consistent with its effect of side spore bacillus brevis STP-B.lat to the general function bacterium.Under lower concentration, offside spore bacillus brevis STP-B.lat promoter action is obvious, along with concentration increases, slowly descends, and its ARA value just is suppressed when high density 1%; To the general function bacterium then is slowly to promote to reach a peak value at 200ppm, along with concentration increases, has begun restraining effect then.Machete is better than the effect of offside spore bacillus brevis STP-B.lat to the restraining effect of general function bacterium.

More than comprehensive, we as can be seen, Londax, Facet play restraining effect to the general function bacterium, Facet just has promoter action at very low (10ppm) situation end; Machete then promotes the general function bacterium, and effect is obvious, and when 200ppm, its ARA value only just shows restraining effect for 150% of contrast under very high concentration.Londax, Machete offside spore bacillus brevis STP-B.lat play a driving role, and Facet then plays restraining effect, and different agricultural chemicals is to influencing difference.From the long and, side spore bacillus brevis STP-B.lat changes milder to the effect of weedicide.

B) influence of mixed herbicide offside spore bacillus brevis STP-B.lat:

The effect of Londax+Facet offside spore bacillus brevis STP-B.lat:

The mixed solution of Londax+Facet is to the active restraining effect that rises of bacterium, and the restraining effect of general function bacterium is better than the effect of offside spore bacillus brevis STP-B.lat, illustrates that side spore bacillus brevis STP-B.lat has certain resistance.But when it should be noted that the high density more than 500ppm, the restraining effect of mixed herbicide offside spore bacillus brevis STP-B.lat is better than the effect to the general function bacterium.

The effect of Londax+Machete offside spore bacillus brevis STP-B.lat:

The mixing of Londax+Machete also plays restraining effect to the bacterium activity, and its restraining effect is milder.

The effect of Machete+Facet offside spore bacillus brevis STP-B.lat:

(10～100ppm) play a driving role the mixing effect of Machete+Facet, and the action effect of offside spore bacillus brevis STP-B.lat is apparently higher than to the general function bacterium under lower concentration.After concentration increases (greater than 100ppm), activity to bacterium shows restraining effect, and the general function bacterium is more consistent with the variation of side spore bacillus brevis STP-B.lat, and when reaching 1000ppm when above, variation tendency is similar to the variation of Londax+Facet.

Comprehensive above result as can be seen, mixed herbicide has only Machete+Facet under lower concentration the general function bacterium to be played a driving role, all the other two kinds of mixing then play restraining effect, the action effect of offside spore bacillus brevis STP-B.lat similar to the general function bacterium, just the variation of side spore bacillus brevis STP-B.lat is relatively milder.

Embodiment 5 side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat increase production Mechanism Study

Potted plant and field experiment shows that the crop yield amplitude is 9.95～53.5%.For verifying volume increase mechanism, we have designed strict pot experiment, triangular flask test, field experiment, are that absorption area and root system vigor, soil plurality of enzymes activity change etc. are measured to the photosynthetic area of test materials and rate of photosynthisis, short taking root.

(1) increases leaf area, increase chlorophyll content

Get the corresponding position blade and take by weighing 1～2g, extraction using alcohol, the spectrophotometer colorimetric reads extinction value, by formula calculates fluid green content (the bright leaf of mg/g)=(C * 25)/G.C＝D ₆₅₂/34.5。The result is as follows:

Experiment 1	Wheat	Corn	Rape
				CK	1.188	0.555	0.918
Test	1.796	0.808	1.569
				Increase (%)	51.2	45.6	70.9

Experiment 2

Wheat

Corn

Rape

CK	65.91	72.09	39.23
				Test	77.59	146.29	66.87
Increase (%)	17.7	102.9	70.5

Crop yield no matter be seed or cauline leaf, all is photosynthetic product, does not have output to say without photosynthesis.Photosynthetic power depends on leaf area size and rate of photosynthisis.Chlorophyll is the material that absorbs luminous energy.The thing chlorophyll content that studies generally increases, amplification 45～70%.Leaf area amplification 17～100%.This has just improved the efficiency of light energy utilization greatly, has increased the synthetic of photosynthate.

(2) enlarge root absorbing area, enhancing root system vigor.

Take by weighing root 1～2g, measure the root system vigor with α-Nai amine oxidation style.Check typical curve according to optical density value and get corresponding α-Nai amine concentration.α-Nai amine amount during the α-Nai amine amount-autoxidation value the during α of root system oxidation-Nai amine amount=on-test-end.Oxidized α-Nai amine amount weighs/hour expression with μ g/ unit root.The result is as follows:

	Wheat		Corn
					Root system vigor (μ g/g/h)	Root fresh weight (g)	Root system vigor (μ g/g/h)	Root fresh weight (g)
	CK	102.12	21.75	53.6					11.0
Test					161.01	26.9	79.79	14.25
	Increase (%)	57.7	23.7	48.7					29.5

Root weighs and the root system vigor all has increase, and amplification is respectively 23～29%, 48～57%.Because the increase of nutrition absorption area and absorption intensity, this just guarantees to offer more nutrient of plant and moisture, makes crop synthesize materials such as more albumen, fat, carbohydrate, promotes plant-growth, metabolism, and then improves crop yield.

(3) strengthen the soil nitrogenase activity:

In the potted plant and field test of paddy rice, get the rhizosphere soil sample in different growing and measure nitrogenase activity.Concrete grammar is: every 17 days with in-situ method sampling and measuring ARA, get rhizosphere cylinder soil sample with the copper pipe of diameter 4cm, high 11cm during sampling and put into wide-necked bottle together with native pipe, sealing, extract 10% gas out, change to 10% acetylene gas again, cultivated 24 hours for 30 ℃, survey ARA, each is handled three times and repeats, and establishes and do not add the acetylene contrast.

The ARA value of the soil in experimental plot is compared according to high always, occurs the peak during heading, and the ARA value of test is apparently higher than contrast.

The increase of soil nitrogenase activity must cause the increase of Soil Nitrogen matter.Just supplying with plant nitrogen increases.

(4) Bs, Bm girth growth effect and mechanism thereof:

Side spore bacillus brevis STP-B.lat the rice root table decide grow:

It is generally acknowledged that growing surely in the success of crop rhizosphere is committed step (OKon, 1985 of performance combination azotobacter growth enhancing effect; Bashan 1986).Single inoculation and combined inoculation dual mode are adopted in this work, offside spore bacillus brevis STP-B.lat is the situation of the growing research of deciding of paddy rice rhizosphere, the result shows: side spore bacillus brevis STP-B.lat has stronger colonization ability at the paddy rice rhizosphere, colonization ability (3.2 * 10 ⁷Individual/CM ²) can exceed an order of magnitude (4.85 * 10 than the general function bacterium ⁶Individual/CM ²).After the inoculation, can significantly improve the rice root table decide grow, the thalline of unit root surface-area can increase several times.Plate count is the result show: the side spore bacillus brevis STP-B.lat that grows surely at the rice root table is 8.34 * 10 ⁸The bright root of/gram is heavy.This result and scanning electron microscopic observation be basically identical as a result.

The stimulatory effect that inoculation is grown to the rice root hair:

By the scanning electron microscope result as can be seen, when not inoculating side spore bacillus brevis STP-B.lat, the root hair at rice root tip is slightly lacked, root approximate number amount in the unit surface is less relatively, after side spore bacillus brevis STP-B.lat inoculated, the root hair becomes elongated, and root approximate number amount increases, and illustrates: inoculation side spore bacillus brevis STP-B.lat has certain promoter action to the growth of rice root hair.This result is consistent with the experimental result of OKon (1997) etc.

Many studies show that, side spore bacillus brevis STP-B.lat can secrete plant hormone, as IAA, GA etc., promotes the root system development (Bacate al 1994) of plant.After Brasil diazotrophic spirillum Cd inoculation corn, the root staple length contrasts twice above (Dubrucovsky et al 1994), and we have also obtained similar result with side spore bacillus brevis STP-B.lat inoculation paddy rice.In side spore bacillus brevis STP-B.lat excretory plant hormone, IAA plays a leading role in the growth enhancing effect to plant, the biosynthesizing of IAA mostly will be with Trp as precursor, at root system of plant, the external source Trp that side spore bacillus brevis STP-B.lat utilizes mainly contains two kinds of sources: proteinic degradation production in root exudates and the dead cell.According to surveying and determination, in the root exudates, soil microorganisms is used for the Trp of synthetic IAA and accounts for 0.28～1.0% of root exudates, and Krerchenko etc. (1995) find that plant rhizosphere has the existence of a small amount of Trp.Side spore bacillus brevis STP-B.lat has higher affinity to Trp, and it can effectively utilize Trp to carry out Metabolic activity.Because floristic difference, root system Trp concentration is also inequality, thereby makes synthetic being subjected to and the host plant kind of its associating and the influence of physiological situation of IAA of rhizosphere side spore bacillus brevis STP-B.lat.

In a word, a large amount of results of laboratory have fully confirmed side spore bacillus brevis STP-B.lat root colonization, fixed nitrogen and the effect of IAA synthetic synergistic blend.

The large scale culturing research of embodiment 6 side spore bacillus brevis STP-B.lat

(1) selection of seed and fermention medium

Characteristic according to strains tested, 5 seed culture mediums and 5 fermention mediums have been designed, through seed culture medium and fermention medium are made up in twos, offside spore bacillus brevis STP-B.lat ferments, and the influence that different substratum combinations are given birth to spore and bacterium number to bacterial strain sees the following form.Fast with thalli growth speed, give birth to spore rate height, production cost is low is target, the substratum combination of selecting for use in screening and the definite bacterial strain production.

Living spore situation and the bacterium number of side spore bacillus brevis STP-B.lat bacterial strain under different substratum combination conditions

The substratum combination		Give birth to spore		The bacterium number
						Seed	Fermentation	Hr	％	Direct census	Plate count
2	4	24	＞90		9.0×10 8
						2	4	16	100	5.5×10 8	4.0×10 8
2	4	17	90		1.0×10 9
						8	5	16	100		5.0×10 9
2	5	17	95	1.1×10 9	1.0×10 9
						2	4	16	100		8.0×10 8
8	5			9.3×10 8	1.4×10 9
						2	5				7.0×10 8

The substratum combination of side spore bacillus brevis STP-B.lat bacterial strain production usefulness

Bacterial strain	Side spore bacillus brevis STP-B.lat
		The seed fermentation substratum	No. 8
The bulk fermentation substratum	No. 5

(2) growth curve

Shake bottle greatly under the speed conditions of 30 ± 1 ℃ of temperature and 200rpm, ferment after 6～10 hours, sampling in per 2～3 hours, examining under a microscope thalline changes, measure the pH value, and with every milliliter of contained bacterium number of fermented liquid of microscope direct-counting method mensuration, the result who proofreaies and correct direct-counting method at last according to colony counting method.Test finds, side spore bacillus brevis STP-B.lat during the fermentation, pH rises to 7.9 faster, does not change later on.With determined substratum, the bacterium number of sampling and measuring is made growth curve.

(3) gemma forms curve:

In fermenting process, offside spore bacillus brevis STP-B.lat microscopy, count results are calculated it and are given birth to the spore percentage, can determine the living spore curve of side spore bacillus brevis STP-B.lat under fixed culture condition, as shown in Figure 1.

(4) influence of inoculum size to fermenting:

By the seed bottle when the big bottle graft kind, test with 5% and 10% inoculum size respectively, with the rotating speed of 200rpm after cultivating 15～20 hours on the shaking table, carry out plate count with the gradient dilution method, through the analysis-by-synthesis of data being thought 5% and 10% inoculum size, increment difference in the preceding several hrs of fermentation beginning, but along with the prolongation of fermentation time, the bacterium number that every milliliter of fermented liquid that the different vaccination amount is handled contains reaches unanimity, and has not had difference during to following jar.Therefore, side spore bacillus brevis STP-B.lat bacterial strain aborning can be with 5% inoculum size.Inoculum size is to the following table that influences of bacterium number.

Side spore bacillus brevis STP-B.lat inoculation amount is to the influence of bacterium number

Inoculum size	Substratum	Bacterium number (colony counting method)
			10％	No. 4	1.9×10 8
5％	No. 4	2.1×10 8

(5) salt resistant character:

(NH with 20% and 30% ₄) ₂SO ₄Solution 10ml, immersion side spore bacillus brevis STP-B.lat5～10 days, side spore bacillus brevis STP-B.lat1 milliliter, distilled water immersion is contrast.Then, soak solution added to be had in the 90ml sterilized water triangular flask of granulated glass sphere, shakes half hour with 30～50rpm speed, gets in the sterilized water test tube that 1ml is added on 9ml to shake up, and repeats to be diluted to 10 successively ⁸Doubly, get 10 ⁶, 10 ⁷, 10 ⁸Times each 0.1ml of bacteria suspension evenly is coated with out with triangular scraper in flat board, cultivates 1～2 day counting for 30 ℃, and survival results sees the following form, and can find out that the salt tolerance of side spore bacillus brevis STP-B.lat bacterial strain is better.

(NH ₄) ₂SO ₄Concentration is to the influence of spore surviving rate

	Soaked 7 days		Soaked 14 days
					Bacterial strain	Concentration 20%	Concentration 30%	Concentration 20%	Concentration 30%
STP-B.lat	99.2	98.1	99.2	97.3

Side spore bacillus brevis STP-B.lat is at different concns (NH ₄) ₂SO ₄Growing state is as follows in the substratum, and+expression can be grown, ++ the expression growth is better, and growth is not seen in-expression.

Table 6: different (NH ₄) ₂SO ₄The influence of concentration offside spore bacillus brevis STP-B.lat growth

Incubation time	5％	10％	20％	30％
					24h	++	++	++	++
48h	++	++	++	++

(6) resistance to elevated temperatures:

With 80 ℃ and 100 ℃ is Temperature Treatment, with 1,3,5 hour was to handle the time, with natural storage temperature is contrast, after offside spore bacillus brevis STP-B.lat carries out high temperature resistant processing, 1 milliliter of 99ml sterilized water triangular flask of putting into granulated glass sphere of weighing, shake half hour with 30～50rpm speed, get in the sterilized water test tube that 1ml is added on 9ml and shake up, repeat to be diluted to 10 successively ⁸Doubly, get 10 ⁶, 10 ⁷, 10 ⁸Times each 0.1ml of bacteria suspension evenly is coated with out with triangular scraper in flat board, cultivates 1～2 day counting for 30 ℃.According to count results temperature is analyzed the influence of gemma survival rate.The gemma survival rate is 93～98% substantially in 1～3 hour the processing of 80 ℃ of temperature down as can be seen, handle 5 hours side spore bacillus brevis STP-B.lat survival rates still up to 90～95%, and 100 ℃ of processing are more than three hours, side spore bacillus brevis STP-B.lat survival rate reduces greatly, and only 20～30%.When as seen heating in the production process, gemma can tolerate 80 ℃ of temperature 3 hours.

(7) technical process and controlled variable:

Operational path and controlled variable are as shown in Figure 2.

Inclined-plane and shake-flask culture culture medium prescription:

Peptone 5.9 ‰

Glucose 4.8 ‰

NaCl????????????3.6‰

KCl?????????????0.9‰

MgSO ₄??????????0.24‰

MnCl ₂??????????0.004‰

Yeast powder 0.5 ‰

Agar 20 ‰ (shaking bottle does not add)

CaCO ₃??????????2‰

The pH nature.

Seeding tank and big jar of production culture medium prescription:

Semen Maydis powder 15 ‰

Soybean cake powder 11.45 ‰

K ₂HPO ₄???????1‰

Yeast extract paste 0.7 ‰

CaCO ₃??????????0.5‰

Corn steep liquor 10 ‰

Trace element 1 ‰

Defoamer

The pH nature.

The influence of culture process parameter offside spore bacillus brevis STP-B.lat

Experimental strain: side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat

Experimental result:

Different Ca CO ₃With the influence of the ratio of bacterium to the bacterium number

Bacterial strain	Handle	The dilution gradient			Bacterium number (hundred million)
						10 -6	10 -7	10 -8
		Side spore bacillus brevis STP-B.lat	Fermented liquid	∞					33	3	38
1∶1+CaCO 3	∞				144.3	25	144.3
			1∶3+CaCO 3	∞				76	21	76
Finished product (1: 3)	∞				105	13.3	105

Different substratum are to giving birth to the influence of spore and bacterium number

Substratum	The spore situation is given birth in growth	Handle	Bacterium number (hundred million)
				No. 4	12h is free spore, does not also arrive growth logarithmic phase 17h100% gemma, the bacterium amount +++, nourishing body ++	CK	8
65℃30’	9
		No. 5	The 12h spore that dissociates +++, nourishing body+17h100% gives birth to the spore rate, the bacterium amount ++ ++			CK	27
65℃30’	42
				No. 9	The 12h spore that dissociates ++ +++, minority nourishing body 17h is most to be free spore, nourishing body is given birth to spore rate 100%	CK	30
65℃30’	51

Foam killer bubble enemy, methyl-silicone oil are to the influence of fermentation culture

Handle	Concentration	Growth and living spore situation	Bacterium number (hundred million)
				The bubble enemy	?0.1％	28h bacterium amount ++, give birth to spore rate 15～20%, free individually 46h bacterium amount ++, nourishing body 80%, give birth to spore and ionization rate each 20%	27
?0.5％	With 0.1%	31
			?0.01％		28h bacterium amount +++, give birth to spore rate 40～45%, the free spore 40～45% of 3～5% free spore 46h, nourishing body and living spore rate respectively account for 20%	30
?0.05％	Close with 0.01% situation	42
			Methyl-silicone oil		?0.1％	The 28h bacterium is measured +++, give birth to spore rate 30～40%, the free spore 50～60% of free individually 46h, nourishing body and living spore rate respectively account for 20%	37
?0.5％	The same close	33
				?0.01％	28h bacterium amount +++, give birth to spore rate 30～40%, the free 46h bacterium amount of minority +++, nourishing body 50～60%, free spore 40%	46
CK		28h bacterium amount ++, give birth to the free spore 60% of spore rate 65～75% 46h, nourishing body includes spore rate 100%					32

The bacterial classification continuous passage is to the influence of bacterium number and living spore

Algebraically goes down to posterity	Growth and living spore situation	The bacterium amount
			1√	22h gives birth to the spore rate more than 98%	+++
2
			3√	21h gives birth to the spore rate more than 95%	+++
4
			5
6√	24h gives birth to the spore rate more than 95%	+++
			7
8
			9√	24h gives birth to the spore rate more than 95%	+++
10
			11
12√	24h gives birth to the spore rate more than 95%	+++
			13

Fermentation algebraically is to the influence of bacterium number and living spore

Algebraically goes down to posterity	Growth and living spore situation	The bacterium amount	Remarks
				1	20h gives birth to the spore rate more than 90%	+++
2	21h gives birth to the spore rate more than 90%	+++
				3	21h gives birth to the spore rate more than 90%	+++
4	21h gives birth to the spore rate more than 90%	+++
				5	25h gives birth to the spore rate more than 90%	+++	Give birth to spore rate 50～60% during 21h
6	24h gives birth to the spore rate more than 95%	+++
				7	26h 80～90% gives birth to the spore rate	++	It is not too neat to give birth to spore
8	28h 75～80% gives birth to the spore rate	++-	The bacterium amount reduces relatively

Fermented liquid adds in trace element and the fermenting process Ensure Liquid liquid to the influence of life spore bacterium number

Handle	Concentration	Growth and living spore situation	The bacterium amount
				Fermented liquid adds trace element	Do not add CK	21h?80～90％	+++
0.1％	21h?90～95％	+++
			0.2％		21h 95% gives birth to the spore rate	++
0.3％	21h gives birth to the spore rate more than 95%	++
			Nutrition (original formulation nutrition 10 times) behind the fermentation 24h		The Ensure Liquid primary fermentation liquid status spore rate 100% of making a living	The 15h nourishing body increases, and mostly is free spore	+++
The 24h thalline is few, and gemma is also few, and assorted bacterium is arranged
		The 45h bacterium rate 50% of mixing is difficult to estimate give birth to the spore situation

Claims (10)

1. a side spore bacillus brevis (Brevibacillus laterosporus) STP-B.lat, its preserving number is CGMCC No.1119.

2. a strain inclined plane that contains microorganism is characterized in that, described microorganism is the described side spore of claim 1 bacillus brevis (Brevibacillus laterosporus) STP-B.lat.

3. strain inclined plane as claimed in claim 2 is characterized in that, it uses the storage medium of being made by the reagent of following weight parts:

Peptone 5.9 ‰

Glucose 4.8 ‰

NaCl??????????3.6‰

KCl???????????0.9‰

MgSO ₄????????0.24‰

MnCl ₂????????0.004‰

Yeast powder 0.5 ‰

Agar 20 ‰

CaCO ₃????????2‰

The pH nature.

4. a fermenting process is characterized in that, the bacterial classification that it uses is the described side spore of claim 1 bacillus brevis (Brevibacillus laterosporus) STP-B.lat.

5. process as claimed in claim 4 is characterized in that, it uses the substratum of being made by the reagent of following weight parts:

Peptone 5.9 ‰

Glucose 4.8 ‰

NaCl??????????3.6‰

KCl???????????0.9‰

MgSO ₄????????0.24‰

MnCl ₂????????0.004‰

Yeast powder 0.5 ‰

CaCO ₃??????2‰

The pH nature.

6. process as claimed in claim 4 is characterized in that, it uses the substratum of being made by the reagent of following weight parts:

Semen Maydis powder 15 ‰

Soybean cake powder 11.45 ‰

K ₂HPO ₄???1‰

Yeast extract paste 0.7 ‰

CaCO ₃??????0.5‰

Corn steep liquor 10 ‰

Trace element 1 ‰

Defoamer is an amount of

The pH nature.

7. a tunning is characterized in that, it contains the described side spore of claim 1 bacillus brevis (Brevibacillus laterosporus) STP-B.lat.

8. a bio-feritlizer is characterized in that, it contains the described side spore of claim 1 bacillus brevis (Brevibacillus laterosporus) STP-B.lat, perhaps contains the described tunning of claim 7.

9. the described side spore of claim 1 bacillus brevis (Brevibacillus laterosporus) STP-B.lat has application in the bio-feritlizer that promotes plant-growth and/or fixed nitrogen function in preparation.

10. the described side spore of claim 1 bacillus brevis (Brevibacillus laterosporus) STP-B.lat has application in the bio-feritlizer of parallel off fungi, nematode function in preparation.

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