CN102399713A - Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving - Google Patents
Bacillus subtilis HL-1 and application thereof in respect of soil phosphate dissolving Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis HL-1 and an application thereof in the respect of soil phosphate dissolving. The strain is collected in China General Microbiological Culture Collection Center (CGMCC) on Aug 24th, 2011, with a collection number of CGMCC No.5175. The 16SrDNA nucleotide sequence of the strain is represented by SEQ ID NO:1. With added diatomite and guava slag adsorbing agent materials, a fermentation broth of the bacillus subtilis HL-1 can be prepared into microbial manure. With the strain provided by the invention, phosphate availability and phosphate fertilizer utilization rate of the soil can be improved, plant growth can be promoted, and soil microbe diversity can be improved. The strain has important significance in reducing chemical fertilizer application, modifying soil micro-ecology, and promoting soil sustainable utilization. The strain has wide application prospect in production practice.
Description
Technical field
The present invention relates to a kind of mikrobe and application thereof, be specifically related to a kind of subtilis (
Bacillus subtilis) HL-1 and decomposing the soil insoluble phosphorus and improving the soil micro-ecosystem structure and the application of aspect such as promotion plant-growth.
Background technology
Phosphorus is one of necessary nutritive element of growth and development of plants, and synthetic and respiration all has material impact to nitrogen fixation, photosynthesis, energy transfer, signal transduction, biomacromolecule.5,700,000,000 hm are arranged in the world
2Soil lacks phosphorus, and phosphorus concentration is from 0.1 ~ 10 μ mol/L in the most of soils, and under perfect condition, the required phosphorus concentration of grass growth is 1-5 μ mol/L, and high to need phosphorus plant such as tomato, pea then be 5 ~ 60 μ mol/L.Do not reach under the perfect condition at phosphorus, plant is with the underproduction 5 ~ 15%.The use of traditional chemical fertilizer can alleviate the shortage of phosphorus in the soil to a certain extent; But as the principal item superphosphate of lime of Chinese phosphate fertilizer and phosphorus ammonium etc.; Still be southern acid soil no matter at northern calcareous soil; Because of the solid phosphorus effect of soil intensive, this season utilization ratio less than 20% causes the wasting of resources.There is 74% the scarce phosphorus of arable soil in China, and the phosphorus in the soil more than 95% is the indissoluble form, and plant is difficult to absorb.And very easily by chemical fixation, form compounds such as the extremely low calcium phosphate of solvability, iron, aluminium after phosphate fertilizer is manured into soil, the plant utilization rate is merely 5 ~ 25%.Even through constantly also not significantly improvement of application of P fertilizer for many years.Increasing the p application rate is the approach of a kind of " high investment, low output ".Simultaneously, the phosphate fertilizer scarcity of resources of China, throughput is difficult to satisfy the demands.Phosphoric in the soil also has no lack of, and through use the accumulation of chemical fertilizer throughout the year, the content of phosphoric in soil is very high, but plant can't absorb these phosphorus, if can improve the utilization ratio of phosphorus in the soil and will bring great variety for Chinese agriculture.Therefore, improve the phosphate fertilizer utilising efficiency, strengthen the activation of soil phosphorus and utilize the vital task that becomes scientific research.
Phosphate solubilizing microorganism is one type of peculiar microorganism function colony that can invalid phosphorus be converted into the available phosphorus that plant can absorb in the soil; Phosphate fertilizer be can improve and phosphate fertilizer utilization efficiency and dissolving soil indissoluble phosphorus fixed, improve; Played keying action in the circulation plain change of natural and agroecological phosphorus biologically, by universally acknowledged for being a kind of environment protection type soil phosphorus activation biological control measure of Cheap highly effective.They are converted into invalid phosphorus through modes such as acidolysis, chelating, displacement and secretion Phosphoric acid esterases can (mainly be HPO by the available phosphorus of plant utilization
4 2-And H
2PO
4 -Form).Big quantity research shows, use the microbial fertilizer that contains phosphate solubilizing bacteria to promote plant-growth, to improve the Soil structure effect remarkable.Utilize to dissolve the phosphorus mikrobe to the utilization ratio of the validity that improves phosphate fertilizer and soil indissoluble phosphorus, reduce the phosphate fertilizer input, production-increasing function and the agricultural sustainable development of bringing into play the limited phosphorus ore of China be significant.In view of the molten phosphorus ability between different types of phosphorus-solubilizing bacteria or the different strains has than big-difference, the screening operation of efficient phosphorus-solubilizing bacteria is particularly important.
The phosphorus decomposing microbial inoculum that is used to make bio-feritlizer at present generally uses vermiculite, the peat composed of rotten mosses, perlite and zeolite powder etc. as absorption carrier; These materials are all Nonrenewable resources; If can seek a kind of renewable resources, make it can partially or completely replace Nonrenewable resources commonly used at present, not only play the effect that economizes on resources; And can promote the recycle of agricultural wastes, thereby reach the purpose of agricultural sustainable development.The piscidia slag is the sub product of the piscidia course of processing, and fibre content is high, in removing waste water, has obtained unusual effect and application prospect aspect dyestuff and the removal heavy metal contamination as sorbent material.Piscidia slag outward appearance and microbial bacteria agent carrier peat are similar, and it is brown brown that quality gentlier shows, and piscidia slag water cut is about 4 ~ 5%, has than the low advantage of peat (about water cut 30%) water cut.Yet, using renewable resources---the piscidia slag replaces Nonrenewable resources commonly used as bio-fertilizer absorbent material, processes the microbial inoculum with efficient phosphorus decomposing effect, does not appear in the newspapers.
Summary of the invention
The objective of the invention is to provides a kind of subtilis according to above-mentioned deficiency of the prior art.
Another object of the present invention provides the application of above-mentioned bacterial strains.
The present invention realizes above-mentioned purpose through following technical scheme:
A kind of subtilis (Bacillus subtilis) HL-1 is called for short the HL-1 bacterium.The classification called after of this bacterial strain: subtilis Bacillus subtilis; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on August 24th, 2011; Deposit number is CGMCC No.5175; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
This bacterial strain screening is gram-positive microorganism in the soil of stove mountain, GuangZhou, Guangdong Province city, on beef-protein medium, cultivate 12h, examines under a microscope thalline with single or paired arrangement; Gemma quantity few (3 ~ 5%) is cultivated 24h on beef-protein medium, thalline is given birth in the gemma or nearly middle giving birth to single, paired or catenation; Gemma is several 80 ~ 90%, and gemma forms the back thalline and do not expand, and thalline is shaft-like; No pod membrane has mobility, and is aerobic.Cultivating the bacterium colony that forms behind the 24h on the nutrient agar is circular, little yellow, and surface irregularity is opaque, and the edge is irregular.
Through sequencing, the 16SrDNA nucleotide sequence of this subtilis HL-1 is shown in SEQ ID NO:1.
The microbial inoculum of processing by above-mentioned subtilis HL-1.
The preparation method of subtilis HL-1 microbial inoculum is to seed culture medium, after shaking table under 30 ℃ of culture temperature, the rotating speed 113rpm condition is cultivated 24h with the activatory inoculation; Obtain primary seed solution; Be transferred to seed tank culture by 10 ~ 15% inoculum sizes then, under 30 ℃, air flow 1000L/h, rotating speed 70rpm condition, cultivate 24h, obtain secondary seed solution with secondary seed medium; Secondary seed solution is inoculated into fermentor tank by 10 ~ 15% inoculum sizes; Cultivate (being enlarged culturing) in the continuous fermentor tank deep layer of fermention medium relaying, culture condition is 30 ℃, air flow 10000L/h, rotating speed 70rpm, cultivates 24h.Fermented liquid after the cultivation mixes fermented liquid with zeyssatite and piscidia slag: zeyssatite: the mass ratio of piscidia slag is 0.6:1:1; After stirring, pulverize, be prepared into said microbial inoculum.
Wherein every 1L seed culture medium contains Carnis Bovis seu Bubali cream 5g, peptone 5g, sodium-chlor 5g, and remainder is a water, pH value 7.1 ~ 7.4; Seed culture medium is through 115 ~ 121 ℃ of sterilization 20min, and is subsequent use.
Every 1L secondary seed medium and fermention medium contain bean cake powder 30g, wheat bran powder 10g, an ammonium nitrate 1.5g, yeast extract paste 3g, sodium-chlor 2.5g, sal epsom 0.2g, potassium hydrogenphosphate 0.3g and manganous sulfate 0.05g, and remainder is a water, pH value 7.1 ~ 7.4.
The concrete preparation process of subtilis HL-1 microbial inoculum is following:
(1) first order seed is cultivated
Seed culture based component: Carnis Bovis seu Bubali cream 5g/L, peptone 5g/L, sodium-chlor 5g/L, zero(ppm) water 1L, pH value 7.1 ~ 7.4.115 ~ 121 ℃ of sterilizations of substratum 20min, the HL-1 bacterium after the access activation, shaking table is cultivated.Culture condition: 30 ℃, rotating speed 113rpm, time 24h.
(2) secondary seed fermentation culture
Secondary seed medium composition: bean cake powder 30g, wheat bran powder 10g, an ammonium nitrate 1.5g, yeast extract paste 3g; Sodium-chlor 2.5g, sal epsom 0.2g, potassium hydrogenphosphate 0.3g, manganous sulfate 0.05g; Water 1L, regulating the pH value is 7.1 ~ 7.4,121 ℃ of sterilization 20min; Wait to be cooled to 30 ℃, insert primary seed solution, inoculative proportion is 1:10 (a 10L secondary seed medium inoculation 1L primary seed solution).It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 10000L/h, and tank pressure remains on 0.02 ~ 0.03MPa.
(3) fermentor tank enlarged culturing
The same secondary seed medium of fermentation culture based component.In the time of 30 ℃, insert secondary seed solution, inoculative proportion is 1:10.It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 10000L/h, and tank pressure remains on 0.02 ~ 0.03MPa.
(4) interpolation of absorption carrier
Bacterium liquid, zeyssatite, piscidia slag after step (3) cultivation are mixed in stirrer by mass ratio 0.6:1:1, process microbial inoculum after the pulverizing.
(5) packing of microbial inoculum and storage
Take by weighing microbial inoculum and be sub-packed in the aluminium matter bag, air seasoning place is deposited in sealing.
Subtilis HL-1 of the present invention is decomposing insoluble phosphorus, promoting plant-growth and/or is increasing the soil microbial community variety and increasing the application in the mikrobe abundance.
Compared with prior art, the present invention has following beneficial effect:
The contriver filters out subtilis through a large amount of experimental studies
Bacillus subtilisHL-1.This bacterial strain not only can improve the validity and the phosphate fertilizer utilization efficiency of indissoluble phosphorus in the soil, thereby promotes plant-growth, improves the soil microorganisms variety, to reducing chemical fertilizer application, improves soil micro-ecosystem and promotes the soil sustainable use to have great importance.
With beans cypress wheat bran etc. is the fermention medium of staple; It is simple to have preparation technology, effective, low cost and other advantages; And use renewable resources---piscidia slag is as absorption carrier, has economizing on resources, turns waste into wealth, promotes the advantages such as recycle of agricultural wastes.
Description of drawings
Fig. 1. HL-1 bacterium gramstaining Photomicrograph;
Fig. 2. HL-1 bacterium spore staining Photomicrograph, figure Smalt partly is gemma;
Fig. 3. different strains produces the comparison diagram of water-soluble phosphorus content under the calcium phosphate substratum, X-coordinate is a strain number, and ordinate zou is a water-soluble phosphorus concentration;
Fig. 4. the comparison diagram of different strains producing microbial phosphorus content under the calcium phosphate substratum, X-coordinate are strain number, and ordinate zou is a microbe-P concentration;
Fig. 5. for the HL-1 bacterium promotes corn growth field effect contrast figure;
Fig. 6. different strains is handled the graphic representation that soil microbial community metabolic activity AWCD value changes, and wherein HL-1 is the HL-1 bacterium, and CK is a control group.
Embodiment
Separation, purifying and the evaluation of embodiment 1 subtilis HL-1
(1) separates
Preparation indissoluble inorganic phosphorus substratum: glucose 10.0g, ammonium sulfate 0.5g, sodium-chlor 0.3g, Repone K 0.3g, iron vitriol 0.03g, four water manganous sulfate 0.03g, yeast extract paste 0.4g, calcium phosphate 5g, zero(ppm) water 1L, pH value 7.0 ~ 7.5.
With the soil of gathering on the stove mountain, Tianhe District, Guangzhou City, Guangdong Province is the screening soil sample; Adopt spread plate accurately to take by weighing soil sample 10.0g to be measured, put into the 250mL triangular flask (adding granulated glass sphere) that the 90mL sterilized water is housed, shaking table vibration 30min; Mikrobe is fully disperseed; Leave standstill 20 ~ 30s, get supernatant and carry out 10 times of dilutions of successively decreasing, adopt 10
3~ 10
5Extent of dilution pipettes diluent 0.1mL respectively with liquid-transfering gun, is coated on the indissoluble inorganic phosphorus culture medium flat plate, is inverted for 28 ℃ and cultivates 7d, observes day by day and dissolves the phosphorus circle, record phosphorus-solubilizing bacteria bacterium colony (d) and molten phosphorus loop diameter (D).The bacterial isolates that filters out the phosphorus decomposing circle is numbered a-4, a-7, and a-15, and b-17, wherein the phosphorus decomposing effect of a-15 is the strongest.After molecular biology identification and Physiology and biochemistry are identified, confirm the a-15 bacterial strain be subtilis (
Bacillus subtilis).
(2) purifying
Nutrient agar: Carnis Bovis seu Bubali cream 3.0g, peptone 5.0g, sodium-chlor 5.0g, agar 18.0g, zero(ppm) water 1L, pH value 7.0 ~ 7.2.
After the bacterial strain that filters out utilized the plate streak purifying, be stored on the nutrient agar slant medium, be called for short the HL-1 bacterium among the present invention.
Embodiment 2 CHARACTERISTICS IDENTIFICATION
(1) morphological features
Gram-positive microorganism has gemma, and gemma forms the back thalline and do not expand.Thalline is shaft-like, and no pod membrane has mobility, and is aerobic.The gramstaining effect is seen Fig. 1, and thalline is dyed bluish voilet, and gemma is not painted.The spore staining effect is seen Fig. 2, and thalline is yellowish brown, and gemma is light blue.
(2) colony morphology characteristic
The bacterium colony that on nutrient agar, forms is circle, little yellow, and surface irregularity is opaque, and the edge is irregular.
(3) physio-biochemical characteristics
According to Biolog microorganism identification appearance, analyze this bacterial strain situation of utilizing to 31 kinds of different carbon sources under aerobic condition, the result sees table 1.
Table 1 HL-1 bacterium is to the situation of utilizing of 31 kinds of different carbon sources
| Characteristic | The result | Characteristic | The result |
| Water | + | Methyl D-glucoside | + |
| D-semi-lactosi lactone | + | D-semi-lactosi lactone | + |
| The pyruvic acid formicester | + | The D-wood sugar | + |
| The D-galacturonic acid | + | Altheine acid | + |
| Polysorbate40 | + | The I-erythritol | + |
| 2-oxybenzene formic acid | - | The L-phenylalanine | + |
| Tween 80 | - | D-N.F,USP MANNITOL | |
| The 4-hydroxy-benzoic acid | + | The L-Serine | + |
| The a-cyclodextrin | + | N-ethanoyl-D-grape amine | + |
| Gamma-hydroxybutyric acid | + | The L-Threonine | + |
| Glycogen | + | D-grape amino acid | + |
| Methylene-succinic acid | + | Glycyl-L-glutamic acid | + |
| The D-cellobiose | + | Cori ester salt | + |
| The a-batanone acid | + | Styroyl amine | + |
| The a-D-lactose | + | D, L-a-glycerine | + |
| The D-oxysuccinic acid | + | Putrescine | + |
Annotate :+be expressed as the positive maybe can utilize;-be expressed as feminine gender maybe can not utilize
(4) molecular biological characteristic
Adopt the test kit method to extract bacteria total DNA.Adopt bacterial 16 S rDNA universal primer F63 (CAG GCC TAA CAC ATG CAA GTC; SEQ ID NO:2) and R1087 (CTC GTTGCG GGA CTT ACC CC, SEQ ID NO:3), the 16S rDNA of pcr amplification bacterium; Tangible band appears near 1000bp; With carrying out sequencing after the pcr amplification product recovery, the dna sequence dna input GenBank with obtaining compares analysis with the Blast program to all sequences in the DB; The result find bacterial strain of the present invention 16S rDNA sequence and with GenBank in subtilis have higher homology, its similarity is 99%.In conjunction with the result of above-mentioned flat-plate bacterial colony characteristic, Biolog microplate cultivation results, 16S rDNA sequence and with reference to " the outstanding Bacteria Identification handbook of uncle is carried out the Physiology and biochemistry experiment, identify this bacterial strain be subtilis (
Bacillus subtilis), called after
Bacillus subtilisHL-1.
The growth characteristics of embodiment 3 HL-1 bacterium under different carbon source environment
Beef-protein medium: Carnis Bovis seu Bubali cream 5g, peptone 5g, sodium-chlor 5g, water 1000ml, pH are 7.1 ~ 7.4.
Respectively with Semen Maydis powder, wheat bran, glucose, peanut press pulp, sucrose, rice bran and 7 kinds of materials of plant amylum as carbon source, concentration is 20g/L, substitutes the Carnis Bovis seu Bubali cream in the beef-protein medium, other components unchanged.Above-mentioned 7 kinds of substratum are boiled 10min, be cooled to room temperature naturally, using the NaOH of 1mol/L or HCl adjusting pH value is 7.1 ~ 7.5, is prepared into fermention medium.Substratum 45mL is respectively organized in packing in the 250mL triangular flask, sterilization, and each is organized substratum and does three repetitions.Inoculum size according to 10% is inoculated into the fermention medium after the above-mentioned sterilization with the HL-1 bacterium, 33 ℃ of temperature, cultivates 24h under the rotating speed 113rpm condition, measures with the method for plate culture count and respectively organizes the viable count in the substratum.The result shows (table 2): the HL-1 bacterium is being under the environment of carbon source with wheat bran, and its growth characteristics are best, and viable count is 5.44 ± 0.22 * 10
8Cfu/mL.
In the fermention medium that with the different substances is carbon source, the grow viable count (* 10 of 24h of table 2:HL-1 bacterium
8Cfu/mL)
| Carbon source | Semen Maydis powder | Wheat bran | Glucose | Peanut press pulp | Sucrose | Rice bran | Plant amylum |
| Viable count | 3.02 ± 0.28d | 5.44 ± 0.22a | 1.20 ± 0.05e | 0.61 ± 0.03f | 4.90 ± 0.38b | 5.14 ± 0.24ab | 4.24 ± 0.51c |
Annotate: data are 5 multiple MV ± standard errors, and the different alphabetical persons of colleague in the table are illustrated in 0.05 level difference significantly (DMRT method).
The growth characteristics of embodiment 4 HL-1 bacterium under the different fermentations culture medium condition
Choose 6 kinds of different medium components and carry out fermentation culture respectively, table 3 is seen in concrete experimental design.6 kinds of substratum in the table 3 are boiled 10min respectively, be cooled to room temperature naturally, using the NaOH of 1mol/L or HCl adjusting pH value is 7.1 ~ 7.4.Accurately measuring 45mL with graduated cylinder cultivates based on the 250mL triangular flask, at 115 ~ 121 ℃ of following high-temperature sterilization 20min.From flat board, the good HL-1 bacterium of activation (method is with the purifying the embodiment 1) is inoculated in the 50mL seed culture medium, places shaking table to cultivate 33 ℃ of temperature, rotating speed 113rpm.Shaking table is seed liquor after cultivating 24h.Get the 5mL seed liquor, the inoculum size according to 10% is inoculated into the fermention medium after the sterilization among the embodiment 3, after shaking table is cultivated 24h, manages the fermented liquid viable count everywhere with the method for plate culture count mensuration, two dilution gradients, and each extent of dilution repeats for three times.Measure result such as table 4, the result shows that the viable count of HL-1 bacterium in No. 4 substratum is the highest, and the viable count of fermentation 24h is 15.03 ± 1.30 * 10
8Cfu/mL.
The experimental design of table 3 fermentation culture
| | Composition | PH | |
| 1 | Dregs of beans 10.0g, starch 32.1g, yeast extract paste 0.2g, potassium hydrogenphosphate 0.3g, sodium-chlor 0.01g, water 1L | 7.1 ~ 7.4 | |
| 2 | Glucose 12g, Semen Maydis powder 18g, dregs of beans 30g, potassium hydrogenphosphate 2g, water 1L | 7.1 ~ 7.4 | |
| 3 | Semen Maydis powder 30g, dregs of beans 30g, sodium-chlor 5g, lime carbonate 1.5g, potassium hydrogenphosphate 0.2g, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.03g, water 1L | 7.1 ~ 7.4 | |
| 4 | Dregs of beans 30g, wheat bran powder 10g, an ammonium nitrate 1.5g, yeast extract paste 3g, sodium-chlor 2.5g, sal epsom 0.2g, potassium hydrogenphosphate 0.3g, manganous sulfate 0.05g, water 1L | 7.1 ~ 7.4 | |
| 5 | Starch 10.0g, sucrose 5.0g, potassium hydrogenphosphate 5.0g, sal epsom .5.0g, yeast extract paste 2.0g, ammonium sulfate 1.0g, dregs of beans 2.0g, zinc sulfate 1.0g, water 1L | 7.1 ~ 7.4 | |
| 6 | Peptone 5.0g, Carnis Bovis seu Bubali cream 5.0g, W-Gum 20g, potassium hydrogenphosphate 0.3g, sal epsom 0.1g, sodium-chlor 5.0g, water 1L | 7.1 ~ 7.4 |
In different substratum, the ferment viable count (* 10 of 24h of table 4:HL-1 bacterium
8Cfu/mL)
| | 1 | 2 | 3 | 4 | 5 | 6 |
| Viable count | 3.37 ± 0.47d | 3.60 ± 0.44cd | 11.67 ± 0.81b | 15.03 ± 1.30a | 4.87 ± 0.45c | 2.93 ± 0.42d |
Annotate: data are 3 multiple MV ± standard errors, and the different alphabetical persons of colleague in the table are illustrated in 0.05 level difference significantly (DMRT method).
The preparation of embodiment 5 HL-1 bacteria agents
(1) first order seed is cultivated
Seed culture based component: Carnis Bovis seu Bubali cream 5g/L, peptone 5g/L, sodium-chlor 5g/L, zero(ppm) water 1L, pH value 7.1 ~ 7.4.115 ~ 121 ℃ of sterilizations of substratum 20min, the HL-1 bacterium after the access activation, shaking table is cultivated.Culture condition: 30 ℃, rotating speed 113rpm, time 24h.
(2) secondary seed is cultivated
Secondary seed medium composition: bean cake powder 30g, wheat bran powder 10g, an ammonium nitrate 1.5g, yeast extract paste 3g; Sodium-chlor 2.5g, sal epsom 0.2g, potassium hydrogenphosphate 0.3g, manganous sulfate 0.05g; Water 1L, regulating the pH value is 7.1 ~ 7.4,121 ℃ of sterilization 20min; Wait to be cooled to 30 ℃, insert primary seed solution, inoculative proportion is 1:10.It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 1000L/h, and tank pressure remains on 0.02 ~ 0.03MPa.
(3) fermentor tank enlarged culturing
The same secondary seed medium of fermentation culture based component.In the time of 30 ℃, insert secondary seed solution, inoculative proportion is 1:3.3.It is 30 ℃ that leavening temperature is set, and rotating speed is 70rpm, and air flow is 10000L/h, and tank pressure remains on 0.02 ~ 0.03MPa.
(4) interpolation of sorbent material
Bacterium liquid, zeyssatite and piscidia slag after step (3) cultivation are mixed in stirrer by mass ratio 0.6:1:1, process microbial inoculum after the pulverizing.
(5) packing of microbial inoculum and storage
Take by weighing the 10.0g microbial inoculum and be sub-packed in the aluminium matter bag, air seasoning place is deposited in sealing.
Embodiment 6 HL-1 bacterium are with the growth characteristics of different sorbent materials as absorption carrier
(1) with single zeyssatite as sorbent material
600mL fermented liquid (embodiment 5 step 3 gained bacterium liquid) and 2kg sterilization zeyssatite uniform mixing are prepared into the dry-powdered microbial inoculum, are positioned over shady and cool sections temperature and preserve.Respectively at the 7th, 14,20,26,31,37d takes by weighing microbial inoculum 10.00g and joins in the 100mL sterilized water that granulated glass sphere is housed, and leaves standstill 20min, the 200rpm 30min that fully vibrates on rotary shaking table measures the viable count of different number of days with the method for plate culture count.The result shows (table 5): the bacterium number is on a declining curve along with the increase of fate, and the viable count of 37d is 0.49 ± 0.06 * 10
8Cfu/mL.
Table 5 zeyssatite is cultivated the viable count (* 10 of HL-1 bacterium in different number of days
8Cfu/mL)
| Fate (d) | 7 | 14 | 20 | 26 | 31 | 37 |
| Viable count | 1.61 ± 0.09 | 0.91 ± 0.06 | 0.80 ± 0.11 | 0.61 ± 0.03 | 0.62 ± 0.03 | 0.49 ± 0.06 |
Annotate: data are 3 multiple MV ± standard errors.
(2) with piscidia slag and zeyssatite mixture as sorbent material.
600mL fermented liquid (embodiment 5 step 3 gained bacterium liquid), 1kg sterilization zeyssatite and 1kg sterilization piscidia slag uniform mixing are prepared into the dry-powdered microbial inoculum, are positioned over shady and cool sections temperature and preserve.Respectively at the 7th, 14,20,26,31,37d takes by weighing microbial inoculum and gets the 10.00g sample and join in the 100mL sterilized water that granulated glass sphere is housed; Leave standstill 20min; The 200rpm 30min that fully vibrates on rotary shaking table; Process into the mother liquor bacteria suspension, measure the viable count of different number of days with the method for plate culture count.The result shows (table 6): as absorption carrier, the viable count of HL-1 bacterium is also more satisfactory with piscidia slag and zeyssatite mixture, and the viable count of 37d is 0.29 ± 0.04 * 10
8Cfu/mL.
Table 6 zeyssatite and piscidia slag are cultivated the viable count (* 10 of HL-1 bacterium different number of days
8Cfu/mL)
| Fate (d) | 7 | 14 | 20 | 26 | 31 | 37 |
| Viable count | 1.80 ± 0.16 | 0.54 ± 0.04 | 0.56 ± 0.08 | 0.48 ± 0.04 | 0.32 ± 0.04 | 0.29 ± 0.04 |
Annotate: data are 3 multiple MV ± standard errors.
(3) HL-1 fermented liquid among the embodiment 5 is packed in the aseptic plastic bottle, seal and be placed in 4 ℃ of refrigerators, respectively the thalline quantity the 1st, 5,10,18, in the 31d analytical unit volume of liquid microbial inoculum.The result shows (table 7): fermented liquid is 2.24 ± 0.07 * 10 at the viable count of 31d
9Cfu/mL.
Table 7 liquid HL-1 microbial inoculum is at the viable count (* 10 of different number of days
9Cfu/mL)
| Fate (d) | 1 | 5 | 10 | 18 | 31 |
| Viable count | 2.76 ± 0.10 | 2.47 ± 0.05 | 2.07 ± 0.06 | 2.29 ± 0.12 | 2.24 ± 0.07 |
Annotate: data are 3 multiple MV ± standard errors.
The effect experiment of the product water-soluble phosphorus of embodiment 8 HL-1 bacterium under insoluble inorganic phosphorus culture medium culturing
The sterilized indissoluble inorganic phosphorus of the 50mL substratum (concentration of calcium phosphate is 5.0g/L) of packing in the triangular flask; Add the a-4 that separating step filtered out, a-7, a-15 (being the HL-1 bacterium) and b-17 bacterium (inoculum size 5 rings) among the embodiment 1 respectively; Process behind the bacterium liquid 28 ℃; 70rpm, shaking table is cultivated 7d.With the bacterium liquid after cultivating at 10000rpm, 4 ℃ of centrifugal 15min; (unit is mg/L with molybdenum antimony resistance colorimetric method mensuration water-soluble phosphorus content to get supernatant; The milligram number of promptly representing contained water-soluble phosphorus in every liter of sample), establish the substratum that does not connect the HL-1 bacterium and be contrast, each handles repetition 3 times.Test-results is seen Fig. 3.The result shows: the water-soluble phosphorus content of HL-1 bacterium is the highest, reaches 63.84mg/L.
The effect experiment of the producing microbial phosphorus of embodiment 9 HL-1 bacterium under insoluble inorganic phosphorus culture medium culturing
Deposition behind embodiment 8 medium centrifugals is handled (the 5mg N,O-Diacetylmuramidase joins in the 1mL TEN solution) with lysate; 37 ℃ of water-bath 1h; 4 ℃ then; The centrifugal 15min of 10000rpm gets supernatant and measures microbe-P content (unit is mg/L, promptly representes the milligram number of contained microbe-P in every liter of sample) with molybdenum antimony resistance colorimetric method.Test-results is seen Fig. 4.The result shows: the microbe-P content of HL-1 bacterium is the highest, reaches 32.58mg/L.
Embodiment 10 HL-1 microbial inoculum promoting growth of plants field effect experiments
Adopt HL-1 bacterium and blank to carry out the effect comparison test; Being divided into 2 treatment group (gets 3.0kg soil for the 1st group and adds HL-1 fermented liquid 3mL; Represent with HL-1; Get HL-1 fermented liquid 3mL that 3.0kg soil adds deactivation as blank, represent for the 2nd group with CK), each treatment group repeats 4 times.Each treatment group soil adds urea 326mg, calcium phosphate 145mg and Repone K 238mg respectively.Corn washing vernalization is soaked into preservation moisture with filter paper, is placed in 30 ℃ of incubators, exposes the bud sowing two days later to the seedbed.About 25cm is high when seedling, transplants seedlings to basin 2 seedlings of every basin.Water every day in right amount, the water yield of every basin is identical, evenly, notes not making water to flow out at the bottom of the basin in order to avoid fertilizer loss and cause error.Measured corn plant height (the most advanced and sophisticated height of straight posterior lobe is smoothed out with the fingers to maize leaf in corn ground) on May 11st, 2011.Measured maize leaf (comprising all blades that grow) on May 9th, 2011.The result shows (table 8): add the HL-1 bacterium and can significantly promote the release of soil available phosphorus and the growth of corn.Compare with contrast, using HL-1 microbial inoculum soil available phosphorus content has increased by 48%.
Table 8 different strains phosphorus decomposing effect reaches the short fruit contrast table that comes into force to corn
| Treatment group | Plant height (cm) | The number of blade (individual) | Soil available phosphorus (mg/kg) | The soil available phosphorus increasing amount |
| HL-1 | 85.7 ± 9.8 a | 6.3 ± 0.8 a | 3.7 ± 0.2 a | 48% |
| CK | 80.9 ± 9.2a | 4.9 ± 0.6b | 2.5 ± 0.3 b |
Annotate: data are 4 multiple MV ± standard errors, and the different alphabetical persons of same column in the table are illustrated in 0.05 level difference significantly (DMRT method).
Embodiment 11 HL-1 bacterium are to the improved effect test of soil microorganisms functional diversity
Concrete operations are following: take by weighing the fresh soil that is equivalent to 10g oven dry soil sample and join in the 250mL triangular flask that 100mL sterile saline (0.85%) is housed; Behind concussion 1min on the vortex concussion machine, in ice-water bath, leave standstill 1min; Repeat 3 times; Leave standstill 2min; Getting the above-mentioned soil extraction of 5mL adds in the 45mL sterile saline (0.85%); Behind the mixing; Repeat this step, the bacterium liquid (the bacterium liquid after the embodiment 5 step 3 gained fermentation culture) after diluting 1000 times is added in the Biolog Eco plate, every hole adds 150 μ L the microplate of inoculation is cultivated at 30 ℃ of incubators; With the soil that do not add bacterial classification as blank, respectively at 0,24,48,72,96h reads the multifarious formula of light absorption value calculating soil microbial community (table 9) as follows 590nm under with ELIASA.10): each handle the AWCD value in time increase and increase; And processing (HL-1 group) soil microbial community variety each item index (the soil microorganisms metabolic activity AWCD that adds bacterial classification; Soil microorganisms diversity index Shannon; Soil microorganisms species richness R ichness) all be significantly higher than the soil (control group is represented with CK) that does not add bacterial classification, reach significant difference (
P<
0.05).Explain that the HL-1 bacterium improved the soil microorganisms variety significantly.See Fig. 6.
Table 9 calculates the formula of microflora's diversity index
Table 10 different treatment soil microbial community diversity index (72h)
| Handle | AWCD | Shannon (H) | Richness |
| CK | 0.65 ± 0.28 a | 1.22 ± 0.09 a | 27.33 ± 1.53 a |
| HL-1 | 0.89 ± 0.09 b | 1.31 ± 0.04 b | 30.33 ± 0.58 b |
Annotate: data are 3 multiple MV ± standard errors, and the different alphabetical persons of same column in the table are illustrated in 0.05 level difference significantly (DMRT method).
SEQUENCE?LISTING
< 110>Agricultural University Of South China
< 120>subtilis HL-1 and the application aspect the soil phosphorus decomposing thereof
<130>
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 1021
<212> DNA
< 213>artificial sequence
<400> 1
aggcgtgcat?gacgacacca?tgcaccacct?gtcactctgc?tcccgaagga?gaagccctat 60
ctctagggtt?ttcagaggat?gtcaagacct?ggtaaggttc?ttcgcgttgc?ttcgaattaa 120
accacatgct?ccaccgcttg?tgcgggcccc?cgtcaattcc?tttgagtttc?agccttgcgg 180
ccgtactccc?caggcggagt?gcttaatgcg?ttaacttcag?cactaaaggg?cggaaaccct 240
ctaacactta?gcactcatcg?tttacggcgt?ggactaccag?ggtatctaat?cctgtttgct 300
ccccacgctt?tcgcgcctca?gtgtcagtta?cagaccagaa?agtcgccttc?gccactggtg 360
ttcctccata?tctctacgca?tttcaccgct?acacatggaa?ttccactttc?ctcttctgca 420
ctcaagtctc?ccagtttcca?atgaccctcc?acggttgagc?cgtgggcttt?cacatcagac 480
ttaagaaacc?acctgcgcgc?gctttacgcc?caataattcc?ggataacgct?tgccacctac 540
gtattaccgc?ggctgctggc?acgtagttag?ccgtggcttt?ctggttaggt?accgtcaagg 600
tgccagctta?ttcaactagc?acttgttctt?ccctaacaac?agagttttac?gacccgaaag 660
ccttcatcac?tcacgcggcg?ttgctccgtc?agactttcgt?ccattgcgga?agattcccta 720
ctgctgcctc?ccgtaggagt?ctgggccgtg?tctcagtccc?agtgtggccg?atcaccctct 780
caggtcggct?acgcatcgtt?gccttggtga?gccgttacct?caccaactag?ctaatgcgac 840
gcgggtccat?ccataagtga?cagccgaagc?cgcctttcaa?tttcgaacca?tgcagttcaa 900
aatgttatcc?ggtattagcc?ccggtttccc?ggagttaccc?cagtcttatg?ggcaggttac 960
ccacgtgtta?ctcacccgtc?cgccgctaac?tcactcgagc?atgctactag?cttttgcccc 1020
g 1021
Claims (9)
1. subtilis HL-1 was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number CGMCC No.5175 on August 24th, 2011.
2. according to the said subtilis HL-1 of claim 1, it is characterized in that its 16SrDNA nucleotide sequence is shown in SEQ ID NO:1.
3. the microbial inoculum that is prepared into by claim 1 or 2 said subtilis HL-1.
4. microbial inoculum according to claim 3 is characterized in that being made up of subtilis HL-1, zeyssatite and piscidia slag.
5. the preparation method of the said microbial inoculum of claim 4 is characterized in that it being that the activatory bacterial strain is inoculated into seed culture medium by 10 ~ 15% inoculum sizes, under 30 ℃ of culture temperature, rotating speed 113rpm condition; Shaking table is cultivated 24h, obtains primary seed solution, is transferred to seed tank culture by 10 ~ 15% inoculum sizes then; Under 30 ℃, air flow 1000L/h, rotating speed 70rpm condition, cultivate 24h with secondary seed medium, obtain secondary seed solution, secondary seed solution is inoculated into fermentor tank by 10 ~ 15% inoculum sizes; In fermention medium, continue enlarged culturing; Under 30 ℃, air flow 10000L/h, rotating speed 70rpm condition, cultivate 24h, the fermented liquid after the cultivation adds zeyssatite and piscidia slag, stirs; Pulverize, be prepared into said microbial inoculum;
Wherein every 1L first order seed substratum contains Carnis Bovis seu Bubali cream 5g, peptone 5g, sodium-chlor 5g, and remainder is a water, pH value 7.1 ~ 7.4;
Every 1L secondary seed medium and fermention medium contain bean cake powder 30g, wheat bran powder 10g, an ammonium nitrate 1.5g, yeast extract paste 3g, sodium-chlor 2.5g, sal epsom 0.2g, potassium hydrogenphosphate 0.3g and manganous sulfate 0.05g, and remainder is a water, pH value 7.1 ~ 7.4.
6. preparation method according to claim 5, the fermented liquid after it is characterized in that cultivating: zeyssatite: the mass ratio of piscidia slag is 0.6:1:1.
7. claim 1 or the 2 said subtilis HL-1 application in decomposing insoluble phosphorus.
8. claim 1 or the 2 said subtilis HL-1 application in promoting plant-growth.
9. claim 1 or 2 said subtilis HL-1 are increasing soil microbial community variety and the application that increases in the mikrobe abundance.
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| CN112625947A (en) * | 2020-12-16 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
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| CN103773709A (en) * | 2013-10-12 | 2014-05-07 | 河北农业大学 | Bacillus subtilis with efficient phosphate solubilizing effect and application thereof |
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| CN110079484B (en) * | 2019-05-28 | 2022-07-08 | 青岛力力惠生物科技股份有限公司 | Bacillus subtilis and application thereof in agricultural production |
| CN110734876A (en) * | 2019-11-04 | 2020-01-31 | 华中农业大学 | Bacillus megatherium FJW1 biocontrol preparation and preparation method and application thereof |
| CN112625947A (en) * | 2020-12-16 | 2021-04-09 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
| CN112625947B (en) * | 2020-12-16 | 2021-08-03 | 江苏省中国科学院植物研究所 | Bacillus subtilis capable of dissolving phosphorus strongly, carbon-based microbial compound fertilizer thereof and application of bacillus subtilis |
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