CN1296474C - Coculturing fermentation method of root nodule bacterial and Bacillus phosphorus - Google Patents
Coculturing fermentation method of root nodule bacterial and Bacillus phosphorus Download PDFInfo
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- CN1296474C CN1296474C CNB2005100361053A CN200510036105A CN1296474C CN 1296474 C CN1296474 C CN 1296474C CN B2005100361053 A CNB2005100361053 A CN B2005100361053A CN 200510036105 A CN200510036105 A CN 200510036105A CN 1296474 C CN1296474 C CN 1296474C
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Abstract
The present invention relates to a co-culture fermentation method of a rhizobium and bacillus megatherium with the function of dissolving phosphor. The present invention is characterized in that first, a co-culture fermentation medium is inoculated with the rhizobium, and the medium is put in a constant temperature rotating shaker to be cultured for 24 to 36 hours; then, the obtained co-culture fermentation medium with the rhizobium is inoculated with the bacillus megatherium with the function of dissolving the phosphor, and the medium is put in the constant temperature rotating shaker to be co-cultured for 48 to 60 hours. The method of the present invention solves the problem that the rhizobium grows slower than bacillus. The yield improvement and fertilizer saving effect of the fermented bacterial preparation in production application is enhanced by mixed synergistic and complementary action of a co-culture technology, so seedlings inoculated with the fermentation liquor have great improvement on high growth, root nodule quantity, root nodule weight, biomass, etc., and the application of the fermented bacterial preparation in fields has no heavy metal pollution of soil. Thus, the method of the present invention is probable to be used for producing a novel generation of multifunctional multi-strain bacterial fertilizers, and has wide application prospects on green agriculture and forestry production.
Description
Technical field
The present invention relates to a kind of cultivation and fermentation method altogether, relate in particular to a kind of root nodule bacterium and be total to the cultivation and fermentation method with the bacillus megaterium that phosphate solubilization is arranged.
Background technology
Phosphorus and nitrogen all are the necessary important nutritive elements of growth and development of plants.The phosphorus overwhelming majority in the soil is organic or inorganic phosphide state, and plant can not directly absorb, and the phosphorus that just plant can be difficult to absorb by phosphate solubilizing bacteria is converted into the phosphorus that plant can absorb.Airborne nitrogen then is molecular state, and plant is difficult to absorb, and the nitrogen fixation by root nodule bacterium can make plant fully absorb airborne nitrogen.Therefore the utilization of root nodule bacterium and phosphate solubilizing bacteria is for promoting that plant-growth is significant, and how the bacillus megaterium with root nodule bacterium and phosphorus decomposing unites two into one, and is current research focus.
At present traditional technology adopts single strain fermentation back directly to use usually, makes up by a certain percentage or mixes and use.If directly use single strain fermentation back, just can only obtain single fixed nitrogen function or phosphorus decomposing function.In proportion two kinds of bacterium are made up after the single strain fermentation or two kinds of bacterium mixing are used, all need to increase the blended operation, thereby pollute easily.Then can produce synergism by common cultivation and fermentation method, as in Chinese patent 98124397.5, the inventor utilizes common cultivation and fermentation method that the effect of dung-produced alkaline bacteria and nitrogen fixing and fixed nitrogen bacillus megaterium is enhanced.Yet this invention only solves the fixed nitrogen problem, and the common cultivation and fermentation method that is used for root nodule bacterium and phosphorus decomposing bacillus megaterium does not have report so far.And because root nodule bacterium are slow than the genus bacillus growth, if according to a conventional method with these two kinds of bacterium mixed culture, exist the growth of the compacting of the genus bacillus faster root nodule bacterium that grow, finally will cause in its fermented liquid a lot of and few phenomenon of root nodule bacterium quantity of genus bacillus quantity.
Yearning between lovers is short period industrial cut stock seeds, and wide adaptability is drought-resistant barren, and fast growing has now become China important fast-growing, high-yield woods and efficient ecological public welfare forest seeds in south.Yearning between lovers belongs to Mimosoideae Acacia seeds, the same with other leguminous plants, can seek symbiotic nitrogen fixation with root nodule bacterium, the molecular nitrogen that plant in the air can not be absorbed is converted into the ammonia-state nitrogen that plant can absorb, promote plant-growth, the amount of nitrogen fixation of leguminous plants and root nodule bacterium symbiotic nitrogen fixation system accounts for half of global biological nitrogen fixation amount, occupies critical role in biological nitrogen fixation.
Summary of the invention
The object of the present invention is to provide a kind of free of contamination root nodule bacterium and the bacillus megaterium that phosphate solubilization is arranged to be total to the cultivation and fermentation method, the fermented liquid that obtains has higher nitrogenase activity and available phosphorus content, is particularly useful for acacia rachii.
The present invention is earlier with root nodule bacterium (Bradyrhizobium elkanii) single culture 24-36 hour, cultivating altogether 48-60 hour in the cultivation and fermentation substratum altogether with the bacillus megaterium that phosphate solubilization is arranged (Bacillus megaterium de Bary) again, the fermented liquid that obtains has higher nitrogenase activity and available phosphorus content, and heavy metal content is low, can not pollute environment, be used for acacia rachii and can improve its increment greatly, root nodule numbers, nodule weight, biomass etc., thus realized purpose of the present invention.
A kind of cultivation and fermentation method altogether of the present invention is characterized in that comprising the steps:
(1) legume inoculation is total in the cultivation and fermentation substratum, places the constant temperature rotary shaker, cultivated 24-36 hour;
(2) the common cultivation and fermentation inoculation of medium that contains root nodule bacterium that obtains in step (1) has the bacillus megaterium of phosphate solubilization, places the constant temperature rotary shaker, cultivates altogether 48-60 hour.
The described root nodule bacterium of step (1) can be the living slowly root nodule bacterium of Erichsen (Bradyrhizobium elkanii) AM01 CCTCC M205074, bacterial strain accounts for yearning between lovers trees (Acacia.mangium Wild) root system root nodule from dragon's cave-stalactite cave forest farm, GuangZhou, Guangdong Province city horse to be separated and gets, and be deposited in Chinese typical culture collection center on July 18th, 2005, be called for short CCTCC, the depositary institution address be loujia hill belongs Wuhan University in the school, deposit number is CCTCC M 205074; The feature of root nodule bacterium AM01 is: cell is shaft-like, 0.5～0.8 μ m * 1.2～3.0 μ m, and born of the same parents contain the Poly-particle, and single flagellum is extremely living, produces abundant exocellular polysaccharide mucus on the carbohydrate substratum.Gram-negative, the bacterium colony circle, opaque, projection, 6.2 hours generation times; The reaction of 3-ketone group lactose, gelatine liquefication, cellulose hydrolysis, Citrate trianion utilization, starch hydrolysis and tyrosine hydrolysis all are negative, alkali is produced in the BTB test, peptone meat soup growth test feminine gender, utilize fructose, inose, maltose, sucrose, glycine and aspartic acid, the nitrate reduction feminine gender is at 100 μ gmL
-1Can grow among erythromycin and the massfraction 1.0%NaCl.
The described root nodule bacterium of step (1) can also be the living slowly root nodule bacterium in Liaoning (Bradyrhizobium Liaoningense) AJ02CCTCC M 205075, bacterial strain obtains from the separation of Ganzhou City, Jiangxi Province black wattle plant (Acacia.meamsii De Wild.) root system root nodule, and be deposited in Chinese typical culture collection center on July 18th, 2005, be called for short CCTCC, the depositary institution address be loujia hill belongs Wuhan University in the school, deposit number is CCTCC M 205075; The feature of root nodule bacterium AJ02 is: cell is shaft-like, 0.6～0.9 μ m * 1.2～3.0 μ m, born of the same parents contain the Poly-particle, single flagellum is extremely living, produces abundant exocellular polysaccharide mucus, Gram-negative on the carbohydrate substratum, the bacterium colony circle, opaque, projection, 6.3 hours generation times; The reaction of 3-ketone group lactose, gelatine liquefication, cellulose hydrolysis, Citrate trianion utilization, starch hydrolysis and tyrosine hydrolysis all are negative, alkali is produced in the BTB test, peptone meat soup growth test feminine gender, utilize fructose and aspartic acid, do not utilize inose, maltose, sucrose, glycine, at 100 μ gml
-1Do not grow among erythromycin and the massfraction 1.0%NaCl.
The described cultivation and fermentation substratum altogether of step (1), total amount counts 100% by massfraction, its composition contains dipotassium hydrogen phosphate 0.08%, sal epsom 0.02%, sodium-chlor 0.02%, Sodium Glutamate 0.05%, glucose 0.5%～0.7%, maltose 0.3%～0.5% and yeast powder 0.15～0.3%, surplus is a distilled water, and to make the pH of common cultivation and fermentation nutritive medium be 6.8～7.0.
The inoculum size of the root nodule bacterium of step (1) preferably is total to 3% of cultivation and fermentation culture volume, and described constant temperature rotary shaker is each model of selling on the market, and its rotating speed is 180～200r/min preferably, preferably 28 ℃～30 ℃ of culture temperature.
Step (2) is described, and the bacillus megaterium of phosphate solubilization is arranged can be bacillus megaterium (Bacillus megaterium deBary) ACCC11107 or bacillus megaterium (Bacillus megaterium de Bary) ACCC11099, two bacterial classifications are embodied in " Chinese agriculture bacterial classification catalogue " (the Chinese agriculture science and technology press that writes at China Committee for Culture Collection of Microorganisms agricultural microorganism center, December calendar year 2001 date of publication, P5), be deposited in China Committee for Culture Collection of Microorganisms agricultural microorganism center, the address is: China, Beijing, No. 12, Zhong Guan-cun Nanjing University street, deposit number is respectively: ACCC11107 and ACCC11099, their strong stress resistances, the phosphorus decomposing ability is strong, the exocytosis thing is abundant, the gemma survival time is long.
The inoculum size of the bacillus megaterium that phosphate solubilization is arranged of step (2) preferably is total to 1% of cultivation and fermentation culture volume in the step (1), the described constant temperature rotary shaker of constant temperature rotary shaker is each model of selling on the market, its rotating speed is 180～200r/min preferably, preferably 28 ℃～30 ℃ of culture temperature.
Root nodule bacterium that the present invention is used and bacillus megaterium all adopt common substratum and cultural method through spawn culture and seed culture.
The present invention is by elder generation single culture root nodule bacterium in being total to the cultivation and fermentation substratum, and then cultivate altogether with the bacillus megaterium that phosphate solubilization is arranged, root nodule bacterium have been overcome than the slow problem of genus bacillus growth, and utilize altogether that the synergistic blend and the complementary action of culture technique have strengthened the volume increase joint fertilizer efficiency fruit of fermenting agent in production application, altogether in the cultivation and fermentation liquid every milliliter contain viable count about 10
9～10
10Nitrogenase activity＞25nmol reductive C behind the individual bacterium, 1 milliliter of second incubation
2H
2/ hour, available phosphorus content＞60%, " microbial fertilizer " that heavy metal content such as lead, mercury, arsenic and cadmium are lower than Ministry of Agriculture issue in the fermented liquid (NT227-94) in the index of defined, fermenting agent can not cause heavy metal contamination to soil in the application in field.Compare with traditional single strain zymotechnique, saved the blended operation.And the fermented liquid of cultivating altogether is inoculated in yearning between lovers and can improves the high growth of nursery stock, root nodule numbers, and nodule weight, biomass, and the ratio that improves is better than single strain fermented liquid or mixed fermentation liquid greatly.Therefore the present invention is expected to be used to produce the bacterial manure of the multi-functional many bacterial strains of a new generation, is with a wide range of applications on green agroforestry are produced.
Description of drawings
Fig. 1: the growth of the living slowly root nodule bacterium AM01 of Erichsen bacterial strain on the bacterium culture medium solid plate
Fig. 2: the growth of bacillus megaterium ACCC11107 bacterial strain on the bacterium culture medium solid plate
Fig. 3: Erichsen living slowly root nodule bacterium AM01 bacterial strain and bacillus megaterium ACCC11107 bacterial strain be cultivation and fermentation liquid doubling dilution and the single colonial morphology that is coated with the root nodule bacterium culture medium flat plate altogether; Macrocolony is for separating Bacillus phosphorus, and small colonies is root nodule bacterium.
Embodiment
Following embodiment further specifies of the present invention, but the invention is not restricted to following embodiment.
Embodiment 1: living slowly root nodule bacterium AM01 of Erichsen and bacillus megaterium ACCC11107 be cultivation and fermentation altogether
Root nodule bacterium AM01 and bacillus megaterium ACCC11107 bacterial classification are cultivated on the bacterium culture medium solid plate respectively.Genus bacillus ACCC11107 on the bacterium culture medium solid plate 28～30 ℃ cultivated 24 hours, substratum is formed and is: peptone 5g, extractum carnis 3g, NaCl 5g, agar 15g, with the distilled water constant volume to 1000mL, medium pH 7.0～7.2.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.Root nodule bacterium AM01 on the bacterium culture medium solid plate 28～30 ℃ cultivated 72 hours, substratum is formed and is: N.F,USP MANNITOL 10g, Sodium Glutamate 0.5g, K
2HPO
40.5g, MgSO
47H
2O 0.2g, NaCl 0.01g, yeast extract paste 0.5g, agar 1.5g uses the distilled water constant volume to 1000mL, medium pH 6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Choosing root nodule bacterium AM01 that above-mentioned spawn culture obtains and the single bacterium colony of bacillus megaterium ACCC11107 respectively is inoculated in the 500mL that fills the 100mL seed culture medium respectively and shakes in the bottle.Bacillus megaterium ACCC11107 seed culture is at 180～200r/min constant temperature rotary shaker, cultivates 48 hours for 30 ℃.Substratum is formed: peptone 5g, and extractum carnis 3g, NaCl 5g uses the distilled water constant volume to 1000mL, medium pH 7.0～7.2.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.The seed culture of root nodule bacterium AM01 is at 180～200r/min constant temperature rotary shaker, cultivates 48 hours for 30 ℃, and the substratum composition is: N.F,USP MANNITOL 10g, Sodium Glutamate 0.5g, K
2HPO
40.5g, MgSO
47H2O 0.2g, NaCl 0.01g, yeast extract paste 0.5g uses the distilled water constant volume to 1000mL, medium pH 6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Changing root nodule bacterium/AM01 seed culture fluid 30mL over to fill the common cultivation and fermentation substratum of 1000mL 3000mL shakes in the bottle, place 180～200r/min constant temperature rotary shaker, cultivate after 24 hours for 30 ℃ and add bacillus megaterium ACCC11107 seed culture fluid 10mL again, place 180～200r/min constant temperature rotary shaker, cultivated altogether 48 hours for 30 ℃.The composition of cultivation and fermentation substratum is altogether: K
2HPO
40.8g, MgSO
47H2O 0.2g, NaCl 0.2g, Sodium Glutamate 0.5g, glucose 5g, maltose 5g, yeast powder 3g uses the distilled water constant volume to 1000mL, pH6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Embodiment 2: living slowly root nodule bacterium AM01 of Erichsen and bacillus megaterium ACCC11107 be cultivation and fermentation liquid biological property altogether
The fermented liquid of getting after embodiment 1 cultivates is altogether measured its nitrogenase activity with known acetylene reduction method, measure its available phosphorus content with known phosphorus molybdenum blue colorimetric method, measure number of viable with known dull and stereotyped dilution method of counting, use the same method in addition and measure root nodule bacterium AM01 single strain fermented liquid respectively, the fermented liquid of bacillus megaterium ACCC11107 single strain fermented liquid and AM01 and ACCC11107 was by 3: 1 mixed nitrogenase activities, and available phosphorus content and number of viable are as a comparison.The results are shown in Table 1.
Table 1 root nodule bacterium AM01 and bacillus megaterium ACCC11107 be cultivation and fermentation liquid altogether, the biological property contrast of single strain fermented liquid and mixed fermentation liquid
| The mensuration project | Root nodule bacterium AM01 single strain fermented liquid | Bacillus megaterium ACCC11107 single strain fermented liquid | The single strain fermented liquid of ACCC1110 and AM01 mixes at 3: 1 | ACCC11107 and AM01 be cultivation and fermentation liquid altogether |
| Nitrogenase activity (nmol ethene/milliliter bacterium liquid/hour) | 21.44 | / | 13.05 | 30.35 |
| Available phosphorus content (mg/L) | / | 62.52 | 30.25 | 83.68 |
| Number of viable (10 8cfu/mL) | 98.7 | 17.3 | 95.1 | 133.7 |
Embodiment 3: living slowly root nodule bacterium AJ02 in Liaoning and bacillus megaterium ACCC11107 be cultivation and fermentation altogether
Root nodule bacterium AJ02 and bacillus megaterium ACCC11107 bacterial classification are cultivated on the bacterium culture medium solid plate respectively.Genus bacillus ACCC11107 on the bacterium culture medium solid plate 28～30 ℃ cultivated 24 hours, substratum is formed and is: peptone 5g, extractum carnis 3g, NaCl 5g, agar 15g, with the distilled water constant volume to 1000mL, medium pH 7.0～7.2.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.Root nodule bacterium AJ02 on the bacterium culture medium solid plate 28～30 ℃ cultivated 72 hours, substratum is formed and is: N.F,USP MANNITOL 10g, Sodium Glutamate 0.5g, K
2HPO
40.5g, MgSO
47H
2O 0.2g, NaCl 0.01g, yeast extract paste 0.5g, agar 1.5g uses the distilled water constant volume to 1000mL, medium pH 6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Choosing root nodule bacterium AJ02 that above-mentioned spawn culture obtains and the single bacterium colony of bacillus megaterium ACCC11107 respectively is inoculated in the 500mL that fills the 100mL seed culture medium respectively and shakes in the bottle.The seed culture of bacillus megaterium ACCC11107 places 180～200r/min constant temperature rotary shaker, cultivates 48 hours for 30 ℃.Substratum is formed: peptone 5g, and extractum carnis 3g, NaCl 5g uses the distilled water constant volume to 1000mL, medium pH 7.0～7.2.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.The seed culture of root nodule bacterium AJ02 places 180～200r/min constant temperature rotary shaker, cultivates 48 hours for 30 ℃, and the substratum composition is: N.F,USP MANNITOL 10g, Sodium Glutamate 0.5g, K
2HPO
40.5g, MgSO
47H
2O 0.2g, NaCl 0.01g, yeast extract paste 0.5g uses the distilled water constant volume to 1000mL, medium pH 6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Changing root nodule bacterium AJ02 seed culture fluid 30mL over to fill the common cultivation and fermentation substratum of 1000mL 3000mL shakes in the bottle, place 180～200r/min constant temperature rotary shaker, cultivate after 36 hours for 30 ℃ and add bacillus megaterium ACCC11107 seed culture fluid 10mL again, place 180～200r/min constant temperature rotary shaker, cultivated altogether 60 hours for 30 ℃.The composition of cultivation and fermentation substratum is altogether: K
2HPO
40.8g, MgSO
47H
2O 0.2g, NaCl 0.2g, Sodium Glutamate 0.5g, glucose 7g, maltose 3g, yeast powder 3g uses the distilled water constant volume to 1000mL, pH6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Embodiment 4: living slowly root nodule bacterium AJ02 in Liaoning and bacillus megaterium ACCC11107 be the cultivation and fermentation biological property altogether
The fermented liquid of getting after embodiment 3 cultivates is altogether measured its nitrogenase activity with known acetylene reduction method, measure its available phosphorus content with known phosphorus molybdenum blue colorimetric method, measure number of viable with known dull and stereotyped dilution method of counting, use the same method in addition and measure root nodule bacterium AJ02 single strain fermented liquid respectively, the fermented liquid of bacillus megaterium ACCC11107 single strain fermented liquid and AJ02 and ACCC11107 was by 3: 1 mixed nitrogenase activities, and available phosphorus content and number of viable are as a comparison.The results are shown in Table 2.
Table 2 root nodule bacterium AJ02 and bacillus megaterium ACCC11107 be cultivation and fermentation liquid altogether, the biological property contrast of single strain fermented liquid and mixed fermentation liquid
| The mensuration project | Root nodule bacterium AJ02 single strain fermented liquid | Bacillus megaterium ACCC11107 single strain fermented liquid | The single strain fermented liquid of ACCC11107 and AJ02 mixes at 3: 1 | ACCC1110 and AM01 be cultivation and fermentation liquid altogether |
| Nitrogenase activity (nmol ethene/milliliter bacterium liquid/hour) | 19.58 | / | 11.2 | 25.76 |
| Available phosphorus content (mg/L) | / | 62.52 | 30.3 | 90.55 |
| Number of viable 10 8The cfu/ milliliter | 87.5 | 17.3 | 79.8 | 110.43 |
Embodiment 5
1 root nodule bacterium AM01 that obtains and bacillus megaterium ACCC11107 cultivation and fermentation liquid altogether will be implemented, and root nodule bacterium AM01 single strain fermented liquid and bacillus megaterium ACCC11107 single strain fermented liquid are inoculated horse respectively and are accounted for the yearning between lovers nursery stock, every young plant inoculation 5mL bacterium liquid, distinguish the height of seedling of experiment with measuring nursery stock then, root nodule numbers, nodule weight and biomass.The results are shown in Table 3.Test-results shows significant difference (P=0.05) through Mathematical Statistics Analysis.
Table 3 root nodule bacterium AM01 and bacillus megaterium ACCC11107 be cultivation and fermentation liquid altogether, and the single strain fermented liquid accounts for the growth-promoting functions contrast of yearning between lovers nursery stock to horse
| The mensuration project | There is not the inoculation contrast | The inoculation of AM01 single strain fermented liquid | The inoculation of ACCC11107 single strain fermented liquid | AMO and ACCC11107 cultivation and fermentation liquid inoculation altogether |
| Height of seedling (cm) | 4.86 | 5.13 | 5.55 | 6.50 |
| Root nodule numbers (individual/strain) | / | 7.19 | / | 8.13 |
| Nodule weight (gram/strain) | / | 0.0290 | / | 0.0336 |
| Biomass (gram/strain) | 0.242 | 0.367 | 0.559 | 0.670 |
Embodiment 6
With implementing 3 root nodule bacterium AJ02 that obtain and bacillus megaterium ACCC11107 cultivation and fermentation liquid altogether, and root nodule bacterium AJ02 single strain fermented liquid and bacillus megaterium ACCC11107 single strain fermented liquid are inoculated horse respectively and are accounted for the yearning between lovers nursery stock, every young plant inoculation 5mL bacterium liquid, distinguish the height of seedling of experiment with measuring nursery stock then, root nodule numbers, nodule weight, over-ground part biomass and underground part biomass.The results are shown in Table 4.Test-results shows significant difference (P=0.05) through Mathematical Statistics Analysis.
Table 4 root nodule bacterium AJ02 and bacillus megaterium ACCC11107 be cultivation and fermentation liquid altogether, and the single strain fermented liquid accounts for the growth-promoting functions contrast of yearning between lovers nursery stock to horse
| The mensuration project | There is not the inoculation contrast | The inoculation of AJ02 single strain fermented liquid | The inoculation of ACCC11107 single strain fermented liquid | AJ02 and ACCC11107 cultivation and fermentation liquid inoculation altogether |
| Height of seedling (cm) | 10.3 | 18.8 | 24.3 | 26.3 |
| Root nodule numbers (individual/strain) | / | 13.5 | / | 19.1 |
| Nodule weight (gram/strain) | / | 0.141 | / | 0.199 |
| Over-ground part biomass (gram/strain) | 0.236 | 0.672 | 0.710 | 0.768 |
| Underground part biomass (gram/strain) | 0.118 | 0.330 | 0.318 | 0.405 |
Embodiment 7: living slowly root nodule bacterium AM01 of Erichsen and bacillus megaterium ACCC11099 be cultivation and fermentation altogether
Root nodule bacterium AM01 and bacillus megaterium ACCC11099 bacterial classification are cultivated on the bacterium culture medium solid plate respectively.Genus bacillus ACCC11099 on the bacterium culture medium solid plate 28～30 ℃ cultivated 24 hours, substratum is formed and is: peptone 5g, extractum carnis 3g, NaCl 5g, agar 15g, with the distilled water constant volume to 1000mL, medium pH 7.0～7.2.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.Root nodule bacterium AM01 on the bacterium culture medium solid plate 28～30 ℃ cultivated 72 hours, substratum is formed and is: N.F,USP MANNITOL 10g, Sodium Glutamate 0.5g, K
2HPO
40.5g, MgSO
47H
2O 0.2g, NaCl 0.01g, yeast extract paste 0.5g, agar 1.5g uses the distilled water constant volume to 1000mL, medium pH 6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Choosing root nodule bacterium AM01 that above-mentioned spawn culture obtains and the single bacterium colony of bacillus megaterium ACCC11099 respectively is inoculated in the 500mL that fills the 100mL seed culture medium respectively and shakes in the bottle.The seed culture of bacillus megaterium ACCC11099 places 180～200r/min constant temperature rotary shaker, cultivates 48 hours for 30 ℃.Substratum is formed: peptone 5g, and extractum carnis 3g, NaCl 5g uses the distilled water constant volume to 1000mL, medium pH 7.0～7.2.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.The seed culture of root nodule bacterium AM01 places 180～200r/min constant temperature rotary shaker, cultivates 48 hours for 30 ℃, and the substratum composition is: N.F,USP MANNITOL 10g, Sodium Glutamate 0.5g, K
2HPO
40.5g, MgSO
47H2O 0.2g, NaCl 0.01g, yeast extract paste 0.5g uses the distilled water constant volume to 1000mL, medium pH 6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Changing root nodule bacterium AM01 seed culture fluid 30mL over to fill the common cultivation and fermentation substratum of 1000mL 3000mL shakes in the bottle, place 180～200r/min constant temperature rotary shaker, cultivate after 24 hours for 30 ℃ and add bacillus megaterium ACCC11099 seed culture fluid 10mL again, place 180～200r/min constant temperature rotary shaker, cultivated altogether 48 hours for 30 ℃.The composition of cultivation and fermentation substratum is altogether: K
2HPO
40.8g, MgSO
47H2O 0.2g, NaCl 0.2g, Sodium Glutamate 0.5g, glucose 5g, maltose 5g, yeast powder 3g uses the distilled water constant volume to 1000mL, pH6.8～7.0.Substratum was sterilized 30 minutes for 121 ℃ at pressure 103kPa.
Embodiment 8: living slowly root nodule bacterium AM01 of Erichsen and bacillus megaterium ACCC11099 be the cultivation and fermentation biological property altogether
The fermented liquid of getting after embodiment 7 cultivates is altogether measured its nitrogenase activity with known acetylene reduction method, measure its available phosphorus content with known phosphorus molybdenum blue colorimetric method, measure number of viable with known dull and stereotyped dilution method of counting, use the same method in addition and measure root nodule bacterium AM01 single strain fermented liquid respectively, the nitrogenase activity of bacillus megaterium ACCC11099 single strain fermented liquid, available phosphorus content and number of viable are as a comparison.The results are shown in Table 5.
Table 5 root nodule bacterium AM01 and bacillus megaterium ACCC11099 be cultivation and fermentation liquid altogether, the biological property contrast of single strain fermented liquid
| Characteristic | Root nodule bacterium AM01 single strain fermented liquid | Bacillus megaterium ACCC11099 single strain fermented liquid | ACCC11099 and AM01 be cultivation and fermentation liquid altogether |
| Nitrogenase activity (nmol ethene/milliliter bacterium liquid/hour) | 21.44 | / | 29.89 |
| Available phosphorus content (mg/L) | / | 68.83 | 96.26 |
| Number of viable 10 8The cfu/ milliliter | 98.7 | 13.2 | 113.0 |
Claims (1)
1. one kind is total to the cultivation and fermentation method, it is characterized in that comprising the steps:
(1) the living slowly root nodule bacterium of the living slowly root nodule bacterium of Erichsen (Bradyrhizobium elkanii) AM01 CCTCC M 205074 or Liaoning (Bradyrhizobium Liaoningense) AJ02 CCTCC M 205075 is inoculated in the common cultivation and fermentation substratum, place the constant temperature rotary shaker of rotating speed 180～200r/min, cultivated 24～36 hours for 28 ℃～30 ℃, the inoculum size of described root nodule bacterium is 3% of a common cultivation and fermentation culture volume, described cultivation and fermentation substratum altogether, total amount counts 100% by massfraction, its composition contains dipotassium hydrogen phosphate 0.08%, sal epsom 0.02%, sodium-chlor 0.02%, Sodium Glutamate 0.05%, glucose 0.5%～0.7%, maltose 0.3%～0.5% and yeast powder 0.15～0.3%, surplus is a distilled water, and to make the pH of common cultivation and fermentation nutritive medium be 6.8～7.0;
(2) the common cultivation and fermentation inoculation of medium that contains root nodule bacterium that obtains in step (1) has bacillus megaterium (the Bacillus megaterium de Bary) ACCC11107 or bacillus megaterium (the Bacillus megaterium de Bary) ACCC11099 of phosphate solubilization, place the constant temperature rotary shaker of rotating speed 180～200r/min, cultivated altogether 48～60 hours for 28 ℃～30 ℃, the described inoculum size that the bacillus megaterium of phosphate solubilization arranged is in the step (1) altogether 1% of the cultivation and fermentation culture volume.
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| CN (1) | CN1296474C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101229986B (en) * | 2007-12-29 | 2010-10-20 | 中国林业科学研究院热带林业研究所 | Microbial fertilizer specially for beef-wood |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762226B (en) * | 2014-12-12 | 2018-07-03 | 中国林业科学研究院热带林业研究所 | A kind of Boluo Autoinducer and its application |
| CN107467074A (en) * | 2017-06-26 | 2017-12-15 | 浦江县昂宝生物技术有限公司 | Growth promotion plant growth regulator |
| CN107494599A (en) * | 2017-06-26 | 2017-12-22 | 浦江县昂宝生物技术有限公司 | Promote conditioning agent of plant establishment and preparation method thereof |
| CN108624528B (en) * | 2018-05-14 | 2021-01-29 | 华中农业大学 | Composite microbial inoculum with growth promoting and yield increasing effects on leguminous plants and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1219584A (en) * | 1998-11-09 | 1999-06-16 | 中国农业科学院原子能利用研究所 | Co-culture and fermentation process for genetic engineering nitrogen fixing dung-produced alkaline bacteria and nitrogen fixing huge bacillus |
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2005
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1219584A (en) * | 1998-11-09 | 1999-06-16 | 中国农业科学院原子能利用研究所 | Co-culture and fermentation process for genetic engineering nitrogen fixing dung-produced alkaline bacteria and nitrogen fixing huge bacillus |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101229986B (en) * | 2007-12-29 | 2010-10-20 | 中国林业科学研究院热带林业研究所 | Microbial fertilizer specially for beef-wood |
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| Publication number | Publication date |
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| CN1757716A (en) | 2006-04-12 |
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