CN1219584A - Co-culture and fermentation process for genetic engineering nitrogen fixing dung-produced alkaline bacteria and nitrogen fixing huge bacillus - Google Patents
Co-culture and fermentation process for genetic engineering nitrogen fixing dung-produced alkaline bacteria and nitrogen fixing huge bacillus Download PDFInfo
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- CN1219584A CN1219584A CN 98124397 CN98124397A CN1219584A CN 1219584 A CN1219584 A CN 1219584A CN 98124397 CN98124397 CN 98124397 CN 98124397 A CN98124397 A CN 98124397A CN 1219584 A CN1219584 A CN 1219584A
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Abstract
A co-culture and fermentation process for azotomegabacillus cereus and genetic engineering modified azotoalcaligenes taecalis and the formula of fermenting nutritive liquid for co-culture are disclosed to greatly increase the IAA secretion of the mixed fermenting liquid, ammonium resistance and the azofication power. It features use of genetic engineering modified engineering azotobacteria and the mixed synergistic function of co-culture technique, resulting in culturing new generation of multifunctional, multi-strain bacterial fertilizer.
Description
The present invention relates to utilize strain of engineering dung-produced alkaline bacteria and nitrogen fixing and fixed nitrogen bacillus megaterium to be total to the technology of cultivation and fermentation together, and be total to cultivation and fermentation nutrition fermentating formula, thereby increase substantially the IAA secretory volume of mixed fermentation liquid, anti-ammonium ability and nitrogenase activity.
Combination azotobacter has fixed nitrogen concurrently and promotes the dual-use function of plant-growth, can be by the nitrogen in the fixed air (a similar miniature organism nitrogen fertilizer plant), secretion plant hormone (stimulating growing of root system and plant), the wide spectrum of common disease insect pest is suppressed or antagonistic action reaches the purpose that joint fertilizer increases production.But directly separation is also lower from the wild azotobacter strain IAA synthesis capability of soil, has NH in environment
4 +Shi Buneng fixed nitrogen, in addition, the competitive capacity behind the inoculation soil is low, and the inoculation benefit is not remarkable.Above-mentioned weakness has limited the application of azotobacteria fertilizer in green agriculture is produced greatly.
At the problems referred to above, this technology is compared two distinguishing features with traditional strain fermentation process: the one, adopt through genetically engineered engineering azotobacter strain to overcome the above-mentioned inherent weakness of wild strain; The 2nd, utilizing altogether, the synergistic blend work of culture technique saves the fertilizer efficiency fruit in order to strengthen the volume increase of fermenting agent in production application.It is existing that details are as follows:
(1) bacterial strain that this technology adopted all has unique good character, as fixed nitrogen bacillus megaterium BM101 strong stress resistance, the exocytosis thing is abundant, the gemma survival time is long, this strains separation is from China's rice in north china soil, identify that through Microbe Inst., Chinese Academy of Sciences the result formally delivers, bacterial strain now is stored in DSMZ of China Agriculture Academe Fertilizer Institute.
Dung-produced alkaline bacteria and nitrogen fixing A1501 separates from China's south rice rhizosphere soil, identify through Microbe Inst., Chinese Academy of Sciences, the result formally delivers, referring to nineteen ninety-five " Journal of Agricultural Biotechnology ", the 3rd volume, the 1st phase, 34-41 page or leaf, bacterial strain now are stored in DSMZ of China Agriculture Academe Fertilizer Institute.This bacterial strain is transformed through the genetic engineering means, obtain following two strain engineering azotobacter strain AC1541 (referring to nineteen ninety-five " Journal of Agricultural Biotechnology ", the 3rd volume, the 1st phase, strains A 1523 in the paper of 34-41 page or leaf) and and AI1581 (referring to " Journal of Agricultural Biotechnology ", the strains A T63 in the paper that supplementary issue in 1998 is the 80th page).The fixed nitrogen positive regulator gene nifA and the general nitrogen metabolism regulatory gene ntrC of constitutive expression have been integrated in the engineering bacteria AC1541 genome, under the synergy of above-mentioned two positive regulator gene products, eliminated the restraining effect that ammonium is expressed nif, engineering bacteria keeps higher nitrogenase activity and Gen Biao field planting ability under the ammonium existence condition, wild-type has then been lost this anti-ammonium ability under the same conditions; Engineering azotobacter strain A1581 obtains through the Tn5 transposon mutagenesis, can synthesize plant growth substance IAA by high level, and secretory volume is 2 times of wild-type A1501.
(2) this technology is total to cultivation and fermentation 24 hours by three strain bacterium, the IAA secretion capacity after mixed fermented liquid is cultivated, and anti-ammonium ability and nitrogenase activity all are higher than the single strain pure growth, show tangible synergistic blend characteristic.Therefore, adopt this technology to be expected to obtain the genetic engineering bacterial manure of the multi-functional many bacterial strains of a new generation.
Cultivation composition and the culture condition of the three strain bacterium that this technology adopted are as follows:
(1) bacterium culture medium (sterilizing 30 minutes for 15 pounds, 121 ℃, down together) KH
2PO
40.4 gram K
2HPO
40.1 gram NaCl 0.1 gram MgSO
4.7H
2O 0.2 gram MnSO
4.H
2O 0.01 gram Fe
2(SO
4)
3.H
2O 0.01 gram Na
2MoO
4.2H
2O 0.01 gram oxysuccinic acid 1.0 gram (NH
4)
2SO
40.4 gram tryptophane 1.0 gram distilled water were cultivated 12-24 hour for 1000 milliliters pH6.830 ℃.Engineering bacteria AC1541 and AI1581 add the kantlex of 50 mcg/ml when cultivating.Add 1.2-1.5% agar in the liquid medium within and promptly prepare solid inclined-plane or flat board
(2) seed culture medium
KH
2PO
40.4 gram K
2HPO
40.1 gram MgSO
4.7H
2O 0.2 gram
(NH
4)
2SO
40.4 gram NaCl 5 gram KCl 1.0 grams
Sucrose 1.0 gram Sodium.alpha.-hydroxypropionates 1.0 gram tryptophanes 1.0 grams
Peptone 2 gram yeast extract pastes 5 grams
1000 milliliters of pH6.8 of distilled water place 180-200 rev/min of constant temperature rotary shaker, cultivate 24 hours for 30 ℃.Engineering bacteria ACl541 and AI1581 add the kantlex of 50 mcg/ml when cultivating.
(3) be total to cultivation and fermentation substratum KH
2PO
40.4 gram K
2HPO
40.1 gram MgSO
4.7H
2O 0.2 gram (NH
4)
2SO
40.4 1000 milliliters of pH6.8 of gram Sodium Benzoate 1.0 gram KCl, 1.0 gram sucrose 1.0 gram Sodium.alpha.-hydroxypropionates, 1.0 gram tryptophane 1.0 gram peptones, 2 gram NaCl 5 gram yeast extract pastes, 5 gram distilled water place 180-200 rev/min of constant temperature rotary shaker, cultivate 24 hours for 30 ℃.
Three bacterial strains respectively on the bacterium culture medium solid plate 30 ℃ leave standstill and cultivated 12-24 hour, choosing single bacterium colony is inoculated in respectively in 3 500 ml shake flasks of containing 90-150 milliliter seed culture mediums, place 180-200 rev/min of constant temperature rotary shaker, 30 ℃ of single strain pure cultures are after 24 hours, change over to the total inoculum size of 6-15% (three strain bacterium respectively inoculate the 30-50 milliliter) and to contain 0.9-1.5 and rise 5 liters of fermention medium and shake in the bottle, place 180-200 rev/min of constant temperature rotary shaker, bacterial strain more than 30 ℃ was cultivated 24 hours altogether.
After testing, altogether in the cultivation and fermentation liquid every milliliter contain the about 1010-1011 of a viable count bacterium; 100 milliliters contain active substance (comprising plant growth excitor substance such as IAA etc.)＞50 micrograms; Nitrogenase activity＞50nmol reductive C after 1 milliliter of second incubation
2H
2/ hour, can keep 50% of nitrogenase activity under the no ammonium condition at (15mM ammonium) under the high ammonium.Chinese Academy Of Preventive Medicine Research Institute Of Nutrition And Food Hygiene's rat peroral acute toxicity test is through the true border of the fermented liquid of this explained hereafter non-toxic substance; " 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.
Fig. 1 shows fixed nitrogen bacillus megaterium BM101 (I) and the growth of dung-produced alkaline bacteria and nitrogen fixing A1501 (II) on the bacterium culture medium solid plate
Fig. 2 shows fixed nitrogen bacillus megaterium BM101 and 2 strain engineering dung-produced alkaline bacteria and nitrogen fixing AC1541 and AI1581 cultivation and fermentation liquid doubling dilution and the single colonial morphology that is coated with after the LB culture medium flat plate is cultivated altogether.White colony is BM101; The brown small colonies is AC1541 or AI1581 (engineering bacteria list colonial morphology all with wild-type indifference).Wherein used LB substratum is:
Peptone 10 gram NaCl 10 grams
Yeast extract paste 5 gram agar 1.2-1.5%
1000 milliliters of pH6.8 of distilled water
30 ℃ leave standstill cultivation 24 hours
Embodiment 1
Adopt fixed nitrogen bacillus megaterium BM101 and 2 strain engineering dung-produced alkaline bacteria and nitrogen fixing AC1541 and AI1581 cultivation and fermentation altogether, its culture condition is:
(1) bacterium culture medium (sterilizing 30 minutes for 15 pounds, 121 ℃, down together) KH
2PO
40.4 gram K
2HPO
40.1 gram NaCl 0.1 gram MgSO
4.7H
2O 0.2 gram MnSO
4.H
2O 0.01 gram Fe
2(Sn)
3.H
2O 0.01 gram Na
2MoO
4.2H
2O 0.01 gram oxysuccinic acid 1.0 gram (NH
4)
2SO
40.4 gram tryptophane 1.0 gram agar 1.5%pH6.8 distilled water were cultivated 24 hours for 1000 milliliters 30 ℃.Engineering bacteria AC1541 and AI1581 add the kantlex of 50 mcg/ml when cultivating.
(2) seed culture medium KH
2PO
40.4 gram K
2HPO
40.1 gram MgSO
4.7H
2O 0.2 gram (NH
4)
2SO
40.4 100 milliliters of pH6.8 of gram NaCl 5 gram KCl 1.0 gram sucrose, 1.0 gram Sodium.alpha.-hydroxypropionate 1.0 gram tryptophanes, 1.0 gram peptone 2 gram yeast extract pastes, 5 gram distilled water place 200 rev/mins of constant temperature rotary shakers, cultivate 24 hours for 30 ℃.Engineering bacteria AC1541 and AI1581 add the kantlex of 50 mcg/ml when cultivating.
(3) fermention medium KH
2PO
40.4 gram K
2HPO
40.1 gram MgSO
4.7H
2O 0.2 gram (NH
4)
2SO
40.4 1000 milliliters of pH6.8 of gram Sodium Benzoate 1.0 gram KCl, 1.0 gram sucrose 1.0 gram Sodium.alpha.-hydroxypropionates, 1.0 gram tryptophane 1.0 gram peptones, 2 gram NaCl 5 gram yeast extract pastes, 5 gram distilled water place 200 rev/mins of constant temperature rotary shakers, cultivate 24 hours for 30 ℃.
1 strain fixed nitrogen bacillus megaterium BM101 and 2 strain engineering dung-produced alkaline bacteria and nitrogen fixing AC1541 and AI1581 respectively on the bacterium culture medium solid plate 30 ℃ leave standstill and cultivated 24 hours, choosing single bacterium colony is inoculated in respectively in 3 500 ml shake flasks of containing 150 milliliters of seed culture mediums, place 200 rev/mins of constant temperature rotary shakers, 30 ℃ of single strain pure cultures are after 24 hours, changing 5 liters of containing 1.5 liters of fermention mediums over to 10% inoculum size (three strain bacterium respectively inoculate 50 milliliters) shakes in the bottle, place 200 rev/mins of constant temperature rotary shakers, bacterial strain more than 30 ℃ was cultivated 24 hours altogether.
Embodiment 2
The comparison of table 1 engineering bacteria and some biological characteristics of wild-type
*: with wild-type is 100% to compare
*: with the nitrogenase activity under the no nitrogen condition is 100% to compare
| Characteristic | The short engineering bacteria A1581 that gives birth to of the wild-type A1501 engineering bacteria of anti-ammonia AC1541 |
| Nitrogenase activity (under the no nitrogen condition) nmol ethene/milligram albumen/minute nitrogenase activity (15mM ammonium) nmol ethene/milligram albumen/minute IAA secretion capacity mcg/ml | 98.0±5.6％????????120±9.3??????????111.6±4.2 (100％)????????????(125％) *4.6±2.1???????????38.4±6.6??????????3.2±1.4 (4.7％) **?????????(32％) **3.9±0.8???????????5.5±1.7???????????8.2±1.0 (100％)??????????????????????????????(210％) * |
Table 2 is total to the comparison of cultivation and fermentation liquid and some biological property of single strain pure culture liquid
*: the nitrogenase activity of 1 milliliter of bacterium liquid second incubation
| Characteristic | Be total to cultivation and fermentation liquid BM101 AC1541 AI1581 |
| Nitrogenase activity (under the no nitrogen condition) *Nmol ethene/milligram albumen/minute nitrogenase activity (15mM ammonium) *Nmol ethene/milligram albumen/minute IAA secretion capacity mcg/ml | ?68.4±2.5???????46.3±10.1??55.2±3.6?????50.1±7.3 ?37.0±3.7???????4.3±0.6????31.4±4.8??????5.9±3.9 ?13.2±1.2???????6.2±0.8?????4.7±2.3??????9.3±2.8 |
Be total to growth-promoting functions and the yield increase effect of cultivation and fermentation liquid in table 3 field plot trial to paddy rice
| The mensuration project | The non-inoculation contrast of cultivation and fermentation liquid BM101 AC1541 AI1581 altogether |
| Plant height (cm) actual output (kilogram/mu) stimulation ratio | ?93.6?????????93.0????92.8????93.3??????90.7 ?477.8????????453.3???465.8???451.5?????437.9 ?9.1??????????3.5?????6.4?????3.1????????/ |
Table 4 cultivation and fermentation liquid is altogether used the yield increase effect of several vegetables
| Vegetable variety | |
| Cucumber tomato autumn cabbage eggplant white turnip shallot fresh kidney beans lettuce | |
| Stimulation ratio (%) | 23％???19％???16.8％?10.7％?23.1％?30.2％?24％??21.2％ |
Claims (2)
1. cultivation and fermentation technology altogether, it is characterized in that with the fixed nitrogen bacillus megaterium and through genetically engineered three kinds of inoculation of dung-produced alkaline bacteria and nitrogen fixing in cultivation and fermentation nutritive medium altogether, place the constant temperature rotary shaker, 28 ℃-30 ℃, cultivated 20-28 hour.
2. cultivation and fermentation technology altogether according to claim 1 is characterized in that described cultivation and fermentation nutrient solution prescription altogether is potassium primary phosphate 0.4 gram, dipotassium hydrogen phosphate 0.1 gram, sodium-chlor 5.0 grams, Repone K 1.0 grams, magnesium sulfate heptahydrate 0.2 gram, ammonium sulfate 0.4 gram, tryptophane 1.0 grams, sucrose 1.0 grams, Sodium.alpha.-hydroxypropionate 1.0 grams, peptone 2.0 grams, yeast extract paste 5 grams, Sodium Benzoate 1.0 grams, 1000 milliliters of distilled water, pH6.8
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|---|---|---|---|
| CN 98124397 CN1120237C (en) | 1998-11-09 | 1998-11-09 | Co-culture and fermentation process for genetic engineering nitrogen fixing dung-produced alkaline bacteria and nitrogen fixing huge bacillus |
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|---|---|---|---|
| CN 98124397 CN1120237C (en) | 1998-11-09 | 1998-11-09 | Co-culture and fermentation process for genetic engineering nitrogen fixing dung-produced alkaline bacteria and nitrogen fixing huge bacillus |
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| CN1120237C CN1120237C (en) | 2003-09-03 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1296474C (en) * | 2005-07-25 | 2007-01-24 | 中国林业科学研究院热带林业研究所 | Coculturing fermentation method of root nodule bacterial and Bacillus phosphorus |
| CN101914468A (en) * | 2010-07-15 | 2010-12-15 | 广西大学 | Nitrogen-fixing bacillus megaterium strain DL7 and application thereof |
| CN102747010A (en) * | 2012-05-09 | 2012-10-24 | 浙江农林大学 | Allethrin degrading bacteria and soil restoration application thereof |
-
1998
- 1998-11-09 CN CN 98124397 patent/CN1120237C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1296474C (en) * | 2005-07-25 | 2007-01-24 | 中国林业科学研究院热带林业研究所 | Coculturing fermentation method of root nodule bacterial and Bacillus phosphorus |
| CN101914468A (en) * | 2010-07-15 | 2010-12-15 | 广西大学 | Nitrogen-fixing bacillus megaterium strain DL7 and application thereof |
| CN102747010A (en) * | 2012-05-09 | 2012-10-24 | 浙江农林大学 | Allethrin degrading bacteria and soil restoration application thereof |
| CN102747010B (en) * | 2012-05-09 | 2013-07-17 | 浙江农林大学 | Allethrin degrading bacteria and soil restoration application thereof |
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| Publication number | Publication date |
|---|---|
| CN1120237C (en) | 2003-09-03 |
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