CN105255757A - Soybean rhizobium culture medium and method for preparing liquid soybean rhizobium inoculant from same - Google Patents
Soybean rhizobium culture medium and method for preparing liquid soybean rhizobium inoculant from same Download PDFInfo
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Abstract
The invention discloses a soybean rhizobium culture medium and a method for preparing liquid soybean rhizobium inoculant from the same. The soybean rhizobium culture medium is an aqueous solution prepared from glycerol, mannitol, glucose, rhamnose, fructose, alpha-ketoglutarate, yeast powder, serine, arginine, ammonium chloride, dipotassium phosphate, monopotassium phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, iron sulfate heptahydrate, sodium chloride, sodium molybdate dihydrate and biotin. The liquid soybean rhizobium inoculant is prepared through the soybean rhizobium culture medium in a fermentation and inoculation mode. The viable count of the inoculant still reaches above 10 billion per liter after the inoculant is preserved for 12 months at 20 DEG C. The soybean rhizobium culture medium and the method for preparing the liquid soybean rhizobium inoculant from the same aim to solve the problems that existing liquid soybean rhizobium inoculant is unbalanced in nutrition and low in thallus activity, and soybean rhizobium prepared through a fermentation technology can not stably survive at room temperature.
Description
Technical field
The present invention relates to root nodule bacterium substratum and adopt it to prepare the method for soybean rhizobium inoculant.
Background technology
Chinese arable land area accounts for 7% of whole world cultivated area, and nitrogen fertilizer amount accounts for 25% of the total consumption in the whole world, is 3.75 times of world's average dose.Produce chemical industry synthetic nitrogen fertile in consuming a large amount of, non-renewable petrochemical materials, as Sweet natural gas, oil and coal etc.Along with the continuous minimizing of the limited reserves of natural energy resources, the price of oil and gas will continue soaring, thus inevitably pull fertilizer price to go up.Chemical fertilizer industry still causes environmental pollution, produces the important source of greenhouse gases.Chemical fertilizer can cause soil compaction after applying field, reduces Land Productivity.The absorption rate of plant to chemical fertilizer only has 30%, this means that the nitrogenous fertilizer of 70% does not only have nourish plants, and enters in underground water source and air, causes tap water and Air quality to decline, and then endangers our healthy.
In fact the nitrogen nutrition needed for soybeans they grow is provided by root nodule bacterium substantially, if do not have root nodule bacterium, will execute the nitrogenous fertilizer demand that more than 100 kilogram of urea could meet per mu yield 180 kilograms of soybean for every mu.
0.07 hectare produces 180 kilograms of soybean, also will produce the stalk of 270 kilograms simultaneously;
Soy bean protein content calculates by 40%: 180 × 0.4=72 kilogram (protein);
Stalk protein content calculates by 10%: 270 × 0.1=27 kilogram (protein);
0.07 hectare is laid eggs white matter: 72+27=99 kilogram;
The nitrogen content of protein is 16%, produces 99 kilogram protein and needs 99 × 0.16=15.84 kilogram of purity nitrogen;
15.84 kilograms of purity nitrogens amount to urea: 15.84 ÷ 0.46=34.43 kilogram urea;
Consider that the utilization ratio of urea is generally below 30%, 34.43 ÷ 0.3=115 kilogram.
In other words, if do not have root nodule bacterium to provide nitrogenous fertilizer, often producing 180 kilograms of soybean needs to execute 115 kilograms of urea and could meet nitrogen demand.The nitrogenous fertilizer of soybean is root nodule bacterium contributions substantially thus.As can be seen here, root nodule bacterium is good and bad most important to the production of soybean.
Why legume inoculation technology fails extensively to adopt in China, and except scientific knowledge is not universal, outside the effect of peasant to root nodule bacterium is had little understanding, main reason is that root nodule bacterium quality product does not pass a test, and the quality guaranteed period is short.The effective ingredient of nitragin is the root nodule bacterium lived, if but preparation do not pass a test, when product often only has a small amount of active root nodule bacterium when a long supply chain arrives in peasant's hand, practical application effect is not obvious.
At present, the substratum adopting document openly to report and the method for normal fermentation technique (being mainly used for fermentation of bacillus) produce soy broth nitragin, there is nutritional imbalance, drawback that microbial activity is low, and, the culture medium carbon source presentate one of current cultivation rihizobium japonicum, although most of substratum can support rhizobial growth well, bacterium number can reach 30-80 hundred million/milliliter, but they are all difficult at room temperature stablize survival substantially, and the viable count that its normal temperature is placed 2 months is only 1 ~ 10%.
Summary of the invention
The rihizobium japonicum that the nutrition of soy broth nitragin is unbalanced, microbial activity is low and prepared by zymotechnique that the object of the invention is to solve existing existence at room temperature can not stablize the problem of survival, and provides soybean nodulation bacterium culture medium and adopt it to prepare the method for soy broth nitragin.
A kind of soybean nodulation bacterium culture medium, is characterized in that: comprise following component: glycerol, N.F,USP MANNITOL, glucose, rhamnosyl, fructose, α-ketoglutaric acid, yeast powder, Serine, arginine, ammonium chloride, dipotassium hydrogen phosphate, potassium primary phosphate, bitter salt, CALCIUM CHLORIDE DIHYDRATE, seven ferric sulfate hydrates, sodium-chlor, two molybdic acid hydrate sodium, vitamin H and water, wherein, the solubility of the relative water of each component is respectively: glycerol concentration is 1 ~ 3 grams per liter, mannitol concentration is 1 ~ 3 grams per liter, glucose concn is 1 ~ 3 grams per liter, rhamnosyl concentration is 1 ~ 3 grams per liter, fructose concentration is 1 ~ 3 grams per liter, α-ketoglutaric acid concentration is 0.5 ~ 1.5 grams per liter, yeast powder concentration is 2.5 ~ 4.5 grams per liters, serine concentration is 0.13 ~ 0.25 grams per liter, arginine concentrations is 0.1 ~ 0.2 grams per liter, ammonium chloride concentration is 0.65 ~ 1.6 grams per liter, dipotassium hydrogen phosphate concentration is 1 ~ 3 grams per liter, biphosphate potassium concn is 0.2 ~ 0.5 grams per liter, bitter salt concentration is 0.1 ~ 0.3 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.1 ~ 0.2 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.005 ~ 0.011 grams per liter, sodium chloride concentration is 0.05 ~ 0.15 grams per liter, two molybdic acid hydrate na concns are 0.01 ~ 0.03 grams per liter, biotin concentration is 0.0005 ~ 0.002 grams per liter, described medium pH is 6.8 ~ 7.5.
Further, described glycerol concentration is 1.5 ~ 2.5 grams per liters, mannitol concentration is 1.5 ~ 2.5 grams per liters, glucose concn is 1.5 ~ 2.5 grams per liters, rhamnosyl concentration is 1.5 ~ 2.5 grams per liters, fructose concentration is 1.5 ~ 2.5 grams per liters, α-ketoglutaric acid concentration is 0.75 ~ 1.25 grams per liter, yeast powder concentration is 3.0 ~ 4.0 grams per liters, serine concentration is 0.15 ~ 0.20 grams per liter, arginine concentrations is 0.125 ~ 0.175 grams per liter, ammonium chloride concentration is 0.8 ~ 1.25 grams per liter, dipotassium hydrogen phosphate concentration is 1.1 ~ 1.6 grams per liters, biphosphate potassium concn is 0.25 ~ 0.4 grams per liter, bitter salt concentration is 0.15 ~ 0.25 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.11 ~ 0.15 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.006 ~ 0.009 grams per liter, sodium chloride concentration is 0.08 ~ 0.12 grams per liter, two molybdic acid hydrate na concns are 0.015 ~ 0.025 grams per liter and biotin concentration is 0.0008 ~ 0.0012 grams per liter.
Further, described glycerol concentration is 2 grams per liters, mannitol concentration is 2.0 grams per liters, glucose concn is 2.0 grams per liters, rhamnosyl concentration is 2.0 grams per liters, fructose concentration is 2.0 grams per liters, α-ketoglutaric acid concentration is 1.0 grams per liters, yeast powder concentration is 3.5 grams per liters, serine concentration is 0.18 grams per liter, arginine concentrations is 0.15 grams per liter, ammonium chloride concentration is 1.1 grams per liters, dipotassium hydrogen phosphate concentration is 1.2 grams per liters, biphosphate potassium concn is 0.34 grams per liter, bitter salt concentration is 0.2 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.13 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.0083 grams per liter, sodium chloride concentration is 0.1 grams per liter, two molybdic acid hydrate na concns are 0.02 grams per liter and biotin concentration is 0.001 grams per liter.
Utilize the method for this rihizobium japonicum medium preparing soy broth nitragin, comprise the following steps:
Step one is 1 ~ 3 grams per liter by glycerol concentration, mannitol concentration is 1 ~ 3 grams per liter, glucose concn is 1 ~ 3 grams per liter, rhamnosyl concentration is 1 ~ 3 grams per liter, fructose concentration is 1 ~ 3 grams per liter, α-ketoglutaric acid concentration is 0.5 ~ 1.5 grams per liter, yeast powder concentration is 2.5 ~ 4.5 grams per liters, serine concentration is 0.13 ~ 0.25 grams per liter, arginine concentrations is 0.1 ~ 0.2 grams per liter, ammonium chloride concentration is 0.65 ~ 1.6 grams per liter, dipotassium hydrogen phosphate concentration is 1 ~ 3 grams per liter, biphosphate potassium concn is 0.2 ~ 0.5 grams per liter, bitter salt concentration is 0.1 ~ 0.3 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.1 ~ 0.2 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.005 ~ 0.011 grams per liter, sodium chloride concentration is 0.05 ~ 0.15 grams per liter, two molybdic acid hydrate na concns are 0.01 ~ 0.03 grams per liter, biotin concentration is that 0.0005 ~ 0.002 grams per liter takes above-mentioned substance, and joins in pure water successively and mix, and regulates pH to 6.8 ~ 7.5, obtains soy broth root nodule bacterium substratum,
Step 2, soy broth root nodule bacterium substratum step one obtained joins in fermentor tank, 121 DEG C of autoclaving 20min, when substratum temperature is down to 30 DEG C, by rihizobium japonicum by volume percentage composition be 5% ~ 10% inoculum size be seeded in the soy broth root nodule bacterium substratum of sterilizing, it is 25 ~ 35 DEG C at leavening temperature, stirring velocity is 50 ~ 150r/min, air flow quantity is 0.3 ~ 0.6v/v/m, tank pressure is 0.05 ~ 0.2kPa, dissolved oxygen is 60% ~ 80%, fermented liquid pH is under the condition of 6.5 ~ 7.5, fermentation 72 ~ 144h, obtain soy broth nitragin.
Further, described in step one is 1.5 ~ 2.5 grams per liters by glycerol concentration, mannitol concentration is 1.5 ~ 2.5 grams per liters, glucose concn is 1.5 ~ 2.5 grams per liters, rhamnosyl concentration is 1.5 ~ 2.5 grams per liters, fructose concentration is 1.5 ~ 2.5 grams per liters, α-ketoglutaric acid concentration is 0.75 ~ 1.25 grams per liter, yeast powder concentration is 3.0 ~ 4.0 grams per liters, serine concentration is 0.15 ~ 0.20 grams per liter, arginine concentrations is 0.125 ~ 0.175 grams per liter, ammonium chloride concentration is 0.8 ~ 1.25 grams per liter, dipotassium hydrogen phosphate concentration is 1.1 ~ 1.6 grams per liters, biphosphate potassium concn is 0.25 ~ 0.4 grams per liter, bitter salt concentration is 0.15 ~ 0.25 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.11 ~ 0.15 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.006 ~ 0.009 grams per liter, sodium chloride concentration is 0.08 ~ 0.12 grams per liter, two molybdic acid hydrate na concns are 0.015 ~ 0.025 grams per liter and biotin concentration is that 0.0008 ~ 0.0012 grams per liter takes above-mentioned substance.
Further, described in step one is 2 grams per liters by glycerol concentration, mannitol concentration is 2.0 grams per liters, glucose concn is 2.0 grams per liters, rhamnosyl concentration is 2.0 grams per liters, fructose concentration is 2.0 grams per liters, α-ketoglutaric acid concentration is 1.0 grams per liters, yeast powder concentration is 3.5 grams per liters, serine concentration is 0.18 grams per liter, arginine concentrations is 0.15 grams per liter, ammonium chloride concentration is 1.1 grams per liters, dipotassium hydrogen phosphate concentration is 1.2 grams per liters, biphosphate potassium concn is 0.34 grams per liter, bitter salt concentration is 0.2 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.13 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.0083 grams per liter, sodium chloride concentration is 0.1 grams per liter, two molybdic acid hydrate na concns are 0.02 grams per liter and biotin concentration is that 0.001 grams per liter takes above-mentioned substance.
Further, the soy broth root nodule bacterium described in step 2 cultivate 70% of fiduciary point fermentor tank cumulative volume.
Further, fermentation condition described in step 2 is: leavening temperature be 29 ~ 31 DEG C, under stirring velocity is 75 ~ 125r/min, air flow quantity is 0.35 ~ 0.55v/v/m, tank pressure is 0.075 ~ 0.18kPa, dissolved oxygen is 65 ~ 75%, fermented liquid pH is the condition of 6.8 ~ 7.3, fermentation 72 ~ 144h.
Further, described in step 2 leavening temperature be 30 DEG C, under stirring velocity is 100r/min, air flow quantity is 0.45v/v/m, tank pressure is 0.1kPa, dissolved oxygen is 70%, fermented liquid pH is the condition of 7.2, fermentation 72 ~ 144h.
Further, the rihizobium japonicum described in step 2 is Slow-growing Soybean rhizobia.
The present invention has following beneficial effect:
The present invention adopts glycerine, N.F,USP MANNITOL, glucose, rhamnosyl, fructose, the material composition mixed carbon sources such as α-ketoglutaric acid, substitute the conventional single carbon source used, accurate control carbon source, the balance of nitrogenous source and mineral nutrient element, and the production process of rihizobium japonicum is controlled with the zymotechnique optimized, the soy broth nitragin prepared is preserved 12 months viable counts and is still reached every milliliter more than 10,000,000,000 in 20 DEG C of constant temperature cabinets, the Ministry of Agriculture's microbial fertilizer and the edible fungus species quality examination center result to microbial inoculum sampling Detection of the present invention is 11,000,000,000/milliliter, miscellaneous bacteria number is 0.The national standard of the agriculture microbiobacterial agent (comprising nitragin) of current China is 200,000,000/gram viable count, compares and improves 50 times.The actual measurement viable count of the best product of external liquid soy nitragin is about 5,000,000,000, and the quality guaranteed period is 12 months, ensures that minimum viable count is not less than 2,000,000,000/milliliter.The viable count of this product is higher than external preferably product.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment 1: be 1.0 grams per liters by glycerol concentration, mannitol concentration is 1.0 grams per liters, glucose concn is 1.0 grams per liters, rhamnosyl concentration is 1.0 grams per liters, fructose concentration is 1.0 grams per liters, α-ketoglutaric acid concentration is 0.5 grams per liter, yeast powder concentration is 2.5 grams per liters, serine concentration is 0.13 grams per liter, arginine concentrations is 0.1 grams per liter, ammonium chloride concentration is 0.65 grams per liter, dipotassium hydrogen phosphate concentration is 1 grams per liter, biphosphate potassium concn is 0.2 grams per liter, bitter salt concentration is 0.1 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.1 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.005 grams per liter, sodium chloride concentration is 0.05 grams per liter, two molybdic acid hydrate na concns are 0.01 grams per liter, biotin concentration is that 0.0005 grams per liter takes above-mentioned substance, and join successively in 1L pure water and mix, regulate pH to 6.8, obtain soy broth root nodule bacterium substratum,
Embodiment 2: by the concentration of glycerol be 2.0 grams per liters, the concentration of N.F,USP MANNITOL is 2.0 grams per liters, the concentration of glucose is 2.0 grams per liters, the concentration of rhamnosyl is 2.0 grams per liters, the concentration of fructose is 2.0 grams per liters, the concentration of α-ketoglutaric acid is 1.0 grams per liters, the concentration of yeast powder is 3.5 grams per liters, the concentration of Serine is 0.18 grams per liter, arginic concentration is 0.15 grams per liter, NH
4the concentration of Cl is 1.1 grams per liters, K
2hPO
4concentration be 1.2 grams per liters, KH
2pO
4concentration be 0.34 grams per liter, MgSO
47H
2the concentration of O is 0.13 grams per liter, CaCl
22H
2the concentration of O is 0.13 grams per liter, FeSO
47H
2the concentration of O is 0.0083 grams per liter, the concentration of NaCl is 0.1 grams per liter, Na
2moO
42H
2the concentration of O is the concentration of 0.02 grams per liter and vitamin H is that 0.001 grams per liter takes above-mentioned substance, and joins successively in 1L pure water and mix, and regulates pH to 7.2, obtains soy broth root nodule bacterium substratum;
Embodiment 3: glycerol concentration is 3.0 grams per liters, mannitol concentration is 3.0 grams per liters, glucose concn is 3.0 grams per liters, rhamnosyl concentration is 3.0 grams per liters, fructose concentration is 3.0 grams per liters, α-ketoglutaric acid concentration is 1.5 grams per liters, yeast powder concentration is 4.5 grams per liters, serine concentration is 0.25 grams per liter, arginine concentrations is 0.2 grams per liter, ammonium chloride concentration is 1.6 grams per liters, dipotassium hydrogen phosphate concentration is 3 grams per liters, biphosphate potassium concn is 0.5 grams per liter, bitter salt concentration is 0.3 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.2 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.011 grams per liter, sodium chloride concentration is 0.15 grams per liter, two molybdic acid hydrate na concns are 0.03 grams per liter, biotin concentration is that 0.002 grams per liter takes above-mentioned substance, and join successively in 1L pure water and mix, regulate pH to 7.5, obtain soy broth root nodule bacterium substratum,
Embodiment 4: the 250mL triangular flask of 100mL embodiment 1 soy broth root nodule bacterium substratum will be housed, life is divided into shaking flask 1, the 250mL triangular flask of 100mL embodiment 2 soy broth root nodule bacterium substratum will be housed, life is divided into shaking flask 2, the 250mL triangular flask of 100mL embodiment 3 soy broth root nodule bacterium substratum will be housed, life is divided into shaking flask 3; In shaking flask 1, shaking flask 2 and shaking flask 3, inoculate the slant strains that a ring Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) activates respectively, temperature be 30 DEG C, rotating speed be the condition of 180r/min under shaking culture 96h, obtain seed liquor; 5mL kind daughter bacteria liquid is gone in new shaking flask 1, shaking flask 2 and shaking flask 3, then temperature be 30 DEG C, rotating speed be the condition of 180r/min under shaking culture 120h, obtain soy broth nitragin.
The Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) of this test, buys from China Agricultural University's Culture Collection, address: No. 2, West Road, Yuanmingyuan Park, Beijing, deposit number: CCBAU15464.
Soy broth nitragin shaking flask 1, shaking flask 2 and shaking flask 3 obtained, under temperature is 20 DEG C of conditions, is placed 3 months, 6 months and 9 months respectively, detects bacterium number, the results are shown in Table 1 by plate count.
Table 1 shake-flask culture rihizobium japonicum testing data
| Starter bacteria number | 20 DEG C/3 months | 20 DEG C/6 months | 20 DEG C/9 months | |
| Shaking flask 1 | 7.51×10 9/mL | 8.33×10 9/mL | 8.14×10 9/mL | 7.51×10 9/mL |
| Shaking flask 2 | 8.37×10 9/mL | 9.10×10 9/mL | 7.58×10 9/mL | 7.19×10 9/mL |
| Shaking flask 3 | 8.63×10 9/mL | 8.54×10 9/mL | 8.43×10 9/mL | 7.11×10 9/mL |
| On average | 8.17×10 9/mL | 8.66×10 9/mL | 8.05×10 9/mL | 7.27×10 9/mL |
* note: because the bottle stopper of triangular flask has ventilation property, storage process has moisture loss, adds sterilized water every month to original volume.
As shown in Table 1, the soy broth nitragin that shake flask fermentation obtains, starter bacteria number is 81.7 hundred million/mL, bacterium number after 3 months is placed higher than the bacterium number at the end of just cultivating under 20 DEG C of conditions, show that root nodule bacterium can slowly increase in shaking flask, bacterium number just slowly declines subsequently, and when 9 months, viable count still has 7,200,000,000/mL.
Embodiment 5: 5 liters of desk-top fermentation cylinder lives that embodiment 1 soy broth root nodule bacterium substratum is housed are divided into the 1st batch, 5 liters of desk-top fermentation cylinder lives that embodiment 2 soy broth root nodule bacterium substratum is housed are divided into the 2nd batch, 5 liters of desk-top fermentation cylinder lives that embodiment 3 soy broth root nodule bacterium substratum is housed are divided into the 3rd batch; The inoculum size of the bacterial classification that Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) is activated by volume percentage composition 5% be inoculated into respectively the 1st batch, the 2nd batch and the 3rd batch sample are housed fermentor tank in, then at the condition bottom fermentation 120h that temperature is 30 DEG C, rotating speed is 250r/min, air flow quantity is 1.58L/min, fermented liquid pH is 6.5 ~ 7.5, obtain soy broth nitragin, aseptically be dispensed in the triangular flask of sterilizing, 20 DEG C store standby inspection.
The Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) of this test, buys from China Agricultural University's Culture Collection, address: No. 2, West Road, Yuanmingyuan Park, Beijing, deposit number: CCBAU15464.
By the 1st batch, the 2nd batch and the 3rd batch of soy broth nitragin obtained under temperature is 20 DEG C of conditions, place 3 months and 6 months respectively, by plate count detection bacterium number, the results are shown in Table 2.
Table 2 lab scale fermentative production rihizobium japonicum testing data
| Starter bacteria number | 20 DEG C/3 months | 20 DEG C/6 months | |
| 1st batch | 1.22×10 10/mL | 1.33×10 10/mL | 1.31×10 10/mL |
| 2nd batch | 1.30×10 10/mL | 1.42×10 10/mL | 1.15×10 10/mL |
| 3rd batch | 9.43×10 9/mL | 1.27×10 10/mL | 1.32×10 9/mL |
| On average | 1.15×10 10/mL | 1.34×10 10/mL | 1.26×10 9/mL |
| YMB contrasts | 2.65×10 9/mL | 6.67×10 8/mL | 3.57×10 7/mL |
* note: because the bottle stopper of triangular flask has ventilation property, storage process has moisture loss, adds sterilized water every month to original volume.
As shown in Table 2, lab scale ferments the soy broth nitragin obtained, starter bacteria number is 11,500,000,000/mL, bacterium number after 3 months is placed higher than the bacterium number at the end of just cultivating under 20 DEG C of conditions, show that root nodule bacterium can slowly increase in shaking flask, bacterium number just slowly declines subsequently, and when 6 months, viable count still has 12,600,000,000/mL.
And existing YMB substratum is the most frequently used root nodule bacterium substratum, it is composed as follows: the bitter salt of the N.F,USP MANNITOL of 10 grams per liters, the dipotassium hydrogen phosphate of 0.5 grams per liter, 0.2 grams per liter, the sodium-chlor of 0.1 grams per liter, the yeast powder of 0.5 grams per liter and 1000 ml pure waters (referring to VincentJ.M.1970Amanualforthepracticalstudyofroot-noduleb acteria.IBPhandbook15.BlackwellScientificPublications, Oxford.).
YMB substratum is adopted to cultivate according to the method for this test the bacterium number that Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) can only obtain 20 ~ 3,000,000,000/milliliter, and at room temperature preserve the bacterium death of after 2 months 90%, cannot scale operation liquid soy nitragin be used as.It can thus be appreciated that 20 DEG C of methods of preserving this test after 6 months cultivate Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) raising more than 35 times compared with YMB culture medium culturing.
Embodiment 6: add the soy broth root nodule bacterium substratum that 210 liters of embodiments 2 obtain in three 300 liters 304 stainless steel fermentor tanks, namely soybean nodulation bacterium culture medium accounts for 70% of fermentor tank cumulative volume, is respectively 1 batch, 2 batches, 3 batches; The inoculum size of the kind daughter bacteria liquid activated by Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) by volume percentage composition 7.5% is inoculated in fermentor tank respectively, then at the condition bottom fermentation 120h that temperature is 30 DEG C, stirring velocity is 100r/min, air flow quantity is 0.45v/v/m, tank pressure is 0.1kPa, dissolved oxygen is 70%, fermented liquid pH is 7.2, obtain liquid soy nitragin, aseptically be dispensed in the double-layer plastic bag of gamma line sterilizing, every packed 225mL, for subsequent use.
V/v/m in this test is the unit of the proprietary expression air flow quantity of fermentation industry, and 0.3 ~ 0.6v/v/m represents that volume of air that per minute enters fermentor tank is 0.3 ~ 0.6 times of culture volume.This test is reached by the change of stirring velocity and controls the object of dissolved oxygen, when dissolved oxygen lower than 70% time Controlling System automatically increase rotating speed, when dissolved oxygen higher than 70% time Controlling System automatically reduce rotating speed.
The Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) of this test, buys from China Agricultural University's Culture Collection, address: No. 2, West Road, Yuanmingyuan Park, Beijing, deposit number: CCBAU15464.
By the 1st batch, the 2nd batch and the 3rd batch of soy broth nitragin obtained under temperature is 20 DEG C of conditions, place 3 months and 6 months respectively, detect viable count by plate count, the results are shown in Table 3.
Rihizobium japonicum testing data produced by table 3 pilot scale fermentation tank
| Starter bacteria number | 20 DEG C/3 months | 20 DEG C/6 months | |
| 1st batch | 9.81×10 9/mL | 1.07×10 10/mL | 8.94×10 9/mL |
| 2nd batch | 1.25×10 10/mL | 1.13×10 10/mL | 8.05×10 9/mL |
| 3rd batch | 1.37×10 10/mL | 1.21×10 10/mL | 9.11×10 9/mL |
| On average | 1.20×10 10/mL | 1.14×10 10/mL | 8.70×10 9/mL |
* note: root nodule bacterium liquid is contained in the plastics bag of sealing, without moisture loss.
As shown in Table 3, the soy broth nitragin that pilot scale fermentation obtains, starter bacteria number is 12,000,000,000/mL, bacterium number after 3 months is placed higher than the bacterium number at the end of just cultivating under 20 DEG C of conditions, show that root nodule bacterium can slowly increase in plastics bag, bacterium number just slowly declines subsequently, and when 6 months, viable count still has 8,700,000,000/mL.
Embodiment 7: the method that this test large scale fermentation prepares soy broth nitragin is as follows:
In three 5 tons 304 stainless steel fermentor tanks, add the soy broth root nodule bacterium substratum that 3.5 tons of embodiments 2 obtain respectively, namely soy broth root nodule bacterium cultivate 70% of fiduciary point fermentor tank cumulative volume, be divided into 1 batch, 2 batches and 3 batches of substratum, then by 1 batch, 2 crowdes and 3 crowdes of substratum high pressure steam sterilization 20min at 121 DEG C of temperature, when substratum temperature is down to 30 DEG C, the bacterial classification activated by Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) the by volume inoculum size of percentage composition 5% is inoculated into respectively and is equipped with 1 batch, in 2 batches and 3 batch sample stainless steel fermentor tanks, then it is 30 DEG C in temperature, stirring velocity is 100r/min, air flow quantity is 0.45v/v/m, tank pressure is 0.1kPa, dissolved oxygen is 70%, fermented liquid pH is the condition bottom fermentation 120h of 7.2, obtain soy broth nitragin, aseptically be dispensed in the double-layer plastic bag of gamma line sterilizing, every packed 225mL, for subsequent use.
V/v/m in this test is the unit of the proprietary expression air flow quantity of fermentation industry, and 0.3 ~ 0.6v/v/m represents that volume of air that per minute enters fermentor tank is 0.3 ~ 0.6 times of culture volume.This test is reached by the change of stirring velocity and controls the object of dissolved oxygen, when dissolved oxygen lower than 70% time Controlling System automatically increase rotating speed, when dissolved oxygen higher than 70% time Controlling System automatically reduce rotating speed.
The Slow-growing Soybean rhizobia (Bradyrhizobiumjaponicum) of this test, buys from China Agricultural University's Culture Collection, address: No. 2, West Road, Yuanmingyuan Park, Beijing, deposit number: CCBAU15464.
By the 1st batch, the 2nd batch and the 3rd batch of soy broth nitragin obtained, often criticize and get 20 bags under temperature is 20 DEG C of conditions, place 3 months, 6 months, 9 months and 12 months respectively, detect bacterium number by plate count, the results are shown in Table 4.
Table 4 large scale fermentation produces rihizobium japonicum testing data
| Starter bacteria number | 20 DEG C/3 months | 20 DEG C/6 months | 20 DEG C/9 months | 20 DEG C/12 months | |
| 1st batch | 1.37×10 9/mL | 1.41×10 10/mL | 1.31×10 10/mL | 1.18×10 10/mL | 1.05×10 10/mL |
| 2nd batch | 1.45×10 10/mL | 1.33×10 10/mL | 1.26×10 10/mL | 1.22×10 10/mL | 9.88×10 9/mL |
| 3rd batch | 1.47×10 10/mL | 1.51×10 10/mL | 1.38×10 10/mL | 1.29×10 10/mL | 1.21×10 10/mL |
| On average | 1.43×10 10/mL | 1.42×10 10/mL | 1.32×10 10/mL | 1.23×10 10/mL | 1.08×10 10/mL |
As can be seen from Table 4, the Controlling System of 5 tons of fermentor tanks is more perfect, and can provide more favorably environment for the growth of root nodule bacterium, viable count is higher.In 20 DEG C of constant temperature cabinets, preserve 12 months viable counts still reach every milliliter more than 10,000,000,000; The result of carrying out sampling Detection to this test by the Ministry of Agriculture's microbial fertilizer and edible fungus species quality examination center is 11,000,000,000/milliliter.
On seed, survival time length weighs another important indicator of liquid nitragin product.If root nodule bacterium are executed after on seed dead rapidly, by the time only have minority bacterium to survive during seed germination, so the effect of Rhizobium Inoculation can be had a greatly reduced quality.
The soy broth nitragin that this test is obtained be substratum with YMB, the soy broth nitragin prepared according to the method for this test carries out simultaneous test: the soy broth nitragin that the soy broth nitragin this test obtained and YMB cultivate is inoculated 2.8 milliliters of nitragins by every kilogram of soybean seeds respectively and then stored under 15 DEG C of conditions, viable count on results of regular determination seed, the results are shown in Table 5; Wherein, YMB substratum is the most frequently used root nodule bacterium substratum, it is composed as follows: the bitter salt of the N.F,USP MANNITOL of 10 grams per liters, the dipotassium hydrogen phosphate of 0.5 grams per liter, 0.2 grams per liter, the sodium-chlor of 0.1 grams per liter, the yeast powder of 0.5 grams per liter and 1000 ml pure waters (referring to VincentJ.M.1970Amanualforthepracticalstudyofroot-noduleb acteria.IBPhandbook15.BlackwellScientificPublications, Oxford.).
Viable count data on table 5 seed
| Viable count on every soybean seeds | This test produce nitragin | YMB produces nitragin |
| 0 hour | 8.5x×10 6CFU/ grain seed | 3.3×10 5CFU/ grain seed |
| 24 hours | 4.5×10 6CFU/ grain seed | 6.2×10 4CFU/ grain seed |
| 72 hours | 1.7×10 6CFU/ grain seed | 2.1×10 4CFU/ grain seed |
| 168 hours | 4.8×10 5CFU/ grain seed | 3.6×10 3CFU/ grain seed |
As shown in Table 5, after 168 hours, this test produce the viable count of nitragin on seed be YMB produce 130 times of root nodule bacterium, it can thus be appreciated that the soy broth nitragin that this test prepares is balanced in nutrition, microbial activity is high, on seed the survival time long.
Claims (10)
1. a soybean nodulation bacterium culture medium, is characterized in that: it comprises glycerol, N.F,USP MANNITOL, glucose, rhamnosyl, fructose, α-ketoglutaric acid, yeast powder, Serine, arginine, ammonium chloride, dipotassium hydrogen phosphate, potassium primary phosphate, bitter salt, CALCIUM CHLORIDE DIHYDRATE, seven ferric sulfate hydrates, sodium-chlor, the aqueous solution of two molybdic acid hydrate sodium and vitamin H, wherein, each component solubility is in aqueous respectively: glycerol 1 ~ 3 grams per liter, N.F,USP MANNITOL 1 ~ 3 grams per liter, glucose 1 ~ 3 grams per liter, rhamnosyl 1 ~ 3 grams per liter, fructose 1 ~ 3 grams per liter, α-ketoglutaric acid 0.5 ~ 1.5 grams per liter, yeast powder 2.5 ~ 4.5 grams per liter, Serine 0.13 ~ 0.25 grams per liter, arginine 0.1 ~ 0.2 grams per liter, ammonium chloride 0.65 ~ 1.6 grams per liter, dipotassium hydrogen phosphate 1 ~ 3 grams per liter, potassium primary phosphate 0.2 ~ 0.5 grams per liter, bitter salt 0.1 ~ 0.3 grams per liter, CALCIUM CHLORIDE DIHYDRATE 0.1 ~ 0.2 grams per liter, seven ferric sulfate hydrate 0.005 ~ 0.011 grams per liters, sodium-chlor 0.05 ~ 0.15 grams per liter, two molybdic acid hydrate sodium 0.01 ~ 0.03 grams per liters, vitamin H 0.0005 ~ 0.002 grams per liter, the pH of described soybean nodulation bacterium culture medium is 6.8 ~ 7.5.
2. soybean nodulation bacterium culture medium according to claim 1, it is characterized in that: each component solubility is in aqueous respectively: glycerol 1.5 ~ 2.5 grams per liter, N.F,USP MANNITOL 1.5 ~ 2.5 grams per liter, glucose 1.5 ~ 2.5 grams per liter, rhamnosyl 1.5 ~ 2.5 grams per liter, fructose 1.5 ~ 2.5 grams per liter, α-ketoglutaric acid 0.75 ~ 1.25 grams per liter, yeast powder 3.0 ~ 4.0 grams per liter, Serine 0.15 ~ 0.20 grams per liter, arginine 0.125 ~ 0.175 grams per liter, ammonium chloride 0.8 ~ 1.25 grams per liter, dipotassium hydrogen phosphate 1.1 ~ 1.6 grams per liter, potassium primary phosphate 0.25 ~ 0.4 grams per liter, bitter salt 0.15 ~ 0.25 grams per liter, CALCIUM CHLORIDE DIHYDRATE 0.11 ~ 0.15 grams per liter, seven ferric sulfate hydrate 0.006 ~ 0.009 grams per liters, sodium-chlor 0.08 ~ 0.12 grams per liter, two molybdic acid hydrate sodium 0.015 ~ 0.025 grams per liters and vitamin H 0.0008 ~ 0.0012 grams per liter.
3. soybean nodulation bacterium culture medium according to claim 1 and 2, it is characterized in that: each component solubility is in aqueous respectively: glycerol 2 grams per liter, N.F,USP MANNITOL 2.0 grams per liter, glucose 2.0 grams per liter, rhamnosyl 2.0 grams per liter, fructose 2.0 grams per liter, α-ketoglutaric acid 1.0 grams per liter, yeast powder 3.5 grams per liter, Serine 0.18 grams per liter, arginine 0.15 grams per liter, ammonium chloride 1.1 grams per liter, dipotassium hydrogen phosphate 1.2 grams per liter, potassium primary phosphate 0.34 grams per liter, bitter salt 0.2 grams per liter, CALCIUM CHLORIDE DIHYDRATE 0.13 grams per liter, seven ferric sulfate hydrate 0.0083 grams per liters, sodium-chlor 0.1 grams per liter, two molybdic acid hydrate sodium 0.02 grams per liters and vitamin H 0.001 grams per liter.
4. utilize the method for the rihizobium japonicum medium preparing soy broth nitragin described in claim 1, it is characterized in that, prepare according to the following steps:
Step one, the preparation of soy broth root nodule bacterium substratum: be 1 ~ 3 grams per liter according to glycerol concentration, mannitol concentration is 1 ~ 3 grams per liter, glucose concn is 1 ~ 3 grams per liter, rhamnosyl concentration is 1 ~ 3 grams per liter, fructose concentration is 1 ~ 3 grams per liter, α-ketoglutaric acid concentration is 0.5 ~ 1.5 grams per liter, yeast powder concentration is 2.5 ~ 4.5 grams per liters, serine concentration is 0.13 ~ 0.25 grams per liter, arginine concentrations is 0.1 ~ 0.2 grams per liter, ammonium chloride concentration is 0.65 ~ 1.6 grams per liter, dipotassium hydrogen phosphate concentration is 1 ~ 3 grams per liter, biphosphate potassium concn is 0.2 ~ 0.5 grams per liter, bitter salt concentration is 0.1 ~ 0.3 grams per liter, CALCIUM CHLORIDE DIHYDRATE concentration is 0.1 ~ 0.2 grams per liter, seven hydrated sulfuric acid concentration of iron are 0.005 ~ 0.011 grams per liter, sodium chloride concentration is 0.05 ~ 0.15 grams per liter, two molybdic acid hydrate na concns are 0.01 ~ 0.03 grams per liter, biotin concentration is that 0.0005 ~ 0.002 grams per liter takes above-mentioned substance, and joins in pure water successively and mix, and regulates pH to 6.8 ~ 7.5, obtains soy broth root nodule bacterium substratum,
Step 2, the preparation of soy broth nitragin: soy broth root nodule bacterium substratum step one obtained joins in fermentor tank, at tank pressure 0.15 ~ 0.2kPa, sterilizing 20min under temperature 121 DEG C of conditions, when substratum temperature is down to 30 DEG C, by rihizobium japonicum by volume percentage composition be 5% ~ 10% inoculum size be seeded in the soy broth root nodule bacterium substratum in fermentor tank, it is 25 ~ 35 DEG C at leavening temperature, stirring velocity is 50 ~ 150r/min, air flow quantity is 0.3 ~ 0.6v/v/m, tank pressure is 0.05 ~ 0.2kPa, dissolved oxygen is 60% ~ 80%, fermented liquid pH is under the condition of 6.5 ~ 7.5, fermentation 72 ~ 144h, obtain soy broth nitragin.
5. the preparation method of soy broth nitragin according to claim 4, it is characterized in that: in step one, each component takes by following concentration requirement: glycerol 1.5 ~ 2.5 grams per liter, N.F,USP MANNITOL 1.5 ~ 2.5 grams per liter, glucose 1.5 ~ 2.5 grams per liter, rhamnosyl 1.5 ~ 2.5 grams per liter, fructose 1.5 ~ 2.5 grams per liter, α-ketoglutaric acid 0.75 ~ 1.25 grams per liter, yeast powder 3.0 ~ 4.0 grams per liter, Serine 0.15 ~ 0.20 grams per liter, arginine 0.125 ~ 0.175 grams per liter, ammonium chloride 0.8 ~ 1.25 grams per liter, dipotassium hydrogen phosphate 1.1 ~ 1.6 grams per liter, potassium primary phosphate 0.25 ~ 0.4 grams per liter, bitter salt 0.15 ~ 0.25 grams per liter, CALCIUM CHLORIDE DIHYDRATE 0.11 ~ 0.15 grams per liter, seven ferric sulfate hydrate 0.006 ~ 0.009 grams per liters, sodium-chlor 0.08 ~ 0.12 grams per liter, two molybdic acid hydrate sodium 0.015 ~ 0.025 grams per liters and vitamin H 0.0008 ~ 0.0012 grams per liter.
6. the preparation method of soy broth nitragin according to claim 5, it is characterized in that: in step one, each component takes by following concentration requirement: glycerol 2 grams per liter, N.F,USP MANNITOL 2.0 grams per liter, glucose 2.0 grams per liter, rhamnosyl 2.0 grams per liter, fructose 2.0 grams per liter, α-ketoglutaric acid 1.0 grams per liter, yeast powder 3.5 grams per liter, Serine 0.18 grams per liter, arginine 0.15 grams per liter, ammonium chloride 1.1 grams per liter, dipotassium hydrogen phosphate 1.2 grams per liter, potassium primary phosphate 0.34 grams per liter, bitter salt 0.2 grams per liter, CALCIUM CHLORIDE DIHYDRATE 0.13 grams per liter, seven ferric sulfate hydrate 0.0083 grams per liters, sodium-chlor 0.1 grams per liter, two molybdic acid hydrate sodium 0.02 grams per liters and vitamin H 0.001 grams per liter.
7. the preparation method of soy broth nitragin according to claim 4, is characterized in that: in step 2, and described soy broth root nodule bacterium cultivate 70% of fiduciary point fermentor tank cumulative volume.
8. the preparation method of soy broth nitragin according to claim 4, it is characterized in that: in step 2, after soy broth root nodule bacterium culture medium inoculated leavening temperature be 29 ~ 31 DEG C, under stirring velocity is 75 ~ 125r/min, air flow quantity is 0.35 ~ 0.55v/v/m, tank pressure is 0.075 ~ 0.18kPa, dissolved oxygen is 65 ~ 75%, fermented liquid pH is the condition of 6.8 ~ 7.3, fermentation 72 ~ 144h.
9. the preparation method of soy broth nitragin according to claim 8, it is characterized in that: in step 2, after soy broth root nodule bacterium culture medium inoculated leavening temperature be 30 DEG C, under stirring velocity is 100r/min, air flow quantity is 0.45v/v/m, tank pressure is 0.1kPa, dissolved oxygen is 70%, fermented liquid pH is the condition of 7.2, fermentation 72 ~ 144h.
10. the preparation method of soy broth nitragin according to claim 4, is characterized in that: in step 2, and described rihizobium japonicum is Slow-growing Soybean rhizobia.
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| CN109169054A (en) * | 2018-09-26 | 2019-01-11 | 浙江海洋大学 | A kind of planting unit and method of organic spinach |
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| CN105176878A (en) * | 2015-10-13 | 2015-12-23 | 王�琦 | Peanut rhizobium culture medium and method for preparing peanut rhizobium inoculant by adopting peanut rhizobium culture medium |
| CN109169054A (en) * | 2018-09-26 | 2019-01-11 | 浙江海洋大学 | A kind of planting unit and method of organic spinach |
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