CN102417890A - Sinorhizobium meliloti and method for applying same for fermenting to produce manganese peroxidase - Google Patents
Sinorhizobium meliloti and method for applying same for fermenting to produce manganese peroxidase Download PDFInfo
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- CN102417890A CN102417890A CN201110407709XA CN201110407709A CN102417890A CN 102417890 A CN102417890 A CN 102417890A CN 201110407709X A CN201110407709X A CN 201110407709XA CN 201110407709 A CN201110407709 A CN 201110407709A CN 102417890 A CN102417890 A CN 102417890A
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Abstract
The invention relates to sinorhizobium meliloti and application thereof, which belong to the technical field of microbial fermentation. The sinorhizobium meliloti J09 is preserved in China Center for Type CultureCollection with the number of CCTCC NO: M 2011318. A method for applying the strain for producing manganese peroxidase through liquid fermentation comprises the following steps of: (1) slant culture; (2) seed culture; and (3) liquid fermentation culture: inoculating the cultured seed into a fermentation medium with the inoculation rate of 1 percent to 2 percent, and culturing at 32-36 DEG C for 96-136 hours with the rotating speed of a table concentrator of 150rpm. The sinorhizobium meliloti provided by the invention has the advantages that: the CCTCC NO: M 2011318 has the capability of producing the manganese peroxidase and higher biological nitrogen fixation activity. Since the strain is used for producing manganese peroxidase through liquid fermentation, the advantages that the production period is short, the production cost is low, the growth conditions are easy to control, and the like are achieved, and the method has potential for industrialization production. At the same time, the high-efficient biological nitrogen fixation activity shows that the strain has the capabilities of improving soil fertility and promoting high yield of crops.
Description
Technical field
One strain Melilotus suaveolens Ledeb. China root nodule bacterium and use the method that manganese peroxidase is produced in its fermentation, a kind of specifically Melilotus suaveolens Ledeb. China Rhizobium strains, and utilize this strain fermentation to produce the method for manganese peroxidase and the potential application aspect biological nitrogen fixation.The invention belongs to the microbial fermentation technology field.
Background technology
Separation of pure dissolves manganese peroxidase (MnP) from Phanerochaete chrysosporium the earliest.It can oxidative lignin etc. all kinds of aromatic compounds; Along with people's is goed deep into aspect researchs such as the cultivation of MnP and separation and purification, structure and function, physico-chemical property; It has high industrial application potentiality and has also obtained continuous exploitation, as aspect the biological degradation of the processing of the bio-bleaching of the decolouring of the degraded of organic pollutant, dyestuff, paper pulp, agricultural wastes, brown coal important application being arranged all.
At present, what can produce manganese peroxidase basic is fungi, especially white-rot fungi, as Phanerochaete chrysosporium (
Phanerochaete chrysosporium), variable color bolt bacterium (
Trametes versicolor), mushroom (
Lentinus edodes), flat mushroom (
Pleurotus pstreatus), Lentinus tigrinus (
Panus tigrinus), dirty cross hair pore fungi (
Dichomitus squalens), worm plan wax bacterium (
Ceriporiopsis subvermispora), penetrate the arteries and veins bacterium (
Phlebia radiata) etc.
Bacterial origin is extensive, growth is quick, and adaptive faculty is strong, is easy to transform and large-scale application, also adopts the fixed cell technology to be beneficial to the application of manganese peroxidase more easily simultaneously.But the research report about producing the manganese peroxidase bacterium is gone back lacking of ten minutes at present, more not about Melilotus suaveolens Ledeb. China root nodule bacterium report in this respect.
Summary of the invention
The object of the present invention is to provide a kind of Melilotus suaveolens Ledeb. China rhizobium strains with product manganese peroxidase, and the method and the application aspect biological nitrogen fixation that utilize this strain liquid fermentation to produce manganese peroxidase.
Technical scheme of the present invention: a strain is produced manganese peroxidase and is had the active bacterial strain of biological nitrogen fixation, called after Melilotus suaveolens Ledeb. China root nodule bacterium (
Sinorhizobium meliloti) J09, in China's typical culture collection center preservation, deposit number is CCTCC NO:M 2011318.
Method with manganese peroxidase is produced in described Melilotus suaveolens Ledeb. China root nodule bacterium CCTCC NO:M 2011318 fermentations, realize through following steps:
(1) slant culture: Melilotus suaveolens Ledeb. China root nodule bacterium CCTCC NO:M 2011318 is inoculated on the slant medium, cultivated 24 hours down at 33 ℃;
(2) seed culture: the long good bacterial classification in inclined-plane is inserted in the seed culture medium, cultivated 150 rev/mins of shaking speed 30 hours at 33 ℃;
(3) liquid fermentation and culture: cultured seed is inserted in the fermention medium, and inoculum size is a volume percent 1% ~ 2%, cultivates 150 rev/mins of shaking speed 72 ~ 96 hours at 32 ~ 36 ℃;
Fermention medium is formed, and unit is a grams per liter: lactose 15 ~ 30, and peptone 5 ~ 20, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015 is settled to 1L with water, and pH 7.0 ~ 8.0,121 ℃ of sterilization 30 min.
Melilotus suaveolens Ledeb. China root nodule bacterium (
Sinorhizobium meliloti) CCTCC NO:M 2011318 screenings, separation and evaluation see embodiment 1 for details.
Slant medium described in the technique scheme is (unit is g/L): sodium-chlor 5, yeast extract paste 5, peptone 10, agar 20,7.0,115 ℃ of sterilizations of pH 20 minutes.
Seed culture medium described in the technique scheme is (unit is g/L): sodium-chlor 5, yeast extract paste 5, peptone 10,7.0,121 ℃ of sterilizations of pH, 30 min.
The mensuration of manganese peroxidase enzyme activity: prepare lactic acid-Sodium.alpha.-hydroxypropionate damping fluid (pH4.5) of 50 mmol/L, the manganese sulfate solution of 1.6 mmol/L, the hydrogen peroxide solution of 1.6 mmol/L, used the same day.Get lactic acid-Sodium.alpha.-hydroxypropionate damping fluid (pH4.5) 3.4 mL of 50 mmol/L; 1.6 manganese sulfate solution 0.1 mL of mmol/L is incubated 10min in 37 ℃ of water-baths; Add an amount of 37 ℃ of insulation 10min enzyme liquid (clear liquid of the centrifugal 10min acquisition of fermented liquid 8000rpm), mixing, hydrogen peroxide solution 0.1 mL that adds 1.6 mmol/L shakes up immediately; Pick up counting, measure 240 nm places absorbancys variation in initial 3 min.PM makes 1 μ mol Mn
2+Be converted into Mn
3+Required enzyme amount is a unit of activity (U).
In process of the present invention, discover that this bacterial strain has higher biological nitrogen fixation activity simultaneously, its nitrogenase activity can reach 667nmol h
-1ML
-1This this bacterial strain of explanation promotes the higher yield of crops aspect to have the potential using value equally at fertilizing soil.
Beneficial effect of the present invention: Melilotus suaveolens Ledeb. China root nodule bacterium CCTCC NO:M 2011318 provided by the present invention not only has the ability of producing manganese peroxidase, but also has higher biological nitrogen fixation activity.It is with short production cycle to utilize this bacteria liquid fermentation product manganese peroxidase to have, and production cost is low, is easy to plurality of advantages such as control growing condition, has the potentiality of suitability for industrialized production fully.Simultaneously, biological nitrogen fixation activity efficiently explains that also this bacterial strain has fertilizing soil fully, promotes the ability of higher yield of crops.
The biological material specimens preservation: a strain is produced manganese peroxidase and is had the active bacterial strain of biological nitrogen fixation, called after Melilotus suaveolens Ledeb. China root nodule bacterium (
Sinorhizobium meliloti) J09, in China's typical culture collection center preservation, be called for short CCTCC, preservation date on September 18th, 2011, deposit number is CCTCC NO:M 2011318.
Embodiment
In described below all specific embodiments,, be this area domestic method like no specified otherwise.
Embodiment 1
Melilotus suaveolens Ledeb. China root nodule bacterium (
Sinorhizobium meliloti) CCTCC NO:M 2011318 screening and separating and evaluation
One, Melilotus suaveolens Ledeb. China root nodule bacterium J09 (
Sinorhizobium melilotiJ09) screening and separating
Melilotus suaveolens Ledeb. China root nodule bacterium J09 bacterial strain is from the wood chip of obtaining from San Yu town, Tongzhou District, Nantong City He Xing village wood working individual business operator, to separate to obtain.Get in 1 gram wood chip to the 250 milliliter sterilization triangular flask, add 100 milliliters of aseptic pH values that contain inorganic salt and be that (contained inorganic salt and concentration thereof are respectively: K in 7.0 the aqueous solution
2HPO
41 g/L; NaH
2PO
41 g/L; (NH
4)
2SO
40.5 g/L; MgSO
40.2 g/L; MnSO
40.02 g/L), under 33 ℃, 150 rev/mins condition, cultivated 12 hours; Get above-mentioned nutrient solution, it is diluted to 10
-5, 10
-6, 10
-7Times diluent, each concentration dilution liquid is got 100 microlitres and is coated on the aniline blue flat board (yeast extract paste 5 g/L, peptone 10g/L, sodium-chlor 5g/L, aniline blue (Azure B) 0.1g/L), and each concentration repeats for three times, cultivates 24-36 hour for 33 ℃.
Produce manganese peroxidase bacterial strain hydrolyzable substratum and form transparent circle.Select repeatedly purifying of the bigger single bacterium colony of transparent circle, to slant medium (yeast extract paste 5 g/L, peptone 10g/L, sodium-chlor 5g/L, agar 20g/L), 33 ℃ of cultivations are put in 4 ℃ of refrigerators and preserve for use after 24-36 hour with the inoculation behind the purifying.4 ℃ of refrigerators are preserved inoculation (glucose 20g/L, peptone 10 g/L, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025 g/L in fermention medium for use; Manganous sulfate 0.015 g/L, pH 7.0), at 33 ℃; Cultivated 120 hours under 150 rev/mins the condition; And the variation of manganese peroxidase enzyme activity in the detection culturing process, from the result, filter out a strain and produce the highest bacterial strain of manganese peroxidase enzyme activity, name and be J09.
Two, Melilotus suaveolens Ledeb. China root nodule bacterium J09 (
Sinorhizobium melilotiJ09) identify
(1) morphological specificity
On the LB culture medium flat plate, cultivate thalline and become shaft-like, do not produce gemma, Gram-negative, ellipse, aerobic.Cultivated 36 hours for 35 ℃, the bacterium colony surface drying, 1～2 millimeter of diameter can spread growth, little yellow.
(2) 16S rDNA measures
Utilize the 16S rDNA sequence of this bacterium of round pcr amplification, its Oligonucleolide primers is:
5 '-GAGTTTGATCCTGGCTCAG-3 ' (upstream primer) and
5 '-CGGTTACC TTGTTACGACTT-3 ' (downstream primer).
Measure the 16S rDNA sequence that increases, its sequence such as SEQ ID NO:1 through automatic dna sequencer (U.S. Applied Biosystems company, model ABI 3130).
Known 16S rDNA sequence among sequencing result and the GeneBank is carried out homology relatively, find with
Sinorhizobium melilotiThe homology of strain KYA71 (number of logining is EU603721) is the highest, is 99%.
(3) physiological and biochemical property
According to " Bergey ' s manual of systematic bacteriology " (second edition; Second volume) (George M. Garrity, Don J. Brenner, Noel R. Krieg; James T. Staley; Springer, 2004) and reference (Polyphasic taxonomy of rhizobia:emendation of the genus sinorhizobium and description of sinorhizobium meliloti comb.nov., sinorhizobium saheli sp.nov.; And sinorhizobium teranga sp.nov.. International journal of systematic bacteriology; 1994,44:715-733) bacterial strain J09 is carried out Physiology and biochemistry and identify (like following table), its physiological and biochemical property and Melilotus suaveolens Ledeb. China root nodule bacterium (
Sinorhizobium meliloti) match.
| Characteristic | J09 | Characteristic | J09 |
| Cell dia＞1.0 μ m | - | Utilization of carbon source: D-lyxose | + |
| Gemma is arranged | - | The D-tagatose | + |
| The pH8 growth | + | The D-trehalose | + |
| Gramstaining | - | The L-sorbose | + |
| Flagellum quantity | 1 | Utilization of carbon source: methyl D-xyloside | + |
| Produce acid on yeast extract paste-N.F,USP MANNITOL | + | Methyl D-glucose glucosides | + |
| Catalase | + | Tetrahydroxybutane | + |
| V-P measures | - | Galactitol | + |
| On leguminous plants, give birth to knurl | + | Xylitol | + |
| Anti-0.1% Brilliant cresyl blue | - | L-arabinose alcohol | + |
| PD | + | The 2-ketogluconic acid | + |
| Gelatin hydrolysis | - | The DL-3-propalanine | + |
| The starch hydrolysis | - | Acetate | - |
| Amino acid utilizes: the L-Xie Ansuan | - | Propionic salt | - |
| The L-Serine | - | Isobutyrate | + |
| The L-Threonine | + | Oxyacetate | - |
| The L-aspartic acid | + | DL-hydroxybutyric acid salt | - |
| L-Methionin | + | Aconitate | - |
| The L-N.delta.-carbamylornithine | - | The p-hydroxybenzoate | - |
| L-Ala | + | Trigonelline | + |
According to thalline gramstaining, colonial morphology observe, the comparison result of Physiology and biochemistry test and 16S rDNA sequence, this bacterial strain matches with Melilotus suaveolens Ledeb. China root nodule bacterium, therefore with its be accredited as the Chinese root nodule bacterium of Melilotus suaveolens Ledeb. (
Sinorhizobium meliloti).
Manganese peroxidase is produced in root nodule bacterium CCTCC NO:M 2011318 fermentations of embodiment 2 Melilotus suaveolens Ledeb.s China
(1) slant culture: Melilotus suaveolens Ledeb. China root nodule bacterium CCTCC NO:M 2011318 is inoculated on the slant medium, cultivated 24 hours down at 33 ℃.Used slant medium is (unit is a grams per liter): sodium-chlor 5, yeast extract paste 5, peptone 10, agar 20,7.0,115 ℃ of sterilizations of pH 20 minutes.
(2) seed culture: the long good bacterial classification in inclined-plane is got an articulating with transfering loop go in the seed culture medium, cultivated 150 rev/mins of shaking speed 30 hours at 33 ℃.Used seed culture medium is (unit is a grams per liter): sodium-chlor 5, and yeast extract paste 5, peptone 10, pH 7.0; The bottled 30 milliliters of seed culture mediums of per 250 milliliters of triangles were sterilized 30 minutes for 121 ℃.
(3) liquid fermentation and culture: used fermention medium is (unit is a grams per liter): lactose 15, peptone 5, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015, pH7.0,121 ℃ of sterilization 30 min.Cultured seed is inserted in 500 milliliters of triangular flasks, and wherein fermention medium is 50 milliliters, and inoculum size is 0.5 milliliter, and 150 rev/mins of shaking speed were cultivated 96 hours at 32 ℃.Cultivate and finish, the manganese peroxidase enzyme of fermented liquid is lived and is the 503U/ liter.
Manganese peroxidase is produced in root nodule bacterium CCTCC NO:M 2011318 fermentations of embodiment 3 Melilotus suaveolens Ledeb.s China
Cultured seed is inserted in 500 milliliters of triangular flasks, and wherein fermention medium is 50 milliliters, and inoculum size is 1 milliliter, cultivates 150 rev/mins of shaking speed 72 hours at 36 ℃.Used fermention medium is (unit is a grams per liter): lactose 30, peptone 20, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015, pH8.0,121 ℃ of sterilization 30 min.Other conditions are cultivated and are finished with embodiment 2, and the manganese peroxidase enzyme of fermented liquid is lived and is the 522U/ liter.
Manganese peroxidase is produced in root nodule bacterium CCTCC NO:M 2011318 fermentations of embodiment 4 Melilotus suaveolens Ledeb.s China
Cultured seed is inserted in 500 milliliters of triangular flasks, and wherein fermention medium is 50 milliliters, and inoculum size is 0.75 milliliter, cultivates 150 rev/mins of shaking speed 82 hours at 33 ℃.Used fermention medium is (unit is a grams per liter): lactose 20, peptone 10, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015, pH7.8,121 ℃ of sterilization 30 min.Other conditions are cultivated and are finished with embodiment 2, and the manganese peroxidase enzyme of fermented liquid is lived and is the 582U/ liter.
Manganese peroxidase is produced in root nodule bacterium CCTCC NO:M 2011318 fermentations of embodiment 5 Melilotus suaveolens Ledeb.s China
Cultured seed is inserted in 500 milliliters of triangular flasks, and wherein fermention medium is 50 milliliters, and inoculum size is 0.75 milliliter, cultivates 150 rev/mins of shaking speed 80 hours at 35 ℃.Used fermention medium is (unit is a grams per liter): lactose 18, peptone 12, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015, pH7.5,121 ℃ of sterilization 30 min.Other conditions are cultivated and are finished with embodiment 2, and the manganese peroxidase enzyme of fermented liquid is lived and is the 551U/ liter.
Manganese peroxidase is produced in root nodule bacterium CCTCC NO:M 2011318 fermentations of embodiment 6 Melilotus suaveolens Ledeb.s China
Cultured seed is inserted in 500 milliliters of triangular flasks, and wherein fermention medium is 50 milliliters, and inoculum size is 1 milliliter, cultivates 150 rev/mins of shaking speed 96 hours at 36 ℃.Used fermention medium is (unit is a grams per liter): lactose 20, peptone 10, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015, pH7.2,121 ℃ of sterilization 30 min.Other conditions are cultivated and are finished with embodiment 2, and the manganese peroxidase enzyme of fermented liquid is lived and is the 509U/ milliliter.
Manganese peroxidase is produced in root nodule bacterium CCTCC NO:M 2011318 fermentations of embodiment 7 Melilotus suaveolens Ledeb.s China
Cultured seed is inserted in 500 milliliters of triangular flasks, and wherein fermention medium is 50 milliliters, and inoculum size is 0.5 milliliter, cultivates 150 rev/mins of shaking speed 72 hours at 32 ℃.Used fermention medium is (unit is a grams per liter): lactose 20, peptone 10, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015, pH7.8,121 ℃ of sterilization 30 min.Other conditions are cultivated and are finished with embodiment 2, and the manganese peroxidase enzyme of fermented liquid is lived and is the 567U/ liter.
The active mensuration of embodiment 8 Melilotus suaveolens Ledeb.s China's root nodule bacterium CCTCC NO:M 2011318 biological nitrogen fixations
Cultured seed is inserted in the serum bottle that fills the 10mL semisolid medium (3 repetitions of each substratum), and inoculum size is 1 milliliter, and used fermention medium is (unit is a grams per liter): oxysuccinic acid 5, potassium hydrogenphosphate 0.5; MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.2, sodium-chlor 0.1, two hydration calcium chloride 0.02, two molybdic acid hydrate sodium 0.002; Dibromothymolsulfonphthalein 5ml, Pottasium Hydroxide 4.5, VITAMINs 0.00001; Agar 2, pH7.5,121 ℃ of sterilization 30 min.Cultivate 48h in 30 ℃ of following incubators, under aseptic condition, change the little bottle cap of serum into rubber plug, extract 10% gas with asepsis injector out; Give every bottle then and inject 10% acetylene; Put again and cultivate 48h in the incubator, from bottle, extract mixed gas 0.2mL inject gas chromatograph (GC-14B, Shimadzu; Japan) in, measure C with the acetylene reduction method
2H
4Growing amount, the result shows that the nitrogenase activity under this bacterium pure culture condition is 667nmol h
-1ML
-1
Below described embodiment of the present invention in detail, can do a lot of improvement and variation obviously for a person skilled in the art and can not deviate from essence spirit of the present invention.All these variations and improvement are all within protection scope of the present invention.
Sequence table
<210> SEQ?ID?NO:?1
<211> 1383
<212> DNA
<213>Melilotus suaveolens Ledeb. China root nodule bacterium (
Sinorhizobium meliloti) J09
<400>1
tcgagcgccc?cgcaagggga?agcggcagac?gggtgagtaa?cgcgtgggaa?tctacccttt 60
tctacggaat?aacgcaggga?aacttgtgct?aataccgtat?acgcccttcg?ggggaaagat 120
ttatcgggaa?aggatgagcc?cgcgttggat?tagctagttg?gtggggtaaa?ggcctaccaa 180
ggcgacgatc?catagctggt?ctgagaggat?gatcagccac?attgggactg?agacacggcc 240
caaactccta?cgggaggcag?cagtggggaa?tattggacaa?tgggcgcaag?cctgatccag 300
ccatgccgcg?tgagtgatga?aggccctagg?gttgtaaagc?tctttcaccg?gtgaagataa 360
tgacggtaac?cggagaagaa?gccccggcta?acttcgtgcc?agcagccgcg?gtaatacgaa 420
gggggctagc?gttgttcgga?attactgggc?gtaaagcgca?cgtaggcgga?catttaagtc 480
aggggtgaaa?tcccggggct?caaccccgga?actgcctttg?atactgggtg?tctagagtat 540
ggaagaggtg?agtggaattc?cgagtgtaga?ggtgaaattc?gtagatattc?ggaggaacac 600
cagtggcgaa?ggcggctcac?tggtccatta?ctgacgctga?ggtgcgaaag?cgtggggagc 660
aaacaggatt?agataccctg?gtagtccacg?ccgtaaacga?tgaatgttag?ccgtcgggca 720
gtttactgtt?cggtggcgca?gctaacgcat?taaacattcc?gcctggggag?tacggtcgca 780
agattaaaac?tcaaaggaat?tgacgggggc?ccgcacaagc?ggtggagcat?gtggtttaat 840
tcgaagcaac?gcgcagaacc?ttaccagccc?ttgacatccc?gatcgcggat?tacggagacg 900
ttttccttca?gttcggctgg?atcggagaca?ggtgctgcat?ggctgtcgtc?agctcgtgtc 960
gtgagatgtt?gggttaagtc?ccgcaacgag?cgcaaccctc?gcccttagtt?gccagcattt 1020
agttgggcac?tctaagggga?ctgccggtga?taagccgaga?ggaaggtggg?gatgacgtca 1080
agtcctcatg?gcccttacgg?gctgggctac?acacgtgcta?caatggtggt?gacagtgggc 1140
agcgagaccg?cgaggtcgag?ctaatctcca?aaagccatct?cagttcggat?tgcactctgc 1200
aactcgagtg?catgaagttg?gaatcgctag?taatcgcaga?tcagcatgct?gcggtgaata 1260
cgttcccggg?ccttgtacac?accgcccgtc?acaccatggg?agttggttct?acccgaaggt 1320
agtgcgctaa?ccgcaaggag?gcagctaacc?acggtagggt?cagcgactgg?ggtgaagtcg 1380
taa 1383
Claims (3)
1. a strain is produced manganese peroxidase and is had the active bacterial strain of biological nitrogen fixation, called after Melilotus suaveolens Ledeb. China root nodule bacterium (
Sinorhizobium meliloti) J09, in China's typical culture collection center preservation, deposit number is CCTCC NO:M 2011318.
2. with the method for the described Melilotus suaveolens Ledeb. of claim 1 China root nodule bacterium CCTCC NO:M 2011318 fermentative prodn manganese peroxidases, it is characterized in that realizing through following steps:
(1) slant culture: Melilotus suaveolens Ledeb. China root nodule bacterium CCTCC NO:M 2011318 is inoculated on the slant medium, cultivated 24 hours down at 33 ℃;
(2) seed culture: the long good bacterial classification in inclined-plane is inserted in the seed culture medium, cultivated 150 rev/mins of shaking speed 30 hours at 33 ℃;
(3) liquid fermentation and culture: cultured seed is inserted in the fermention medium, and inoculum size is a volume percent 1% ~ 2%, cultivates 150 rev/mins of shaking speed 72 ~ 96 hours at 32 ~ 36 ℃;
Fermention medium is formed, and unit is g/L: lactose 15 ~ 30, and peptone 5 ~ 20, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.025, manganous sulfate 0.015, water are settled to 1 liter, and pH 7.0 ~ 8.0,121 ℃ of sterilization 30 min.
3. the described Melilotus suaveolens Ledeb. of claim 1 China root nodule bacterium CCTCC NO:M 2011318, it is characterized in that having higher biological nitrogen fixation activity, reach 667nmol h
-1ML
-1
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| CN103275888A (en) * | 2013-04-09 | 2013-09-04 | 南京师范大学 | Ensifer meliloti and its use in degradation of chloronicotinyl insecticide thiacloprid |
| CN104611271A (en) * | 2015-02-09 | 2015-05-13 | 河北大学 | Efficient nitrogen-fixing alfalfa rhizobium strain and method for screening molecular marker |
| CN105349456A (en) * | 2015-11-12 | 2016-02-24 | 甘肃省科学院生物研究所 | Sinorhizobium sp. and its application |
| CN109370995A (en) * | 2018-12-11 | 2019-02-22 | 中国农业科学院饲料研究所 | Manganese peroxidase CsMnP and its encoding gene and application |
| CN115747103A (en) * | 2022-11-04 | 2023-03-07 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Sinorhizobium meliloti LQR-15 capable of promoting growth of traditional Chinese medicinal materials, microbial inoculum and application |
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| CN103275888A (en) * | 2013-04-09 | 2013-09-04 | 南京师范大学 | Ensifer meliloti and its use in degradation of chloronicotinyl insecticide thiacloprid |
| CN103275888B (en) * | 2013-04-09 | 2014-11-05 | 南京师范大学 | Ensifer meliloti and its use in degradation of chloronicotinyl insecticide thiacloprid |
| CN104611271A (en) * | 2015-02-09 | 2015-05-13 | 河北大学 | Efficient nitrogen-fixing alfalfa rhizobium strain and method for screening molecular marker |
| CN105349456A (en) * | 2015-11-12 | 2016-02-24 | 甘肃省科学院生物研究所 | Sinorhizobium sp. and its application |
| CN105349456B (en) * | 2015-11-12 | 2020-04-07 | 甘肃省科学院生物研究所 | Sinorhizobium and application thereof |
| CN109370995A (en) * | 2018-12-11 | 2019-02-22 | 中国农业科学院饲料研究所 | Manganese peroxidase CsMnP and its encoding gene and application |
| CN109370995B (en) * | 2018-12-11 | 2021-05-25 | 中国农业科学院北京畜牧兽医研究所 | Manganese peroxidase CsMnP and coding gene and application thereof |
| CN115747103A (en) * | 2022-11-04 | 2023-03-07 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Sinorhizobium meliloti LQR-15 capable of promoting growth of traditional Chinese medicinal materials, microbial inoculum and application |
| CN115747103B (en) * | 2022-11-04 | 2024-03-22 | 宁夏农林科学院植物保护研究所（宁夏植物病虫害防治重点实验室） | Sinorhizobium meliloti LQR-15 capable of promoting growth of traditional Chinese medicinal materials, microbial inoculum and application |
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