CN105420169A - Gamma-polyglutamic acid producing strain and method for producing gamma-polyglutamic acid organic fertilizer - Google Patents
Gamma-polyglutamic acid producing strain and method for producing gamma-polyglutamic acid organic fertilizer Download PDFInfo
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- 238000004519 manufacturing process Methods 0.000 title abstract description 17
- 239000003895 organic fertilizer Substances 0.000 title abstract description 7
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- 241000894006 Bacteria Species 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 25
- 230000035558 fertility Effects 0.000 claims abstract description 22
- 108700022290 poly(gamma-glutamic acid) Proteins 0.000 claims abstract description 15
- 239000002154 agricultural waste Substances 0.000 claims abstract description 13
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000013922 glutamic acid Nutrition 0.000 claims abstract description 10
- 239000004220 glutamic acid Substances 0.000 claims abstract description 10
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- 229960002989 glutamic acid Drugs 0.000 description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
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- 241000311115 Bacillus paralicheniformis ATCC 9945a Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
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- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 244000046109 Sorghum vulgare var. nervosum Species 0.000 description 1
- 101710097943 Viral-enhancing factor Proteins 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C05F9/00—Fertilisers from household or town refuse
- C05F9/04—Biological compost
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/80—Soil conditioners
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention relates to a gamma-polyglutamic acid producing strain and a method for producing organic fertilizer for preserving moisture and fertility by means of mixed fermentation of the strain and a cellulose degradation bacterium, and belongs to the field of microbial fermentation engineering. The Bacillus sp. J3CGMCC No.11582 is adopted, and the strain can be independently fermented on the low-concentration glutamic acid substrate condition or mixed with the cellulose degradation bacterium to be fermented to obtain the high-yield gamma-PGA. Straw, excrements of livestock and other agricultural wastes are utilized for mixed fermentation to prepare the organic fertilizer for preserving moisture and fertility, the agricultural wastes are recycled and turned into wealth, pressure of the agricultural wastes on the environment is relieved, resources are saved, and the method is of great significance on sustainable development of agriculture.
Description
Technical field
The present invention relates to a strain gamma-polyglutamic acid generating bacterium, be specifically related to a kind of gamma-polyglutamic acid generating bacterium and a kind of cellulose-degrading bacteria and utilize this bacterial strain mixed fermentation to produce the method for preserve moisture and fertility fertilizer, belonging to microbial fermentation engineering field.
Background technology
China is large agricultural country, and be again the country that water resources lacks, arid, semiarid zone account for the over half of area, and arid is the important factor that restriction China gets agricultural profit, and the loss caused thus is positioned at first of various natural disaster.For ensureing China's agricultural foison, need suitably to irrigate, but China's water resources wretched insufficiency, and skewness, Nan Duobei is few, and Xia Duodong is few.The mode adopting flood irrigation at present more, waste is serious; After irrigation, most of water and fertilizer evaporate or run off, and can not play the effect of expection; A large amount of fertilizer loss must cause the Overdosing problems that applies fertilizer simultaneously, both exacerbates widespread pollution from the overuse of fertilizers and pesticides in rural area, and wastes ample resources again; Excessive fertilising also results in farmland ecosystem and is destroyed, the soil organism declines, and crumb structure performance reduces, and soil compaction phenomenon is aggravated, fertilizer conservation water retention capacity reduces, a large amount of microbial death, structure of soil microbial community changes, and many beneficial microorganisms become secondary population from dominant population, easily there is all kinds of disease in crop, the utilization ratio of fertilizer is lower simultaneously, and quality of agricultural product declines, and production cost increases.
For many years, in order to improve the utilising efficiency of fertilizer and moisture, the synergistic factor of people to fertilizer and moisture has carried out large quantifier elimination.Wherein have that water-soluble height, adsorptivity are strong, good biocompatibility, nontoxic, harmless, noresidue, can promote that water-reataining fertilizer-keeping agent that crop nutrition content absorbs is subject to the attention of Chinese scholars, develop hundreds of water-reataining fertilizer-keeping agent.A certain amount of water-reataining fertilizer-keeping agent is added in soil, not only can improve the water retention property of soil, save irrigation water, water-reataining fertilizer-keeping agent and mixing with soil can improve the crumb structure of soil simultaneously, make soil become soft ventilative, and can combine with the nutritive element such as the nitrogen in soil, phosphorus, potassium and various trace element, minimizing nutrient loss, reduce fertilizer application amount, strengthen the resistance against diseases of farm crop.Gamma-polyglutamic acid-(γ-polyglutamicacid, γ-PGA) be combined into by the amido linkage between alpha-amino group and γ-carboxyl by D-Glu or Pidolidone, be the outer water miscible polymer aminoacid polymers of a kind of born of the same parents that some bacillus (Bacillus) is synthesized.γ-PGA has excellent water-soluble, superpower adsorptivity and biodegradability, degraded product is the L-glutamic acid of natural nuisance-free, it is a kind of excellent environment-friendly type macromolecule material, gamma-polyglutamic acid-both can increase the output of crop further, good slow releasing function can also be played to fertilizer and moisture, there is significant preserve moisture and fertility, volume increase joint fertilizer efficiency fruit.
The preparation mainly biological fermentation process of current γ-PGA, namely obtains product by cultivating the bacterial classification producing polyglutamic acid.Domestic and international for studying typical strain mainly BacilluslicheniformisATCC9945a and BacillussubtilisIFO3335 etc. that gamma-polyglutamic acid-is produced at present, fermentation manufacturing technique has liquid fermenting and solid fermentation two kinds usually.At present, gamma-polyglutamic acid-zymotechnique is based on liquid submerged fermentation, but it is longer still to there is the production cycle, and production efficiency is low, production cost higher etc. problem.Obtain a bacillus licheniformis BacilluslicheniformisDou-6 as (publication number CN104017760A) such as Li Peipei is separated, its substrate glutamic acid transformation efficiency is only 60%.Be compared to liquid fermenting, solid fermentation has the advantages such as power consumption is low, device simple, cost are low, generation waste is few, raw material sources are extensive, therefore is usually used in the middle of the fermentable industrial production such as fertilizer, feed, wine brewing.On the other hand; large-scale cultivation produces the agricultural wastes such as the stalk of feces of livestock and poultry and agricultural planting generation and throws aside everywhere; not only cause serious pollution of area source, also significant wastage a large amount of nutrients resources (C, N, P, K, S and trace element).At present, also have with the research of agricultural wastes solid state fermentation production gamma-polyglutamic acid-fertilizer and report successively, " have biological organic fertilizer of preserve moisture and fertility effect and preparation method thereof " as CN102674986 discloses one, the method utilizes single Bacillus strain to carry out gamma-polyglutamic acid-organic fertilizer production in human and animal excreta, domestic refuse or straw.CN101417894 discloses " a kind of production method of the fertilizer additive containing gamma-polyglutamic acid-", the method is fermentation strain with single subtilis (Bacillussubtilis) PGA-7CCTCCM206102, produces the fertilizer additive containing gamma-polyglutamic acid-with amino acid fermentation waste liquid, dregs of beans, groundnut meal, wheat bran, rice straw powder etc. for dominant fermentation base-material carries out solid fermentation.
In sum, gamma-polyglutamic acid-organic fertilizer production mostly rests on ferments based on gamma-polyglutamic acid-single strain, and the productive rate of gamma-polyglutamic acid-is lower, therefore needs the high new strains of the productive rate of developing a kind of gamma-polyglutamic acid-badly and utilize this bacterial strain high yield to produce the method for gamma-polyglutamic acid-fertilizer.
Summary of the invention
The object of this invention is to provide a strain gamma-polyglutamic acid generating bacterium, through being accredited as genus bacillus (Bacillussp.) J3CGMCCNo.11582, this bacterial strain can under L-glutamic acid concentration of substrate comparatively low condition separately or with a strain cellulose-degrading bacteria mixed fermentation high yield γ-PGA.
For achieving the above object, the present invention is by the following technical solutions:
From pedotheque, screening obtains a bacillus (Bacillussp.) J3, this bacterial strain is preserved in China Microbial Culture Preservation Commission's common micro-organisms center (CGMCC), depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is: CGMCCNo.11582, and preservation date is: on November 5th, 2015.This bacterium properties and characteristics is as follows:
(1) morphological specificity
On peptone nutrient agar, vegetative cell is the bacillus of 0.6 ~ 1.0 × 1.2 ~ 2.5 μm of sizes, and cultivate for 37 DEG C and form gemma in 3 ~ 5 days, gemma size 0.6 ~ 0.9 × 1.0 ~ 1.5 μm is Long Circle or cylindrical.
(2) cultural property
Extractum carnis, glucose, nutrient agar slat chain conveyor: cultivating for 30 ~ 40 DEG C 1 day can raised growth, bacterium colony rat, smooth corrugationless, and bacterium colony is transparent, and edge be regular circle shapes, and bacterium colony viscosity greatly, has been provoked thread.
One strain cellulose-degrading bacteria---Thermomonospora (Thermomonosporasp.) GC, from straw compost sample, separation screening obtains, this bacterial strain is preserved in China Microbial Culture Preservation Commission's common micro-organisms center, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is: CGMCCNo.1364, and preservation date is: on April 28th, 2005.
Another object of the present invention be to provide a kind of produce gamma-polyglutamic acid-method and utilize agricultural wastes to carry out method that gamma-polyglutamic acid-preserve moisture and fertility fertilizer is produced in solid-state mixed fermentation, namely be matrix with agricultural wastes, gamma-polyglutamic acid generating bacterium and cellulose-degrading bacteria mixed fermentation is utilized to produce the method for preserve moisture and fertility fertilizer, utilize this fertilizer can improve the absorption property of soil to moisture and fertilizer, promote water retention in soil and water and fertilizer utilization, reduce rate of fertilizer application and irrigation volume, alleviate widespread pollution from the overuse of fertilizers and pesticides in rural area, reduce agriculture production cost, to reach the Sustainable development of agricultural.Preparation method is as follows:
(1) bacterial strain uses therefor is that genus bacillus J3 and thermomonospora fusca GC, thermomonospora fusca GC obtain from the purchase of China Microbial Culture Preservation Commission's common micro-organisms center;
(2) substratum
1) activation medium
Genus bacillus J3 activation medium (beef-protein medium): extractum carnis 3g, peptone 10g, NaCl5g, agar 18 ~ 20g, moisturizing is to 1000mL, pH7.0 ~ 7.4;
Thermomonospora fusca GC activation medium (Gause I substratum): Zulkovsky starch 20g, yeast powder 2g, glucose 5g, KNO
31g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, agar 18 ~ 20g, moisturizing are to 1000mL, pH7.2 ~ 7.6;
Genus bacillus J3 and thermomonospora fusca GC is accessed respectively activate in the beef extract-peptone and Gause I slant medium that sterilizing (121 DEG C, 30min) processes for subsequent use;
2) liquid culture
By beef extract-peptone liquid nutrient medium and Gause I liquid nutrient medium high-temperature sterilization (121 DEG C, 30min) after process, by step 1) the genus bacillus J3 that activated and thermomonospora fusca GC accesses in beef extract-peptone liquid nutrient medium and Gause I liquid nutrient medium respectively, 35 DEG C, 150r/min shaking culture 28h and 50h;
3) solids mixing fermention medium: add stalk and ight soil ratio in urea or adjustment matrix in agricultural waste fermentation matrix, make the mol ratio of carbon nitrogen in matrix be 15 ~ 35:1; Add Sodium Glutamate 0.5 ~ 2.0%, glucose or sucrose 1 ~ 3%, K in mass ratio again
2hPO
40.1 ~ 1%, MgSO
47H
2o0.01 ~ 0.1%, by Sodium Glutamate, glucose or sucrose, K
2hPO
4, MgSO
47H
2o mixing is soluble in waterly sprayed in above-mentioned matrix, regulates fermented substrate water ratio to be 50 ~ 65%, pH value 7.0 ~ 8.0, and stirs and be made into solids mixing fermention medium;
(3) mixed bacterium inoculation volume ratio
Genus bacillus J3: thermomonospora fusca GC=1 ~ 3:5 ~ 10;
(4) mixed bacterium seed liquor step (3) obtained according to solid medium total mass 0.1 ~ 2% inoculum size access sterilize solid mixed fermentive culture medium in, 30 ~ 37 DEG C of aerated culture 20 ~ 40d, stop fermentation, obtain containing γ-PGA preserve moisture and fertility fertilizer.
The present invention improves liquid nutrient medium, beef extract-peptone liquid nutrient medium after improvement and the formula of Gause I liquid nutrient medium are respectively: the beef extract-peptone liquid culture based formulas after improvement: extractum carnis 5g, peptone 10g, glucose 5g, NaCl5g, moisturizing is to 1000mL, pH7.0 ~ 7.4; Gause I liquid culture based formulas after improvement: Zulkovsky starch 15g, yeast powder 2g, glucose 10g, KNO
31g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, moisturizing is to 1000mL, pH7.2 ~ 7.6.
By the beef extract-peptone liquid nutrient medium after improvement and the Gause I liquid nutrient medium high-temperature sterilization (121 DEG C after improving, 30min) after process, by above-mentioned steps 1) the genus bacillus J3 that activated and thermomonospora fusca GC accesses in the beef extract-peptone liquid nutrient medium after improvement and the Gause I liquid nutrient medium after improving respectively, 35 DEG C, 150r/min shaking culture 24h and 48h.
Above-mentioned steps 3) stalk and ight soil ratio in matrix can be adjusted in the following manner: with solid fermentation substratum total mass for benchmark, in 10 ~ 40% straw powder and 5 ~ 10% wheat brans or 5 ~ 10% dregs of beans, add the feces of livestock and poultry of 5 ~ 50%.
Described agricultural wastes are material common in the agriculture production such as stalk, feces of livestock and poultry, and wherein stalk can be one or more in cornstalk, wheat-straw, paddy rice stalk, kaoliang stalk; Feces of livestock and poultry can be one or more in pig manure, chicken manure, cow dung, also can be that stalk mixes with feces of livestock and poultry.
Superior part of the present invention is:
(1) gamma-polyglutamic acid-is the degradable macromolecular material of a kind of polyamino acid class multifunctional bio, has water-soluble height, and adsorptivity is strong, good biocompatibility, nontoxic, harmless, noresidue, promotes the advantages such as crop nutrition content absorption.Enter after in soil, can natural decomposition, generate amino acid, be finally absorbed and used by plants, the secondary pollution of environment can not be caused.
(2) utilize the agricultural wastes such as stalk, feces of livestock and poultry to carry out mixed fermentation and prepare preserve moisture and fertility fertilizer, agricultural wastes are carried out resource reutilization, turned waste into wealth, namely alleviates agricultural wastes and the pressure of environment is again saved resource, played an important role to the Sustainable development of agricultural.
(3) preserve moisture and fertility fertilizer can improve the utilization ratio of farm crop for moisture and fertilizer, reduces the loss of moisture and nutrient; Energy slowly-releasing nutrient, reduces rate of fertilizer application simultaneously, can reduce the function of production cost, prevention soil compaction.
(4) preserve moisture and fertility fertilizer can increase the yield and quality of unit surface farm crop, increases the income of people.
(5) product polyglutamic acid bacterium used in the present invention under compared with hypoglutamatergic concentration of substrate and the polyglutamic acid of cellulose-decomposing bacterium mixed fermentation synthesis high yield, can reduce the production cost of fertilizer.
(6) the inventive method is easy to use, and the preserve moisture and fertility fertilizer of production easily forms commercialization, convenient transport and preservation.
(7) the present invention has done improvement to traditional liquid substratum, uses the substratum after improving to cultivate bacterial strain, can save incubation time.
Embodiment
Embodiment 1
The selection of gamma-polyglutamic acid generating bacterium, step is as follows:
(1) soil taken from by 10g around the Gourmet Powder Factory of Sichuan grinds to be suspended from 90mL sterile distilled water and shakes evenly, boil 15min, gradient dilution after cooling, and coat in screening culture medium, 24h is cultivated at 37 DEG C, obtain bacterium colony, screening culture medium is consisting of of LB solid medium (g/L): glucose 10, peptone 10, NaCl5.0, agar 20, pH=7.0, moisturizing is to 1000mL.
(2) select and come in every shape, grow vigorous, smooth surface and bacterium colony 9 strain of thickness, under identical culture condition, this few strain bacterium is carried out repeatedly streak culture more than three times, to obtain pure bacterial strain, be inoculated in LB liquid nutrient medium, at 37 DEG C, cultivate 24h, obtain pure bacterial strain seed liquor.
(3) by pure bacterial strain seed liquor according to 2% ~ 5% inoculum size be inoculated in the 500ml triangular flask containing 100ml liquid fermentation medium, in 30 DEG C, 200r/min fermentation culture 72h.Fermention medium (g/L) consists of: citric acid 13.5, glycerine 75, NH
4cl7.0, Sodium Glutamate 10, K
2hPO
43H
2o1.0, FeCl
36H
2o0.02, CaCl
22H
2o0.2, MgSO
47H
2o0.5, MnSO
4h
2o0.05, pH=7.5, moisturizing is to 1000mL.
(4) fermented liquid of No. 2, No. 3 and No. 8 bacterium that selection viscosity is higher is in the centrifugal 10min of 8000r/min, supernatant liquor 4 times of alcohol settling spend the night, in 8000r/min centrifugal l0min, throw out is dissolved in aliquots of deionized water again, get 2.0mL lysate and put into hydrolysis pipe, add the HCl of 3.0mL6mol/L, 110 DEG C of vacuum hydrolysis 24h, obtain γ-PGA hydrolysate.
(5) use L-glutamic acid standard substance in contrast, said hydrolyzed product is carried out derivatize HPLC analysis, the hydrolysate obtaining No. 3 bacterium is L-glutamic acid, and No. 3 bacterium hydrolysate content of glutamic acid is the highest, is 14g/L; In conjunction with its fermented liquid viscosity, tentatively judge that its γ-PGA synthesis capability is the strongest, called after J3.
The Physiology and biochemistry character of table 1J3
Embodiment 2
1, by beef extract-peptone and Gause I solid slant culture base high-temperature sterilization (121 DEG C, 30min) for subsequent use after process, wherein beef extract-peptone solid culture based formulas is: extractum carnis 3g, peptone 10g, NaCl5g, agar 18 ~ 20g, moisturizing is to 1000mL, pH7.0 ~ 7.4; Gause I solid culture based formulas is: Zulkovsky starch 20g, yeast powder 2g, glucose 5g, KNO
31g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, agar 18 ~ 20g, moisturizing is to 1000mL, pH7.2 ~ 7.6.
2, bacterial classification J3 and GC is accessed respectively activate in the beef extract-peptone of sterilising treatment (121 DEG C, 30min) and Gause I slant medium for subsequent use.
3, by modified version beef extract-peptone liquid nutrient medium and modified version Gause I liquid nutrient medium high-temperature sterilization (121 DEG C, 30min) for subsequent use after process, wherein modified version beef extract-peptone liquid culture based formulas is: extractum carnis 5g, peptone 10g, glucose 5g, NaCl5g, moisturizing is to 1000mL, pH7.0 ~ 7.4; Modified version Gause I liquid culture based formulas is: Zulkovsky starch 15g, yeast powder 2g, glucose 10g, KNO
31g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, moisturizing is to 1000mL, pH7.2 ~ 7.6.
4, bacterial classification J3 and GC activated is accessed respectively (35 DEG C, 150r/min) shaking culture 24h and 48h in modified version beef extract-peptone and modified version Gause I liquid nutrient medium, then cultured two strain bacterium liquid mixed according to the volume ratio of J3:GC=1:10 and obtain mixed seeds liquid.
5, solids mixing fermentation: with solids mixing fermention medium total mass for benchmark, adds the pig manure of 8%, makes the mol ratio of carbon nitrogen in matrix be 15:1 in 10% straw powder and 5% wheat bran; Add Sodium Glutamate 2.0% in mass ratio again, glucose 1%, K
2hPO
40.1%, MgSO
47H
2o0.05%, by Sodium Glutamate, glucose, K
2hPO
4, MgSO
47H
2o mixing is soluble in waterly sprayed in above-mentioned matrix, regulates fermented substrate water ratio to be 50 ~ 55%, pH value 7.0 ~ 8.0, and stirs and be made into solids mixing fermention medium; By aforementioned mixed seeds liquid according in 0.1% inoculum size access solid fermentation substratum, 115 ~ 121 DEG C of sterilizing 30min, be cooled to room temperature, then be stacked in sterilized isoperibol, 30 ~ 37 DEG C of aerated culture 20 ~ 40d, stop fermentation, the complete solid materials that ferments is containing polyglutamic acid fertilizer.
Embodiment 3
1, the activation of bacterial strain J3: with embodiment 2.
2, by for subsequent use after glutamic acid fermentation substratum high-temperature sterilization (121 DEG C, 30min) process, wherein glutamic acid fermentation culture medium prescription is: citric acid 13.5g, glycerine 75g, NH
4cl7.0g, Sodium Glutamate 5g, K
2hPO
43H
2o1.0g, FeCl
36H
2o0.02g, CaCl
22H
2o0.2g, MgSO
47H
2o0.5g, MnSO
4h
2o0.05g, pH=7.2 ~ 7.5, moisturizing is to 1000mL.
3, by the bacterial strain J3 activated according to glutamic acid fermentation substratum quality 2% ~ 5% inoculum size be inoculated in the 500mL triangular flask that 100mL glutamic acid fermentation substratum is housed, in 30 DEG C, the shaking table of 200r/min stops fermentation after cultivating 72h.
4, fermented liquid is in the centrifugal 10min of 8000r/min, supernatant liquor 4 times of alcohol settling spend the night, in 8000r/min centrifugal l0min, throw out is dissolved in aliquots of deionized water again, get 2.0mL lysate in 20mL hydrolysis pipe, add the HCl of 3.0mL6mol/L, 110 DEG C of vacuum hydrolysis 24h, obtain γ-PGA hydrolysate.
5, said hydrolyzed product is carried out derivatize HPLC analysis, recording γ-PGA concentration is 8.5g/L, and substrate glutamic acid sodium utilization is 170%, reaches high level.At present, L-glutamic acid (Sodium Glutamate) utilization ratio of research report is generally lower than 100%, such as " the selection systems of a strain gamma-polyglutamic acid-synthesis bacterium of the report such as Yao Jun, microbiology is circulated a notice of, 2011, 38 (2): 164-168 " 52% Sodium Glutamate utilization ratio, " screening of gamma-polyglutamic acid generating bacterium and the fermentation condition of the report such as Sang Li, process engineering journal, 2004, 4 (5): 462-466) 47.3% Sodium Glutamate utilization ratio ", " the fermention medium optimization of gamma-polyglutamic acid generating bacterium such as Li Wenjing, food and fermentation industries, 2010, 36 (3): 108-116) 64.5% Sodium Glutamate utilization ratio ", " the mutagenic and breeding of gamma-polyglutamic acid generating bacterium such as Wang great Yun, 2013, 32 (8): 25-29) 65.7% Sodium Glutamate utilization ratio ", " the seed selection of gamma-polyglutamic acid-Producing Strain such as Wang Hao, 2011, 100% Sodium Glutamate utilization ratio 3:3-6) ".
Embodiment 4
1, the activation of bacterial strain J3: with embodiment 2.
2, by modified version beef extract-peptone liquid nutrient medium and modified version Gause I liquid nutrient medium high-temperature sterilization (121 DEG C, 30min) for subsequent use after process, wherein modified version beef extract-peptone liquid culture based formulas is: extractum carnis 5g, peptone 10g, glucose 5g, NaCl5g, moisturizing is to 1000mL, pH7.0 ~ 7.4; Modified version Gause I liquid culture based formulas is: Zulkovsky starch 15g, yeast powder 2g, glucose 10g, KNO
31g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, moisturizing is to 1000mL, pH7.2 ~ 7.6.
3, bacterial strain J3 and GC activated is accessed respectively (35 DEG C, 150r/min) shaking culture 24h and 48h in modified version beef extract-peptone and modified version Gause I liquid nutrient medium, then cultured two strain bacterium liquid mixed according to the volume ratio of J3:GC=1:2 and obtain mixed seeds liquid.
4, solids mixing fermentation: with solid fermentation substratum total mass for benchmark, adds pig manure and 1% urea of 5%, makes the mol ratio of carbon nitrogen in matrix be 35:1 in 30% straw powder and 5% wheat bran; Add Sodium Glutamate 1.0% in mass ratio again, sucrose 1.0%, K
2hPO
40.5%, MgSO
47H
2o0.01%, by Sodium Glutamate, sucrose, K
2hPO
4, MgSO
47H
2o mixing is soluble in waterly sprayed in above-mentioned matrix, regulates fermented substrate water ratio to be 60 ~ 65%, pH value 7.0 ~ 8.0, and stirs and be made into solid fermentation substratum; Pressed by aforementioned mixed seeds liquid in the inoculum size access solid fermentation substratum of 1% of solid fermentation substratum total mass, 121 DEG C of sterilizing 30min, are cooled to room temperature, are then stacked in sterilized isoperibol, 35 DEG C of aerated culture 25d, stop fermentation.
5, the content detecting γ-PGA in fermentation materials after fermentation ends is 10.5g/Kg, and organic content is 35.6%.
Embodiment 5
1, the activation of bacterial strain J3: with embodiment 2.
2, by modified version beef extract-peptone liquid nutrient medium and modified version Gause I liquid nutrient medium high-temperature sterilization (121 DEG C, 30min) for subsequent use after process, wherein modified version beef extract-peptone liquid culture based formulas is: extractum carnis 5g, peptone 10g, glucose 5g, NaCl5g, moisturizing is to 1000mL, pH7.0 ~ 7.4; Modified version Gause I liquid culture based formulas is: Zulkovsky starch 15g, yeast powder 2g, glucose 10g, KNO
31g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, moisturizing is to 1000mL, pH7.2.
3, bacterial classification J3 and GC activated is accessed respectively (35 DEG C, 150r/min) shaking culture 24h and 48h in modified version beef extract-peptone and modified version Gause I liquid nutrient medium, then cultured two strain bacterium liquid mixed according to the volume ratio of J3:GC=1:3 and obtain mixed seeds liquid.
4, solids mixing fermentation: with solid fermentation substratum total mass for benchmark, adds the pig manure of 40%, makes the mol ratio of carbon nitrogen in matrix be 20 ~ 25:1 in 40% straw powder and 10% dregs of beans; Add Sodium Glutamate 0.5% in mass ratio again, glucose 3.0%, K
2hPO
41%, MgSO
47H
2o0.1%, by Sodium Glutamate, glucose or sucrose, K
2hPO
4, MgSO
47H
2o mixing is soluble in waterly sprayed in above-mentioned matrix, regulates fermented substrate water ratio to be 55 ~ 60%, pH value 7.0 ~ 8.0, and stirs and be made into solid fermentation substratum; Aforementioned mixed seeds liquid is pressed in the inoculum size access solid fermentation substratum of 2% of solid fermentation substratum total mass, and 121 DEG C of sterilizing 30min, are cooled to room temperature, are then stacked in sterilized isoperibol, 35 DEG C of aerated culture 30d, stop fermentation.
5, the content detecting γ-PGA in fermentation materials after fermentation ends is 12.3g/Kg, and organic content is 45.8%.
Embodiment 6
Test containing γ-PGA preserve moisture and fertility fertilizer pot test effect: testing potted plant used is corn, first through hole-ground film, maize seedling is put into hole, then by the hole on film, around every strain maize seedling rhizosphere, add the preserve moisture and fertility fertilizer of preparation in 50g embodiment 5 or common fertilizer, then water 300ml normal root water.
Setup Experiments three treatment group, are respectively:
(1) control group, does not use any fertilizer;
(2) common fertilizer group, uses common fertilizer;
(3) preserve moisture and fertility fertilizer group, use preserve moisture and fertility fertilizer prepared by embodiment 5, wherein γ-PGA content is 12.3g/Kg, and organic content is 45.8%.
After maize seedling transplants 30 days, measure 0 ~ 10cm soil moisture content immediately below corn plants and measure the fresh weight of corn plants.Measurement result is as shown in table 2 below, uses common fertilizer and preserve moisture and fertility fertilizer can both increase the water content of 0 ~ 10cm soil and the fresh weight of milpa significantly.Relative to control treatment group, common fertilizer treatment group soil moisture content and plant fresh weight add 15.2% and 18.9% respectively, and water retention organic fertilizer treatment group soil moisture content and plant fresh weight add 32.7% and 55.6% respectively.Preserve moisture and fertility fertilizer can increase significantly maize seedling strain rhizosphere soil water content and seedling stage plant fresh weight, there is the function that preserve moisture and fertility improves moisture and utilization rate of fertilizer.
Table 2 preserve moisture and fertility fertilizer experimental result
| Testing index | Control group | Common fertilizer group | Preserve moisture and fertility fertilizer group |
| 0 ~ 10cm soil moisture content (%) | 15.4 | 17.7 | 20.4 |
| Milpa fresh weight (g/ basin) | 85.4 | 101.5 | 132.9 |
Claims (5)
1. a bacillus (Bacillussp.) J3, its preserving number is: CGMCCNo.11582.
2. produce a method for gamma-polyglutamic acid-, it is characterized in that, comprise the following steps:
(1) bacterial strain activation: the activation of genus bacillus J3 beef-protein medium is for subsequent use;
(2) by for subsequent use after the process of glutamic acid fermentation substratum high-temperature sterilization;
(3) by the genus bacillus J3 activated according to 2 ~ 5% inoculum size be inoculated in glutamic acid fermentation substratum, shaking table cultivate;
(4) get fermented liquid supernatant liquid 4 times of alcohol settling to spend the night, centrifugal, throw out is dissolved in aliquots of deionized water, gets lysate in hydrolysis pipe, add HCl, vacuum hydrolysis, obtain gamma-polyglutamic acid-hydrolysate.
3. produce a method for gamma-polyglutamic acid-fertilizer, it is characterized in that, comprise the following steps:
(1) bacterial strain activation: genus bacillus J3 and thermomonospora fusca GC, respectively with beef-protein medium and the activation of Gause I substratum;
(2) liquid culture: after beef extract-peptone liquid nutrient medium and the process of Gause I liquid nutrient medium high-temperature sterilization, the genus bacillus J3 activated step (1) and thermomonospora fusca GC accesses in beef extract-peptone liquid nutrient medium and Gause I liquid nutrient medium respectively, 35 DEG C, 150r/min shaking culture 28h and 50h;
(3) solids mixing fermention medium is prepared: in agricultural waste fermentation matrix, add stalk and ight soil ratio in urea or adjustment matrix, make the mol ratio of carbon nitrogen in matrix be 15 ~ 35:1; Again by Sodium Glutamate 0.5 ~ 2.0%, glucose or sucrose 1 ~ 3%, K
2hPO
40.1 ~ 1%, MgSO
47H
2o0.01 ~ 0.1% mixes and is soluble in waterly sprayed in above-mentioned matrix, regulates fermented substrate water ratio to be 50 ~ 65%, pH value 7.0 ~ 8.0, and stirs and be made into solids mixing fermention medium;
(4) mixed bacterium inoculation proportioning: be that 1 ~ 3:5 ~ 10 mix by volume by cultured in step (2) genus bacillus J3: thermomonospora fusca GC, obtain mixed bacterium seed liquor;
(5) by mixed bacterium seed liquor according to solid fermentation substratum total mass 0.1 ~ 2% inoculum size access sterilize solid mixed fermentive culture medium in, 30 ~ 37 DEG C of aerated culture 20 ~ 40d, stop fermentation, obtain the preserve moisture and fertility fertilizer containing γ-PGA.
4. a kind of method of producing gamma-polyglutamic acid-fertilizer as claimed in claim 3, it is characterized in that, beef extract-peptone liquid nutrient medium in described step (2) and the formula optimization of Gause I liquid nutrient medium are: beef extract-peptone liquid culture based formulas: extractum carnis 5g, peptone 10g, glucose 5g, NaCl5g, moisturizing is to 1000mL, pH7.0 ~ 7.4; Gause I liquid culture based formulas: Zulkovsky starch 15g, yeast powder 2g, glucose 10g, KNO
31g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o0.5g, NaCl0.5g, FeSO
47H
2o0.01g, moisturizing is to 1000mL, pH7.2 ~ 7.6; The genus bacillus J3 activated and thermomonospora fusca GC is accessed respectively in the beef extract-peptone liquid nutrient medium after improvement and the Gause I liquid nutrient medium after improving, 35 DEG C, 150r/min shaking culture 24h and 48h.
5. a kind of method of producing gamma-polyglutamic acid-fertilizer as claimed in claim 3, it is characterized in that, described step (3) adjusts stalk and ight soil ratio in matrix in the following manner: with solid fermentation substratum total mass for benchmark, adds the feces of livestock and poultry of 5 ~ 50% in 10 ~ 40% straw powder and 5 ~ 10% wheat brans or 5 ~ 10% dregs of beans.
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| CN108102968A (en) * | 2018-01-16 | 2018-06-01 | 民勤县恒盛贸易有限公司 | A kind of wheat planting is with microorganism and preparation method thereof |
| CN110845253A (en) * | 2019-11-20 | 2020-02-28 | 中国科学院成都生物研究所 | Preparation and application method of gamma-polyglutamic acid biological organic fertilizer |
| CN110845253B (en) * | 2019-11-20 | 2022-02-08 | 中国科学院成都生物研究所 | Preparation and application method of gamma-polyglutamic acid biological organic fertilizer |
| CN113584097A (en) * | 2021-06-28 | 2021-11-02 | 杨凌未来中科环保科技有限公司 | Fermentation method and device for producing gamma-polyglutamic acid by reflux type solid fermentation |
| CN113583895A (en) * | 2021-06-28 | 2021-11-02 | 杨凌未来中科环保科技有限公司 | Bacillus subtilis, organic fertilizer, production method and application |
| CN113583895B (en) * | 2021-06-28 | 2023-02-17 | 杨凌未来中科环保科技有限公司 | Bacillus subtilis, organic fertilizer, production method and application |
| CN113584097B (en) * | 2021-06-28 | 2023-08-18 | 杨凌未来中科环保科技有限公司 | Fermentation method and device for producing gamma-polyglutamic acid through reflux type solid fermentation |
| CN113698244A (en) * | 2021-08-31 | 2021-11-26 | 武汉光华时代生物科技有限公司 | Application of poly-gamma-glutamic acid as fertilizer synergist in crops |
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