CN101294143B - Bacterial strain for preparing 3-hydroxy butanone and uses thereof - Google Patents
Bacterial strain for preparing 3-hydroxy butanone and uses thereof Download PDFInfo
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- CN101294143B CN101294143B CN2008101224907A CN200810122490A CN101294143B CN 101294143 B CN101294143 B CN 101294143B CN 2008101224907 A CN2008101224907 A CN 2008101224907A CN 200810122490 A CN200810122490 A CN 200810122490A CN 101294143 B CN101294143 B CN 101294143B
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- Prior art keywords
- oxobutanol
- culture
- bacterial strain
- medium
- acid
- Prior art date
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- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
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- 238000009825 accumulation Methods 0.000 description 1
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- 239000012620 biological material Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a strain for generating 3-hydroxy butanone and the application thereof, which belong to the field of bioengineering technology. The stain is named as Paenibacillus polymyxa ME-J25 and is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCC No.2505. The strain can be fermented to generate 3-hydroxy butanone with higher concentration, higher transformation efficiency and extremely low content of the reduced byproduct 2,3-butanediol of 3-hydroxy butanone, and is suitable for industrial production of 3-hydroxy butanone.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of bacterial strain and application thereof of the 3-of producing oxobutanol.
Background technology
3-oxobutanol (3-Hydroxybutanone), have another name called acetoin (Acetoin), methyl acetylcarbinol (Acetylmethylcarbinol), natural being present in dairy products and some fruit, it is a kind of widely used flavouring agent, it is edible that China GB2760-86 stipulates that it allows, U.S. food and extraction association (FEMA) security number be 2008 (Ling Guanting. foodstuff additive handbook (second edition) [M]. Beijing: Chemical Industry Press, 1997,301-301).The 3-oxobutanol is extremely extensive as the fragrance applications scope, and consumption is also bigger, so extremely perfumer's favor, its topmost application is the production (Hu Mingyi that is used for spices such as cream, dairy products, sour milk and strawberry type, among the king. meticulous and specialty chemicals, 2002,10 (1): 20-21).In addition, the 3-oxobutanol can also be widely used in industries such as daily use chemicals food, pharmacy, chemical industry, coating and IT as a kind of hardware and software platform compound.2004, USDOE is classified it one of as hardware and software platform compound that 30 kinds of preferential developments utilize (Werpy T, Petersen G.Top value added chemicals from biomass:VolumeI-Results of screening for potential candidates from sugars and synthesis gas[EB/OL] .http: //www1.eere.energy.gov/biomass/pdfs/35523.pdf, 2004-08-06), in recent years, along with the continuous growth of people to 3-oxobutanol demand, the production method of relevant 3-oxobutanol and applied research have caused people's extensive concern (Ji Xiaojun, yellow and, Du Jun, Deng. modern chemical industry, 2008,28 (4): 18-22).
At present, the synthetic method of 3-oxobutanol mainly contains 3 kinds: chemical method, enzyme transforming process, microbe fermentation method.
Chemical method is produced the 3-oxobutanol and is mainly contained three kinds of technologies: and (1) dimethyl diketone partial hydrogenation reducing process (Zhang Xiaozhou, Ceng Chongyu, Ren Xiaoqian. Jiangsu chemical industry, 2001,29 (2): 29-31); (2) 2,3-butyleneglycol selective oxidation processes (Zhang Xiaozhou, Ceng Chongyu, Ren Xiaoqian. Jiangsu chemical industry, 2001,29 (2): 29-31); (3) butanone chlorination hydrolysis process (Hu Mingyi is among the king. meticulous and specialty chemicals, 2002,10 (1): 20-21).These three kinds of technology ubiquities product yield and yield is lower, and shortcoming such as environmental pollution is more serious, and quality product is difficult to reach the maximum consumer field of present 3-oxobutanol---the requirement of flavouring agent; The raw material sources of these three kinds of technologies that even more serious is are limited, caused utilizing chemical method to produce the 3-oxobutanol and have been difficult to extensive development.Employed raw material dimethyl diketone and butanone all come from non-renewable fossil resource---oil at present in technology (1) and (3), soaring day by day along with the worsening shortages of petroleum resources and oil price, the cost of these two operational paths will be more and more higher, will certainly further increase the production cost of 3-oxobutanol; Raw material 2 in the technology (2), the 3-butyleneglycol can be by chemical method production, also can pass through Production by Microorganism Fermentation (Ji Xiaojun, Zhu Jianguo, Gao Zhen, etc. modern chemical industry, 2006,26 (8): 23-27), produce 2 by chemical method, the 3-butyleneglycol equally also is faced with the pressure that petroleum resources shortage and environmental pollution etc. bring; And by 2 of Production by Microorganism Fermentation, 3-butyleneglycol itself also is a spices, but not large Chemicals, therefore the raw materials cost of this operational path is very high, add under the existing state of the art, 2, the transformation efficiency that 3-butyleneglycol selective oxidation obtains the 3-oxobutanol is lower, therefore uses this operational path to produce the trend that the 3-oxobutanol does not meet current Sustainable development equally.
It is similar with chemosynthesis that enzyme transforming process is produced the 3-oxobutanol, is with dimethyl diketone or 2, and the 3-butyleneglycol is a raw material, generate 3-oxobutanol (Hummel W through intravital enzyme partial reduction of microorganism or oxidation, Kula M R, Boermann F.US5164314,1992-11-17; De Faveri D, Torre P, Molinari F, etal.Enzyme and MicrobialTechnology, 2003,33 (5): 708-719), difference is that the enzyme process efficiency of pcr product is higher, do not have other by product, and product has specific rotation, but obtain relatively difficulty of a large amount of specific enzymes, and its substrate source is restricted.Therefore, in essence, enzyme transforming process fails fundamentally to change the dual-pressure of the resource and environment that is faced in the 3-oxobutanol production process, so this route does not still possess the potentiality of large-scale industrial production.
More than the chemical synthesis production of Jie Shaoing still be enzyme transforming process all be with dimethyl diketone or 2,3-butyleneglycol etc. is a raw material, dimethyl diketone and 2, the 3-butyleneglycol also is a synthetic perfume, but not large Chemicals, therefore raw material sources and price all are restricted, and this may also be one of reason of better not researched and developed of present 3-oxobutanol, so active development is produced the 3-oxobutanol with the saccharic for the prepared using microbial fermentation and is expected to change this situation.
The certain micro-organisms of occurring in nature has the ability of small amount of accumulation 3-oxobutanol, comprise that mainly Klebsiella (Klebisella), enterobacter (Enterobacter), bacillus (Bacillus), series bacillus in the bacterium belong to (Paenibacillus), serratia (Serratia) and lactococcus (Xiao Z J such as (Lactococcus), Liu P H, Qin J Y, et al.Applied Microbiology and Biotechnology, 2007,74 (1): 61-68).In most of bacterial strain metabolic processes, the 3-oxobutanol is as 2, the by product of 3-butyleneglycol and a small amount of (Juni E.Journal ofBiological Chemistry, 1952,195 (2): 715-726 that exist; Xiao Z J, Xu P.Critical Reviews inMicrobiology, 2007,33 (2): 127-140), during the fermentation, accumulated concentrations is lower.This has directly caused being difficult to utilize these microbial strains industrial fermentations to produce the 3-oxobutanol.Therefore, filter out to follow from occurring in nature and go back ortho states by product 2, the 3-oxobutanol high yield microorganism strains that the 3-butyleneglycol yields poorly, and to work out a kind of fermentation process that can production high density 3-oxobutanol be necessary.
Summary of the invention
The bacterial strain that the purpose of this invention is to provide a kind of energy fermentative production higher concentration 3-oxobutanol.
Another object of the present invention provides the application of this bacterial strain in fermentative production 3-oxobutanol.
The objective of the invention is to realize by following technical measures:
The screening of microorganism strains and evaluation:
In the present invention, set up a model that screens high yield 3-oxobutanol bacterial strain fast and effectively.According to sporiferous bacillus and series bacillus genus is this generally acknowledged fact of bacterial classification that two classes can accumulate the 3-oxobutanol, design from soil sample and filter out sporiferous bacterium by the heating enrichment, again by V-P (Vagex-Proskauer) experimental identification, filter out V-P positive strain (can accumulate the 3-oxobutanol), isolate 2 by the isolation medium that contains vinylcarbinol and butenol then, the bacterial strain (2 that 3-butanediol dehydrogenation enzyme activity is lower, 3-oxobutanol and 2 in the 3-butanediol dehydrogenation enzyme catalysis microbe, the reversible reaction between the 3-butyleneglycol; Vinylcarbinol and butenol can be reduced to propenal and crotonaldehyde by alcoholdehydrogenase respectively, and propenal and crotonaldehyde have the intensive toxic action to microorganism cells, therefore, the active high bacterial strain of alcoholdehydrogenase can not be survived in the isolation medium that contains vinylcarbinol and crotonaldehyde, the bacterial strain that the alcoholdehydrogenase activity is not high (can significantly accumulate the 3-oxobutanol) then can survive therein), detect by the fermentation checking at last, finishing screen is selected to follow and is gone back ortho states by product 2, the 3-oxobutanol superior strain that 3-butyleneglycol output is extremely low.
The present invention takes by weighing 1.0～5.0 grams from the face of land, Pukou District flag of a commander in chief farm orchard, Nanjing soil sampling, and 80～100 ℃ of baking 24～48h join in 50～200mL sterilized water and make suspension; Get 0.1～1mL bacteria suspension and be forwarded in the 150mL triangular flask, the enrichment medium liquid amount is 40～60mL in the triangular flask, 30～37 ℃ carry out enrichment culture 18～36h after, get nutrient solution and carry out gradient dilution to 10
-6, getting 0.05～0.25mL diluent and coat on the flat board that contains enrichment medium, behind 30～37 ℃ of cultivation 18～36h, picking list bacterium colony carries out the V-P The effects one by one.With the V-P positive strain that obtains, toothpick with the bacterium of going out is forwarded on the screening flat board that isolation medium is housed one by one, after cultivating 36～48h, the bacterial strain that picking can be survived on the screening flat board is cultivated 24～48h respectively in containing the 10mL culture tube of 3～5mL fermention medium; Get fermented liquid 1.5mL after cultivating end, centrifugal 5～the 10min of 8000～12000r/min, measure the content of 3-oxobutanol in the supernatant liquor with the HPLC method, filter out tens strains and produce the bacterial strain of 3-oxobutanol, wherein the ability of bacterial strain ME-J25 product 3-oxobutanol is the strongest, and follow and go back ortho states by product 2,3-butyleneglycol output is extremely low.
(deposit number is CGMCC No.2505 to the bacterial strain ME-J25 that can effectively accumulate the 3-oxobutanol that the present invention's screening obtains, down together), carrying out Physiology and biochemistry according to " common bacteria system identification handbook " identifies, think and belong to genus bacillus (Bacillaceae) section, the Paenibacillus polymyxa (Paenibacilluspolymyxa) that series bacillus (Paenibacillus) belongs to.The cell of this bacterium becomes shaft-like, and thick gemma wall is arranged, and gemma is expansion shape, ellipse, and is positioned at the end of thalline.Big, the mattness of bacterium colony is the mucus shape in nutrient broth medium, produces the smell of fruit perfume (or spice).Gram-positive, amphimicrobian.This bacterial strain can be grown in containing the substratum of glucose and be produced the 3-oxobutanol, can also utilize the hydrolyzed solution of sucrose, fructose, maltose, lactose, wood sugar, pectinose, semi-lactosi, whey, starch and cassava, corn, beet or lignocellulose or syrup etc. as substrate.Main metabolites is the 3-oxobutanol, also produces a spot of acetate and lactic acid, compare with other Paenibacillus polymyxa, and by-product alcohols 2,3-butyleneglycol and ethanol content are extremely low.
Utilize described strain fermentation to produce the method for 3-oxobutanol, comprise the following steps:
(1) medium preparation
Seed culture medium adds agar for the liquid nutrient medium that carbon source, nitrogenous source and inorganic salt can be provided of pH 6.0～7.0 during slant culture;
Fermention medium has added VITAMIN on the basis of the liquid nutrient medium that carbon source, nitrogenous source and inorganic salt can be provided of pH 6.0～7.0;
(2) seed preparation
Slant culture: the bacterial strain that will be numbered ME-J25 carries out plate streaking in slant medium after, cultivate in incubator, temperature is 30～37 ℃, cultivates 24～48h, is used for seed culture medium inoculation or bacterial strain and preserves;
Seed culture: the seed culture medium liquid amount is 40～60mL in the 150mL triangular flask, 115～121 ℃ of sterilization 15～20min, the bacterial strain that is numbered ME-J25 of 1～2 ring slant culture is inserted in the cooling back, culture temperature is 30～37 ℃, shaking speed is 200～300r/min, incubation time is 10～16h, is used for the fermention medium inoculation;
(3) shake flask fermentation is cultivated
The fermention medium liquid amount is 50～80mL in the 250mL triangular flask, 115～121 ℃ of sterilization 15～20min, the bacterial strain that is numbered ME-J25 through seed culture is inserted in the cooling back, inoculum size is 3～7% (v/v), culture temperature is 30～37 ℃, and shaking speed is 200～300r/min, and incubation time is 30～70h, after cultivating end, separation and Extraction 3-oxobutanol; Perhaps,
(4) the 5L ferment tank is cultivated
5L fermentation cylinder for fermentation culture volume is 2～4L, inoculum size is 3～7% (v/v), culture temperature is 30～37 ℃, mixing speed is 200～300r/min, bubbling air, air flow 1～2vvm, control pH is 6.0～7.0 in the fermenting process, incubation time is 30～70h, after cultivation finishes, and separation and Extraction 3-oxobutanol.
Carbon source in the used substratum of aforesaid method is the hydrolyzed solution of glucose, fructose, maltose, sucrose, lactose, wood sugar, pectinose, semi-lactosi, whey, starch and cassava, corn, beet or lignocellulose or in the syrup one or more; Nitrogenous source is the organic or inorganic nitrogenous compound, wherein inorganic nitrogen-containing compound is one or more in ammonium sulfate, ammonium chloride, primary ammonium phosphate, Secondary ammonium phosphate, the ammonium nitrate, and nitrogen-containing organic compound is one or more in urea, corn steep liquor, peptone, yeast extract paste and the extractum carnis; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, manganese salt, ferrous salt, the phosphoric acid salt; VITAMIN is folic acid, vitamin H, vitamins B
12, Thioctic Acid, nicotinic acid, pantothenic acid, VitB1, p-aminophenyl first, sour vitamins B
6With in the riboflavin one or more.
In the aforesaid method in the preferred sodium-chlor of inorganic salt, potassium primary phosphate, Sodium phosphate dibasic, magnesium chloride, calcium chloride, Manganous chloride tetrahydrate, the iron protochloride one or more.
The seed culture medium formula optimization is in the above-mentioned method of utilizing bacterial strain ME-J25 fermentative production 3-oxobutanol: yeast extract paste 2.0～10.0g/L, peptone 2.0～10.0g/L, NaCl 0.5～5.0g/LL, KH
2PO
44～12g/L, Na
2HPO
49.0～15.0g/, adding agar during slant culture, to make its concentration be 15～20g/L, and all the other are water, and pH 6.0～8.0;
Fermentative medium formula is preferably in the above-mentioned method of utilizing bacterial strain ME-J25 fermentative production 3-oxobutanol: glucose 40～150g/L, yeast extract paste 10～50g/L, peptone 10～50g/L, MnCl
20.05～0.5g/L, MgCl
26H
2O0.05～0.5g/L, FeCl
20.05～0.5g/L, CaCl
20.05～0.5g/L, KH
2PO
41.0～10.0g/L, folic acid 5～20mg/L, vitamin H 5～20mg/L, vitamins B
125～20mg/L, Thioctic Acid 5～20mg/L, nicotinic acid 5～20mg/L, pantothenic acid 5～20mg/L, VitB1 5～20mg/L, para-amino benzoic acid 5～20mg/L, vitamins B
65～20mg/L, riboflavin 5～20mg/L, all the other are water, pH 6.0～7.0.
Analytical procedure:
Residual substrate glucose, product 3-oxobutanol and various by product such as acetate, lactic acid and 2 in the fermented liquid, 3-butyleneglycol and ethanol etc. adopt DIONEX summit P680 high performance liquid chromatograph to measure.Chromatographic column is AminexHPX-87H post (Bio-Rad), and column temperature is 60 ℃, and detector is a SHODEX RI-101 refractive power differential detector, and moving phase is 0.005mol/L H
2SO
4, flow velocity is 0.2mL/min, sample size is 20 μ L.Various product standardized solution (target product and the various by product: 3-oxobutanol (Fluka), acetate (Sigma), lactic acid (Fluka), 2 of redistilled water preparation different concns, 3-butyleneglycol (Sigma), ethanol (Sigma)) (on behalf of the trade mark of standard substance, Fluka here and Sigma limit, represent that it is the chromatographically pure standard substance), according to the concentration relationship production standard curve of peak area and various product standardized solution, fermented sample after centrifugal dilution according to the content of the various products of calculated by peak area.
The solvent of the used substratum of the present invention does not all adopt water as solvent when clearly indicating.
The per-cent of inoculum size is meant the ratio of seed liquor and the volume of fermention medium.
The pH regulator mode: when ferment tank was cultivated, by the monitoring of automatic fermenter pH probe, the corresponding acid of auto-feeding or alkali were realized pH is realized regulating that acid or alkali generally adopt NaOH and H then to feed back to control center
2SO
4, can adopt concentration is that the NaOH and the concentration of 10～30% (weight) is the H of 10～30% (volumes)
2SO
4
Beneficial effect of the present invention:
The bacterial strain Paenibacillus polymyxa ME-J25 (CGMCC No.2505) that the present invention filtered out has higher 3-oxobutanol synthesis capability, compare with disclosed other Paenibacillus polymyxa of document, ortho states by product 2 is gone back in following of 3-oxobutanol, the output of 3-butyleneglycol is very low, thereby 3-oxobutanol transformation efficiency is higher, reach 25-50g/L, be up to more than the 50g/L, be suitable for suitability for industrialized production, and can utilize cheap corn, reproducible agricultural byproducts such as cassava are that fermenting raw materials is produced the 3-oxobutanol, be a kind of continuable production method, can alleviate the nervous problem of petrochemical industry resource of 3-oxobutanol chemosynthesis, environmentally friendly.
Biomaterial preservation information:
Paenibacillus polymyxa, its classification called after Paenibacillus polymyxa (Paenibacillus polymyxa) ME-J25, (be called for short: CGMCC in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation date on 05 19th, 2008, deposit number is CGMCC No.2505.
Embodiment
The following examples elaborate to the present invention, but to the present invention without limits.
Embodiment 1
Gather soil sample from the face of land, Pukou District flag of a commander in chief farm orchard, Nanjing, take by weighing 3.0 grams, 90 ℃ of baking 36h join in the 100mL sterilized water and make suspension; Get the 0.5mL bacteria suspension and be forwarded in the 150mL triangular flask, the enrichment medium liquid amount is 50mL in the triangular flask, 35 ℃ carry out enrichment culture 24h after, get nutrient solution and carry out gradient dilution to 10
-6Getting the 0.1mL diluent coats on the flat board that contains enrichment medium, behind 35 ℃ of cultivation 24h, picking list bacterium colony, carry out V-P (Vagex-Proskauer) The effects one by one, specific practice is: with inoculation to be measured in the 10mL culture tube that 5mLV-P experiment substratum is housed, in 35 ℃ of cultivations 4 days, get nutrient solution 2.5mL, add the straight alcohol solution 0.6mL that contains 5% naphthyl alcohol earlier, add 40% potassium hydroxide aqueous solution 0.2mL again, shake 3min, positive bacteria presents redness immediately, if redfree occurs, be statically placed in 35 ℃ of thermostat containers, negative as still not manifesting red decidable in the 2h.Through the V-P The effects, from single bacterium colony of enrichment, filter out 77 strain V-P positive strains altogether.
Wherein:
Enrichment medium (g/L): NaCl2, K
2HPO
45, NaH
2PO
45, MgCl
26H
2O 0.1, CaCl
2H
2O 0.15, (NH
4)
2SO
41.5, yeast extract paste 5, peptone 5, glucose 20.Adding agar during dull and stereotyped the cultivation, to make its final concentration be 20g/L.
V-P tests substratum (g/L): peptone 5; Glucose 5; Sodium-chlor 10.
Embodiment 2
77 strain V-P positive strains with embodiment 1 acquisition, toothpick with the bacterium of going out is forwarded on the screening flat board that isolation medium is housed one by one, in incubator, cultivate, temperature is 37 ℃, after cultivating 24h, find to have the corresponding bacterial strain of 28 strains can normal growth, collect this 28 single bacterium colonies respectively, numbering is labeled as: ME-J1～ME-J28, and in containing the 10mL culture tube of 5mL fermention medium, cultivate 24h respectively; After cultivating end, the centrifugal 5min of 12000r/min with the content of 3-oxobutanol in the HPLC method mensuration supernatant liquor, finds that most bacterial strain lactic acid producing and acetate are more, and it is more that the minority bacterial strain produces the 3-oxobutanol, and it is the most remarkable that wherein bacterial strain ME-J25 produces the 3-oxobutanol.
The meta-bolites of table 1 V-P positive strain distributes
Annotate: ND represents " not measuring ".
Wherein:
Isolation medium (g/L): NaCl2, K
2HPO
45, NaH
2PO
41, MgCl
26H
2O 0.1, CaCl
2H
2O 0.15, (NH
4)
2SO
41.5, yeast extract paste 5, peptone 5, glucose 20, vinylcarbinol 0.5, butenol 0.7, adding agar during dull and stereotyped the cultivation, to make its final concentration be 20g/L;
Fermention medium (g/L): glucose 70, yeast extract paste 25, peptone 25, MnCl
20.25, MgCl
26H
2O 0.2, FeCl
20.15, CaCl
2H
2O 0.3, K
2HPO
45.0, folic acid 0.01, vitamin H 0.01, vitamins B
120.005, Thioctic Acid 0.015, nicotinic acid 0.01, pantothenic acid 0.02, VitB1 0.005, para-amino benzoic acid 0.005, vitamins B
60.005, riboflavin 0.005, pH 6.5.
Embodiment 3
The ME-J25 bacterial strain that screening among the embodiment 2 obtains is pressed " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying, Deng. common bacteria system identification handbook (first version). Beijing: Science Press, 2001, p 59) carry out physio-biochemical characteristics and identify (table 2), relatively bacterial strain ME-J25 is accredited as Paenibacillus polymyxa (Paenibacillus polymyxa) according to morphology in the table 2 and physio-biochemical characteristics, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserved and be numbered CGMCC No.2505
Table 2 bacterial strain ME-J25 physiological and biochemical property qualification result synopsis
| Index | The Paenibacillus polymyxa reference culture | ME-J25 | Index | The Paenibacillus polymyxa reference culture | ME-J25 |
| The gemma shape | Oval | Oval | Degrade chitin | - | - |
| Sporangiocyst expands | + | + | Depolymerized pectin | + | + |
| Anaerobic growth | + | + | The degraded amylopectin | + | + |
| Catalase | + | + | Degradation of xylan | + | + |
| Oxydase | - | - | Degraded β-1, the 2-dextran | - | - |
| Nitrate reduction | + | + | Fixed nitrogen | + | + |
| Produce acetyl methyl carbinol | + | + | Utilize Citrate trianion | - | - |
| Produce indoles | - | - | Utilize succinate | - | - |
| Produce otan | + | + | Optimum temperuture (℃) | 30 | 30 |
| The pH of V-P meat soup | 4.5～6.8 | 4.0～7.0 | Growth pH 5.6 | + | + |
| Decompose tyrosine | - | - | Grow 50 ℃ | - | - |
| Hydrolysed casein | + | + | Grow in 0.001% N,O-Diacetylmuramidase | + | + |
| Starch | + | + | Grow in 5%NaCl | - | - |
| Urea | - | - | Fermentation D-glucose | ++ | ++ |
| Alginate | - | - | The fermentation L-arabinose | ++ | ++ |
| Yolk lecithin | - | - | Fermentation D-wood sugar | ++ | ++ |
| Ferment glycerin | ++ | ++ |
Annotate :+:＞90% bacterial strain is positive;-:＞90% bacterial strain is negative; ++: produce acid, aerogenesis
Embodiment 4
Step 1: medium preparation
Seed culture medium (g/L): yeast extract paste 5.0, peptone 5.0, NaCl 2.5, KH
2PO
48.0, Na
2HPO
410.0 pH 7.0.Adding agar during slant culture, to make its final concentration be 20g/L.
Fermention medium (g/L): glucose 60～120, yeast extract paste 25, peptone 25, MnCl
20.25, MgCl
26H
2O0.2, FeCl
20.15, CaCl
2H
2O 0.3, KH
2PO
45.0, folic acid 0.01, vitamin H 0.01, vitamins B
120.005, Thioctic Acid 0.015, nicotinic acid 0.01, pantothenic acid 0.02, VitB1 0.005, para-amino benzoic acid 0.005, vitamins B
60.005, riboflavin 0.005, pH 6.5.
Step 2: seed preparation
Slant culture: with deposit number is after the bacterial strain of CGMCC No.2505 is rule in slant medium, to cultivate in incubator, and temperature is 35 ℃, cultivates 36h, is used for seed culture medium inoculation or bacterial strain and preserves;
Seed culture: seed culture medium is 25mL in the 150mL triangular flask, 121 ℃ of sterilization 15min, and the deposit number that 1 ring slant culture is inserted in the cooling back is the bacterial strain of CGMCC No.2505, culture temperature is 35 ℃, shaking speed is 200r/min, and incubation time is 12h, is used for the fermention medium inoculation;
Step 3: shake flask fermentation is cultivated
Fermention medium is 60mL in the 250mL triangular flask, 121 ℃ of sterilization 15min, the deposit number that the cooling back is inserted through seed culture is the bacterial strain of CGMCC No.2505, inoculum size is 5% (v/v), culture temperature is 35 ℃, and shaking speed is 220r/min, and incubation time is 30～54h (exhausting in glucose), fermented liquid is measured the wherein content of 3-oxobutanol with the HPLC method after centrifugal.
When glucose concn is 80g/L, fermentation 36h, 3-oxobutanol concentration reaches 35.85g/L in the fermented liquid, when glucose concn is 100g/L, fermentation 48h, 3-oxobutanol concentration reaches 43.5g/L in the fermented liquid.
Embodiment 5
Step 1: with embodiment 4
Step 2: with embodiment 4
Step 3:5L ferment tank is cultivated
5L fermentation cylinder for fermentation substratum liquid amount is 3L, and inoculum size is 5% (v/v), and culture temperature is 35 ℃, and mixing speed is 200r/min, and bubbling air, air flow are 1.5vvm, and fermenting process adopts 30%NaOH and 30%H
2SO
4PH is 6.5 in control, and incubation time is 30～54h (exhausting in glucose), and fermented liquid is measured the wherein content of 3-oxobutanol with the HPLC method after centrifugal.
When glucose concn is 80g/L, fermented 36 hours, 3-oxobutanol concentration reaches 33.6g/L in the fermented liquid, when glucose concn is 100g/L, ferments 48 hours, and 3-oxobutanol concentration reaches 41.5g/L in the fermented liquid.
Embodiment 6
With the glucose in the fermention medium among 100g/L sucrose or fructose or maltose or lactose or wood sugar or pectinose or semi-lactosi or the starch replacement embodiment 4, and insert cultured CGMCC No.2505 seed, fermentation 48h, with the content of 3-oxobutanol in the HPLC method mensuration fermented liquid supernatant, the content of 3-oxobutanol is as shown in table 3:
Table 3 CGMCC No.2505 bacterial strain utilizes different carbon source through fermentation to produce the 3-oxobutanol
| Carbon source | 3-oxobutanol content (g/L) |
| Sucrose | 42.2 |
| Fructose | 41.6 |
| Maltose | 40.8 |
| Lactose | 38.7 |
| Wood sugar | 36.5 |
| Pectinose | 35.2 |
| Semi-lactosi | 34.9 |
| Starch | 28.5 |
Among the present invention, the carbon source in the fermention medium can also be the hydrolyzed solution or the syrup of cassava, corn, beet or lignocellulose, and the initial reducing sugar total concn of fermented liquid is 60～120g/L.
Claims (6)
1. bacterial strain that produces the 3-oxobutanol, its classification called after Paenibacillus polymyxa (Paenibacillus polymyxa) ME-J25, in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.2505.
2. the application of the described bacterial strain of claim 1 in fermentative production 3-oxobutanol.
3. application according to claim 2 is characterized in that the method for utilizing this strain fermentation to produce the 3-oxobutanol may further comprise the steps:
(1) medium preparation
Seed culture medium is the liquid nutrient medium that carbon source, nitrogenous source and inorganic salt can be provided of pH 6.0～7.0, and adds agar during slant culture;
Fermention medium has added VITAMIN on the basis of the liquid nutrient medium that carbon source, nitrogenous source and inorganic salt can be provided of pH 6.0～7.0;
(2) seed preparation
Slant culture: with deposit number is the bacterial strain of CGMCC No.2505 carries out plate streaking in slant medium after, to cultivate in incubator, and temperature is 30～37 ℃, cultivates 24～48h, is used for seed culture medium inoculation or bacterial strain and preserves;
Seed culture: the seed culture medium liquid amount is 40～60mL in the 150mL triangular flask, 115～121 ℃ of sterilization 15～20min, the deposit number that 1～2 ring slant culture is inserted in the cooling back is the bacterial strain of CGMCC No.2505, culture temperature is 30～37 ℃, shaking speed is 200～300r/min, incubation time is 10～16h, is used for the fermention medium inoculation;
(3) shake flask fermentation is cultivated or the cultivation of 5L ferment tank
Shake flask fermentation is cultivated: the fermention medium liquid amount is 80～120mL in the 250mL triangular flask, 115～121 ℃ of sterilization 15～20min, the deposit number that the cooling back is inserted through seed culture is the bacterial strain of CGMCC No.2505, inoculum size is 3～7% (v/v), culture temperature is 30～37 ℃, shaking speed is 200～300r/min, and incubation time is 30～70h, cultivates and finishes back separation and Extraction 3-oxobutanol;
The 5L ferment tank is cultivated: 5L fermentation cylinder for fermentation culture volume is 2～4L, inoculum size is 3～7% (v/v), culture temperature is 30～37 ℃, mixing speed is 200～300r/min, bubbling air, air flow 1～2vvm, control pH is 6.0～7.0 in the fermenting process, incubation time is 30～70h, after cultivation finishes, and separation and Extraction 3-oxobutanol.
4. application according to claim 3 is characterized in that carbon source is the hydrolyzed solution of glucose, sucrose, fructose, maltose, lactose, wood sugar, pectinose, semi-lactosi, whey, starch and cassava, corn, beet or lignocellulose or in the syrup one or more; Nitrogenous source is the organic or inorganic nitrogenous compound, wherein inorganic nitrogen-containing compound is one or more in ammonium sulfate, ammonium chloride, primary ammonium phosphate, Secondary ammonium phosphate, the ammonium nitrate, and nitrogen-containing organic compound is one or more in urea, corn steep liquor, soybean cake powder, peptone, yeast extract paste and the extractum carnis; Inorganic salt are one or more in sodium salt, sylvite, magnesium salts, calcium salt, manganese salt, ferrous salt, the phosphoric acid salt; VITAMIN is folic acid, vitamin H, vitamins B
12, Thioctic Acid, nicotinic acid, pantothenic acid, VitB1, para-amino benzoic acid, vitamins B
6With in the riboflavin one or more.
5. application according to claim 4 is characterized in that inorganic salt are one or more in sodium-chlor, potassium primary phosphate, Sodium phosphate dibasic, magnesium chloride, calcium chloride, Manganous chloride tetrahydrate, the iron protochloride.
6. application according to claim 3 is characterized in that the seed culture based formulas is: yeast extract paste 2.0～10.0g/L, peptone 2.0～10.0g/L, NaCl 0.5～5.0g/L, KH
2PO
44～12g/L, Na
2HPO
49.0～15.0g/L, adding agar during slant culture, to make its concentration be 15～20g/L, and all the other are water, and pH 6.0～7.0; Fermentative medium formula is: glucose 40～150g/L, yeast extract paste 10～50g/L, peptone 10～50g/L, MnCl
20.05～0.5g/L, MgCl
26H
2O0.05～0.5g/L, FeCl
20.05～0.5g/L, CaCl
2H
2O 0.05～0.5g/L, K
2HPO
41.0～10.0g/L, folic acid 5～20mg/L, vitamin H 5～20mg/L, vitamins B
125～20mg/L, Thioctic Acid 5～20mg/L, nicotinic acid 5～20mg/L, pantothenic acid 5～20mg/L, VitB1 5～20mg/L, para-amino benzoic acid 5～20mg/L, vitamins B
65～20mg/L, riboflavin 5～20mg/L, all the other are water, pH 6.0～7.0.
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| CN103060255B (en) * | 2013-01-24 | 2014-12-03 | 盐城工学院 | Genetically engineering bacterium capable of producing S-3-hydroxy-butanone as well as construction method and application thereof |
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| CN103483167B (en) * | 2013-08-27 | 2016-06-08 | 山东鲁北药业有限公司 | A kind of purification process of 3-Hydroxybutanone |
| CN103627698A (en) * | 2013-12-05 | 2014-03-12 | 江南大学 | Breeding of acetoin high-tolerance bacterial strain and acetoin fermentation production with bacterial strain |
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