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Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate

A Bacillus subtilis, environmentally safe technology, applied in the direction of bacteria, microorganisms, and methods based on microorganisms, can solve the problems of restricting industrial applications, difficult industrial production, and difficult operation, etc., and achieve high raw material utilization rate, easy control, The effect of mild reaction conditions

Inactive Publication Date: 2010-10-06
JIANGNAN UNIV
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Problems solved by technology

2,3-BDO can be produced through chemical synthesis and microbial production. Due to the special structure of 2,3-butanediol, chemical production of 2,3-butanediol is mainly based on four-carbon carbons produced during petroleum cracking Hydrogen compounds are obtained by hydrolysis under high temperature and high pressure, which is costly, cumbersome, and difficult to operate, so it has been difficult to achieve large-scale industrial production
At present, the industrialization of 2,3-butanediol is mainly the fermentation method. The relevant researches were carried out earlier in foreign countries, and the achievements obtained are relatively remarkable. The domestic research is still in its infancy. Most of the bacteria commonly used in research are Klebsiella, and Klebsiella has potential pathogenicity, which limits its industrial application

Method used

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  • Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate
  • Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate
  • Environmentally-friendly breeding of bacillus subtilis for producing 2,3-butanediol by fermentation with glucose substrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the screening of bacterial strain:

[0029] Screening of target strains: Enrich the collected samples in seed medium (glucose 20g / L, yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L pH 7.0), shake at 37°C 150r / min Bed for 10h, take different dilutions to spread and culture. Pick a single colony on the plate separation medium (yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, agar powder 18g / LpH 7.0) for streak separation, culture at 37°C for 1d-2d, microscopic examination, The strains to be tested were preserved in the slant preservation medium for primary screening.

[0030] The seeds cultivated to the logarithmic phase with the seed medium were added to the fermentation medium containing 15% glucose according to the inoculum amount of 5%, and then placed at 37°C and 150r / min for shaking culture for 72h. The fermentation broth was collected, centrifuged, and the supernatant was taken for detection.

Embodiment 2

[0031] Embodiment 2: 2,3-BDO qualitative detection:

[0032] Take 2ml of the supernatant, extract with 2ml of ethyl acetate, and then use gas chromatography-mass spectrometry (GC-MS) to identify whether the extract contains 2,3-BDO. GC-MS detection conditions: The temperature control program of the chromatographic column is as follows: first, keep warm at 60°C for 1min, then raise the temperature to 90°C at a rate of 6°C / min and keep it for 2min, and finally increase the temperature at a rate of 30°C / min. To 180 ℃, constant temperature 2min. The temperature of the injection port is 250°C, the injection volume is 1 μL, the carrier gas is high-purity helium, the flow rate of the carrier gas is 1.2mL / min, and the detector temperature is 240°C;

[0033] TraceMS mass spectrometry conditions: EI+ bombardment source, full scan mode, scan mass range of 30-500amu, emission current of 200μA, electron energy of 70eV, mass spectrometry detection spectrum library is NIRT98 spectrum librar...

Embodiment 3

[0035] Embodiment 3: Detection of the ability to synthesize 2,3-butanediol of bacterial strains:

[0036]The 2,3-butanediol content in the fermented liquid obtained in Example 1 was determined by liquid chromatography. Waters liquid chromatography, sugar column Sugarpakl (6.5mmid × 300mm)); column temperature: 85 ° C; mobile phase: pure water, flow rate 0.4ml / min; injection volume is 10 μ L; detector: differential refractive index detector. The retention times of different configurations of 2,3-BDO were 19.3min and 22.5min, respectively.

[0037] 2,3-BDO standard HPLC diagram Figure 6 As shown, 6-7 uses glucose as the substrate 72h fermentation broth HPLC spectrum as shown in Figure 12;

[0038] After 72 hours of fermentation of a selected strain of Bacillus subtilis, the residual glucose in the fermentation liquid was about 1.5g / L, and the content of 2,3-butanediol was about 50g / L.

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Abstract

2,3-BDO is important industrial chemicals and a medicinal precursor, for example, the 2,3-BDO can form 1,3-butadiene through dehydration and can be used for synthesizing rubber, polyester and polyurethane; the esterified 2,3-BDO is a precursor of synthetic polyimine, and can be applied to medicaments, cosmetics, washing liquor liquid and the like; and in addition, the 2,3-BDO can also be used as a flavor substance to be added into distilled spirit and cream. The 2,3-BDO can be prepared by a chemical synthesis method and a microorganism production method, and due to a special structure of 2,3-butanediol, in the chemical method for producing the 2,3-butanediol, the 2,3-butanediol is mainly prepared by hydrolyzing four-carbon hydrocarbon generated during the splitting decomposition of petroleum at the high temperature and under the high pressure and has the disadvantages of high cost, complicated processes and difficult operation, so the large-scale industrial production is difficult to realize all the time. With the increasing rise in oil price, people pay close attention to the production of the 2, 3-butanediol by using the microbial fermentation method and the developmental research of derivatives thereof gradually. The fermentation method for producing the 2,3-butanediol has the following advantages that: firstly, the fermentation method has the superiority in cost, and the chemical method has high production cost; secondly, the fermentation method has an environmental-friendly characteristic, and the 2,3-butanediol is used as an organic synthesis intermediate to occupy markets of a number of compounds and other petroleum chemicals, has wide prospect, obvious social and environmental benefits, and is particularly favorable for the extensive application of the products; and thirdly, in the fermentation method for producing the 2,3-butanediol, renewable resources are used as raw materials, so the fermentation method does not depend on the petroleum chemicals.

Description

technical field [0001] The invention relates to the breeding of an environment-safe Bacillus subtilis which uses glucose as a substrate to ferment and produce 2,3-butanediol, and belongs to the technical field of fermentation in bioengineering. Background technique [0002] 2,3-butanediol, the English name is 2,3-butanediol, abbreviated as 2,3-BDO, it is colorless crystal or liquid, melting point is 23-27°C, boiling point is 170-182°C, it is water-absorbing and can be mixed with Water and alcohol are miscible with each other and can be dissolved in alcohol and ether. [0003] 2,3-BDO is an important chemical raw material and pharmaceutical precursor, for example, it can be dehydrated to form 1,3-butadiene, which can be used to synthesize rubber, polyester and polyurethane; 2,3 in the esterified form -BDO is a precursor for the synthesis of polyimine, which can be used in drugs, cosmetics, lotions, etc. In addition, 2,3-BDO can also be added to liquor and cream as a flavor ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/125
Inventor 饶志明林清张显杨套伟张术聪
Owner JIANGNAN UNIV
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