Method for immobilizing clostridium acetobutylicum by use of bacterial cellulose membrane

A technology of bacterial cellulose membrane and Clostridium acetobutylicum, applied in the direction of immobilized on/in organic carriers, bacteria, fermentation, etc., can solve the problem of poor thermal stability and mechanical stability affecting the stability of immobilized materials, bacteria Single cellulose structure, unfavorable bacteria adsorption and other problems, to achieve the effect of shortening fermentation time, reducing production cost and increasing product concentration

Active Publication Date: 2015-09-09
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, bacterial cellulose has a single structure and high hydrophilicity, which is not conducive to the adsorption of bacteria, and its poor thermal and mechanical stability affects its stability as an immobilized material.

Method used

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  • Method for immobilizing clostridium acetobutylicum by use of bacterial cellulose membrane
  • Method for immobilizing clostridium acetobutylicum by use of bacterial cellulose membrane
  • Method for immobilizing clostridium acetobutylicum by use of bacterial cellulose membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Acetobacter xylinum preserved at -80°C (purchased from ATCC, the number of the bacterium is ATCC 23769) was inoculated on the slant medium, and the slant medium was: glucose 100g / L, yeast powder 10g / L, calcium carbonate 20g / L, agar 15g / L, pH=6.8; at 30°C, culture statically for 24 hours, after the bacterial lawn grows, scrape a ring and transfer it to 100ml seed medium, the seed medium is: glucose 20g / L, peptone 10g / L, yeast powder 5g / L, disodium hydrogen phosphate 2.7g / L, citric acid 1.2g / L, magnesium sulfate 0.25g / L, pH6.0, high temperature sterilization for 15min, 150r / min , 30°C, cultured for 18 hours; inoculated into 200ml fermentation medium with 5% inoculum size, the fermentation medium was: glucose 20g / L, peptone 10g / L, yeast powder 5g / L, disodium hydrogen phosphate 2.7g / L, citric acid 1.2g / L, magnesium sulfate 0.25g / L, pH 6.0, 30°C, static culture for 72h, until the bacterial cellulose film is formed, take out the bacterial cellulose film, rinse with distilled...

Embodiment 2

[0058] According to the methods shown in Table 1, the modified bacterial cellulose membranes modified by acetylation, esterification, silane coupling agent, polymer and organic phosphine were prepared respectively. The addition amount of bacterial cellulose in the following modification method is 0.5g.

[0059] Table 1 Modification method of bacterial cellulose membrane

[0060]

[0061]

[0062] See the experimental results image 3 , 4 .

[0063] Such as image 3 , It can be seen from the characteristic peaks in the infrared spectrum: the characteristic peak of bacterial cellulose is 3450nm, O—H stretching vibration, 2920nm, CH2—CH stretching vibration, 900nm, glycosidic bond, 1059nm, C=O stretching vibration .

[0064] After organic phosphine modification, the peak at 2100nm disappeared, the peaks at 1500nm and 1400nm appeared, and the peak at 1000-1300nm was enhanced, indicating that the phosphoester bond appeared and strengthened. CO at 2400nm 2 absorption pe...

Embodiment 3

[0067] Preparation of Clostridium acetobutylicum cell liquid: Clostridium acetobutylicum B3 (CGMCC No.5234) preserved at -80°C was inoculated on the plate medium, and the plate medium was: glucose 5g / L, Peptone 5g / L, yeast powder 3g / L, ammonium acetate 2g / L, MgSO 4 ·7H 2 O 3g / L, NaCl 2g / L, K2HPO4 1g / L, KH2PO4 1g / L, FeSO4 7H 2 O 0.1g / L, agar powder 15-20g / L, pH 6.0, 121°C, sterilized for 15 minutes; at 37°C, anaerobic culture for 24 hours, after the bacterial lawn grows, scrape the bacterial lawn and transfer to another plate After 12 hours, transfer again, scrape the lawn after 12 hours of cultivation, and transfer to 100ml seed medium, the seed medium is: glucose 5g / L, peptone 5g / L, yeast powder 3g / L, acetic acid Ammonium 2g / L, MgSO 4 ·7H 2 O 3g / L, NaCl 2g / L, K2HPO4 1g / L, KH2PO4 1g / L, FeSO4 7H 2 O 0.1g / L, pH 6.0, high temperature sterilization for 15min, 37°C, static culture for 12h; inoculate 100ml fermentation medium with 10% inoculum size, the fermentation medium is: ...

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Abstract

The invention discloses a method for immobilizing clostridium acetobutylicum by use of a bacterial cellulose membrane. The method comprises the following steps: (1) hydrophobic modification of a bacterial cellulose membrane: conducting acid treatment on the bacterial cellulose membrane, and then cleaning and drying to obtain a bacterial cellulose membrane A; conducting alkaline treatment on the bacterial cellulose membrane A, and then cleaning and drying to obtain a bacterial cellulose membrane B; conducting acetylation, esterification, silane-coupling, polymeric modification or organic phosphate coupling on the surface of the bacterial cellulose membrane B to prepare a modified bacterial cellulose membrane; (2) immobilization of clostridium by use of the modified bacterial cellulose membrane: fixing the modified bacterial cellulose membrane obtained in the step (1) in a continuous fermentation tank, pumping in a clostridium acetobutylicum bacterial suspension for self-circulation culture to enable clostridium acetobutylicum to be adsorbed onto the bacterial cellulose membrane so as to obtain clostridium acetobutylicum immobilized onto the bacterial cellulose membrane. According to the method, after hydrophobic modification, clostridium is immobilized for continuous fermentation, the production efficiency is improved and the cost is lowered.

Description

technical field [0001] The invention belongs to the field of continuous fermentation of immobilized cells, and in particular relates to a method for immobilizing Clostridium with a modified bacterial cellulose membrane as a carrier. Background technique [0002] Among Clostridium microorganisms, Clostridium acetobutylicum is often used to ferment and produce butanol, but the traditional free fermentation has a long production cycle and low efficiency, and it cannot be fermented continuously for a long time, and it is difficult to separate the product from the bacteria. Immobilized cells can overcome the above problems, realize continuous batch fermentation, improve production efficiency and reduce costs. [0003] Bacterial cellulose has a unique ultra-fine mesh fiber structure. The material has a high specific surface area and contains a large number of pore structures, which is conducive to cell adsorption and fixation. However, bacterial cellulose has a single structure a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/12C12P7/16C12N1/20
Inventor 应汉杰杨静庄伟吴菁岚周精卫陈勇朱晨杰柳东牛欢青
Owner NANJING TECH UNIV
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