Clostridium beijerinckii for producing butanol from xylose and its application

A technology for the production of butanol by Clostridium beijerinckii, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of reducing the utilization rate of cellulose substrates, losing the ability to produce butanol, and weak xylose utilization ability , to achieve breakthroughs in low substrate utilization, high-efficiency production, and solve the effect of low substrate utilization

Active Publication Date: 2020-04-03
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the anaerobic Clostridia have good ability to utilize glucose, but their ability to utilize xylose is very weak, which will greatly reduce the utilization rate of cellulose substrates. Moreover, most of the strains that have been reported are under passage After several times, it loses the ability to produce butanol, and the stability is poor

Method used

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  • Clostridium beijerinckii for producing butanol from xylose and its application
  • Clostridium beijerinckii for producing butanol from xylose and its application
  • Clostridium beijerinckii for producing butanol from xylose and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] The breeding steps of high-yield butanol strain (Clostridium beijerinckii) 51543 are as follows:

[0018] (1) Collection of soil samples: Use a small shovel to dig off the soil surface, collect about 20g of soil at about 10cm from the surface, then put the collected soil samples in disposable gloves and seal them, and use toilet paper before and after each soil sample collection Wipe the small shovel clean and collect a total of 100 soil samples (32 of which were taken from the surrounding areas of Tianjin and 68 from all over China).

[0019] (2) Enrichment of strains: At room temperature, weigh about 1g from 100 parts of soil and inoculate them into 100 parts of 10ml screw-lock test tubes containing 10ml of xylose medium (xylose medium (1L): Yeast powder 5g, CaCO 3 3g, superphosphate 0.7g, (NH 4 )2SO 4 3g xylose, 30g xylose, natural pH, 121°C for 21min), mix upside down, heat shock at 80°C for 10min, cool to room temperature with running water, and incubate in a 30...

Embodiment 2

[0058] Comparison of the growth of the 51543 strain with the original strain under static conditions

[0059] 1. Test materials: 51543 strain; original strain (Clostridium beijerinckii) CH5.

[0060] 2. Experimental method:

[0061] Seed medium (g / l): yeast powder 5, CaCO 3 3. Superphosphate 0.7, (NH 4 ) 2 SO 4 3. Natural pH, sterilized at 121°C for 21 minutes, adding xylose to make the final concentration reach 30g / l before inoculation. Separate sterilization: xylose mother liquor 200g / l, sterilized at 110°C for 18min.

[0062] Fermentation medium (g / l): beef extract 2, yeast powder 2, tryptone 6, ammonium acetate 3, K 2 HPO 4 0.5, pH 6.5, sterilized at 121°C for 21 minutes, added before fermentation to make the final concentration of xylose 40g / l. Separate sterilization: xylose mother liquor 200g / l, sterilized at 110°C for 18min, MgSO 4 ·7H 2 O 0.2, FeSO 4 ·7H 2 O 0.01 filter sterilized.

[0063] Inoculate 500ul of the 51543 strain and the starting strain (store...

Embodiment 3

[0069] Comparison of the fermentation curves of the 51543 strain with the original CH5 strain under static conditions

[0070] 1. Experimental material: same as embodiment 2

[0071] 2. Experimental method: seed culture medium, fermentation medium are the same as embodiment 2

[0072] Inoculate 500ul of the 51543 strain and the starting strain (stored in a glycerol tube at -20°C) into 10ml seed medium, heat shock at 80°C for 10min, cool with running water and then culture at a constant temperature of 30°C for 16h, the OD is about 2. Inoculate in TYA medium with 5% inoculum amount, incubate at 30°C for 16 hours, OD is 2. Inoculate in a 250ml screw-lock conical flask containing 150ml with initial cell concentration OD600=0.1, and inoculate at 30°C Cultivate and measure the concentration of xylose, butanol, isopropanol, ethanol, butyric acid, and acetic acid in the bacteria.

[0073] 3. Analytical method: xylose concentration, butanol concentration, isopropanol concentration, e...

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Abstract

The invention discloses Clostridium beijerinckii for producing butanol by using xylose and an application thereof. The Clostridium beijerinckii for producing butanol by using xylose (Clostridium beijerinckii) 51543 is preserved in the China Center for Type Culture Collection with the preservation number CCTCC M2017031. The present invention uses xylose as the only substrate to screen the Clostridium beijerinckii strain through the combination of natural selection and chemical mutagenesis. The bacterial strain can efficiently utilize xylose for fermentation, realize the efficient production of butanol, and solve the problem of The problem of low substrate utilization. In addition, the strain still has a butanol production capacity similar to that of the non-passaged strain after 11 transfer passages for 60-70 generations, and has a certain stability. The invention breaks through the problem of low utilization rate of the cellulose substrate by the bacterial strain, simultaneously breaks through the limitation of the instability of the bacterial strain, and solves the bottleneck of the glucose repression effect.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and specifically relates to a Clostridium beijerinckii 51543 with high butanol yield obtained by using xylose as the only substrate through natural selection combined with chemical mutagenesis. Background technique [0002] ABE (acetone-butanol-ethanol) fermentation was once the second largest industrial fermentation industry after ethanol fermentation in history. However, because the fermentation production of butanol is more costly and less efficient than the traditional chemical production method, the development is so slow that it is finally replaced by the chemical production method. In recent years, due to the sharp increase in oil prices, the greenhouse effect and the increasing environmental pollution, some scientists have been forced to seek some renewable biomass fuels instead of oil to alleviate the current environmental and economic problems. Anaerobic Clostridium spp can produce ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/16C12R1/145
CPCC12P7/16C12P2203/00C12N1/205C12R2001/145Y02E50/10
Inventor 马媛媛冯春迎洪解放张敏华邹少兰
Owner TIANJIN UNIV
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