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Culture method for improving activity of clostridium thermoceuum cellulase

A culture method and a technology for Clostridium thermocellum are applied in the fields of microbial culture and fermentation engineering to achieve the effects of promoting growth period, increasing enzyme activity and reducing cost

Inactive Publication Date: 2014-02-05
BIOGAS SCI RES INST MIN OF AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to overcome the above-mentioned problem existing in the cellulase activity of existing solution Clostridium thermocellum, provide a kind of culture method that improves Clostridium thermocellum cellulase activity

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The invention uses multiple carbon sources as the components of the culture medium, induces the improvement of cellulase activity through the stimulation and addition of carbon sources, and increases the enzyme activity of the fermentation broth under the condition of not adding additional carbon sources. This method mainly comprises the following steps:

[0032]1. The base liquid medium is: ammonium sulfate 1.30 g / L, magnesium chloride 2.60 g / L, potassium dihydrogen phosphate 1.43 g / L, dipotassium hydrogen phosphate 5.50 g / L, calcium chloride 0.13 g / L, iron sulfate 1.10 mg / L L, cysteine ​​0.25 g / L, yeast extract 4.50 g / L, resazurin 1.0 mg / L, microcrystalline cellulose 10.00 g / L, or cellobiose 5.00 g / L. Add the primary bacterial seed liquid to the basic medium, the volume ratio of the primary bacterial seed liquid to the basic medium is 1:10 to 1:20, and ferment for 48-72 hours.

[0033] 2. Fermentation medium: ammonium sulfate 1-2 g / L magnesium chloride 2-3 g / L, pot...

Embodiment 2

[0043] 50ml anaerobic bottle fermentation

[0044] 1． C.thermocellum DSM1237 T Freeze-dried powder bacteria are configured as a first-grade strain solution.

[0045] 2． Activated C.thermocellum DSM1237 T Inoculate 5 ml of the primary strain solution into 50 ml of the basal medium, and incubate at a constant temperature of 60°C for 48 hours to obtain the secondary strain liquid. Inoculate the secondary bacterial seed liquid into the fermentation medium at an inoculation ratio of 1:10.

[0046] 3． Configure medium according to formula, fermentation medium: ammonium sulfate 1-2 g / L magnesium chloride 2-3 g / L, potassium dihydrogen phosphate 1-2 g / L, dipotassium hydrogen phosphate 5-6 g / L, calcium chloride 0.1-0.3 g / L, iron sulfate 1-2 mg / L, cysteine ​​0.2-0.5 g / L, yeast extract 4-6 g / L, resazurin 1.0 mg / L, carbon source 10.0-12.0 g / L. Boil, cool to room temperature to adjust pH 6.5-7.5, and continuously ventilate the air in the bottle with nitrogen, dispense 50ml into ...

Embodiment 3

[0053] 1L anaerobic bottle fermentation

[0054] 1． Prepare C.thermocellum DSM1237 according to the method described in Example 2 T The secondary strain liquid is 100ml, the inoculum size is 1:10, and the total volume of the fermentation broth is 1L.

[0055] 2． According to the formula configuration medium, (NH 4 ) 2 SO 4 1.30 g / L MgCl 2 x 6 H2O 2.60 g / L, KH 2 PO 4 1.43g / L,K 2 HPO 4 5.50 g / L, CaCl 2 x 2 H 2 O 0.13 g / L, FeSO 4 x 7 H 2 O 1.10 mg / L, Cysteien hydrochloride 0.5 g / L, Yeast extract 2.50 g / L, Resazurin 1.0 mg / L, carbon source 10.0 g / L. Boil, cool to room temperature to adjust pH 6.5-7.5, and continuously ventilate the air in the bottle with nitrogen, dispense 50ml into 150ml culture bottles, and seal with polybutylate stoppers to ensure an anaerobic environment in the bottle. Sterilize at 121°C for 30 minutes. Calcium, magnesium, and iron salts were made into 100% mother liquor, and added after sterilization.

[0056] 3． After static cultur...

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Abstract

The invention discloses a culture method for improving the activity of clostridium thermoceuum cellulase, which comprises the following steps of (a) preparing a strain DSM1237T into a first-level strain liquid; (b) adding the first-level strain liquid into a base culture medium, wherein the volume ratio of the first-level strain liquid to the base culture medium is (1:10)-(1:20); performing fermentation culture for 48-72 hours to obtain a second-level strain liquid; (c) adding the cultured second-level strain liquid into the prepared fermentation culture medium, wherein the volume ratio of the second-level strain liquid to the fermentation culture medium is (1:30)-(1:10); (d) performing fermentation culture at a temperature of 50-70 DEG C under an anaerobic condition at a pressure of (-0.1)-0.1MPa, and standing, wherein the pH value is 6.5-8.0, and the culture time is 48-72 hours. According to the method disclosed by the invention, under the anaerobic condition, the energy consumption for introducing oxygen or compressed air in the fermentation process can be saved. Under the condition of same culture cost, the activity of the fermentation liquid cellulase is remarkably improved, and the economic benefits are remarkably increased.

Description

technical field [0001] The invention relates to the field of microorganism culture and fermentation engineering, in particular to a culture method for improving the cellulase activity of Clostridium thermocellum. Background technique [0002] The energy and environmental crisis is a major problem faced by the whole world, and it is a hot topic of research in various countries at present. The energy development and utilization of biological resources is crucial to solving energy shortages, food crises and reducing CO 2 Gas emissions have profound implications (Tilman et al, 2009; Regalbuto et al, 2009). Bioethanol is currently a promising alternative energy source, but at present, grain fermentation is mostly used. Due to population expansion and food crisis, starch ethanol is subject to many restrictions. [0003] In my country, the annual output of straw agricultural waste is more than 700 million tons, which is the largest resource and the highest utilization potential, ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12R1/01
CPCC12N9/2437C12Y302/01004
Inventor 贺静马诗淳代莉蓉陈璐邓宇周正
Owner BIOGAS SCI RES INST MIN OF AGRI
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