Method for rapidly screening thermophilic anaerobic ethanol microbe high-yield ethanol bacterial strain

Abstract

The invention relates to a method for rapidly screening thermophilic anaerobic ethanol microbe high-yield ethanol bacterial strain, comprising the following steps: 1) wild strains are cultured continuously in a fluid nutrient medium containing ethanol, the ethanol tolerance of the thermophilic anaerobic ethanol microbe is evolved directionally; 2) ethylmethylsulfone mutagenesis is carried out to ethanol-tolerance bacterial strains, and the mutagenesis bacterium liquid is coated on the solid culture medium containing the ethanol; 3) after individual bacterial colony is formed, the individual bacterial colony with large radius is dropwise added in 2,3,5-triphenyl chlorination tetrazole developing liquid, and single spots with deep color development are taken for re-screening on a CaCO3 rescreening plate containing high substrate concentration; 4) bacterial colonies with large radius and small radius of a transparent ring, pH indicators are dropwise added for eliminating false positive; 5) the ethanol concentration is tested by a gas chromatography or an ethanol quantitative kit after fermentation is carried out, and the bacterial strains with high ethanol output are cultured for at least 20 generations under a non-selection condition, and the bacterial strain which still has high yield is destination bacterial strain.

Description

technical field [0001] The invention belongs to the field of mutation and chemical mutagenesis screening of the physiological properties of specific microorganisms. Specifically, it adopts the means of combining directed evolution and chemical mutagenesis to mutate thermophilic anaerobic ethanol-producing microorganisms, and quickly screens high-yield ethanol through color reaction. strain. Background technique [0002] With the rapid development of the global economy, non-renewable energy sources such as oil, coal, and natural gas are increasingly depleted, and a large amount of greenhouse gases emitted by burning fossil energy have seriously damaged the ecological environment ([Cifuentes, L., Borja-Aburto, V.H., Gouveia, N., Thurston, G., Davis, D.L.2001.Climate change.Hidden health benefits of greenhouse gas mitigation.Science 293,1257-1259.], [Lashof,D.A.,Ahuja,D.R.1990.Relative Contributions of Greenhouse Gas Emissions to Global Warming.Nature 344,529-531.]), countries...

AI Technical Summary

Problems solved by technology

This method has a large workload, takes a long time, and has low screening efficiency, and is not suitable for thermophilic anaerobic ethanol-producing microorganisms, because ethanol tolerance is the main factor restricting yeast alcohol production, but for thermophilic anaerobic ethanol-producing microorganisms, such as Thermoanaerobacter ethanolicus ([Wiegel J., L.L.G.1981. Thermoanaerobacter ethanolicus gen.nov., spec.nov., a new, extreme thennophilic, anaerobic bacterium. Arch Microbiol.128, 343-348.]), which There are many types of products in the intracellular glycolysis pathway. When the substrate concentration is low, the main fermentation product is ethanol, but once the substrate concentration exceeds 1% (w / v), the fermentation product changes from ethanol to acetic acid and lactic acid. product