Bacillus with relatively high tolerance to methanol

A Bacillus, methanol technology, applied in bacteria, microorganism-based methods, microorganisms and other directions, can solve the problem that microorganisms cannot grow normally, and achieve broad industrial application prospects and good passage stability.

Active Publication Date: 2021-12-31
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although methanol has many advantages as a substrate, there are also some key problems: microorganisms cannot grow normally in a certain concentration of methanol. For example, Wen Dongming and others obtained a strain that can grow in 7% methanol through gene mutation and screening. MB200 strain
However, there are no reports of methanol-tolerant Bacillus

Method used

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  • Bacillus with relatively high tolerance to methanol
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  • Bacillus with relatively high tolerance to methanol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The isolation and identification of embodiment 1 bacterial strain ZSL-8

[0034] (1) Isolation of strains

[0035] Soils from different areas were collected for screening. The soils came from relevant places on the campus of Nanjing University of Technology and near the factories of related environmental protection enterprises in Nanjing. The soil was collected from a distance of 5 cm from the soil surface, and the soil samples were collected and stored in sterile sealed bags in a refrigerator at 4°C in the laboratory. Weigh 5 g of soil, put it into a sterile Erlenmeyer flask, add 45 mL of sterile water, tie it tightly with a sterile air-permeable sealing film, and place it in a shaker at 37°C and 200 rpm for 20 minutes. Take 1 mL from the soil suspension for serial dilution to 10 7 times, take 10 from each 5 、10 6 、10 7 10 μL of the diluted sample was applied to the screening medium, and each gradient was coated in three parallels, and the plate was placed upside ...

Embodiment 2

[0060] The construction of embodiment 2 bacterial strain ZSL-MS

[0061] Pick a single colony from a fresh plate of ZSL-8 and inoculate it in liquid LB medium, and culture it at 200 rpm at 37°C for 12 hours as the seed solution. In the ultra-clean bench, transfer the inoculation amount of 5% to the acclimatization medium with methanol content of 15g / L, place the inoculated Erlenmeyer flask at 37°C, and cultivate it in a shaker at 200rpm for 12h, then inoculate with 5% The inoculum was transferred to the acclimatization medium with a methanol content of 15g / L, and continued to cultivate for 12 hours. The main purpose was to make the strain grow stably at this concentration. In the next step, directly transfer the bacterium fluid stably grown in the acclimatization medium containing 15g / L methanol to the acclimatization medium containing 20g / L methanol at an inoculum size of 5%. After the same steps inoculated to methanol content of 30g / L, 40g / L, 50g / L, 60g / L, 70g / L, 80g / L, 90g...

Embodiment 3

[0062] Example 3 Methanol tolerance comparison of different bacterial strains

[0063] The strains constructed in Example 2, the model strain Bacillus subtilis 168, and Bacillus amyloliquefaciens NB (disclosed in published papers (J Agric Food Chem 67, 282-290 (2019)), were respectively added to liquid culture media containing different concentrations of methanol The growth situation contrast at the time of 48h.The results are as follows Figure 5 As shown, when the methanol concentration is 50g / L, the OD of strain ZSL-MS 600 7.86, the OD of Bacillus subtilis 168 and Bacillus amyloliquefaciens NB 600 1.48 and 1.16, respectively. When the concentration of methanol is 150g / L, the OD of strain ZSL-MS 600 6.98, the OD of Bacillus subtilis 168 and Bacillus amyloliquefaciens NB 600 are 0.21 and 0.14, respectively.

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Abstract

The invention discloses bacillus with relatively high tolerance to methanol and belongs to the technical fields of microbial culture, molecular biology and application. The strain disclosed by the invention has been preserved in the China Center for Type Culture Collection on April 6, 2021, and the preservation number is CCTCC NO: M 2021326. The strain can tolerate 20% or even higher methanol, has good passage stability, still keeps methanol tolerance after 50 generations of passage, can be applied to construction of methylotrophication strains and synthesis of high-added-value products by using methanol as a substrate, and has wide industrial application prospects.

Description

technical field [0001] The invention relates to a Bacillus strain with high tolerance to methanol, which belongs to the field of microbial culture, molecular biology and application technology, and specifically relates to the screening, identification and adaptive domestication of Bacillus with high methanol tolerance. Background technique [0002] With the advancement of social development and industrialization, various chemically synthesized products and subsequent extraction operations have caused serious pollution to the environment, and people's attention has begun to shift to green biomanufacturing with biorenewable resources as substrates. . Whereas among commonly used feedstocks, lignocellulosic biomass requires costly pretreatment due to glucose competing with the food supply, methanol will hopefully be used as an alternative feedstock. Methanol is an abundant and pure raw material that can be used directly in bacterial fermentation processes. At the same time, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12R1/01
CPCC12N1/20
Inventor 罗正山潘斐徐虹胡毅朱逸凡严一凡李莎续晓琪王瑞徐铮
Owner NANJING UNIV OF TECH
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