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High selenate tolerant bacteria, and screening method and application thereof

A screening method, selenate technology, applied in the direction of bacteria, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of low yield

Inactive Publication Date: 2020-03-27
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It took more than 36 h to produce red nano-elemental selenium by direct fermentation of two strains of endophytic Helicospirinus tea plants under the condition of 200 mM selenate, and the yield was low.

Method used

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  • High selenate tolerant bacteria, and screening method and application thereof
  • High selenate tolerant bacteria, and screening method and application thereof
  • High selenate tolerant bacteria, and screening method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Inoculate the endophyte WT00F strain of Endophyte spirochete WT00F in the laboratory into LB medium (10 g tryptone, 5 g yeast extract, 5 g NaCl, add water to 1000 mL), and incubate at 37 °C and 200 rpm for 12 h. Cells were collected by centrifugation, suspended in PBS (1.44g disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, 8g sodium chloride, 0.2g potassium chloride, pH7.4), and then treated with sodium selenate. The specific method is as follows: Endospira WT00F was treated with 200 mM sodium selenate at room temperature for 2 h, and the cultured bacteria were screened in 200 mM sodium selenate LB medium at a ratio of 1:100.

[0032] In order to further improve bacterial selenate tolerance, 250 μl of the bacterial culture obtained from sodium selenate treatment was taken out and inoculated into 25 ml of LB medium containing 200 mM sodium selenate (200 mM sodium selenate, 1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH7.2), 37 ℃, 200 rpm shaking culture...

Embodiment 2

[0035]Using the method of forced evolution, the high selenate-tolerant Heliospirinus NTC00F was successfully obtained. In order to further verify whether the obtained NTC00F strain is still Helicospirillum spirulina, we used the bacterial 16S rRNA gene universal primer pair 5'-AGAGTTTGATCATGGCTCAG-3' and 5'-ACGGTTACCTTGTTACGACTT-3' to PCR amplify the 16SrRNA gene of the NTC00F strain and cloned it into Plasmid pUC18 was sent to a DNA sequencing company for sequencing. The results of sequencing showed that the 16S rRNA gene sequence of NTC00F strain was completely consistent with the 16S rRNA gene sequence of Endospira WT00F. The 100% identical 16S rRNA gene indicated that the high selenate-tolerant Helicospirillum NTC00F was indeed derived from the Endospira WT00F of the tea tree.

Embodiment 3

[0037] In order to test the tolerance of high selenate tolerance of the spirulina spirulina NTC00F to selenate, inoculate 250ml of the spirulina spirulina NTC00F containing 0, 50, 100, 200, 400, 800 mM sodium selenate at a ratio of 1:100 LB liquid medium, cultured with shaking at 37°C and 200rpm. Samples were taken at different incubation times and the OD was measured on a UV / Vis spectrophotometer 600 value. Each point was repeated 3 times to obtain the average. Finally, the growth curve of the bacteria was compiled. At the same time, under the same conditions, test the tea plant endophyte WT00F as a control. Endophytic Helicospirinus WT00F in tea trees can grow in the medium up to 400 mM sodium selenate, but the growth cycle needs more than 80 hours. When selenate was higher than 400 mM, bacteria could not grow. Even in a medium with 200 mM sodium selenate, the growth cycle takes 32 hours (see Figure 1A ). The high-selenate-tolerant Helispirulina spirulina NTC00F is d...

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Abstract

The invention provides a strategy for obtaining a novel high selenate tolerant strain by utilizing selenate to treat tea endophytic herbaspirillum spp WT00F and combining with a forced evolution method, and a method of the novel high selenate tolerant strain to produce red elemental selenium (Se<0>). Through selenate treatment and forced evolution, the tea endophytic herbaspirillum spp can be repeatedly transferred and cultured in a 200 mM selenate culture medium, so that the stable novel high selenate tolerant strain- tea Herbaspirillum sp.NTC00F can be finally formed, and selenate tolerancecan reach 800 mM. The tea Herbaspirillum sp.NTC00F is placed into a culture medium containing 400 mM of selenate to ferment; and the bacteria can reduce the selenate and efficiently produce the red nano elemental selenium, and the yield of the red nano elemental selenium can reach 1,000 [mu]g / ml. The prepared pure red nano elemental selenium can be used for pharmaceutical, agricultural and food processing, and is simple in technical operation, low in cost, green, environmentally-friendly and pollution-free.

Description

technical field [0001] What the present invention relates to is to utilize tea tree endophytic spirulina WT00F to repeatedly transfer and cultivate under the condition of high concentration selenate (200 mM) and obtain high selenate tolerance bacterium-the tea tree spirulina NTC00F bacterial strain, especially A technical method for co-cultivating and fermenting high-selenate-tolerant spirulina spirulina NIC00F and high-concentration selenate (400 mM) to produce red nanometer elemental selenium. Background technique [0002] Selenium is an essential trace non-metallic element for animals and plants. In nature, selenium often exists in inorganic and organic forms. The organic forms of selenium usually exist in animal and plant cells, such as selenocysteine, selenomethionine and selenoprotein; inorganic selenium usually has 4 valence states: selenate (Se 6+ ), selenite (Se 4+ ), selenide (Se 2- ) and elemental selenium (Se 0 ), where Se 2- It is often used as a raw mater...

Claims

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Application Information

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IPC IPC(8): C12N1/04C12N1/20C12P3/00C12R1/01
CPCC12N1/04C12N1/20C12P3/00C12N1/205C12R2001/01
Inventor 王行国倪雪晨雷佳田金宝周佳卉李亚东
Owner HUBEI UNIV
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