A saline-alkali-tolerant growth-promoting bacterium and its application in saline-alkali land improvement
A technology of saline-alkali land and pure bacteria, applied in the field of environmental microorganisms, can solve problems such as high cost and secondary salinization of land
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[0071] The third aspect of the present invention provides a method for preparing a liquid bacterial agent, which at least includes the following steps: after activating and culturing the pure bacteria of Halomonas aquamarina DB01 in seawater, inoculate it in LB liquid medium, and inoculate it at 30°C for 180r / min cultured for 48h to obtain liquid bacterial agent.
[0072] Wherein, the LB liquid medium: peptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L.
[0073] The fourth aspect of the present invention provides a solid bacterial agent, the solid bacterial agent includes Halomonas aquamarina DB01, the concentration of Halomonas aquamarina DB01 is at least 1 × 10 9 cfu / g.
[0074] The fifth aspect of the present invention provides a method for preparing a solid bacterial agent, which at least includes the following steps: drying the liquid bacterial agent containing Halomonas aquamarina DB01 to obtain a solid microbial agent.
[0075] Furthermore, spray drying, vacuum...
Embodiment 1
[0088] Example 1: Isolation, screening and performance determination of saline-alkali-resistant growth-promoting bacteria DB01
[0089] (1) Collect saline-alkali soil from different regions, take 1-5g sample and put it into a conical flask filled with 100mL sterile water, shake at 180r / min at 30°C for 30min to fully disperse the microorganisms in the tested soil in the sterile water Bacterial water to obtain a soil mixture.
[0090] (2) The obtained mixed solution is diluted according to the gradient, and the concentration gradient is selected to be 10 -2 、10 -3 、10 -4 、10 -5 、10 -6Soil mixture solution, in high-salt and high-alkali LB solid medium (peptone 10g / L, yeast extract 5g / L, sodium chloride 100g / L, agar 15g / L, adjust the pH to 10 with sodium carbonate after sterilization) Coating on the plate is used for primary screening to isolate saline-alkali-resistant microorganisms, and the plate is inverted and placed in a constant temperature incubator at 30°C for 48h to ...
Embodiment 2
[0098] Embodiment 2: Molecular biological identification of saline-alkali-tolerant growth-promoting bacteria DB01
[0099] The strains were identified by 16S rDNA sequence alignment. The genomic DNA of the strain DB01 was extracted according to the prior art, and a pair of universal primers (27F, 1492R) was used to amplify the 16S rRNA of the strain using this as a template.
[0100] The upstream primer is 27F (5'-AGAGTTTGATCCTGGCTCA-3'),
[0101] The downstream primer is 1492R (5'-GGTTACCTTGTTACGACTT-3').
[0102] The PCR reaction system (20 μL) is as follows: template DNA 0.5 μL, PCR Taqmix 10 μL, upstream and downstream primers 0.6 μL, add ddH 2 O to 20 μL of the reaction system.
[0103] PCR program: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min and 30 s, and a total of 30 cycles of the above, extension at 72°C for 10 min, and finally storage at 4°C.
[0104] PCR products were purified and seq...
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