A kind of Citrobacter and its application in the treatment of sulfate-containing wastewater and the isolation and identification method of Citrobacter
A technology of citric acid bacteria and sulfate, which is applied in mining wastewater treatment, biological water/sewage treatment, microbial-based methods, etc., can solve the problems of cumbersome screening process and long cycle, and achieve simple screening process, short cycle, and easy The effect of cultivation
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Embodiment 1
[0043] The sediment samples collected from the Hengshihe River Basin in Dabaoshan Mining Area of Guangdong Province were used for enrichment culture of sulfate-reducing bacteria in Postgate B-type medium under anaerobic conditions. In order to simulate the acidic environment of acid mine wastewater, the ratio of phosphate was adjusted to make the medium pH 5.6. Use a spoon to pick up 0.1-0.5 grams of sediment sample, transfer it to a 25mL anaerobic tube in an ultra-clean bench, and add 20mL of Postgate B-type medium that has been autoclaved after deoxygenation. The rubber stopper is compacted and sealed before aluminum sealing to ensure that Isolated from outside air. The whole operation process is carried out in an anaerobic operation box. After inoculation, the cells were transferred to a constant temperature incubator with a temperature of 25°C for 7 days. During the cultivation process, it was judged whether the enrichment was successful by observing whether its color ...
Embodiment 2
[0046] Dilute the flora collected in the dialysis bag isolation group to 10 -3 , 10 -4 , 10 -5 In the anaerobic operation box, take each dilution of the bacterial suspension and apply it to the solid Postgate B-type medium. The incubation temperature was 30°C under both aerobic and aerobic conditions, and the incubation time under both anaerobic and aerobic conditions was 3 d until colonies grew. Pick a single colony, inoculate it on the liquid medium, and culture it anaerobically in a shaker at 30°C and 150rpm for 5-7 days. Select the anaerobic tube that turns black, and then continue to streak the bacterial liquid in the anaerobic tube with a flat plate. After 3 times of alternate screening of solid-liquid medium, under anaerobic conditions, Fe-containing 2+ and SO 4 2- On the solid medium of , the target strain produces black colonies after one week; the colonies are white under aerobic culture conditions, and the reasons for this may be as follows.
[0047] 1) This s...
Embodiment 3
[0050] Take the bacterial liquid (OD) that grows to logarithmic phase 600 0.3), centrifuge at 4°C and 8000 × g for 5 min, and discard the supernatant. After adding 5 mL of 2.5 wt% pentanediol solution, it was fully shaken, and then placed in a refrigerator at 4°C for 2-4 hours. After washing 3 times with 10mmol / L phosphate buffer, dehydrate with ethanol gradient, followed by 30%, 50%, 70%, 85%, and 95% ethanol once each (gradient concentration ethanol is configured with phosphate buffer). ), dehydrated twice with 100% ethanol, and finally washed twice with isoamyl acetate. Each step needs to be fully shaken and kept for more than 20 min, and finally centrifuged at 8000 × g for 5 min, discarded the supernatant, and proceeded to the next step. After the treated bacterial cells were freeze-dried, a small amount of bacterial cells were taken and placed on the sample tray covered with conductive glue, and then treated with gold spray for 120 s, and observed by field emission scan...
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