Clostridium kogasensis EO-08 and application thereof in degrading ethylene oxide
An ethylene oxide, carboxyl-like technology, applied in the field of biodegradation, can solve the problems of easy explosion, low absorption saturation, high biological toxicity, etc.
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Embodiment 1
[0068] Enrichment, screening and identification of EO-08 original strain
[0069] Weigh 10.0 g of sludge sample, add 100 mL of 0.03 mol / L phosphate buffer solution, mix and clarify for 120 min, and remove large particles of sediment. Take 1mL of the suspension and add 10mL of enrichment enrichment liquid medium (containing preferably 100mg / L low-concentration ethylene oxide), and place it on a shaker for anaerobic enrichment culture at 200r / min, anaerobic at 37°C Cultured for 24-48h to observe the growth.
[0070]Bacteria enrichment medium: tryptone 17g, soybean peptone 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, distilled water 1000ml, adjust pH to 7.0-7.5, pack into 500mL Erlenmeyer flask, each Erlenmeyer flask 250mL. Sterilize at 121°C for 20 minutes, cool to room temperature, take out the ice box from the refrigerator, and place reagent pure ethylene oxide on the ice box. Use a sealed syringe to draw 28 μL of ethylene oxide liquid into the...
Embodiment 2
[0086] Ethylene oxide-induced domestication of strain EO-08
[0087] Ethylene oxide tolerance induced acclimatization: Using the streak method, inoculate the EO-08 original strain in the 100mg / L ethylene oxide tolerance acclimatization medium, inoculate the culture plate and the 2.5L anaerobic gas generating bag at the same time Place in a 2.5L round-bottom vertical anaerobic culture bag, seal the culture bag, and place it in a 37°C incubator for anaerobic culture for 48 hours; select the single colony with the largest colony radius on the plate, and subculture until it contains 200mg / L ethylene oxide Alkane tolerance acclimation medium, 37°C constant temperature anaerobic culture for 48 hours; select the single colony with the largest colony radius on the plate, subculture to 500 mg / L ethylene oxide tolerance acclimation medium, 37°C constant temperature anaerobic culture for 48 hours; Select the single colony with the largest colony radius on the plate, subculture to the 800...
Embodiment 3
[0095] Identification of Ethylene Oxide Degradation Effect of EO-08 Strains
[0096] Microbial culture and activation: Take out EO-08 strains and EO-08 original strains from the -80°C refrigerator, take 10 μL and inoculate in 100 mL Sabouraud liquid medium, and culture anaerobically at 37°C, 200 rpm for 48 hours. The number of bacteria in the culture solution is 10 10 ~10 12 cfu / mL.
[0097] Sabouraud liquid medium: Glucose 40g, casein trypsin digest, animal tissue gastric enzyme digest equal mixture 10g, pH 5.4-5.8, distilled water 1000mL, divided into 500mL Erlenmeyer flasks, each Erlenmeyer flask 250mL . Sterilize at 121°C for 20 minutes and cool to room temperature.
[0098] Sabouraud's liquid induction medium: 10g of peptone, 12g of glucose, 1000mL of distilled water, aliquoted into 400mL, sterilized at 121°C for 20min, cooled to room temperature and stored. Before use, add 160 mg of ethylene oxide with a sealed syringe to make a liquid medium containing a carbon sou...
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