Team:DTU-Denmark/Methods/Bioreactor

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== Procedure ==
== Procedure ==
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# Put carefully one long needle in the vessel for injecting N<sub>2</sub>
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In this anaerobic experiment we add nitrite in our cultures with the Nir transformed cells, in order to test how nitrite is converted to nitrous oxide.
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# Put one short needle in the vessel for removing the containing oxygen
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# Open the pressure valve of the Nitrogen gas container
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# Make an overnight culture with the Nir transformed cells and then pour 100 ml in each of the 3 bioreactor vessels with 800 ml DM modified minimal medium. 
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# Turn round (anticlockwise) the nitrogen valve to open it until there is a flow of gas.
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# Set the bioreactor at 37<sup>o</sup>C.
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# Sparge with N<sub>2</sub> by connecting the N<sub>2</sub> gas tube with the long needle.  
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# Add 0.8 ml of 0.916 M ammonium chloride solution in each of the three vessels.
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# After 3/ 5 min remove first the small needle and after 2 seconds the long needle.
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# Add 8 ml of 50mM of nitrite stock solution to each of the three vessels.
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# Turn off the nitrogen valve.  
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# Take 100 mL as the first sample t=0 for both colorimetric analysis and nitrous oxide measurement, from each of the 3 vessels.
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# Turn off the pressure valve.
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# After 10 hours take another 100 ml of sample to measure nitrous oxide and make the colorimetric analysis, from each of the 3 vessels.
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# After taking the samples pour them in 200 ml sealed vessels with syringes in order to keep the conditions anaerobic.
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#To finish gathering data, make the colorimetric measurements for nitrite and ammonium for each of the samples collected by using the protocols [[Team:DTU-Denmark/Methods/Nitrite colorimetric measurements|Nitrite colorimetric measurements]] and [[Team:DTU-Denmark/Methods/Ammonium colorimetric measurements|Ammonium colorimetric measurements]], respectively.
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Latest revision as of 13:47, 4 October 2013

Bioreactor

Procedure

In this anaerobic experiment we add nitrite in our cultures with the Nir transformed cells, in order to test how nitrite is converted to nitrous oxide.

  1. Make an overnight culture with the Nir transformed cells and then pour 100 ml in each of the 3 bioreactor vessels with 800 ml DM modified minimal medium.
  2. Set the bioreactor at 37oC.
  3. Add 0.8 ml of 0.916 M ammonium chloride solution in each of the three vessels.
  4. Add 8 ml of 50mM of nitrite stock solution to each of the three vessels.
  5. Take 100 mL as the first sample t=0 for both colorimetric analysis and nitrous oxide measurement, from each of the 3 vessels.
  6. After 10 hours take another 100 ml of sample to measure nitrous oxide and make the colorimetric analysis, from each of the 3 vessels.
  7. After taking the samples pour them in 200 ml sealed vessels with syringes in order to keep the conditions anaerobic.
  8. To finish gathering data, make the colorimetric measurements for nitrite and ammonium for each of the samples collected by using the protocols Nitrite colorimetric measurements and Ammonium colorimetric measurements, respectively.


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