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I am working on a project for my General Chemistry class.

I am trying to compare the EtOH production (and the rate of EtOH production) between seven types of yeast strains by fermenting the YPD media, apple juice, or both, if necessary. I can describe the difference between the yeast strains if asked, but I don't think it is necessary right now.

Right now, I have only tested plain YPD media, which is 1% yeast extract, 2% peptone and 2% d-glucose mixture. All strains yield around 8-9 mg/mL of EtOH, and the fermentation stops after ~24 hours. My next step is to test YPD + apple juice media (because the project asks for "fun" element), and I was wondering if I should use the same method for analyzing this YPD + apple juice fermentation.

The method I used for YPD media is:

  1. Grow each strain (total of 7 strains) in ~5 mL YPD media at 30 degrees shaker ~250 rpm overnight.
  2. Subculture each strain into new fresh YPD media so that the new media is 50 mL and has 0.5 OD at 600 nm. Use the "special" flask designed for anaerobic growth. This flask is a 250 mL flask with a special cap. The cap is plastic at the outer radius and rubber at the inside. This allows me to grow yeast anaerobically and poke a needled syringe and retrieve small amount of sample without letting the air in.
  3. Flush all flasks with nitrogen gas for four minutes.
  4. After 6 hour, 12 hour, and 24 hour growths, use syringe to retrieve ~1.5 mL of sample.
  5. Measure OD600 of each sample. (This is used to get the number of yeast cells in the media)
  6. Centrifuge samples for 8 minutes in 14,000 rpm.
  7. Pipette 1 mL of the supernatant into a GC vial.
  8. Use HPLC to obtain concentration of ethanol. I am using refractive index detector, ion exclusion column(?) (I forgot the type of column used at this moment), and sulfuric acid as mobile phase.

For YPD + apple juice fermentation, I was thinking of doing the same procedure with 50 mL YPD + 50 mL apple juice in each "special flask". Will this work? Will this be good for the same HPLC setting?

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Thank you for an especially clear and detailed question!

For YPD + apple juice fermentation, I was thinking of doing the same procedure with 50 mL YPD + 50 mL apple juice in each "special flask". Will this work?

I think this would work but it seems like a better comparison to your earlier experiments to keep using 50 mL of total medium, i.e., mix 25 mL of YPD with 25 mL of apple juice. That way the volume of the fermentation is the same in each experiment.

Will this be good for the same HPLC setting?

Probably, although it depends on the components of the apple juice you use. Have you injected just apple juice (unfermented) into your HPLC? Have you verified that this negative control injection contains no ethanol or other interfering peaks in the region of interest? If not, those are things you should do to make sure the HPLC method you have developed for YPD medium will still work in apple juice-based media.

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