[Beef]

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

I am trying to compare the ethanol production (and the rate of ethanol production) between seven types of yeast strains by fermenting the YPD media (1% yeast extract, 2% peptone and 2% d-glucose mixture), 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. All strains yield around 8-9 mg/mL of ethanol, 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?

I have posted the same question at Stack Exchange: http://chemistry.stackexchange.com/questions/29009/yeast-fermentation-with-ypd-apple-juice

[Arkcon]

I'd like to know what your expected ranges are for content.  Can you build some standard concentrations so you can determine the accuracy and resolution of your method and instrument setup?

[Beef]

My calibration curve was constructed from external ethanol standards: 1, 2, 3, 5, 7, 10, 15, 20 mg/mL so that the determination of ethanol concentration is good for 1-20 mg/mL. The R^2 value was 1.00000

The link, http://link.springer.com/article/10.1007%2Fs00217-002-0505-2, suggests that the glucose concentration of apple juice is about 9.3-32 mg/ml, so I suppose I can make my mixture 5 mL apple juice + 45 mL YPD so that ethanol concentration stays within the range.

Using 50mL YPD (2% w/v glucose) gave me around 8-9 mg/mL of EtOH

[Furanone]

What detector are you using?  Likely Differential Refractive Index, but others include ELSD & UV (<210nm, likely not as weak absorbance)?

Also, is this the column you are using (Bio-Rad Aminex Fermentation Monitoring Column)?

http://www.perkinelmer.com/content/relatedmaterials/productnotes/sep_hplcfermentationbio-radcolumn.pdf

I use this column and method (0.001 M H2SO4 as solvent) connected with multiple detectors in tandem -- UV, DRI, then ELSD. For more complex food samples, the refractive index detector can become convoluted since it detects practically everything. The ELSD detects very sensitively the sugars but not the organic acids or ethanol (too volatile), while the UV preferentially detects the organic acids with low sensitivity for most sugars. This helps in deconvoluting the data and in identification of unknown peaks.

Luckily for you, if using a DRI detector, the ethanol peak comes out late so there will be little interferences near it, but you may see additional peaks from adding the apple juice elute earlier such as sugars other than glucose (fructose, sucrose) and malic acid. Otherwise, I cannot see any reason the same sample prep method would not work for the apple juice. If you still see turbidity in solution after centrifugation, I would suggest filtering through 0.45 um syringe filter before injecting.