Using Robots to Make Glycerol

Of course, by 'robots' we are speaking of using microörganisms - specifically bakers/brewers yeast (aka: Saccharomycese cerevisiae). Normally yeast ferment sugar to CO2 and ethanol, but if we add a chemical, NaHSO₃, that blocks one intermediary step (by removing acetaldehyde), the cell is forced to recycle its NADH via another route - reducing glyceraldehyde phosphate, GAP, eventually to glycerol. (Print out the structures of the chemicals discussed in this unit.)

The first thing we must do in order to know how much starting compound should be used is to write out the equation of the expected reaction. And we must also realize that yeast prefer sugar concentrations to be no more than about 12%. If the culture mixture is too hypertonic the yeast won't grow.

Sucrose + 2 NaHSO₃ → 2 CO₂ + 2 acetaldehyde:HSO₃Na + 2 glycerol

This means that you can expect to convert half of the weight of the input sucrose to glycerol. Another thing you will notice that there is no oxygen gas in this reaction. Pasteur defined "fermentation" as growing in the absence of O₂. So your fermentation set-up must exclude O₂!

Now assemble your fermentation vessel as depicted next to the title, above.

Find an appropriate 500 mL conical or round flask (with flattened bottom)
Find a flexible, unstressed, 1-hole rubber stopper that will fit the flask snuggly.
Find a 6-inch length of glass tubing that will snuggly fit the hole in the stopper. (Previously you have used copper tubing through stoppers, but you shouldn't this time because the copper is toxic such that any drip-back into the culture will kill it. Without your yeast robot chemists, you will not produce glycerol.)
Learn glass-blowers' techniques. Watch the instructor demonstrate to you how to do it.
- Horizontally hold the tubing horizontally with both hands on each end and put the center in the flame with constant rolling. (Your fingers will not get burned as glass is a very poor conductor of heat.) Constantly roll the tubing in your fingers so that it is heated all around the circumference. Soon the flame will turn orange as sodium atoms from the glass begin evaporating. Keep rolling the tubing. It will become flexible at that point of heating. Don't overheat so that it droops. Remove the tubing from the flame, quickly bend the tubing to an angle slightly less than 90°, and lay the hot tubing on the desk so that the angle will be flat. In about 5 seconds the glass will harden. (But it is still very HOT!) And will take as much as 10 minutes to be safe to touch with bare fingers. Do NOT cool in water: the glass will shatter!)
Find some lanoline or KY-jelly, and put a very little of it around one end of the "L". You need a non-toxic water-soluble lubricant that is non-toxic for yeast, and vasoline, mineral oil and stop-cock grease are not water soluble.
Now comes the MOST DANGEROUS part of the SEMESTER. Read this complete step before attempting the insertion of the tubing into the stopper. Many fingers and hands get pierced and prepared for the plastic surgeon in the attempt IF not done properly. Do NOT hold the bent tubing by any portion except the straight portion with the slippery end. Slowly twist the top of the stopper onto the tubing. If it is more than moderately difficult, withdraw the glass and add more lubricant. Work the stopper onto the tubing so that eventually it protrudes about 3 mm out of the bottom of the stopper. The instructor will demonstrate added safety features. Do pay attention so that you won't be spurting blood all over the lab. The dragons in the biology department's basement pen go berserk at the smell of blood.
In similar fashion attach a length of rubber tubing to the other end of the "L". And finally attach a 6-inch length of tubing to the other end of the tubing. This will be later inserted into a small flask of water to complete what is called the water-trap that excludes atmospheric O₂. The value of this second straight bit of tubing is that its weight will keep it from jumping out of the water-trap.
Fill your fermentation flask with TAP water (yeast need minerals just like you do!) so that there is about 8 cm of free space between the meniscus and the stopper.
Pour the flask's water into a graduated cylinder, note the volume, and then pour the contents into a beaker and add the right amounts of sucrose and NaHSO₃. How much is that? You need twice as many moles of bisulfite as you will have added in moles sucrose to make a 10% solution (i.e.: 10% means 10 grams of sucrose made up to 100 mL; or multiples of that).
Run a glycerol test on a mL of this to make sure that there are no cross-reacting materials.
Determine the initial optical activity of your mix as a test of your sucrose concentration.
Make the solution to 0.1% with 95% ethanol, which will act as an antifoaming agent.
Stir in about 1/4 pkg of yeast.
Add the mix to the fermentation vessel.
Assemble your apparatus in the next lab so that it is out-of-the-way of other classes who use the lab. Be sure to put water in the air-trap flask.

Question

As you put together your apparatus, you took no precautions to prevent extraneous bacteria from invading your culture. Since prokaryotic bacteria can usually grow much faster than eukaryotic yeasts, how can be sure that the reaction will go as planned? Ans.: while fungi such as yeast can grow in hypertonic solutions, most prokaryots cannot. Hence, the salting of meat and fish, and the making of highly sweet jellies and jams.