In 1906 Harden and Young, in a series of classic studies on the fermentation of glucose to ethanol and CO2 by extracts of brewer's yeast, made the following observations.

(A) Inorganic phosphate was essential to fermentation; when the supply of phosphate was exhausted, fermentation ceased before all the glucose was used.
(B) During fermentation under these conditions, ethanol, CO2, and a sugar phosphate accumulated.
(C) When arsenate was substituted for phosphate, no sugar phosphate acumulated, but the fermentation proceeded until all the glucose was converted to ethanol and CO2.

The fermentation of ethanol in yeast has the following overall equation Glucose 2ADP 2Pi88n2 ethanol 2CO22ATP 2H2O which makes it clear that phosphate is required for the continued operation of glycolysis and formation of ethanol . In extracts to which glucose is added, fermentation proceeds until ADP and Pi(present in the extracts) are exhausted.(a)Phosphate is required in the glyceraldehyde 3-phosphate dehydrogenase reaction, and glycolysis will stop at this step when Piis exhausted. Because glucose remains, it will be phosphorylated by ATP, but Piwill not be released.(b)Fermentation in yeast cells produces ethanol and CO2rather than lactate . Without these reactions (in the absence of oxygen), NADH would accumu-late and no new NADwould be available for further glycolysis ). Thehexose bisphosphate that accumulates is fructose 1,6-bisphosphate; in terms of energet-ics, this intermediate lies at a “low point” or valley in the pathway, between the energy-input reactions that precede it and the energy-payoff reactions that follow.(c)Arsenate replaces Piin the glyceraldehyde 3-phosphate dehydrogenase reaction to yieldan acyl arsenate, which spontaneously hydrolyzes. This prevents formation of fructose1,6-bisphosphate and ATP but allows formation of 3-phosphoglycerate, which continuesthrough the pathway.