The Fermentation Process

From Mystical Spirit to Cellular Biochemistry — ATP, Glycolysis, and Why Yeast Ignores the Krebs Cycle

Active fermentation — yeast at work converting sugar into alcohol and CO₂.

For most of human history, the transformation of a sweet liquid into alcohol was genuinely inexplicable. People knew it happened — they relied on it, celebrated it, built economies and religions around it — but the mechanism was entirely invisible to them. The answer, when it finally came, turned out to be one of the most consequential discoveries in the history of biology.

We've established that mashing produces a complex, sugar-rich liquid ready for fermentation. Now comes the biological heart of whiskey production — where living organisms take center stage and the language shifts from chemistry to biochemistry. Fermentation is one of the most well-understood metabolic processes in biology, and understanding it in detail is essential to understanding where whiskey flavor actually comes from.

Louis Pasteur and the End of Spontaneous Generation

Louis Pasteur's dismantling of this theory, beginning in 1857, was one of the most important contributions to both biology and practical science in the 19th century. Working on an industrial problem — the occasional souring of beet sugar fermentations in Lille — Pasteur examined samples from both successful alcoholic fermentations and failed ones under the microscope and found something unambiguous: different microorganisms were responsible for different fermentation outcomes. The alcoholic fermentations contained yeast; the soured fermentations contained bacteria.

His conclusion — that fermentation was a biological process driven by the specific metabolic activity of specific living organisms — overturned the dominant chemical theory and laid the foundation for germ theory, pasteurization, modern microbiology, and ultimately modern medicine. The "spirit" in fermented beverages had been identified: it was Saccharomyces cerevisiae, a single-celled fungus whose cellular biochemistry is now among the best-understood biological systems in existence.

Glycolysis: The Universal Energy-Extraction Pathway

To understand how yeast produces ethanol, we must first understand the pathway by which it extracts energy from sugar — a process called glycolysis. Glycolysis is not unique to yeast; it is one of the most ancient and universally conserved metabolic pathways in biology, present in virtually every living cell from bacteria to human neurons. It is the first stage of cellular respiration, and in fermentation, it is also essentially the last.

Glycolysis is a ten-step enzymatic pathway in the yeast cell cytoplasm that converts one molecule of glucose (C₆H₁₂O₆) into two molecules of pyruvate (C₃H₄O₃), generating a net gain of two molecules of ATP (adenosine triphosphate — the cell's fundamental energy currency) and two molecules of NADH (a reduced electron carrier).

The Krebs Cycle: Why Yeast Bypasses 34 ATP to Make Your Whiskey

This is where the biochemistry of fermentation diverges from what most people learn in general biology — and where an understanding of cellular energetics explains why fermentation produces alcohol instead of simply growing more yeast.

In aerobic conditions (where oxygen is present), pyruvate — the end product of glycolysis — is transported into the mitochondria of the yeast cell and converted to acetyl-CoA by the enzyme pyruvate dehydrogenase. Acetyl-CoA then enters the Krebs cycle (also called the citric acid cycle or TCA cycle), a circular series of eight enzymatic reactions that completely oxidize the acetyl group to two molecules of CO₂ while generating NADH, FADH₂, and GTP. The NADH and FADH₂ then donate their electrons to the electron transport chain, which drives ATP synthesis — generating approximately 34 additional ATP molecules per glucose molecule beyond what glycolysis produces.

Total aerobic respiration: ~36–38 ATP per glucose. This is enormously more efficient than fermentation's 2 ATP per glucose, which is why yeast grows explosively when oxygen is available — it can extract 18–19× more energy from each sugar molecule.

Why Ethanol Happens: The NAD⁺ Regeneration Imperative

Here is the central biochemical logic of alcoholic fermentation, stated plainly: glycolysis requires NAD⁺ (the oxidized form of the electron carrier nicotinamide adenine dinucleotide) to function. Each turn of glycolysis converts two NAD⁺ molecules into two NADH molecules. If those NADH molecules are not re-oxidized back to NAD⁺, glycolysis halts — the cell runs out of the electron acceptors it needs to keep running. In aerobic conditions, the electron transport chain accepts electrons from NADH and regenerates NAD⁺ (while also producing the bulk of the cell's ATP). In anaerobic conditions, where the electron transport chain cannot function (no oxygen to serve as the final electron acceptor), the cell needs an alternative way to regenerate NAD⁺.

Yeast's solution is elegant: convert acetaldehyde (itself produced from pyruvate by pyruvate decarboxylase) into ethanol, using the NADH as the electron donor. The alcohol dehydrogenase reaction — acetaldehyde + NADH + H⁺ → ethanol + NAD⁺ — regenerates the NAD⁺ needed to keep glycolysis running. The production of ethanol is thus not the goal of yeast fermentation from the cell's perspective; it is a metabolic necessity for maintaining glycolytic NAD⁺ supply under anaerobic conditions. The ethanol is effectively a waste product of yeast's effort to survive without oxygen. Which is convenient for us.

Saccharomyces cerevisiae cells budding — the biological engine of fermentation.

Congeners: The Flavor Library Built During Fermentation

The equation above (glucose → ethanol + CO₂ + ATP) is accurate but incomplete. Alongside ethanol production, yeast generates a vast library of secondary metabolic compounds called congeners — flavor-active molecules present in the parts per million or parts per billion range that nonetheless define the character of the new make spirit.

Yeast Selection: Distiller's Yeast vs. Brewer's Yeast

Every yeast strain carries its own biochemical fingerprint — its preferred temperature range, fermentation rate, alcohol tolerance, and characteristic congener profile. The yeast used in a whiskey fermentation is one of the most powerful flavor variables in the entire process — and one of the least discussed in popular whiskey writing.

At Seven Stills, our philosophy is rooted in the brewing side of this equation — we start by making beer and then distilling it, which means the yeast decisions we make directly shape the flavor compounds that carry through into the still and into the finished spirit. An expressive ale yeast producing isoamyl acetate during a long, cool fermentation will contribute detectable fruity character to the new make spirit — and, ultimately, to the aged whiskey. It's a production philosophy that treats fermentation as a flavor-building tool, not just an alcohol-generating mechanism.

Why Whiskey Has No Hops — And Why That's Federal Law

In the beer world, hops are fundamental: they provide bitterness, aroma, and most importantly, act as a natural antiseptic preservative. The alpha acids in hops (humulone, cohumulone, adhumulone) are bacteriostatic — they inhibit the gram-positive bacteria (primarily Lactobacillus and Pediococcus) that would otherwise compete with yeast and sour the beer before it reaches the consumer.

In whiskey production, hops are not used — and this is not merely stylistic. Under US TTB Standards of Identity, the fermentable materials and flavoring agents in whiskey must be grain-derived. Botanical additions like hops fall outside this definition and are explicitly prohibited. The practical rationale is straightforward: the beer/wash is going to be distilled, which destroys all microorganisms and concentrates the alcohol — rendering bacterial contamination of the wash a non-issue for the finished product. The sour mash process used in bourbon production even deliberately employs controlled lactic acid fermentation (from backset — spent wash from a previous distillation) to acidify the mash and improve fermentation conditions, without any negative impact on the finished spirit.