Fermentation

Eukaryotic aerobic cellular respiration is the process of producing ATP by capturing energy that is released when food molecules are oxidized, with mitochondria playing a central role and oxygen acting as the final oxidizer. This is one of the core topics of Bio 6B. We'll go into this in detail, with explanations from the textbook, lecture videos, and other sources. I've created several pages related to cellular respiration:

The word fermentation can refer to a wide range of catabolic biochemical processes in both prokaryotes and eukaryotes; on this page, I'll describe the process in which eukaryotic cells can generate ATP using glycolysis without oxidative phosphorylation (OXPHOS).

Objectives

By the time you complete this unit, you should be able to explain:

  • How fermentation pathways keep glycolysis going without oxidative phosphorylation.
  • How fermentation pathways relate to cellular respiration in yeast and human cells.

Fermentation keeps glycolysis going without OXPHOS

As you’ve seen, aerobic cellular respiration with oxidative phosphorylation is an efficient way to generate ATP. It works because of the huge difference in redox potential between glucose (the electron source) and H2O (where the electrons end up). However, eukaryotic cells don’t always generate all their ATP via OXPHOS. In situations where the rate of OXPHOS is limited, fermentation pathways can supplement or replace OXPHOS, generating ATP by glycolysis alone without the citric acid cycle, the electron transport chain, or ATP synthase.

Glycolysis

You've already seen glycolysis as a part of aerobic cellular respiration.

You've already seen glycolysis as a part of aerobic cellular respiration.

Summary of glycolysis inputs and outputs

The most important outputs of glycolysis in cellular respiration are pyruvate, which feeds the citric acid cycle (and thus the electron transport chain) and NADH, which feeds electrons into the ETC. Some ATP is produced in glycolysis, but it's a small amount compared to the amount produced through OXPHOS. In cellular respiration, the NADH and pyruvate contribute much more to the cell's ATP production than does the small amount of ATP produced directly in glycolysis.

However, under certain circumstances, OXPHOS can't produce ATP rapidly enough to meet cells' needs. The rate of OXPHOS can be limited by low oxygen concentration, by the number of mitochondria, or by other factors such as the rate of transport of substances across mitochondrial membranes. Under such circumstances, many cells can keep glycolysis going without OXPHOS, by way of fermentation pathways.

As with any biochemical pathway, the rate of glycolysis depends on the concentrations of reactants and products. If you put some glucose, ADP, phosphate, NAD+ into a test tube, along with all the necessary enzymes, you could get glycolysis going in the test tube. (The term for this is in vitro, meaning "in glass," as opposed to in vivo, meaning inside a living cell.) However, your test tube glycolysis would be short-lived, even if you add plenty of glucose. Several things would happen that would start pushing the reaction back toward the left, until it reached equilibrium:

  • The concentration of pyruvate would increase, slowing the forward reaction.
  • The NAD+ would be reduced to NADH; lack of NAD+ would slow the forward reaction.
  • The ADP would be phosphorylated to ATP; lack of ADP and high [ATP] would again slow the forward reaction.

The only way to keep glycolysis going would be to continually supply more reactants (glucose, NAD+, and ADP) and remove the products (pyruvate, NADH, and ATP). In cellular respiration, OXPHOS oxidizes the NADH and pyruvate from glycolysis, and cellular work converts the ATP back to ADP.  Fermentation pathways achieve the same result by other means.

Ethanol fermentation

Yeasts are eukaryotes like us, and they can use oxygen and perform OXPHOS. However, they can also live without oxygen. In other words, they are facultative anaerobes. For example, in winemaking, yeast cells are trapped in a bottle with plenty of sugar but limited oxygen. They begin catabolizing the sugars using OXPHOS, but as O2 runs low, they begin using the ethanol fermentation pathway:

Ethanol fermentation pathway. Pyruvate reduced to ethanol by NADH.

The ethanol fermentation pathway includes glycolysis and some subsequent steps. This pathway achieves two essential things that keep glycolysis going when the rate of OX-PHOS is limited:

  • Removes pyruvate by converting it to ethanol, an uncharged waste product that can freely diffuse out of the cell. (Ethanol is considered to be a waste product in fermentation when it is produced much faster than it can be used. However, in the human body and in other organisms, ethanol can be used both as a source of energy and as a substrate for synthesizing larger molecules. These pathways depend on the cell's redox state, which in turn is strongly dependent on oxygen availability. For more on ethanol metabolism and human health, see this article by Zakhari.)
  • Oxidizes NADH to NAD+. NADH is a valuable electron carrier, but without O2, it can't be used in OXPHOS. The NAD+ is needed to keep glycolysis going. 

In this pathway, each pyruvate is converted to a 2-carbon intermediate (called acetaldehyde). Note that one of pyruvate's carbons, shown in red, is already fully oxidized. It's a carboxyl group, or COO-. This carbon is chopped off and released as CO2, which can freely diffuse out of the cell.

Lactate fermentation

Human cells don't normally perform ethanol fermentation, but they can perform a similar pathway, called lactate fermentation.

Lactate fermentation pathway. Pyruvate gets reduced by NADH to form lactate.

The lactate fermentation pathway also includes glycolysis and some subsequent steps. Like the ethanol fermentation pathway, lactate fermentation achieves two essential things that keep glycolysis going when the rate of OXPHOS is limited: it removes pyruvate and regenerates NAD+. One key difference is the waste product; unlike ethanol, lactate is charged and can't diffuse out of the cell without a transport protein.

No CO2 is produced in lactate fermentation.

Fermentation in a facultative anaerobe

As I described above, baker’s yeast cells can switch from aerobic cellular respiration to fermentation when the O2 level runs low. Because yeast cells can survive with or without O2, they’re called facultative anaerobes. But what actually controls the switch from aerobic to anaerobic pathways? Glycolysis is the same in either case, but the fate of pyruvate is different in fermentation compared to OXPHOS. In OXPHOS, pyruvate would enter the mitochondrion, get oxidized by pyruvate dehydrogenase, and the remaining carbons (the acetyl group) would be passed to the citric acid cycle. None of these steps require oxygen, so why would they stop when O2 runs out?

The only step in OXPHOS that directly uses O2 is at the very end of the electron transport chain, when O2 pulls electrons away from Complex IV, forming H2O. Cells don't need O2 for anything else. Without oxygen, electrons can't travel down the ETC. NADH can't deliver its electrons to the ETC, so NADH accumulates and NAD+ level runs low. The citric acid cycle can't work without NAD+, so it slows down. Ultimately, the accumulation of pyruvate and NADH pushes the cell toward fermentation.

Fermentation in humans

You’re not a facultative anaerobe. Without oxygen, you can’t survive (in other words, you’re an obligate aerobe). However, some human cells do perform a substantial amount of lactate fermentation. For example, erythrocytes (red blood cells) don’t have mitochondria, so they can’t perform OXPHOS and must rely on fermentation. Muscles, which are abundantly supplied with mitochondria and specialized for high rates of respiration, also perform lactate fermentation alongside OXPHOS.

The traditional diagram in Campbell and other biology textbooks, implying that muscle cells switch from OXPHOS to fermentation when oxygen runs out, isn’t correct. In fact, working muscle cells perform some fermentation all the time, even oxygen is abundant.  

Lactate misconceptions

The role of lactate fermentation in muscle cells during exercise was completely misunderstood for many years, and is still controversial. In the past, a simple (but erroneous) story about muscle fermentation was widely accepted:

  • Muscle cells switch from respiration to lactate fermentation when oxygen runs out.
  • Lactic acid is a toxic byproduct of fermentation, and it accumulates in muscles that are starved for oxygen during exercise. Eventually it causes fatigue and “the burn” feeling during intense exercise.
  • Trained athletes produce less lactate at a given rate of exercise, because they supply more O2 to their muscles and perform OXPHOS. 

All the ideas above are wrong. More recent research has given us a truer, more complex and interesting picture:

  • Working muscle cells perform lactate fermentation all the time, even oxygen is abundant. Muscles don’t switch to fermentation when oxygen runs out. In fact, oxygen partial pressure in muscle cells may increase at the beginning of exercise, due to increased blood flow. Meanwhile, lactate production increases with exercise.
  • Fermentation produces lactate, not lactic acid, in its last step. Look closely at the conversion of pyruvate to lactate. Lactate has two more hydrogens than pyruvate. One hydrogen comes from NADH (one proton and two electrons), and one proton comes from the surrounding solution. This step actually reduces acidity. However, other steps of catabolism, along with the accumulation of CO2, cause muscles to become more acidic during intense exercise.
  • Lactate isn’t a waste product; it’s a valuable fuel. Remember, lactate is reduced compared to pyruvate, so it has even more redox potential. Some lactate is shuttled into mitochondria, where it can be converted back to pyruvate, producing NADH in the process. (This reaction, catalyzed by lactate dehydrogenase, is the reverse of the last step of fermentation.) The pyruvate can then be oxidized and fed into the citric acid cycle (even during intense exercise, muscles normally have enough O2 to oxidize it). Some lactate is also released from muscles into the blood, and can be absorbed and used as an energy source for other cells.
  • Trained athletes such as runners are likely to have a high rate of lactate turnover during exercise: they rapidly generate lactate, then quickly metabolize it. They don’t avoid lactate, they make better use of it.

If cells don’t switch to lactate fermentation due to running out of oxygen, what controls the relative rates of fermentation and respiration? As it turns out, this question has become harder to answer as researchers have learned more, and the experts are still arguing over how it works. It's an important question, especially since many cancer cells preferentially use fermentation over OXPHOS (this tendency is called the Warburg effect, and it's one of the metabolic hallmarks of cancer cells). Meanwhile, we’ve learned that lactate isn’t a waste product, and isn’t only a fuel molecule; it plays a fundamental role in helping cells balance their competing catabolic pathways. 

Some people may be disappointed to find out that the clear and simple textbook story isn’t quite true, and I’m afraid that you’ll find out that this is often the case. The bad news is that you’ll never fully understand anything. The good news is that most things in biology become more interesting and more complex as your knowledge grows. There will always be something to surprise you.

Review

Terms & concepts

  • Ethanol
  • Facultative anaerobe
  • Fermentation
  • Lactate
  • NAD+/NADH: its role in glycolysis and fermentation
  • Pyruvate

Review questions

  1. Fermentation pathways include glycolysis plus some additional steps. Why are those steps necessary to keep glycolysis going when OX-PHOS is not happening? Why are those steps not necessary to keep glycolysis going when OX-PHOS is happening?
  2. Compare and contrast ethanol fermentation and lactate fermentation.
  3. In the fermentation steps that occur after glycolysis, what gets reduced and what gets oxidized?
  4. Compare and contrast the fate of pyruvate in OXPHOS and fermentation.
  5. What is the direct mechanism that causes yeast cells to switch to fermentation when oxygen runs out?
  6. Take a close look at the lactate fermentation pathway. Can you see how the molecular structures show that lactate is more reduced than pyruvate?
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