Bis2A_Singer_Fermentation

Fermentation and regeneration of NAD+

Section summary

This section discusses the process of fermentation. Due to the heavy emphasis in this course on central carbon metabolism, the discussion of fermentation understandably focuses on the fermentation of pyruvate. Nevertheless, some of the core principles that we cover in this section apply equally well to the fermentation of many other small molecules.

The "purpose" of fermentation

The oxidation of a variety of small organic compounds is a process that is utilized by many organisms to garner energy for cellular maintenance and growth. The oxidation of glucose via glycolysis is one such pathway. Several key steps in the oxidation of glucose to pyruvate involve the reduction of the electron/energy shuttle NAD⁺ to NADH. You were already asked to figure out what options the cell might reasonably have to reoxidize the NADH to NAD⁺ in order to avoid consuming the available pools of NAD⁺ and to thus avoid stopping glycolysis. Put differently, during glycolysis, cells can generate large amounts of NADH and slowly exhaust their supplies of NAD⁺. If glycolysis is to continue, the cell must find a way to regenerate NAD⁺, either by synthesis or by some form of recycling.

In the absence of any other process—that is, if we consider glycolysis alone—it is not immediately obvious what the cell might do. One choice is to try putting the electrons that were once stripped off of the glucose derivatives right back onto the downstream product, pyruvate, or one of its derivatives. We can generalize the process by describing it as the returning of electrons to the molecule that they were once removed, usually to restore pools of an oxidizing agent. This, in short, is fermentation. As we will discuss in a different section, the process of respiration can also regenerate the pools of NAD⁺ from NADH. Cells lacking respiratory chains or in conditions where using the respiratory chain is unfavorable may choose fermentation as an alternative mechanism for garnering energy from small molecules.

An example: lactic acid fermentation

An everyday example of a fermentation reaction is the reduction of pyruvate to lactate by the lactic acid fermentation reaction. This reaction should be familiar to you: it occurs in our muscles when we exert ourselves during exercise. When we exert ourselves, our muscles require large amounts of ATP to perform the work we are demanding of them. As the ATP is consumed, the muscle cells are unable to keep up with the demand for respiration, O₂ becomes limiting, and NADH accumulates. Cells need to get rid of the excess and regenerate NAD⁺, so pyruvate serves as an electron acceptor, generating lactate and oxidizing NADH to NAD⁺. Many bacteria use this pathway as a way to complete the NADH/NAD⁺ cycle. You may be familiar with this process from products like sauerkraut and yogurt. The chemical reaction of lactic acid fermentation is the following:

Pyruvate + NADH ↔ lactic acid + NAD⁺

Figure 1. Lactic acid fermentation converts pyruvate (a slightly oxidized carbon compound) to lactic acid. In the process, NADH is oxidized to form NAD⁺. Attribution: Marc T. Facciotti (original work)

Energy story for the fermentation of pyruvate to lactate

An example (if a bit lengthy) energy story for lactic acid fermentation is the following:

The reactants are pyruvate, NADH, and a proton. The products are lactate and NAD⁺. The process of fermentation results in the reduction of pyruvate to form lactic acid and the oxidation of NADH to form NAD⁺. Electrons from NADH and a proton are used to reduce pyruvate into lactate. If we examine a table of standard reduction potential, we see under standard conditions that a transfer of electrons from NADH to pyruvate to form lactate is exergonic and thus thermodynamically spontaneous. The reduction and oxidation steps of the reaction are coupled and catalyzed by the enzyme lactate dehydrogenase.

A second example: alcohol fermentation

Another familiar fermentation process is alcohol fermentation, which produces ethanol, an alcohol. The alcohol fermentation reaction is the following:

Figure 2. Ethanol fermentation is a two-step process. Pyruvate (pyruvic acid) is first converted into carbon dioxide and acetaldehyde. The second step converts acetaldehyde to ethanol and oxidizes NADH to NAD⁺. Attribution: Marc T. Facciotti (original work)

In the first reaction, a carboxyl group is removed from pyruvic acid, releasing carbon dioxide as a gas (some of you may be familiar with this as a key component of various beverages). The second reaction removes electrons from NADH, forming NAD⁺ and producing ethanol (another familiar compound—usually in the same beverage) from the acetaldehyde, which accepts the electrons.

Fermentation pathways are numerous

While the lactic acid fermentation and alcohol fermentation pathways described above are examples, there are many more reactions (too numerous to go over) that Nature has evolved to complete the NADH/NAD⁺ cycle. It is important that you understand the general concepts behind these reactions. In general, cells try to maintain a balance or constant ratio between NADH and NAD⁺; when this ratio becomes unbalanced, the cell compensates by modulating other reactions to compensate. The only requirement for a fermentation reaction is that it uses a small organic compound as an electron acceptor for NADH and regenerates NAD⁺. Other familiar fermentation reactions include ethanol fermentation (as in beer and bread), propionic fermentation (it's what makes the holes in Swiss cheese), and malolactic fermentation (it's what gives Chardonnay its more mellow flavor—the more conversion of malate to lactate, the softer the wine). In Figure 3, you can see a large variety of fermentation reactions that various bacteria use to reoxidize NADH to NAD⁺. All of these reactions start with pyruvate or a derivative of pyruvate metabolism, such as oxaloacetate or formate. Pyruvate is produced from the oxidation of sugars (glucose or ribose) or other small, reduced organic molecules. It should also be noted that other compounds can be used as fermentation substrates besides pyruvate and its derivatives. These include methane fermentation, sulfide fermentation, or the fermentation of nitrogenous compounds such as amino acids. You are not expected to memorize all of these pathways. You are, however, expected to recognize a pathway that returns electrons to products of the compounds that were originally oxidized to recycle the NAD⁺/NADH pool and to associate that process with fermentation.

Figure 3. This figure shows various fermentation pathways using pyruvate as the initial substrate. In the figure, pyruvate is reduced to a variety of products via different and sometimes multistep (dashed arrows represent possible multistep processes) reactions. All details are deliberately not shown. The key point is to appreciate that fermentation is a broad term not solely associated with the conversion of pyruvate to lactic acid or ethanol. Source: Marc T. Facciotti (original work)

A note on the link between substrate-level phosphorylation and fermentation

Fermentation occurs in the absence of molecular oxygen (O₂). It is an anaerobic process. Notice there is no O₂ in any of the fermentation reactions shown above. Many of these reactions are quite ancient, hypothesized to be some of the first energy-generating metabolic reactions to evolve. This makes sense if we consider the following:

The early atmosphere was highly reduced, with little molecular oxygen readily available.
Small, highly reduced organic molecules were relatively available, arising from a variety of chemical reactions.
These types of reactions, pathways, and enzymes are found in many different types of organisms, including bacteria, archaea, and eukaryotes, suggesting these are very ancient reactions.
The process evolved long before O₂ was found in the environment.
The substrates, highly reduced, small organic molecules, like glucose, were readily available.
The end products of many fermentation reactions are small organic acids, produced by the oxidation of the initial substrate.
The process is coupled to substrate-level phosphorylation reactions. That is, small, reduced organic molecules are oxidized, and ATP is generated by first a red/ox reaction followed by the substrate-level phosphorylation.
This suggests that substrate-level phosphorylation and fermentation reactions coevolved.

Consequences of fermentation

Imagine a world where fermentation is the primary mode for extracting energy from small molecules. As populations thrive, they reproduce and consume the abundance of small, reduced organic molecules in the environment, producing acids. One consequence is the acidification (decrease in pH) of the environment, including the internal cellular environment. This can be disruptive, since changes in pH can have a profound influence on the function and interactions among various biomolecules. Therefore, mechanisms needed to evolve that could remove the various acids. Fortunately, in an environment rich in reduced compounds, substrate-level phosphorylation and fermentation can produce large quantities of ATP.

It is hypothesized that this scenario was the beginning of the evolution of the F₀F₁-ATPase, a molecular machine that hydrolyzes ATP and translocates protons across the membrane (we'll see this again in the next section). With the F₀F₁-ATPase, the ATP produced from fermentation could now allow for the cell to maintain pH homeostasis by coupling the free energy of hydrolysis of ATP to the transport of protons out of the cell. The downside is that cells are now pumping all of these protons into the environment, which will now start to acidify.

-------------------------------- BONUS READING ON COMMON REDOX CHEMISTRY ISSUES -------------------------------

Alternative View of Some Common Confusing Issues in Basic Redox Chemistry for Biology

This reading tries to break down some of the more challenging topics that students run into when studying redox chemistry in General Biology. This reading is not a substitute for your main reading but rather a complement to it that revisits some of the same topics through a different lens.

Finding ΔE

Students often struggle with finding the ∆E for a given redox reaction. One of the main barriers to developing this skill seems to be associated with developing a picture of the redox reaction itself. From the context of most biological redox reactions it is useful to imagine/picture a redox reaction as a simple exchange of electrons between two molecules, an electron donor and an electron acceptor that accepts electrons from the donor.

An analogy with kiwi fruit: To help build this mental picture we offer an analogy. Two people are standing next to one another. At the start, one person is holding a kiwi fruit in their hand and the second person's hands are empty. In this reaction, person 1 gives the kiwi to person 2. At the end of the reaction person 2 is holding a kiwi and person 1 is not. We can write this exchange as we might a chemical reaction:

person 1(kiwi) + person 2() <-> person 1() + person 2(kiwi).

start/initial state <-> final/end state

If we read this "reaction" from left to right, person 1 is a kiwi donor and person 2 is a kiwi acceptor. We can extend this analogy a little by proposing that person 1 and person 2 have different desire and ability to grab and hold kiwi fruit - we'll call that kiwi-potential. We can then propose to set up a situation where person 1 and person 2 compete for a kiwi. Let's propose that person 2 has a higher "kiwi-potential" than person 1 - that is, person 2 has a stronger desire and ability to grab and hold wiki than person 1.

If we set up a competition where person 1 starts with the kiwi and person 2 competes for it, we should expect that after some time the kiwi will be exchanged to person 2 and stay there most often. At the end of the reaction the kiwi will be with person 2. Due to the difference in "kiwi-potential" between person 1 and person 2, we can say that the spontaneous direction of kiwi flow is from person 1 to person 2. If we ever observed the kiwi flow from person 2 to person 1 we could probably conclude that person 1 required some extra help/energy to make that happen - flow from person 2 to person 1 would be non-spontaneous.

Let's call the "kiwi-potential", Kp. In our analogy, Kp_person₁ < Kp_person₂. We can calculate ∆Kp, the difference in Kp between the two people, and that will tell us something about how likely we can expect to see kiwi exchange hands between these two people. The bigger the difference in Kp the more likely the kiwi will move from the person who has a lower Kp to the person who has the higher Kp.

By definition, to calculate ∆Kp we obtain the solution to ∆Kp = Kp_final_/end - Kp_initial_/start. Since the kiwi is with person 2 at the end of the reaction and it starts with person 1 at the beginning of the reaction we would calculate ∆Kp = Kp_person₂ - Kp_person₁.

Doing it with electrons instead of kiwi fruit: To find ∆E for a redox reaction we can translate this analogy to the molecular space. Instead of people we have two molecules. Instead of a kiwi, we have electrons. Different molecules have different inherent abilities to grab and hold electrons and this can be measured by the value E. If two molecules exchange one or more electrons we can imagine that electrons will flow spontaneously from a molecule with lower E₀ to one with a higher E₀. We can write a familiar reaction with those substitutions.

molecule 1(electron) + molecule 2() <-> person 1() + molecule 2(electron).

start/initial state <-> final/end state

To find ΔE₀, you solve for ΔE₀ = E_0-final/end - E_{0-initial/start}. Alternatively, you can think of it as ΔE₀ = E_0-acceptor - E_0-donor.

When evaluating a redox reaction for ∆E you therefore need to:

First, find which of the reactants is the electron donor. The donor can also be associated with the initial state because it is the molecule that initially (before the start of the reaction) has the electron(s) to donate. This will always be one of the reactants and will be the molecule that gets oxidized (i.e. the molecule that loses electrons).
Second, find which of the reactants is the electron acceptor. This will also always be a reactant and will be the molecule that becomes reduced by the reaction (i.e. gains electrons). This molecule can be also associated with the final state since, in its reduced form, it is the molecule that has the electrons at the end of the reaction.
Third, calculate ΔE₀ = E_0-acceptor - E_0-donor or if you prefer, ΔE₀ = E_0-final/end - E_{0-initial/start}.

In the example above, we can examine the reactants and determine that NAD⁺ is the oxidized form of the electron carrier - it can, therefore, not be the donor. This means that H₂ must act as the electron donor in this reactant. During the reactantion electrons flow onto NAD⁺from the donor H₂creating the reduced product NADH and oxidized product H⁺. To calculate ∆E₀ we say that at the start of the reaction the exchanged electrons are on the donor H₂. We say that at the end of the reaction the electrons are found on NADH. Calculating ∆E₀requires us to evaluate the difference:

E_0-acceptor - E_0-donor

or equivalently,

E_0-final/end - E_{0-initial/start}.

Using a redox table to find E₀ values for the start and end molecules shows us that NAD⁺/NADH has an E₀ of -0.30 while H⁺/H₂ has an E₀ of -0.42.

Therefore, ΔE₀ = (-0.30) - (-0.42) = 0.12 V.

We can see intuitively that this reaction is spontaneous: electrons are flowing from a molecule that "wants" electrons less (E₀ of H⁺/H₂ = -0.42) to a molecule that wants them more (E₀ of NAD⁺/NADH = -0.30).

Reading Different Looking Redox Towers

Novice students of redox chemistry will all undoubtedly run across different ways of representing a redox tower. These different representations may look different but contain the same information. Without explanation, however, reading these tables - when they look different - can be confusion. We will compare and contrast different common forms of redox towers.

Redox Tower: Type 1

Figure 1. A Generic redox tower with oxidized/reduced couple listed with its reduction potential (E₀^') .

Attribution: Caidon Iwuagwu

In this type of redox tower, the oxidized and reduced forms of a molecule are separated by a slash. There is a line drawn from each half-reaction to its redox potential E₀ reported on the vertical axis.

Redox Tower: Type 2

Electron Acceptor	Electron Donor	E₀ (eV)
CO₂ + 24e^- →	glucose	- 0.43
2H⁺ + 2e^- →	H₂	- 0.42
CO₂ + 6e^- →	methanol	- 0.38
NAD⁺ + 2e^- →	NADH	- 0.32
CO₂ + 8e^- →	acetate	- 0.28
S₀ + 2e^- →	H₂S	- 0.28
SO₄²^- + 8e^- →	H₂S	- 0.22
Pyruvate + 2e^- →	lactate	- 0.19
S₄O₆²^- + 2e^- →	S₂O₃²^-	+ 0.024
Fumarate + 2e^- →	succinate	+ 0.03
Cytochrome b_ox + 1e^- →	Cytochrome b_red	+ 0.035
Ubiquinone_ox + 2e^- →	Ubiquinone_red	+ 0.11
Fe³⁺ + 1e- → (pH 7)	Fe²⁺	+ 0.2
Cytochrome c_ox + 1e^- →	Cytochrome c_red	+ 0.25
Cytochrome a_ox + 1e^- →	Cytochrome a_red	+ 0.39
NO₃^- + 2e^- →	NO₂^-	+ 0.42
NO₃^- + 5e^- →	1/2 N₂	+ 0.74
Fe³⁺ + 1e^- → (pH 2)	Fe²⁺	+ 0.77
1/2 O₂ + 2e^- →	H₂O	+ 0.82

In this type of redox tower, each row consists of a half-reaction. The oxidized form of a molecule is shown in the first column, the reduced form of the molecule is shown in the second column. Finally, the E₀ value of the molecule is listed in the third column from the left. The number of electrons transferred to reduce the oxidized form of the molecule is shown in column 1. While the format of the table looks different from Type 1 tower, both contain the exact same information.

Redox Tower: Type 3

oxidized form	reduced form	n (electrons)	Eo´ (volts)
CO₂	glucose	24	-0.43
2H⁺	H₂	2	-0.42 (at [H⁺] = 10^-7; pH=7) Note: at [H⁺] = 1; pH=0 the Eo' for hydrogen is ZERO. You will see this in chemistry class.
CO₂	methanol	6	-0.38
NAD⁺+ 2H⁺	NADH + H⁺	2	-0.32
CO₂	acetate	8	-0.28
S⁰	H₂S	2	-0.28
SO₄²^-	H₂S	8	-0.22
Pyruvate + 2H⁺	lactate	2	-0.19
S₄O₆²^-	S₄O₆²^-	2	0.024
Fumarate	succinate	2	0.03
Cytochrome b_ox	Cytochrome b_red	1	0.035
Ubiquinone; (ox)	Ubiquinone; (red)	2	0.1
Fe³⁺(pH = 7)	Fe²⁺(pH = 7)	1	0.20
Cytochrome c; Fe³⁺	Cytochrome c; Fe²⁺	1	0.25
Cytochrome a	Cytochrome a	1	0.39
Nitrate	nitrite	2	0.42
Nitrate	1/2 N₂	5	0.74
Fe³⁺(pH = 2)	Fe²⁺(pH = 2)	1	0.77
1/2 O₂ + 2H⁺	H₂O	2	0.816

In this redox tower, the oxidized form of a molecule is in the leftmost column, its reduced form is in the second column from the left, the number of electrons transferred is in the third column from the left, and the E₀ is in the far right column.

Again, all of these towers contain the exact same information and are used in an identical manner.

Special note: If you have studied redox chemistry in a formal chemistry course, you might notice two key differences between the towers you use in a biology setting and those used by chemists.

1. In chemistry, the redox towers are flipped relative to those in biology: In chemistry, the molecules with the most positive E₀ are listed starting at the top of the table and the compounds with the most negative E₀ are listed at the bottom. In bioloigal redox tables molecules with the largest E₀ are listed at the bottom while those with the smallest E₀ are listed starting at the top. The biology orientation has the advantage of making it easy to picture electrons spontaneously falling down the table from molecules that "want" the electrons less (lower E₀) to molecules that "want" electrons more (higher E₀).

2. In chemistry, the redox potential for hydrogen (H⁺/H₂)is listed as 0. This is because (a) redox potentials for chemistry are measured under a set of non-biologically relevant standard conditions and (b) hydrogen is being used as the common standard redox potential against which all other redox potentials are measured. In biology, the redox potential for hydrogen (H⁺/H₂)is listed as -0.42. This difference between the chemistry and biology tables comes about because the redox potential for (H⁺/H₂) in biology is measured at a physiological pH of 7.0.

Familiarize yourself with how to read and interpret all three types of redox towers!

Chemistry and Biology Teach Redox Differently

For students who have been taught redox chemistry in a formal chemistry course, biological lessons in redox can sometimes seem like they're talking about something completely different. Not surprisingly, chemists tend to teach the most proper and universally applicable approach to evaluating redox reaction. This approach consists of using a set of rules to formally evaluate whether atoms in a molecule have undergone a change in oxidation state. Meanwhile, biologists tend to approach the discussion of redox reactions by thinking about electron transfers between molecules. It turns out that the approach biologists take is not as rigorous as how chemists approach redox reactions and can sometimes not identify bona fide redox reactions that wouldn't be missed using the chemist's approach. However, since the vast majority of biological redox reactions do involve a transfer of electron (and therefore change in redox states).

Let's look at a specific example to see the differences in approach.

The Chemistry Approach (oxidation numbers):

To evaluate/solve redox reactions in CHEMISTRY, we use the concept of oxidation states/numbers (we will just be saying oxidation number here). The oxidation number of an element refers to how the electrons are shared between atoms in a chemical compound and they tell us about the movement of the electrons in the redox reaction. There are specific rules to assigning oxidation numbers, we will not be going over all of them since they will not be applicable to the redox reactions you will see in General Biology, but here are a few:

A single element has an oxidation number of 0
Fluorine ALWAYS has an oxidation number of -1
Hydrogen has an oxidation number of +1 with nonmetals and -1 with metals.

etc.

For more on calculating oxidation numbers see: <https://chem.libretexts.org/Bookshel...ation_Numbers)>

So to find which elements are reduced/oxidized when given a redox reaction, you must track the change of the oxidation numbers between the reactants and the products. Here is an example:

In the unbalanced reaction NO₃^-+ FADH₂⟶ NO₂^-+ FAD⁺

Using the rules, we observe that in NO₃^-, the oxidation number of Nitrogen is +5. In NO₂^-, the oxidation number of Nitrogen is +3. So because +5 ⟶ +3, N is reduced in this reaction.
We could conduct a similar calculation for key atoms on FAD⁺ and FADH₂ to discover that FADH₂ is oxidized in the reaction.

The Biology/Biochemistry Approach (electron flow):

To evaluate/solve redox reactions in biology/biochemistry, we typically do not use or assign oxidation numbers to evaluate redox reactions. Rather we follow the exchange of electrons between molecules. Fortunately, biology tends to reuse a limited number of electron carriers and redox towers tell us which form of a compound is reduced or oxidized. As mentioned above, the basic approach to redox in biology is to define oxidation as: the loss of electrons. Reduction is defined as: the gain of electrons. Here is an example:

NO₃^-+ FADH₂⟶ NO₂^-+ FAD⁺

Here we examine the reactants and immediately spot the common electron carrier FADH₂, the reduced form of the electron carrier. In the products we observe the oxidized form of the electron carrier FAD⁺. We conclude that FADH₂lost electrons (became oxidized) in the reaction. Since the electrons had to go somewhere they were likely accepted by NO₃^-which then became reduced to NO₂^-. In this case the biologist's model arrives at the same conclusion as the chemist's approach through a more intuitive approach that doesn't require memorizing numerous rules and how to apply them.

In our General Biology class, we take the biology/biochemistry approach to redox. You will not need to know how to calculate redox states in this course.

DISCLAIMER: DO NOT WORRY IF YOU HAVE NOT TAKEN CHEMISTRY YET !! WE WILL NOT BE USING THE CHEMISTRY APPROACH WHEN IT COMES TO REDOX REACTIONS IN OUR CLASS. THE PURPOSE OF THIS IS JUST TO DISTINGUISH AND HOPEFULLY CLARIFY THE TWO APPROACHES FOR STUDENTS THAT MAY HAVE ALREADY TAKEN A CHEMISTRY COURSE!!