Dissimilation – Fermentation

Overview of Fermentative Dissimilation

In biological systems, fermentation is a form of dissimilation in which organic nutrients (typically carbohydrates) are broken down to release some usable energy without using an external electron acceptor such as oxygen. Instead, organic molecules derived from the nutrient itself serve as both electron donors and electron acceptors.

Fermentation:

• Operates under anaerobic conditions (no oxygen required; sometimes inhibited by oxygen).
• Yields much less ATP per glucose than respiration.
• Produces characteristic end products (e.g., ethanol, lactate, organic acids, gases) that still contain considerable chemical energy.
• Is widespread in microorganisms and also occurs in some animal tissues (e.g., muscle during intense exercise).

This chapter concentrates on the types, pathways, energetics, and significance of fermentation, assuming that the basic ideas of metabolism, ATP, redox coenzymes (e.g., NAD⁺/NADH), and glycolysis have been introduced elsewhere in the course.

General Principles of Fermentation

Redox Balance and the Role of NAD⁺/NADH

In many central metabolic pathways (e.g., glycolysis), oxidation of nutrients is coupled to reduction of the coenzyme NAD⁺ to NADH:

$$
\text{nutrient} + \text{NAD}^+ \rightarrow \text{oxidized product} + \text{NADH} + \text{H}^+
$$

In respiration, NADH is reoxidized to NAD⁺ via an electron transport chain. In fermentation, there is no external electron transport chain; instead:

• NADH is reoxidized by transferring its electrons back to an organic intermediate derived from the original substrate.
• This produces a reduced organic end product (such as ethanol or lactate), while regenerating NAD⁺ needed to keep glycolysis and ATP production running.

A simple scheme for fermentation of glucose is:

1. Glucose is partially oxidized to an intermediate (e.g., pyruvate) with formation of ATP and NADH.
2. Pyruvate (or a derivative) is reduced by NADH to yield the fermentation end product, regenerating NAD⁺.

The key chemical feature of fermentation is thus an internal redox balance: the overall oxidation state of the carbon atoms in the products is similar to that in the substrate, because electrons are not exported but redistributed among organic molecules.

ATP Production in Fermentation

Fermentation typically yields ATP only by substrate-level phosphorylation:

$$
\text{ADP} + \text{Pi} \rightarrow \text{ATP}
$$

This occurs at specific steps in pathways like glycolysis. Because fermentation lacks oxidative phosphorylation, the ATP yield per glucose molecule is low (commonly 2 ATP/glucose for many pathways involving glycolysis).

Major Types of Fermentation

Fermentations are classified by:

• The main end products formed.
• The organisms that perform them.
• The metabolic pathway used.

Below are some important fermentations relevant in biological systems and in everyday life.

Lactic Acid Fermentation

Homolactic Fermentation

In homolactic fermentation, the main (often exclusive) end product is lactate (lactic acid in its protonated form). This process is important in:

• Animal tissues, especially muscle cells during intense, short-term exercise when oxygen supply is insufficient.
• Lactic acid bacteria (e.g., Lactobacillus, Streptococcus species), common in food fermentations.

A typical overall reaction (from glucose) is:

$$
\text{C}_6\text{H}_{12}\text{O}_6 \; (\text{glucose}) \rightarrow 2 \, \text{CH}_3\text{–CH(OH)–COOH} \; (\text{lactic acid})
$$

Mechanistically (building on glycolysis):

1. Glucose is converted to 2 pyruvate, producing 2 ATP and 2 NADH.
2. Each pyruvate is reduced to lactate:

$$
\text{pyruvate} + \text{NADH} + \text{H}^+ \rightarrow \text{lactate} + \text{NAD}^+
$$

No additional ATP is generated in this reduction step; its purpose is to regenerate NAD⁺.

Heterolactic and Mixed Acid Lactic Fermentation

Some bacteria produce not only lactate but also ethanol, acetate, CO₂, and other products. These pathways involve branch points after glucose breakdown and different intermediates (e.g., the phosphoketolase pathway).

Characteristic features:

• Lower lactate yield per glucose.
• Formation of gases (CO₂) and other organic acids.
• Important in food fermentations such as sauerkraut, kefir, and some cheeses, where complex flavor and texture depend on a mixture of acids and other metabolites.

Alcoholic Fermentation (Ethanol Fermentation)

Alcoholic fermentation produces ethanol and CO₂ as the principal end products. It is characteristic of:

• Many yeasts (e.g., Saccharomyces cerevisiae).
• Some types of bacteria and plant tissues (usually under low-oxygen conditions).

Overall stoichiometry from glucose:

$$
\text{C}_6\text{H}_{12}\text{O}_6 \; (\text{glucose}) \rightarrow 2 \, \text{CH}_3\text{–CH}_2\text{–OH} \; (\text{ethanol}) + 2 \, \text{CO}_2
$$

Key biochemical steps after glycolysis:

1. Decarboxylation of pyruvate to acetaldehyde:

$$
\text{pyruvate} \rightarrow \text{acetaldehyde} + \text{CO}_2
$$

2. Reduction of acetaldehyde to ethanol, coupled to NADH oxidation:

$$
\text{acetaldehyde} + \text{NADH} + \text{H}^+ \rightarrow \text{ethanol} + \text{NAD}^+
$$

ATP balance:

• Glycolysis yields 2 ATP per glucose.
• The conversion of pyruvate to ethanol itself does not produce additional ATP.
• As with lactic acid fermentation, the reduction step serves to recycle NAD⁺.

This pathway underlies the production of alcoholic beverages, leavened bread (CO₂ causes dough to rise; ethanol mostly evaporates), and certain biofuels.

Mixed Acid, Propionic, and Other Microbial Fermentations

Many microorganisms use complex fermentative pathways that yield mixtures of end products. Some important types include:

Mixed Acid Fermentation

Typical of many enteric bacteria (e.g., Escherichia coli). End products may include:

• Lactic acid
• Acetic acid
• Formic acid
• Ethanol
• CO₂, H₂

Mixed acid fermentation:

• Involves several branching routes from pyruvate.
• Maintains redox balance and regenerates NAD⁺ using different end-product combinations.
• Is used in microbiological diagnostics (e.g., tests that detect specific fermentation products).

Propionic Acid Fermentation

Performed by Propionibacteria, important in certain cheeses (e.g., Emmental/Swiss cheese). Example overall scheme (from lactate):

$$
3 \, \text{lactate} \rightarrow 2 \, \text{propionate} + \text{acetate} + \text{CO}_2 + \text{H}_2\text{O}
$$

The CO₂ produced forms the characteristic "holes" (eyes) in Swiss-type cheeses. Propionate also contributes to flavor and can be used as a preservative.

Butyric and Butanol Fermentation

Members of the genus Clostridium can convert sugars into:

• Butyric acid
• Butanol
• Acetone
• CO₂ and H₂

These pathways are more complex, involving:

• Extended chains of reactions from pyruvate to four-carbon acids or alcohols.
• Multiple redox steps to balance NADH and other reduced cofactors.

Such fermentations are important in anaerobic environments (e.g., sediments, intestinal tracts) and have been exploited historically for industrial solvents (acetone–butanol–ethanol (ABE) fermentation).

Fermentation of Other Substrates

Although glucose is a central example, many fermentations involve other substrates:

• Carbohydrates derived from polysaccharides such as starch, cellulose, or glycogen are first hydrolyzed to monosaccharides, which then enter fermentative pathways.
• Some organisms ferment amino acids (e.g., Stickland reactions, in which one amino acid is oxidized and another is reduced).
• In various ecosystems (e.g., rumen, large intestine), fermentation of complex plant materials by microbial communities produces short-chain fatty acids (e.g., acetate, propionate, butyrate) that are then absorbed and used by the host.

Energetics and Efficiency of Fermentation

Comparison with Aerobic Respiration

Using glucose as an example:

• Aerobic respiration can yield up to about 30–32 ATP per glucose (depending on organism and conditions), owing to oxidative phosphorylation.
• Typical fermentative pathways yield only 2 ATP per glucose (from glycolysis via substrate-level phosphorylation).

Reasons for the low ATP yield in fermentation:

• Carbon is only partially oxidized. End products like lactate and ethanol still hold considerable chemical energy.
• There is no external electron acceptor (e.g., O₂) to support a high-energy proton gradient and ATP synthase activity.

Nevertheless, fermentation can still be advantageous:

• It allows ATP production when oxygen is absent or limited.
• It enables survival in anaerobic niches where competitors relying on respiration cannot thrive.
• It often operates at high rates, compensating in part for the low ATP yield per molecule of substrate.

Thermodynamic Considerations

Fermentative reactions:

• Are typically exergonic overall (negative Gibbs free energy change, $\Delta G < 0$) under physiological conditions, making them capable of driving ATP formation via coupled substrate-level phosphorylation steps.
• Rely heavily on maintaining redox balance (conversion of NADH back to NAD⁺). If NAD⁺ is not regenerated, glycolysis halts, stopping ATP production.

The particular set of end products produced by an organism is tuned so that:

• Electron flow from reduced intermediates to more oxidized intermediates yields a net negative $\Delta G$.
• The overall electron balance (from substrate to all products) maintains cellular redox homeostasis.

Physiological and Ecological Roles of Fermentation

Fermentation in Animals and Humans

In animal muscle cells:

• During short bursts of intense activity, oxygen delivery may not keep pace with ATP demand.
• Cells rapidly metabolize glucose via glycolysis, and pyruvate is reduced to lactate:
• This allows continued ATP production in the absence of sufficient oxygen.
• Lactate can later be reconverted to pyruvate and further oxidized, or used in the Cori cycle (conversion to glucose in the liver).

Consequences:

• Accumulation of lactate and protons in muscle contributes to metabolic acidosis in the tissue, which may be associated with fatigue.
• Once oxygen becomes available again, respiration can restore redox balance more completely.

In certain tissues and microorganisms within the human body (e.g., gut microbiota), fermentation:

• Produces short-chain fatty acids that can be absorbed and used as an energy source.
• Influences pH and nutrient availability in the local environment.

Fermentation in Microbial Communities

In many ecosystems, fermentation is a central process:

• In anaerobic sediments, wetlands, and animal digestive tracts, fermentative microorganisms break down complex organic matter.
• Their end products often serve as substrates for other organisms, such as:
• Methanogens that convert fermentation products (e.g., hydrogen, CO₂, acetate) into methane.
• Sulfate-reducing bacteria using fermentation products as electron donors.

This leads to syntrophic relationships, where different organisms depend on each other to keep the overall metabolism thermodynamically favorable.

Fermentation in Food and Biotechnology

Fermentation is widely exploited in food production and industrial biotechnology:

• Food fermentations:
• Bread (yeast ethanol/CO₂ fermentation).
• Beer, wine, and other alcoholic beverages (ethanol fermentation).
• Yogurt, kefir, sauerkraut, kimchi, cheese (lactic acid, propionic acid, and mixed fermentations).
• Industrial products:
• Organic acids (lactic acid, citric acid).
• Solvents (e.g., butanol, historically acetone).
• Bioethanol as a fuel.

In such applications, specific strains are selected and conditions are optimized to:

• Channel metabolism toward the desired end product.
• Control pH, temperature, nutrient levels, and oxygen exclusion.
• Influence flavor, texture, and preservation properties.

Chemical Features of Common Fermentation Products

Fermentation end products are chemically diverse but share some features:

• They are typically small organic molecules (2–4 carbon atoms) that are:
• Alcohols (ethanol, butanol).
• Carboxylic acids (lactic acid, acetic acid, propionic acid, butyric acid).
• Aldehydes or ketones (e.g., acetaldehyde as an intermediate).
• They often appear in conjugate base form at physiological pH (e.g., lactate, acetate instead of lactic acid, acetic acid).
• Their formation redistributes oxidation states between carbon atoms rather than fully oxidizing them to CO₂.

Examples of structural changes:

• Lactic acid: a $\beta$-hydroxycarboxylic acid, derived from reduction of the keto group in pyruvate:
• Pyruvate: $\text{CH}_3\text{–CO–COO}^-$
• Lactate: $\text{CH}_3\text{–CH(OH)–COO}^-$
• Ethanol: a primary alcohol, derived from reduction of acetaldehyde, which itself is formed by decarboxylation of pyruvate.

These structural modifications correspond directly to redox changes: reduction of carbonyl groups to alcohols, or rearrangements that allow proton and electron transfers while maintaining overall charge and redox balance.

Summary

Fermentation is a central form of dissimilation in biological systems under anaerobic conditions. Its key features are:

• Internal redox balance using organic molecules as both electron donors and acceptors.
• Substrate-level phosphorylation as the only direct source of ATP.
• Formation of characteristic end products (e.g., lactate, ethanol, organic acids and alcohols) that still contain significant chemical energy.
• Wide occurrence in microorganisms, animal tissues, and natural and engineered ecosystems.
• Crucial practical importance in food technology, biotechnology, and energy production.

Understanding fermentation at the chemical level involves tracking:

• The oxidation state of carbon atoms in substrates and products.
• The flow of electrons through coenzymes such as NAD⁺/NADH.
• The coupling between exergonic redox reactions and ATP formation via substrate-level phosphorylation.