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Metabolism - Fermentation

©2000 Timothy Paustian, University of Wisconsin-Madison

Fermentation is defined as an energy yielding process whereby organic molecules serve as both electron donors and electron accepters. The molecule being metabolized does not have all its potential energy extracted from it. In other words, it is not completely oxidized.

General Concepts

Microorganisms are capable of an amazing array of different types of fermentation. Most natural compounds are degraded by some type of microbe and even many man-made compounds can be attacked by bacteria. In environments devoid of oxygen (or other suitable inorganic electron accepter), this degradation involves fermentation.

Despite the many methods bacteria employ to ferment organic compounds, there are some unifying concepts that are true of all fermentations.

• NAD⁺ is almost always reduced to NADH

  Remember that metabolism involves the oxidation of the substrate. These electrons are removed from the organic molecule and most often given to NAD. (This is true both in fermentation and respiration). Below is shown an example of NAD reduction.

  

  Figure 1 - oxidation of glyceraldehyde-3-phosphate to 1,3 bisphosphoglycerate. Electrons are removed from the carbon denoted in red and donated to NAD⁺. An inorganic phosphate is attached to the carbon shown

• Fermentation results in a excess of NADH

  Accumulation of NADH causes a problem for anaerobes. They have too much of it and it prevents further oxidation of substrate due to a lack of an NAD⁺ pool to accept electrons. In many fermentation pathways, the steps after energy generation are performed in part to get rid of the NADH.

• Pyruvate is often an important intermediate

  Many of the reactions that we will look at eventually end up making pyruvate. Pyruvate is a valuable intermediate because it can be used for cell synthesis and many different enzymes can act on it. It gives the microbe flexibility.

• Energy is derived from Substrate-Level Phosphorylation (SLP)

  The substrate is converted to a phosphorylated compound and in subsequent reactions the high energy phosphate is transferred to ATP.

  

  Figure 2 - Phosphoenolpyruvate is converted to pyruvate with the formation of ATP. The phosphate hilited in blue is transferred from PEP to ATP.

• Energy yields are low

  SLP is an inefficient process and much of the energy of the electrons is lost. Typically energy yields are 1-4 ATP per substrate molecule fermented.

• Oxygen is not involved

Fermentation can involve any molecule that can undergo oxidation. Typical substrates include sugars (such as glucose) and amino acids. Typical products depend upon the substrate but can include organic acids (lactic acid, acetic acid), alcohols (ethanol, methanol, butanol), ketones (acetone) and gases (H₂ and CO₂)

Glycolysis - Embden-Meyerhoff-Parnas pathway (EMP)

EMP is the most commonly used series of reactions for oxidizing glucose to pyruvate and many bacteria, animals and plants employ this pathway in their catabolism. EMP is so ubiquitous that is worthwhile to use it as an example of a typical fermentation. It is an essential part of many organisms catabolism, even yours! However, it is not the only method for the fermentation of glucose. Remember that bacteria are remarkably creative and other pathways are present in different species.

EMP can be divided into 3 stages, activation of glucose, hexose splitting and energy extraction

Activation of Glucose

Glucose is a relatively stable molecule and in order to degrade it, it must first be destabilized by adding high energy phosphates. In the first step a phosphate is donated from ATP (or phosphoenolpyruvate - the source of the phosphate depends on the species of microbe you look at) to glucose to form glucose-6-phosphate. The molecule is isomerized to fructose-6-phosphate (another sugar) and a second phosphate is added. Fructose-1,6-bisphosphate is easier to attack than glucose and is ready to be split.

Figure 3 - Activation of glucose by phosphorylation with ATP.

Hexose Splitting

Fructose bisphosphate aldolase then breaks the phosphate loaded fructose into two 3 carbon compounds, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetonephosphate (DAP). This is the crucial step in the EMP pathway, converting the 6 carbon glucose molecule to two 3 carbon molecules that will eventually become pyruvate.

Figure 4 - Splitting of Fructose by aldolase.

Energy Extraction

In the next reaction, DAP is converted into GAP, which can be acted on by the rest of the EMP. The next step is a very important one. Inorganic phosphate is added to GAP to make 1,3-bisphosphoglycerate (BPG). No energy is required and in fact electrons are transferred from GAP to NAD⁺. This reaction is the payback for running the pathway and the phosphates added here are later transferred to ADP to make ATP. After several enzymatic rearrangements, the final product of the EMP pathway is pyruvate.

Figure 5 - Extraction of Energy. Note the two reactions hilighted in blue that yield energy. Remember that each glucose molecule that comes into glycolysis generates two GAP molecules that can then proceed down the latter half of the pathway.

The total reaction can be summarized as follows

2 ATP + glucose + 4 ADP + 2 P_i + 2 NAD⁺ 2 ADP + 2 pyruvate + 4 ATP + 2 NADH

The blue highlight denotes energy put into the reaction. Subtracting this from the energy extracted, the net energy gain is 2 ATP per glucose. That is a lot of work for just 2 ATP. Fermentations do not yield large amounts of energy and this explains why fermenting microbes go through so much substrate without much growth.

Some of the NADH that is generated can be used for cell biosynthesis, but there is a large excess or reducing power. Fermenting bacteria must find a way to get rid of these extra electrons and they do it by adding them to pyruvate to form end products.

End Product Formation

One of the more familiar fermentations is conversion of glucose to ethanol to form alcoholic beverages. After the formation of pyruvate, ethanol is formed by two simple reactions. First, CO₂ is removed from pyruvate to form acetaldehyde. Then acetaldehyde is reduced by, you guess it, NADH

Figure 6 - Oxidation of NADH. Acetaldehyde is reduced to ethanol (the active ingredient in alcoholic beverages). This is the final step in yeast fermentation of glucose to ethanol.

Another favorite microbial fermentation is the formation of lactic acid. This is performed by the lactic acid bacteria. Homofermentative lactic acid bacteria use the EMP pathway to make pyruvate and then reduce it to lactate using up their excess NADH in the process. Other bacteria use alternative pathways to generate lactate from glucose. Close examination of the heterofermentative pathway reveals that it does not use EMP at all. The take home message is, EMP is common, but there are many other ways of doing business.

Figure 7 - Formation of lactate by homofermentative bacteria. The pathway used is identical to glycolysis. The final end product is lactate which is excreted by the cells into their environment.

Figure 8 - Fermentation of glucose by heterofermentative bacteria. In this pathway the top part of the glycolytic pathway is not used. Note the recycling of NADH and the low yield. Only one ATP is generated per glucose fermented.

Microorganisms perform many more fermentations than what is covered here, but these examples give you a general idea. In the next section we look at some of the processes used to make fermented food products.

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