Supplemental Lecture (98/04/03 update) by Stephen T. Abedon (abedon.1@osu.edu)

1. Chapter title: Glycolysis and Fermentation
1. A list of vocabulary words is found toward the end of this document
2. "In the living world, energy passes from the sun to photosynthetic organisms and then to other organisms in the form of the potential energy contained in the bonds of chemical compounds. To obtain energy in a usable form, a cell must have an electron (or hydrogen) donor, which serves as an initial energy source within the cell. Electron donors can be as diverse as photosynthetic pigments, glucose or other organic compound, elemental sulfur, ammonia, or hydrogen gas. . . . In aerobic respiration, oxygen serves as the final electron acceptor. In anaerobic respiration, inorganic substances other than oxygen, such as nitrate ions (NO₃^-) or sulfate ions (SO₄^2-) , serve as the final electron acceptors. In fermentation, organic compounds serve as the final electron acceptor. In aerobic respiration and anaerobic respiration, a series of electron carriers called an electron transport chain releases energy that is used . . . to synthesize ATP. Regardless of their energy sources, all organisms use similar oxidation-reduction reactions to transfer electrons and similar mechanisms to use the energy released to produce ATP." (p. 129, Tortora et al., 1995)
3. As noted, these catabolic processes, whereby ATP is produced from ADP and Pi, may be separated into two fundamentally different groups: Those utilizing an electron transport system (cellular respiration) and those that do not (glycolysis). By far and away the simpler process is glycolysis, which leads to fermentation in the absence of, for example, molecular oxygen (cellular respiration and electron transport systems utilize the end products of glycolysis).
4. In this lecture we will discuss glycolysis in terms of the expression of fermentation by microorganism such as bacteria and yeasts, especially in terms of the production of useful organic compounds. Then, we will elaborate on these mechanisms specifically in describing how glycolysis occurs in mammalian and microorganismal systems.
2. Fermentation, the word
1. Anaerobic byproduct production:
1. The word fermentation has a number of related meanings, depending on context.
2. For the most part these meanings describe similar processes that involve either energy production in the absence of air or the associated production of byproducts (a.k.a. wastes, but often quite valuable to you or me).
2. Numerous definitions:
1. That is, historically (if not actually) all of the following have at their root fermentation as defined in its strictest (i.e., most scientific) sense (see last definition, below; p. 124, Tortora et al., 1995).
2. Thus, fermentation is . . .
1. Any process that produces alcoholic beverages or acidic dairy products (general use).
2. Any spoilage of food by microorganism (general use).
3. Any large-scale microbial process occurring with or without air (common definition used in industry).
4. Any energy-releasing metabolic process that takes place only under anaerobic conditions (becoming more scientific).
5. All metabolic processes that release energy from a sugar or other organic compound, do not require molecular oxygen or an electron transport system, and use an organic compound as the final electron acceptor.
3. We will consider this last definition of fermentation in much greater detail in the course of this lecture.
3. Both electron transport systems and the use of molecular oxygen as a final electron acceptor will be considered upon discussion of cellular respiration.
3. Fermentation, the process
1. Anaerobic byproduct production:
1. Fermentation includes all metabolic processes that do at least three^* of the following:
1. release energy from a sugar or other organic compound
2. do not require molecular oxygen
3. do not require an electron transport system
4. use an organic compound as the final electron acceptor.
2. *Three of the four because a lack of requirement for oxygen (item 2) is implied by items 3 and 4.
2. Fermentation, because many energy-rich bonds are not broken, tends to produce much less ATP than does cellular respiration.
3. Wasteful (chemically):
1. Fermentation also tends to produce waste products that can accumulate in the extracellular environment.
2. By contrast, the wastes left over after ATP production by aerobic respiration are limited to CO₂ and H₂O.
4. Numerous end products:
1. Various microorganism can fermentation various substrates resulting in the generation of various waste products.
2. Different methods of fermentation are readily distinguishable and referred to based on their products as, for example:
1. lactic acid fermentation
2. alcohol fermentation
3. mixed acid fermentation
4. etc.
3. The type of fermentation employed constitutes some degree of compromise between:
1. substrate (nutrient) availability
2. waste product tolerance
3. energy yield
5. Muscles-lactic acid:
1. Fermentation occurs in mammals under circumstances where ATP is required but molecular oxygen is lacking (for example, during sprints).
2. Such fermentation follows a normal biochemical pathway called glycolysis and results in the production of lactic acid.
6. Genetic limitations:
1. Different microorganism tend to have a characteristic fermentation pathway (rather than an unlimited potential to drastically switch fermentation pathways depending upon utility).
2. That is, different organisms differ in their fermentative starting material, product, and the conditions under which they ferment.
7. Knowledge of the substrate fermented and the waste product produced can be utilized in the identification of microorganisms.
4. Fermentation ecology
1. Some microorganisms yield comparatively large amounts of ATP from a given fermentation pathway, but only, for instance, when substrate and nutrients are abundant and wastes are either well buffered or are removed.
2. For other microorganisms, growth may be achieved even in environments containing only minimal nutrients (and even in which one wallows in one's own wastes and those of others), though perhaps via fermentation pathways which convert substrate to ATP with lower efficiency.
3. Succession:
1. Given the presence of different types of fermenters, an environment will tend to become dominated by those organisms whose fermentation pathway and other attributes make them best suited to microbial growth in that environment at that point in time.
2. The environment will then tend to change over time, minimally in response to ongoing fermentation. Often this will result in a change in the microorganism whose growth dominates in the environment.
3. Any given environment may go through multiple cycles of succession such as this.
5. Electron (hydrogen) donor [reducing agent]
1. The source of high energy electrons, i.e., reduced bonds (e.g., C-H bonds).
2. The breaking of these reduced bonds may be coupled directly (i.e., via substrate level phosphorylation) or indirectly (i.e., via an electron transport system) to the production of ATP.
3. Hydrogen donors donate their electrons to electron acceptors.
6. Electron acceptor [oxidizer]
1. In the oxidation of an electron donor, the electron acceptor is the oxidizing agent and therefore is reduced, that is, the electron acceptor accepts electrons.
7. Final (terminal) electron acceptor
1. The place (molecule) where energy-rich electrons in, for example, C-H and C-C bonds ultimately end up.
2. In fermentation the place where electrons end up is a less reduced (more oxidized) organic compound.
3. In aerobic respiration (which may be distinguished from fermentation by the former's use of an electron transport system), the place where electrons end up is the atoms of molecular oxygen (water is thus made).
4. In anaerobic respiration (which also may be distinguished from fermentation by the former's use of an electron transport system), something other than oxygen or an organic compound serves as the final electron acceptor.
8. Substrate level phosphorylation
1. Glycolysis' ATP generation:
1. Generally, substrate-level phosphorylation is the means by which fermentation processes lead to ATP generation.
2. See glycolysis for more details of this process.
2. Substrate level phosphorylation is a way of stating that ATP generation is directly coupled to the breaking of energy-rich, reduced bonds (specifically ones between carbon and phosphate groups found in the electron donor molecule).
3. Substrate level phosphorylation contrasts with the utilization of an electron transport system (chemiosmosis).
9. Glycolysis
1. Glucose to ATP and pyruvate:
1. Glycolysis is a multistep reaction that converts glucose (a six carbon molecule) to two pyruvate (a three carbon molecule) during which two ATP are produced via substrate level phosphorylation.
2. Note that all of the steps of glycolysis are enzymatically catalyzed.
2. "The glycolytic reaction sequence is thought to have been among the earliest of all biochemical processes to evolve. It uses no molecular oxygen and occurs readily in an anaerobic environment. All of its reactions occur free in the cytoplasm; none is associated with any organelle or membrane structure. Except for a few . . . bacteria, every living creature is capable of carrying out the glycolytic sequence." (p. 163, Raven & Johnson, 1995)
3. Overall stoichiometry of glycolysis:

C6H12O6 + 2ADP  + 2Pi + 2NAD+ --- 

2C3H4O3 + 2ATP +2NADH + 2H+

4. where C₆H₁₂O₆ is glucose and 2C₃H₄O₃ is pyruvic acid (pyruvate).
5. The efficiency of glycolysis is only about 2% (that is, amount of energy harnessed as ATP divided by the total amount of energy present in glucose if it were burned--oxidized--completely to CO₂ and H₂O).
6. However, this calculation is misleading since in the process of glycolysis, glucose is not oxidized completely to CO₂ and H₂O.
7. In addition, for many organisms, the wastes of glycolysis may be utilized by an electron transport system to generate (much) more ATP.
10. NAD+ [nicotinamide adenine dinucleotide, NADH]
1. NAD⁺:
1. NAD⁺ is nicotinamide adenine dinucleotide.
2. It serves as an intracellular oxidizing agent (a.k.a., electron acceptor).

NAD+ + 2H+ + 2e- --- NADH + H+

2. NADH:
1. NADH is reduced NAD⁺.
2. NADH serves as means by which cells transfer reducing power around (a reducing power intermediate) similar in concept to the way cells employ ATP as an energy intermediate.
11. NAD+ regeneration [NADH recycling]
1. The reactions of glycolysis are accomplished at the expense of the reduction of NAD⁺ to NADH + H⁺.
2. I say expense because in order for further glycolysis to occur, a source of NAD⁺ must be established. This source is typically NADH which is first induced to donate its reducing electrons to a subsequent molecule (e.g., a final electron acceptor).
3. In aerobic respiration this subsequent molecule is molecular oxygen with the Krebs cycle and an electron transport system serve as intermediates in this process
4. In fermentation (i.e., when molecular oxygen is absent or in microorganisms which are unable to take advantage of molecular oxygen when it is present), pyruvate serves as the final electron acceptor resulting in the formation of lactic acid, ethanol, etc.
5. In general,

organic molecule (e.g., pyruvate) + NADH à
 reduced organic molecule + NAD+

6. See lactic acid fermentation, alcohol fermentation, and mixed acid fermentation.
12. Lactic acid fermentation [homolactic-acid fermentation, lactic acid]
1. Final electron acceptor:
1. Fermentation where pyruvate serves as the final electron acceptor to form lactic acid.
2. Overall lactic acid fermentation occurs as follows:

C6H12O6 + 2NAD+ + 2ADP + 2Pi à
 2C3H6O3 + 2NAD+ + 2ATP

2. (C₆H₁₂O₆ is glucose; C₃H₆O₃ is lactic acid).
3. The structure of lactic acid is as follows:

    H   OH       
    |   |        
H - C - C - C = O
    |   |   |    
    H   H   OH   

4. The simplest of fermentations, lactic acid fermentation is carried out by a number of bacteria
5. Lactic acid fermentation is all but identical to glycolysis.
6. Makes things taste sour:
1. Lactic acid is an acid and therefore sour to the taste.
2. Lactic acid fermentation, consequently, tends to make things sour such as in the production of yogurt or during the spoilage of milk.
7. Lactic acid fermentation produces no gas, which is unusual among fermenation pathways.
13. Alcohol fermentation [ethanol]
1. Final electron acceptor:
1. Fermentation where pyruvate serves as the final electron acceptor to form ethanol.
2. Compare with lactic acid fermentation.
3. The structure of ethanol is as follows:

    H   H     
    |   |     
H - C - C - OH
    |   |     
    H   H     

2. Typical of yeasts:
1. Alcohol fermentation is a characteristic of yeasts and rare among bacteria.
2. Alcohol fermentation, of course, supports a certain beverage industry.
3. Carbon dioxide produced:
1. The carbon dioxide produced by alcohol fermentation is the gas involved in the rising of yeast breads as well as supplying the bubbles in champagne.
2. Note the carbon lost going from pyruvate to ethanol; this is where the carbon in the CO₂ comes from.
14. Mixed acid fermentation
1. Mixed acid fermentation is a characteristic fermentation of members of family Enterobacteriaceae
2. Mixed acid fermentation results in the formation of:
1. lactic acid
2. ethanol
3. formic acid
4. succinic acid
15. Gas formation
1. Mixed acid fermentation lowers pHs as acidic waste products accumulate.
2. Gas formation is a characteristic of Escherichia coli and results from the conversion of some of the various acidic waste products (e.g., formic acid, HCOOH) to gas consisting of CO₂ and H₂.
3. Gas formation occurs in gas formers when environmental pHs are low (i.e., <= 6).
16. Biochemistry of glycolysis (outline)
1. (see lecuture: Glycolysis in Detail for a detailed overview of that information presented in this section below)
2. Considering only carbons (e.g., C₆ is glucose), phosphate, and high energy bonds (~):
1. C₆ + ATP à C₆-Pi + ADP
2. C₆-Pi ß à C₆-Pi
3. C₆-Pi + ATP à Pi-C₆-Pi + ADP
4. Pi-C₆-Pi ß à C₃-Pi + C₃-Pi
5. C₃-Pi + C₃-Pi ß à 2C₃-Pi
6. 2C₃-Pi + 2Pi + 2NAD⁺ ß à 2Pi~C₃-Pi + 2NADH + 2H⁺
7. 2Pi~C₃-Pi + 2ADP ß à 2C₃-Pi + 2ATP
8. 2C₃-Pi ß à 2C₃-Pi
9. 2C₃-Pi ß à 2<a href="C₃~Pi + 2H₂O
10. 2C₃~Pi + 2ADP à 2C₃ + 2ATP
3. Note the priming of glucose in step (1), the addition of Pi to form glucose_6_phosphate: C₆-Pi.
4. Note the formations of high energy bonds with phosphate and their subsequent transfer to ADP (i.e., substrate level phosphorylation).
5. Note the reduction of NAD⁺ to NADH. See NAD⁺ NAD⁺ regeneration, below.
6. Note that steps 2, 5, and 8 are isomerization steps.
7. Note how many of these reactions are reversible indicating that differences in energy between various intermediates are no greater than that supplied by thermal energy. Note also that the overall reactions are driven forward (i.e., to the right) by the presence of reactants and that over all glycolysis is driven forward by the presence of glucose and a requirement for ATP.
8. Note that in the above depiction we are omitting details of structure, some reactions, required enzymes, control of enzymes, structure of enzymes, side reactions, and subtle (and perhaps not so subtle) variations in the scheme observed between species.
17. Structures of glycolytic intermediates
1. C₆: glucose:

     H   H   H   OH  H   H    
     |   |   |   |   |   |    
HO - C - C - C - C - C - C = 0
     |   |   |   |   |        
     H   OH  OH  H   OH  
     

2. C₆-Pi: glucose-6-phosphate:

     H   H   H   OH  H   H    
     |   |   |   |   |   |    
Pi - C - C - C - C - C - C = 0
     |   |   |   |   |        
     H   OH  OH  H   OH  
     

3. In the course of glycolysis, glucose_6_phosphate is isomerized into fructose-6-phosphate (note, this reaction is reversible):

     H   H   H   OH      H     
     |   |   |   |       |     
Pi - C - C - C - C - C - C - OH
     |   |   |   |   ||  |     
     H   OH  OH  H   O   H  
   

4. Pi-C₆-Pi: fructose-1,6-diphosphate:

     H   H   H   OH      H     
     |   |   |   |       |     
Pi - C - C - C - C - C - C - Pi
     |   |   |   |   ||  |     
     H   OH  OH  H   O   H     

5. C₃-Pi: glyceraldehyde-3-phosphate:

     H   H     
     |   |     
Pi - C - C - OH
     |   |     
     H   C = 0 
         |     
         H    
 

6. Note how glyceraldehyde-3-phosphate (left, below) resembles glycerol (right, below), hence the "glycer" in its name (the double-bonded oxygen, aldehyde group is on the first carbon, bottom, below, left). This resemblance (three carbons, each bound to an -OH group or modified -OH group) is consistent for all of the subsequent three carbon intermediates of glycolysis up to but not including phosphoenolpyruvate.

    H        --       H     
    |        --       |     
H - C - Pi   --   H - C - OH
    |        --       |     
H - C - OH   --   H - C - OH
    |        --       |     
    C = O    --   H - C - OH
    |        --       |     
    H        --       H     

7. A second C₃ is produced along with glyceraldehyde-3-phosphate called dihydroxyacetone phosphate. This intermediate is then converted in an isomerization reaction into glyceraldehyde-3-phosphate. Note resemblance of this intermediate to glycerol:

     H   H     
     |   |     
Pi - C - C = O 
     |   |     
     H   C - 0H
         |     
         H     

8. Pi~C₃-Pi: 1,3-diphosphoglycerate (note resemblance to glycerol):

     H   H     
     |   |     
Pi - C - C - OH
     |   |     
     H   C ~ Pi
         ||    
         O     

9. C₃-Pi: 3-phosphoglycerate (note that this is a different molecule from that produced two steps previous, glyceraldehyde-3-phosphate; note resemblance to glycerol):

     H   H     
     |   |     
Pi - C - C - OH
     |   |     
     H   C = 0 
         |     
         OH    

10. C₃~Pi: also 2-phosphoglycerate (note resemblance to glycerol):

     H   H     
     |   |     
H0 - C - C - Pi
     |   |     
     H   C = 0 
         |     
         OH    

11. C₃~Pi: also phosphoenolpyruvate (note that with the far left carbon bound only to carbons and hydrogens there is less of a resemblance to glycerol and this difference is reflected in its name not indicating that it is a glycerol derivative):

 H - C = C ~ Pi
     |   |     
     H   C = O 
         |     
         OH  
  

12. C₃: pyruvate:

    H        
    |        
H - C - C = O
    |   |    
    H   C = O
        |    
        OH  

18. Vocabulary
1. Alcohol fermentation
2. Electron donor
3. Final electron acceptor
4. Fermentation
5. Fermentation ecology
6. Fermentation, the word
7. Gas formation
8. Glycolysis
9. Glycolysis (biochemistry of)
10. Homolactic-acid fermentation
11. Lactic acid
12. Lactic acid fermentation
13. Mixed acid fermentation
14. NAD⁺
15. NADH
16. NAD⁺ regeneration
17. NADH recycling
18. Nicotinamide adenine dinucleotide
19. Substrate level phosphorylation
19. Practice questions
1. Given an unlimited source of glucose, what is the limiting factor in the glycolytic reaction? [PEEK]
2. Name two differences between respiration and fermentation (short answer).[PEEK]
3. Under what circumstances might a facultative microorganism employ fermentation rather than respiration (be general; short word answer)?[PEEK]
4. A not well mixed culture growing at high density in broth media containing only the sugar lactose as a carbon and energy source is producing gas. Name the organism. [PEEK]
5. In lactic acid fermentation, pyruvate serves as the final electron acceptor in a reaction required for the regeneration of what? [PEEK]
6. What is the basic purpose of glycolysis, especially as performed in the absence of subsequent cellular respiration? (less than 10 word answer) [PEEK]
7. Name three products of glycolysis or fermentation. [PEEK]
8. What is the basic purpose of fermentation (i.e., the post-glycolysis production of organic waste)? (less than 10 word answer) [PEEK]
9. In the course of ecological succession, the dominant organisms present in a fermentation tend to start out being ones which are able to extract more ATP per unit nutrient, but are less able to withstand significant build ups of waste. Subsequent organisms are less efficient in their extraction of useable energy (i.e., ATP) but are better able to withstand greater build ups of organic wastes. Why, specifically, are organic wastes building up? [PEEK]
10. Distinguish anaerobic respiration and fermentation by at least two criteria. [PEEK]
11. In fermentation, in general, the place where electrons end up is __________. [PEEK]
12. Which of the following is associated with gas formation? [PEEK]
1. glycolysis
2. lactic acid fermentation
3. yeast aerobic growth
4. ethanol fermentation
5. muscle anaerobic fermentation
6. none of the above
13. Describe succession with regard to fermentation ecology. Do so in such a way that convinces me that you know what the word means. [PEEK]
20. Bio 113 only:
1. Substrate-level phosphorylation occurs twice in glycolysis. This involves the donation of inorganic phosphates from carbon compounds to ADP. How many carbons do these carbon compounds consist of? [PEEK]
2. Considering only the carbon atoms of intermediates found during the glycolytic conversion of glucose to pyruvate, draw an example of substrate level phosphorylation (you may draw actual structures if you find that helpful). [PEEK]
3. What do you suppose causes the "burn" in your muscles during intense sprinting or weight lifting? (less than 10 word answer) [PEEK]
21. Practice question answers
1. NAD⁺ regeneration
2. Respiration makes lots of ATP, uses inorganic final electron acceptor (such as molecular oxygen), and uses an electron transport chain. fermentation makes only a little ATP, does not use an electron transport chain, and uses organic final electron acceptor. Note that in either case "waste" molecules are given off into the environment.
3. For example, when molecular oxygen concentrations are very low.
4. E. coli
5. NAD⁺
6. production of ATP.
7. ATP, NADH + H⁺, ethanol, lactic acid, pyruvate.
8. regeneration of NAD⁺.
9. The organic wastes are fermentation products. Fermentation products are created in the course of the regeneration of NAD⁺ from NADH.
10. Anaerobic respiration uses an electron transport chain, produces far more ATP, and employs an inorganic final electron acceptor.
11. in a less reduced organic compound, e.g., pyruvate is converted to lactate
12. iv, ethanol fermentation
13. the fermentation of a substance by a complex combination of microorganisms will typical follow a path whereby one organism type's growth dominates under certain conditions, that dominance leads to a change conditions, which results in a subsequent organism type coming to dominate. This succession from one organism dominating growth to another as conditions change is formerly described as ecological succession and it is equivalent to the succession which occurs all around us as used up farm land yields fields which in turn yields forest by pioneer tree species and ultimately results with the climax tree species (mostly maple-beech forests in the Mansfield area)
22. Bio 113 only:
1. three
2. C₃~Pi + ADP --- C₃ + ATP
3. lactic acid from lactic acid fermentation following glycolytic production of ATP under anaerobic conditions.
23. References
1. Black, J.G. (1996). Microbiology. Principles and Applications. Third Edition. Prentice Hall. Upper Saddle River, New Jersey. pp. 116-120.
2. Keeton, W.T. (1976). Biological Science. Third Edition. W.W. Norton and Co., New York, pp. 163-167.
3. Stryer, L. (1981). Biochemistry. Second Edition. W.H. Freeman and Co., San Francisco, pp. 255-282.
4. Raven, P.H., Johnson, G.B. (1995). Biology (updated version). Third Edition. Wm. C. Brown publishers, Dubuque, Iowa. pp. 135-166.
5. Tortora, G.J., Funke, B.R., Case, C.L. (1995). Microbiology. An Introduction. Fifth Edition. The Benjamin/Cummings Publishing, Co., Inc., Redwood City, CA, pp. 111-116.