Quiz Me!	Catabolism: glycolysis & fermentation
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Last revised: Wednesday, February 16, 2000
Ch. 9 (p. 164-168; 174-176) in Prescott et al, Microbiology, 4th Ed.

Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!

Copyright 2000. Thomas M. Terry

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Breakdown of glucose to pyruvate

1. Embden-Meyerhof (glycolytic) pathway (see handout)

• most common pathway -- found in all microbial groups
• View animation of glycolysis; overview, without chemical structures. (Note: Macromedia's shockwave plug-in is required).
• 3 stages: View glycolysis pathway
  1. 6-carbon phase: add two phosphate groups from ATP (net cost = 2 high-energy phosphate groups, or 2 ~P), then split into two 3-carbon molecules
  2. oxidation phase (energy release): glyceraldehyde-3-phosphate is oxidized. Lots of energy is released (~ 100 kcal). Some is temporarily trapped in electrons (+ protons) as NADH (reduced). Some is used to add inorganic phosphate (Pi) to the 3-C molecule, forming 1,3-bis-glyceric acid (this is substrate level phosphorylation, or SLP). Rest is released as heat.
  3. energy harvest phase: when phosphate groups are configured to high energy state, they can be passed to ADP ----> ATP. This happens twice for each 3-C molecule. Since one glucose produces two 3-C molecules, total yield at this stage is 4 ATP. The resulting 3-C molecule is pyruvic acid, or pyruvate.
  Net result:
  glucose + 2 ADP + 2 P_i + 2 NAD⁺ --->
  2 pyruvate + 2 ATP + 2 (NADH + H⁺)
  
• Note: total energy of glucose available by aerobic oxidation = 688 kcal/mole. Total energy yield of 2 ATP = 2 x 7.3 = 14.6 kcal/mole. This is ~ 2% efficient compared to aerobic respiration possibilities --- 98% of energy potentially available in glucose is not available to cell.

2. Entner-Doudoroff pathway (see handout)

• Another pathway for glucose breakdown discovered by Entner & Doudoroff. Has evolutionary, taxonomic significance.
• Found in Pseudomonas, Rhizobium, Azotobacter, others. Almost all Gram-negatives, and all aerobes (require oxygen, make their energy mainly from respiration).
• See Fig. 9.6 in text.
  Glucose-6-P goes to 6-phosphogluconate instead of Fructose 6-P, in an oxidation reaction that produces NADPH, not NADH.
  6-phosphogluconate then goes to 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDGP is split into two 3-C molecules. One is pyruvate; the other is glyceraldehyde-3-P (same 3-C compound as in Embden-Meyerhof pathway. Subsequent reactions are same as in E-M pathway and produce 1 more pyruvate.
• Note that only E-D pathway produces only 1 ATP net gain, instead of 2 as in E-M pathway.

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Problem: what do with electrons removed by oxidation reactions?

The critical role of NAD and other temporary electron carriers

NADH (and NADPH) are present in very small amounts. Unless quickly oxidized back to NAD⁺ (or NADP⁺), will stop all further oxidation reactions that need these as coenzymes.

Must find some terminal electron acceptor to get rid of electrons ---> waste products to be excreted from cell. What are options?

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Solution 1: Fermentation

• basic schema: use organic molecule derived from foodstuffs being metabolized as electron acceptor
• Pyruvate (or molecules derived from pyruvate) is available from glucose breakdown. Many cells use this as terminal electron acceptor, create waste products to be dumped out of cell.
• Note: these wastes are still high in energy content if they could be further oxidized; must be excreted in enormous concentrations because each molecule of glucose yields so little energy (only 2 ATP or so, roughly 2-3% efficient when looking at potential 688 kcal available by complete aerobic oxidation of glucose.)
• Actually, efficiency of fermentation is not bad considered by itself:
  • Glucose -------> 2 lactic acid
  • delta G_o' = -29 kcal/mole
  • since we harvest 2 ATP, we recover ~ 14.6 kcal, almost 50% efficient!
• Not as efficient as respiration; but does allow catabolism to continue in absence of oxygen, better than nothing at all.
• Some bacteria (e.g. Lactic acid bacteria, including streptococci and lactobacilli) gain all their from fermentation, have no ability to respire.

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Lactic acid fermentation

• pyruvate + NADH -------> lactic acid + NAD⁺
• found in many bacteria: lactic acid bacteria, Bacillus, also in some protozoa, water molds, even human skeletal muscle
• Responsible for souring of milk products ---> yogurt, cheese, buttermilk, sour cream, etc. Excellent keeping properties.
• Some bacteria produce only lactic acid = Homolactic fermenters
• Other bacteria produce other products as well; ethanol, CO₂, lactate, etc. = Heterolactic fermenters

Alcoholic fermentation (2 steps)

• pyruvate --------> acetaldehyde + CO₂
• acetaldehyde + NADH -------> ethanol + NAD⁺
• Found in many fungi, yeasts, some bacteria.
• Very important in human applications. Bread, alcoholic spirits.

Note: WWI -- German biochemist Neuberg solved critical problem of glycerol shortage caused by Allied blockade, needed for explosives.

Add sodium bisulfite to fermenting yeast; adds to acetaldehyde, blocks its use as electron acceptor. Yeasts adapt, use DHAP as electron acceptor, produce glycerol-3-phosphate, then glycerol as waste product.

Formic acid and mixed acid fermentations

• pyruvate (3-C) + CoA -------> Acetyl-CoA (2-C) + formic acid (1-C)
• HCOOH ------> CO₂ + H₂
• found in many bacteria, very common in enterics (Gram-negative faculative anaerobic rods, include E. coli and other common intestinal tract denizens).

useful in identification: 2 common variants

1. Mixed acid fermentation: some bacteria use several pathways, produce ethanol, formic acid, acetic acid, lactic acid, succinic acid, CO₂, and H₂. Note lots of acid, lower pH than many other fermentations.

   Note: ATP yield via mixed acid is ~2.5 ATP/glucose, a bit higher than straight lactic acid fermentation

2. Butanediol fermentation: butanediol produced, also much more CO₂, and H₂

Why not get rid of hydrogen directly as H₂ gas?

• Certain bacteria can do this.
• Clostridia (obligate anaerobes): can oxidize pyruvate to acetyl-CoA, reduce ferredoxin (has very low redox potential, E_o' = -420mv). Hydrogen gas can be liberated directly. Responsible for gas produced by botulism (C. botulinum) that causes swollen cans in improperly sterilized canning process.
• Another possibility: can oxidize NADH directly, using ferredoxin as reductant. But this involves change to a more negative redox potential (from E_o' = -320mv for NADH to E_o' = -420mv for ferredoxin). Can only happen if H₂ is removed, so its concentration remains low. This does occur in nature when certain hydrogen using bacteria are present (e.g. methanogens) and hydrogen doesn't have a chance to accumulate.
• Note: hydrogen production in biosphere is significant.

Roles of fermentation in nature

• Fermentations play major role.
• large part of cellulose ingested by herbivores is excreted in undigested form.
• Wherever organic matter accumulates, bacteria can grow and remove oxygen (by respiration), leading to anaerobic conditions that favor fermentation.
• Even in lab cultures (test tubes of media), bacteria eat up all available oxygen, rely largely on fermentation unless vigorous aeration is maintained! Bacteria are pigs, gorge themselves at every opportunity!
• Beside bacteria, fermentations also carried out be protozoa, fungi, even animal muscle tissues (only works as temporary energy supplement).

What substances can be fermented?

• must have intermediate oxidation state (o.s.)
• if totally oxidized (-CO)n cannot be fermented
• if totally reduced (-CH₂)n, cannot be fermented
• must be convertible to a substrate for substrate level phosphorylation (usually into some glycolytic step)
• Many sugars can be fermented. Also amino acids (e.g. by Clostridia, oxidizing one amino acid and using a different amino acid as electron acceptor.)

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