Catabolism: Fermentation & Respiration

Reading: Chapter 9 in Prescott et al, Microbiology 4th Ed.
Last revised: Wednesday, January 5, 2000

Note: These lecture notes were not written, and should not be used, as a substitute for the lecture. Notes are presented here only as an aid to students who missed lecture, or whose notes may be incomplete in certain particulars.

If you wish a record of these notes, you may save them as a text file and use them on your own computer, or print them in the computer lab (see lab supervisor for details).

Copyright 1999. 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:
  1. View glycolysis pathway
  2. 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
  3. 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.
  4. 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.

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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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Solution 2: Respiration

• Use an external electron acceptor. Oxygen as prototype.
• The "problem" with fermentation is that, by using an organic molecule as a terminal electron acceptor to be discarded as waste, cell is losing out on potential to further oxidize organic molecule, get more energy.
• Alternative solution is to use some non-organic molecule that has a low redox potential, can accept electrons and become some reduced molecule. Oxygen is perfect for this, has extremely low redox potential, and becomes reduced to water, the "perfect" waste product for an aqueous environment.
• To transfer electrons (and protons, H⁺) to oxygen, need special oxidase enzyme. In mitochondria, this is a cytochrome, cyt a. In bacteria, different cytochromes; in E. coli, cyt o or d.
• Note: respiration depends on availability of external electron acceptor. As soon as this is used up, respiration ceases.

Electron transport system (ETS)

• Although cells could transfer electrons directly from NADH to oxygen, this would liberate all energy in NADH directly as heat.
• NADH possesses lots of energy. If electrons are transferred directly to oxygen:
  NADH + O₂ -------> NAD + H₂O, delta G_o' = - 52 kcal/mole
  (Note: calculate this from redox tower -- see handout)
• If NADH has ~52 kcal of energy, and it only takes 7.3 kcal to make one ATP, could conceivably make 52/7.3 = ~ 7 ATP per NADH if energy conversion were 100% efficient.
• In practice, cells have evolved ways to get up to 40% efficiency (~ 3 ATP/NADH) under optimal circumstances.
• Electron transport system (ETS) = membrane-bound pathway transferring electrons from organic molecules to oxygen.
• ETS moves both electrons and protons:electrons are passed from carrier to carrier in the membrane, while protons are moved from inside to outside of membrane
• Net result: electrons enter ETS from carriers like NADH or FADH, wind up at terminal oxidase, get attached to oxygen.
• ETS consists of 4 complexes, connected by mobile carriers (Coenzyme Q, cytochrome c) that shuttle between complexes in membrane
• View animation of electron transport system

Specific carriers of ETS:

1. mitochondria (in eucaryotes): NADH ---> (Flavoprotein ---> Iron sulfur proteins ---> Quinone ---> cytochrome b ---> cytochrome c ---> cytochrome a ---> cytochrome a₃ ---> oxygen
2. bacteria (prokaryotes) have different ETS carriers, shorter chains. In E. coli, can have two different terminal oxidases, one functions at high oxygen levels, one at lower oxygen levels. Cytochromes involved include: b₅₅₈, b₅₉₅, b₅₆₂, d, and o

proton gradient and oxidative phosphorylation (oxphos)

Chemiosmotic hypothesis (Peter Mitchell, 1961)

• As electrons flow through ETS, at certain steps protons (H+) are moved from inside to outside of the membrane.
• This builds up proton gradient; since + charges are removed from inside of cell, - charge remains inside, mainly as OH^- ions.
• pH just outside membrane can reach 5.5, pH just inside membrane can reach 8.5 ---> difference of 3 pH units, or 1000x concentration differential of H+ across membrane. This represents potential energy stored up in proton gradient = protonmotive force.
• Membrane is basically impermeable to protons, so gradient doesn't get squandered away by leaky reentry.
• ATP synthase protein complex contains only channels for proton entry. As protons push in through channel, ADP + P_i ---> ATP. This can be called chemiosmotic phosphorylation (assuming chemiosmotic hypothesis is correct), or oxidative phosphorylation (makes no assumption about mechanism).

differences between respiration in mitochondria (eucaryotes) and bacteria (procaryotes)

1. In Eukaryotes:
   • ETS located in inner mitochondrial membrane. Proton gradient develops across inner mitochondrial membrane.
   • Mitochondria are very efficient at generating proton gradient. Can measure how many ~P bonds (in ATP) are made for each O₂ consumed = P/O ratio.
   • With NADH as electron donor, P/O ratio can be 3 (means 3 ATP made per NADH).
   • But with FADH as electron donor, P/O ration only 2 (fewer protons are transported, less proton gradient).
   • Overall efficiency of respiration in mitochondria: ~ 40% (means that about 40% of energy in glucose actually gets converted to ATP).
2. In Prokaryotes:
   • ETS located in cytoplasmic membrane. Proton gradient develops across this membrane.
   • Bacteria are not as efficient. ETS chains are shorter, P/O ratios are lower.
   • As a ballpark estimate, P/O ratios for NADH are only ~2. Overall efficiency of glucose oxidation is closer to 28%, not 40%.

Inhibitors of Oxidative Phosphorylation

• Several chemicals can block electron transfer in ETS, or transfer of electrons to oxygen. All are strong poisons. Some examples:
  • Carbon monoxide -- combines directly with terminal cytochrome oxidase, blocks oxygen attachment
  • Cyanide (CN^-) and Azide (N₃^-) bind to cytochrome iron atoms, prevent electron transfer.
  • Antimycin A (an antibiotic) inhibits electron transfer between cyt b and c.

Anaerobic respiration:

• Use of acceptors other than oxygen.
• Most common in bacteria. Most alternative electron acceptors are inorganic molecules, but some organic molecules can serve.
• As with aerobic respiration, anaerobic respiration uses ETS, membrane localization, proton gradient, and ATP synthase.
• Processes are of great importance both ecologically and industrially.

Examples of anaerobic respiration:

1. Nitrate (NO₃^-).
   • Process called denitrification. Also called dissimilative nitrate reduction. Reduced waste products are excreted in significant amounts.
   • Redox potential is + 0.42 v (compared to + 0.82 v for oxygen). So organisms respiring anaerobically gain less energy than with oxygen.
   • Requires new terminal oxidase called nitrate reductase. Enzyme is repressed by oxygen, synthesis turned on in absence of oxygen.
   • Process can have several steps, proceed in two different directions:
     
     1. (A) nitrate (NO₃^-) ---> nitrite (NO₂^-) ---> ---> ---> ammonia (NH₃)
        
     2. (B) nitrate (NO₃^-) ---> nitrite (NO₂^-) ---> nitrous oxide (N₂O) ---> ---> dinitrogen gas (N₂)
        
   • Second process is major pathway for loss of nitrogen compounds from soil, return of nitrogen to atmosphere.
   • Pseudomonas species are common denitrifiers, widespread in soils. When fertilized soils become flooded, oxygen is rapidly depleted, pseudomonads switch to anaerobic respiration and can use up soil nitrate, leaving field in unfertile state.
   • Note: Studied this in lab. Media must contain nitrate in addition to nutrients, otherwise won't work. Also, in scavenger hunt at end of course, one target microbe will be Pseudomonas, enrichment culture depends on its ability to grown anaerobically using nitrate reduction.
2. 2. Sulfate (SO₄^2-).
   • Process called sulfate reduction.
   • Sulfate (SO₄^2-) ---> ---> ---> ---> Hydrogen Sulfide (H₂S)
   • Small group of bacteria carry out this reaction; all obligate anaerobes.
   • Have unique cytochrome c3.
   • Sulfate is common in seawater. Often, H₂S combines with iron, forms insoluble FeS ----> black sediments. Common in estuaries.
3. 3. Carbon dioxide (CO₂).
   • One of most common inorganic ions.
   • Methanogens: most important group of CO₂ reducers. Obligate anaerobes, archaebacteria. Produce methane as waste product.
   • Reaction: CO₂ + H₂ + H⁺ ---> CH₄ + H₂O
   • Note: reaction also requires Hydrogen gas. Methanogens typically live alongside bacteria that produce hydrogen by fermentation, remove hydrogen as it is made.

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TCA cycle: further catabolism of pyruvate

formation of acetyl-CoA

• Oxidation of pyruvate (3-C) + NAD⁺ -------> Acetyl-CoA (2-C) + CO₂ + NADH
• Carried out by pyruvate dehydrogenase (multienzyme system)
• Note: Acetyl-CoA can also be produced by breakdown of lipids or certain amino acids -- important focal point of central metabolism

net effects of TCA cycle (see handout)

• To start cycle:
  Acetyl-CoA (2-C) + oxalacetate (4-C) -------> citric acid (6-C)
• Subsequent steps:
  1. Convert citrate to isocitrate (still 6-C)
     
  2. Oxidize -------> alpha-ketoglutarate (5-C) + CO₂ + NADH
     
  3. Oxidize -------> succinyl-CoA (4-C) + CO₂ + NADH
     
  4. SLP reaction: succinyl-CoA (4-C) + GDP -------> succinate (4-C) + GTP (Note: GTP can be interconverted with ADP to form ATP)
     
  5. Oxidize -------> fumarate (4-C) + FADH₂ -- convert fumarate to malate
  6. (6)oxidize again -------> oxalacetate (4-C) + NADH
• Net yield: Acetyl-CoA (2-C) + 3 NAD⁺ + FAD -------> 2 CO₂ + 3 NADH + FADH2 + ATP
• TCA cycle completes the oxidation of carbons in pyruvate to most oxidized form (CO₂); removes electrons originally in C-H bonds to electron carriers NADH and FADH for use in respiration machinery.

Catabolism of substances other than glucose

• Many other possible C-sources for catabolism beside glucose. In general, must convert these into molecules that can enter into central metabolism, either in glycolysis or TCA cycle.
  1. carbohydrates
     • Most abundant C-sources in most environments, most in various polysaccharides (cellulose, starch, lignin, etc.)
     • To gain access to sugars, must first secrete hydrolytic enzymes that break down glycosidic bonds in polysaccharides, produce mono- and disaccharides that can be transported into cells.
     • Starch, glycogen -- easily hydrolyzed by amylases
     • Cellulose -- difficult to digest, very insoluble, tightly folded. Many fungi, some bacteria produce cellulases.
     • Agar -- some marine bacteria produce agars
     • Once mono- or di-saccharides are available, they are transported into cell, converted into some typical glycolytic intermediate such as glucose-6-phosphate, catabolized by glycolytic enzymes.
  2. lipids
     • Biological lipids common as triglycerides, diglycerides.
     • To catabolize, bacteria secrete lipases, hydrolyze glycerides to free fatty acids and glycerol.
     • Fatty acids attacked by Beta-oxidation pathway.
     • Using FAD and NAD+ to remove electrons, 2-C units are removed as Acetyl-CoA, feed directly into central metabolism at TCA cycle entry. Glycolysis pathway not involved (except for use in synthesizing sugars needed for cell wall, running sections of pathway in reverse).
  3. proteins
     • Proteins must first be hydrolyzed by protease enzymes, to get individual amino acids which can be transported into cells.
     • Amino acids all have common structure: NH₂ - RCH - COOH.
     • 1st step in catabolism is to remove amino group (deamination), often by swapping it with another substrate (transamination).
     • Typical example: glutamic acid (an AA) + pyruvate ---> alpha-ketoglutarate + alanine (= pyruvate + amino group). Now alpha-KG can be oxidized in TCA cycle, since it is a TCA cycle compound.
     • As excess amino groups accumulate, must be secreted as waste products, possibly as ammonium ion (leads to alkaline pH).

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