III. Fermentation and Respiration Overview An emphasis on the flow of carbon and other elements in fermentation and respiration 3.8 Glycolysis 3.9 Fermentative Diversity and the Respiratory Option 3.10 Respiration: Electron Carriers 3.11 Respiration: The Proton Motive Force 3.12 Respiration: Citric Acid and Glyoxylate Cycle 3.13 Catabolic Diversity

III. Fermentation and Respiration Overview Two key metabolic pathways Complementary Overlapping Definition depends on context-industrial, medical, biochemical

Fermentation In food science fermentation can refer to the production of foods such as yogurt In chemical engineering it can refer to the production of ethanol as an additive for gasoline In microbiology it refers to the breakdown of carbon compounds (eg glucose) to smaller compounds with a limited harvest of energy through substrate level phosphorylation and no oxygen used

Respiration In medicine or exercise science respiration refers to breathing In microbiology respiration refers to the removal of electrons from a substance and their transfer to a terminal acceptor with a significant harvest of energy through oxidative phosphorylation (redox reactions). Oxygen may be used as the terminal acceptor (aerobic respiration) or not (anaerobic respiration).

Fermentation: substrate-level phosphorylation; ATP is directly synthesized from an energy-rich intermediate Respiration: oxidative phosphorylation; ATP is produced from proton motive force formed by transport of electrons

Fermentation A basic and important process for microorganisms A sugar is the starting material and the end product depends on the species Three Stages (I) Preparation, (II) Energy Harvesting, (III) Reboot Reboot stage is very diverse!

3.8 Glycolysis Stage I and Stage II (Figure 3.14) called Glycolysis (Embden–Meyerhof-Parnas or EMP pathway): a common pathway for catabolism of glucose End product of glycolysis is pyruvate (pyruvic acid) In fermentation pyruvate is processed through Stage III It accepts electrons so is reduced

Figure 3.14 Fermentation with lactic acid produced Figure 3.14 Embden–Meyerhof–Parnas pathway (glycolysis). Figure 3.14

3.9 Fermentative Diversity and the Respiratory Option Fermentations may be classified by products formed (See Sec.13.12) in Stage III Ethanol Lactic acid (homolactic vs heterolactic) Propionic acid “Mixed acids” Butyric acid (extra ATP generated) Butanol

3.9 Fermentative Diversity and the Respiratory Option Fermentations may be classified by substrate fermented (See Sec.13.12) Usually NOT glucose Amino acids Purines and pyrimidines Aromatic compounds

3.9 Fermentative Diversity and the Respiratory Option Fermentation Helps detoxify and eliminate waste products Provides metabolites for other microbes in the environment May help to recover additional ATP Maintains redox balance (page 87 and Fig. 3.14) of NAD and NADH.

3.9 Fermentative Diversity and the Respiratory Option Fermentation Helps detoxify and eliminate waste products Provides metabolites for other microbes in the environment May help to recover additional ATP Maintains redox balance (page 87 and Fig. 3.14) AND……. Helps to generate precursor metabolites for anabolism

Broad Overview of Metabolism Prokaryotes will not make something if they can import it There are only a few key precursor molecules (but lots of ways to make them) Energy sources vary

3.9 Fermentative Diversity and the Respiratory Option Pentose Phosphate Pathway (Shunt) “Alternate” pathway Runs “parallel” to glycolysis Different reactions thus different intermediates Generates different precursor metabolites Generates reducing power

3.12 Respiration: Citric Acid and Glyoxylate Cycle Citric acid cycle (CAC): pathway through which pyruvate is completely oxidized to CO2 (Figure 3.22a) (aka Krebs or TCA cycle) Initial steps (glucose to pyruvate) same as glycolysis Subsequently 6 CO2 molecules released and NADH and FADH generated Plays a key role in both catabolism AND anabolism…why?

Figure 3.22a The citric acid cycle.

3.12 Respiration: Citric Acid and Glyoxylate Cycle The citric acid cycle generates many compounds available for biosynthetic purposes - α-Ketoglutarate and oxaloacetate (OAA): precursors of several amino acids; OAA also converted to phosphoenolpyruvate, a precursor of glucose Succinyl-CoA: required for synthesis of cytochromes, chlorophyll, and other tetrapyrrole compounds Acetyl-CoA: necessary for fatty acid biosynthesis

In other words-the citric acid cycle generates key precursor metabolites As well as harvesting energy

3.12 Respiration: Citric Acid and Glyoxylate Cycle The Citric Acid Cycle is also a key collection point and entry point for metabolites C4-C6 citric acid cycle intermediates (e.g., citrate, malate, fumarate, and succinate) are common natural plant and fermentation products and can be readily catabolized through the citric acid cycle alone Fatty acids metabolized via Acetyl-CoA

3.12 Respiration: Citric Acid and Glyoxylate Cycle A variation of the citric acid cycle with glyoxylate as a key intermediate Shares enzymes with citric acid cycle Allows utilization of C2-C3 organic acids if larger molecules not available (Figure 3.23) Isocitrate to glyoxylate and succinate For anabolism or catabolism

Figure 3.23 The glyoxylate cycle.

3.10 Respiration: Electron Carriers Important electron carriers embedded in membranes include: NADH dehydrogenases Flavoproteins Cytochromes (heme) Iron-sulfur proteins Quinones

Respiration is much more productive than fermentation Aerobic respiration is the most productive of all Figure 3.22b

13.5 Autotrophic Pathways (pp 390-391) Carbon fixation is reduction of CO2 to carbohydrate-a key feature of autotrophy At least 6 pathways exist in various Archaea and Bacteria But the Calvin cycle (Figure 13.16, 13.17) is the most important

13.5 Autotrophic Pathways (pp 390-391) Named for its discoverer, Melvin Calvin Fixes CO2 into cellular material for autotrophic growth Requires NADPH, ATP, CO2 and special enzymes e.g. ribulose bisphophate carboxylase (RubisCO), 6 molecules of CO2 are required to make 1 molecule of glucose (Figure 13.17)

Figure 13.17 The Calvin cycle.

13.5 Autotrophic Pathways: RUBISCO Responsible for most carbon fixation on planet Unusual enzyme-”weak”- when CO2 is low easily inhibited by oxygen Often found sequestered in carboxysomes to increase CO2 and lower O2

3.13 Catabolic Diversity Chemolithotrophy Uses inorganic chemicals as electron donors Examples include hydrogen sulfide (H2S), hydrogen gas (H2), ferrous iron (Fe2+), ammonia (NH3) Begins with oxidation of inorganic electron donor Uses an electron transport chain and transmembrane ion gradient

Dissimilative Iron Oxidizers are chemolithotrophs (13.9 and 14.15) Oxidize Fe2+ to Fe3+ Very widely distributed in many environments where Fe2+ is available Autotrophic or heterotrophic Aerobic or anaerobic Archaea or Bacteria

Acidithiobacillus ferrooxidans is a representative iron oxidizer Acidophile at pH 2-3 Acid environments with Fe2+ Fe2+ -> Fe3+ -> FeOH3

Figure 13.24 (pH 2) (pH 6) + ATP ATP e– e– Out Outer membrane cyt c Electron transport generates proton motive force. Rusticyanin e– Periplasm Reverse e– flow e– NAD+ Q cyt bc1 cyt c cyt aa3 Figure 13.24 Electron flow during Fe2+ oxidation by the acidophile Acidithiobacillus ferrooxidans. In (pH 6) + ATP NADH Cell material ADP ATP Figure 13.24

Figure 13.23 Iron-oxidizing bacteria.

3.17 Nitrogen Fixation (Sec 3.17 also pp 438-439) Living systems require nitrogen in the form of NH3 or R-NH2 “Fixed” or “reduced” nitrogen, not N2 Only some prokaryotes can fix atmospheric nitrogen: diazotrophs

3.17 Nitrogen Fixation Some nitrogen fixers are free-living, and others are symbiotic Cyanobacteria are free-living nitrogen fixers Soybean root nodules contain endosymbiotic Bradyrhizobium japonicum

3.17 Nitrogen Fixation Energetically expensive (8 ATP per N atom) Requires electron donor, often pyruvate Reaction is catalyzed by nitrogenase Sensitive to the presence of oxygen Fe plus various metal cofactors Can catalyze a variety of reactions