CHE 309 Introductory Bioengineering

Chapter 5 Metabolism and Physiology of Microbial Cells

Objectives of Chapter 5
Identify functions and control sites of metabolic pathways Relate the major metabolic pathways for cell growth to their nutrition requirements Any changes due to metabolic engineering?

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Definitions
Metabolism = the sum of all biochemical reactions inside cells utilizing or making molecules Catabolism = intracellular process of degrading a compound into smaller and simpler products (e.g., glucose into CO2 and H2O) produces energy Anabolism = synthesis of more complex compounds from simple ones (e.g., glucose into glycogen) requires energy
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Bioenergetics
Energy sources (organic and inorganic materials) to produce ATP
Nitrates or forms of sulfur (elemental sulfur, sulfites, and H2S) - lithotrophs Light energy (photosynthesis) - autotrophs organic compounds (mostly carbohydrates, fats, and proteins) - heterotrophs

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Some Reactions in Bacterial Cells

Class I Fueling reactions Class II Biosynthesis reactions Class III Polymerization reactions

Glucose

ATP

Carbon skeletons

Amino acids Fatty acids

Chromosomes Envelope Storage

CO2 , waste organics

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NH4+ , SO42Figure 5.1 of textbook

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Adenosine Tri-Phosphate (ATP)

Active form is complexed with Mg2+ ATP + ADP + AMP = 215mM in cells Cytoplasm, organelles

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Nicotinamide Adenine Dinucleotide (NADH+)

Adenine

Donates or accepts electrons (or H atoms) in oxidation-reduction reactions

NADH supplies hydrogen for CO2 fixation

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3 mol ATP / 1 mol NADH in respiratory metabolism Chapter 3

Carbon Metabolism
Glycolysis
Embden-Meyerof-Parnas (EMP) pathway Ferments glucose into pyruvate

TCA (Tri Carboxylic Acid) cycle

Krebs cycle Citric acid cycle Converts pyruvate into CO2 and NADH Electron transport chain Formation of ATP Transfers electrons from NADH to an electron acceptor
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Respiration

Transport across membrane-1

Energy-independent uptake
Passive diffusion

Facilitated diffusion carrier molecule (protein)

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Transport across membrane-2

Energy-dependent uptake
Active transport against concentration gradient
Proton-motive force (pH gradient) Secondary gradients (Na+ or other ions)

Group translocation
Chemical modification phosphotransferase system Energy = phosphoenolpyruvate (PEP)
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Glycolysis
Cell membrane 1 = feedback inhibition 1

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Glycolysis
1. 2. 3. All reactions in cytoplasm

Dihydroxyacetone 2 mol ATP formed / mol of glucose (anaerobic)

Glyceraldehyde-3-phosphate

1. Control point: enzyme activated by ADP and Pi but inactivated by ATP Pasteur effect high oxygen concentration reduces rate of glycolysis ATPanaerobic < ATPaerobic
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TCA (Krebs) Cycle

1

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Krebs cycle
In mitochondria Converts pyruvate from glycolysis to CO2 and NADH

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Respiration
Feedback inhibition: cyanide, azide, carbon monoxide, Antibiotics (amytal)

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Respiration, contd.
Also called Electron transport chain Role = regenerate NADs for glycolysis and ATPs for biosynthesis Formation of ATP from respiration = oxidative phosphorylation FADH2 acts similarly to NADH + H+

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Respiration - eukaryotes
3 mol ATP / 1 mol NADH + H+ and 2 mol ATP / 1 mol FADH2
Table 1 Formation of NADH, FADH2 and ATP during aerobic catabolism of 1 mol glucose
NADH FADH2 ATP Total ATP1
Glycolysis 2 2 62 Oxidative decarboxylation of 2 6 pyruvate TCA cycle 6 2 2 24 Total 10 mol 2 mol 4 mol 36 mol 1 Assuming P/O = 3 for NADH, and P/O = 2 for FADH 2 2 2 NADH are converted to 2 FADH for transfer of acetyl CoA into mitochondria 2

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Eukaryotes versus prokaryotes

glucose + 36 Pi + 36 ADP + 6 O2 6 CO2 + 6 H2O + 36 ATP (eukaryotes)
Eukaryotes mol ATP generated from 1 mol NADH + H+ 3 (or 2.5) Prokaryotes 2

mol ATP generated from 1 mol FADH2 Yield of ATP from 1 mol glucose
Formation of 36 mol ATP (kcal / mol glucose) Gglucose oxidation (kcal / mol glucose) glycolysis (std. cond.) glycolysis (non-std. cond.)
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2 36 (or 30)
263 686 38% > 60%

1 < 24

< eukaryotes
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Nitrogen Metabolism
Oxidation level: carbohydrates > nitrogenous compounds > lipids So: nitrogenous compounds nitrogen, carbon, energy source

Protein 1 Peptides 1 Amino acids 2

Organic acids +

NH3

(medium )
1 proteases 2 deamination 3 transamination 4 hydrolysis

Protein

Peptides

Amino acids 3

Organic acids ATP (energy)

NH3

Protein, nucleic acids

Organic acids 4

Amino acids Purines / pyrimidines

Nucleic acids

Ribose / Phosphoric + acid deoxyribose ATP, phospholipids, nucleic acids

Cell
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CO2 , H2O aerobic

Urea, acetic acid

NH3, CO2
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Nitrogen Fixation
Air a) Anaerobic conditions: Facultative anaerobes: Bacillus, Klebsiella, Rhodopseudomonas, Rhodospirillum Strict anaerobes: Clostridia b) Aerobic conditions: Cyanobacteria: Anabaena sp. (to fungi) c) Low oxygen concentration (soil): Symbiotic Bacteria: Azotobacter, Azotomonas, Non-symbiotic Azotococcus, Biejerinckia, Symbiotic Rhizobium

Dissolved air
1

N2 + 6 H+ + 6 e 2 NH3
Cell
1 nitrogenase inhibited by O2

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Hydrocarbon Metabolism
Air

Dissolved oxygen Hydrocarbon

Medium

Ex., Gramnegative bacteria

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Hydrocarbon Metabolism, contd.

Bioremediation Pseudomonas, Mycobacteria, certain yeasts and molds Low solubility of hydrocarbons in water is barrier to rapid metabolism Metabolism rates in aerobic conditions
Aliphatic > aromatic

Anaerobic < aerobic

Only few organisms Yield methane
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Central and Intermediary Metabolism

glucose 3-phosphoglycerate serine, glycine, cystine aromatic amino acids valine, leucine, alanine fatty acids

aspartate, asparagine, threonine, isoleucine, methionine lysine O2 tyrosine, phenylalanine

phosphoenolpyruvate
pyruvate

lactate glycerides

acetyl-CoA
oxaloacetate malate fumarate citrate iso-citrate

acetate aromatic & branched amino acids

glutamate, glutamine, proline, arginine, NH3
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-ketoglutarate

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methionine, valine, isoleucine

succinyl-CoA
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histidine porphyrins

Pentose Phosphate Pathway

Also called Hexose Monophosphate Pathway NADPH is reducing power for biosynthesis

The 3-7 carbon units are converted into ribose, purines, coenzymes, and aromatic amino acids
Better picture in textbook, Figure 5.7
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Anaerobic Metabolism
In waste-water treatment processes, etc.

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Anaerobic Metabolism, Fermentation

Balanced series of oxidative and reductive reactions NADH = NAD + H+ Other examples: Acetone-butanol fuel Propionic acid Swiss cheese Acetic acid vinegar 2,3-butanediol fuel; rubber Isopropanol cosmetics Glycerol humectant in foods; soaps

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Major End Products from Pyruvate

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Anaerobic Metabolism Entner-Doudoroff Pathway

Alternate formation of pyruvate In Zymomonas fermentation to ethanol, 1 mol ATP / mol glucose (recall EM pathway yielded 2!) more glucose into ethanol than growth

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Anaerobic Metabolism Xylose Pathway

Sweetener for toothpaste and gum

Examples: Zymomonas, Pichia stipitis, etc.

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Autotrophic Metabolism Calvin-Benson Cycle

Reduction of fixed CO2
Energy from light (photosynthesis; ex., cyanobacteria, green sulfurbacteria and some purple bacteria) or chemicals (nitrifying or sulfur-oxidizing bacteria)

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Regeneration of the CO2 acceptor

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Photosynthesis
Overall reaction
6 CO2 + 6 H2Olight C6H12O6 + 6 O2

Light phase
6 H2O + 6 NADP+ + 6 Pi + 6 ADPlight oxygen + 6 NADPH + 6 H+ + 6 ATP

Dark phase
6 CO2 + 6 NADPH + 6 H+ + 6 ATP C6H12O6 + 6 NADP+ + 6 Pi + 6 ADP
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