Biochemical Engineering

8CH01

Dr. Sudhir Ranganath

Contact Hours/Week: 3 (Lecture) Credits: 3.0

CIE Marks: 50
Total Lecture Hours: 39 SEE Marks: 50
UNIT IV
KINETICS OF CELL GROWTH & PRODUCT FORMATION

Major Cellular metabolic pathways

Growth kinetics in batch culture (Monod)

Substrate-limited growth kinetics

Kinetic models with growth inhibitors

Transient cell growth kinetics

Cell growth in continuous culture

Cellular Metabolic Pathways
The total of all chemical reactions in a cell is Metabolism

Factors affecting metabolism

Genetic factors
Differences in responses to environmental changes
Nutritional changes

Metabolic control via nutritional & environmental regulation is critical

Eg: S. cerevisiae produces ethanol in anaerobic conditions, but produces yeast
cells (baker’s yeast) in aerobic conditions.

Aerobic metabolism
Anaerobic metabolism

Catabolism: Intracellularly degrading a compound into smaller products

and produces energy for the cell.
Glucose to CO2 and H2O
Anabolism: Synthesis of complex compounds & needs energy
Glucose to Glycogen
Bioenergetics
Fueling reactions (breakdown of nutrients)
Biosynthesis reactions (formation of building blocks)
Polymerization reactions (formation of macromolecules)

ATP + H2O ↔ ADP + Pi ΔGo = -7.3 kcal/mol

ADP + H2O ↔ AMP + Pi ΔGo = -7.3 kcal/mol

Bioenergetics
ATP analogs are GTP, UTP and CTP
High energy phosphate compounds such as phoshphoenol pyruvate and
1,3-diphosphoglycerate
Transfer of P group into ATP
Transfer of P group from ATP into low-energy phosphate compounds such
as glucose-6-phosphate & glycerol-3-phosphate
Bioenergetics
Use of Hydrogen atoms in Redox reactions
NAD+ and NADP+ are electron (or H atoms) acceptors
NADH and NADPH are electron donors
Reducing power: NADH & NADPH supply H atoms for biosynthesis
Eg: CO2 fixation by autotrophic organisms

CO2 + 4H → CH2O + H2O

ATP formation in respiratory metabolism:

NADH donates H (electrons) to oxygen via intermediates (respiratory
chain)
Energy released results in the formation of three ATP molecules
In the absence of O2, other electron acceptors such as NO3- could be
used (anaerobic respiration)
Glucose metabolism: Glycolysis & TCA cycle
Catabolism of glucose
Glycolysis or EMP pathway for fermentation of glucose to pyruvate
Krebs, TCA or citric acid cycle for conversion of pyruvate to CO2 and
NADH
Respiratory or electron transport chain for ATP formation by transferring
electrons from NADH to an electron acceptor
Glycolysis or Embden-Meyerhof-Parnas (EMP) Pathway

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Glycolysis or EMP Pathway
Overall reaction in glycolysis is

Glucose + 2 ADP + 2 NAD+ + 2 Pi Pyruvate + 2 ATP + 2(NADH + H+)

Pyruvate is a key metabolite

Glycolysis takes place in the cytoplasm

Anaerobic conditions: Pyruvate converts into lactic acid, ethanol, acetic

acid, butanol or acetone, etc……FERMENTATION

Aerobic conditions: Pyruvate converts into CO2 and NADH through the
TCA cycle
Kreb’s Cycle or Tricarboxylic Acid (TCA) Cycle
Pyruvate transfers its reducing power to NAD+ via Kreb’s cycle
Kreb’s cycle takes place in the matrix of mitochondria
Entry into Kreb’s cylce is as follows

Pyruvate + NAD+ + CoA-SH → acetyl CoA + CO2 + NADH + H+

Kreb’s Cycle or Tricarboxylic Acid (TCA) Cycle
Kreb’s cycle produces NADH, H+ and FADH2 which have reducing power
Reducing power used either for biosynthesis or ATP generation through the
electron transport chain
Overall reaction for TCA cycle is:

acetyl CoA + 3NAD+ + FAD + GDP + Pi + H2O →

CoA + 3(NADH + H+) + FADH2 + GTP + 2 CO2

Major roles of TCA cycle are:

Provide electrons (NADH) for electron transport chain & biosynthesis
Supply C skeletons for amino acid synthesis (succinic acid, oxaloacetic
acid, a-ketoglutaric acid)
Generate energy
Respiration (Electron Transport Chain)
Formation of ATP from electron transport chain
Oxidative phosphorylation
Electron carried by NADH + H+ transferred to oxygen via a series of
electron carriers…forming ATP
3 ATPs are formed from 1 (NADH + H+)
2 ATPs are formed from 1 FADH2
Major roles of electron transport chain
Regenerate NADs for glycolysis
Produce ATPs for biosynthesis
Overall reaction of aerobic glucose catabolism

Glucose + 36 Pi + 36 ADP + 6 O2 → 6 CO2 + 6 H2O + 36 ATP

Effect of dissolved O2 or O2 partial pressure on rate of glycolysis is called

Pasteur Effect
Rate of glycolysis under anaerobic conditions > under aerobic conditions
Metabolism of Nitrogenous Compounds
Nitrogenous compounds: source for N, C and energy
Proteases hydrolyze proteins into amino acids
Amino acids are converted to organic acids by deamination

O
||
R-CH-COOH + HO + NAD+ → R-C-COOH + NH + NADH + H+
|
NH2

Ammonia released utilized in protein & nucleic acid synthesis as N source

Organic acids utilized for energy production (ATP)

Transamination also used for conversion of amino acids to organic acids &
other amino acids

glutamic acid + oxaloacetic acid → a-ketoglutaric acid + aspartic acid

Metabolism of Nitrogenous Compounds
Nucleic acids utilized as C, N & energy sources by many organisms
Nucleases hydrolyze DNA and RNA producing ribose/deoxyribose,
phosphoric acid, and purine/pyrimidine bases
Phosphoric acids used in ATP, phospholipid & nucleic acid synthesis
Purines/pyrimidines degrade into urea & acetic acid and then to ammonia
and CO2

adenine → CO2 + NH3 + acetic acid + urea → 5 NH3 + 5 CO2

Nitrogen Fixation
Under reductive conditions, MOs fix atmospheric nitrogen
Eg: Azotobacter, Azotomonas, Azotococcus, etc
Catalyzed by Nitrogenase (which is inhibited by oxygen)
MOs sequester Nitrogenase in compartments protected from oxygen

N2 + 6 H+ + 6 e → 2 NH3
Azotobacter (in soil) fixes atm N2 & provides NH3 for plants
Metabolism of Nitrogenous Compounds
Nitrogen Fixation
Azotobacter (in soil) fixes atm N2 & provides NH3 for plants
Facultative anaerobes Rhodospirillum, Bacillus, Klebsiella fix N2 under strict
anaerobic conditions
Strictly anaerobic microbes Clostridia fix N2
Cyanobacteria fix N2 in aerobic conditions
Rhizobium (found in roots of leguminous plants) fixes N2 under low oxygen
pressure and provides ammonia to plants
Rhizobium used as fertilizers
Metabolism of Hydrocarbons
Metabolism of aliphatic hydrocarbons
Few organisms such as Pseudomonas, mycobacteria, etc can metabolize
hydrocarbons
Low solubility of hydrocarbons in water slows rapid metabolism

Steps in the metabolism of aliphatic HCs

Oxygenation of HCs into alcohols by Oxygenase by the incorporation of
oxygen into the end of the carbon skeleton
Alcohol oxidized to aldehyde and then to an organic acid and finally to
acetyl CoA
Acetyl CoA is then metabolized by the TCA cycle

Steps in the metabolism of aromatic HCs

Slower compared to aliphatic HCs

benzene → cathecol → cis-cis-muconate → b-keto adipate → acetyl CoA +

succinate
Biosynthesis
Synthesis of amino acids, nucleic acids, lipids and polysaccharides

Hexose-Monophosphate Pathway (HMP) or Pentose-

Phosphate Pathway
Provides carbon skeletons for biosynthetic reactions via NADPH
Reducing power for supporting anabolism

Provides an array of small organic

compounds with 3 to 7 C atoms

Important for synthesis of ribose,

purines, coenzymes & aromatic amino
acids

Lipid & polysaccharide synthesis via

acetyl CoA and CO2
Biosynthesis
Gluconeogenesis
Production of glucose for synthesis of polysaccharides

Reverse of glycolysis gives glucose…but most reactions in the EMP

pathway are irreversible

Pyruvate synthesis is the starting point & forms phosphoenolpyruvate (PEP)

pyruvate + 2 ATP + H2O → PEP + 2 ADP + 2 H+

Fructose-1,6-diphosphatase & glucose-6-phosphatase are needed to

complete gluconeogenesis
Anaerobic Metabolism
Production of energy in the absence of oxygen
Same pathways as aerobic metabolism used but with different electron
acceptors

Eg: NO3- and its product N2O- as electron acceptors

Denitrification is an environmental friendly process used in biological waste

water treatment

Fermentation
Energy generation without the electron transport chain is called
fermentation

Hence, the organic substrate must undergo a balanced series of oxidative &
reductive reactions

Rates of conversion of NAD+ & NADP+ to NADH & NADPH must equal the
rates of conversion of NADH & NADPH to NAD+ & NADP+
Examples are lactic acid and ethanol production
Anaerobic Metabolism

Other partially oxidized by-products of fermentation are:

acetone-butanol fermentation, propionic acid, acetic acid, isopropanol,
2,3-butanediol and glycerol
Anaerobic Metabolism

No one organism is able to make all these products.

Hence it is important to screen wide varieties of them & select the ones
with maximum yield of a desired product

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Autotrophic Metabolism
Use of CO2 as the carbon source
Fix CO2 via the Calvin-cycle

Photoautotrophic
Chemoautotrophic

Eg: Photosynthesis

Overall reaction

light
6 CO2 + 6 H2O → C6H12O6 + 6 O2

light
Light Phase: H2O + NADP + Pi + ADP → oxygen + NADPH + H + ATP

Dark Phase: CO2 + NADPH + H+ + ATP → 1/6 glucose + NADP + ADP + Pi

Autotrophic Metabolism
Procaryotes: Photosynthesis in stacked membranes
Eucaryotes: Photosynthesis in Chloroplasts
Chlorophyll is the light absorbing molecule
Electronic excitation & returns to normal
state by emitting a photon (quanta)
called as fluorescence
Excited chlorophyll donates an
electron to enzymes and ATP is made
Photophosphorylation
Thank you