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Biochemistry of Fermentation Processes
Biochemistry of Fermentation Processes
Biochemistry of Fermentation Processes
Processes
David A. Boyles
Professor of Chemistry
Department of Chemistry and Chemical Engineering
South Dakota School of Mines and Technology
I. Overview of Fermentation
II. Biochemistry of Fermentation
Fermentation Background
Known since antiquity
Earliest use of term referred to natural
fermentation by wild and unidentified
microbes
Distinguish two kinds
Indigenous Fermentations
Technological Fermentations
INDIGENOUS FERMENTATIONS
Fermentation originally used to produce foods and
beverages
Many products have been standardized and
commercialized
Alesnatural yeasts
Cheesesnatural fungi
Winesnatural yeasts
Many others are produced commercially in
limited quantities for specialized markets, or
remain uncommercialized and are products
of indigenous, local cultures
kefir, kim-chi, sauerkraut, yoghurt, San
Francisco sourdough bread
Fermentation: Current
Definitions
In the strict biochemical sense of the term fermentation
involves the action of anaerobic organisms on organic
substrates
Modern usage extends definition to the microbiological
formation of smaller organic molecules, whether
aerobic or anaerobic
The component products of fermentation may be
isolated from the feedstock and purveyed as pure
substances, unlike fermentation of antiquity: eg.,
ethanol versus wine
Technological Fermentation:
Features
Large scale reactors for commercial production
Carefully controlled conditions
Optimized yields of pure products
Pure strains of microbes
Genetically engineered microbes by
recombinant technologies allowing
production of rare natural products such as
insulin, growth hormones, enzymes
Variety of Isolated
Fermentation Products
Classical Fermentation Products Before 1950
Organic molecules of six or fewer
carbons
Current Fermentation Products
Amino acids, and even (loosely) includes
proteins such as insulin, HGH,
polysaccharides
demand
Reliable
supply
Technological
Profitability
Downstream
Dateline
1859
Edwin Drake
Oil industry began in Titusville, Pennsylvania
1865
Louis Pasteur
1865 process to inhibit fermentation of wine and milk
1903
Henry Ford founds Ford Motor Company in 1903
Model T Automobile: By 1927, 15 million had been sold
1910
to 1919
WWI
1939
to 1945
WWII
Classic Fermentation
Products
from Technology
Ethanol
Acetone
Fermentation: Scale
Production will never replace
petroleum-based chemicals
Not enough agricultural biomass available
Biomass is oxygen-rich, unlike petroleum
which is carbon-rich, reducing mass
Production will serve to augment petroleumbased chemicals
Ethanol
Classic Fermentation
Products I
industrial solvent,
beverage, fuel
Saccharomyces
cerevisiae
Glycerol
food and
pharmaceutical use
Lactobacillus
delbrukki, bulgaricus
Acetone-Butanol
solvent
Clostridium
acetobutylicum
2,3-Butanediol
synthetic rubber
Bacillus polymyxa,
Acetobacter aerogenes
Classic Fermentation
Products II
Organic Acids
Acetic AcidSaccharomyces sp., Acetobacter
Lactic AcidLactobacillus delbruckii
Citric AcidAspergillus niger
Itaconic AcidAspergillus itaconicus
Ethanol
C2
1906
Organic Acids
Vinegar C2
French
Process: 1670
Organic Acids
Lactic Acid C3
Principal
Glycerol
C3
oils
Diverse use in explosives, foods, beverages,
cosmetics, plastics, paints, coatings
First identified by Pasteur
WWI demand exceeded supply, esp. in
Germanybecame leader in fermentation
At least one integrated plant took directly to
nitroglycerine
True,
Acetone-Butanol
C3 and C4
2,3-Butanediol
C4
Major
Organic Acids
Itaconic Acid C5
Resin
Organic Acids
Citric Acid C6
Made
Biochemistry of Fermentation
A.
B.
Overall Strategy
Bioenergetics
Energy transfer from highly negative G to
less negative G
Harvesting of electrons
Temporary energy storage
C.
A. Overall Strategy
Organic
atoms
electrons
Living
Organic
B. Bioenergetics
storage molecule
Electrons
Energy
ATP
ATP has
an intermediate energy of
hydrolysis
G of hydrolysis is 7.3 kcal/mol
Low compared to some, high compared to other
hydrolyses
ATP levels
Electron Carriers
Electrons
Two
NAD
Both
NAD
FAD
Both
carrier molecule
Electron
carrier molecules
Organic
dioxide
Particular
No
Two
Kinds of Metabolism
chemical transformations
Each
A
The
E1
E2
Metabolism: Specific
Pathways and Cycles
Glycolysis
Citric Acid
Electron
Cycle
Transport Chain
Glycolysis
Central
Embden-Meyerhof
Begins
with glucose C6
Requires
Ends
Pathway
10 discrete steps
with pyruvate 2 X C3
Anaerobic
pathway--primitive
Glycolysis: Features
Textbook,
page 133
One
The
Final
Fates depend on
Organism
Conditions
Tissue
Conversion by
Decarboxylation to ethanol 2C and carbon dioxide
1C
Decarboxylation to Acetyl CoA 2C and carbon
dioxide
Reduction by NADH to lactate 2C; regenerates
NAD+
Sequence:
pyruvate
acetaldehyde
ethanol
2 Pyruvate
O2
-2CO2
2 Acetyl CoA
Anaerobic conditions
2 Lactate
Some organisms,
contracting muscle
O2
Citric Acid Cycle: Aerobic conditionsanimal, plant,
microbial cells
4CO2 and 4 H2O
Glycolysis Energetics
Standard
Glycolytic
TCA: Background
Krebs Cycle,
Regarded
Is
TCA: Function
To continue
To produce
To continue
To serve
TCA: Schematic
Pyruvate 3C
Amino acids
Fatty acids
Acetyl CoA 2C
Oxaloacetate 4C
Citrate 6C
Isocitrate
Malate
Note: Sequence
is Clockwise
+ NADH
+ FADH2
Fumarate
Succinate
+2 carbon dioxi
Alpha-ketoglutarate
Succinyl CoA
Cytochromescopper
containing
FeS Centers
Coenzyme Q: a quinone
NADH
FMN
10
CoQ
cyt b
Eo
20
+0.2
+0.4
+0.8
kcal
30
cyt c
Protons are pumped across
membrane at each incremental
drop
40
cyt a
??
50
Reduction Potentials
measure the natural
(inherent) tendency of
substances to gain
electrons (be reduced)
Some substances naturally
gain electons more easily
than others: in the electron
transport chain, oxygen
gains them most easily of all
Synthesis of ATP
Proton
Gradient
ATP Bookkeeping
ATP Synthesis:
Proton Pumping During
Course of ETC
As electrons are passed from one carrier to
another along the chain, protons are pumped to
the OUTSIDE of the membrane
Protons build up outside the membrane,
lowering pH
A chemical gradient is thus produced
ATP Bookkeeping
Each NADH molecule produced in any pathway is
ultimately responsible for the production of 3 ATP
NADH
FADH2
Substrate
Level Phos.
Total
ATP
Glycolysis
0 produced
= 0 ATP
2
= 2 ATP
2 ATP
6 ATP
Pyruvate to
Acetyl CoA
2 produced
= 6 ATP
0 ATP
6 ATP
2
= 2 ATP
2 ATP
(GTP)
24 ATP
Total 36 ATP
Overall Energetics
36 ATP produced upon complete oxidation of
glucose
Multiplied times
-7.3 kcal/mol per each ATP (energy of hydrolysis of
ATP to ADP and inorganic phosphate)
EQUALS TOTAL STORAGE OF 263 kcal
ENERGY FROM GLUCOSE
(263 kcal/686 kcal)/100 = 38% of energy in
SUMMARY
1.
2.
3.
4.
FINALLY
Additional Pathways I
Pentose-Phosphate Pathway
Serves to harvest electrons
Is an alternative glucose pathway
Produces 5C sugar intermediates critical for
Additional Pathways II
Amino Acid Anabolism: From TCA intermedicates