Transcript History of Fermentation Processes and Their Fundamental

Biochemistry of Fermentation
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
Ales—natural yeasts
Cheeses—natural fungi
Wines—natural 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…
Advantages of Indigenous Products
Unique flavor profile
Enhanced storage
Disadvantages of Indigenous Products
Quality control—natural variations over
time, possibility of contamination
Difficult to mass produce
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
Criteria for Potential Industrial
Chemical Products and
Transformations

Favorable demand
Reliable
supply
eg., Citric acid
eg., petroleum, starch
Technological Knowledge
eg., intellectual capital
Profitability
eg., value added
Downstream Utilization
eg., food additive
eg., ‘THIS IS IT!’
Merchandising
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 and n-Butyl Alcohol
 Organic Acids
– Citric Acid
– Acetic Acid
– Lactic Acid
– Itaconic Acid
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
Classic Fermentation
Products I
Ethanol
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 Acid—Saccharomyces sp., Acetobacter
Lactic Acid—Lactobacillus delbruckii
Citric Acid—Aspergillus niger
Itaconic Acid—Aspergillus itaconicus
Ethanol
C2

1906 in US Industrial Act—denatured product was
legalized in the US
 WWII: demands for industrial product
increased—use for synthetic rubber and smokeless
gunpowder
 Whole grains, starches, sulfite liquors or
saccharine materials are used as feed stocks
 Saccharomyces cerevesiae cannot ferment starch
directly—amylases must first break down starch to
sugars
Organic Acids
Vinegar C2

French name vin + aigre
 Condiment and preservative
 Feedstock: sugary or starchy
 Slow Process: Orleans or French method
--”mother of vinegar”

Generator Process: 1670
--fast process, maximum air exposure

Cider (apples), wine (grapes), malt (barley),
sugar, glucose, spirit (grain) used for
biomass
Organic Acids
Lactic Acid C3



1790 by Scheele from milk
Present in sour milk, sauerkraut, bread, muscle
tissue, principal organic soil acid
1881 Commercial production by Chas. Avery,
Littleton, Mass
as substitute for cream of tartar

Dextrose, maltose, lactose, sucrose, whey
Starch, grapefruit, potatoes, molasses, beet juice

Dimerizes to lactide upon heating
PURAC for applications
Glycerol
C3

Principal source is saponification of fats and
oils
 Diverse use in explosives, foods, beverages,
cosmetics, plastics, paints, coatings
 First identified by Pasteur
 WWI demand exceeded supply, esp. in
Germany—became leader in fermentation
 At least one integrated plant took directly to
nitroglycerine






Acetone-Butanol
C3 and C4
True, anaerobic fermentation by Clostridium
Major development during WWI: used for
synthetic rubber via butadiene; critical commodity
for cordite
WWII production was solely by fermentation
1861 Pasteur first observed formation; 1905
Schardinger
1916 Chaim Weizmann procedure first industrial
use in Canada, Terre Haute for WWI production
1926 Demand for lacquers: Peoria
– 96 fermentors in use, cap. 50,000 gallons each
2,3-Butanediol
C4





Major interest in WWII by US and Canada
Northern Regional Research Laboratory of USDA
in Peoria
Uses as antifreeze, butadiene synthesis
1936, Julius Nieuwland of Notre Dame with
DuPont’s Wallace Carothers--DuPrene (neoprene)
from it and later from petroleum sources
Fermentation sources never commercialized
Organic Acids
Itaconic Acid C5








Resin and detergent industries
Polymerizable alkene
Competition with methacrylate
Also produced by pyrolysis of citric acid
Commercial production since 1940s
Surface culture method—shallow pans
Submerged culture method—vats
Corn steep liquor: mixture of aa and sugars
Organic Acids
Citric Acid C6

Made today by mold fermentation
 1893: Carl Wehmer discovery
 1917: Currie surface fermentation method
 1945 Commercial, Landenburg Germany
 Molasses, cane blackstrap molasses, sugar
 Remarkable increase in production over
past 60 years—huge sales to China
 Originally produced directly from citrus
fruit
Biochemistry of Fermentation
A.
B.
Overall Strategy
Bioenergetics
– Energy transfer from highly negative DG to
less negative DG
– Harvesting of electrons
– Temporary energy storage
C.
Major metabolic pathways and cycles
A. Overall Strategy

Organic molecules “contain” energy
– True interest is twofold
atoms
electrons

Living organisms strip organic foodstuffs of
electrons and successively oxidize foodstuffs in
order to carry out life processes

Organic foodstuffs become successively more
oxidized and may be released to atmosphere
ultimately as CO2
B. Bioenergetics

Energy must be stored in temporary, highly
available chemical form
– Adenosine triphosphate is the universal energy
storage molecule

Electrons must be transported by organic
molecules in the form of utilizable
“reducing equivalents”
– Nicotinamide adenine dinucleotide and flavin
adenine dinucleotide are the universal electron
carriers
ATP

Energy of organic molecules is not useable
to living organisms—requires conversion
into the “currency” of the cell, ATP,
adenosine triphosphate

ATP has an intermediate energy of
hydrolysis



DG of hydrolysis is –7.3 kcal/mol
Low compared to some, high compared to other
hydrolyses
ATP levels must be kept constant in all cells
for life processes to continue to occur
Electron Carriers

Electrons stripped from foodstuffs must be
transported

Two universal electron carriers are used
– Nicotinamide adenine dinucleotide
NAD
– Flavin adenine dinucleotide FAD

Both are found in conjuction with enzymes,
thus are termed “coenzymes”

NAD accepts two electrons and a proton
(H+) to form NADH

FAD accepts two electrons and two protons
to form FADH2

Both NADH and FADH2 are termed
“reducing equivalents” since they carry
electrons
In Summary Have Three Players
To Consider in ALL Metabolic
Pathways

Energy carrier molecule

Electron carrier molecules

Organic compounds at various oxidation
states along the way
– Glucose to A to B to C to D to E to carbon
dioxide
C. Major Metabolic Pathways
and Cycles

Definition

Particular pathways and cycles
Metabolism: Definition and
Types

Metabolism is a sequence of discrete chemical
transformations (chemical reactions)

No reaction is at all foreign to organic chemistry

Two Kinds of Metabolism
– Catabolic—complex organics to simpler
– Anabolic—simpler organics to complex
– Both operate simultaneously by different sequences of
chemical transformations

A
Each reaction in the sequence requires a
specific enzyme
E1
E2
B
C
The linked sequence is a ‘pathway’
 Each enzyme is specific for its substrate
 Regulation of the pathway is possible since
some enzymes can be activated, and others
inhibited

Metabolism: Specific
Pathways and Cycles

Glycolysis

Citric Acid Cycle

Electron Transport Chain
Glycolysis

Central pathway in most organisms

Embden-Meyerhof Pathway

Begins with glucose C6

Requires 10 discrete steps

Ends with pyruvate 2 X C3

Anaerobic pathway--primitive
Glycolysis: Features

Textbook, page 133

One glucose is ‘split’ (glucose + lysis =
glycolysis)

The splitting step is a reverse aldol
condensation

Final pyruvate has several possible fates
– 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+
One Fate: Alcoholic
Fermentation

Yeast ferment glucose to ethanol and carbon
dioxide, rather than to lactate

Sequence:
pyruvate
acetaldehyde
ethanol
Glycolysis: Summary Schematic from Pyruvate Onward
Glucose
10 marvelous steps!
Anaerobic conditions
2 Pyruvate
O2
2 EtOH + 2 CO2
Alcoholic fermentation
-2CO2
2 Acetyl CoA
Anaerobic conditions
2 Lactate
Some organisms,
contracting muscle
O2
Citric Acid Cycle: Aerobic conditions—animal, plant,
microbial cells
4CO2 and 4 H2O
Glycolysis Energetics

Standard Free Energy for calorimetric oxidation of
glucose to carbon dioxide and water is –686
kcal/mol

Glycolytic degradation of glucose to two lactate
(DG = -47.0 kcal/mole)
(47/686) X 100 = 6.9 percent of the total energy that can
be set free from glucose
This does NOT mean anaerobic glycolysis is wasteful, but
only incomplete to this point of metabolism!
Citric Acid Cycle

Background

Function

Schematic
TCA: Background

Kreb’s Cycle, Tricarboxylic Acid Cycle
– Sir Hans Krebs 1930’s

Regarded as the most single important
discovery in the history of metabolic
biochemistry

Is a true cycle: not a linear pathway
TCA: Function

To continue to strip remaining energy from
pyruvate on its way to carbon dioxide which is
released to atmosphere

To produce organic molecules which may be
drained off the cycle for anabolic purposes

To continue to harvest electrons from pyruvate

To serve as a central collecting pool for foodstuffs
originating from molecules other than glucose
TCA: Schematic
Pyruvate 3C
Amino acids
Fatty acids
Acetyl CoA 2C
Oxaloacetate 4C
Citrate 6C
Isocitrate
Malate
Note: Sequence
is Clockwise
+ NADH
Fumarate
+ FADH2
Succinate
+2 carbon dioxi
Alpha-ketoglutarate
Succinyl CoA
Electron Transport Chain
Organization of “Chain”
Electron Carriers in Chain
Electron Carriers: Free Energy Changes
Direction of Flow via Electron Carriers
Ultimate Fate of Electrons and Protons
ETC: Organization of “Chain”

The physical electron carriers are molecules
embedded in the cell membrane as freefloating bodies
See Figure 5.6 page 137 in your textbook
• Likened to buoys that bob and move
to carry electrons from one carrier to
the other
• Also often likened to a bucket
brigade
ETC: Electron Carriers in Chain
A ‘carrier’ both accepts and then donates electrons
Thus, carriers undergo reversible oxidation and
reduction
Variety of electron carriers are used, eg.
Flavoproteins
Cytochromes—copper
containing
FeS Centers
Coenzyme Q: a quinone
Electron Carriers: Free-Energy
Changes
Electrons flow from electronegative toward
electropositive “carriers”
This is the result of the loss of free energy,
since electrons always move in such a direction
that the free energy of the reacting system:
DECREASES! The free energy decreases
for spontaneous changes!
Electrons move spontaneously from negative to more
positive standard reduction potentials
Direction of Electron Flow via Electron Carriers
-0.4
0.0
NADH
0
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
Direction of Electron Flow is Consistent with Thermodynamics
Direction of Electron Flow is Consistent with Thermodynamics
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
That is, oxygen has the
most positive reduction
potential of all electron
acceptors in the chain
The more positive
the reduction
potential, the more
the substance wants
to gain electrons
Reduction potentials
are easily related to free
energy changes by the
Faraday equation
ETC: Fate of Electrons
Oxygen O2 is the ultimate electron and
proton acceptor
Since this is the only stage of metabolism
at which oxygen (O2) is used, the electron
transport chain is referred to as the

RESPIRATORY TRANSPORT CHAIN
Synthesis of ATP

Proton Pumping During ETC Processes

Gradient Released via ATPase

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 Synthesis: Gradient
Released via ATPase
The proton gradient formed during the electron
transport chain is used to do work
The protons are pumped back through an enzyme in the
membrane, a process which catalyzes the formation of
ATP
(This concept of proton gradient used to do work is known as Peter
Mitchell’s ‘chemiosmotic hypothesis’)
This constitutes THE mechanism by which ATP is
continuously provided for the steady-state storage of utilizable
energy
OXIDATIVE
The process is known as
PHOSPHORYLATION
ATP Bookkeeping
Each NADH molecule produced in any
pathway is ultimately responsible for the
production of 3 ATP
Each FADH2 molecule produced is
ultimately responsible for the production
of 2 molecules of ATP
nb: These ratios of 1:3 and 1:2 vary
depending on organism (cf. page 137)
ETC: Balance Sheet per Glucose
Molecule Start to Finish
Metabolic
Stage
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
0 ATP
6 ATP
2
= 2 ATP
2 ATP
(GTP)
24 ATP
Kreb’s Cycle 6 produced
= 18 ATP
Cf. Table 5.1 page 138 Textbook
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
glucose conserved as ATP
SUMMARY
1.
The function of metabolism is to ensure the life of the
organism
2.
Oxidative pathways—first glycolysis, then the
Kreb’s cycle—use electron carriers to harvest
electrons
3.
The electrons are passed through the electron
transport chain, leading to a proton gradient
4.
The proton gradient is used to do work by converting
gradient energy to chemical energy in the form of
high-energy ATP
FINALLY
Additional Pathways I
Pentose-Phosphate Pathway
–Serves to harvest electrons
–Is an alternative glucose pathway
–Produces 5C sugar intermediates critical for
DNA and RNA synthesis (anabolism)
These are referred to as purines in textbook, pg. 139
Figure 5.7
Additional Pathways II
Amino Acid Anabolism: From TCA intermedicates
Amino acids must be supplied for the growth
requirements of all cells
Example: Oxaloacetate to form glutamate
Chemically, this is the reductive amination of
a ketone to produce an amine