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
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

Advantages of Indigenous Products

Unique flavor profile
Enhanced storage

Disadvantages of Indigenous Products

Quality controlnatural 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

Technological
Profitability
Downstream

eg., Citric acid

eg., petroleum, starch

Knowledge eg., intellectual capital

eg., value added

Utilization eg., food additive

Merchandising
eg., THIS IS IT!

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

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

in US Industrial Actdenatured 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
directlyamylases 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

 Principal

Glycerol
C3

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
Germanybecame leader in fermentation
At least one integrated plant took directly to
nitroglycerine

 True,

Acetone-Butanol
C3 and C4

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
DuPonts 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 methodshallow pans
Submerged culture methodvats
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 yearshuge sales to China
Originally produced directly from citrus
fruit

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.

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

 Energy

ATP

of organic molecules is not useable

to living organismsrequires conversion
into the currency of the cell, ATP,
adenosine triphosphate

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

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

Cataboliccomplex organics to simpler

Anabolicsimpler organics to complex
Both operate simultaneously by different sequences of

chemical transformations

 Each

reaction in the sequence requires a

specific enzyme

A
The

E1

E2

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
Electron

Cycle

Transport Chain

Glycolysis
Central

pathway in most organisms

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

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 EtOH + 2 CO2
Alcoholic fermentation

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

Free Energy for calorimetric oxidation of

glucose to carbon dioxide and water is 686 kcal/mol

Glycolytic

degradation of glucose to two lactate (G

= -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
Krebs Cycle,

Tricarboxylic Acid Cycle

Sir Hans Krebs 1930s

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 serve

to harvest electrons from pyruvate

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
+ FADH2

Fumarate
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

Cytochromescopper
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

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
Mitchells 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 ATP

6 ATP

2
= 2 ATP

2 ATP
(GTP)

24 ATP

Krebs 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 pathwaysfirst glycolysis, then the

Krebs cycleuse 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