Biochemistry of Fermentation Biochemistry of Fermentation ProcessesProcesses

David A. Boyles

Professor of Chemistry

Department of Chemistry and Chemical Engineering

South Dakota School of Mines and Technology

II. Biochemistry of Fermentation

I. Overview of Fermentation

Fermentation BackgroundFermentation Background

Earliest use of term referred to natural fermentation by wild and unidentified microbes

Known since antiquity

Distinguish two kinds

•Indigenous Fermentations

•Technological Fermentations

Fermentation originally used to produce foods and beverages

Ales—natural yeastsCheeses—natural fungiWines—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…

Many products have been standardized and commercialized

INDIGENOUS FERMENTATIONS

Advantages of Indigenous Products

Disadvantages of Indigenous Products

Unique flavor profile

Enhanced storage

Quality control—natural variations over time, possibility of contamination

Difficult to mass produce

Fermentation: Current Fermentation: Current DefinitionsDefinitions

In the strict biochemical sense of the term fermentation involves the action of anaerobic organisms on organic substrates

The component products of fermentation may be isolated from the feedstock and purveyed as pure substances, unlike fermentation of antiquity: eg., ethanol versus wine

Modern usage extends definition to the microbiological formation of smaller organic molecules, whether aerobic or anaerobic

Technological Fermentation: Technological Fermentation: FeaturesFeatures

•Carefully controlled conditions

•Pure strains of microbes

•Optimized yields of pure products

•Large scale reactors for commercial production

•Genetically engineered microbes by recombinant technologies allowing production of rare natural products such as insulin, growth hormones, enzymes

Variety of Isolated Variety of Isolated Fermentation ProductsFermentation 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 Criteria for Potential Industrial Chemical Products and Chemical Products and

TransformationsTransformationsFavorable demand eg., Citric acid

Reliable supply eg., petroleum, starch

Technological Knowledge eg., intellectual capital

Profitability eg., value added

Downstream Utilization eg., food additiveMerchandising eg., ‘THIS IS IT!’

DatelineDateline1859

– 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 Classic Fermentation ProductsProducts

from Technologyfrom TechnologyEthanolAcetone and n-Butyl AlcoholOrganic 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 petroleum-based chemicals

Classic Fermentation Classic Fermentation Products IProducts I

Ethanol Acetone-Butanol

Glycerol 2,3-Butanediol

industrial solvent, beverage, fuel

Saccharomyces cerevisiae

solvent

Clostridium acetobutylicum

synthetic rubber

Bacillus polymyxa, Acetobacter aerogenes

food and pharmaceutical use

Lactobacillus delbrukki, bulgaricus

Classic Fermentation Classic Fermentation ProductsProducts IIII

Organic AcidsAcetic Acid—Saccharomyces sp., Acetobacter

Lactic Acid—Lactobacillus delbruckii

Citric Acid—Aspergillus niger

Itaconic Acid—Aspergillus itaconicus

EthanolEthanol

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

C2C2

Organic AcidsOrganic Acids

French name vin + aigreCondiment and preservativeFeedstock: sugary or starchySlow Process: Orleans or French method

--”mother of vinegar”Generator Process: 1670

--fast process, maximum air exposureCider (apples), wine (grapes), malt (barley),

sugar, glucose, spirit (grain) used for biomass

Vinegar C2Vinegar C2

Organic AcidsOrganic Acids

1790 by Scheele from milk Present in sour milk, sauerkraut, bread, muscle

tissue, principal organic soil acid 1881 Commercial production by Chas. Avery,

Littleton, Massas substitute for cream of tartar

Dextrose, maltose, lactose, sucrose, whey Starch, grapefruit, potatoes, molasses, beet juice

Dimerizes to lactide upon heating

Lactic Acid C3Lactic Acid C3

PURAC for applications

GlycerolGlycerol

Principal source is saponification of fats and oils

Diverse use in explosives, foods, beverages, cosmetics, plastics, paints, coatings

First identified by PasteurWWI demand exceeded supply, esp. in

Germany—became leader in fermentationAt least one integrated plant took directly to

nitroglycerine

C3C3

Acetone-ButanolAcetone-Butanol

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

C3 and C4C3 and C4

2,3-Butanediol2,3-Butanediol

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

C4C4

Organic AcidsOrganic Acids

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

Itaconic Acid C5Itaconic Acid C5

Organic AcidsOrganic Acids

Made today by mold fermentation1893: Carl Wehmer discovery1917: Currie surface fermentation method1945 Commercial, Landenburg GermanyMolasses, cane blackstrap molasses, sugar Remarkable increase in production over

past 60 years—huge sales to ChinaOriginally produced directly from citrus

fruit

Citric Acid C6Citric Acid C6

Biochemistry of FermentationBiochemistry of Fermentation

A. Overall Strategy

B. Bioenergetics– Energy transfer from highly negative G to

less negative G– Harvesting of electrons– Temporary energy storage

C. Major metabolic pathways and cycles

A.A. Overall StrategyOverall 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.B. BioenergeticsBioenergetics 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

ATPATPEnergy 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

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 CarriersElectron CarriersElectrons 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 In Summary Have Three Players To Consider in ALL Metabolic To Consider in ALL Metabolic

PathwaysPathwaysEnergy 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 C. Major Metabolic Pathways and Cyclesand Cycles

Definition

Particular pathways and cycles

Metabolism: Definition and Metabolism: Definition and TypesTypes

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

Each reaction in the sequence requires a specific enzyme

A B C

The linked sequence is a ‘pathway’Each enzyme is specific for its substrateRegulation of the pathway is possible since

some enzymes can be activated, and others inhibited

E1 E2

Metabolism: Specific Metabolism: Specific Pathways and CyclesPathways and Cycles

Glycolysis

Citric Acid Cycle

Electron Transport Chain

GlycolysisGlycolysis 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: FeaturesGlycolysis: 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 One Fate: Alcoholic FermentationFermentation

Yeast ferment glucose to ethanol and carbon dioxide, rather than to lactate

Sequence:

pyruvate acetaldehyde ethanol

Glucose

10 marvelous steps!

2 Pyruvate

2 Acetyl CoA

Citric Acid Cycle: Aerobic conditions—animal, plant, microbial cells

4CO2 and 4 H2O

2 EtOH + 2 CO22 Lactate

Anaerobic conditionsAnaerobic conditions

Alcoholic fermentation Some organisms, contracting muscle

O2

-2CO2

O2

Glycolysis: Summary Schematic from Pyruvate Onward

Glycolysis EnergeticsGlycolysis 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 CycleCitric Acid Cycle

Background

Function

Schematic

TCA: BackgroundTCA: 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: FunctionTCA: 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: SchematicTCA: SchematicPyruvate 3C

Acetyl CoA 2C

Fatty acidsAmino acids

Citrate 6COxaloacetate 4C

+2 carbon dioxide

Isocitrate

Alpha-ketoglutarate

Succinyl CoASuccinate

Fumarate

Malate

+ NADH

+ FADH2

Note: Sequence is Clockwise

Electron Transport ChainElectron 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”ETC: Organization of “Chain”

The physical electron carriers are molecules embedded in the cell membrane as free-floating 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 ChainETC: Electron Carriers in Chain

Variety of electron carriers are used, eg.

A ‘carrier’ both accepts and then donates electrons

Flavoproteins

FeS Centers

Cytochromes—copper containing

Coenzyme Q: a quinone

Thus, carriers undergo reversible oxidation and reduction

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

Eo’

Direction of Electron Flow is Consistent with Thermodynamics

NADH FMN

CoQ cyt b

cyt c

cyt a

??

-0.4

0.0

+0.4

+0.2

+0.8

0

10

20

40

30

50

kcal

Direction of Electron Flow via Electron Carriers

Protons are pumped across membrane at each incremental drop

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 ElectronsETC: 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 ATPSynthesis of ATP

Proton Pumping During ETC Processes

Gradient Released via ATPase

ATP Bookkeeping

ATP Synthesis:ATP Synthesis: Proton Pumping During Proton Pumping During

Course of ETC 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 ATP Synthesis: Gradient Released via ATPaseReleased 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 constitutes THE mechanism by which ATP is continuously provided for the steady-state storage of utilizable energy

The process is known asOXIDATIVE PHOSPHORYLATION

(This concept of proton gradient used to do work is known as Peter Mitchell’s ‘chemiosmotic hypothesis’)

ATP BookkeepingATP 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 ETC: Balance Sheet per Glucose Molecule Start to FinishMolecule Start to Finish

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

Kreb’s Cycle 6 produced

= 18 ATP

2

= 2 ATP

2 ATP

(GTP)

24 ATP

Metabolic Stage NADH FADH2

Substrate Level Phos.

Total 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

Additional Pathways IAdditional Pathways I

Pentose-Phosphate Pathway

FINALLY

–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. 139Figure 5.7

Additional Pathways IIAdditional Pathways IIAmino 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