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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.

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biochemistry of fermentation processes
Biochemistry of Fermentation Processes

David A. Boyles

Professor of Chemistry

Department of Chemistry and Chemical Engineering

South Dakota School of Mines and Technology

slide2

I. Overview of Fermentation

II. Biochemistry of Fermentation

fermentation background
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
slide4

INDIGENOUS FERMENTATIONS

Fermentation originally used to produce foods and beverages

Many products have been standardized and commercialized

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…

slide5

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
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
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
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
Criteria for Potential Industrial Chemical Products and Transformations
  • Favorable demand eg., Citric acid
  • Reliable supply eg., petroleum, starch
  • Technological Knowledge eg., intellectual capital
  • Profitability eg., value added
  • Downstream Utilization eg., food additive
  • Merchandising eg., ‘THIS IS IT!’
dateline
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
Classic Fermentation Productsfrom Technology
  • Ethanol
  • Acetone and n-Butyl Alcohol
  • Organic Acids
    • Citric Acid
    • Acetic Acid
    • Lactic Acid
    • Itaconic Acid
slide12

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 products i
Classic Fermentation Products I

industrial solvent, beverage, fuel

Saccharomyces cerevisiae

solvent

Clostridium acetobutylicum

food and pharmaceutical use

Lactobacillus delbrukki, bulgaricus

synthetic rubber

Bacillus polymyxa, Acetobacter aerogenes

classic fermentation products ii
Classic Fermentation ProductsII
  • Acetic Acid—Saccharomyces sp., Acetobacter
  • Lactic Acid—Lactobacillus delbruckii
  • Citric Acid—Aspergillus niger
  • Itaconic Acid—Aspergillus itaconicus
ethanol
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
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 acids17
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
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
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
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 acids21
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 acids22
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
Biochemistry of Fermentation

A. Overall Strategy

B. 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
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
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
slide26
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
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”
slide28
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
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
C. Major Metabolic Pathways and Cycles
  • Definition
  • Particular pathways and cycles
metabolism definition and types
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
slide32
Each reaction in the sequence requires a specific enzyme

A 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

E2

E1

metabolism specific pathways and cycles
Metabolism: Specific Pathways and Cycles
  • Glycolysis
  • Citric Acid Cycle
  • Electron Transport Chain
glycolysis
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
Glycolysis: Features
  • Textbook, page 133
  • One glucose is ‘split’ (glucose + lysis = glycolysis)
  • The splitting step is a reverse aldol condensation
slide36
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
One Fate: Alcoholic Fermentation
  • Yeast ferment glucose to ethanol and carbon dioxide, rather than to lactate
  • Sequence:

pyruvate acetaldehyde ethanol

slide38

Glycolysis: Summary Schematic from Pyruvate Onward

Glucose

10 marvelous steps!

2 Pyruvate

Anaerobic conditions

Anaerobic conditions

O2

-2CO2

2 Lactate

2 EtOH + 2 CO2

Some organisms, contracting muscle

Alcoholic fermentation

2 Acetyl CoA

O2

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

4CO2 and 4 H2O

glycolysis energetics
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
Citric Acid Cycle
  • Background
  • Function
  • Schematic
tca background
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
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
TCA: Schematic

Pyruvate 3C

Fatty acids

Amino acids

Acetyl CoA 2C

Oxaloacetate 4C

Citrate 6C

Isocitrate

Malate

Note: Sequence is Clockwise

+2 carbon dioxide

Fumarate

+ NADH

+ FADH2

Alpha-ketoglutarate

Succinyl CoA

Succinate

electron transport chain
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
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 chain
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

slide47

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

slide48

Direction of Electron Flow via Electron Carriers

0

-0.4

NADH

FMN

10

0.0

CoQ

cyt b

Eo’

20

+0.2

kcal

cyt c

30

Protons are pumped across membrane at each incremental drop

+0.4

40

cyt a

??

+0.8

50

Direction of Electron Flow is Consistent with Thermodynamics

slide49

Direction of Electron Flow is Consistent with Thermodynamics

Reduction Potentials measure the ‘natural’ (inherent) tendency of substances to gain electrons (be reduced)

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

Some substances “naturally” gain electons more easily than others: in the electron transport chain, oxygen gains them most easily of all

Reduction potentials are easily related to free energy changes by the Faraday equation

etc fate of electrons
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
Synthesis of ATP
  • Proton Pumping During ETC Processes
  • Gradient Released via ATPase
  • ATP Bookkeeping
atp synthesis proton pumping during course of etc
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
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 PHOSPHORYLATION

The process is known as

atp bookkeeping
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
ETC: Balance Sheet per Glucose Molecule Start to Finish

Metabolic Stage

Substrate Level Phos.

Total ATP

NADH

FADH2

Cf. Table 5.1 page 138 Textbook

Total 36 ATP

slide56

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

slide57

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 i

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
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