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
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