CHE 366

INDUSTRIAL MICROBIOLGY

WEEK 5
Microbial Metabolism

Dr. Burcu ÖZTÜRK

Food Engineering Department
Office No: B116/A
[email protected]
 MICROBIAL METABOLISM
Metabolism : the Greek metabole, meaning change.
✓ the sum of the biochemical reactions required for energy generation
✓ +
✓ the use of energy to synthesize cell material from small molecules in the
environment.
environmental conditions: pH, moisture, temperature, salinity

Carbon
cell

energy cell

metabolic byproducts
nutrients
electron acceptor
Why do we must know the metabolism of microorganisms?
Because we want to know how to inhibit or stop bacterial growth and want to control their
metabolism.

✓ The microbial physiology of microorganisms are common to many living systems;

✓ carbohydrates, fatty acids, amino acids, nitrogeneous bases occur in all cells.

Sources of energy for all living cells(biological systems) :

1- Chemical sources
2-Sunlight

➢ A cell is a miniature factory

➢ A large number of chemical reactions are occurring
➢ A (reactants) + B (reactants) ----> C (products)
➢ Chemical reactions are either endogonic (req. energy) or exogonic (release energy)
 METABOLIC DIVERSITY

Cellular metabolism is classified into nutritional groups on the basis of three major criteria:

1. Source of energy, used for growth

2. Source of carbon, and
3. Source of electron donors used for growth.
SOURCES OF BIOLOGICAL ENERGY, ENERGY CONVERSION and USE

Sources of energy Common Energy Biosynthetic reactions

Currency

Organic Compounds Chemoorganotrophs Protein Synthesis

Light Phototrophs NADH DNA Synthesis

ATP

Inorganic Compounds Chemolithotrophs Carbohydrates

Proteins
Lipids
Nucleotides
 ENERGY – capacity to do work or cause change

Chemical energy is the energy released when organic or inorganic compounds are oxidized.

First law of thermodynamics : energy can be converted from one form to another, but can neither be
created nor destroyed.

In biology most common energy units are kcal and kilojoule.

1 kilocalorie : amount of heat necessary to raise the temperature of

1kg of water by 1 degrees celcius.

1kcal = 4.184 kilojoules

 ENERGY
Chemical reactions are accompanied by changes in energy.

The amount of energy involved in a chemical reaction is expressed in terms of gain or loss of energy
during a chemical reaction.

There are two expressions used to define the amount of energy release during a chemical reaction:

H : enthalpy – total amount of energy released during a chemical reaction

Some of the energy released is lost as heat energy and can not be used to do useful work.

G : free energy – amount of energy available to do useful work.

When molecules react or undergo change from one form to another, energy is

1)released as heat: these are exergonic reactions with a –ΔG (catabolism –break down)
or

2)trapped in the chemical bonds of different compounds : these are endergonic reactions with a

+ΔG (anabolism – biosynthesis).

ENERGY
Exergonic chemical reactions from which microorganisms gain energy :

Glucose C6H12O6 + 6O2 ―›6H2O + 6CO2 ΔG: -686 kcal/mole

Lactic acid C3H6O3 + 3O2 ―›3H2O + 3CO2 ΔG: -318 kcal/mole
Acetic acid C2H4O2 + 2O2 ―›2H2O + 2CO2 ΔG: -214 kcal/mole
Amonia NH4 + 2O2 ―›NO3 + 2H2O + 2H ΔG: -82.5 kcal/mole
 ENERGY

✓Catabolic reactions or sequences produce energy as ATP

(adenosine triphosphate) , which can be utilized in anabolic
reactions to build cell material from nutrients in the environment.
 ENERGY FROM ATP

ATP(adenosine triphosphate)
3 part molecule consisting of
➢adenine – a nitrogenous base
➢ribose – a 5-carbon sugar
➢3 phosphate groups

ATP contains energy that can be easily released

(high-energy or unstable energy bond)

Required for anabolic reactions

REACTIONS

Cells capture energy released ATP + H2O ―› ADP + H3PO4 ΔG: -7.3 kcal/mole
from chemical reactions by ADP + H2O ―› AMP + H3PO4 ΔG: -7.3 kcal/mole
forming energy rich bonds in ATP
and other related compounds. ADP + H3PO4 ―› ATP + H2O ΔG: +7.3 kcal/mole
ATP - ADP -AMP
 REDUCED PYRIDINE NUCLEOTIDES
NAD (Nicotineamide Adenine Dinucleotide)

This molecule is a coenzyme made from:

➢ Adenine nucleotides

➢ Ribose

➢ Vitamin niacin Properties

Molecular formula C21H27N7O14P2
Molar mass 663.43 g/mol

NAD is a strong reductant, participating in many enzyme catalysed oxidation reduction reactions
essential to both catabolism and biosynthesis.
 ENERGY FROM OXIDATION-REDUCTION REACTIONS

• Oxidation–reduction reactions,
commonly known as redox reactions,
• Basic reaction
are reactions that involve the transfer
of electrons from one species to
another.

• The species that loses electrons is

said to be oxidized, while the species
• Biological
reaction that gains electrons is said to be
reduced.
• Biological reactions such as cellular
respiration and photosynthesis are the
best examples of Redox reactions.
 ENERGY FROM OXIDATION-REDUCTION REACTIONS

• Always occur in pairs.

• There is an electron donor and electron acceptor which constitute a redox pair.
• Oxidation is the loss of electrons or an increase in oxidation state by a molecule, atom, or ion.
• Reduction is the gain of electrons or a decrease in oxidation state by a molecule, atom, or ion.
• Released energy can be captured to phosphorylate ADP or another compound.
-2H +2H
C3H6O3-------------------›C3H4O3-----------------›C3H6O3
Lactic acid oxidation Pyruvic acid reduction Lactic acid

A hydrogen atom is composed of a proton and an electron.

The hydrogen atoms in these reactions are transferred between compounds by electron
carriers such as NAD.

C3H4O3 + NADH + H -------------›C3H6O3 + NAD+

Pyruvic acid Lactic acid
NADH is oxidized to NAD, while pyruvic acid is reduced to lactic acid.
ENERGY FROM OXIDATION-REDUCTION REACTIONS

The more reduced an electron carrier is, the greater its potential to give electrons and the more
energy it contains.

The ability of a reduced compound such as NADH or H2 to give electrons can be measured
electrically as the electromotive force and expressed in volts.

The more –ve the E0, the stronger the compounds reducing power.
ENZYMES
• Enzymes are proteins that function as catalyts – they speed up the rate of a reaction without
being consumed in the process.

• Like all catalysts, enzymes increase the reaction rate by lowering its activation energy.

• The amount and kinds of enzymes present in a cell determine its metabolic capability.

• Enzymes are active within certain ranges of temperature and pH.

For most of them typical optimum temperature : 30-37ºC

typical optimum pH : 4.5 – 8.5

• Most chemical reactions are temperature dependant.

• Although at high temperatures the reaction rates are higher, above a certain temperature
enzymes are deactivated.
• Enzymes lower the activation energy of a
reaction - that is the required amount of
energy needed for a reaction to occur.
• They do this by binding to a substrate and
holding it in a way that allows the reaction
to happen more efficiently.
• The part of the enzyme where the
substrate binds is called the active site.
• Here, the enzyme changes shape
slightly, fitting tightly with the
substrate and forming
the enzyme/substrate complex.
ENZYMES

Naming of Enzymes - most are named by adding “ase” to the substrate

Naming according to the substrate

• Lipases Lipids
• Sucrases Sucrose
• Ureases Urea
• Proteases Proteins
• DNases DNA

Naming according to the type of reaction catalyzed

• removes a hydrogen Dehydrogenase
• removes a phosphate Phosphotase
 TYPES OF ENZYMES
1.Oxidoreductase Oxidation reduction in which hydrogen or oxygen Cytochrome oxidase, lactate
are gained or lost dehydrogenase
2.Transferase Transfer of functional groups, e.g. amino, acetyl or Acetate kinase, alanine deaminase
phosphate groups
3.Hydrolase Hydrolysis – addition of water Lipase, sucrase
4.Lyase Removal of atoms without addition of water Oxalate decarboxylase, isocitrate lyase
5.Isomerase Rearrangement of atoms within a molecule Glucose phosphate isomerase, alanine
racemase
6.Ligase Joining of two molecules Acetyl-CoA synthetase, DNA ligase

For example, hexokinase

(EC 2.7.1.1)
ENZYME COMPONENTS

An inactive enzyme, without the cofactor is called an Apoenzyme, while the complete
enzyme with cofactor is the Holoenzyme.
 ENZYME COMPONENTS
Cofactors

• non-protein chemical compound bound to a protein (mostly enzymes)

• These may be attached temporarily to the enzyme through ionic or hydrogen bonds, or
permanently through stronger covalent bonds.

• Common cofactors include inorganic ions such as iron and magnesium

• For example, the enzyme that builds DNA molecules, DNA polymerase, requires
magnesium ions to function
 ENZYME COMPONENTS
Cofactors

◼ may accept or donate atoms removed from the substrate or donated to the
substrate

◼ may act as electron carriers

◼ often derived from vitamins

◼ e.g. NAD and NADP – electron carriers derived from nicotinic acid
Cofactors
Metal Cofactors

• inorganic metal ion

• called a metal ion activator.
• Zn+2, Mg+2, Mn+2, Fe+2, Cu+2, K+1, Na+1
• metal ions may be bonded through coordinate covalent bonds.

• major reason for the nutritional requirement for minerals

Coenzymes
• Coenzymes are a subset of cofactors that are organic (carbon-based) molecules.
• For example, vitamin C is a coenzyme for several enzymes that take part in building the protein
collagen, a key part of connective tissue.
• "helper molecules"
• assist in biochemical transformations.
• required for the protein's biological activity
• activate the protein by changing its geometric shape, or by actually participating in the overall reaction.
Every cell contains a small quantity of coenzymes – they are used repeatedly. not specific to a single
enzyme –
recognized by many enzymes
enzymes competes for its availability.

stable to heat (unlike enzymes)

Coenzymes participate in:
Oxidation-reduction rxns
Transfer of chemical groups
Molecular rearrangement rxns
Coenzymes
Many coenzymes are derived from vitamins

1. Niacin(nicotinic acid)
➢NAD (Nicotinamide adenine dinucleotide)
2. Riboflavin(vitamin B2)
➢FAD (Flavin adenine dinucleotide)
3. Pantothenic Acid
➢CoEnzyme A (CoA-SH)
4. Thiamine(vitamin B1)
➢TPP (thiamine pyrophosphate)
5. Cyanocobolamin (vitamin B12)
➢Coenzyme B12
 ENZYMATIC REACTIONS
• begins with the binding of the substrate (or substrates) to the active site on the enzyme.

• active site

the specific region of the enzyme which combines with the substrate.

• binding of the substrate to the enzyme

• causes changes in the distribution of electrons in the chemical bonds of the substrate and

• causes the reactions that lead to the formation of products.

• release of products from the enzyme surface

• regeneration the enzyme for another reaction cycle.

• Enzymes are specific

• The active site has a unique geometric shape that is complementary to the geometric shape
of a substrate molecule. This means that enzymes specifically react with only one or a
very few similar compounds.
ENZYMATIC REACTIONS

Lock and Key Theory

First postulated in 1894 by Emil Fischer.

• The lock is the enzyme and the key is the substrate.

Only the correctly sized key (substrate) fits into
the key hole (active site) of the lock (enzyme).
ENZYMATIC REACTIONS

Induced Fit Theory

• This theory was given by Daniel Koshland in 1958.

• assumes that the substrate plays a role in determining the final

shape of the enzyme and that the enzyme is partially flexible.

• This explains why certain compounds can bind to the enzyme but
do not react because the enzyme has been distorted too much.
Other molecules may be too small to induce the proper alignment
and therefore cannot react.

• Only the proper substrate is capable of inducing the proper

alignment of the active site.
 ENZYMATIC REACTIONS
Inhibitors

Enzyme Inhibitors

• molecules that interact in some way with the enzyme

• prevent it from working in the normal manner.

Types of inhibitors

➢ nonspecific

➢ irreversible,

➢ reversible - competitive and

➢ noncompetitive.
Nonspecific Inhibitors

• affect all enzymes in the same way.

• physical or chemical changes which denatures the protein portion of

the enzyme
• therefore irreversible.

➢ Temperature:
➢ Usually, the reaction rate increases with temperature,

➢ At high temperatures the protein part of the enzyme begins to denature, thus
inhibiting the reaction.

➢ Acids and Bases:

➢ Enzyme activity is also controlled by pH.

➢ As the pH is decreased or increased, the nature of the various acid and amine
groups on side chains is altered with resulting changes in the overall shape &
structure of the enzyme.
Specific Inhibitors

➢ Specific inhibitors exert their effects upon a single enzyme.

➢ Most poisons work by specific inhibition of enzymes.

➢ Many drugs also work by inhibiting enzymes in bacteria, viruses, or

cancerous cells.
Specific Inhibitors / Competitive

• any compound which closely resembles the chemical structure and

molecular geometry of the substrate.

• The inhibitor competes for the same active site as the substrate
molecule.

• may interact with the enzyme at the active site, but no reaction
takes place. The inhibitor is "stuck" on the enzyme and prevents
any substrate molecules from reacting with the enzyme.

• reversible if sufficient substrate molecules are available to

displace the inhibitor.

• the amount of enzyme inhibition depends upon the inhibitor

concentration, substrate concentration, and the relative
affinities of the inhibitor and substrate for the active site.
Specific Inhibitors / Non competitive

• a substance that interacts with the enyzme, but usually not

at the active site.

• reacts either away from or very close to the active site.

• net effect:
• change the shape of the enzyme and thus the active site, so that the
substrate can no longer interact with the enzyme to give a reaction.

Non competitive inhibitors are usually reversible, but are not

influenced by concentrations of the substrate as is the case
for a reversible competitive inhibitor.
Ireversible Inhibitors

• They form strong covalent bonds with an enzyme.

• These inhibitors may act at, near, or away from the active site.

• They may not be displaced by the addition of excess substrate.

• The basic structure of the enzyme is modified to the degree that it

ceases to work.
 MICROBIAL METABOLISM

Metabolism

Catabolism Anabolism
- produce energy (ATP) - use energy (ATP)
- breakdown molecules - synthesize macromolecules
(ex. glycolysis) (ex. DNA, lipids, etc.)
MICROBIAL METABOLISM
MICROBIAL METABOLISM
CATABOLISM
series of reactions involving the oxidation

Two types of catabolism:

1.) respiration :
-aerobic( with oxygen);
molecular oxygen serves as the terminal electron acceptor
C6H12O6 + 6 O2 -------> 6 CO2 + 6 H2O
38 ADP + 38 P 38 ATP

- anaerobic respiration (without oxygen) ;

other inorganic compounds serve as the terminal electron acceptor

2.) fermentation (does not use oxygen): oxidation reduction process occurs in the absence of any added
terminal electron acceptor
AEROBIC RESPIRATION

It is the process of cellular respiration

Organic compound energy is released by splitting the glucose molecules

(oxidation)
CO2
with the help of oxygen gas. At the end of the
Carbon flow
Electron chemical reaction, energy, water molecules, and carbon
flow dioxide gas are released as the by-products or end

O2 products of the reactions.

(reduction)

Biochemical Pathways Involved

✓ Glycolysis
✓ TCA
✓ ATP synthesis mech.: oxidative phosphorylation
 The different cycles involved in aerobic respiration
➢ such as glycolysis,
➢ Krebs cycle,
➢ electron transport chain

•Glycolysis – Partial breakdown of Glucose to Pyruvic acid (Anaerobic)

•Krebs Cycle – Complete oxidation of Pyruvate to release Carbon dioxide (Aerobic respiration)
•Electron Transport system – Oxidation of NADH and FADH2 to generate ATP
Steps of Aerobic Respiration

1. Glycolysis
It is the primary step of aerobic respiration is glycolysis and takes
place within the cytosol of the cell. During the glycolysis process,
the glucose molecules are splitting and separated into two ATP and
two NADH molecules, which are later used in the process of
aerobic respiration.
2. Formation of Acetyl Coenzyme A
The second step in aerobic respiration is the formation of acetyl
coenzyme A. In this process, pyruvate is oxidized in the
mitochondria and 2-carbon acetyl group is produced. The newly
produced 2-carbon acetyl group binds with coenzyme A, producing
acetyl coenzyme A.
Steps of Aerobic Respiration

3. Citric Acid Cycle

The third step in aerobic respiration is the citric acid cycle, which is
also called the Krebs cycle. In this stage of Aerobic respiration, the
oxaloacetate combines with the acetyl-coenzyme A and produces citric
acid. The citric acid cycle undergoes a series of reactions and
produces 2 molecules of carbon dioxide, 1 molecule of ATP, and
reduced forms of NADH and FADH.
4. Electron Transport Chain
This is the last step in aerobic respiration. In this phase, the large
amounts of ATP molecules are produced by transferring the electrons
from NADH and FADH. A single molecule of glucose creates a total of
34 ATP molecules
Key Points on Aerobic Respiration

•Aerobic respiration is the process of utilisation of oxygen to breakdown glucose, amino acids, fatty

acids to produce ATP.

•The pyruvate is then converted into acetyl CoA in the mitochondrial matrix.

•The Kreb’s cycle occurs twice per glucose molecule.

•The protein complexes are arranged on the inner mitochondrial matrix so that the electrons pass

from one reacting molecule to the other. This is known as the electron transport chain.

•ATP synthase produces ATP from ADP and inorganic phosphate

ANAEROBIC RESPIRATION

Organic compound
It is a process which takes place in the
CO2
Carbon flow absence of oxygen gas. In this process, the
Electron energy is obtained by the breakdown of
flow (reduction)
glucose in the absence of oxygen. One of the
NO2 S best examples of anaerobic respiration is
NO3 SO4
the process of fermentation in yeast.

Biochemical Pathways Involved

✓ Glycolysis
✓ TCA
✓ ATP synthesis mech.: electron transfer phosphorylation
Anaerobic respiration produces a relatively lesser amount of energy as compared to
aerobic respiration, as glucose is not completely broken down in the absence of oxygen.

Obligate anaerobes : intolerant to oxygen, they grow only in the absence of oxygen.
Facultative anaerobes : they grow both in aerobic and anaerobic environments.
Some anaerobic habitats
deep wells, buttom of ponds, stomach, rumen, large intestines
 FERMENTATION

Organic Compound

◼A
ADP +P
Oxidized Organic Compound
Pyruvate (Glycolysis) Electron Carriers
ATP

Biochemical Pathways Present

Reduced Organic Compund ◼ Glycolysis
Fermentation Product ◼ ATP synthesis mech.: substrate level
phosphorylation
 Fermentation occurs in yeast cells and bacteria and also in the muscles of animals.
It is an anaerobic pathway in which glucose is broken down.

Types of Fermentation
There are three different types of fermentation:
1. Lactic Acid Fermentation
In this, starch or sugar is converted into lactic acid by yeast strains and bacteria. During exercise,
energy expenditure is faster than the oxygen supplied to the muscle cells. This results in the
formation of lactic acid and painful muscles.
2. Alcohol Fermentation
Pyruvate, the end product of glycolysis is broken down into alcohol and carbon dioxide. Wine and
beer are produced by alcoholic fermentation.
3. Acetic Acid Fermentation
Starch and sugar present in grains and fruits ferment into vinegar and condiments. E.g. apple cider
vinegar.
Any type of cellular respiration begins with glycolysis where a 3-C molecule, pyruvic acid is formed
as the end product.
the pyruvic acid formed by partial oxidation of glucose is converted to ethanol and carbon dioxide
(CO2). This anaerobic condition is called alcoholic or ethanol fermentation. The whole reaction is
catalyzed by the enzymes, pyruvic acid decarboxylase and alcohol dehydrogenase.
In certain bacteria and animal muscle cells, under anaerobic conditions, the pyruvic acid is reduced
to lactic acid by lactate dehydrogenase.
n the alcoholic and lactic acid fermentation, NADH+H+ is the reducing agent which is oxidized to
NAD+. The energy released in both the processes is not much and the total sum of ATP molecules
produced during fermentation is two, which is very less as compared to aerobic respiration.
However, this is commercially employed in the food and beverage industries, and pharmaceutical
industries.
FERMENTATION may result in
numerous end products

1. Type of organism
2. Original substrate
3. Enzymes that are present and active
FERMENTATION END PRODUCTS
Glycolysis ◼Glycolysis

◼Respiration ◼Fermentation

◼TCA
◼Cycle

◼Organic products
◼Oxidative phosphorylation
◼Electron transfer phosphorylation

Glycolysis is the process in which glucose is broken down to produce energy. It produces two
molecules of pyruvate, ATP, NADH and water. The process takes place in the cytoplasm of a cell
and does not require oxygen. It occurs in both aerobic and anaerobic organisms.
Glycolysis is the primary step of cellular respiration, which occurs in all organisms. Glycolysis is
followed by the Krebs cycle during aerobic respiration. In the absence of oxygen, the cells make
small amounts of ATP as glycolysis is followed by fermentation.
Stage 1
•A phosphate group is added to glucose in the cell cytoplasm, by the action of enzyme hexokinase.
•In this, a phosphate group is transferred from ATP to glucose forming glucose,6-phosphate.
Stage 2
Glucose-6-phosphate is isomerised into fructose,6-phosphate by the enzyme phosphoglucomutase.
Stage 3
The other ATP molecule transfers a phosphate group to fructose 6-phosphate and converts it into
fructose 1,6-bisphosphate by the action of the enzyme phosphofructokinase.
Stage 4
The enzyme aldolase converts fructose 1,6-bisphosphate into glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate, which are isomers of each other.
Step 5
Triose-phosphate isomerase converts dihydroxyacetone phosphate into glyceraldehyde 3-phosphate
which is the substrate in the successive step of glycolysis.
Step 6
This step undergoes two reactions:
•The enzyme glyceraldehyde 3-phosphate dehydrogenase transfers 1 hydrogen molecule from glyceraldehyde phosphate to
nicotinamide adenine dinucleotide to form NADH + H+.
• Glyceraldehyde 3-phosphate dehydrogenase adds a phosphate to the oxidised glyceraldehyde phosphate to form 1,3-
bisphosphoglycerate.
Step 7
Phosphate is transferred from 1,3-bisphosphoglycerate to ADP to form ATP with the help of phosphoglycerokinase. Thus
two molecules of phosphoglycerate and ATP are obtained at the end of this reaction.
Step 8
The phosphate of both the phosphoglycerate molecules is relocated from the third to the second carbon to yield two
molecules of 2-phosphoglycerate by the enzyme phosphoglyceromutase.
Step 9
The enzyme enolase removes a water molecule from 2-phosphoglycerate to form phosphoenolpyruvate.
Step 10
A phosphate from phosphoenolpyruvate is transferred to ADP to form pyruvate and ATP by the action of pyruvate kinase.
Two molecules of pyruvate and ATP are obtained as the end products.
Key Points of Glycolysis

•It is the process in which a glucose molecule is broken down into two molecules of pyruvate.

•The process takes place in the cytoplasm of plant and animal cells.

•Six enzymes are involved in the process.

•The end products of the reaction include 2 pyruvate, 2 ATP and 2 NADH molecules.
KREB’S CYCLE (TCA)

• Provides for complete oxidation of pyruvate to CO2 and H2O.

• Provides for oxidation of lipid and amino acid intermediates to CO2 and H2O.
• Provides materials for amino acid synthesis.
• Not a stand alone pathway; materials must be fed in from other metabolic pathways such as the
glycolysis.
• Carbohydrate enters in the form of Acetyl-CoA (a 2 carbon fragment)
• Acetyl-CoA is produced from pyruvate that comes from the glycolysis.
• 2 pyruvates yield 2 Acetyl-CoA’s
• 1 Acetyl-CoA yields 1 turn of the Krebs cycle -so-2 pyruvates from the Glycolysis-EMP yields 2
Acetyl-CoA’s which in turn yield 2 turns of the Krebs cycle (for each glucose to undergo oxidation
by the EMP)
• Krebs cycle yields
➢ 36 ATP/glucose (procaryotes)
➢ 34 ATP/glucose (eucaryotes)
 THE ELECTRON TRANSPORT CHAIN (ETC)
• Electrons released by oxidation are passed down an Electron Transport Chain
• oxygen being the Final Electron Acceptor
• within the cell membrane of procaryotes
• within the inner mitochondrial membrane of eucaryotes

• The electron carriers (NADH and FADH2) bring

electrons and protons (H+) to the ETC.

• Carrier molecules in the membrane of the mitochondria

pass electrons from one to another and ultimately to
final electron acceptor.
The electron transport chain is composed of :
Flavoproteins Non heme iron sulfur proteins
Quinones NADH dehydrogenases
Cytochromes
 ATP FORMATION

• When O2 is the final electron acceptor , the mechanism is called oxidative phosphorylation

(aerobic respiration)

• When inorganic compounds other than O2 is the final electron acceptor, the mechanism is

called electron transfer phosphorylation (anaerobic respiration).

• When an organic compound is the final electron acceptor, the mechanism of ATP formation

is called substrate level phosphorylation (fermentation).

ENERGY YIELD FROM AEROBIC RESPIRATION

Complete oxidation of glucose

C6H12O6 + 6 O2 -------> 6 CO2 + 6 H2O
686 kcal/mole glucose.

AEROBIC RESPIRATION
C6H12O6 + 6 O2 -------> 6 CO2 + 6 H2O
38 ADP + 38 P 38 ATP

38x7.3 kcal/mole ATP = 277 kcal/mole glucose.

Yield: 277/ 686 x100 = 40 %
The cell stores only 40% of the total energy released as ATP.
METABOLIC STRATEGIES

Pathways Final e- acceptor ATP yield

Aerobic respiration Glycolysis, TCA, O2 38
ETC

Anaerobic respiration Glycolysis, TCA, NO3-, SO4-2, CO3-3 variable

ETC

Fermentation Glycolysis Organic molecules 2

LIPID METABOLISM

• Lipids are essential to the structure and function of membranes.

• Lipids also function as energy reserves, which can be utilized as

sources of carbon.

• 90% of this lipid is “triacyglycerol”.

triacyglycerol lipase glycerol + 3 fatty acids

• The major fatty acid metabolism is “β-oxidation”

LIPID CATABOLISM

• Glycerol is oxidised by glycolysis and the TCA

cycle

• Lipids are broken down to 2 carbon acyl units

where they enter the TCA cycle
PROTEIN CATABOLISM
• Intact proteins cannot cross bacterial plasma membrane,
• so bacteria must produce extracellular enzymes called proteases and peptidases

• Many of the amino acids are used in building bacterial proteins,

• Some may also be broken down for energy.

• broken down to some form that can enter the Kreb’s cycle.
• These reactions include:
1. Deamination
—the amino group is removed, converted to an ammonium ion, and excreted.
2. Decarboxylation
—the ---COOH group is removed
3. Dehydrogenation
—a hydrogen is removed

• Tests for the presence of enzymes that allow various amino acids to be broken down are used in identifying bacteria in
the lab.
CATOBOLISM OF ORGANIC FOOD
MOLECULES

• Proteins,lipids and carbohydrates are degraded by

secreted enzymes
• proteases, lipases and amylases.
PHOTOSYNTHESIS

• Light energy is harvested by photosynthetic pigments and transferred to the reaction

center (photosystem) of chlorophyll molecules.
• The light energy is used to remove electrons from an electron donor (the electron donor
goes from a reduced to an oxidized state).
• The electrons are transferred through a series of electron carriers from high energy state
to a low energy state.
• During this process, ATP is formed.

SOURCE of energy : LIGHT

AUTOTROPHY

Microorganisms that derive all their nutritional needs from inorganic compounds are called autotrophs.

These organisms are different from the heterotrophs, which gain their energy and nutritional needs
from organic compounds.

All autotrophs can use CO2 as their only source of carbon by combining it with a sugar
molecule, in a process called the Calvin Cycle.

There are two subgroups of autotrophs:

-photolithotrophic autotrophy (photoautotrophs) : gain their energy
from sunlight
-chemolithotrophic autotrophy (chemoautotrophs) : gain their energy
from the oxidation of inorganic compounds.
 AUTOTROPHY

Chemoautotrophs
Photoautotrophs
CO2+Reduced inorganic compounds
Oxygenic Photosynthesis Calvin Cycle

CO2+H2O Glucose + O2 Glucose + oxidation prdts+H2O

Calvin Cycle

Anoxygenic Photosynthesis
CO2+Reduced inorganic compounds Glucose+oxidation prdts+H2O
Calvin Cycle
CALVIN CYCLE
This cycle is present in :
✓ Photoautotrophs
(oxygenic and anoxygenic photosynthesis)
✓ Chemoautotrophs

This cycle is an enzymatic pathway :

1-CO2 is combined with the 5 carbon sugar ribulose 1,5 diphosphate.
2-Three carbon trioses are formed.
3-Trioses are converted to 6 carbon hexoses.

Overall
Glucose is produced from 6 moles of CO2
18 moles of ATP consumed
12 moles of NADP+ are produced
 AUTOTROPHY / PHOTOAUTOTROPHS

Photoautotrophs contain photosynthetic pigments (chlorophylls and caretonoids) in the chloroplasts that
participate in using light energy.

Procaryotes Cyanobacteria and Cyanophyta

Eucaryotes Green plants and Algea

WE DEPEND ON GREEN PLANTS, ALGEA, CYANOBACTERIA TO MAINTAIN A BALANCE IN THE WORLD’S SUPPLY OF
OXYGEN.

• Oxygenic photosynthesis :
• O2 from water by the following reaction :
6 CO2 + 6 H2O + sunlight -----> C6H12O6 + 6 O2
• Absorption of light energy activates electrons
• they participate in generating ATP and NADPH
• needed to reduce CO2 into CHO via the Calvin
Cycle.
 AUTOTROPHY / PHOTOAUTOTROPHS
Anoxygenic photosynthesis :
Some bacteria capture the energy of light only in anaerobic environments.

Oxygen inhibits their photosynthetic growth.

Found in green and purple bacteria: electron donor

• Green sulfur bacteria (e.g. Chlorobium) H2S or S
• Purple sulfur bacteria (e.g. Chromatium) H2S or S
• Green nonsulfur bacteria (e.g. Chloroflexus) H2 or organic cpds
• Purple nonsulfur bacteria (e.g. Rhodobacter H2 or organic cpds

These bacteria posses a single reaction center and use this center for the
light driven oxidation of reduced sulfur compounds, such as H2S or other
reduced organic carbon (R-H2) compounds.
CO2 + 2H2S + sunlight -----> (CH2O)n + 2S + H2O

Primary function is ATP production.

 AUTOTROPHY / CHEMOAUTOTROPHS
Examples of electron donors Examples of electron acceptors
• Ammonia (NH4+) → Nitrite (NO2-) • Oxygen (O2) → Water (H2O)
in Nitrosomonas in many organisms
• Nitrite (NO2-) → Nitrate (NO32-) • Carbon dioxide (CO2) → Methane (CH4)
in Nitrobacter in the methanogenic bacteria

• Hydrogen sulfide (H2S) → Sulfur (So)

in Thiobacillus and Beggiatoa

• Sulfur (So) → Sulfate (SO42-)

in Thiobacillus

• Hydrogen (H2) → Water (H2O)

in Alcaligenes
AUTOTROPHY / CHEMOAUTOTROPHS

Features of Chemoautotrophs

• Electrons are removed from a reduced inorganic electron donor

• Chemoautotrophs gain their energy from the oxidation of reduced

inorganic compounds and use this energy to produce organic
compounds by fixing CO2 in the Calvin Cycle.

• Many of the oxidations are essential to the recycling of inorganic

compounds.
BIOSYNTHESIS OF CARBOHYDRATES
• Gluconeogenesis
• generation of glucose from non-carbohydrate carbon substrates
• pyruvate, lactate, glycerol, glucogenic amino acids, and fatty acids.

• Cells need CHO’s to make :

• cell walls, glycoproteins, nucleic acids, starch, PHB’s, slime layers and capsules.
• As glucose is utilized
• organic biomolecules necessary for growth and reproduction from this compound.
• specific intermediates are taken from the catabolic pathway
• used in biosynthetic pathways.
• The pathways for the metabolism of glucose are connected with the biosynthetic
pathways for making essential biomolecules.
• Glucose is metabolized by glycolysis, pentose phosphate cycle, TCA cycle to produce
the starting organic molecule for the biosynthetic reactions.
• Gluconeogenesis presents in plants, animals, fungi, bacteria, and other microorganisms
BIOSYNTHESIS OF AMINO ACIDS
• Biological systems need 20 amino acids for synthesizing proteins.
• Amino acids are synthesized by 20 different pathways,
• There are two aspects of amino acid biosynthesis :
- the synthesis of the carbon skeleton
- the incorporation of the amino group.
• The attachment of the amino group
after the complete synthesis of the carbon skeleton
at some intermediate step.
The source of the amino group for many organisms are ammonia.
Once ammonia is incorporated into the amino group of glutamate, the amino group can be transferred
to other carbon skeletons by enzymes called transaminases.
BIOSYNTHESIS OF LIPIDS

Lipids are synthesized from glycerol phosphate formed from glycolytic

pathway and from fatty acids.

BIOSYNTHESIS OF NUCLEOTIDES

Nucleotides are synthesized by adding carbon and nitrogen groups from

amino acids, formic acid and CO2 to ribose 5-phosphate in a complex
series of reactions
 BIOCHEMICAL COMPONENTS OF CELLS

• Water: 80 % of wet weight

• Dry weight
• Protein 40-70 %
• Nucleic acid 13-34%
• Lipid 10-15 %
• Also monomers, intermediates and inorganic ions

• To obtain energy and construct new cellular components, organisms must have a supply of raw
material or nutrients.

• Nutrients are required for anabolic and catabolic processes of the cell and are required for growth.

Function of the nutrients :

Generation of energy
Synthesis of cellular materials
 NUTRIENTS
2 types of nutrients
1. Macronutrients – needed in large quantities for cellular metabolism & basic cell structure
➢ Major elements :C, H, O, N, S, P (g/l culture medium)
➢ Minor elements : K, Ca, Mg, Fe (mg/l culture medium)
2. Micronutrients – needed in small quantities; more specialized (enzyme & pigment structure &
function)
➢ Trace elements : Mn, Zn, Co, Ni, Cu (чg/l culture medium)
3. Growth factors – required in very small amounts, can not be synthesized by some cells.
These are vitamind and organic molecules.

Fastidious Bacteria: microbes that require other complex - nutrients/growth factors ( i.e., Vitamins or AAs)

Essential nutrients (basic bioelements needed for bacterial cell growth)

✓H2O: universal solvent; hydrolyzing agent
✓Carbon: food & energy source; in form of protein, sugar, lipid
✓Nitrogen: for protein synthesis; nucleic acid synthesis (purines & pyrimidines)
✓Sulfur (sulfate): amino acid synthesis (i.e., Cystine)
✓Phosphate: key component of DNA & RNA, ATP, and inner & outer membrane phospholipids
✓Minerals: associated with proteins (i.e., Fe:PRO); common component of enzymes.
NUTRIENTS

Element Usual form in the environment

C Carbon dioxide (CO2), organic compounds
H organic compounds
O Water(H2O), oxygen gas(O2)
N Ammonia(NH3), nitrate(NO3-), organic compounds such as amino acids

P Phosphate(PO4 3-)
S Hydrogen sulfide(H2S), sulfate(SO4 2-), organic compounds such as
cysteine
K K+
Mg Mg2+
Ca Ca2+
Na Na2+
Fe Fe3+, organic iron complexes
NUTRITION
 MICROBIAL CULTIVATION
• The process of growing microorganisms in culture by

• taking microorganisms from their natural habitat (environment) and/or

• the infection site (in vivo) and

• grow them in an artificial environment in the laboratory (in vitro)

• Laboratory growth requires proper nutrients and environmental conditions.

• When a microorganism is cultured, it replicates and increases in number and/or mass.

• We can get an idea of what microorganisms do by studying their activities in the laboratory in
pure cultures.

• A pure culture is a culture consisting of only one type of microorganism.

FACTORS AFFECTING MICROBIAL CULTIVATION
Conditions for microbial growth in the lab.

• Availability of Nutrients & H2O – culture(cultivation medium)

• Temperature

• Atmosphere – O2 & CO2

• H-ion concentration

• Moisture & drying

• Osmotic effects

• Radiation

• Mechanical & sonic stress.

pure culture if only one type of organism is present

mixed culture / coculture if populations of different organisms are present.

 CULTURE (CULTIVATION) MEDIA

Primary ingredients required by all living organisms include:

• carbon source,
• nitrogen source,
• minerals,
• water
GROWTH FACTORS
• Some cells require certain organic compounds in minute quantities – Growth Factors.

• They are needed for coenzymes or functional groups of certain enzymes and protein
synthesis.

• Most of microorganisms can synthesize these growth factors, but some require them from
the culture medium.

• The need for a growth factor results from either

- a blocked metabolic pathway or
- a missing metabolic pathway
• Auxotroph : a mutated microorganism that lacks the ability to synthesize an essential
nutrient and therefore must obtain it from its environment.
Auxotrophs require growth factors for survival.

• Prototroph : a microorganism requiring the same nutrients as most members of its species.

A Prototroph may mutate and change to an Auxotrophic microorganism.

Growth factors : Growth factors stated below take part in :

• Vitamin B
complex Vitamins → coenzymes

• thiamine Amino acids → protein synthesis

Purines and pyrimidines → RNA&DNA synthesis

• riboflavine

• nicotinic acid

• pyridoxine

• folic acid &

• Vitamin B 12
 CULTURE (CULTIVATION) MEDIA

The ingredients in a medium will affect the chemical nature of the medium.
This is important because organisms vary in their requirement for different
environments.
One such property is:
pH (which is a measure of the amount of hydrogen ions in a particular medium).

This has to be monitored during the preparation of media since this will
influence the kind of organisms that are able to grow in the medium.

The pH of the medium will thus determine which organisms are able to grow on
the medium.
For example, fungi prefer acidic media for their growth while bacteria
grow on neutral pH media.
MICROBIAL CULTIVATION
Due to their high rate of growth, it is easy to obtain in a short period of time a pure culture starting
with only a single cell.

To obtain a pure culture, growth must be done in the lab!!!

Therefore, it is essential to keep the other microorganisms from entering the pure culture.

Pure cultures are used to study the properties of the organism.

When pathogenic(disease causing) microorganisms are studied, special precautions must be taken to
prevent infection of the people nearby.

Microbiological techniques(aseptic technique) used in the labs avoid contamination of pure cultures.
Unwanted organisms are called contaminants.

Aseptic technique : manipulation of any microbial culture in such a way that contamination
does not occur.
ASEPTIC TECHNIQUE
Aseptic technique is a must in microbiology labs.

Most common problem in the labs : air borne contaminants.

Air contains dust particles that have a population of microorganisms.

To solve the contamination problem during lab. manipulations :

- All bottles, test tubes, flasks, petri plates etc. are opened at an angle to reduce the surface
exposed to air.
- Transfer of cultures are done with an inocolating loop or needle which is sterized by inciniration
in a flame.
- All the work is done near a flame, in a laminar flow hood with hepa filters that retains dust
particles and microorganisms or in dust free rooms without air currents.
ASEPTIC TECHNIQUE

Laminar flow hood

ASEPTIC TECHNIQUE
Aseptic technique is a must in microbiology labs.

Most common problem in the labs : air borne contaminants.

Air contains dust particles that have a population of microorganisms.

To solve the contamination problem during lab. manipulations :

- All bottles, test tubes, flasks, petri plates etc. are opened at an angle to reduce the surface
exposed to air.
- Transfer of cultures are done with an inocolating loop or needle which is sterized by inciniration in a
flame.
- All the work is done near a flame, in a laminar flow hood with hepa filters that retains dust
particles and microorganisms or in dust free rooms without air currents.
 Aseptic transfer
Inoculation needle

Aseptic transfer
Inoculation loop
AUTOCLAVES