ENVIRONMENTAL
MICROBIOLOGY
-LABORATORY MANUALPREPARED FOR ENVIRONMENTAL MICROBIOLOGY
IV
BIOCHEMICAL ACTIVITY OF
MICROORGANISM
E N V I RO N M E N TA L M A N A G E M E N T T E C H N O L O G Y
FAC U L T Y O F C I V I L A N D E N V I RO N M E N TA L E N G I N E E R I N G
ITB, 2010
Terms
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Metabolism
Anabolism
Catabolism
Catalytic
Catalyst
Enzyme
Apoenzyme
Holoenzyme
Coenzym
Endoenzyme
Exoenzyme
Enzyme activity
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Enzyme system
Substrate
Active Site
Oxidation
Reduction
Hydrolysis
Glycolysis
Fermentation
Aerobic respiration
Anaerobic
respiration
Peptide bonding
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Peptonization
Starch
Carbohydrate
Amino acid
Lipid
Fatty acid
Protein
Casein
Pyruvic acid
ATP, ADP
Metabolism
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Metabolism refers to all chemical reactions that
occur within a living organism.
Catabolic:
Degradative reactions
that release energy
by BREAKING DOWN
large, complex
molecules into smaller
ones. E.g. Hydrolysis
Anabolic:
Biosynthetic reactions
that BUILD large
complex molecules
from simpler ones;
requires energy
Anabolic and Catabolic Reactions are Linked by ATP in Living
Organisms
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Catalyst = an agent that accelerates chemical
reaction without itself being destroyed or used up
ENZYME
Enzyme
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Organic catalyst (elaborated by living cell)
Protein mollecular
Thermolabile (denaturated by heat)
Precipitated by ethanol and high concentration
inorganic salts
Non-dialyzable (does not go through semipermeable membrane)
CHARACTERISTIC OF ENZYMES
Specificity
• Enzymes are
highly specific
and usually
catalyze only
one or a few
closely related
reactions
Effectivity
• Enzymes are
extremely
efficient.
• Speed up
reaction up to
10 billion times
more than
without enzyme.
Energy of Activation
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Energy of activation: The
amount of energy
required to trigger a
chemical reaction.
Enzymes speed up
chemical reactions by
decreasing their energy
of activation without
increasing the
temperature or pressure
inside the cell.
Extracellular
Enzyme
Intracellular
Enzyme
• Exoenzymes
• works outside
cell
• Altering nutrient
in vicinity of cell
• Endoenzymes
• works inside cell
• Synthesizing
cellular material
to provide
required energy
Enzyme Components
 Some
enzymes consist of protein only.
 Others : Holoenzyme = Apoenzyme + Cofactor
 Enzyme cofactors may be a metal ion, an organic
molecule, or derived from vitamins.
Examples:
 NAD+: Nicotinamide adenine dinucleotide
 NADP+: Nicotinamide adenine dinucleotide phosphate
are both cofactors derived from niacin (B vitamin).
 Coenzyme A is derived from panthotenic acid.
Components of a Holoenzyme
Mechanism of Enzymatic Action
Surface of enzyme contains an active site that binds
specifically to the substrate.
1. An enzyme-substrate complex forms.
2. Substrate molecule is transformed by:
Rearrangement of existing atoms
 Breakdown of substrate molecule
 Combination with another substrate molecule
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3. Products of reaction no longer fit the active site and are
released.
4. Unchanged enzyme is free to bind to more substrate
molecules.
Mechanism of Enzymatic Action
Factors that Affect Enzyme Activity: pH,
Temperature, and Substrate Concentration
Denaturation of a Protein Abolishes its Activity
Denaturation: Loss of three-dimensional protein structure. Involves breakage
of H and noncovalent bonds.
REDOX
Redox Reactions: Reactions in which both
oxidation and reduction occur.
OXIDATION
• Removal of electrons or H atoms
• Addition of oxygen
• Associated with loss of energy
REDUCTION
• Gain of electrons or H atoms
• Loss of oxygen
• Associated with gain of energy
Oxidation-Reduction Reactions
Aerobic Respiration is a Redox Reaction
C6H12O6 + 6 O2 -----> 6 CO2 +
6 H2O + ATP
Glucose oxygen oxidized reduced
Hydrolysis
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Hydrolysis is a chemical reaction during which
molecules of water (H2O) are split into hydrogen
cations (H+) (conventionally referred to as protons)
and hydroxide anions (OH−) in the process of a
chemical mechanism.
Carbohydrate Catabolism
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Most microorganisms use glucose or other
carbohydrates as their primary source of energy.
Lipids and proteins are also used as energy sources.
Two general processes are used to obtain energy
from glucose:
 cellular
respiration
 fermentation
Cellular Respiration : AEROBIC
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ATP generating process in which food molecules are oxidized.
Final electron acceptor is oxygen.
Aerobic Respiration
C6H12O6 + 6 O2 -----> 6 CO2 + 6H2O + ATP
Glucose
oxygen
oxidized
reduced
Aerobic Respiration occurs in three stages:
1. Glycolysis
2. Kreb’s Cycle
3. Electron Transport & Chemiosmosis
Three Stages of Aerobic Respiration
Fermentation
Releases energy from sugars or other organic molecules.
 Does not require oxygen, but may occur in its presence.
 Does not require an electron transport chain.
 Final electron acceptor is organic molecule.
 Inefficient: Produces a small amount of ATP for each
molecule of food.
 End-products are energy rich organic compounds:
 Lactic acid
 Alcohol
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Aerobic Respiration versus Fermentation
CELLULAR RESPIRATION : ANAEROBIC
Final electron acceptor is not oxygen.
 Instead it is an inorganic molecule:
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Nitrate (NO3-): Pseudomonas and Bacillus. Reduced to nitrite
(NO2-):, nitrous oxide, or nitrogen gas.
 Sulfate (SO42-): Desulfovibrio. Reduced to hydrogen sulfide
(H2S).
 Carbonate (CO32-): Reduced to methane.
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Inefficient (2 ATPs per glucose molecule).
Only part of the Krebs cycle operates without oxygen.
 Not all carriers in electron transport chain participate.
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Anaerobes tend to grow more slowly than aerobes.
EXPERIMENTS
BASIC PRINCIPLES
When identifying a suspected organism, you inoculate
a series of differential media. Then incubated.
You observe each medium to see if specific end
products of metabolism are present, by adding
indicators to the medium that react specifically with
the end product being tested, giving some form of
visible reaction such as a color change.
The results of these tests on the suspected
microorganism are then compared to known results for
that organism to confirm its identification.
Exp.
M.O.
Reagent
Reaction
Enzyme
18.A Starch Hydrolysis
E. coli, B.subtilis
Lugol
Polysaccharide monosaccharide
Amilase
18.B Lipid Hydrolysis
-
-
Tryglyseride  glycerol + fatty acid
Lipase
18.C Casein Hydrolysis
E. coli, B.subtilis
Lugol
Proteinpeptide amino acid
Protease
19.AFermentation of
Carbohydrates
E.coli, P. Aeruginosa
Red Phenol
e.g.Glucose  pyruvic acid
Various
19.B Reduction of Nitrate
E.coli, P. aeruginosa
Sulfanilic Acid,
Alphanaphtylamine
Nitrate  Nitrite
Nitrate reductase
19.C Catalase Reaction
S.aureus, S.lutea, B.subtilis
3 % H 2 O2
H 2 O2  H 2 O + O 2
Catalase,
superoxide
dismutase
19.D Oxidation Test
E.coli, P. Aeruginosa,
A.faecalis
p-aminodimethylanyline
oxalate
Form of oxydase cytochrome
Oxydase
20.A1 Indol Reaction Test
E.coli, B.cereus
Kovac
Tryptophane  indole + pyruvic acid +
ammonia
Tryptophanase
20.A2 Methyl Red Reaction
E.coli, E.aerogenes
Methyl red
Glucose + H2O  acid + CO2 + H2
various
20.A3 Vogus-Proskauer
Reaction
E.coli, E.aerogenes
Barrit
Glucose + O2  acetic acid 
acetymethylcarbinol + CO2 + H2
20.A4 Use of Citrate
E.coli, E.aerogenes
Bromthymol blue
Citrate  oxaloacetate acid + acetat 
pyruvic acid + CO2
20.B Triple Sugar Iron Agar
Test
E.coli, P. Aeruginosa,
E.aerogenes
Phenol red
Carbohydrate + O2  acid
21.A Litmus Milk Reaction
E.coli, P. Aeruginosa,
A.faecalis, S.lactis,
E.aerogenes
Litmus
Lactose  glucose + galactose  pyruvic acid
 lactate acid
B-galactosidase
21.B Urease Test
E.coli, P.vulgaris
Phenol red
Amide substances  ammonia
urease
21.C H2S Production
E.coli, P.vulgaris
Ferrous sulphate
Cysteine  pyruvic acid + H2S + ammonia
Cysteine desulfurase
Citrate permease
Exp. 18 – Acivities of Extracelullar Enzymes
Hydrolysis of Starch and Casein
Hydrolysis of Starch
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Starch  dextrin /
monohydrates
Amylase
Starch Agar  the
presence of starch in
medium
Iodine  indicator, if
starch is still present,
blue (-), if does not
present, transparent (+)
Hydrolysis of Casein
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Protein  various amino acids
linked together in long chains by
means of peptide bonds
Protein  peptide  amino
acid
Prior use as nutrition material 
needs to be degraded into
simpler substances through
peptonization or proteolysis
process using protease enzyme
(breaking CO-NH bonding)
Proteolytic zone  transparent
Hydrolysis of Starch & Casein
E. coli, B.subtilis
Carbohydrate Agar
Milk Agar
Streak
Streak
Incubate 37◦
Incubate 37◦
Iodine
Observe for transparent area (+)
Observe for color change
Blue (-), Transparent (-)
Exp. 19 – Acivities of Intracelullar Enzymes : Fermentation
Test and Oxidation
19.A. Fermentation of Carbohydrates
19.B. Reduction of Nitrate
19.C. Catalase Reaction
19.D. Oxidation Test
19.A. Fermentation of Carbohydrates
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A wide variety of carbohydrates may be fermented
in order to obtain energy and the types of
carbohydrates which are fermented by a specific
organism can serve as a diagnostic tool for the
identification of that organism.
End products of fermentation.
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acid end products.
acid and gas end products.
Red phenol  red in normal pH, yellow in acid
condition
19.A. Fermentation of Carbohydrates
E.coli, P. Aeruginosa
Lactose broth tube
+ Durham
Sucrose broth
tube+ Durham
Glucose broth
tube+ Durham
Mannitol broth
tube+ Durham
Inoculate, control
Inoculate, control
Inoculate, control
Inoculate, control
Incubate 37◦
Incubate 37◦
Incubate 37◦
Incubate 37◦
Red phenol
Red phenol
Red phenol
Red phenol
(+) red,
(-) yellow
Observe
for gas
(+) red,
(-) yellow
Observe
for gas
(+) red,
(-) yellow
Observe
for gas
(+) red,
(-) yellow
Observe
for gas
19.B. Reduction of Nitrate
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Bacteria can reduce nitrate
Anaerobic condition
Nitrate reductase enzyme
NO3- + 2e- + 2H-  NO2- + H2O
Reagent A Sulfanilic acid + Reagen B
alphanaphtylamine, if nitrite is presence (+), red
If (-), zinc will reduce nitrate, bring red
color,indicates that nitrate did not reduced before,
if transparent (+)
19.B. Reduction of Nitrate
E.coli, P. Aeruginosa
nitrate broth tube
inoculate
Incubate 37◦
Reagent A + B
If broth turns red (+), no color changes(-)
If (-) add zinc, shake. If nitrate is
presence, broth will turn red (-)
19.C. Catalase Reaction
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Aerobic reaction  hydrogen peroxide, reactive,
destructing enzyme
Catalase  preventing damage, turning H2O2 into
free H20 and O2
Superoxide dismutase  in species which has no
catalase
19.C. Catalase Reaction
S.aureus, S.lutea, B.subtilis
Nutrition Agar plate
streak
Incubate 37◦
3 % H2O2
Air bubble (+)  the presence of
catalase
19.D. Oxidase Test
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Oxidase enzyme  Electron transport system in
aerobic resp.
p-aminodimethylanyline oxalate  artificial
substrate, donating electrone and be oxidized into
black substances if oxydase and free oxygen are
present
(+)  pink – maroon-black
(-)  no color change
19.D. Oxidase Test
E.coli, P. Aeruginosa, A.faecalis, A, B
Trypticase soy in plate
streak
Incubate 37◦
p-aminodimethylanyline oxalate
Blackening (+), no color change (-)
Exp. 20 – Acivities of Intracelullar Enzymes : IMViC and TSI
Test
20.A.1 Indol Reaction Test
20.A.2 Methyl Red Reaction
20.A.3 Vogus-Proskauer Reaction
20.A.4 Use of Citrate
20.B. Triple Sugar Iron Agar Test
A. IMVic Test
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Enterobactericeae  G.I tract
Identification is important in preventing
contamination to food and water supply
Pathogenic, sometimes pathogenic, normal flora
Non Enteric
Fermenting
lactose
Enteric
Not fermenting
lactose
20.A.1 Indol Reaction Test
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Indol =a component of
tryptophane, an essential amino
acid
Will not occur if carbohydrate
that needs to be degraded is
exsist  low pH
Specific characteristic of
intestinal bacteria
Indol + Kovac reagent  red
cherry on the surface of the test
tube
20.A.1 Indol Reaction Test
E.coli, B.cereus
Trypton 1 % broth tube
Trypton 1 % + glucose 1 % broth tube
streak
Streak
Incubate 37◦
Incubate 37◦
Kovac reagent
Kovac reagent
Red colors on tube surface (+)
Observe for red ring
20.A.2 Methyl Red Reaction
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Glucose  primary energy source for enteric
Some turn glucose into acid (glucose fermentation)
 low pH
Important in differentiating E.coli and E.aerogenes
Methyl red
 red
: pH 4
 Yellow : pH6
20.A.2 Methyl Red Reaction
E.coli, E.aerogenes
MR-VP broth tube
inoculate
Incubate 37◦
Methyl red
Red (+), yellow (-)
The Methyl Red Test: Left
to Right: positive, positive,
negative, control.
20.A.3 Vogus-Proskauer Reaction
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Some fermentative organisms do not produce enough
stable acids to lower the pH of the medium.
To detect the ability of m.o. In producing non acid
substance acetymethylcarbinol
Characteristic of E. aerogenes
m.o. culture
MR-VP broth tube
inoculate
Incubate 37◦
1 mL Barrit reagent
Wait 5-30 min, Observe for red color
(+), copper color or no changes (-)
Voges-Proskauer Test
Left: uninoculated control
Right: negative (copper
color)
Left: uninoculated control
Right: positive (red color)
20.A.4 Use of Citrate
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When carbohydrate does not present, citrate is used as
carbon source in providing energy
Citrate permease  facilitating citrate transport
Citrate  oxalatoacetate acid + acetate  pyruvic
acid + CO2  during this reaction medium will turn to
alkaline condition
Bromthymol blue indicator will turn from green at neutral
pH (6.9) to blue when a pH higher than 7.6 is reached
(basic or alkaline).
If the citrate is utilized, the resulting gowth will produce
alkaline products (pH >7.6), changing the color of the
medium from green to blue.
E.coli, E.aerogenes, A, B
Simmons citrate slant agar
Stab or streak
Incubate 37◦
Observe
Citrate Utilization
Enterobacter cloacae: positive
Eschericia coli: negative
Klebsiella pneumoniae: positive
20.B. Triple Sugar Iron Agar Test
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To differentiate Enterobactericeae (bacil, gram -,
fermenting glucose, producing acid) vs non
Enterobactericeae
TSI  glucose + lactose + sucrose + phenol red
Phenol red as pH indicator  red (alkaline), yellow
(acid)
Surface : red, bottom : yellow  glucose fermentation only
 Surface and bottom : yellow  lactose and sucrose
fermentation also occur
 Surface and bottom : red  no carb fermentation
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20.B. Triple Sugar Iron Agar Test
E.coli, P. Aeruginosa,
E.aerogenes
TSI slant agar
Stab and streak
Incubate 37◦
Observe
Exp. 21 – Other Biochemical Activities
21.A. Litmus Milk Reaction
21.B. Urease Test
21.C. H2S Production
21.A. Litmus Milk Reaction
Several milk substrate reactions using litmus in media:
 Glucose fermentation  litmus act as pH indicator 
purple : normal pH, pink : acid, formation of gas
 Litmus reduction  litmus act as acceptor to bond
hydrogen ion  purple : oxidized, white : reducted
 Curd formation  acid type (solid) and rennet type
(semi solid)
 Proteolysis  forming of ammonia  litmus act as pH
indicator
 Alcaline reaction  litmus act as pH indicator
POSSIBLE REACTION
A. Acid/Reduction/Cu
rd
B. Reduction/Curd
(arrow denotes gas
pocket)
C. Uninoculated
Control
D. Acid Formation
E. Proteolysis of casein
F. Alkaline Reaction
21.A. Litmus Milk Reaction
E.coli, P. Aeruginosa, A.faecalis,
S.lactis, E.aerogenes
Litmus milk broth tube
inoculate
Incubate 37◦
Observe
21.B. Urease Test
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Identifying P.vulgaris with urease enzyme
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Amide substances  ammonia

Phenol red as pH indicator
21.B. Urease Test
E.coli, P.vulgaris
Urea broth tube
inoculate
Incubate 37◦
Dark pink (+)
21.B. H2S Production
Some bacteria are capable of breaking down
sulfur or reducing inorganic sulfur-containing
compounds to produce hydrogen sulfide (H2S).
 For identifying Proteus and Salmonella.
 a medium with a sulfur-containing compound
and iron salts  If the sulfur is reduced and
hydrogen sulfide is produced, it will combine
with the iron salt to form a visible black ferric
sulfide (FeS) in the tube.

21.B. H2S Production
E.coli, P.vulgaris
H2S deep agar
stab
Incubate 37◦
Observe for blackening
Why all these efforts ?
“To identify bacteria, we must rely heavily
on biochemical testing. The types of
biochemical reactions each organism
undergoes act as a "thumbprint" for its
identification.”