Physiology of microorganisms

Assoc. Prof. Dr. Tomas Kačergius

Institute of Biomedical Sciences, Faculty of Medicine
Vilnius University
 Nutrition types of microorganisms
The ways different microorganisms obtain carbon and capture energy can be
classified as autotrophy (“self-feeding”) and heterotrophy (“other-feeding”).

Autotrophs – use carbon dioxide as a carbon source to synthesize organic

molecules:
• Photoautotrophs – obtain energy from light by photosynthesis
(e.g., cyanobacteria).
• Chemoautotrophs – obtain energy from oxidizing simple inorganic
substances such as sulfides and nitrites (e.g., nitrifying bacteria).

Heterotrophs – get their carbon from ready-made organic molecules, which

they obtain from other organisms (dead or living):
• Chemoheterotrophs – obtain chemical energy from breaking down
ready-made organic compounds:
• Metatrophs – obtain carbon from glycogen, starch, cellulose.
• Paratrophs (parasites) – obtain carbon from soluble carbohydrates
(saccharose, glucose), and nitrogen – from amino acids.
• Photoheterotrophs – obtain chemical energy from light but require organic
substances as alcohols, fatty acids, or carbohydrates as carbon sources.
 Types of bacteria according to their dependence on oxygen
• Aerobes: bacteria, which require molecular oxygen for the metabolism and
growth (e.g., Mycobacterium tuberculosis). Aerobes use oxygen for
the oxidation of nutrients (e.g., carbohydrates) and production of
energy. For aerobic bacteria, oxygen is the final acceptor of electrons
in the electron transport chain.
• Anaerobes: bacteria, which do not require molecular oxygen for the metabolism
and growth (e.g., Clostridium botulinum). Oxygen is toxic for these
bacteria. Oxygen containing inorganic molecules (nitrates, nitrites,
sulfates) are final acceptors of electrons in electron transport chain.
• Facultative anaerobes: bacteria, which grow in either the presence or the
absence of oxygen (e.g., Staphylococcus aureus). They
can generate ATP in aerobic conditions, when oxygen
is present in environment, and in anaerobic conditions,
when oxygen is absent in environment.
• Microaerophiles: bacteria, which require an oxygen to survive, however at
much lower levels (2–10%) than it is present in atmosphere
(e.g., Campylobacter jejuni). Most of them are capnophiles
because they need the elevated amount of carbon dioxide.
 Bacterial enzymes
Bacterial enzymes are classified into endoenzymes located intracellularly, and
exoenzymes secreted to the environment (e.g., collagenase, hyaluronidase).
Endoenzymes are subdivided into six groups according to their catalyzed
reactions:
• Oxidoreductases: participate in the oxidation-reduction reactions, when
oxygen and hydrogen are added or released, e.g.,
cytochrome oxidase.
• Transferases: transfer the specific functional groups (amine, acyl) from one
molecule to another molecule, e.g., alanine transaminase.
• Hydrolases: participate in the hydrolysis reactions (addition of water), e.g.,
saccharases.
• Lyases: transfer the groups of atoms without hydrolysis reaction, e.g., oxalate
decarboxylase.
• Isomerases: reorganize atoms inside the molecule, e.g., glucose phosphate
isomerase.
• Ligases: joins two large molecules by using ATP energy, e.g., DNA ligase.
 Uptake of nutrients by the bacterial cell

• Passive diffusion: occurs by the concentration gradient of substances. In this

diffusion, transmembrane proteins – porins are involved that
form pores in bacterial cytoplasmic membrane and cell wall,
as well as they assist in the transport function (facilitated
diffusion). Pores and channels allow entry of ions and small
hydrophilic molecules by the passive diffusion.

• Active transport: occurs against the concentration gradient of substances.

The enzymes – permeases are involved in active transport.
Permeases form the phosphotransferase system. This
transport system uses energy obtained from the high-energy
phosphoenolpyruvate (PEP) molecules. When PEP is present
in the cytoplasm, it can provide energy and a phosphate
group to a permease in the membrane. Then the permease
transfers the phosphate to a sugar molecule and at the same
time moves the sugar across the membrane. A phosphorylated
sugar is thus transported inside the cell and is prepared to
undergo metabolism.
 Bacterial metabolism

• Metabolism is the sum of all chemical processes occurring in the bacterial cell.
• Bacterial metabolism consists of:
• catabolism – chemical reactions that release energy by breaking
complex molecules into simpler ones.
• anabolism – chemical reactions that require energy to synthesize
complex molecules from simpler ones.
• Catabolic reactions provide energy for bacteria that is required for bacterial
life processes: movement, active transport of nutrients, and the synthesis of
complex molecules.
• Anabolic reactions are needed for the bacterial growth, reproduction, and
repair of cellular structures.
• All catabolic reactions involve electron transfer, which allows energy to be
captured in high-energy bonds in the ATP and similar molecules.
• Electron transfer is directly related to oxidation (loss or removal of electrons)
and reduction (gain of electrons).
 Energy production in bacteria

Bacteria produce energy in the forms of adenosine triphosphate (ATP) and

reduced nicotinamide adenine dinucleotide (NADH) from glucose using three
major metabolic pathways in its catabolism: glycolysis, tricarboxylic acid cycle
and pentose phosphate pathway.

• Glycolysis occurs under both aerobic and anaerobic conditions yielding 2 ATP
molecules, 2 NADH molecules and 2 pyruvate molecules from 1 glucose
molecule. The main chemical processes that occur during glycolysis:
1. Substrate-level phosphorylation – that is, transfer of phosphate groups
from ATP to glucose.
2. Breakdown of six-carbon glucose into two three-carbon molecules.
3. Transfer of two electrons to the coenzyme NAD.
4. Capture of energy in the ATP molecules.
 Energy production in bacteria

• Tricarboxylic acid cycle (the Krebs cycle) – that is a sequence of chemical

reactions in which acetyl groups are oxidized to carbon dioxide. Hydrogen
atoms are also removed, and their electrons are transferred to coenzymes
that serve as electron carriers. The hydrogens are eventually combined with
oxygen to form water.

• Each reaction in the Krebs cycle is controlled by a specific enzyme, and the
molecules are passed from one enzyme to the next as they go through the cycle.
The main chemical processes that occur during the Krebs cycle:
1. The oxidation of carbon.
2. The transfer of electrons to coenzymes.
3. Capture of energy in the ATP molecules.
 Energy production in bacteria

• Tricarboxylic acid cycle occurs only under aerobic conditions and allows the
bacteria to generate substantially more energy than is possible from glycolysis
alone. In total, the aerobic metabolism can produce 38 ATP molecules from
glucose, that is, 19 times more than anaerobic metabolism (2 ATP molecules).

• The importance of tricarboxylic acid cycle:

1. It is the most efficient mechanism for the generation of ATP.
2. It serves as the final common pathway for the complete oxidation of amino
acids, fatty acids and carbohydrates.
3. It supplies key intermediates (i.e., α-ketoglutarate, pyruvate, oxaloacetate)
for the ultimate synthesis of amino acids, lipids, purines and pyrimidines.
 Energy production in bacteria

• Pentose phosphate pathway occurs along with glycolysis in bacteria, when

not only glucose but also the five-carbon carbohydrates (pentoses) are broken
down.

• The function of this pathway is to provide nucleic acid precursors and reducing
power in the form of NADPH for use in biosynthesis.

• Pentose phosphate pathway can generate only 1 ATP molecule for each oxidized
glucose molecule.
Energy production in bacteria using amino acids
(Stickland reaction)

ALANINE PROLINE
NAD+
NH3 NADH
Pyruvate 5-Aminovalerate

Acetyl-CoA

Acetyl-phosphate
ADP
ATP
Acetate

Copyright © ASM Press

 Purposes of the microorganisms’ cultivation in medicine

• Laboratory diagnosis of the infectious diseases.

• Production of the biologically active substances (biopharmaceuticals),

vaccines and antimicrobial agents.

• Research for understanding the pathogenesis of infectious diseases as

well as development of the effective treatment and preventive measures.
Approaches for the cultivation of microorganisms

• Culture media.

• Cell line cultures (primary; diploid; tumor or immortalized).

• Embryonated chicken eggs.

• Laboratory animals.
General requirements for culture media

• Sterile

• Isotonic

• Neutral pH

• Translucent (transparent)
 Types of culture media
• Nonselective media (e.g., blood agar) are designed to support the growth
of most microorganisms without fastidious growth requirements.

• Selective media (e.g., brilliant green bile agar) are designed for the recovery
of specific microorganisms that may be present in a mixture of other
microorganisms (e.g., an enteric pathogen in stool). The media are
supplemented with inhibitors that suppress the growth of unwanted
microorganisms.

• Differential-diagnostic media made from selective media by adding

specific ingredients that allow the identification of a microorganism in a
mixture (e.g., addition of lactose and a pH indicator to detect lactose
fermenting bacteria).

• Specialized (special formulation) media are designed for the detection of

specific microorganisms that may be fastidious or typically present in large
mixtures of microorganisms.

• Chemically defined media are used for the cultivation of mammalian cells.
 Types of culture media
• Transport media are formulated to preserve a specimen and minimize
bacterial overgrowth from the time of collection to the time it is received
at the laboratory to be processed.

• Storage media are used for storing the microorganisms for a long period of time.
Enterobacteria growing on the nonselective medium
(Columbia blood agar)

Copyright © 2016 E&O Laboratories Ltd. All rights reserved.

Escherichia coli growing on the selective medium
(MacConkey agar)

Copyright © 2016 E&O Laboratories Ltd. All rights reserved.

 Bacterial cell division

Copyright © by Elsevier Inc. All rights reserved.

 Bacterial growth

• Bacteria reproduce asexually by means of simple transverse binary fission.

• Their numbers (n) increase logarithmically (n = 2G). The time required for
a reproduction cycle (G) is called the generation time (g) and can vary greatly
from species to species.

• Fast-growing bacteria cultivated in vitro have a generation time of 15–30 min.

The same bacteria may take hours to reproduce in vivo. Obligate anaerobes
grow much more slowly than aerobes; this is true in vitro as well. Tuberculosis
bacteria have an in vitro generation time of 12–24 h. The generation time also
depends on the nutrient content of the medium.
Phases of bacterial growth in the culture medium

A – lag phase
B – acceleration phase
C – log (exponential) phase
D – deceleration phase
E – stationary phase
F – death phase

Kayser, Medical Microbiology © 2005 Thieme. All rights reserved.

 Phases of the bacterial growth curve

• The lag phase (A) is characterized by an increase in bacterial mass per unit
of volume, but no increase in cell count. During this phase, the metabolism
of the bacteria adapts to the conditions of the nutrient medium.

• In the following log (or exponential) phase (C), the cell count increases
logarithmically up to about 109/ml. This is followed by growth deceleration (D)
and transition to the stationary phase (E) due to exhaustion of the nutrients
and the increasing concentration of toxic metabolites. Finally, death phase (F)
processes begin.
 Literature

1. Black J. G., Black L. J. Microbiology: principles and explorations.

9th edition. Willey, New Jersey, 2015.

2. Murray P. R., Rosenthal K. S., Phaller M. A. Medical microbiology.

8th edition. Elsevier Inc., Philadelphia, 2015.

3. Lamont R. J., Hajishengallis G. N., Jenkinson H. F. Oral microbiology

and immunology. 2nd edition. ASM Press, Washington, DC, 2014.

4. Kayser F. H., Bienz K. A., Eckert J., Zinkernagel R. M. Medical microbiology.

Thieme Publishers, Stuttgart, New York, 2005.