LESSON 1: The Nature of Microbial

Physiology

Physiology – the study of the vital life processes of

organisms, especially how these processes normally
function in living organisms.

Microbial Physiology – concerns the vital life processes of

microorganisms specifically bacteria.

➔ The study of how microbial cell structures, growth,

and metabolism function in living organisms.

➔ The study of microbial cell functions which includes

the structure of microbial growth, microbial Functional Anatomy of Prokaryotes
metabolism, and microbial structure.
Prokaryotic Cell
➔ Structure-function relationships in microorganisms,
especially how microbes respond to their • Lacks nucleus or
environment membrane-bound
organelles
Microbial Metabolism - Sum of chemical reactions in a • Includes Bacteria
microorganism. • Simplest cell
• Single, Circular
Chromosome
• Nucleoid region (center)
Why study Microbial Physiology? contains the DNA.
• Surrounded by cell membrane & cell wall
• Play an important role in both fundamental research (peptidoglycan).
and in industrial applications of microorganisms. • Contain ribosomes (no membrane) in their cytoplasm to
• To analyze the role of individual components (genes make proteins.
or proteins) in the overall cell function.
 Cytoplasm
WHY BACTERIA? • Also known as proto-plasm.
• Gel-like matrix of water, enzymes, nutrients, wastes,
 Inexpensive to maintain in the lab and gases and contains cell structures.
 Take up little space • Location growth, metabolism, and replication.
 Reproduce quickly
 Morphology, nutrition, and metabolism are easily
observed  Granules
• Bacteria’s way of storing nutrients.
Microorganisms in the Physiological World • Staining of some granules aids in identification.

• Biosphere has been shaped both by physical events and

by interactions with the organisms that occupy it.  Cytoskeleton
• Prokaryotes are much more metabolically diverse than • Cellular “scaffolding” or skeleton within the
eukaryotes and can also thrive under a variety of cytoplasm.
extreme conditions where eukaryotes cannot. • Major advance in prokaryotic cell biology in the last
decade has been discovery of the prokaryotic
• This is possible because of the wealth of genes, cytoskeleton.
metabolic pathways, and molecular processes that are • Up until recently, thought to be a feature only of
unique to prokaryotic cells. eukaryotic cells.
• For this reason, prokaryotes are very important in the
cycling of elements, including carbon, nitrogen, sulfur,
and phosphorus, as well as metals and metalloids such
as copper, mercury, selenium, arsenic, and chromium.
• A full understanding of the complex biological
phenomena that occur in the biosphere therefore
requires a deep knowledge of the unique biological
processes that occur in this vast prokaryotic world.
 Ribosomes tooth enamel (yuck mouth and one of the causes of
cavities).
• Found within cytoplasm or attached to
plasma membrane.  Capsule
• Made of protein & rRNA. • Polysaccharides firmly attached to the cell wall.
• Composed of two subunits. • Capsules adhere to solid surfaces and to nutrients
• Cell may contain thousands. in the environment.
• Site for protein synthesis. • Adhesive power of capsules is a major factor in the
initiation of some bacterial diseases.
• Capsule also protect bacteria from being
 Plasma Membrane phagocytized by cells of the hosts immune system.
• Separates the cell from its environment.
• Phospholipids molecules -> hydrophilic water-loving  Chromosome
heads directed outward and hydrophobic water- • one, circular chromosome attached to the inside of
hating tails directed inward. the cell membrane.
• Proteins embedded in two layers of lipids (bilayer).
 Endospores vs. Exospores
 Bacterial Cell Wall • Dormant, tough, non-reproductive structure
• From the peptidoglycan produced by small number of bacteria.
inwards all bacteria are very • Resistant to radiation, desiccation, lysozyme,
similar. Going further out, temperature, starvation, and chemical disinfectants.
the bacterial world divides • Endospores are commonly found in soil and water,
into two major classes (plus where they may survive for very long periods of time.
couple of odd types).
• Peptidoglycan is a huge
polymer of interlocking
chains of alternating
monomers.
• Provides rigid support while
freely permeable to solutes.
• Backbone of peptidoglycan
molecule composed of two
amino sugar derivatives of glucose. Surface Appendages:
The “glycan” part of peptidoglycan:  Flagella
- N-acetylglucosamine (NAG) • Some prokaryotes have distinct appendages that
- N-acetylmuramic acid (NAM) allow them to move about or adhere to solid
• NAG/NAM strands are connected by interlocking peptide surfaces. • Consists of delicate stands of proteins.
bridges. The “peptid” part of peptidoglycan. • Long, thin extensions that allow some bacteria to
move about freely in aqueous environments
(singular: flagellum)

 Axial Filament (Endoflagella)

• Wind around bacteria, causing movement in waves.

 Surface Fimbriae/Fimbria
• Most Gram-negative bacteria have these short, fine
appendages surrounding the cell. Gram+ bacteria
don’t have.
 Cell Envelope • No role in motility
• The bacterial plasma membrane and cell wall • Help bacteria adhere to solid surfaces.
together are called the cell envelope. • Major factor in virulence.

 Glycocalyx
• Some bacteria have an additional layer outside of Cell Shapes
the cell wall called glycocalyx. Most bacteria are classified according to shape:
• These additional layers can come in two forms:
capsule and slime 1. Bacillus (pl. bacilli) = rod – shaped
2. Coccus (pl. cocci …. Sounds like cox-eye) = spherical
 Slime Layer 3. Spiral shaped
• Glycoproteins loosely associated with the cell wall. a. Spirillum (pl. spirilla) = spiral with rigid cell
• Slime layer causes bacteria to adhere to solid wall, flagella
surfaces and helps prevent the cell from drying out. b. Spirochete (pl. spirochetes) = spiral with
• Streptococcus - The slime layer of Gram+ flexible cell wall, axial filament
Streptococcus mutans allows it to accumulate on
• There are many more shape beyond these basic ones. A LESSON 2: Microbial Growth and Nutrition
few examples:
➢ Coccobacilli = elongated coccal form Microbial Growth
➢ Filamentous = bacilli in long threads
➢ Vibrios = short, slightly curved rods • Growth takes place on two conditions:
➢ Fusiform = bacilli with tapered ends ➢ Cell synthesizes new cell components and
increase in size.
Cell Arrangements ➢ Number of cells in the population increases
• Growth of most microorganisms occurs by binary fission.
• Cell division and chromosome replication are
coordinately regulated.

Microbial growth is affected by two major factors:

1. Environmental: temperature, pH, osmotic conditions
2. Chemicals: proper concentrations of C, H, O, N, P, S,
some trace elements, and some organic cofactors.

Population Growth – can be measured through the

increase of the number of microbial cells or increase in the
number of microbial mass. It is studied by analyzing the
growth curve of a microbial culture.
Growth Rate – change in cell number or cell mass per unit
time.
• Bacteria sometimes occur in groups, rather than singly.
• Bacilli divide along a single axis, seen in pairs or chains. Generation – interval for the formation of two cells from one.
• Cocci divides on one or more planes, producing cells in: Generation Time – time required for the formation of two
➢ Pairs (diplococci) cells from one.
➢ Chains (streptococci)
Binary Fission - Asexual form of reproduction
➢ Packets (sarcinae)
- Growth of an individual cell continues unit cell
➢ Clusters (staphylococci) divides into two new cells; a cell divides into two, two
to four, four to eight, etc.
• Size, shape and arrangement of cells often first clues in
identification of a bacterium. Generation or doubling time
• Many “look-alikes”, so shape and arrangements not enough • The time required for a complete fission cycle
for id of genus and species • Each new fission cycle or generation increases the
population by a factor of 2.
• As long as the environment is favorable, the doubling effect
continues at a constant rate.
• The length of the generation time – a measure of the
growth rate of an organism.
• Average generation time – 30 to 60 minutes under
optimum conditions.
• Can be as short as 10 to 12 minutes
• This growth pattern is termed exponential.

4 Phases of Microbial Growth

1. Lag Phase
• No immediate increase in cell number occurs however
cells in the culture are synthesizing new components.
Variety of reasons for Lag Phase:
• Cells may be old and depleted of ATP;
• Co-factors and ribosomes must be synthesized before
growth can begin.
• Medium may be different from the one the microorganism
was growing in previously.
• New enzymes would be needed to use different nutrients
• Possibly the microorganisms have been injured and
require time to recover.
• Whatever the causes, eventually the cells begin to replicate
their DNA, increase in mass, and finally divide.
 2. Exponential/Log Phase ➢ Cell wall strength
• Microorganisms are growing and dividing at the maximal ➢ Dps (DNA-binding protein from starved cells)
rate protein protects DNA
➢ Genetic potential • Starved cells become harder to kill
➢ Nature of the medium • More resistant to:
➢ Environmental conditions ➢ Starvation
• Growth rate is constant ➢ Damaging temperature changes
• Completing the cell cycle and doubling in number at regular ➢ Oxidative and osmotic damage
intervals ➢ Toxic chemicals such as chlorine.
• Population is uniform in terms of chemical and physiological
properties
• These changes are so effective that some bacteria can
• Exponential phase cultures are usually used in biochemical
survive starvation for years.
and physiological studies.
• There is even evidence that Salmonella enterica serovar
typhi and some other bacterial pathogens become more
• Balanced Growth
virulent when starved.
➢ All cellular constituents are manufactured at • These considerations are of great practical importance in
constant rates relative to each other. medical and industrial microbiology.
• Unbalanced Growth – change in nutrient level or
environmental condition.
➢ Rates of synthesis of cell components vary 4. Death Phase
relative to one another until a new balanced state is • Decline in viable cells following the stationary phase.
reached. • It was assumed that detrimental environmental change
• Shift – up – culture is transferred from a nutritionally poor such as:
medium to a richer one. ➢ Nutrient deprivation
➢ There is a lag while the cells first construct new ➢ Buildup of toxic wastes caused irreparable
ribosomes to enhance their capacity for protein harm and loss of viability
synthesis. • That is, even when bacterial cells were transferred to fresh
• Shift - down – culture is transferred from a rich medium to medium, no cellular growth was observed.
a poor one • Because loss of viability was often not accompanied by a
➢ There is a lag in growth because cells need time loss in total cell number, it was assumed that cells died but
to make the enzymes required for the biosynthesis did not lyse.
of unavailable nutrients.
• Once the cells are able to grow again, balanced growth is
resumes and the culture enters the exponential phase. Microbial Nutrition
• These shift-up and shift-down experiments demonstrate
that microbial growth is under precise, coordinated control • Uptake of nutrients that are acquired from the
and responds quickly to changes in environmental environment and are used for growth and metabolism
conditions. • Microorganisms (or microbes) vary significantly in terms
of the source, chemical form, and amount of essential
3. Stationary Phase elements they need.
• Population growth eventually ceases and the growth curve
becomes horizontal Basic Nutrients for Microbial Growth
• Total number of viable microorganisms remains constant
• Result from a balance between cell division and cell death  ATP for cellular processes
or  Carbon is necessary for the production of many
• Population may simply cease to divide but remain macromolecules (proteins, lipids, and
metabolically active. carbohydrates)
 Oxygen for metabolism nitrogen for amino acid
Reasons for Stationary Phase: synthesis
 Sulfur for vitamins, amino acids, structural stability
• Nutrient limitation of proteins
• Accumulation of toxic waste products  Phosphorous makes ATP and membranes
• Response to starvation for survival  Trace elements are used for metabolic reaction in
➢ Morphological changes such as endospore the cell and cell component stabilization.
formation Ex. Cobalt (Co), Potassium (K), Molybdenum (Mo),
• Decrease somewhat in overall size Magnesium (Mg), Manganese (Mn), Calcium, Iron, Zinc.
➢ Protoplast shrinkage  Organic growth factors such as vitamins, amino
➢ Nucleoid condensation acods, and nucleic acids some growth factors
• The more important changes during starvation are in cannot be synthesized by own cellular processes.
gene expression and physiology.  Water – water activity
• Starving bacteria frequently produce a variety of starvation
proteins which make the cell much more resistant to damage
➢ Increase peptidoglycan crosslinking
How to Obtain Nutrients? Lesson 3 Energy and Enzymes
• Heterotroph: uses organic carbon source
➢ They are unable to manufacture their own organic
food and hence are dependent on external source.
➢ Cannot use CO2 as a carbon source.
• Autotroph: uses inorganic carbon dioxide
➢ They are able to synthesize their own organic
food from inorganic substances.
➢ Can use CO2 as a sole carbon source (Carbon
Fixation).

Energy sources for bacteria:

• Phototroph: uses visible light as energy source
- photosynthesis
- Cyanobacteria, purple/green sulfur bacteria
 Enriched Media – added nutrient encourage the growth
• Chemotroph: uses chemical compound, org or inorg
of microorganism.
- chemosynthesis
 Selective Media – selects form a microorganism while
- Sulfur-oxidizing, nitrogen-fixing bacterias
inhibiting most other
➢ Saprobe
➢ Phenol Ethanol Agar – is a selective medium
➢ Parasite used to cultivates Gram positive organism.
➢ Deoxycholate Agar – solid bacteriological
growth medium used for isolation of enteric
Culturing Microorganisms
pathogens.
• Inoculum introduced into medium (broth or solid).  Differential Media – allows for the differentiation of
➢ Environmental specimens microorganisms based on action occurs on the media or
➢ Clinical specimens a color change within the media that is based on a pH
change.
➢ Stored specimens
➢ Mannitol Salt Agar – The MSA will select for
• Culture – refers to act of cultivating microorganisms or the organisms such as Staphylococcus species
microorganisms that are cultivated. which can love in areas of high salt
concentration.
Special Culture Techniques ➢ MacConkey Agar – it is designed to selectively
• Techniques developed for culturing microorganisms isolate Gram-negative and enteric bacteria
➢ Animal and cell culture and differentiate them based on lactose
fermentation. Lactose fermenters turn red or pink
➢ Low-oxygen culture
on McConkey Agar, and nonfermenters do
➢ Enrichment culture not change color.

Culture Media
Growth medium is used to grow microorganisms.
• It contains everything bacteria need to grow outside the
body and under laboratory conditions.
• Provides nutrients for the microorganisms.
 Chemically defined – medium is a growth medium
suitable for the microorganisms in which all of the
chemical components are known.
 Natural – the exact chemical composition is not known
is called natural or empirical culture media.
➢ Examples: milk, urine, diluted blood, vegetables
juice, meat extracts, beef and tomato juice, blood etc.
 Living – consists of living cells or tissue; strictly for
parasitic bacteria and viruses which cannot be culture in
non – living culture.
 LESSON 3: Microbial Metabolism: Energy
Generation and Conservation
Autotrophy in Hydrogen Bacteria
- Capacity of certain types of bacteria to manufacture their
Metabolism own organic materials for nutrition (self-feed), from hydrogen
- Sum of all chem reactions within organism - Do not depend on external sources, only hydrogen
- energy-balancing act – gives and acquires energy
Ex. Ralstonia eutropha – a chemolithoautotroph that utilizes
2 types of Metabolism Processes hydrogen gas to create organic compounds from CO2
➢ Anabolic (Biosynthetic) – Photosynthesis, DNA
- energy-requiring reactions (dehydration, endergoni Oxidation of Reduced Sulfur Compounds
- building complex molecules from simple ones - Sulfur containing compounds with extra electrons
- consume more energy than what they produce (reduced), undergo reactions to lose electrons (oxidized)
---- lower oxidation state to higher, by removing electrons
➢ Catabolic (Degenerative) – Glycolysis, Krebs - For treating sulfur-containing pollutants.
- enzyme-regulated reactions (hydrolytic, exergonic) This research holds substantial promise, potentially
- breakdown of complex org compounds to simple ushering in new avenues to:
- releases more energy than what they consume ➔ Effectively eliminate RSCs from the environment,
including within anaerobic wastewater treatment systems.
3 Stages: ➔ Innovate biofuel production by harnessing RSCs as a
1. Stage of Digestion valuable resource.
2. Release of Energy
➔ Enhance our understanding of the global sulfur cycle.
3. Energy Stored

Oxidation-Reduction (Redox Reaction) 3.3

- oxidized – electrons removed/taken away Enzymes and Catalysis
- reduction – electrons gained
- always coupled Structure of Enzymes
• Electron Donors – gives away/loses electron • Biological catalysts that speeds up biological reactions
• Electron Acceptors – receives/gains electron • Large globular protein
• Linear chain of amino acids for 3D structure
3.2 • Dents on surface where binding process takes place.
Energy and Enzymes Deformity is due to few amino acids.

ENERGETICS AND ENZYMES 2 Parts:

- important for the prediction of growth yields ➢Apoenzymes
- bacteria can synthesize ATP - Inactive Portion
- Methane, Biogas when bacteria generate energy - Determines the specificity of enzyme toward substr

Energetics of Chemolithotrophy ➢Cofactor

- Chemolithotrophic bacteria with the ability to use - non-protein chem compound that can be loosely or
inorganic sources tightly bound to proteins or;
- ‘litho’ means stones, so the are also called Stone-eaters - enzymes composes of org and inorg compounds
- essential for micropollutant cleaning such as hygienic
wastes, leftover medication, and synthetic hormones • Coenzyme – E. coli synthesizes vitamin B12
- could grow in unfavorable environment. • Prosthetic groups – binds enzymes permanently
Ex. Hydrogenovibrio crunogenus, Gammaproteobacteria
species Thiomicrospira crunogena (sulfur-oxidizing from Haloenzyme – Apoenzyme and Cofactor as whole
thermal vents)

Energetics of Hydrogen Oxidation

- Energy changes when hydrogen molecules reacts with
oxygen-producing water
- Highly exothermic reaction significant amount of energy
- important for assessing the efficiency of hydrogen-
based energy systems

Involves:
• Thermodynamics – explores energy levels of reactants
and products and asses overall energy change enthalpy
• Kinetics – rate of the reaction, speed and efficient inter

Knaligas bacteria – derives energy from hydrogen by oxidat

Classification of Enzymes Enzyme Regulation
- International Union of Biochemists (IUB) - activity where the controlling of individual molecules
bound to the enzyme is capable of preventing or promoting
= 6 Functional Groups = enzyme functionality

1. Oxidoreductases  Activators – increase activity

- oxidation-reduction (transfer electrons) • Allosteric Activator
- pyruvate dehydrogenase - link themselves to a part of enzyme that is not active site

2. Transferases  Inhibitor – Decrease activity

- role in moving functional groups • Allosteric Inhibitor
- catalyzes the transfer or chemical group - attaches to active site, decreases its functionality
• Reversible Inhibitor -
3. Hydrolases ➢ Competitive Inhibitor
- hydrolytic activity - active site of the enzymes with the present
- hydrolysis by introducing water to break hydrogen bonds substrates, and only one can attach.
➢ Non-Competitive Inhibitor
4. Lyases – attach any site, does not obstruct binding of subst
- add or remove water, CO2, or ammonia • Feedback Inhibition
- eliminates double bonds - system where products are programmed to stop production
5. Isomerases  Allosteric Regulation – binding with another site
- structural changes allosteric site
- alters shape, catalyzes isomer formation
 Allosteric Enzymes – maintains number of active sites
6. Ligases Enzyme Compartmentalization dictates that the enzyme
- Charge catalysis of ligation process assigned to a particular job can be limited to its particular
- Ligase in DNA joining two DNA fragments place in a cell
Mechanisms of Enzyme Activity and Regulation
(inhibitor) Mechanisms of Energy Production and
Conservation
Active Site or Catalytic Site
- location where substrates binds to active site
through weak forces, altering enzymes shape.  Generation of Adenosine Triphosphate (ATP)
- ATP is the main energy currency in all life forms.
• Induced Fit - Holds energy like battery.
- mutual interaction results in enzyme-substrate complex - Glucose is the main energy in Cellular Metabolism
- highly specific interaction, substrate fit like hand in glove
EX. Glycolysis, Krebs Cycle, Oxidative Phosphorylation

 Chemiosmotic Mechanism of ATP Generation

Chemiosmosis
- movement of ions across selectively permeable membrane
– electron carriers NADH and FADH donate electrons to
electron transport chain, causing conformational changes.
Enzymatic Reaction Process - generate 90% ATP during aerobic glucose catabolism.
• Lock and Key Theory
 Differences in Electron Transport
- Enzymes have a specific shape that fits the substrate
- like the lock and key! Electron Transport Chain
- series of 4 protein complexes that couple redox creating
electrochemical gradient leading to ATP creation (OPP)
- sequential oxidation/reduction (redox) reactions

 Role of Oxygen in Electron Transport

– Oxygen is used as the end electron acceptor for the
electron transport in cellular respiration.
- Allows electrons to be transferred through the Electron
Transport Chain in order to create an electrochemical
gradient for hydrogen to create ATP.
Factors that influence Activity of an Enzyme - In cellular respiration, oxygen is used in the inner
• Temperature mitochondrial membrane in the electron transport chain to
• pH facilitate oxidative phosphorylation.
• Substrate Concentration - In cellular respiration is to act as the end electron acceptor
  Phosphorylation
- Addition of Phosphoryl (PO3) group to some organic
compound
- Maintains the state of homeostasis by regulating chemical
reactions that require energy.
- Vital for glycolysis.
- It is also used for protein-protein interaction, protein
degradation, also regulates the inhibition of enzymes.

Types of Phosphorylation

1. Oxidative Phosphorylation
- Process by which the cells store and release chemical
energy in the body.

2. Glucose Phosphorylation
- Also undergoes phosphorylation as the first step of its
catabolism. For instance, the very first step of glycolysis of
D-glucose is the conversion of D-glucose into D-glucose-6-
phosphate

3. Protein Phosphorylation
- Addition of a phosphoryl group to an amino acid which is
normally serine, however, the process can also occur on
histidine (in prokaryotes) and threonine and tyrosine (in
eukaryotes).

 Substrate level phosphorylation

- This process involves an enzyme (a type of protein) which

transfers a phosphate group from a substrate (in this case, a
carbon-based molecule from food) to ADP.

- The ATP generated during glycolysis and the citric acid

cycle come about in this way, accounting for 4 ATPs per
glucose molecule.

● Glycolysis - (from glycose, an older term for glucose + -

lysis degradation) is the metabolic pathway that converts
glucose into pyruvate

● Pentose Phosphate - (PPP — also known as the hexose

monosphosphate shunt) is a cytosolic pathway that
interfaces with glycolysis. In this pathway, no ATP is directly
produced from the oxidation of glucose 6-phosphate; instead
the oxidative portion of the PPP is coupled to the production
of NADPH. *In addition to generating NADPH, which is
essential for detoxification reactions and fatty acid synthesis,
it also produces five-carbon sugars required for nucleotide
synthesis.

● Entner Duodoroff - describes an alternate series of

reactions that catabolize glucose to pyruvate using a set of
enzymes different from those used in either glycolysis or the
pentose phosphate pathway.