Microbial physiology and enzymology

Prof. Dr. Mohammad Magdy

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2
Introduction
Microbial physiology: what and why

from Ancient Greek φύσις (physis), meaning "nature, origin", and - logia),
meaning "study of") is the scientific study of function in living systems. A
sub-discipline of biology, its focus is in how organisms, organ systems,
organs, cells, and bio-molecules carry out the chemical or physical functions
that exist in a living system.

What is the broad definition of physiology?

all the structures of organism and related
functions which leads to basic processes.

What is the narrow definition of physiology?

study of the relationship between structure and
function.
Broad definition of physiology encompasses....?

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all of life. biochemistry, nutrition, adaptation

and responses to environmental conditions,
metabolism, genetics.

Cell Structure and Organisation

The basic unit of all living things is the cell. The cell theory is one of the
fundamental concepts of biology; it states that: all organisms are made up
of cells, and that all cells derive from other, pre-existing cells. As we shall see
in this chapter, there may exist within a cell many smaller, subcellular
structures, each with its own characteristics and function, but these are not
capable of independent life. An organism may comprise just a single cell
(unicellular), a collection of cells that are not morphologically or functionally
differentiated (colonial), or several distinct cell types with specialised
functions (multicellular). Among microorganisms, all The names given to the
two cell types derive from Greek words: Procaryotic = ‘before nucleus’
Eucaryotic = ‘true nucleus’ bacteria and protozoans are unicellular; fungi
may be unicellular or multicellular, while algae may exist in all three forms.
There is, however, one way that organisms can be differentiated from each
other that is even more fundamental than whether they are uni- or
multicellular. It is a difference that is greater than that between a lion and a
mushroom or between an earthworm and an oak tree, and it exists at the
level of the individual cell. All organisms are made up of one or other
(definitely not both!) of two very distinct cell types, which we call procaryotic
and eucaryotic cells, both of which exist in the microbial world. These differ
from each other in many ways, including size, structural complexity and

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organisation of genetic material. The most fundamental difference between

procaryotic and eucaryotic cells is reflected in their names; eucaryotic cells
possess a true nucleus, and several other distinct subcellular organelles that
are bounded by a membrane. Procaryotes have no such organelles. Most of
these differences only became apparent after the development of electron
microscopy techniques. The procaryotes comprise the simpler and more
primitive types of microorganisms; they are generally single celled, and arose
much earlier in evolutionary history than the eucaryotes. Indeed, as
discussed later in this chapter, it is widely accepted that eucaryotic cells
actually arose from their more primitive counterparts.
Organelles of cells
- The cell is the fundamental unit of life.
- The modern ‘Cell theory’ states :
i) All living organisms are composed of cells.
ii) All new cells are derived from other cells.
iii) Cells contain the hereditary material of an
organism which is passed from parent to
daughter cells.
iv) All metabolic process take place within
cells.
Cell wall
A. Strong structural layer that lies outside the plasma membrane but does
not include capsule or slime layer (cell wall + capsule = cell envelope)
B. Function
1. shape
2. protection from osmotic lysis: pressure inside the cell = 300 lbs/in2
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3. can be virulence factor (LPS in gram-negatives)

4. defense against host immune response (LPS in gram-negatives)
5. protection from some toxic substances

Cell membranes
- They are described as selectively permeable, since apart from small
molecules, such as water, larger molecule e.g. glucose, amino acids, glycerol
and ions can diffuse slowly through them. And they also exert a measure of
active control over what substances they allow through.
- As organic solvent (alcohol) penetrate membranes even more rapidly than
water, this suggested that membranes have non-polar portions; in other
words they contain lipids.
- After careful chemical analysis, it is found that membranes are comprised
almost entirely of proteins and lipids (phospholipids, glycolipids and
sterols).
- The fluid-mosaic model can be used to describe the detailed structure of
plasma membrane.
The fluid-mosaic model (Singer-Nicholson model ) :
It was put forward in the early 1970s by S.J. Singer and G.L. Nicholson.
The protein molecules vary in size and have a much less regular
arrangement. Some proteins occur on the surface of the phospholipid layer,
while others extend into it, some even extend completely across it.

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Viewed from the surface, the proteins are dotted throughout the
phospholipid bilayer in a mosaic arrangement. The hydrophilic phosphate
heads of the phospholipids face outwards into the aqueous environment
inside and outside the cell. The hydrocarbon tails face inwards and create a
hydrophobic interior. Hydrophilic molecules will be repelled by the
hydrophobic tails of the phospholipid, they can pass the membrane only
through the pores formed by proteins that spans the membrane or by protein
carriers.
The phospholipids are fluid and move about rapidly by diffusion in their own
layers. Membranes also contain cholesterol which disturbs the close packing
of phospholipids and keeps them more fluid. This can be important for
organisms living at low temperatures when membranes can solidify.
Cholesterol also increase flexibility and stability of membranes. Without it
membranes break up.
- Function
1. separate contents of cells from their external environment;
2. controlling exchange of substances between two cells;

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3. form separate compartments inside cells in which specialised

metabolic pathways can take place, i.e. not interfering each
other;
4. as receptor sites for recognizing external stimuli;
5. the glycoproteins on the surface act as cell identity markers, i.e.
antigens;
6. site for reaction to take place, e.g. protein on the membrane of
chloroplast and mitochondria take part in the energy transfer
system.

Endoplasmic reticulum (ER)

- It is a complex network of double membranes extending throughout the
cytoplasm of all eukaryotic cells
- It is an extension of the outer nuclear membrane
- Types of ER :
i) Rough ER : the ER lined with ribosomes; it is used for transporting the
proteins synthesized from the ribosomes.
ii) Smooth ER : they are lacking ribosomes and concerned with the
synthesis and transport of lipids.
- Functions of ER
1. Biosynthesis : the sER may assemble fats, steroids and
carbohydrates and the rER produce proteins, especially enzymes
2. Transportation : it is a complex network of passageways that
extends throughout the cytoplasmic fluid, therefore, wastes and
nutrients are transported intracellularly

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3. Support : it forms a sort of cytoskeleton that to maintain the

shape of the cell

4. Increase surface area of the cell : network-like ER provides a lot

of surface area for the biochemical reactions to occur
5. Storage : in striated muscle cells, there are a highly specialized
sER called sarcoplasmic reticulum which stores calcium ions and
is involved in the muscle contraction
6. Detoxification : in the liver cells both rough and smooth ER are
involved in detoxification of various drugs

Golgi apparatus :
- It is a secretory organelle
- It has a similar structure to sER but is more compact
- It consists of flattened, membrane-bound sacs called cisternae, together
with a system of associated vesicles called Golgi vesicles.

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- In plant cells a number of separate stacks called dictyosomes are found

while in animal cells a single larger stack is thought to be more usual
- At one end of the stack new cisternae are constantly being formed by fusion
of vesicles which are probably derived from buds of smooth ER. This ‘outer’
or ‘forming’ face is convex, whilst the other end is the concave ‘inner’ or
‘maturing’ face where the cisternae break up into vesicles once more
(forming lysosome or secretory vesicle)

Ribosomes
Apart from the nucleoid, the principal internal structures of procaryotic cells
are the ribosomes. These are the sit of protein synthesis, and there may be
many thousands of these in an active cell, lending a speckled appearance to
the cytoplasm. Ribosomes are composed of complex of protein and RNA ,
and are the site of protein synthesis in the cell.
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Although they carry out a similar function , the ribosomes of procarytic cells
are smaller and lighter than their eukaryotic counterparts.
Ribosomes are mea – sured in Svedberg units ( s ) , a function of their size
and shape, and determind by their rate of sedimentation in a centrifuge;
prokaryotic ribosomes are 70 s, while those of eukaryotes are 80 s.

Genetic material
Although it occupies awell defined area within the cell, the genetic material
of prokaryotes is not present as a true nucleus, as it lacks a surrounding
nuclear membrane (eukaryotic nucleus). The nucleoid or bacterial
chromosome comprises aclosed circle of double stranded DNA, many times
the length of the cell and highly folded and compacted. Escherichia coli is
around 3-4 µm in length, but contains a DNA molecule some 1400 µm in
length!) The DNA may be associated with certain bacterial pro- teins but
these are not the same as histones found in eukaryotic chromosomes .
common laboratory

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Some bacteria contain additional DNA in the form of small, self replicating
extrachrosomal elements called plasmids. These do not carry any genes
essential for growth and reproduction, and thus the cell may survive without
them. They can be very important however, as they may include genes
encoding toxins or resistance to antibiotance, and can be passed from cell.

Plasmids
Plasmids are autonomous extrachromosomal replicons, which are
widespread amongst the bacteria and offer metabolic and physiological
flexibility of the organism’s response to environmental changes and stresses.
. Plasmids are present in host cells as either single copies or multiple copies
in excess of 40 per cell. Plasmids may encode one or more phenotypic
features, which may in turn be expressed by the host. These features include
antibiotic resistance toxin production, substrate degradation including
xenobiotics, adherence antigens such as encoded by the K88 plasmid of E.
coli, bacteriocin production), and sex pilus and conjugation.

A representation of R100 is included as a typical plasmid in Figure 4. In

addition to the genes encoding these features, plasmids contain the
necessary regulatory and replicative elements to ensure replication to correct
copy number and segregation into daughter cells.

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3
Microbial Nutrition and cultivation

Microbial Nutrition
• Nutrition is a process of acquiring chemical substances from the
environment
• The absorbed nutrients are used
– for energy yielding processes
– growth
• The chemical elements absolutely needed - essential nutrients
– Macronutrients: C, H, O…
– Micronutrients: Mn, Zn, Ni…

Sources of Essential Nutrients

Carbon
• Structural backbone of living matter
– 50% of microbial dry weight is C
– Autotrophs derive C from CO2
– Heterotrophs derive C from organic matter

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Nitrogen
• 14% of microbial dry weight is N
– required for protein, DNA, RNA, ATP synthesis
• Microorganisms derive N by:
– Breaking down proteins into amino acids (reuse of amino acids)
– NH4, – ammonium ions
– NO3 – nitrate
– N2 – nitrogen fixers
• Free-living
• Symbionts with plants

Other Elements
• Sulfur - synthesis of sulfur-containing amino acids
• Phosphorus - synthesis of DNA, RNA, ATP and phospholipids of cell
membrane
• Trace elements – minerals needed as enzyme cofactors
• Growth factors – organic chemicals that cannot be synthesized by certain
organisms (vitamins, certain amino acids…)

Heterotrophs
• Chemoheterotrophs
– Energy and Carbon source from organic molecules
• Saprobes derive nutrients from dead organic material
– Opportunistic pathogene – a saprobe infecting the
compromised host
• Parasites derive nutrients from living organisms
– Pathogenes – harm the host (Streptococcus)
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– Obligate intracellular parasites (Rickettsias,

Chlamydias, Viruses)

How microbes eat?

• Absorb nutrients that are dissolved

• The molecules need to be small
• The big molecules are degraded by extracellular enzymes
• Diffusion of water molecules through a selectively permeable membrane
• Water molecules will move from the side that has more water to the side with
less water Until equilibrium is reached

Endocytosis
• Engulfing particles and molecules from the outside with the cell
membrane

Pinocytosis
• Absorbing liquids (oils)

Phagocytosis
• White blood cells can ingest whole cells - bacteria

Nutritional Type Energy Source Carbon Source Examples

Photoautotrophs Light CO2 Cyanobacteria, some

Purple and Green

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Bacteria

Light Organic Some Purple and Green

Photoheterotrophs
compounds Bacteria

Chemoautotrophs or Inorganic CO2 A few Bacteria and many

Lithotrophs compounds, e.g. Archaea
(Lithoautotrophs) H2, NH3, NO2,
H2S

Chemoheterotrophs Organic Organic Most Bacteria, some

or Heterotrophs compounds compounds Archaea

Uptake of Nutrients by the Microorganisms

Uptake of Nutrients

In order to support its’ activities, a cell must bring in nutrients from the external
environment across the cell membrane. In bacteria and archaea, several different
transport mechanisms exist.

Passive Diffusion

Passive or simple diffusion allows for the passage across the cell membrane of
simple molecules and gases, such as CO2, O2, and H2O. In this case, a
concentration gradient must exist, where there is higher concentration of the
substance outside of the cell than there is inside the cell. As more of the substance is

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transported into the cell the concentration gradient decreases, slowing the rate of
diffusion.

Facilitated Diffusion

Facilitated diffusion also involves the use of a concentration gradient, where the
concentration of the substance is higher outside the cell, but differs with the use of
carrier proteins (sometimes called permeases). These proteins are embedded
within the cell membrane and provide a channel or pore across the membrane
barrier, allowing for the passage of larger molecules. If the concentration gradient
dissipates, the passage of molecules into the cell stops. Each carrier protein typically
exhibits specificity, only transporting in a particular type of molecule or closely
related molecules.

Active Transport

Many types of nutrient uptake require that a cell be able to transport substances
against a concentration gradient (i.e. with a higher concentration inside the cell than
outside). In order to do this, a cell must utilize metabolic energy for the transport of
the substance through carrier proteins embedded in the membrane. This is known as
active transport. All types of active transport utilize carrier proteins.

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Active Transport Versus Facilitated Diffusion

Primary active transport

Primary active transport involves the use of chemical energy, such as ATP, to
drive the transport. One example is the ABC system, which utilizes ATP-Binding
Cassette transporters. Each ABC transporter is composed of three different
components: 1) membrane-spanning proteins that form a pore across the cell
membrane (i.e. carrier protein), 2) an ATP binding region that hydrolyzes ATP,
providing the energy for the passage across the membrane, and 3) a substrate-
binding protein, a peripheral protein that binds to the appropriate substance to be
transporter and ferries it to the membrane-spanning proteins. In gram negative
bacteria the substrate-binding protein is located in the cell’s periplasm, while in
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gram positive bacteria the substrate-binding protein is attached to the outside of the
cell membrane.

ABC Transporter Structure.

Secondary active transport

Secondary active transport utilizes energy from a proton motive force (PMF). A
PMF is an ion gradient that develops when the cell transports electrons during
energy-conserving processes. Positively charged protons accumulate along the
outside of the negatively charged cell, creating a proton gradient between the outside
of the cell and the inside.

There are three different types of transport events for simple transport: uniport,
symport, and antiport and each mechanism utilizes a different protein porter.
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Uniporters transport a single substance across the membrane, either in or out.

Symporters transport two substances across the membrane at the same time,
typically a proton paired with another molecule. Antiporters transport two
substances across the membrane as well, but in opposite directions. As one
substance enters the cell, the other substance is transported out.

Uniport, Synport and Antiport.

Group Translocation

Group translocation is a distinct type of active transport, using energy from an

energy-rich organic compound that is not ATP. Group translocation also differs
from both simple transport and ABC transporters in that the substance being
transported is chemically modified in the process.

One of the best studied examples of group translocation is the

phosphoenolpyruvate: sugar phosphotransferase system (PTS), which uses
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energy from the high-energy molecule phosphoenolpyruvate (PEP) to transport

sugars into the cell. A phosphate is transferred from the PEP to the incoming sugar
during the process of transportation.

Group Translocation via PTS.

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Iron Uptake

Siderophores and Receptor Sites.

Iron is required by microbes for the function of their cytochromes and enzymes,
resulting in it being a growth-limiting micronutrient. However, little free iron is
available in environments, due to its insolubility. Many bacteria have evolved
siderophores, organic molecules that chelate or bind ferric iron with high affinity.
Siderophores are released by the organism to the surrounding environment, whereby
they bind any available ferric iron. The iron-siderophore complex is then bound by a
specific receptor on the outside of the cell, allowing the iron to be transported into
the cell.

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4
Microbial Growth
Provided with the right conditions (food, correct temperature, etc) microbes can
grow very quickly. Depending on the situation, this could be a good thing for
humans (yeast growing in wort to make beer) or a bad thing (bacteria growing in
your throat causing strep throat). It’s important to have knowledge of their growth,
so we can predict or control their growth under particular conditions.

While growth for muticelluar organisms is typically measured in terms of the

increase in size of a single organism, microbial growth is measured by the increase
in population, either by measuring the increase in cell number or the increase in
overall mass.

Bacterial Division
Bacteria and archaea reproduce asexually only, while eukartyotic microbes can
engage in either sexual or asexual reproduction. Bacteria and archaea most
commonly engage in a process known as binary fission, where a single cell splits
into two equally sized cells. Other, less common processes can include multiple
fission, budding, and the production of spores.

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The process begins with cell elongation, which requires careful enlargement of the
cell membrane and the cell wall, in addition to an increase in cell volume. The cell
starts to replicate its DNA, in preparation for having two copies of its chromosome,
one for each newly formed cell. The protein FtsZ is essential for the formation of a
septum, which initially manifests as a ring in the middle of the elongated cell. After
the nucleoids are segregated to each end of the elongated cell, septum formation is
completed, dividing the elongated cell into two equally sized daughter cells. The
entire process or cell cycle can take as little as 20 minutes for an active culture of E.
coli bacteria.

Growth Curve

Since bacteria are easy to grow in the lab, their growth has been studied extensively.
It has been determined that in a closed system or batch culture (no food added, no
wastes removed) bacteria will grow in a predictable pattern, resulting in a growth
curve composed of four distinct phases of growth: the lag phase, the exponential or
log phase, the stationary phase, and the death or decline phase. Additionally, this
growth curve can yield generation time for a particular organism – the amount of
time it takes for the population to double.

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The details associated with each growth curve (number of cells, length of each
phase, rapidness of growth or death, overall amount of time) will vary from
organism to organism or even with different conditions for the same organism. But
the pattern of four distinct phases of growth will typically remain.

Lag phase

The lag phase is an adaptation period, where the bacteria are adjusting to their new
conditions. The length of the lag phase can vary considerably, based on how
different the conditions are from the conditions that the bacteria came from, as well
as the condition of the bacterial cells themselves. Actively growing cells transferred
from one type of media into the same type of media, with the same environmental
conditions, will have the shortest lag period. Damaged cells will have a long lag
period, since they must repair themselves before they can engage in reproduction.

Typically cells in the lag period are synthesizing RNA, enzymes, and essential
metabolites that might be missing from their new environment (such as growth
factors or macromolecules), as well as adjusting to environmental changes such as
changes in temperature, pH, or oxygen availability. They can also be undertaking
any necessary repair of injured cells.

Exponential or Log phase

Once cells have accumulated all that they need for growth, they proceed into cell
division. The exponential or log phase of growth is marked by predictable
doublings of the population, where 1 cell become 2 cells, becomes 4, becomes 8 etc.
Conditions that are optimal for the cells will result in very rapid growth (and a
steeper slope on the growth curve), while less than ideal conditions will result in

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slower growth. Cells in the exponential phase of growth are the healthiest and most
uniform, which explains why most experiments utilize cells from this phase.

Bacterial Growth Rates.

Due to the predictability of growth in this phase, this phase can be used to
mathematically calculate the time it takes for the bacterial population to double in
number, known as the generation time (g). This information is used by
microbiologists in basic research, as well as in industry. In order to determine
generation time, the natural logarithm of cell number can be plotted against time
(where the units can vary, depending upon speed of growth for the particular
population), using a semilogarithmic graph to generate a line with a predictable
slope.

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The slope of the line is equal to 0.301/g. Alternatively one can rely on the fixed
relationship between the initial number of cells at the start of the exponential phase
and the number of cells after some period of time, which can be expressed by:

where N is the final cell concentration, N0 is the initial cell concentration, and n is
the number of generations that occurred between the specified period of time.
Generation time (g) can be represented by t/n, with t being the specified period of
time in minutes, hours, days, or months. Thus, if one knows the cell concentration at
the start of the exponential phase of growth and the cell concentration after some
period of time of exponential growth, the number of generations can be calculated.
Then, using the amount of time that growth was allowed to proceed (t), one can
calculate g.

Stationary Phase

All good things must come to an end (otherwise bacteria would equal the mass of
the Earth in 7 days!). At some point the bacterial population runs out of an essential
nutrient/chemical or its growth is inhibited by its own waste products (it is a closed
container, remember?) or lack of physical space, causing the cells to enter into the
stationary phase. At this point the number of new cells being produced is equal to
the number of cells dying off or growth has entirely ceased, resulting in a flattening
out of growth on the growth curve.

Physiologically the cells become quite different at this stage, as they try to adapt to
their new starvation conditions. The few new cells that are produced are smaller in
size, with bacilli becoming almost spherical in shape. Their plasma membrane

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becomes less fluid and permeable, with more hydrophobic molecules on the surface
that promote cell adhesion and aggregation. The nucleoid condenses and the DNA
becomes bound with DNA-binding proteins from starved cells (DPS), to protect
the DNA from damage. The changes are designed to allow the cell to survive for a
longer period of time in adverse conditions, while waiting for more optimal
conditions (such as an infusion of nutrients) to occur. These same strategies are used
by cells in oligotrophic or low-nutrient environments. It has been hypothesized that
cells in the natural world (i.e. outside of the laboratory) typically exist for long
periods of time in oligotrophic environments, with only sporadic infusions of
nutrients that return them to exponential growth for very brief periods of time.

During the stationary phase cells are also prone to producing secondary
metabolites, or metabolites produced after active growth, such as antibiotics. Cells
that are capable of making an endospore will activate the necessary genes during
this stage, in order to initiate the sporulation process.

Death or Decline phase

In the last phase of the growth curve, the death or decline phase, the number of
viable cells decreases in a predictable (or exponential) fashion. The steepness of the
slope corresponds to how fast cells are losing viability. It is thought that the culture
conditions have deteriorated to a point where the cells are irreparably harmed, since
cells collected from this phase fail to show growth when transferred to fresh
medium. It is important to note that if the turbidity of a culture is being measured as
a way to determine cell density, measurements might not decrease during this phase,
since cells could still be intact.

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It has been suggested that the cells thought to be dead might be revived under
specific conditions, a condition described as viable but nonculturable (VBNC).
This state might be of importance for pathogens, where they enter a state of very low
metabolism and lack of cellular division, only to resume growth at a later time, when
conditions improve.

It has also been shown that 100% cell death is unlikely, for any cell population, as
the cells mutate to adapt to their environmental conditions, however harsh. Often
there is a tailing effect observed, where a small population of the cells cannot be
killed off. In addition, these cells might benefit from their death of their fellow cells,
which provide nutrients to the environment as they lyse and release their cellular
contents.

The Mathematics of Growth

CALCULATION:

The generation time can be calculated from the growth curve(Fig 3).

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Calculation of generation time

The exactly doubled points from the absorbance readings were taken and, the points
were extrapolated to meet the respective time axis.

Generation Time = (Time in minutes to obtain the absorbance 0.4) – (Time in

minutes to obtain the absorbance 0.2)

= 90-60

= 30 minutes

Let No = the initial population number

Nt = population at time t

N = the number of generations in time t

Therefore,

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Therefore,

The growth rate can be expressed in terms of mean growth rate constant (k), the
number of generations per unit time.

Mean generation time or mean doubling time (g), is the time taken to double its size.

Therefore,

Substituting equation 4 in equation 3

(Since the population doubles t= g)

Therefore,

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Mean growth rate constant,

Mean generation time,

How we can use the growth curve parameters in biotechnological

application of microorganisms: (e.g The Continuous Culture of
Microorganisms)

Two major types of continuous culture systems commonly are

used:

(1) Chemostats
A chemostat is constructed so that sterile medium is fed into the
culture vessel at the same rate as the media containing microorganisms
is removed (figure 6.9). The culture medium for a chemostat possesses
an essential nutrient (e.g., an amino acid) in limiting quantities.
Because of the presence of a limiting nutrient, the growth rate is
determined by the rate at which new medium is fed into the growth
chamber, and the final cell density depends on the concentration of the
limiting nutrient. turbidostats.
(2) Turbidostat
The second type of continuous culture system, the turbidostat, has a
photocell that measures the absorbance or turbidity of the culture in
the growth vessel. The flow rate of media through the vessel is
automatically regulated to maintain a predetermined turbidity or cell
density. The turbidostat differs from the chemostat in several ways.
The dilution rate in a turbidostat varies rather than remaining
constant, and its culture medium lacks a limiting nutrient. The

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turbidostat operates best at high dilution rates; the chemostat is most

stable and effective at lower dilution rates.
Continuous culture systems are very useful because they provide a constant
supply of cells in exponential phase and growing at a known rate. They make
possible the study of microbial growth at very low nutrient levels,
concentrations close to those present in natural environments. These
systems are essential for research in many areas—for example, in studies on
interactions between microbial species under environmental conditions
resembling those in a freshwater lake or pond. Continuous systems also are
used in food and industrial microbiology.

Factors affecting microbial growth

Environmental Factors

What do real estate agents always say? Location, location, location! It’s all about
where you live, or at least adapting to where you live. At least it is for microbes.

Competition is fierce out in the microbial world (non-microbial world, too!) and
resources can be scarce. For those microbes that are willing and able to adapt to
what might be considered a harsh environment, it can certainly mean less
competition.

So what environmental conditions can affect microbial growth? Temperature,

oxygen, pH, water activity, pressure, radiation, lack of nutrients…these are the
primary ones. We will cover more about metabolism (i.e. what type of food can they

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eat?) later, so let us focus now on the physical characteristics of the environment and
the adaptations of microbes.

Osmolarity

Cells are subject to changes in osmotic pressure, due to the fact that the plasma
membrane is freely permeable to water (a process known as passive diffusion).
Water will generally move in the direction necessary to try and equilibrate the cell’s
solute concentration to the solute concentration of the surrounding environment. If
the solute concentration of the environment is lower than the solute concentration
found inside the cell, the environment is said to be hypotonic. In this situation water
will pass into the cell, causing the cell to swell and increasing internal pressure. If
the situation is not rectified then the cell will eventually burst from lysis of the
plasma membrane. Conversely, if the solute concentration of the environment is
higher than the solute concentration found inside the cell, the environment is said to
be hypertonic. In this situation water will leave the cell, causing the cell to
dehydrate. Extended periods of dehydration will cause permanent damage to the
plasma membrane.

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Hypertonic vs. Hypotonic Solutions.

Cells in a hypotonic solution need to reduce the osmotic concentration of their

cytoplasm. Sometimes cells can use inclusions to chemically change their solutes,
reducing molarity. In a real pinch they can utilize what are known as
mechanosensitive (MS) channels, located in their plasma membrane. MS channels
open as the plasma membrane stretches due to the increased pressure, allowing
solutes to leave the cell and thus lowering the osmotic pressure.

Cells in a hypertonic solution needing to increase the osmotic concentration of their

cytoplasm can take up additional solutes from the environment. However, cells have
to be careful about what solutes they take up, since some solutes can interfere with
cellular function and metabolism. Cells need to take up compatible solutes, such as
sugars or amino acids, which typically will not interfere with cellular processes.

There are some microbes that have evolved to extreme hypertonic environments,
specifically high salt concentrations, to the point where they now require the
presence of high levels of sodium chloride to grow. Halophiles, which require a
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 Microb. Physio. 2024-2025

NaCl concentration above 0.2 M, take in both potassium and chloride ions as a way
to offset the effects of the hypertonic environment that they live in. Their evolution
has been so complete that their cellular components (ribosomes, enzymes, transport
proteins, cell wall, plasma membrane) now require the presence of high
concentrations of both potassium and chloride to function.

pH

pH is defined as the negative logarithm of the hydrogen ion concentration of a

solution, expressed in molarity. The pH scale ranges from 0 to 14, with 0
representing an extremely acidic solution (1.0 M H+) and 14 representing an
extremely alkaline solution (1.0 x 10-14 M H+). Each pH units represents a tenfold
change in hydrogen ion concentration, meaning a solution with a pH of 3 is 10x
more acidic than a solution with a pH of 4.

Typically cells would prefer a pH that is similar to their internal environment, with
cytoplasm having a pH of 7.2. That means that most microbes are neutrophiles
(“neutral lovers”), preferring a pH in the range of 5.5 to 8.0. There are some
microbes, however, that have evolved to live in the extreme pH environments.

Acidophiles (“acid lovers”), preferring an environmental pH range of 0 to 5.5, must

use a variety of mechanisms to maintain their internal pH in an acceptable range and
preserve the stability of their plasma membrane. These organisms transport cations
(such as potassium ions) into the cell, thus decreasing H+ movement into the cell.
They also utilize proton pumps that actively pump H+ out.

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 Microb. Physio. 2024-2025

Alkaliphiles (“alkaline lovers”), preferring an environmental pH range of 8.0 to

11.5, must pump protons in, in order to maintain the pH of their cytoplasm. They
typically employ antiporters, which pump protons in and sodium ions out.

Temperature

Microbes have no way to regulate their internal temperature so they must evolve
adaptations for the environment they would like to live in. Changes in temperature
have the biggest effect on enzymes and their activity, with an optimal temperature
that leads to the fastest metabolism and resulting growth rate. Temperatures below
optimal will lead to a decrease in enzyme activity and slower metabolism, while
higher temperatures can actually denature proteins such as enzymes and carrier
proteins, leading to cell death. As a result, microbes have a growth curve in relation
to temperature with an optimal temperature at which growth rate peaks, as well as
minimum and maximum temperatures where growth continues but is not as
robust. For a bacterium the growth range is typically around 30 degrees.

The psychrophiles are the cold lovers, with an optimum of 15oC or lower and a
growth range of -20oC to 20oC. Most of these microbes are found in the oceans,
where the temperature is often 5oC or colder. They can also be found in the Arctic
and the Antarctic, living in ice wherever they can find pockets of liquid water.
Adaptation to the cold required evolution of specific proteins, particularly enzymes,
that can still function in low temperatures. In addition, it also required modification
to the plasma membrane to keep it semifluid. Psychrophiles have an increased
amount of unsaturated and shorter-chain fatty acids. Lastly, psychrophiles produce
cryoprotectants, special proteins or sugars that prevent the development of ice

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crystals that might damage the cell. Psychrotophs or cold tolerant microbes have a
range of 0-35oC, with an optimum of 16oC or higher.

Humans are best acquainted with the mesophiles, microbes with a growth optima of
37oC and a range of 20-45oC. Almost all of the human microflora fall into this
category, as well as almost all human pathogens. The mesophiles occupy the same
environments that humans do, in terms of foods that we eat, surfaces that we touch,
and water that we drink and swim in.

On the warmer end of the spectrum is where we find the thermophiles (“heat
lovers”), the microbes that like high temperatures. Thermophiles typically have a
range of 45-80oC, and a growth optimum of 60oC. There are also the
hyperthermophiles, those microbes that like things extra spicy. These microbes
have a growth optima of 88-106oC, a minimum of 65oC and a maximum of 120oC.
Both the thermophiles and the hyperthermophiles require specialized heat-stable
enzymes that are resistant to denaturation and unfolding, partly due to the presence
of protective proteins known as chaperone proteins. The plasma membrane of
these organisms contains more saturated fatty acids, with increased melting points.

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Oxygen Concentration

The oxygen requirement of an organism relates to the type of metabolism that it is

using. Energy generation is tied to the movement of electrons through the electron
transport chain (ETC), where the final electron acceptor can be oxygen or a non-
oxygen molecule.

Organisms that use oxygen as the final electron acceptor are engaging in aerobic
respiration for their metabolism. If they require the presence of atmospheric oxygen
(20%) for their metabolism then they are referred to as obligate aerobes.
Microaerophiles require oxygen, but at a lower level than normal atmospheric
levels – they only grow at levels of 2-10%.

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 Microb. Physio. 2024-2025

Organisms that can grow in the absence of oxygen are referred to as anaerobes,
with several different categories existing. The facultative anaerobes are the most
versatile, being able to grow in the presence or absence of oxygen by switching their
metabolism to match their environment. They would prefer to grow in the presence
of oxygen, however, since aerobic respiration generates the largest amount of energy
and allows for faster growth. Aerotolerant anaerobes can also grow in the presence
or absence of oxygen, exhibiting no preference. Obligate anaerobes can only grow
in the absence of oxygen and find an oxygenated environment to be toxic.

While the use of oxygen is dictated by the organism’s metabolism, the ability to live
in an oxygenated environment (regardless of whether it is used by the organism or
not) is dictated by the presence/absence of several enzymes. Oxygen by-products
(called reactive oxygen species or ROS) can be toxic to cells, even to the cells
using aerobic respiration. Enzymes that can offer some protection from ROS include
superoxide dismutase (SOD), which breaks down superoxide radicals, and
catalase, which breaks down hydrogen peroxide. Obligate anaerobes lack both
enzymes, leaving them little or no protection against ROS.

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Oxygen and Bacterial Growth.

Pressure

The vast majority of microbes, living on land or water surface, are exposed to a
pressure of approximately 1 atmosphere. But there are microbes that live on the
bottom of the ocean, where the hydrostatic pressure can reach 600-1,000 atm. These
microbes are the barophiles (“pressure lovers”), microbes that have adapted to
prefer and even require the high pressures. These microbes have increased
unsaturated fatty acids in their plasma membrane, as well as shortened fatty acid
tails.

Radiation

All cells are susceptible to the damage cause by radiation, which adversely affects
DNA with its short wavelength and high energy. Ionizing radiation, such as x-rays
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and gamma rays, causes mutations and destruction of the cell’s DNA. While
bacterial endospores are extremely resistant to the harmful effects of ionizing
radiation, vegetative cells were thought to be quite susceptible to its impact. That is,
until the discovery of Deinococcus radiodurans, a bacterium capable of completely
reassembling its DNA after exposure to massive doses of radiation.

Ultraviolet (UV) radiation also causes damage to DNA, by attaching thymine

bases that are next to one another on the DNA strand. These thymine dimers inhibit
DNA replication and transcription. Microbes typically have DNA repair
mechanisms that allow them to repair limited damage, such as the enzyme
photolyase that splits apart thymine dimers.

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5
Enzymes
Enzymes are biologic polymers that catalyze the chemical reactions

which make life as we know it possible. The presence and maintenance

of a complete and balanced set of enzymes is essential for the breakdown

of nutrients to supply energy and chemical building blocks; the

assembly of those building blocks into proteins, DNA, membranes, cells,

and tissues; and the harnessing of energy to power cell motility and

muscle contraction. With the exception of a few catalytic RNA

molecules, or ribozymes, the vast majority of enzymes are proteins.

Deficiencies in the quantity or catalytic activity of key enzymes can

result from genetic defects, nutritional deficits, or toxins.

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 Microb. Physio. 2024-2025

General concepts of enzymology

Substrate is the molecule that the enzyme acts upon to form

product.

E+S E-S E-P E+P

Apoenzyme The protein part of the enzyme without co-factors. It

may be active itself or inactive.

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Co-factors Small organic or inorganic (non-protein chemical)

molecules that is bound to a protein (enzyme) and activate it. It can

be considered as helper molecules.

a- Co-enzymes: loosely-bound cofactors (linked to enzyme by

noncovalent bonds).

b- Prosthetic group: tightly-bound cofactors (Bind to enzyme by a

covalent bond).

c- Activators: Metal ion cofactors, also called metalloenzymes (Ex:

K+, Fe++, Cu++, Mg++, Ca++).

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 Microb. Physio. 2024-2025

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 Microb. Physio. 2024-2025

It can be divided into 3 classes:

Holoenzyme The active enzyme (protein moiety _enzyme_+ co-factor).

Substra
te Binding site (active site) is a particular arrangement of chemical

groups on the enzyme surface that is specifically formulated to bind

a specific substrate (the place where the Substrate binds and at

which catalysis occurs).

Allosteric site is the site where small molecules – not substrate- bind

and induce changes in the active site. It may make enzyme more

active by making binding site has a greater affinity to the substrate,

or inactivation of the enzyme by making the active site has less

affinity to the substrate.

Enzyme classification

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 Microb. Physio. 2024-2025

According to IUBMB (International Union of Biochemistry and

Molecular Biology), there are six classes of enzymes can be

expressed by E.C number (Enzyme Commission number) which can be

defined as a numerical classification scheme for enzymes, based on

the chemical reactions they catalyze. As a system of enzyme

nomenclature, every EC number is associated with a recommended

name for the respective enzyme.

Every enzyme code consists of the letters "EC" followed by four

numbers separated by periods. Those numbers represent a

progressively finer classification of the enzyme.

Each of the six classes has more specific subclasses as well. The key

to using this classification scheme is to look at the reaction the

enzyme catalyzes, decide which type of reaction it is, and apply the

appropriate name.

Specific enzyme names are systematically derived by specifiying the

substrate (the molecule being acted upon -- the reactant), the type

of reaction, and appending the suffix ase. Alcohol dehydrogenase

thus is an enzyme which acts on an alcohol and takes hydrogen

(oxidizes) from it: it is therefore classified as an oxidoreductase. We

can tell a lot about what an enzyme does from its name.

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 Microb. Physio. 2024-2025

No. Class Biochemical Properties Reaction& Example

E.C.1 Oxido- These are enzymes which AH + B → A + BH

reductases catalyze the reduction or (reduced)
oxidation of a molecule. A + O → AO
Remember that oxidation is (oxidized)
the reverse of reduction and
that an enzyme has to
catalyze the forward and Example:

reverse reactions to the same Dehydrogenase,

degree. Any enzyme which oxidase
catalyzes a reduction has to
also catalyze the reverse
(oxidation) reaction, thus the
double-barreled name
"oxidoreductase."

2 Transferases These enzymes catalyze the AB + C → A + BC

transfer of a group of atoms
Transaminase, kinase
from one molecule to
another. A common example
involves transfer of a
phosphate between ATP and
a sugar molecule.

3 Hydrolases As the name suggests, these AB + H2O → AOH +

enzymes catalyze hydrolysis BH

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 Microb. Physio. 2024-2025

reactions (and their reverse

reactions). The hydrolysis of
Lipase, amylase,
an ester would be an
peptidase
example of such a reaction.

4 Isomerases These enzymes catalyze the AB → BA

conversion of a molecule
Ex: Isomerase,
into an isomer. The cis-trans
mutase, cis-trans,
interconversion of maleate
Keto-enol conversion
and fumarate is an example.
enzymes.

5 Lyases cleave various bonds by RCOCOOH →

means other than hydrolysis RCOH + CO2 or [x-
& Oxidation as adding small A-B-Y] → [A=B + X-
molecules as water or Y]
ammonia.

Decarboxylase

6 Ligases These enzymes catalyze X + Y+ ATP → XY +

reactions which make bonds ADP + Pi
to join together (ligate)
smaller molecules to make
larger ones. Synthetase

Enzyme specificity

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 Microb. Physio. 2024-2025

In general, there are four distinct types of specificity:

1- Absolute specificity - the enzyme will catalyze only one reaction

(Each Enzyme is usually very specific as to which reaction it catalyze

and the substrate that is involved in the reactions. For example:

glucose oxidase oxidize only glucose but not galactose).

2- Group specificity - the enzyme will act only on molecules that

have specific functional groups, such as amino, phosphate and methyl

groups such as hexokinase which can catalyzes the phosphorylation of

glucose, mannose, glucosamine, 2-deoxyglucose and fructose.

3- Linkage specificity - the enzyme will act on a particular type of

chemical bond regardless of the rest of the molecular structure such

as endopeptidase which can split certain types of peptide linkage in

the peptide chain.

4- Stereochemical specificity - the enzyme will act on a particular

steric or optical isomer (D-amino acid oxidase bind only D-amino acids

and not L-amino acids).

Though enzymes exhibit great degrees of specificity, cofactors may

serve many apoenzymes. For example, nicotinamide adenine

dinucleotide (NAD) is a coenzyme for a great number of

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 Microb. Physio. 2024-2025

dehydrogenase reactions in which it acts as a hydrogen acceptor.

Among them are the alcohol dehydrogenase, malate dehydrogenase

and lactate dehydrogenase reactions.

Factors affect enzyme activity

The rate at which an enzyme converts its substrate into product is

called its velocity (v), and is affected by a variety of factors.

Temperature

The rate of any chemical reaction increases with an increase in

temperature due to the more rapid movement of molecules, and so it

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 Microb. Physio. 2024-2025

is with enzyme-catalysed reactions, until a peak is reached (the

optimum temperature) after which the rate rapidly falls away. causes

this drop in the velocity? the very ordered secondary and tertiary

structure of a protein molecule is due to the existence of numerous

weak molecular bonds, such as hydrogen bonds. Disruption of these by

excessive heat results in denaturation that is, an unfolding of the

three-dimensional structure. In the case of an enzyme, this leads to

changes in the configuration of the active site, and a loss of catalytic

properties. The effect of temperature on enzyme activity is shown in

the Figure. The graph can be thought of as a composite of two lines,

one increasing with temperature due to the rise in thermal energy of

the substrate molecules, and one falling due to denaturation of the

protein structure. Before the optimum temperature it is the former

which dominates, then the effect of the latter becomes more

pronounced, and takes over completely.

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 Microb. Physio. 2024-2025

pH

Enzyme velocity is similarly affected by the prevailing pH. Once

again, this is due to alterations in three-dimensional protein

structure. Changes in the pH affect the ionization of charged „R‟-

groups on amino acids at the active site and elsewhere, causing

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 Microb. Physio. 2024-2025

changes in the enzyme‟s precise shape, and a reduction in catalytic

properties. As with temperature, enzymes have an optimum value at

which they operate most effectively; when the pH deviates

appreciably from this in either direction, denaturation occurs, leading

to a reduction of enzyme activity Microorganisms are able to operate

in a variety of physicochemical environments, a fact reflected in the

diversity of optimum values of temperature and pH encountered in

their enzymes.

Substrate concentration

Under conditions where the active sites of an enzyme population are

not saturated, an increase in substrate concentration will be

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 Microb. Physio. 2024-2025

reflected in a proportional rise in the rate of reaction. A point is

reached, however, when the addition of further substrate has no

effect on the rate. This is because all the active sites have been

occupied and the enzymes are working flat out; this is called the

maximum velocity (Vmax). A measure of the affinity an enzyme has

for its substrate (i.e. how tightly it binds to it) is given by its

Michaelis constant (Km). This is the substrate concentration at which

the rate of reaction is half of the Vmax value. Values of Vmax and Km

are more easily determined experimentally by plotting the reciprocals

of [S] and V to obtain a straight line.

Enzyme inhibitors:

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 Microb. Physio. 2024-2025

Many substances are able to interfere with an enzyme‟s ability to

catalyse a reaction. Enzyme inhibition forms the basis of several

methods of microbial control, so a consideration of the main types of

inhibitor is appropriate here. Perhaps the easiest form of enzyme

inhibition to understand is competitive inhibition. Here, the inhibitory

substance competes with the normal substrate for access to the

enzyme‟s active site; if the active site is occupied by a molecule of

inhibitor, it can‟t bind a molecule of substrate, thus the reaction will

proceed less quickly.

and inhibitor. If the inhibitor is only present at a low concentration,

its effect will be minimal, since the number of enzyme–inhibitor

interactions will be greatly outweighed by reactions with the „proper‟

substrate. The Vmax value for the enzyme is not reduced,

but it is only reached more gradually. The apparent affinity of the

enzyme for its substrate is decreased, reflected by an increase in

the Km (Figure 6.11).

Not all inhibitors act by competing for the active site, however. Non-

competitive inhibitors act by binding to a different part of the

enzyme and in so doing alter its three-dimensional configuration

(Figure 6.10b). Although they do not affect substrate binding, they

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 Microb. Physio. 2024-2025

do reduce the rate at which product is formed. Vmax cannot be

reached; however, the value of Km, is unchanged (Figure 6.12). Such

inhibitors may bind to either the enzyme–substrate complex or to

free enzyme. Both competitive and non-competitive forms of

inhibition are reversible, since the inhibitor molecule is relatively

weakly bound and can be displaced.

Irreversible inhibition, on the other hand, is due to the formation of

a strong covalent linkage between the inhibitor and an amino acid

residue on the enzyme. As a result of its binding, the inhibitor

effectively makes a certain percentage of the enzyme population

permanently unavailable to catalyse substrate conversion.

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 Microb. Physio. 2024-2025

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 Microb. Physio. 2024-2025

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 Microb. Physio. 2024-2025

Feedback Inhibition
The end-products of metabolic
pathways are important reversible enzyme inhibitors • inhibit 1st

enzyme in pathway, turning the pathway “off” • provide an important way

of regulating end-product levels • can be competitive or allosteric

inhibition

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Factors affect enzyme activity

The rate at which an enzyme converts its substrate into product is

called its velocity (v), and is affected by a variety of factors.

Temperature

The rate of any chemical reaction increases with an increase in

temperature due to the more rapid movement of molecules, and so it

is with enzyme-catalysed reactions, until a peak is reached (the

optimum temperature) after which the rate rapidly falls away. causes

this drop in the velocity? the very ordered secondary and tertiary

structure of a protein molecule is due to the existence of numerous

weak molecular bonds, such as hydrogen bonds. Disruption of these by

excessive heat results in denaturation that is, an unfolding of the

three-dimensional structure. In the case of an enzyme, this leads to

changes in the configuration of the active site, and a loss of catalytic

properties. The effect of temperature on enzyme activity is shown in

the Figure. The graph can be thought of as a composite of two lines,

one increasing with temperature due to the rise in thermal energy of

the substrate molecules, and one falling due to denaturation of the

protein structure. Before the optimum temperature it is the former

which dominates, then the effect of the latter becomes more

pronounced, and takes over completely.

 Microb. Physio. 2024-2025

pH

Enzyme velocity is similarly affected by the prevailing pH. Once

again, this is due to alterations in three-dimensional protein

structure. Changes in the pH affect the ionization of charged „R‟-

groups on amino acids at the active site and elsewhere, causing

-1-
 Microb. Physio. 2024-2025

changes in the enzyme‟s precise shape, and a reduction in catalytic

properties. As with temperature, enzymes have an optimum value at

which they operate most effectively; when the pH deviates

appreciably from this in either direction, denaturation occurs, leading

to a reduction of enzyme activity Microorganisms are able to operate

in a variety of physicochemical environments, a fact reflected in the

diversity of optimum values of temperature and pH encountered in

their enzymes.

Substrate concentration

Under conditions where the active sites of an enzyme population are

not saturated, an increase in substrate concentration will be

-2-
 Microb. Physio. 2024-2025

reflected in a proportional rise in the rate of reaction. A point is

reached, however, when the addition of further substrate has no

effect on the rate. This is because all the active sites have been

occupied and the enzymes are working flat out; this is called the

maximum velocity (Vmax). A measure of the affinity an enzyme has

for its substrate (i.e. how tightly it binds to it) is given by its

Michaelis constant (Km). This is the substrate concentration at which

the rate of reaction is half of the Vmax value. Values of Vmax and Km

are more easily determined experimentally by plotting the reciprocals

of [S] and V to obtain a straight line.

Enzyme inhibitors:

-3-
 Microb. Physio. 2024-2025

Many substances are able to interfere with an enzyme‟s ability to

catalyse a reaction. Enzyme inhibition forms the basis of several

methods of microbial control, so a consideration of the main types of

inhibitor is appropriate here. Perhaps the easiest form of enzyme

inhibition to understand is competitive inhibition. Here, the inhibitory

substance competes with the normal substrate for access to the

enzyme‟s active site; if the active site is occupied by a molecule of

inhibitor, it can‟t bind a molecule of substrate, thus the reaction will

proceed less quickly.

and inhibitor. If the inhibitor is only present at a low concentration,

its effect will be minimal, since the number of enzyme–inhibitor

interactions will be greatly outweighed by reactions with the „proper‟

substrate. The Vmax value for the enzyme is not reduced,

but it is only reached more gradually. The apparent affinity of the

enzyme for its substrate is decreased, reflected by an increase in

the Km (Figure 6.11).

Not all inhibitors act by competing for the active site, however. Non-

competitive inhibitors act by binding to a different part of the

enzyme and in so doing alter its three-dimensional configuration

(Figure 6.10b). Although they do not affect substrate binding, they

-4-
 Microb. Physio. 2024-2025

do reduce the rate at which product is formed. Vmax cannot be

reached; however, the value of Km, is unchanged (Figure 6.12). Such

inhibitors may bind to either the enzyme–substrate complex or to

free enzyme. Both competitive and non-competitive forms of

inhibition are reversible, since the inhibitor molecule is relatively

weakly bound and can be displaced.

Irreversible inhibition, on the other hand, is due to the formation of

a strong covalent linkage between the inhibitor and an amino acid

residue on the enzyme. As a result of its binding, the inhibitor

effectively makes a certain percentage of the enzyme population

permanently unavailable to catalyse substrate conversion.

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 Microb. Physio. 2024-2025

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 Microb. Physio. 2024-2025

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 Microb. Physio. 2024-2025

Feedback Inhibition
The end-products of metabolic
pathways are important reversible enzyme inhibitors • inhibit 1st

enzyme in pathway, turning the pathway “off” • provide an important way

of regulating end-product levels • can be competitive or allosteric

inhibition

NON-CANONICAL ENZYMES
Although, the term non-canonical enzyme has not been used in

enzymology, yet we used here to represent the enzyme like molecules,

which have been associated with catalytic reactions in biological systems

in some non-conventional manner. Until now we learnt that, enzymes are

usually proteins, but new concepts emerged several years after the

establishment of enzymology particularly with a landmark discovery of

ribozymes by Chech and Altman. Beyond ribozymes, DNazymes have

also been reported. Interestingly, some antibodies, specific to the

transition state complex are also able to catalyse the reaction, in a much

specific manner compared to enzymes and they are named abzymes.

More recently, it has been possible to create chemical entities that

catalyses reactions like enzymes, and they are named chemzymes or

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 Microb. Physio. 2024-2025

synzymes, which lead to the convergence of chemical catalysts and

biological catalysts. Also, a sub-set of synzymes, particularly size

restricted molecules, which are reduced to nanoscale and show catalysis

similar to biological reactions are known as nanozymes. Most of the

areas of research in this domain are still very active and new additions

are continuously being made. In this chapter we will discuss, about all

these non-canonical enzymes in detail.

RIBOZYME
Ribozymes are RNA molecules that are capable of catalyzing specific

biochemical reactions, similar to the action of protein enzymes. Thomas

R. Cech and Sidney Altman shared the Nobel Prize in chemistry for

their discovery of catalytic properties of RNA in the year 1989. The

term ribozyme, however, was first introduced by Kelly Kruger et al. in

1982. Cech discovered that introns in a ribosomal RNA gene in

Tetrahymena thermophile (a ciliated protozoan and a model organism)

are catalytic in splicing events and Sidney Altman, found that tRNA

molecules are processed in the cell via an enzyme called RNase-P, which

is responsible for conversion of a precursor tRNA into the active tRNA.

Some common examples of ribozymes include the hammerhead ribozyme,

VS ribozyme, leadzyme and the hairpin ribozyme. However, a

classification scheme of ribozymes has also be been proposed on the

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 Microb. Physio. 2024-2025

basis of their secondary structure and functions. Table 8.1 summarizes

common of its classes

is generally agreed that the percentage of cerium atoms in the reduced

state increases with decreasing particle size. It is observed that an

increase in Ce3+ concentration from 17 to 44% as particle size

decreased from 30 to 3 nm. With this reason scaling down the cerium

oxide to nanosize proved beneficial in biological models. Another striking

feature of the cerium oxide at nanoscale is that its lattice expands as

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 Microb. Physio. 2024-2025

the particles become smaller leading to decrease in oxygen release and

re-absorption, therefore containing a larger fraction of reduced ceria

atoms.

Well before the biological activity of cerium oxide was known, it begun

to be used as an abrasive catalyst in chemical mechanical planarization/

polishing, as a catalyst in fuel cell power generation and catalytic

converters, and in fuel borne additives. Studies demonstrating

successful therapeutic or prophylactic effects of nanoceria have

exponentially grown over the past decade. Aforesaid, switching between

Ce3+ and Ce4+oxidation states have been identified as enzymes has

been improved significantly. Infact, artificial enzymes have also been

synthesized successfully and represent the frontiers of enzyme

technology.

In the following section we will discuss the various processes of enzyme

technology and some commercial applications.

EXTRACTION AND PURIFICATION OF ENZYMES

Commercial production of enzymes is much different from the lab-scale

synthesis, due to the fact that industrial production should be cost

effective and handling of larger volumes and processes needs

automation. The initial steps of the enzyme purification begin

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 Microb. Physio. 2024-2025

laboratories with the establishment of enzyme producing strain of a

microbe. In earlier days extraction of organs/ tissues and their

homogenization was done, but now most of the enzyme can be genetically

engineered and may be produced in various host systems. After the

establishment of culture and characterization of stain, the scaling up of

the process is done that requires a set up called bioreactor. The science

of producing and designing biological products at commercial levels is

known as bioprocess technology or bioprocess engineering, which

requires use of principles of physics, mathematics, electronics and

computer sciences besides biology of microbes.

In most of the commercial processes a crude extract is first obtained

by either breaking the cells (if the enzyme is intracellular) or collecting

the culture media (if the enzyme is extracellular).

The process of further processing and purification is also referred to as

downstream processing in bioprocess technology.

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 Microb. Physio. 2024-2025

ribozymes.

Methods of Enzyme Purification

After the cells have been disrupted or culture media has been collected

(in case of secreted enzyme) the lysate may contain a number of

components other than the desired enzymes. The process of enzyme

extraction from this mixture and final purification is a tedious job. The

lysate that is obtained from the source is known as crude extract. The

volume is significantly reduced during the process due to the loss of

impurities in subsequent steps with a concurrent increase in the specific

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 Microb. Physio. 2024-2025

activity of enzyme (described later in this chapter). There are several

methods of

enzyme purification from the crude extract.

purification methods is also very critical. If one begins with affinity

chromatography in crude extract the columns may be clogged and lead

to poor yield. In general, the sequence of events of purification is as

follows:

 Mincing or chopping of tissue or homogenization of cells

 Lysis of cells using lysis buffer

 Precipitation of proteins using ammonium precipitate (an

ultracentrifugation separation can also be performed).

 Ion exchange chromatography (cation exchange or anion exchange

or both types may be used sequentially)

 Size exclusion chromatography (size based separation)

 Affinity purification (separation on preparative polyacrylamide gel

can also performed)

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5
Energetics & Redox Reactions

Introduction to Metabolism

Metabolism refers to the sum of chemical reactions that occur within a cell.
Catabolism is the breakdown of organic and inorganic molecules, used to release
energy and derive molecules that could be used for other reactions. Anabolism is
the synthesis of more complex molecules from simpler organic and inorganic
molecules, which requires energy.

Energetics

While some energy is lost as heat in chemical reactions, the measurement of interest
for cells is the amount of free energy (G), or the energy available to do work. Cells
perform three different types of work: chemical work (such as anabolism),
transport work (such as nutrient uptake), and mechanical work (such as the
rotation of a flagellum).

The change in free energy is typically denoted as ΔG°’, which indicates the change
in free energy under standard conditions of pH 7, 25oC, 1 atmosphere pressure (also
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known as the standard free energy change). A reaction that generates a positive
ΔG°’ indicates that the reaction requires energy and is endergonic in nature. A
reaction that generates a negative ΔG°’ indicates that the reaction releases energy
and is exergonic in nature. Reactions that are exergonic release energy that can be
conserved by the cell to do work.

Adenosine triphosphate (ATP)

Adenosine triphosphate or ATP is a high-energy molecule used by all cells for

energy currency, partly because it readily donates a phosphoryl group to other
molecules. An exergonic reaction will release energy, driving the synthesis of ATP
from the addition of a phosphate molecule (orthophosphate or Pi) to adenosine
diphosphate or ADP. An exergonic reaction, which requires energy, will couple
with the hydrolysis of ATP to ADP + Pi, using the released energy to drive the
reaction.

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Redox Reactions

Cells conserve energy in the form of ATP by coupling its synthesis to the release of
energy via oxidation-reduction (redox) reactions, where electrons are passed from
an electron donor to an electron acceptor. The oxidation of a molecule refers to
the loss of its electrons, while the reduction of a molecule refers to its gain of
electrons. Organic chemists often refer to the process by the mnemonic OIL RIG:
Oxidation Is Loss, Reduction Is Gain. A molecule being oxidized is acting as an
electron donor, while the molecule being reduced is acting as an electron acceptor.
Since electrons represent energy, a substance with many electrons to donate can be
thought of as energy-rich.
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Conjugate Redox Pair.

Electrons do not exist freely in solution, they must be coupled with atoms or
molecules. Every redox reaction consists of two half reaction, where one substance
donates electrons and thus becomes an oxidized product while another substance
accepts the electrons and thus becomes a reduced product. Conjugate redox pair
refers to the acceptor and donor of a half reaction.

A substance can be either an electron donor or an electron acceptor, dependent upon

the other substances in the reaction. A redox couple represents both forms of a
substance in a half reaction, with the oxidized form (the electron acceptor) always
placed on the left and the reduced form (the electron donor) on the left. An example
would be ½ O2/H2O, where H2O could serve as an electron donor and O2 could
serve as an electron acceptor. Each half reaction is given a standard reduction
potential (E’0) in volts or millivolts, which is a measurement of the tendency of the
donor in the reaction to give up electrons. A substance with greater tendency to
donate electrons in the reduced form has a more negative E’0, while a substance

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with a weak tendency to donate electrons in the reduced form has a less negative or
even positive E’0. A substance with a negative E’0 makes a very good electron
donor, in the reduced form.

Redox Tower

The information regarding standard reduction potentials for various redox couples is
displayed in the form of a redox tower, which lists the couples in a vertical form
based on their E’0. Redox couples with the most negative E’0 on listed at the top
while those with the most positive E’0 are listed on the bottom. The reduced
substance with the greatest tendency to donate electrons would be found at the top of
the tower on the right, while the oxidized substance with the greatest tendency to
accept electrons would be found at the bottom of the tower on the left. Redox
couples in the middle can serve as either electron donors or acceptors, depending
upon what substance they partner with for a reaction. The difference between
reduction potentials of a donor and an acceptor (ΔE’0) is measured as acceptor E’0
minus donor E’0. The larger the value for ΔE’0, the more potential energy for a cell.
Larger values are derived when there is the biggest distance between the donor and
the acceptor (or a bigger fall down the tower).

While ΔE’0 is proportional to ΔG°’, the number of electrons that a substance has to
donate is important too. The actual formula is:

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where n is the number of electrons being transferred and F is the Faraday constant
(23,062 cal/mole-volt, 96, 480 J/mole-volt).

Electron Carriers

The transference of electrons from donor to acceptor does not occur directly, since
chemically dissimilar electron donors and acceptors might never interact with one
another. Instead, many cellular intermediates participate in the process, with the
possibility for energy capture occurring along the way. These intermediates are
called electron carriers and they go back and forth between a reduced form (when
they are carrying an electron) and an oxidized form (after they have passed the
electron on), without being consumed in the reaction themselves.

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In order for the reaction to be energetically favorable for the cell, the carriers must
be arranged in order of their standard reduction potential (i.e. going down the redox
tower), with an electron being passed from a carrier with the most negative E’0 to a
carrier with a less negative E’0. It is important to note that some carriers accept both
electrons and protons, while other carriers accept electrons only. This fact will
become of crucial importance later, in the discussion of how energy is generated.

While there are many different electron carriers, some unique to specific organisms
or groups of organisms, let us cover some of the more common ones:

Nicotinamide adenine dinucleotide (NAD+/NADH) – a co-enzyme that carriers

both electrons (e-) and protons (H+), two of each. A closely related molecule is
nicotinamide adenine dinucleotide phosphate (NADP+/ NADPH), which accepts
2 electrons and 1 proton.

Flavin adenine dinucleotide (FAD/FADH) and flavin mononucleotide

(FMN/FMNH) – carry 2 electrons and 2 protons each. Proteins with these
molecules are called flavoproteins.

Coenzyme Q (CoQ)/ubiquinone – carries 2 electrons and 2 protons.

Cytochromes – use iron atoms as part of a heme group to carry 1 electron at a time.

Iron-sulfur (Fe-S) proteins, such as ferredoxin – use iron atoms not part of heme
group to carry 1 electron at a time.

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Electron Transport Chain

The process starts with an initial electron donor, a substance from outside of the cell,
and ends with a final electron acceptor, another substance from outside of the cell. In
the middle the electrons are passed from carrier to carrier, as the electrons work their
way down the electron tower. In order to make the process more efficient, most of
the electron carriers are embedded within a membrane of the cell, in the order that
they are arranged on a redox tower. These electron transport chains are found
within the cell membrane of bacteria and archaea, and within the mitochondrial
membrane of eukaryotes.

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Chemoorganotrophy

Chemoorganotrophy is a term used to denote the oxidation of organic chemicals to

yield energy. In other words, an organic chemical serves as the initial electron
donor. The process can be performed in the presence or absence of oxygen,
depending upon what is available to a cell and whether or not they have the enzymes
to deal with toxic oxygen by-products.

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Aerobic Respiration

To start, let us focus on the catabolism of organic compounds when it occurs in the
presence of oxygen. In other words, oxygen is being used as the final electron
acceptor. When the process utilizes glycolysis and the tricarboxylic acid (TCA)
cycle to completely oxidize an organic compound down to CO 2, it is known as
aerobic respiration. This generates the most ATP for a cell, given the large amount
of distance between the initial electron donor (glucose) and the final electron
acceptor (oxygen), as well as the large number of electrons that glucose has to
donate.

Organic Energy Sources

In chemoorganotrophy, energy is derived from the oxidation of an organic

compound. There are many different organic compounds available to a cell, such as
proteins, polysaccharides, and lipids. But cellular pathways are arranged in such a
way to increase metabolic efficiency. Thus, the cell funnels reactions into a few
common pathways. By convention, glucose is used as the starting molecule to
describe each process.
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Glycolysis

Glycolysis is a nearly universal pathway for the catabolism of glucose to pyruvate.

The pathway is divided into two parts: part I, which focuses on modifications to the
6-carbon sugar glucose, and part II, where the 6-carbon compound is split into two
3-carbon molecules, yielding a bifurcated pathway. Part I actually requires energy in
the form of 2 molecules of ATP, in order to phosphorylate or activate the sugar. Part
II is the energy conserving phase of the reaction, where 4 molecules of ATP are
generated by substrate-level phosphorylation, where a high-energy molecule
directly transfers a Pi to ADP.

The net yield of energy from glycolysis is 2 molecules of ATP for every molecule of
glucose. In addition, 2 molecules of the carrier NAD+ are reduced, forming NADH.
In aerobic respiration, these electrons will ultimately be transferred by NADH to an
electron transport chain, allowing the cell to capture more energy. Lastly, 2
molecules of the 3-carbon compound pyruvate are produced, which can be further
oxidized to capture more energy for the cell.

The tricarboxylic acid (TCA) cycle picks up at the end of glycolysis, in order to
fully oxidize each molecule of pyruvate down to 3 molecules of CO 2, as occurs in
aerobic respiration. It begins with a type of connecting reaction before the molecules
can enter the cycle proper. The connecting reaction reduces 1 molecule of NAD+ to
NADH for every molecule of pyruvate, in the process of making citrate.

The citrate enters the actual cycle part of the process, undergoing a series of
oxidations that yield many different products, many of them important precursor
metabolites for other pathways. As electrons are released, carriers are reduced,

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yielding 3 molecules of NADH and 1 molecule of FADH2 for every molecule of

pyruvate. In addition, 1 molecule of GTP (which can be thought of as an ATP-
equivalent molecule) is generated by substrate-level phosphorylation

Tricarboxylic acid (TCA) cycle

Taking into account that there were two molecules of pyruvate generated from
glycolysis, the net yield of the TCA cycle and its connecting reaction are: 2
molecules of GTP, 8 molecules of NADH, and 2 molecules of FADH2. But where
does the ATP come from? So far we only have the net yield of 2 molecules from
glycolysis and the 2 molecules of ATP-equivalents (i.e. GTP) from the TCA cycle.
This is where the electron transport chain comes into play.

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Oxidative Phosphorylation

The synthesis of ATP from electron transport generated from oxidizing a chemical
energy source is known oxidative phosphorylation. We have already established
that electrons get passed from carrier to carrier, in order of their standard reduction
potential. We have also established that some carriers accept electrons and protons,
while others accept electrons only. What happens to the unaccepted protons? And
how does this generate ATP for the cell? Welcome to the wonderful world of the
proton motive force (PMF) and ATP synthase!

ATP Generation.

Anaerobic Chemoorganotrophy

Certainly oxygen is a wonderful final electron acceptor, particularly when paired

with glucose as an initial electron donor. It is part of the lowest redox couple on an
electron tower, with an extremely positive standard electron potential. But what does
a microbe do, if oxygen is not available or it lacks the protections necessary from
toxic oxygen by-products? Let us focus on the generation of energy in the absence

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of oxygen, using a different electron acceptor, when an organic chemical is still

being used as the initial electron donor. Examples of anaerobic chemoorganotrophy
include anaerobic respiration and fermentation.

Anaerobic Respiration

Anaerobic respiration starts with glycolysis as well and the pyruvate can be
shunted off to the TCA cycle, just like in aerobic respiration. In fact, oxidative
phosphorylation is used to generate most of the ATP, which means the use of an
ETC and ATP synthase. The key difference is that the final electron acceptor will
not be oxygen.

There are a variety of possible final electron acceptors that can be used in anaerobic
respiration, allowing microbes to live in a wide variety of locations. The best
electron acceptor will be the one that is lowest down on the electron tower, in an
oxidized form (i.e. on the left-hand side of the redox couple). Some common
electron acceptors include nitrate (NO3-), ferric iron (Fe3+), sulfate (SO42-),
carbonate (CO32-) or even certain organic compounds, like fumarate.

How much ATP is generated by anaerobic respiration? That will depend upon the
final electron acceptor being used. It will not be as much as is generated during
aerobic respiration, since we know that oxygen in the best possible electron
acceptor. Selection of an electron acceptor other than oxygen pushes an organism up
the electron tower, shortening the distance between the electron donor and the
acceptor, reducing the amount of ATP produced.

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Fermentation

No matter what they might teach you in a biochemical class, fermentation and
anaerobic respiration are not the same thing, at least not to a microbiologist.

Fermentation is catabolism of glucose in the absence of oxygen as well and it does

have some similarities to anaerobic respiration. Most obviously, it does not use
oxygen as the final electron acceptor. It actually uses pyruvate, an organic
compound. Fermentation starts with glycolysis, a process which we have already
covered, that also starts off both aerobic respiration and anaerobic respiration. What
does it yield? Two net molecules of ATP by substrate-level phosphorylation and 2
molecules of NADH. Organisms doing either aerobic or anaerobic respiration would
then utilize oxidative phosphorylation in order to increase their ATP yield.
Fermenters, however, lack an ETC or repress synthesis of their ETC when oxygen is
not available, so they do not use the TCA cycle at all.

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Without the use of an ETC (or a PMF or ATP synthase), no further ATP is
generated beyond what was synthesized during glycolysis. But organisms using
fermentation cannot just stop with glycolysis, since eventually all their molecules of
NAD+ would become reduced. In order to re-oxidize this electron carrier they use
pyruvate as a final electron acceptor, yielding a variety of fermentation products
such as ethanol, CO2, and various acids.

Fermentation products, although considered waste products for the cell, are vitally
important for humans. We rely on the process of fermentation to produce a variety
of fermented foods (beer, wine, bread, cheese, tofu), in addition to using the
products for a variety of industrial processes.

Chemolithotrophy

Chemolithotrophy is the oxidation of inorganic chemicals for the generation of

energy. The process can use oxidative phosphorylation, just like aerobic and
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anaerobic respiration, but now the substance being oxidized (the electron donor) is
an inorganic compound. The electrons are passed off to carriers within the electron
transport chain, generating a proton motive force that is used to generate ATP with
the help of ATP synthase.

Chemolithotrophy Pathways.

Electrons donors

Chemolithotrophs use a variety of inorganic compounds as electron donors, with the

most common substances being hydrogen gas, sulfur compounds (such as sulfide
and sulfur), nitrogen compounds (such as ammonium and nitrite), and ferrous iron.

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Hydrogen oxidizers – these organisms oxidize hydrogen gas (H2) with the use of a
hydrogenase enzyme. Both aerobic and anaerobic hydrogen oxidizers exist, with
the aerobic organisms eventually reducing oxygen to water.

Sulfur oxidizers – as a group these organisms are capable of oxidizing a wide

variety of reduced and partially reduced sulfur compounds such as hydrogen sulfide
(H2S), elemental sulfur (S0), thiosulfate (S2O32-), and sulfite (SO32-). Sulfate
(SO42-) is frequently a by-product of the oxidation. Often the oxidation occurs in a
stepwise fashion with the help of the sulfite oxidase enzyme.

Nitrogen oxidizers – the oxidation of ammonia (NH3) is performed as a two-step

process by nitrifying microbes, where one group oxidizes ammonia to nitrite (NO2-)
and the second group oxidizes the nitrite to nitrate (NO3-). The entire process is
known as nitrification and is performed by small groups of aerobic bacteria and
archaea, often found living together in soil or in water systems.

Iron oxidizers – these organisms oxidize ferrous iron (Fe2+) to ferric iron (Fe3+).
Since Fe2+ has such a positive standard reduction potential, the bioenergetics are
not extremely favorable, even using oxygen as a final electron acceptor. The
situation is made more difficult for these organisms by the fact that Fe2+
spontaneously oxidizes to Fe3+ in the presence of oxygen; the organisms must use it
for their own purposes before that happens.

Electron acceptors

Chemolithotrophy can occur aerobically or anaerobically. Just as with either type of

respiration, the best electron acceptor is oxygen, to create the biggest distance
between the electron donor and the electron acceptor. Using a non-oxygen acceptor

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allows chemolithotrophs to have greater diversity and the ability to live in a wider
variety of environments, although they sacrifice energy production.

Amount of ATP generated

Just as both the electron donors and acceptors can vary widely for this group of
organisms, the amount of ATP generated for their efforts will vary widely as well.
They will not make as much ATP as an organism using aerobic respiration, since the
largest ΔE0’ is found using glucose as an electron donor and oxygen as an electron
acceptor. But how much less than 32 molecules of ATP greatly depends upon the
actual donor and acceptor being used. The smaller the distance between the two, the
less ATP that will be formed.

Chemolithoautotrophs vs chemolithoheterotrophs

Most chemolithotrophs are autotrophs (chemolithoautotrophs), where they fix

atmospheric carbon dioxide to assemble the organic compounds that they need.
These organisms require both ATP and reducing power (i.e. NADH/NADPH) in
order to ultimately convert the oxidized molecule CO2 into a greatly reduced
organic compound, like glucose. If a chemolithoautotroph is using an electron donor
with a higher redox potential than NAD+/NADP, they must use reverse electron
flow to push electrons back up the electron tower. This is energetically unfavorable
to the cell, consuming energy from the proton motive force to drive electrons in a
reverse direction back through the ETC.

Some microbes are chemolithoheterotrophs, using an inorganic chemical for their

energy and electron needs, but relying on organic chemicals in the environment for

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their carbon needs. These organisms are also called mixotrophs, since they require
both inorganic and chemical compounds for their growth and reproduction.

Nitrogen Metabolism

The nitrogen cycle depicts the different ways in which nitrogen, an essential element
for life, is used and converted by organisms for various purposes. Much of the
chemical conversions are performed by microbes as part of their metabolism,
performing a valuable service in the process for other organisms in providing them
with an alternate chemical form of the element.

Nitrogen Cycle.

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Nitrogen Fixation
Nitrogen fixation describes the conversion of the relatively inert dinitrogen gas
(N2) into ammonia (NH3), a much more useable form of nitrogen for most life
forms. The process is performed by diazotrophs, a limited number of bacteria and
archaea that can grow without an external source of fixed nitrogen, because of their
abilities. Nitrogen fixation is an essential process for Earth’s organisms, since
nitrogen is a required component of various organic molecules, such as amino acids
and nucleotides. Plants, animals, and other organisms rely on bacteria and archaea to
provide nitrogen in a fixed form, since no eukaryote is known that can fix nitrogen.

Nitrogen fixation is an extremely energy and electron intensive process, in order to

break the triple bond in N2 and reduce it to NH3. It requires a particular enzyme
known as nitrogenase, which is inactivated by O2. Thus, nitrogen fixation must
take place in an anaerobic environment. Aerobic nitrogen-fixing organisms must
devise special conditions or arrangements in order to protect their enzyme. Nitrogen-
fixing organisms can either exist independently or pair up with a plant host:

1. Symbiotic nitrogen-fixing organisms: these bacteria partner up with a plant, to

provide them with an environment appropriate for the functioning of their
nitrogenase enzyme. The bacteria live in the plant’s tissue, often in root
nodules, fixing nitrogen and sharing the results. The plant provides both the
location to fix nitrogen, as well as additional nutrients to support the energy-
taxing process of nitrogen fixation. It has been shown that the bacteria and the
host exchange chemical recognition signals that facilitate the relationship.

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One of the best known bacteria in this category is Rhizobium, which partners up
with plants of the legume family (clover, soybeans, alfalfa, etc).
2. Free-living nitrogen-fixing organisms: these organisms, both bacteria and archaea,
fix nitrogen for their own use that ends up being shared when the organisms
dies or is ingested. Free-living nitrogen-fixing organisms that grow
anaerobically do not have to worry about special adaptations for their
nitrogenase enzyme. Aerobic organisms must make adaptations. Cyanobacteria,
a multicellular bacterium, make specialized cells known as heterocysts in which
nitrogen fixation occurs. Since Cyanobacteria produce oxygen as part of their
photosynthesis, an anoxygenic version occurs within the heterocyst, allowing
the nitrogenase to remain active. The heterocysts share the fixed nitrogen with
surrounding cells, while the surrounding cells provide additional nutrients to
the heterocysts.

Assimilation

Assimilation is a reductive process by which an inorganic form of nitrogen is

reduced to organic nitrogen compounds such as amino acids and nucleotides,
allowing for cellular growth and reproduction. Only the amount needed by the cell is
reduced. Ammonia assimilation occurs when the ammonia (NH3)/ammonium ion
(NH4+) formed during nitrogen fixation is incorporated into cellular nitrogen.
Assimilative nitrate reduction is a reduction of nitrate to cellular nitrogen, in a
multi-step process where nitrate is reduced to nitrite then ammonia and finally into
organic nitrogen.

Nitrification
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As mentioned above, nitrification is performed by chemolithotrophs using a reduced

or partially reduced form of nitrogen as an electron donor to obtain energy. ATP is
gained by the process of oxidative phosphorylation, using a ETC, PMF, and ATP
synthase.

Denitrification

Denitrification refers to the reduction of NO3- to gaseous nitrogen compounds,

such as N2. Denitrifying microbes perform anaerobic respiration, using NO3- as an
alternate final electron acceptor to O2. This is a type of dissimilatory nitrate
reduction where the nitrate is being reduced during energy conservation, not for the
purposes of making organic compounds. This produces large amounts of excess
byproducts, resulting in the loss of nitrogen from the local environment to the
atmosphere.

Anammox

Anammox or anaerobic ammonia oxidation is performed by marine bacteria,

relatively recently discovered, that utilize nitrogen compounds as both electron
acceptor and electron donor. Ammonia is oxidized anaerobically as the electron
donor while nitrite is utilized as the electron acceptor, with dinitrogen gas produced
as a byproduct. The reactions occur within the anammoxosome, a specialized
cytoplasmic structure which constitutes 50-70% of the total cell volume. Just like
denitrification, the anammox reaction removes fixed nitrogen from a local
environment, releasing it to the atmosphere.

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Example of physiological adaptation in microbes

Resistance of Microorganisms to Extreme Environmental

n this section we explore the diversity of extremophilic microbes, their mechanisms

of adaptation, and examples of extreme environmental conditions where they are
found.
1. Thermophiles and Hyperthermophiles
Thermophiles are microorganisms that thrive at relatively high temperatures,
between 45 °C and 80 °C. Hyperthermophiles are particularly extreme thermophiles
for which the optimal temperatures are above 80 °C.
The best known and well-studied geothermal areas are in North America
(Yellowstone National Park), Iceland, New Zealand, Japan, Italy, and the former
Soviet Union. These locations are usually rich in reduced chemicals from inside the
Earth, and hence, many thermophiles are chemoautotrophic, reacting hydrogen,
ferrous iron, or reduced sulfur compounds with electron acceptors like oxygen or
nitrate. As a consequence of extracting energy by oxidizing sulfur compounds, these
reactions produce sulfuric acid, thus often making the geothermal waters very
acidic. Consequently, many heat-loving microbes are also adapted to highly acid
conditions. Indeed, hyperthermophilic extreme acidophiles, with pH optima for
growth at or below 3.0 (like the archaea genera Sulfolobus, Sufurococcus,
Desulfurolobus, and Acidianus), produce sulfuric acid from the oxidation of
elemental sulfur or sulfidic ores in solfataras of Yellowstone National Park.
Deep sea hydrothermal vent communities are found near subsurface volcanoes and
at the boundary between seawater and magma, usually kilometers beneath the ocean
surface. Since no light is available and the content of oxygen is very low, a large

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majority of thermophilic isolates from these deep sea locals are chemoautotrophic
anaerobes. The present record of high-temperature growth is held by two Archaea
isolated from these environments: Strain 121 and Methanopyrus kandleri can
survive and reproduce at 121 °C and 122 °C, respectively [18,19].
The molecular basis for adaptations to extreme environments has been studied more
intensively for high temperatures than for any other parameter. At high
temperatures, biomolecules, such as enzymes, denature, losing their function and
hence, stopping the metabolism. Also, the fluidity of membranes increases
significantly, disrupting the cell.
To prevent denaturation and degradation, thermophiles present a variety of cellular
adaptations. Their membrane lipids contain more saturated and straight chain fatty
acids than do mesophiles (which grow typically between 15 °C and 40 °C). This
allows thermophiles to grow at higher temperatures by providing the right degree of
fluidity needed for membrane function. Thermophilic proteins appear to be smaller
and in some cases more basic, which may also result in increased stability. Another
method used to improve the stability of proteins is through the action of chaperones,
which help to refold denatured proteins. Furthermore, monovalent and divalent salts
enhance the stability of nucleic acids because these salts screen the negative charges
of the phosphate groups, and KCl and MgCl2 protect the DNA from depurination
and hydrolysis. Another way to stabilize DNA is through the employment of DNA-
binding proteins and the compaction of the genome into chromatin.

2. Psychrophiles
Psychrophiles are microorganisms that grow at or below 0°C and which have an
optimum growth temperature of 15°C and an upper limit of 20°C. They are found in
a variety of cold environments, from the stratosphere to the deep-sea.
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Most of the deep sea is at a constant temperature of 2 °C, while around the polar ice
caps, liquid seawater may even be cooled to below 0 ºC, as the typical salt content of
sea water (3.4%) lowers the freezing point to −1.8°C . When the seawater freezes,
the salt becomes increasingly concentrated in small pockets. Under these conditions,
the freezing point of water may be depressed to −20°C. At such frozen temperatures,
natural microbial metabolism has been measured. For example, the bacterium
Psychrobacter cryopegella can grow at −10 ºC, stay alive, and even keep
metabolizing at −20 °C [28]. For this reason, many psychrophiles are also halophiles
(microbes that grow in elevated salt concentrations).
To survive and flourish at low temperatures, psychrophiles have to overcome some
problems related to permanent cold environments. At low temperatures, enzymes
become very rigid, and solute concentrations are at high, perhaps toxic levels.
Furthermore, once the water is frozen, ice crystals may pierce the cell membranes,
destroying cellular integrity.
Membranes of psychrophiles contain increased levels of unsaturated fatty acids that
further increase with the reduction in temperature in order to modulate membrane
fluidity. Psychrophiles produce cold-adapted enzymes that have high specific
activities at low temperatures. These enzymes have the ability to support
transcription and translation at low temperatures. Studies have also revealed the
presence of certain genes active at low temperature. Moreover, antifreeze proteins
have been identified in cold adapted microbes. Such proteins have the ability to bind
to ice crystals through a large complementary surface, and hence prevent these
crystals from piercing the cell membrane.

3. Acidophiles

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Acidophiles are microorganisms that grow optimally at pH values of 2.0. Acidic

environments are especially interesting because, in general, the low pH of the habitat
is the consequence of microbial metabolism, and not a condition imposed by the
system, as is the case for other extreme environments.
Acidophiles oxidize the elemental sulfur (in volcanic areas) or sulfidic minerals (in
mine drainage) to obtain energy, which produces extreme acidic environments.
Indeed, most of the characterized strict acidophilic microorganisms have been
isolated from volcanic areas or acid mine drainage. For example, Archaea
Picrophilus oshimae and P. torridus were isolated from volcanically heated, dry
soils in Japan. They thrive at pH 0.7 and 60 °C. These microorganisms maintain the
intracellular pH value at 4.6, whereas the other acidophiles maintain their pH at 6.0.
Ferroplasma acidarmanus was isolated from acid mine drainage in Iron Mountain,
California, and is able to grow at a pH of 0.
Environments with low pH condition are a challenge to cellular biochemistry since
extreme acidity leads to the denaturation of proteins. Acidophiles protect their
proteins by including more amino acids with neutral side-groups and by actively
pumping protons out of the cell to maintain constant intracellular pH levels.

4. Alkaliphiles
Alkaliphiles are microorganisms that grow optimally at pH values above 9.0, often
with pH optima around 10.0, while showing little or no growth at near neutral pH
values. Alkaline environments may be found in places with high amounts of Ca2+
generated by the serpentinization of silicate minerals, as exemplified by the
hyperalkaline spring waters seen in Jordan and in the soda lakes and soda deserts of
arid and semi-arid areas on Earth, including the high desert in the west of the United

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States, the East African Rift Valley, and the plateaus of Mongolia, Inner Mongolia,
and Tibet.
A diversity of microorganisms can live at a pH of 10.5. Microbial communities live
at a pH of 12.9 in the soda lakes of Maqarin, Jordan . Alkaliphiles are often isolated
from natural environments that also tend to have high concentrations of NaCl; these
are thereby called haloalkalophiles.
Under alkaline conditions, the concentrations of hydrogen ions are very low and
cells have trouble using ATP-synthase to produce energy and other essential ions,
such as magnesium and calcium, which precipitate out of the water as salts (and
hence are available only at very low levels). Base-loving microbes circumvent these
problems by actively pumping in these ions and by exporting others to maintain
their interior at near-neutrality. Furthermore, the cell wall of alkalophiles acts as a
defense barrier from extreme environmental conditions.

5. Halophiles
Halophiles are microorganisms that grow in elevated salt concentrations, starting
from approximately 10% sodium chloride to saturation, and some of them can even
survive in salt crystals.
The environments where halophilic microorganisms are found include aquatic
habitats of varying salinity, salt marshes, surface salt lakes, subterranean salt lakes,
and some other places. Two of the largest and best-studied modern hypersaline
environments are The Great Salt Lake in Utah and the Dead Sea in the Middle East.
Permanently cold hypersaline evaporation ponds are found in dry regions of
Antarctica, including Deep Lake, Organic Lake, and Lake Suribati. In these regions,
the high salt content may maintain the water liquid at temperatures as low as −20°C.

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In response to the salt, all these adapted microorganisms maintain very high
concentrations of other solutes in their cytoplasm to keep their insides in osmotic
balance with the outside world. Halophilic Archaea keep extremely high
concentrations of potassium chloride in their cells. All the proteins in a halophile
have to be optimally folded and functional under saturated salt conditions, in much
the same way the proteins of hyperthermophiles remain active near 100 °C.
Researchers have therefore studied the amino acid sequences, structures, and
functional characteristics of halophilic proteins in comparison with thermophilic and
mesophilic proteins in order to gain some insights into the evolutionary strategies
used to adapt proteins to stress conditions, but the picture remains far from
complete.

6. Piezophiles
Piezophiles are microorganisms that have adapted to high-pressure environments
and can grow more easily under high hydrostatic pressure conditions than at
atmospheric pressure. Piezophiles are widespread in the seafloor and deep within the
Earth’s crust. These microbes were isolated from the deepest part of the ocean at a
depth of 10.5 Km, and are adapted to pressures of up to 110 Mpa at 2 °C and of 40
Mpa at temperatures above 100 °C.
In subsurface habitats deep within the Earth’s crust, microbes are adapted to high
temperatures and extremely limited resources. For example, two different species of
thermophilic iron-reducing bacteria were isolated from the granite of Siijan
(Sweden) at a depth of 6.7 km. Complex ecosystems have been reported within
rocks freshly harvested from 3 km down in South African gold mines. Furthermore,
methanogenic microbes have been collected from several hundred meters within
basalt rocks in the Columbia River basin (Washington State, USA.
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These microbes grow at extremely slow rates and live at low densities. However,
considering the vast volume of the upper crust on Earth, this could still constitute a
substantial mass of living material. The ―deep biota‖ has been estimated, by some
researchers, to exceed the sum total of all surface living systems. In many ways,
these subterranean fissures are ideal habitats since they provide a stable ambient
with constant flow of chemical energy. Additionally, these environments protect
microbes from UV or energetic cosmic radiation, and from the most devastating
catastrophes above.
The difficulty in obtaining samples from deep-sea habitats, along with the
challenges of conducting biochemical experiments under high pressure conditions in
the laboratory, have conspired to make this research field one of the less
comprehensively studied. However, recent studies are in progress. Protein-protein
interactions are very sensitive to pressure increases, which can be the reason for
enzyme dissociation [60]. Pressure is also known to alter gene expression.
Furthermore, when pressure increases, or temperature decreases, the molecules in
lipid membranes pack tighter, resulting in decreased membrane fluidity. Organisms
often circumvent this problem by increasing the proportion of unsaturated fatty acids
in their membranes.

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