1. Lecture 2 & 3
Role of microbial systems, microbial metabolism
and growth kinetics – Principle and
characteristics

2. Microbes and metabolism
Metabolic pathways (Michal, 1992) are interlinked to produce what can
develop into an extraordinarily complicated network, involving several levels
of control
Interaction of natural cycles and represent the biological element of the
natural geobiological cycles
Impinge on all aspects of the environment, both living and nonliving
Carbon cycle as an example, carbon dioxide in the atmosphere is
returned by dissolution in rainwater, and also by the process of
photosynthesis to produce sugars, which are eventually metabolised
to liberate the carbon once more

3. In addition to constant recycling through metabolic pathways,
carbon is also sequestered in living and nonliving components such as in
trees in the relatively short term, and deep ocean systems or ancient
deposits, such as carbonaceous rocks, in the long term
Cycles which involve similar principles of incorporation into biological
molecules and subsequent re-release into the environment operate for
nitrogen, phosphorus and sulphur.
All of these overlap in some way, to produce the metabolic pathways
responsible for the synthesis and degradation of biomolecules.
Superimposed, is an energy cycle, ultimately driven by the sun, and
involving constant consumption and release of metabolic energy.

4. Microbes are referred to as such, simply because they cannot be seen by the naked eye.
Many are bacteria or archaea, term ‘microbe’ also encompasses some eukaryotes,
including yeasts, which are unicellular fungi, as well as protozoa and unicellular plants.
In addition, there are some microscopic multicellular organisms, such as rotifers, which
have an essential role to play in the microsystem ecology of places such as sewage
treatment plants.
An individual cell of a eukaryotic multicellular organism like a higher plant or animal, is
approximately 20 microns in diameter, while a yeast cell, also eukaryotic but unicellular,
is about five microns in diameter.
Although bacterial cells occur in a variety of shapes and sizes, depending on the species,
typically a bacterial cell is rod shaped, measuring approximately one micron in width and
two microns in length.

5. At its simplest visualisation, a cell,
be it a unicellular organism, or one
cell in a multicellular organism, is a
bag, bounded by a membrane,
containing an aqueous solution in
which are all the molecules and
structures required to enable its
continued survival. In fact, this ‘bag’
represents a complicated
infrastructure differing distinctly
between prokaryotes and
eukaryotes
Microorganisms may live as free individuals or as communities, either as a clone of
one organism, or as a mixed group.
Biofilms are examples of microbial communities, the components of which may
number several hundred species.
Represent the structure of microbial activity in many relevant technologies such as
trickling filters

6. A biofilm is a mixed
population of
microbes live in close
proximity which may
be mutually
beneficial
Such consortia can
increase the habitat
range, the overall
tolerance to stress and
metabolic diversity of
individual members of
the group.
Recalcitrant pollutants are
eventually degraded due to
combined contributions of
several of its members
Bacterial transformation
Bacterium may absorb free
deoxyribonucleic acid (DNA), the
macromolecule which stores genetic
material, from its surroundings
released by other organisms, as a result
of cell death
Process is dependent on the ability, or
competence, of a cell to take up DNA, and
concentration of DNA in the surrounding
environment. This is commonly referred to
as horizontal transfer as opposed to vertical
transfer which refers to inherited genetic
material, either by sexual or asexual
reproduction.
Bacterial transformation is the transfer of free DNA released from a donor bacterium
into the extracellular environment that results in assimilation and usually an expression
of the newly acquired trait in a recipient bacterium.

7. Sliminess often associated with biofilms is usually attributed to
excreted molecules often protein and carbohydrate in nature,
which may coat and protect the film
Biofilm may proliferate at a rate to cause areas of anoxia at the
furthest point from the source of oxygen, thus encouraging the
growth of anaerobes
Composition of the biofilm community is likely to change with time

8. Microbial Metabolism
Energy required to carry out all cellular processes is obtained
from ingested food in the case of chemotrophic cells, additionally
from light in the case of phototrophs and from inorganic
chemicals in lithotrophic organisms
Definition
All biological macromolecules contain the element
carbon, a dietary source of carbon is a requirement
Ingested food is therefore, at the very least, a source of
energy and carbon, the chemical form of which is
rearranged by passage through various routes called
metabolic pathways

9. One purpose of this reshuffling is to produce, after addition or removal of
other elements such as hydrogen, oxygen, nitrogen, phosphorous and
sulphur, all the chemicals necessary for growth.
Other is to produce chemical energy in the form of adenosine triphosphate
(ATP), also one of the ‘building blocks’ of nucleic acids. Where an organism
is unable to synthesize all its dietary requirements, it must ingest them, as
they are, by definition, essential nutrients.
The profile of these can be diagnostic for that organism and may be used in
its identification in the laboratory.
An understanding of nutritional requirements of any given microbe, can
prove essential for successful remediation by bioenhancement.

10. Microbial Growth Kinetics
Growth of a microorganism is the basis of biotechnological exploitation of
microflora for production of desired product
Optimization of growth of microorganism in a particular media is desirable
due to economical and availability of particular growth constituent in a
region
Some microorganisms have specific requirement and they grow in a
particular growth media
 Common media for growth of different microorganism
 Bacterial cell division
 Methods of measuring growth
 Different phase in bacterial growth and
 Growth kinetics

11. Bacterial Cell Division
Binary division
Binary division is the most common mode of cell
division in bacteria. In this mode of cell division, a
single bacteria cell grows transversely with the
synthesis of chromosomal DNA. A transverse
septum appears in the middle of the cell body
that divides the bacterial cell into the two with a
distribution of chromosomal DNA, ribosome and
other cellular machinery.
Budding
In this mode of cell division, chromosomal DNA
divides to form two copies. Sister chromosomal
DNA moves to one side of the cell and this
portion of the cells protrude from main body to
form bud. Eventually bud grows in size and get
separated from main cell to develop a new cell.

12. Fragmentation
This mode of asexual division is more common in filamentous bacteria. In this mode,
filament of the growing cell gets fragmented into small bacillary or coccoid cells, these
cellular fragments eventually develop into new cell.
Measuring Bacterial growth
Microscopic count - Bacterial cells can be counted easily on a “petroff-hausser
counting chamber” The chamber has a ruling to make square (1/400 mm2) of
equivalent volume. A glass slide is placed (~1/50mm height) to make a chamber
filled with bacterial cell suspension. Volume of each chamber is 1/20,000 mm3.
This chamber can be used to observe bacteria with phase contrast microscope.
For example, if each chamber has 8 bacteria then there are 8x20,000,000 or
1.6x108 bacteria/ml. A very high or low concentration of bacterial sample cannot
be counted accurately.

13. Plate count method
A defined amount of bacterial culture suspension is introduced onto solid support
media to grow and give colonies. If number of colonies on solid media is too high,
then serial dilution of original stock can be plated on solid media and number of
colony can be counted with a colony counter. A manual colony counter has lamp at
the bottom, a grid to divide the bacterial culture plate and a magnifying glass to
visualize and count single colony. A plate with colony count of 30-300 can be used to
determine the number of bacteria present in original stock
Number of bacteria per ml= Number of colonies counted on plate X dilution of
sample

14. Turbidimetric methods
This method is based on light scattering principles of particulate matter such as
bacteria. A bacteria cell suspension is placed in test cuvette and corresponding media
in reference cuvette. The optical density or absorbance of the bacterial suspension is
used to measure the number of bacteria number. This method can not distinguish
between live or dead bacteria as both form contribute to the turbidity

15. Nitrogen content and Dry weight
A bacterial cell mass can be measured by direct measurement of dry weight of
culture or nitrogen content
Growth cycle of bacteria
The most common method of bacteria division is binary fission and by this method,
one bacteria cell gives two daughter cells. The time a bacteria takes to complete
one division is called as generation time and it depends on bacteria species and
media properties

16. Glycolysis via the Embden-Meyerhof-Parnas
Glycolytic Pathway
Glycolysis is the almost universal pathway that converts glucose into
pyruvate along with the formation of nicotinamide adenine dinucleotide
(NADH) and adenosine triphosphate (ATP). It primarily occurs in the
cytoplasm of the cell
Under aerobic conditions, the pyruvate passes into the mitochondria where it is
completely oxidized by O2 into CO2 and H2O and its chemical energy largely
conserved as ATP.
Pyruvate generated via aerobic glycolysis feeds into the TCA or Kreb’s Cycle
In the absence of sufficient oxygen, the pyruvate is reduced by NADH via
anaerobic glycolysis or fermentation to a wide range of products, routinely
lactate in animals and ethanol in yeasts

18. Starting molecule for glycolysis is glucose, a simple and abundant sugar found
in carbohydrates, which provides the energy for most cells.
Carbohydrates synthesized during photosynthesis act as the main storage
molecules of solar energy.
When ingested, complex carbohydrates are enzymatically hydrolyzed to
monosaccharides, such as starch to D(+)-glucose.
Catabolism of glucose is the primary energy source for short-term
requirements.
Embden-Meyerhof-Parnas (EMP) pathway, name of the discoverers, Gustav
Embden, Otto Meyerhof, and Jakub Karol Parnas.

19. 10 STEPS IN THE GLYCOLYTIC PATHWAY AND
ENZYMES OF GLYCOLYSIS

20. Reaction Enzyme IUBMB EC Number
1. Phosphorylation of glucose. D(+)-Glucose is phosphorylated with ATP
to give glucose-6-phosphate.
Hexokinase EC 2.7.1.1
2. Isomerization of glucose-6-P to fructose-6-P. The isomerization of
glucose-6-phosphate in the second reaction to fructose-6-phosphate
occurs via ring-opening and subsequent keto-enol-tautomerization.
Glucose-6-
phosphate
isomerase
EC 5.3.1.9
3. Phosphorylation of fructose-6-P. The third reaction is another
phosphorylation with ATP, whereby fructose-6-phosphate is converted to
fructose-1,6-bisphosphate.
6-P-Fructokinase EC 2.7.1.11
4. Fructose-1,6-bisphosphate to glyceraldehyde phosphate and
dihydroxyacetone phosphate. A key branching reaction is the fourth
reaction: a ring-opening reaction of fructose-1,6-bisphosphate, which is
cleaved in a retro-aldol reaction into D-glyceraldehyde-3-phosphate, and
dihydroxyacetone phosphate.
Fructose-
bisphosphate
aldolase
EC 4.1.2.13
5. Isomerization of dihydroxyacetone-P to glyceraldehyde-P. The branch
via dihydroxyacetonephosphate is channelled back into D-
glyceraldehyde-3-phosphate in the fifth reaction by an isomerization.
Triose-phosphate
isomerase
EC 5.3.1.9
6. Glyceraldehyde phosphate oxidation & phosphorylation to 1,3-
bisphosphoglycerate. In the sixth reaction, the combined D-
glyceraldehyde- 3-phosphate from both routes is oxidized at the C1 to a
carboxylic acid and then phosphorylated in the 1-position to yield 1,3-
bisphospho-D-glycerate.
Glyceraldehyde
phosphate
dehydrogenase
EC 1.2.1.12
7. ATP formation. This phosphate group in the 1-position is transferred in
the seventh reaction from the carboxyl group to ADP to give 3-phospho-
D-glycerate.
Phosphoglycerate
kinase
EC 2.7.2.3

21. 8. 3-Phosphoglycerate to 2-phosphoglycerate. The eighth reaction is an
isomerization of 3-phospho-D-glycerate to 2-phospho-D-glycerate.
Phosphoglycerate
mutase
EC 5.4.2.1
9. 2-Phosphoglycerate to phosphonenolpyruvate. The next metabolite,
phosphoenolpyruvate, is formed in a dehydration reaction from 2-
phospho-D-glycerate.
Enolase EC 4.2.1.11
10. Formation of pyruvate & ATP. The glycolysis pathway from D(+)-
glucose to two molecules of pyruvate is concluded by the tenth reaction,
which transfers a phosphate group from phosphoenolpyruvate to ADP,
thereby giving ATP and pyruvate.
Pyruvate kinase EC 2.7.1.40

22. TCA cycle is usually described beginning with acetyl-CoA

23. TCA cycle description
1. TCA cycle begins with an enzymatic aldol addition reaction of acetyl CoA to
oxaloacetate, forming citrate
2. Citrate is isomerized by a dehydration-hydration sequence to yield (2R,3S)-isocitrate
3. Further enzymatic oxidation and decarboxylation gives 2-ketoglutarate
4. After another enzymatic decarboxylation and oxidation, 2-ketoglutarate is
transformed into succinyl-CoA
5. Hydrolysis of this metabolite to succinate is coupled to the phosphorylation of
guanosine diphosphate (GDP) to guanosine triphosphate (GTP)
6. Enzymatic desaturation by flavin adenine dinucleotide (FAD)-dependent succinate
dehydrogenase yields fumarate
7. After stereospecific hydration, fumarate catalyzed by fumarase is transformed to
L-malate
8. Last step of NAD-coupled oxidation of L-malate to oxaloacetate is catalyzed by malate
dehydrogenase and closes the cycle.

24. Glyoxylate Cycle
 Glyoxylate cycle is an anabolic pathway that is considered a variation of the
tricarboxylic acid (TCA) cycle.
 TCA cycle occurs in plants, bacteria and fungi and acetyl - CoA is converted
into succinate
 Glyoxylate cycle was thought not to occur in animals due to the absence of
these enzymes isocitrate lyase and malate synthase, however, this hypothesis
is being explored
 Glyoxylate cycle occurs in glyoxysomes, which are specialized peroxisomes.
 There are no decarboxylation reactions in the glyoxylate cycle.
 Glyoxylate cycle allows cells to utilize 2 carbon units of acetate, and convert
them into 4 carbon units, succinate, for energy production and biosynthesis
 Additionally, each turn of the cycle produces a molecule of FADH2 and NADH

25. Function in plants
Seeds cannot carry out photosynthesis as they lack chloroplasts.
However, seeds have specific peroxisomes known as glyoxysomes, where the
glyoxylate cycle can occur.
Glyoxylate cycle occurs in seeds during germination so that:
 Lipids stored in seeds can be used as an energy source for the
formation of carbohydrates for the growth and development of the
shoot.
 Acetate is converted to acetyl - CoA, which in turn is:
Utilized as a source of carbon and energy
Used to produce NADPH, which drives ATP synthesis in the electron
transport chain

26. Reactions, Yield, and Energy Balance
Plants, fungi and bacteria require carbohydrates for energy and cell wall
synthesis (e.g., cellulose, chitin, and glycans). The glyoxylate cycle enables organisms
to producecarbohydrates using acetyl - CoA from the β-oxidation of fatty acids.
Reactions
The pathway begins with 2 molecules of acetyl – CoA
Citratesynthase converts 1 of the acetyl - CoA molecules to citrate.
Citrate is converted to isocitrate by the enzyme aconitase.
Isocitrate is converted to glyoxylate and succinate.
Succinate is converted to fumarate by succinate dehydrogenase.
The next step involves the formation of 2 molecules of malate:
1 molecule of malate is formed by the combination of acetyl - CoA and
glyoxylate.
The 2nd molecule is formed by the conversion of fumarate to malate in the
presence of fumarase.
Malate dehydrogenase converts 2 malate molecules into 2 oxaloacetate molecules.
1 molecule of oxaloacetate is converted to citrate, and 1 molecule of oxaloacetate is
used for gluconeogenesis.