NUTRITION, LABORATORY CULTURE, AND METABOLISM OF MICROORGANISMS

Microbial Nutrition
o Microbial nutrition is an aspect of microbial physiology that deals with the supply of
monomers (or the precursors of monomers) that cells need for growth. Collectively, these
required substances are called nutrients.
o Different organisms need different complements of nutrients, and not all nutrients are
required in the same amounts.
Microbial Nutrition
o Some nutrients, called macronutrients, are required in large amounts, while others, called
micronutrients, are required in trace amounts.
o All microbial nutrients originate from the chemical elements; however, just a handful of
elements dominate biology

Carbon and Nitrogen

o All cells require carbon, and most prokaryotes require organic compounds as their source
of carbon. On a dry weight basis, a typical cell is about 50% carbon, and carbon is the major
element in all classes of macromolecules.
o Following carbon, the next most abundant element in the cell is nitrogen. A bacterial cell
is about 12% nitrogen (by dry weight), and nitrogen is a key element in proteins, nucleic
acids, and several other cell constituents.
Other Macronutrients: P, S, K, Mg, Ca, Na
o After C and N, other essential elements are needed in smaller amounts.
 o Phosphorus is required by the cell primarily for synthesis of nucleic acids and
phospholipids.
o Sulfur is required because of its structural role in the amino acids cysteine and methionine
and because it is present in several vitamins, including thiamine, biotin, and lipoic acid, as
well as in coenzyme A
Other Macronutrients: P, S, K, Mg, Ca, Na
o Potassium is required by all organisms. Many enzymes, including some necessary for
protein synthesis, require potassium for activity.
o Magnesium functions to stabilize ribosomes, membranes, and nucleic acids and is also
required for the activity of many enzymes.
o Calcium helps stabilize cell walls in many microorganisms and plays a key role in the heat
stability of endospores
o Sodium is required by some but not all microorganisms and is typically a reflection of the
habitat (marine microorganisms usually require sodium for growth. By contrast,
freshwater species are usually able to grow in the absence of sodium)
Iron and Trace Metals
o Microorganisms require various metals for growth. Among these is iron, which plays a
major role in cellular respiration. Iron is a key component of cytochromes and ironsulfur
proteins involved in electron transport reactions
o Under anoxic conditions, iron is generally in the ferrous (Fe2+) form and soluble. However,
under oxic conditions, iron is typically in the ferric (Fe3+) form in insoluble minerals.
o To obtain iron from such minerals, cells produce iron-binding agents called siderophores
that bind iron and transport it into the cell. Many other iron-binding agents are known.
Some bacteria produce phenolic siderophores called enterobactins
Iron and Trace Metals
o Some organisms can grow in the absence of iron. For example, the bacteria Lactobacillus
plantarum and Borrelia burgdorferii do not contain detectable iron. In these bacteria,
Mn2+ substitutes for iron as the metal component of enzymes that normally contain Fe2+.
o Many other metals are required or otherwise metabolized by microorganisms. Like iron,
these micronutrients are called trace elements. Micronutrients typically play a role as
components of enzymes, the cells catalysts
Growth Factors
o Growth factors are organic compounds required in only small amounts and then only by
certain organisms.
o Growth factors include vitamins, amino acids, purines, and pyrimidines.
Culture Media
o Culture medium: an aqueous solution of various nutrients suitable for the growth of
microorganisms
o Two broad classes of culture media are used in microbiology:
Defined medium: a culture medium whose precise chemical composition is
known
Complex medium: a culture medium composed of digests of chemically
undefined substances such as yeast and meat extracts
 o For growing many organisms, knowledge of the exact composition of a medium is not
essential. In these instances complex media may suffice and may even be advantageous.
Complex media employ digests of animal or plant products, such as casein (milk protein),
beef (beef extract), soybeans (tryptic soy broth), yeast cells (yeast extract), or any of a
number of other highly nutritious yet impure substances.
o In clinical microbiology, culture media are often made to be selective or differential (or
both).
o A selective medium contains compounds that selectively inhibit the growth of some
microorganisms but not others.
o A differential medium is one in which an indicator, typically a dye, is added that allows for
the differentiation of particular chemical reactions that have occurred during growth.
Differential media are quite useful for distinguishing between species of bacteria
o Different microorganisms can have vastly different nutritional requirements. Thus, for
successful culture of any microorganism it is necessary to understand its nutritional
requirements and then supply the nutrients in the proper form and in the proper
proportions in a culture medium.
Laboratory Culture of Microorganisms
o Once a culture medium has been prepared and made sterile (free of microorganisms),
organisms can be inoculated (added to it) and the culture incubated under conditions that
will support growth.
o In a laboratory, inoculation will typically be of a pure culture, a culture containing only a
single kind of microorganism.
o It is essential to prevent other organisms from entering a pure culture. Such unwanted
organisms, called contaminants, are ubiquitous, and microbiological techniques are
designed to avoid contamination.
Solid versus Liquid Culture Media
o Culture media are sometimes prepared in a semisolid form by the addition of a gelling
agent to liquid media. Such solid culture media immobilize cells, allowing them to grow
and form visible, isolated masses called colonies
o Bacterial colonies can be of various shapes and sizes depending on the organism, the
culture conditions, the nutrient supply, and several other physiological parameters. Some
bacteria produce pigments that cause the colony to be colored
o Colonies permit the microbiologist to visualize the purity of the culture. Plates that contain
more than one colony type are indicative of a contaminated culture. The appearance and
uniformity of colonies on a Petri plate has been used as one criterion of culture purity for
over 100 years
Microbial metabolisms
o Catabolism: break down molecules into smaller units and release energy
o Anabolism: Build up molecules from smaller units and require energy
Enzymes
o A catalyst is a substance that lowers the activation energy of a reaction, thereby increasing
the reaction rate
o Biological catalysts are called enzymes. Enzymes are proteins (or in a few cases, RNAs) that
are highly specific in the reactions they catalyze.
 oThe catalytic power of enzymes is remarkable. Enzymes increase the rate of chemical
reactions anywhere from 108 to 1020 times the rate that would occur spontaneously.
Adenosine Triphosphate (ATP)
o The most important energy-rich phosphate compound in cells is adenosine triphosphate
(ATP). ATP consists of the ribonucleotide adenosine to which three phosphate molecules
are bonded in series
o The reactions ATP ADP + Pi and ADP AMP + Pi each release roughly 32 kJ/mol of
energy. The synthesis of one mole of ATP is on the order of 55 to 60 kJ.
Coenzyme A
o In addition to energy-rich phosphate compounds, cells can produce other energy-rich
compounds including derivatives of coenzyme A (for example, acetyl-CoA)
o Coenzyme A derivatives contain thioester bonds. Upon hydrolysis, these yield sufficient
free energy to drive the synthesis of an energy-rich phosphate bond.

Acetyl-S-CoA + H2O + ADP + Pi acetate + HS-CoA + ATP + H+

Long-Term Energy Storage

o ATP is a dynamic molecule in the cell; it is continuously being broken down to drive
anabolic reactions and resynthesized at the expense of catabolic reactions.
o For longer-term energy storage, microorganisms typically produce insoluble polymers that
can be oxidized later for the production of ATP.
o Examples of energy storage polymers in prokaryotes include glycogen, poly--
hydroxybutyrate and other polyhydroxyalkanoates, and elemental sulfur, stored from the
oxidation of H2S by sulfur chemolithotrophs.
o In eukaryotic microorganisms, polyglucose in the form of starch or lipids in the form of
simple fats are the major reserve materials.
Respiration
o Glycolysis
o Krebs/Citric cycle
o Oxydative phosphorylation
o Fermentation

o Glycolysis occurs in the cytoplasm.

o It begins catabolism by breaking glucose into two molecules of pyruvate.
o The Krebs cycle occurs in the mitochondrial matrix.
o It degrades pyruvate to carbon dioxide.
o Several steps in glycolysis and the Krebs cycle transfer electrons from substrates to NAD +,
forming NADH.
o NADH passes these electrons to the electron transport chain.
o As they are passed along the chain, the energy carried by these electrons is stored in the
mitochondrion in a form that can be used to synthesize ATP via oxidative phosphorylation.
o Oxidative phosphorylation produces almost 90% of the ATP generated by respiration
Glycolysis
o During glycolysis, glucose, a six carbon-sugar, is split into two, three-carbon sugars.
 o These smaller sugars are oxidized and rearranged to form two molecules of pyruvate.
o Each of the ten steps in glycolysis is catalyzed by a specific enzyme.
o These steps can be divided into two phases: an energy investment phase and an energy
payoff phase.
o The net yield from glycolysis is 2 ATP and 2 NADH per glucose.
No CO2 is produced during glycolysis.
o Glycolysis occurs whether O2 is present or not.
If O2 is present, pyruvate moves to the Krebs cycle and the energy stored in NADH
can be converted to ATP by the electron transport system and oxidative
phosphorylation.
Kreb cycle (citric cycle)
o If oxygen is present, pyruvate enters the mitochondrion where enzymes of the Krebs cycle
complete the oxidation of the organic fuel to carbon dioxide.
o As pyruvate enters the mitochondrion, a multienzyme complex modifies pyruvate to acetyl
CoA which enters the Krebs cycle in the matrix.
o The Kreb cycle has 8 steps, each catalyzed by a specific enzyme .
2 C enter in the relatively reduced form of acetate (step 1), and
2 C leave in the completely oxidized form of CO2 (steps 3 and 4).
Subsequent steps decompose the citrate back to oxaloacetate, giving off CO2 as
"exhaust." It is this regeneration of oxaloacetate that accounts for the "cycle" in
the Krebs cycle.
Most of the energy harvested by the oxidative steps of the cycle is conserved in
NADH
For each acetate that enters the cycle, 3 NAD+ are reduced to NADH (steps 3, 4,
and 8).
In one oxidative reaction (step 6) electrons are transferred to FAD (flavin adenine
dinucleotide, derived from riboflavin, a B vitamin). The reduced form, FADH2,
donates its electrons to the electron transport chain, as does NADH.
Step 5, that forms 1 ATP directly by substrate-level phosphorylation,
But most of the ATP output of respiration results from oxidative phosphorylation,
when the NADH and FADH2 produced by the Krebs cycle relay the electrons
extracted from food to the electron transport chain
Electron Transport Chain
o Electrons removed from food (during glycolysis and the Krebs cycle) are transferred by
NADH to flavoprotein, the first molecule of the electron transport chain. This molecule is
a prosthetic group called flavin mononucleotide
o In the next redox reaction, the flavoprotein returns to its oxidized form as it passes
electrons to an iron-sulfur protein (FeS ), one of a family of proteins with both iron-sulfur
tightly bound.
o The iron-sulfur protein then passes the electrons to ubiquinone (Q ). This electron carrier
is a lipid, the only member of the electron transport chain that is not a protein.
o Most of the remaining electron carriers between Q and O2 are proteins called
cytochromes (cyt).
 o Another source of electrons for the transport chain is FADH2 (the other reduced product
of the Krebs cycle) adds its electrons to the electron transport chain at a lower energy
level than NADH does.
Anaerobic Respiration
o A form of respiration in which oxygen is absent and alternative electron acceptors are
reduced
o Some of the electron acceptors used in anaerobic respiration include nitrate (NO3), ferric
iron (Fe3+), sulfate (SO42), carbonate (CO32), and even certain organic compounds
o Because of their positions on the redox tower, less energy is released when these electron
acceptors are used instead of oxygen
o Anaerobic respirations are very important for supporting the growth of prokaryotes in
anoxic environments
Fermentation
o Fermentation consists of glycolysis plus reactions that regenerate NAD+ by transferring
electrons from NADH to pyruvate or derivatives of pyruvate.
o The NAD+ can then be reused to oxidize sugar by glycolysis, which nets 2 ATP by substrate-
level phosphorylation
o There are many types of fermentation, differing in the waste products formed from
pyruvate. Two common types are alcohol fermentation and lactic acid fermentation.
Energy Conservation
o Two reaction series are linked to energy conservation in chemoorganotrophs:
fermentation and respiration
Fermentation: Anaerobic catabolism in which an organic compound is both an
electron donor and an electron acceptor, and ATP is produced by substrate-level
phosphorylation
Respiration: The process in which a compound is oxidized with O2 (or an O2
substitute) as the terminal electron acceptor, usually accompanied by ATP
production by oxidative phosphorylation
o In both forms of energy conservation, ATP synthesis is coupled to energy release in
oxidationreduction reactions
o However, the redox reactions that occur in fermentation and respiration differ
significantly.
In fermentation the redox process occurs in the absence of exogenous electron
acceptors.
In respiration, on the other hand, molecular oxygen or some other exogenous
electron acceptor serves as a terminal electron acceptor.
o Fermentation and respiration differ in the mechanism by which ATP is synthesized
o In fermentation, ATP is produced by substrate-level phosphorylation. In this process, ATP
is synthesized directly from an energy-rich intermediate during steps in the catabolism of
the fermentable compound
o In oxidative phosphorylation, ATP is produced at the expense of the proton motive force.
Biosynthesis of Sugars and Polysaccharides
o In Bacteria, the peptidoglycan cell wall has a polysaccharide backbone
 o In addition, cells often store carbon and energy reserves in the form of the polysaccharides
glycogen or starch
o Ribose and deoxyribose, present in the backbone of RNA and DNA
o In prokaryotes, polysaccharides are synthesized from either uridine diphosphoglucose
(UDPG) or adenosine-diphosphoglucose (ADPG), both of which are activated forms of
glucose
Biosynthesis of Amino Acids and Nucleotides
o The monomeric constituents of proteins and nucleic acids are amino acids and nucleotides,
respectively.
o Their biosyntheses are often long, multistep pathways requiring many individual enzymes
to complete.
o The amino group of amino acids is typically derived from some inorganic nitrogen source
in the environment, such as ammonia (NH3).
o Purine is the precursor of the purine nucleotides adenine and guanine. Pyrimidine is the
precursor of the pyrimidines thymine, cytosine, and uracil
Biosynthesis of Fatty Acids and Lipids
o Fatty acids are biosynthesized two carbon atoms at a time with the help of a protein called
acyl carrier protein (ACP).
o Each two-carbon unit is donated from the three-carbon compound malonate, which is
attached to the ACP to form malonyl-ACP.
o As each malonyl residue is donated, one molecule of CO2 is released
Lipids
o In the final assembly of lipids in Bacteria and Eukarya, fatty acids are added to glycerol.
o For simple triglycerides (fats), all three glycerol carbons are esterified with fatty acids.
o In complex lipids, one of the glycerol carbon atoms contains a molecule of phosphate,
ethanolamine, a sugar, or some other polar substance
Regulation of Activity of Biosynthetic Enzymes
o There are hundreds of different enzymatic reactions that occur in these anabolic
processes, and many of the enzymes that carry these out are highly regulated.
o There are two major modes of enzyme regulation, one that controls the amount (or even
the complete presence or absence) of an enzyme in the cell and another that controls the
activity of a preexisting enzyme by temporarily inactivating the protein.
Feedback Inhibition
o A major mechanism for the control of enzymatic activity is feedback inhibition.
o Feedback inhibition is a mechanism for turning off the reactions in an entire biosynthetic
pathway, such as a biosynthetic pathway of an amino acid or nucleotide.
Isoenzymes
o Some biosynthetic pathways controlled by feedback inhibition employ isoenzymes (iso
means same).
o Isoenzymes are different enzymes that catalyze the same reaction but are subject to
different regulatory controls.
Enzyme Regulation by Covalent Modification
o Some biosynthetic enzymes are regulated by covalent modification, typically by attaching
or removing some small molecule to the protein.
o Common modifiers include the nucleotides adenosine monophosphate (AMP) and
adenosine diphosphate (ADP), inorganic phosphate (PO42), and methyl (CH3) groups.