Microbial Nutrition

Microbial Nutrition

 macroelements or macronutrients--carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron

 micronutrients or trace elements---—manganese, zinc, cobalt, molybdenum, nickel, and copper(Micronutrients are normally a part of enzymes and cofactors, and they aid in the catalysis of reactions and maintenance of protein structure)
 zinc (Zn2+) is present at the active site of some enzymes but is also involved in the association of regulatory and catalytic subunits in E. coli aspartate carbamoyltransferase.
 Manganese (Mn2+) aids many enzymes catalyzing the transfer of phosphate groups.
 Molybdenum (Mo2+) is required for nitrogen fixation.
 cobalt (Co2+) is a component of vitamin B12

Particular requirements

 Diatoms need silicic acid (H4SiO4) to construct their beautiful cell walls of silica [(SiO2)n].
 Although most bacteria do not require large amounts of sodium, many bacteria growing in saline lakes and oceans (see pp. 123, 461) depend on the presence of high concentrations of sodium ion (Na+).

Requirements for Carbon, Hydrogen,and Oxygen

 Carbon is needed for the skeleton or backbone of all organic molecules, and molecules serving as carbon sources normally also contribute both oxygen and hydrogen atoms

 They are the source of all three(C,H,O) elements. Because these organic nutrients are almost always reduced and have electrons that they can donate to other molecules, they also can serve as energy sources.
 Indeed, the more reduced organic molecules are, the higher their energy content (e.g., lipids have a higher energy content than carbohydrates).

 electron transfers release energy when the electrons move from reduced donors with more negative reduction potentials to oxidized electron acceptors with more positive potentials.

 One important carbon source that does not supply hydrogen or energy is carbon dioxide (CO2). This is because CO2 is oxidized and lacks hydrogen.

 only autotrophs can use CO2 as their sole or principal source of carbon

 The reduction of CO2 is a very energy-expensive process. Thus many microorganisms cannot use CO2 as their sole carbon source but must rely on the presence of more reduced, complex molecules such as glucose for a supply of carbon

 most heterotrophs use reduced organic compounds as sources of both carbon and energy.
 For example, the glycolytic pathway produces carbon skeletons for use in biosynthesis and also releases energy as ATP and NADH.

aboratory experiments indicate that there is no naturally occurring organic molecule that cannot be used by some microorganism.
• Actinomycetes will degrade amyl alcohol, paraffin, and even rubber.

• Burkholderia cepacia can use over 100 different carbon compounds.
• some bacteria are exceedingly fastidious and catabolize only a few carbon compounds. Cultures of methylotrophic bacteria metabolize methane, methanol, carbon monoxide, formic acid, and related one-carbon molecules.


• Parasitic members of the genus Leptospira use only long-chain fatty acids as their major source of carbon and energy.


Indigestible molecules sometimes are oxidized and degraded in the presence of a growthpromoting nutrient that is metabolized at the same time, a process called cometabolism .The products of this breakdown process can then be used as nutrients by other microorganisms.











Nutritional Types of Microorganisms

Carbon Sources


Autotrophs CO2 sole or principal biosynthetic carbon source

Heterotrophs Reduced, preformed, organic molecules from other organisms

Energy Sources
Phototrophs Light
Chemotrophs Oxidation of organic or inorganic compounds

Electron Sources
Lithotrophs Reduced inorganic molecules
Organotrophs Organic molecules

Major Nutritional Types of Microorganisms



Major Nutritional Types Sources of Energy, Hydrogen/Electrons, and Carbon Representative Microorganisms
Photolithotrophic autotrophy

(Photolithoautotrophy) • Light energy
• Inorganic hydrogen/electron (H/e–) donor Purple and green sulfur bacteria CO2 carbon source • Algae
• Purple and green sulfur bacteria
• Cyanobacteria
Photoorganotrophic heterotrophy (Photoorganoheterotrophy) • Light energy

• Organic H/e– donor Organic carbon source (CO2 may also be used) • Purple nonsulfur bacteria

• Green nonsulfur bacteria
Chemolithotrophic autotrophy (Chemolithoautotrophy) • Chemical energy source (inorganic)

• Inorganic H/e– donor CO2 carbon source • Sulfur-oxidizing bacteria Hydrogen bacteria

• Nitrifying bacteria Iron-oxidizing bacteria
Chemoorganotrophic heterotrophy
(Chemoorganoheterotrophy) • Chemical energy source (organic

• Organic H/e– donor Organic carbon source • Protozoa
• Fungi
• Most nonphotosynthetic bacteria (including most pathogens)


 Eucaryotic algae and cyanobacteria employ water as the electron donor and release oxygen

 Purple and green sulfur bacteria cannot oxidize water but extract electrons from inorganic donors like hydrogen, hydrogen sulfide, and elemental sulfur.

 It should be noted that essentially all pathogenic microorganisms are chemoheterotrophs.

 These photoorganotrophic heterotrophs (photoorganoheterotrophs) are common inhabitants of polluted lakes and streams. Some of these bacteria also can grow as photoautotrophs with molecular hydrogen as an electron donor.


 The chemolithotrophic autotrophs (chemolithoautotrophs), oxidizes reduced inorganic compounds such as iron, nitrogen, or sulfur molecules to derive both energy and electrons for biosynthesis.

 Chemolithotrophs contribute greatly to the chemical transformations of elements (e.g., the conversion of ammonia to nitrate or sulfur to sulfate) that continually occur in the ecosystem.


Some microorganism changes their mode of nutrition on course of environment

 many purple nonsulfur bacteria act as photoorganotrophic heterotrophs in the absence of oxygen but oxidize organic molecules and function chemotrophically at normal oxygen levels. When oxygen is low, photosynthesis and oxidative metabolism may function simultaneously.

 bacteria such as Beggiatoa that rely on inorganic energy sources and organic (or sometimes CO2) carbon sources. These microbes are sometimes called mixotrophic because they combine chemolithoautotrophic and heterotrophic metabolic processes.




Requirements for Nitrogen, Phosphorus, and Sulfur


 Many microorganisms can use the nitrogen in amino acids, and ammonia often is directly incorporated through the action of such enzymes as glutamate dehydrogenase or glutamine synthetase and glutamate synthase

 Almost all microorganisms use inorganic phosphate as their phosphorus source and incorporate it directly.

 Low phosphate levels actually limit microbial growth in many aquatic environments.

 Phosphate uptake by E. coli has been intensively studied. This bacterium can use both organic and inorganic phosphate.

 Some organophosphates such as hexose 6-phosphates can be taken up directly by transport proteins.

 Other organophosphates are often hydrolyzed in the periplasm by the enzyme alkaline phosphatase to produce inorganic phosphate, which then is transported across the plasma membrane.

 At high phosphate concentrations, transport probably is due to the Pit system. When phosphate concentrations are low, the PST, (phosphate-specific transport) system is more important.

 The PST system has higher affinity for phosphate; it is an ABC transporter and uses a periplasmic binding protein.

 Sulfur is needed for the synthesis of substances like the amino acids cysteine(reduced form) and methionine, some carbohydrates, biotin, and thiamine.

Growth Factors:

 There are three major classes of growth factors:
(1) amino acids,
(2) purines and pyrimidines
(3) vitamins.


 Other growth factors are also seen; heme (from hemoglobin or cytochromes) is required by Haemophilus influenzae, and some mycoplasmas need cholesterol

Functions of Some Common Vitamins in Microorganisms

Vitamin Functions
Biotin Carboxylation (CO2 fixation) One-carbon metabolism
Cyanocobalamin (B12) Molecular rearrangements One-carbon metabolism—carries methyl groups
Folic acid One-carbon metabolism
Lipoic acid Transfer of acyl groups
Pantothenic acid Precursor of coenzyme A—carries acyl groups
(pyruvate oxidation, fatty acid metabolism)
Pyridoxine (B6) Amino acid metabolism (e.g., transamination)
Niacin (nicotinic acid) Precursor of NAD and NADP—carry electrons
and hydrogen atoms
Riboflavin (B2) Precursor of FAD and FMN—carry electrons or hydrogen atoms
Thiamine (B1) Aldehyde group transfer (pyruvate decarboxylation, -keto acid oxidation)



 Good examples of such vitamins and the microorganisms that synthesize them are:
• Riboflavin by (Clostridium, Candida, Ashbya, Eremothecium)
• coenzyme A by(Brevibacterium)
• vitamin B12 by (Streptomyces, Propionibacterium, Pseudomonas)
• vitamin C by (Gluconobacter, Erwinia, Corynebacterium)
• B-carotene (Dunaliella)
• vitamin D (Saccharomyces).


Uptake of Nutrients by the Cell
1. facilitated diffusion,
2. active transport,
3. group translocation.

Eucaryotic microorganisms do not appear to employ group translocation but take up nutrients by the process of endocytosis .

A. Passive diffusion
 Passive diffusion, often simply called diffusion, is the process in which molecules move from a region of higher concentration to one of lower concentration because of random thermal agitation.
 The rate of passive diffusion is dependent on the size of the concentration gradient between a cell’s exterior and its interior
 A fairly large concentration gradient is required for adequate nutrient uptake by passive diffusion (i.e., the external nutrient concentration must be high)
 Very small molecules such as H2O, glycerol,O2, and CO2 often move across membranes by passive diffusion.


 The rate of diffusion across selectively permeable membranes is greatly increased by using carrier proteins, sometimes called permeases, which are embedded in the plasma membrane.

• Because a carrier aids the diffusion process, it is called facilitated diffusion. The rate of facilitated diffusion increases with the concentration gradient much more rapidly and at lower concentrations of the diffusing molecule than that of passive diffusion

The two most widespread MIP- (major intrinsic protein) channels in bacteria are aquaporins that transport water and glycerol facilitators, which aid glycerol diffusion


B. Active Transport
 Active transport is the transport of solute molecules to higher concentrations, or against a concentration gradient, with the use of metabolic energy input.


 Binding protein transport systems or ATP-binding cassette transporters (ABC transporters) are active in bacteria, archaea, and eucaryotes.

 ABC transporters employ special substrate binding proteins, which are located in the periplasmic space of gram-negative bacteria) or are attached to membrane lipids on the external face of the gram-positive plasma membrane.

 These binding proteins, which also may participate in Chemotaxis

 E. coli transports a variety of sugars (arabinose, maltose, galactose, ribose) and amino acids (glutamate, histidine, leucine) by this mechanism.

 It should be noted that eucaryotic ABC transporters are sometimes of great medical importance.

 Some tumor cells pump drugs out using these transporters.
 Cystic fibrosis results from a mutation that inactivates an ABC transporter that acts as a chloride ion channel in the lungs.

 linked transport of two substances in the same direction is called symport.(lactose &H+ in E.coli)

 linked transport in which the transported substances move in opposite directions is termed antiport.(Na ion &H+ in E.coli))

C. Group Translocation

 A process in which a molecule is transported into the cell while being chemically altered (this can be classified as a type of energy-dependent transport because metabolic energy is used).


 The best-known group translocation system is the phosphoenolpyruvate: sugar phosphotransferase system (PTS).

 It transports a variety of sugars into procaryotic cells while phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor

PEP +sugar (outside) pyruvate + sugar +P (inside)

 The PTS is quite complex. In E. coli and Salmonella typhimurium, it consists of two enzymes and a low molecular weight heat-stable protein (HPr). HPr and enzyme I (EI) are cytoplasmic. Enzyme II (EII) is more variable in structure and often composed of three subunits or domains.

 PTSs are widely distributed in procaryotes. Except for some species of Bacillus that have both glycolysis and the phosphotransferase system, aerobic bacteria seem to lack PTSs.
 Besides their role in transport, PTS proteins can act as chemoreceptors for chemotaxis


D. Iron Uptake

 Almost all microorganisms require iron for use in cytochromes and many enzymes.

 Iron uptake is made difficult by the extreme insolubility of ferric iron (Fe3+) and its derivatives, which leaves little free iron available for transport
 Many bacteria and fungi have overcome this difficulty by secreting siderophores [Greek for iron bearers].
 Siderophores are low molecular weight molecules that are able to complex with ferric iron and supply it to the cell.
 These iron-transport molecules are normally either hydroxamates or phenolatescatecholates.
 Ferrichrome is a hydroxamate produced by many fungi; enterobactin is the catecholate formed by E. coli






Culture Media

Synthetic or Defined Media
 Such a medium in which all components are known is a defined medium or synthetic medium.
 Media that contain some ingredients of unknown chemical composition are complex media.
 complex media often are needed because the nutritional requirements of a particular microorganism are unknown, and thus a defined medium cannot be constructed.
 This is the situation with many fastidious bacteria, some of which may even require a medium containing blood or serum.
 Complex media contain undefined components like peptones, meat extract, and yeast extract.

• Peptones are protein hydrolysates prepared by partial proteolytic digestion of meat, casein, soya meal, gelatin, and other protein sources.
o They serve as sources of carbon, energy, and nitrogen.
• Beef extract and yeast extract are aqueous extracts of lean beef and brewer’s yeast, respectively.
o Beef extract contains amino acids, peptides, nucleotides, organic acids, vitamins, and minerals.
• Yeast extract is an excellent sourceof B vitamins as well as nitrogen and carbon compounds.



Three commonly used complex media are :
(1) nutrient broth
(2) tryptic soy broth
(3) MacConkey agar.


 Agar is a sulfated polymer composed mainly of D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic acid. It usually is extracted from red algae.
 Agar is well suited as a solidifying agent because after it has been melted in boiling water, it can be cooled to about 40 to 42°C before hardening and will not melt again until the temperature rises to about 80 to 90°C.
 Agar is also an excellent hardening agent because most microorganisms cannot degrade it..
 Other solidifying agents are sometimes employed. For example, silica gel is used to grow autotrophic bacteria

 Types of Media

 Media such as tryptic soy broth and tryptic soy agar are called general purpose media because they support the growth of many microorganisms.

 Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious heterotrophs. These specially fortified media (e.g., blood agar) are called enriched media.

 Selective media favor the growth of particular microorganisms.

 Bile salts or dyes like basic fuchsin and crystal violet favor the growth of gram-negative bacteria by inhibiting the growth of gram-positive bacteria without affecting gram-negative organisms.
 Endo agar, eosin methylene blue agar, and MacConkey agarthree media widely used for the detection of E. coli and related bacteria in water supplies and elsewhere, contain dyes that suppress gram-positive bacterial growth.
 MacConkey agar also contains bile salts.

 Differential media are media that distinguish between differentgroups of bacteria and even permit tentative identification of microorganisms based on their biological characteristics.


 Blood agar is both a differential medium and an enriched one. It distinguishes between hemolytic and nonhemolytic bacteria.
 Hemolytic bacteria (e.g., many streptococci and staphylococci isolated from throats) produce clear zones around their colonies because of red blood cell destruction.

 MacConkey agar is both differential and selective. Since it contains lactose and neutral red dye, lactose-fermenting colonies appear pink to red in color and are easily distinguished from colonies of nonfermenters.

Isolation of Pure Cultures

 pure culture, a population of cells arising from a single cell, to characterize an individual species(development of pure culture techniques by the German bacteriologist Robert Koch).
1. The Spread Plate
2. Streak Plate
3. The Pour Plate----------Extensively used with bacteria and fungi, a pour plate also can yield isolated colonies. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating


 Generally the most rapid cell growth occurs at the colony edge. Growth is much slower in the center, and cell autolysis takes place in the older central portions of some colonies. These differences in growth appear due to gradients of oxygen, nutrients, and toxic products within the colony

 At the colony edge, oxygen and nutrients are plentiful. The colony center, of course, is much thicker than the edge. Consequently oxygen and nutrients do not diffuse readily into the center, toxic metabolic products cannot be quickly eliminated, and growth in the colony center is slowed or stopped. Because of these environmental variations within a colony, cells on the periphery can be growing at maximum rates while cells in the center are dying.

SUMMARY


• Microorganisms require nutrients, materials that are used in biosynthesis and energy production.

• Macronutrients or macroelements (C, O, H, N, S, P, K, Ca, Mg, and Fe) are needed in relatively large quantities; micronutrients or trace elements (e.g., Mn, Zn, Co, Mo, Ni, and Cu) are used in very small amounts.
• Autotrophs use CO2 as their primary or sole carbon source; heterotrophs employ organic molecules.
• Microorganisms can be classified based on their energy and electron sources.
• Phototrophs use light energy, and chemotrophs obtain energy from the oxidation of chemical compounds. Electrons are extracted from reduced inorganic substances by lithotrophs and from organic compounds by organotrophs.
• Nitrogen, phosphorus, and sulfur may be obtained from the same organic molecules that supply carbon, from the direct incorporation of ammonia and phosphate, and by the reduction and assimilation of oxidized inorganic molecules.
• Probably most microorganisms need growth factors. Growth factor requirements make microbiological assays possible.
• Although some nutrients can enter cells by passive diffusion, a membrane carrier protein is usually required.
• In facilitated diffusion the transport protein simply carries a molecule across the membrane in the direction of decreasing concentration, and no metabolic energy is required .
• Active transport systems use metabolic energy and membrane carrier proteins to concentrate substances actively by transporting them against a gradient. ATP is used as an energy source by ABC transporters. Gradients of protons and sodium ions also drive solute uptake across membranes .
• Bacteria also transport organic molecules while modifying them, a process known as group translocation. For example, many sugars are transported and phosphorylated simultaneously .
• Iron is accumulated by the secretion of siderophores, small molecules able to complex with ferric iron . When the ironsiderophore complex reaches the cell surface, it is taken inside and the iron is reduced to the ferrous form.
• Culture media can be constructed completely from chemically defined components (defined media or synthetic media) or may contain constituents like peptones and yeast extract whose precise composition is unknown (complex media).
• Culture media can be solidified by the addition of agar, a complex polysaccharide from red algae.
• Culture media are classified based on function and composition as general purpose media, enriched media, selective media, and differential media.
• Pure cultures usually are obtained by isolating individual cells with any of three plating techniques: the spread-plate, streak-plate, and pour-plate methods.
• Microorganisms growing on solid surfaces tend to form colonies with distinctive morphology. Colonies usually grow most rapidly at the edge where larger amounts of required resources are available.























JSR