Nutrition and Cultivation of Microorganisms

Bacterial Nutrition

Nutrition- the kind of food used and the methods by which it is

assimilated and utilized

Two-fold purpose:1. to synthesize (build up) protoplasm 2. to supply energy for all life processes

Microorganisms require nutrients like:1. Carbon source

a. Heterotrophs- microbes that used organic cpdsas major source of C

- obtain such organic molecules by absorbing them from the environment, orby ingesting autotrophs or other heterotrophs

b. Autotrophs- microbes that used carbon dioxide (mostoxidized form of carbon) as their major or evensole source of carbon

-can live exclusively on relatively simple inorganic molecules and ions absorbed from the environment

2. Nitrogen Source3. Certain inorganic ions4. Essential metabolites (vitamins; possibly amino acids)5. Water

*Differences between the nutrition of animals and that of bacteria1. Holozoic nutrition –wherein solid food is ingested by the

organism (eg. Typical of animals and protozoa)2. Holophytic – bacteria cannot ingest solid food as well as

true fungi so they must receive their nutrients in asolution of water, which means digestion must take place outside the organism.

Some nutrients, called MACRONUTRIENTS, arerequired in large amounts, like C, H, N, O

- needed by cell but in small amounts (P, S, K,Mg, Ca, Na)

while others MICRONUTRIENTS, are required in just trace amounts. - Iron and Other Trace Metals

*siderophores –produce by cell as iron-binding molecules

- function to bind Fe3+ and transport it into the cell. A major group of siderophores is the hydroxamic acids, organic molecules that chelate Fe3+ strongly.

Type Function Examples

Carbon Source Provides energy through oxidation and provides the structural components of the cell wall

May include virtually any carbon containing compound; varies from CO2 in the air to very complex organic substances

Nitrogen Source Provides nitrogen for the synthesis of amino acids, nucleic acids and coenzymes

Some species use N2 of air, others inorganic compounds such as NO3

- or NH4+; others

require organic sources of nitrogen such as glutamine or asparagine

Inorganic ions Necessary cofactors for enzymes; storage of energy; electron transport system

Mg2+, Mn2+, Fe2+, PO42-, Na+,

K+ and even Mo for organisms fixing gaseous nitrogen

Essential metabolites To provide complex organic compounds which an organism is unable to synthesize

Vitamins, amino acids, purines, pyrimidines, coenzymes, heme

Table 4 Summary of Bacterial Nutrients

Nutritional Classification of MicroorganismsChemotrophs – organisms that use chemical compounds for energyAutotrophs - organisms that depend primarily on radiant energy

(light)*by combining these terms with those of C sources, the following

groupings emerge1. Chemoautotrophs – organisms use chemical substances (inorganic)

as source of energy + carbon dioxide as the main source ofcarbon

2. Chemoheterotrophs- organisms that use chemical substance(organic) as source of energy + organic compounds as themain source of carbon

3. Photoautotrophs – organisms that use light (E source) + CO2 (carbonsource)

4. Photoheterotrophs- organisms use light (E source)+ organic cpds (carbon source)

Nutritional Group Carbon source Energy source Examples

Chemoautotrophs Carbon dioxide Inorganic compounds

Nitrifying, hydrogen, iron and sulfur bacteria

Chemoheterotrophs Organic compounds Organic compounds Most bacteria, fungi, protozoa and animals

Photoautotrophs Carbon dioxide Light Purple sulfur and Green sulfur bacteria, algae, cyanobacteria and plants

Photoheterotrophs Organic compounds light Purple nonsulfur and green nonsulfur bacteria

Table 4.1 Nutritional Classification of Bacteria and other organisms

*some species of microorganisms are versatile in their nutritional needs; they cannot be categorized exclusively into one of the four groupings (eg. Certain phototrophic bacteria can also grow as chemotrophs)

in the absence of oxygen (anaerobic conditions), Rhodospirillum rubrum depends on light as its energy source and lives a photoheterotrophs but in the presence of oxygen (aerobic conditions), it can grow in the dark as a chemoheterotroph

*one organism can help another to grow

-when 1 organism produces a waste product that another organism uses as food (eg. The good relationship of three species of bacteria- Streptococcus thermophilus, Lactobacillus bulgaricus, andPropionibacterium shermanii –used in the manufacture of Swiss cheese; strptococci and lactobacilli ferment the lactose in milk and produce the lactic acid as waste product and the propionibacteria can then grow on lactic acid to produce propionic acid as their waste which gives the characteristic nutlike flavor of Swiss cheese

-Two organisms can benefit equally when each makes an essential nutrient required by the other (Bacillus polymyxa and Proteus vulgaricus will not grow in lab culture medium lacking the vitamins niacin and biotin but they can grow together in such medium as a mixed culture because B. polymyxa makes the niacin required by the P. vulgaricus and P. vulgaricus makes the biotin needed bythe B. polymyxa)

-relationship between the bacteria rhizobia and leguminous plants

Media used for cultivating microorganismsChemically defined media

-used to determine the precise nutritional requirements of amicroorganism

Bacterial strain 2 –prototrophic (it does not require organic supplements since it can grow on the minimal medium plate “control”)

Bacterial strains 1 & 3- auxotrophic (need organic supplements in the minimal medium before they can grow (no growth on the “control” plate)

*Control medium has glucose and salts only

Biosynthesis of tryptophan:Minimal medium anthranilic acid indole tryptophan ingredients

A B C

For routine lab cultivation abc study of heterotrophs, complex culture media prepared from natural products are used, such media are chemically undefined.

Examples of natural products added to media include:1. Meat extract(an aqueous beef extract concentrated to a paste)2. peptone (proteins that have been partially degraded by enzymes,

milk-casein hydrolysate and soybean-protein hydrolysate)3. yeast extract4. blood serum5. milk6. soil extract7. bovine rumen fluid

*there are media that are commercially available

Fig 4. Different commercially prepared agar media in Petri dishes are shown streaked with bacteria to obtain isolated colonies

API 20E System -for identificationof bacteria in the Enterobacteriaceae family and Gram- bacteria

0.85% NaCl Saline suspension of E. coli cells

Media for the Growth of Bacteria

A. Chemically defined medium for a chemoautotrophic bacteriumIngredient Function Amount

(NH4)2SO4 Nitrogen as well as energy source

0.5 g

NaHCO3 Carbon source in the form of CO2 in aqueous solution

0.5 g

Na2HPO4 Buffer and essential ions 13.5 g

KH2PO4 Buffer and essential ions 0.7 g

MgSO4.7H2O Essential ions 0.1 g

FeCl.6H2O Essential ions 0.014 g

CaCl2.2H2O Essential ions 0.18 g

water Solvent 1000 ml

The 3M™ Petrifilm™ E.coli/Coliform Count Plate identifies both E. coli and other coliforms with confirmed results in just 24-48 hours.

3M™ Petrifilm™ Aerobic Count Plates

B. Chemically Defined Medium for a Heterotrophic bacterium

Ingredient Function Amount

Glucose Carbon and energy source 1 g

NH4H2PO4 Nitrogen source, buffer and essential ions

5 g

K2HPO4 Buffer and essential ions 1 g

NaCl Essential ions 5 g

MgSO4.7H2O Essential ions 0.2 g

water solvent 1000 ml

*The above ingredients represent the minimum constituents in a medium for anonfastidious bacterium such as the wildtype E. coli. For a fastidious speciessuch as Lactobacillus acidophilus, additional substances such as amino acids andvitamins have to be added to the medium

Solid and Liquid Culture Media

C. Composition of Nutrient Broth, a complex medium for the growth of heterotrophic bacteria

Ingredient Function Amount

Beef extract Water-soluble substances of animal tissue; carbohydrates, organic nitrogen compounds, vitamins, salts

3 g

Peptone Organic nitrogen 5 g

Sodium chloride Ions and osmotic requirements

8 g

Water solvent 1000 ml

•If a solid medium is required, agar (15 g) is added, the medium is then called nutrient agar

Media for the Growth of Fungi- Have higher sugar concentration (4%) and a lower pH range(3.8 to 5.6) than mediaFor bacterial growth which is generally pH 6.5 to 7.5.

D. Composition of a General Purpose Medium, Sabouraud’s Agar for the isolation andgrowth of fungi

Ingredient Function Amount

Peptone Source of Carbon, nitrogen elements

10 g

Glucose Carbon and energy source; high concentration favors growth of fungi but inhibits growth of bacteria

40 g

Agar Solidifying agent 15 g

water solvent 1000 ml

pH Low pH suppresses bacterial growth but enhances fungal growth

5.6

Media for the Growth of the Protozoa-have pH range of 6 to 8 for optimal growth-protozoa are aerobic heterotrophs with complex nutritional requirements

Media for the Growth of Algae - algae use light for energy and require only carbon dioxide, water and various

soluble inorganic ions for growth (they are photoautotrophs) -some undefined media for algae usually contain supplements such as soil

extract, a rich source of nutrients Other types 1. Special-Purpose media

-when microbiologists want to isolate, identify or count microbesa. Media for Anaerobes

anaerobes( organisms that tolerate little or no oxygen and do notuse oxygen to obtain energy)

-for years, anaerobic bacteria were grown in agar medium deeps or media in tall test tubes(bacteria could grow in the bottom of these tubes

bec. the top layer of agar excluded atmospheric O2; other refinementsincluded the addition of Sodium thioglycolate

Tube 1 = strict aerobeTube 2 = facultative Tube 3 = aerotolerantTube 4 = strict anaerobe

Sodium thioglycolate –reducing agent (that would remove oxygen to makewhat we called reduced media)

2. Selective media- are designed to enhance the growth of particular kind of microorganism

or suppress the growth of other kinds of microorganisms(some may do both)

eg. Sabouraud’s agar

3. Differential Media-microbiologists use differential media when they want to differentiate

among various kinds of microorganisms on an agar plate

4. Selective/Differential-some culture media are both selective and differential.eg. MacConkey agar which contains bile salt and crystal violet dye to inhibit the growth of Gram+ bacteria and allow Gram_ bacteria to grow

5. Enrichment Media-when a species of special interest are present but only in very small

numbers, microbiologists use this medium. The medium favors the growth

of that species, but not the growth of the others present in the mixed population

MacConkey’s agar-Selective for gram negative Bacteria (growth of G+ is inhi-bited by the crystal violet dye & bile salts in the media)

-differential for lactose fer-mentation(neutral red pHindicator turns red in thepresence of acid by-productsof lactose fermentation)

Microbiological Assay Media- specific microorganisms can be used to measure the concentrations of

Substances such as antibiotics and vitamins-blood serum or other tissue fluids can be assayed for antibiotics by using

Microorganisms known to be susceptible to those antibiotics. This type of assayInvolves the measurement of growth inhibition caused by antibiotic.

Tissue Culture Media and MethodsTissue cultures are plant or animal cells grown in the lab in specialized

media-methods were developed to cultivate viruses in vitro

Bacillus subtilis

gram positive, sporeforming rods produce colonies which are dry, flat, and irregular, with lobate margins.

Circular, pinhead colonies which are convex with entire margins. This gram positive coccus often produces colonies which have a golden-brown color.

Staphylococcus aureus.

Micrococcus luteus. Circular, pinhead colonies which are convex with entire margins. This gram positive coccus produces a bright yellow, non-diffusable pigment. Rhodospirillum rubrum. Pinpoint circular colonies which are convex with entire margins. This gram negative spirillum produces a non-diffusable red pigment. Serratia marcescens. These gram negative rods produce mucoid colonies which have entire margins and umbonate elevation. Note that there are both red and white colonies present on this plate. Some strains of S. marcescens produce the red pigment prodigiosin in response to incubation at 30o C, but do not do so at 37o C. This is an example of temperature-regulated phenotypic expression.

Escherichia coli. This gram negative rod (coccobacillus) forms shiny, mucoid colonies which have entire margins and are slightly raised. Older colonies often have a darker center. Enterobacter aerogenes. This gram negative rod is a common contaminant of vegetable matter which forms shiny colonies with entire margins and convex elevation.

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