Lect. 3 (microbial nutrition and cultivation)

Microbial Growth Conditions1. Macronutrients

2. Micronutrients

3. Growth factors

4. Environmental factors: temperature; pH; Oxygen etc.

Nutrients: Substances in the environment used by organisms for catabolism and anabolism.

1. Macronutrients: required in large amounts, including: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus (Components of carbonhydrates, lipids, proteins, and mucleic acids ); potassium, calcium, magnesium and iron (cations and part of enzymes and cofactors).

2. Micronutrients: Microbes require very small amounts of other mineral elements, such as iron, copper, molybdenum, and zinc; these are referred to as trace elements. Most are essential for activity of certain enzymes, usually as cofactors.

Microorganisms require about ten elements in large quantities, because they are used to construct carbohydrates, lipids, proteins, and nucleic acids. Several other elements are needed in very small amounts and are parts of enzymes and cofactors.

Nutrient requirementsNutrient requirements

Amino acids are needed for protein synthesis,

purines and pyrimidines for nucleic acid synthesis.

Vitamins are small organic molecules that usually make up all or part enzyme cofactors, and only very small amounts are required for growth.

The Common Nutrient Requirements

Macroelements (macronutrients)• C, O, H, N, S, P, K, Ca, Mg, and Fe• required in relatively large amounts

Micronutrients (trace elements)• Mn, Zn, Co, Mo, Ni, and Cu• required in trace amounts• often supplied in water or in media components

Microbial NutritionMicrobial Nutrition

A.A. Nutrient RequirementsNutrient Requirements

B.B. Nutritional types of microorganisms Nutritional types of microorganisms (Nutritional categories)(Nutritional categories)

C.C. Nutrient Transport Processes Nutrient Transport Processes ((How do How do nutrient get into the microbial cell?)nutrient get into the microbial cell?)

D.D. Culture Media Culture Media (How to cultivate (How to cultivate microorganisms?)microorganisms?)

E.E. Isolation of Pure CulturesIsolation of Pure Cultures

Autotroph and Heterotroph

All organisms require Carbon, Hydrogen, and Oxygen. Carbon is needed for the backbone of all organic molecules.

In addition all organisms require a source of electrons. Electrons are involved in oxidation-reduction reactions in the cell, electron transport chains, and pumps that drive molecules against a concentration gradient on cell membranes.

Organic molecules that supply, carbon, hydrogen, and oxygen are reduced and donate electrons for biosynthesis.

Autotrophs

CO2 is used by many microorganisms as the source of Carbon.

Autotrophs have the capacity to reduce it , to form organic molecules.

Photosynthetic bacteria and microalgae are Photoautotrophs that are able to fix CO2 and use light as their energy source.

Heterotrophs

Organisms that use organic molecules as their source of carbon are Heterotrophs. The most common heterotrophs use organic compounds for both energy and their source of carbon.

Microorganisms are versatile in their ability to use diverse sources of carbon. Burkholderia cepacia can use over 100 different carbon compounds.

Methylotrophic bacteria utilize methanol, methane, and formic acid.

Requirements for Nitrogen, Phosphorus, and Sulfur

Needed for synthesis of important molecules (e.g., amino acids, nucleic acids)

Nitrogen supplied in numerous ways Phosphorus usually supplied as inorganic phosphate Sulfur usually supplied as sulfate via assimilatory

sulfate reduction

Requirements for Nitrogen

Nitrogen is required for the synthesis of amino acids that compose the structure of proteins, purines and pyrimidines the bases of both DNA and RNA, and for other derivative molecules such as glucosamine.

Many microorganisms can use the nitrogen directly from amino acids. The amino group ( NH2) is derived from ammonia through the action of enzymes such as glutamate dehydrogenase.

Most photoautotrophs and many nonphotosynthetic microorganisms reduce nitrate to ammonia and assimilate nitrogen through nitrate reduction. A variety of bacteria are involved in the nitrogen cycle such as Rhizobium which is able to use atmospheric nitrogen and convert it to ammonia. ( Found on the roots of legumes like soy beans and clover) These compounds are vital for the Nitrogen cycle and the incorporation of nitrogen into plants to make nitrogen comounds.

Sources of nitrogen

Organic nitrogenOrganic nitrogen• Primarily from the catabolism of amino acids

OxidizedOxidized forms of inorganicinorganic nitrogen• Nitrate (NO

32-) and nitrite (NO

2-)

Reduced inorganic Reduced inorganic nitrogen• Ammonium (NH

4+)

Dissolved nitrogen gas Dissolved nitrogen gas (N2) (Nitrogen

fixation)

Requirements for Phosphorous

Phosphorous is present in phospholipids( membranes), Nucleic acids( DNA and RNA), coenzymes, ATP, some proteins, and other key cellular components.

Inorganic phosphorous is derived from the environment in the form of phosphates. Some microbes such as E. coli can use organophosphates such as hexose – 6-phosphates .

Phosphate sourcePhosphate source• Organic phosphate

• Inorganic phosphate (H2PO4- and HPO4

2-)

Sulfur sourceSulfur source

• Organic sulfur

• Oxidized inorganic sulfur

• Sulfate (SO42-)

• Reduced inorganic sulfur

• Sulfide (S2- or H2S)

• Elemental sulfur (So)

Requirements for Sulfur

Synthesis of proteinSynthesis of protein Synthesis of vitaminsSynthesis of vitamins Involved in cellular respiration and phtosynthesisInvolved in cellular respiration and phtosynthesis

Growth Factors (Growth Factors (Special requirements)Special requirements)

organic compounds essential cell components (or their

precursors) that the cell cannot synthesize must be supplied by environment if cell is

to survive and reproduce

e.ge.g..•Amino acids•Nucleotide bases•Enzymatic cofactors or “vitamins”

Classes of growth factors amino acids

• needed for protein synthesis purines and pyrimidines

• needed for nucleic acid synthesis vitamins

• function as enzyme cofactors

Practical importance of growth factors Development of quantitative growth-response assays for

measuring concentrations of growth factors in a preparation.

Industrial production of growth factors by microorganisms.

Nutritional types of microorganisms Nutritional types of microorganisms (Nutritional categories)(Nutritional categories)

Energy SourceEnergy Source• PhototrophPhototroph (Uses light as an energy source)• ChemotrophChemotroph (Uses energy from the oxidation of

reduced chemical compounds)

Electron (Reduction potential) SourceElectron (Reduction potential) Source• OrganotrophOrganotroph (Uses reduced organic

compounds as a source for reduction potential)• LithotrophLithotroph (Uses reduced inorganic

compounds as a source for reduction potential)

• PrototrophPrototrophA species or genetic strain of microbe capable of growing on a minimal medium consisting a simple carbohydrate or CO2 carbon source, with inorganic sources of all other nutrient requirements

• AuxotrophAuxotrophA species or genetic strain requiring one or more complex organic nutrients (such as amino acids, nucleotide bases, or enzymatic cofactors) for growth

Chemical energy – source organic, Inorganic H/e- donor, Organic carbon source

• MixotrophyMixotrophy

Carbon sourceCarbon source• AutotrophAutotroph (Can use CO

2 as a sole carbon source)

• HeterotrophHeterotroph (Requires an organic carbon source; cannot use CO

2 as a carbon source)

Photolithotrophic autotrophs

Use light energy and have CO2 as their carbon source.

Cyanobacteria uses water as the electron donor and release oxygen

Purple and green sulfur bacteria use inorganic donors like hydrogen and hydrogen sulfide for electrons

Chemoorganotrophic heterotrophs

Use organic compounds as sources of energy,hydrogen, electrons and carbon

Pathogenic organisms fall under this category of nutrition

Photoorganoheterotrophs Common inhabitants of polluted streams. These bacteria

use organic matter as their electron donor and carbon source.

They use light as their source of energy Important ecological organisms

Chemolithotrophic autotrophs Autotrophs Oxidize reduce inorganic compounds such as iron,

nitrogen, or sulfur molecules Derive energy and electrons for biosynthesis Carbon dioxide is the carbon source

Uptake of Nutrients by the Cell

Some nutrients enter by passive(simple) diffusion Most nutrients enter by:

• facilitated diffusion• active transport• group translocation

1. Phagocytosis – Protozoa

2. Permeability absorption – Most microorganisms

C. Nutrient Transport Processes C. Nutrient Transport Processes How do nutrient get into the microbial cell?How do nutrient get into the microbial cell?

D. Culture MediaD. Culture MediaHow to cultivate microorganisms?How to cultivate microorganisms?

preparations devised to support the growth (reproduction) of microorganisms

can be liquid or solid

• Liquid medium• Components are dissolved in water and sterilized

• Semisolid medium• A medium to which has been added a gelling agent• Agar (most commonly used)• Gelatin• Silica gel (used when a non-organic gelling agent is

required)

• solid media are usually solidified with agar

important to study of microorganisms

Microbiological MediaMicrobiological Media Chemically defined vs. complex mediaChemically defined vs. complex media

• Chemically defined media• The exact chemical composition is known

• e.g. minimal media used in bacterial genetics experiments

• Complex media• Exact chemical composition is not known

• Often consist of plant or animal extracts, such as soybean meal, milk protein, etc.

• Include most routine laboratory media, e.g., tryptic soy broth

Synthetic or Defined Media

all components and their concentrations are known

Complex Media

contain some ingredients of unknown composition and/or concentration

Some media components

peptones• protein hydrolysates prepared by partial digestion of

various protein sources extracts

• aqueous extracts, usually of beef or yeast agar

• sulfated polysaccharide used to solidify liquid media

Types of Media

General purpose media• support the growth of many microorganisms

• e.g., tryptic soy agar

Enriched media• general purpose media supplemented by blood or other

special nutrients

• e.g., blood agar

Types of media…Types of media…

Selective media• Favor the growth of some microorganisms and

inhibit growth of others• MacConkey agar

• selects for gram-negative bacteria

• Inhibits the growth of gram-positive bacteria

Types of media…

Differential media• Distinguish between different groups of

microorganisms based on their biological characteristics

• Blood agar• hemolytic versus nonhemolytic bacteria

• MacConkey agar• lactose fermenters versus nonfermenters

Streak plate method• Developed in the 1870s by Koch and his co-workers

• The objective: to obtain isolated colonies – spots of microbial growth that come from a single parent cell

• The method: streak the sample on semisolid medium, containing a gelling agent

• Agar: the most commonly used gelling agent

E. Isolation of Pure CulturesE. Isolation of Pure Cultures

Spread plating & pour plating Limiting dilution

Pure Culture TechniquePure Culture Technique

The Spread Plate and Streak Plate

Involve spreading a mixture of cells on an agar surface so that individual cells are well separated from each other

Each cell can reproduce to form a separate colony (visible growth or cluster of microorganisms)

1. dispense cells ontomedium in petri dish

2. - 3. sterilize spreader

4. spread cellsacross surface

Spread-plate technique

inoculatingloop

Streak plate technique

Isolation of Pure Cultures

Pure culture• population of cells arising from a single cell

Spread plate, streak plate, and pour plate are techniques used to isolate pure cultures

The Pour Plate

Sample is diluted several times

Diluted samples are mixed with liquid agar

Mixture of cells and agar are poured into sterile culture dishes

Colony growth

Most rapid at edge of colony• oxygen and nutrients are more available at edge

Slowest at center of colony In nature, many microorganisms form

biofilms on surfaces

Terms

1. Colony shape and size: round, irregular, punctiform (tiny)2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed)3. Elevation: convex, umbonate, flat, raised4. Color: color or pigment, plus opaque, translucent, shiny or dull5. Texture: moist, mucoid, dry (or rough).

Preserving Bacterial Cultures

Deep-freezing: –50° to –95°C Lyophilization (freeze-drying): Frozen (–

54° to –72°C) and dehydrated in a vacuum