Microbial Nutrition Gary Andersen Reference Chapter 2,5,6, 7, 8, 9 Microbiology by Tortora, Funke & Case Chemical Substances Metabolism Growth Cell.

Microbial Nutrition

Gary AndersenReference Chapter 2,5,6, 7, 8, 9 Microbiology by

Tortora, Funke & Case

ChemicalSubstances

Metabolism

Growth

Cell

What do microbes eat?

Composition and Nutrition of Cells

• 96% of cells are composed of HCNOPS.• Macronutrients are nutrients required in

larger quantities. (carbohydrates, proteins, fats and other CHO molecules)

• Micronutrients are nutrients required in trace amounts. (Mn, Zn, Cu, Ni)

Where are the Elements Used?

Sources of Nutrients

• Carbon – CO2 in air and rocks organisms• Nitrogen – N2 in air, NO2, NO3, NH4 in soil and

water organisms (NH3)• Oxygen – O2 in air, inorganic salts SO4, PO4, NO3,

H2O• Hydrogen – Water, Organic compounds in

organisms• Phosphorus – Rocks and minerals organisms• Sulfur – Rocks and minerals organisms

Microbe Nutritional Types• Autotrophs – “Self feeder”

– Photoautotrophs – Photosynthetic (energy from light)

– Chemoautotrophs – Energy from simple inorganic chemicals

• Methanogens – Metabolize H2 and CO2 into CH4 and H2O

• Heterotrophs – “Other feeder”– Chemoheterotroph – Obtain carbon and energy from

organic compounds. CnH2nOn + O2 CO2 + H2O + ATP (Adenosine tri-phosphate)

• Saprobe – Free living organisms that feed on dead organisms

• Parasite – Derive nutrients from the tissues of hosts.

Microbial Clean-Up: The 1989 Exxon Valdez oil spill left great quantities of pooled oil on sites in the Gulf of

Alaska, such as on Green Island

Microbial Clean-Up: Bioremediation in 1989, by the application of nutrients (nitrogen and phosphorus) to the shoreline accelerated the bacterial biodegradation of the oil into carbon dioxide and water

Microbial Clean-Up: In 1991, the area was surveyed and found to be mostly cleared of oil, with no further treatment recommended

How do Microbes Eat?

Transport Mechanisms• Passive Transport

– Diffusion – Movement of molecules from a high concentration to a low concentration.

– Facilitated Diffusion- diffusion assisted by conformational change in a protein molecule.

– Osmosis – Diffusion of water through a semipermeable membrane

• Active Transport – Moving particles against the diffusion gradient using membrane proteins and expending energy.– Endocytosis – Engulfing with cell membrane and forming a

vacuole.• Phagocytosis – Engulfing of cells or particles by the cell membrane• Pinocytosis – Engulfing of liquids by the cell membrane

Passive Transport• Osmosis animation:

http://www.tvdsb.on.ca/westmin/science/sbi3a1/Cells/Osmosis.htm

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Comparing Solutions• Hypertonic Solution – Higher solute concentration.

Cells in hypertonic solutions lose water and the cell membrane shrinks away from the cell wall. (Salt on a slug)

• Hypotonic Solution – Lower solute concentration. Cells in hypotonic solutions take on water and swell. (Prune wrinkles of skin)

• Isotonic Solutions – Solutions that have reached an equilibrium with a cell or another solution. The concentration of solute is equal and the diffusion of water proceeds at equal rates.

(See page 93 of text for what happens to a cell in hypertonicand hypotonic solutions.)

Animations of Passive and Active Transport

• http://programs.northlandcollege.edu/biology/Biology1111/animations/transport1.html

• http://highered.mcgraw-hill.com/olc/dl/120068/bio02.swf

How do microbes metabolize nutrients?

Fermentation and Respiration

Enzymes• Provide a surface on which reactions take place

• Active site: the area on the enzyme surface where the enzyme forms a loose association with the substrate

• Substrate: the substance on which the enzyme acts

• Enzyme-substrate complex: formed when the substrate molecule collides with the active site of its enzyme

• Enzymes generally have a high degree of specificity

• Endoenzymes (intracellular)/exoenzymes (extracellular)

The Action of Enzymes on Substrates to Yield Products

Each substrate binds to an active site, producing an enzyme-substrate complex. The enzyme helps

a chemical reaction occur, and one or more products are formed

Competitive Regulation and Inhibition of Enzymes

Noncompetitive (allosteric) inhibition of enzymes

http://highered.mcgraw-hill.com/olc/dl/120070/bio10.swf

Factors Influencing Enzymes

• Temperature

• pH

• Concentration of substrate, product, and enzyme

Relationship between temperature and enzyme activity

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Microbes and Environmental Factors

• Temperature• pH• Oxygen• PressureExtremophiles – Organisms that can survive under

extreme environmental conditions. An interesting source of chemical products.

Interesting Website on Extremophiles and Chemical Products: http://www.mediscover.net/Extremophiles.cfm

Temperature

Classification

• Psychrophile• Mesophile• Thermophile

Temperature

• Thermophiles – organisms that grow at >45 degrees C.

• Pyrococcus fumarii is an example of a thermophile that can survive at 113 C.

Thermal pool

Grand Prismatic Spring

pH

• Acidophiles – grow at low pH levels. (1-2)

• Alkalinophiles – live at high pH levels. (9-10)

14 Alkaline13121110987 Neutral6543210 Acidic

Pressure

• Barophiles – organisms that grow at elevated pressure (3-1000 x air pressure). (Found in ocean depths often in thermal vents)

Presence of Oxygen• Aerobe – Organism able to use O2 in metabolism.• Anaerobe – Organism unable to use O2 in metabolism.

Obligate aerobes - oxygen mandatoryObligate anaerobes - oxygen toxic

Facultative anaerobes – Aerobe that can also live without O2

Microaerophiles - low oxygen levels required.

Aerotolerant - anaerobic metabolism, oxygen not toxic

Microbial Processing of Oxygen• Step 1: O2

- + O2- + 2H+ H2O2 + O2 (Catalyzing

enzyme is Superoxide dismutase)

• Step 2: H2O2 + H2O2 2H2O + O2 (Catalyzing enzyme is

Catalase)

Living without Oxygen….Glucose Fermentation Pathways

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Glucose FermentationNet and practical results

• Cells get chemical energy (ATP)

• Fermentation products are natural waste products useful to humans:

1. Fermented beverages

2. Bread

3. Cheese

4. Yogurt

Using fermentation metabolism to identify microbes: A positive (yellow) mannitol-fermentation test. This test

distinguishes the pathogenic Staphylococcus aureus

(MSA)TestMannitolSaltsAgar

Comparing Aerobic Respiration with Anaerobic Fermentation

Fermentation• Fermentation yields small amount of ATP (2)• Partial oxidation of carbon atoms (6 C 3 C)

Respiration • Substrate molecules are completely oxidized to

C02 (6 C 1 C)• Far higher yield of ATP (36)• The Krebs Cycle and Electron Transport Chain

Final Electron Acceptors: Aerobic respiration, anaerobic respiration, and fermentation have

different final electron acceptors

Microbe Growth

• Binary or transverse fission

• Generation or Doubling Time – the time required for parent cell to form two new daughter cells.

Microbe Growth

• Lag – new cells require adjustment and enlargement. The cells are not multiplying rapidly.

• Log or exponential – maximum rate of growth

Microbe Growth 2

• Stationary – death and multiplication balance out. Depleted nutrients and waste buildup.

• Death – limiting factors intensify. May last a long time.

Calculating Growth of Cells

Nf = (Ni)2nNf = Final population

Ni = Initial population

2n = # cells in generation

n = generation number

Use the table in the handout from the Talaro Appendix A-2 to calculate the number of cells in the generation.

Measuring growthturbidityplate counts

Measuring Growth Serial Dilutions, Plate Counts and Turbidity

Calculation of the number of bacteria per milliliter of culture using serial dilution

Pour plate: made by first adding 1.0ml of diluted culture to 9ml of molten agar

Spread plate: made by adding 0.1ml of diluted culture to surface of solid medium

Counting colonies using a bacterial colony counter

Bacterial colonies viewed through the magnifying glass against a colony-counting grid

Which of these plates would be the correct one to count? Why?

Countable number of colonies

(30 to 300 per plate)

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The Petroff-Hausser Counting Chamber

Turbidity, or a cloudy appearance, is an indicator of bacterial growth in urine in the tube on the left

A Spectrophotometer: This instrument can be used to measure bacterial growth by determining the degree of light transmission through the culture

The Streak Plate Method uses agar plates to prepare pure cultures

A Streak Plate of Serratia marcescens. Note the greatly reduced numbers of growth /colonies in each successive region

Types of Culture Media

• Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter

• Synthetic medium: A medium prepared in the laboratory from material of precise or reasonably well-defined composition

• Complex medium: contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)

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Commonly Used Media

• Yeast Extract

• Casein Hydrolysate

• Serum

• Blood agar

• Chocolate agar

Selective, Differential, and Enrichment Media

• Selective medium: encourages growth of some organisms but suppresses growth of others (e.g. antibiotics)

• Differential medium: contains a constituent that causes an observable change (e.g. MacConkey agar)

• Enrichment medium: contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified

Three species of Candida can be differentiated in mixed culture when grown on CHROMagar Candida plates

Identification of urinary tract pathogens with

differential media (CHROMagar)

Ecological Associations

Mutualism Commensalism Parasitism

Symbiotic

Synergism Antagonism

Non-Symbiotic

Microbial Associations

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Symbiosis (Mutualism)

• Obligatory• Both organisms

benefit.• Examples: algae +

fungus = lichen, termites and trychonympha (a protist)

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Symbiosis (Commensalism)

• One organism benefits and the other is not harmed.

• Examples: Non-pathogenic bacteria on our skin; satellitism between bacteria colonies.

Symbiosis (Parasitism)

• One organism benefits and the other is harmed.

• Examples: Pathogenic organisms on their host. Plasmodium vivax a protozoan parasite causing malaria.

Non Symbiotic (Synergism)

• Free living organisms.• Both benefit• The relationship is

optional.• Examples: Shared

metabolism; nitrogen fixing bacteria in the soil and plants

Substance A

Substance B

Substance C

Microorganism 1

Microorganism 2

End Product used by all threemicroorganisms

Microorganism 3

Non Symbiotic (Antagonism)

• Free living organisms• Organisms compete

for resources.• One organism secretes

a substance toxic to the other.

• Example: Ruminal cellulose digesting bacteria and fungi

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End of Microbial Nutrition Slides