MicroBio Mod. 3

Microbial Growth and Metabolism

Bacterial Growth

an increase in the number of cells in a population rather than the growth of individual cells

• primarily occurs through a process called binary fission,

Requirements for bacterial growth: physical

Optimal temperature temperature

• minimum, optimal, and maximum growth temperatures

pH

• most bacteria like pH 6.5-7.5

• molds and yeasts like 5-6

• acidophiles grow in acidic environments

Osmotic Pressure:

• hypertonic environments (increased salt or sugar) cause plasmolysis

• so, salt and sugar increase osmotic pressure of the environment

• extreme/obligate halophiles require high osmotic pressure while facultative halophiles tolerate it since they’re able to adapt to environmental changes

Requirements for bacterial growth: chemical

Carbon:

• energy source

• chemoheterotrophs use organic carbon sources (cannot use carbon directly to make their own organic compounds)

• autotrophs use CO2 (synthesize their own organic molecules)

• like humans, bacteria are made primarily of carbon based organic molecules

Nitrogen:

• in amino acids, proteins; nucleic acids

• most bacteria decompose proteins

• some bacteria use NH4+ or NO3- for energy

• a few use N2 in nitrogen fixation

Sulfur:

• In amino acids (cys and met), thiamine, biotin

• Most bacteria decompose proteins

• Some bacteria use SO42 or H2S for energy

Phosphorus:

• In DNA, RNA, ATP, and membranes

• PO43 is a source of phosphorus

Oxygen

Optimum growth temperatures

Psychrophiles:

• about 15 degrees C

Psychrotrophs:

• about 25 (20-30) degrees C

Mesophiles: (where most human pathogens belong to)

• about 37 (30-40) degrees C

Thermophiles:

• about 65 degrees C

Hyperthermophiles:

• above 80 degrees C

Obligate aerobes

require oxygen

• sit at the top of a culture broth tube

Facultative anaerobe

prefer oxygen but can grow without it

• mostly at the top of the culture broth tube but also suspended throughout

Obligate anaerobes

find oxygen toxic

• it can cause cell death

• found at the bottom of the culture broth tube

aerotolerant anaerobes

don’t need oxygen to grow but it won’t harm them

• found suspended throughout the entire culture broth tube

microaerophiles

grow only when oxygen is present at a very specific quantity or in a very small amount

Osmotic pressure and plasmolysis relationship

When the solute concentration (salt and/or sugar) in the environment around the cell is higher than the solute concentration inside the cell (called a hypertonic solution, which has a high osmotic pressure), water will rush out of the cell to dilute the concentration. This causes the cytoplasm to shrink back, causing the cell itself to shrivel.

Toxic forms of oxygen

• singlet oxygen

• superoxide free radicals

• peroxide anion

• hydroxyl radical

Superoxide dismutase (SOD)

converts superoxide free radicals (which are very unstable and highly reactive) to molecular oxygen and peroxide (which is toxic by itself)

O2 - + 2H+ —→ H2O2 + O2

Catalase

enzyme organisms use to detoxify peroxide which generates oxygen and water from peroxide

2H2O2 —→ 2H2O + O2

Peroxidase follows:

H2O2 + 2O+ —→ 2H2O

Bacterial Growth Curve

Lag Phase:

• bacteria hasn’t begun to reproduce yet (could last anywhere from 1-24 hours

Log phase:

• bacteria growing and increasing in number rapidly

• exponential growth period

Stationary phase:

• number of cells dying = number of cells alive and still dividing

Death phase:

• number of cells dying is greater than number of cells dividing and growing

• decreasing at a logarithmic rate

It is best to observe bacteria in log or stationary phases because they are most active/most representative of the culture.

Methods for counting living bacteria

Plate counts:

• series of serial dilutions from an original inoculum

• 1:10 dilution factor each time

• transfer dilutions to petri plates using either the pour plate method or spread plate method and incubate them

• count colonies on plates containing 25-250 colonies and multiply that number by the dilution factor to get the number of colonies in the original inoculum

Biofilms

Microbial communities forming along solid surfaces and sticking to each other

• Form slime or hydrogels if excessively hydrated with water

• form slimy extracellular matrix secreted outside of the cells (made of macromolecules like polysaccharides, proteins, lipids)

• Bacteria communicate by chemicals via quorum sensing, altering their behavior based on the factors/communication (they behave in unison)

• natural, industrial, and hospital settings (e.g. teeth plaque)

• share nutrients

• sheltered from harmful factors (environmental waste products, antibiotics, host body immune system)

Enzymes

Encoded for by genes

Biological catalyst (protein)

• specific for a chemical reaction; not used up in that reaction or permanently altered

• acts on a specific substance or reactant, called a substrate (becomes the product)

• speeds up a chemical reaction by reducing activation energy

• enzyme + substrate = product

• each catalyzes only one reaction

The turnover number is generally 1-10,000 molecules per second

How enzymes work

1. The substrate contacts the active site on the enzyme to form an enzyme–substrate complex.

2. The enzyme orients the substrate into a position that increases the probability of reaction, which enables the collisions to be more effective.

3. The substrate is then transformed/broken down into products, the products are released because they no longer fit the active site, and the enzyme is recovered unchanged, free to interact with other substrate molecules.

Collision theory

States that chemical reactions can occur when atoms, ions, and molecules collide in just the right way

• collide with enough force to transfer to the bonds which break and create new bonds

activation energy is needed to disrupt electron configurations

• increases collisions in a cell

Reaction rate

the frequency of collisions with enough energy to bring about a reaction

• can be increased by enzymes or by increasing temperature or pressure

Apoenzyme

the protein portion of an enzyme

• inactive and cannot perform catalytic function until attached to the cofactor

cofactor

nonprotein portion of an enzyme

• activates the apoenzyme when attached to it

• examples: Ions of iron, zinc, magnesium, or calcium

• coenzyme - cofactor made of organic molecules (NAD+,NADP+, FAD, Coenzyme A)

Holoenzyme

when the apoenzyme and cofactor come together to form a whole, active enzyme

Protein denaturation

The shape of the protein changes

• the protein unfolds

• prevents enzyme from working properly (thereby stopping the part of metabolism related to that enzyme)

• can lead to cell death

Causes of protein denaturation:

• temperature (35-40 degrees C)

• pH (4-6)

• substrate concentration (enzyme activity plateaus as substrate concentration increases because an enzyme cannot work any faster than it can function in the molecular environment)

Competitive inhibition

inhibitor fits into the active site and blocks the substrate

• competition between inhibitor and substrate

Noncompetitive binding

inhibitor fits somewhere else on the protein except the active site

• changes the shape of the active site so the substrate cannot bind at the proper location

Feedback inhibition

the final product of a metabolic pathway can itself inhibit the pathway by binding to an enzyme early on in the pathway

• a normal mechanism of cellular control because the cell can regulate its own metabolism

• when enough product is made, the metabolic pathway will stop to preserve energy and resources

• turns back on once product is depleted by freeing the enzymes and allowing them to work again

Metabolism

sum of all the chemical reactions that occur within a cell or organism

Catabolism

The breakdown of complex organic molecules into simpler molecules such as glucose, amino acids, glycerol, and fatty acids

• provides the building blocks and energy for anabolism

• the energy released from chemical bonds is transferred to molecules of ATP for storage or lost as heat

Anabolism

The building up of complex molecules from simpler ones

• requires energy

• generates material required for cellular growth

Metabolic pathway

All the build up and breakdown of molecules that occurs through an intricate series of steps

• sequences of enzymatically catalyzed chemical reactions in a cell that in total become the cell’s metabolism

• determined by enzymes because all chemical reactions need to occur rapidly

Glycolysis

The oxidation/breakdown of glucose into pyruvic acid

• first stage of carbohydrate catabolism (cell respiration)

• starting block for Krebs cycle (a process in cell respiration) and fermentation

Splits glucose, a six-carbon sugar, into two three-carbon sugars. These sugars are then oxidized, releasing energy, and their atoms are rearranged to form two molecules of pyruvic acid.

Electron transport chain

(Part of cell respiration, not fermentation)

• requires O2

• produces most of an aerobic cell’s energy/ATP

Aerobic respiration

The final electron acceptor in the electron transport chain is molecular oxygen (O2)

• occurs when oxygen is present

• produces 32-36 ATP/Glucose

Anaerobic respiration

The final electron acceptor in the electron transport chain is NOT molecular oxygen (O2)

• occurs when oxygen is NOT present

• yields less energy than aerobic respiration because only part of the Krebs cycle operates under anaerobic conditions.

• cells will use other inorganic molecules like nitrate, sulfate, and carbonate (as well as some organic molecules like acid and alcohol) instead of O2.

Fermentation

• releases energy from oxidation of organic molecules

• does not require oxygen

• does not use Krebs cycle or an electron transport chain

• use of organic molecules as the final electron acceptor

• yields the least amount of energy

Anaerobic respiration vs Fermentation

Organisms use anaerobic respiration when:

• They possess an electron transport chain.

• Alternative electron acceptors are available in the environment.

• They benefit from obtaining more ATP per glucose.

Organisms use fermentation when:

• They lack an electron transport chain.

• Suitable external electron acceptors are unavailable.

• Simplicity is advantageous.

• Energy demands are relatively low.

Common products produced by fermentation:

Performed by:

• yeasts

• bacteria

• molds

• animal muscle cells

Products:

• sugar into lactic acid: sauerkraut, kimchi, yogurt, pickles, kefir

• Yeasts convert sugar into ethanol and carbon dioxide: beer, wine, cider, leavened bread

• Uses molds and bacteria to break down proteins: soy sauce, miso.

Cell respiration: How it works

1. Glycolysis is the oxidation of glucose to pyruvic acid with the production of some ATP and energy-containing NADH.

2. The Krebs cycle is the oxidation of acetyl CoA (a derivative of pyruvic acid) to carbon dioxide, with the production of some ATP, energy-containing NADH, and another reduced electron carrier, FADH2 (the reduced form of flavin adenine dinucleotide).

3. In the electron transport chain (system), NADH and FADH2 are oxidized, contributing the electrons they have carried from the substrates to a “cascade” of oxidation-reduction reactions involving a series of additional electron carriers. Energy from these reactions is used to generate a considerable amount of ATP. In respiration, most of the ATP is generated in the third step.

Final electron acceptors:

• O2 if aerobic

• inorganic molecules if anaerobic