Microbial Requirements

Chemical Analysis of Bacterial Cells

  • Composition of bacterial cells:

    • 70% water

    • Proteins

    • 96% of cell composed of 10 elements = Macroelements

      • Components of Carbohydrates (CHO), Nucleic acids (NA), Lipids, Proteins, needing in Gram quantities

        • Carbon (C)

        • hydrogen (H)

        • oxygen (O)

        • phosphorus (P)

        • sulfur (S)

        • nitrogen (N)

  • Nutrient requirements:

    • needed in milligram (mg) quantities (1 mg = 0.001 g)

      • potassium (K)

      • calcium (Ca)

      • magnesium (Mg)

      • iron (Fe)

Other Nutrients Important in Microbial Metabolism

  • Nutrients necessary for microbial metabolism:

    • Potassium: essential for protein synthesis and membrane function

    • Calcium: stabilizer for cell walls and endospores

    • Magnesium: stabilizer for membranes and ribosomes

    • Iron: component of the electron transport chain (ETC) important for ATP production

Trace Elements

  • Trace elements comprise about 4% of microbial needs:

    • Essential minerals include:

      • Manganese

      • Zinc

      • Cobalt

      • Molybdenum

      • Nickel

      • Copper

    • Functions of trace elements:

      • are normally part of enzymes and cofactors

      • Aid in maintenance of protein structure

      • Needed in microgram (µg) quantities (1 µg = 0.000001 g) and usually obtained from air and water

Nutritional Complexity

  • The number of nutrients an organism must acquire is determined by the kind and number of its enzymes: (more enzymes = decrease of extra nutrients)

    • Enzymes facilitate metabolic reactions.

    • If a specific enzyme is absent, the organism cannot synthesize certain substances and must obtain them from the environment.

    • A higher number of enzymes may decrease extrinsic nutrient requirements.

E. coli Nutritional Sources

  • E. coli has over 5000 different compounds but requires only a few compounds from the environment (e.g., glucose, trace elements, H2O).

  • This implies E. coli has numerous enzymes facilitating diverse metabolism with limited basic compounds.

    • ecoli had at least 607 enzymes = uses just a few compounds to metabolize ~ 5000 compounds

Nutrient Classifications

  • Inorganic Nutrients: Molecules containing combinations of atoms other than carbon (C) and hydrogen (H).

    • Can have neither C or H

    • Can have C or H- just cannot have both

      • Examples include metals and their salts (e.g., magnesium sulfate, ferric nitrate) and gases (O2, CO2).

  • Organic Nutrients:

    • Molecules containing both carbon and hydrogen, usually derived from living organisms.

    • Can have other elements present

      • Examples include methane (CH4), carbohydrates (CHO), lipids, proteins, and nucleic acids.

    • All organic compounds have a carbon backbone

    • molecules serving as carbon sources usually also contribute both hydrogen & Oxygen

Environmental Factors Affecting Microbes

  • Environmental factors fundamentally affect the function of metabolic enzymes

    • Temperature

    • pH

    • Gas requirements (e.g., oxygen)

    • Osmotic pressure

    • Radiation

    • Barometric pressure

  • Enzymes drive metabolic reactions = no/inactive enzymes → Cell death

  • Understanding microbial ecological niches = ability to control microbial growth

Cardinal Temperatures

  • Cardinal temperatures define microbial growth curve

  • Most single cell organisms are poikilothermic = assume ambient T

    1. Minimum temperature: lowest temperature permitting growth and metabolism

    2. Maximum temperature: highest temperature permitting growth and metabolism

    3. Optimum temperature: temperature promoting the fastest growth and metabolism.

      1. cardinal temperatures are not “rigidly fixed” because influenced by other environmental factors

Temperature Adaptation Groups

  1. Psychrophiles (cold-loving):

    • Growth range: -10°C to 20°C (opt T 10 to 13°C)

    • Thrive in ocean temperatures ~5°C; non-pathogenic to humans.

    • Membrane lipids are highly unsaturated, increasing fluidity at low temperatures.

  2. Psychrotolerant:

    • Growth range: 4°C to 35°C (opt T 15 to 30°C)

    • Slow growth in cold conditions.

  3. Mesophiles:

    • Growth range: 10°C to 45°C (opt T 20°C to 40°C)

    • Human pathogens and normal flora, environmental microbes.

  4. Thermophiles (heat-loving):

    • Growth range: 45°C to 80°C (opt T 67°C to 72°C)

    • Found in compost piles and hot-water heaters.

  5. Extreme Thermophiles:

    • Growth above 70°C

    • Thrive in hot springs and deep ocean vents.

    • Archaea - unique enzymes , DNA ^ C+G%, no PG, lipids in CM = highly saturated = no double bonds = more resistant to heat

Effects of pH on Microbial Growth

  • pH is a measure of the hydrogen activity of a solution defined as the degree of acidity or alkalinity of a solution on a 0 to 14 scale:

    • Pure water pH is 7 (neutral).

    • Each species has a definite pH growth range

    • Most microorganisms grow optimally between pH 6 to 8 because acid and base can be

      • damaging to proteins - especially enzymes.

      • damaging to cell membrane and other parts of the cell (even DNA)

    • The effects of pH=

      • related to the concentration of acid int he medium and

      • to the protection that bacterial cell walls sometimes provide

    • Changes in pH can lead to

      • denaturing of enzymes and other proteins and

      • can interfere with pumping ions at the cell membrane because fo the lack of electrical gradient

    • many bacteria produce large quantities of acids as they metabolize and grow = leads to high acid concentration = toxic environment

    • acidic = high H+

Effects of pH

  • Majority of microorganisms grow at a pH from 6-8

  • Acidophiles - grow at extreme acid pH (pH 0 to 5.5)

    • archaea in hot spring change in pH is very bad

  • Neutrophiles - growth between pH 6-8

  • Alkaliphiles - growth between pH 8.5-12

  • Helicobacter pylori (gm - spirochete, high mortality rate)

    • found in the stomach ~pH 2.5

    • causes peptic ulcers, gastric and esophageal cancer

    • is not ACID tolerant

    • Produces toxins that cause inflammation and damage

      • disease symptoms made worse but increase in stress and environmental factors and diet

    • Protects itself from the stomach by growing in protective mucus layers of the stomach

    • breaks down urea in the stomach = pdces NH4+ basic = neutralize the microenvironment

    • treat via antibiotics

Gas Requirements for Microbes

  • Oxygen is vital for many microbes but can also produce toxic by-products:

    • Singlet oxygen (O2), superoxide ion (O2^-), hydrogen peroxide (H2O2), hydroxyl radicals (OH^-).

    • Protective enzymes neutralize these toxic products (e.g., catalase, superoxide dismutase).

  • If a microbe is not capable of dealing with toxic oxygen= lacks protective enzymes

    • so it is forced to live in oxygen free habitats = anaerobe

Categories of Oxygen Requirement

  1. Aerobe: Utilizes oxygen and detoxifies it, Has protective enzymes

    • Obligate aerobe: cannot grow without oxygen.

      • must have O2 to grow

    • Facultative anaerobe: prefers oxygen but can grow without it.

      • E.Coli

    • Microaerophilic: requires a small amount of oxygen (~2-10% O2).

      • grows best at 5% O2

  2. Anaerobe: Does not utilize oxygen to make ATP

    • lacks protective enzyme that protect form reactive O species

    • Obligate anaerobe: cannot survive in oxygen due to lack of detoxifying enzymes.

    • Aerotolerant anaerobes: can grow in presence of oxygen but do not use it for metabolism.

      • Has different protective mechanisms - uses metal ions

Osmotic Pressure

  • Availability of water influences microbial life and depends on water amount and solute concentration (osmotic pressure).

    • Most microbes thrive under hypotonic or isotonic conditions.

    • halophiles - require a high concentration of salt or they will burst

    • Osmotolerant - does not require high concentration of solutes but can tolerate them when it occurs.

  • Water activity (Aw) is a measure of the water that is avaliable for use by an organism

  • Aw is lowered by adding solutes to a solution thus

    • Increase the solute concentrations of the solution and

    • thus increase osmotic pressure and lower Aw (inversely related)

    • Enzymes require an aqueous environment in order to be functional

    • decrease Aw → Decrease enzyme function → decrease in metabolism → death of cell

  • Aw and osmotics pressure are inversely related with an Increase osmotic pressure then you have a decrease in Aw

  • The aw of pure water is ~1.00

  • most organisms require an Aw of .90-1.00 for metabolic activity and growth

    • ex: Staphlyococcus aureus → can grow at ~0.85Aw = grows on our skin which is fairly salty

    • Fungi → can grow at ~0.70 Aw

  • Salts/Sugars = Decrease Aw= used as food preservative

Radiation Effects on Microbial Life

  • Ultraviolet (UV) light induces DNA mutations (T-T dimers = toxic) and even kill organisms

  • Some organisms have enzyme systems that can repair certain kinds of DNA damage

  • Ionizing radiation can be used to sterilize items Endospores can survive large doses of ionizing radiation

  • Bacillus stearothermophilus endospores=

    • Purple sugar broth in ampule & spores on a piece of filter paper

    • Autoclave and then crush ampule to release broth to coat filter paper with spores

    • If turns yellow, spores germinated and grew & produced acids= so spores not killed= Sterilization did not take place!.

Pressure Factors on Microbial Life

  • Many orgs spend their lives on land or on the surface of water so are subjected to a pressure of 1 atm so are never affected significantly by pressure.

  • Hydrostatic pressure is the pressure exerted by a water column as a result of the weight of the column with each 10 m of water depth equivalents to 1 atm

  • Hydrostatic pressures of > 200 atm generally inactivate enzymes and disrupts Cell membranes and transport enzymes cell membrane systems

  • Hydrostatic pressure in the deep ocean can reach > 1000 atm (with a temp of 2-3C). Despite these extremes bacteria survive and adapt.

  • These Barophiles will actually rupture when exposed to normal atmospheric pressure

    • Barophile enzymes need pressure to maintain their 3-D shape.

    • No 3-D shape = enzyme is non-functional

Applications in Microbiology

  • Understanding microbial communities and their ecological interactions can aid in biotechnology, medicine, and environmental applications.

Ecological Associations Among Microorganisms

  • Symbiosis

    • Organisms living in a close relationship

    • at least one member is dependent on (requires) the relationship

      1. Mutualism: Obligatory, dependent; both organisms benefit.

        1. uing host metabolic byproducts and actively producing vitamins

      2. Commensalism: commensal member is dependent and benefits, while the other is not harmed.

        1. using host metabolic byproducts to their benefit

      3. Parasitism: one benefits at the expense of the other, host is harmed

        1. antibiotics wipe out much of normal flora

        2. sporeformers germinate and produce toxins = tissue damage

  • Mutualism = an association between fungus and a photosynthetic microbe (either an algae or cyanobacteria)

    • the fungus gets it organic carbon from the photosynthetic organism and in return

      • protect it from excessive light intensities

      • and supplies water and mineral

      • while providing a support structure

    • Lichens are resistant to Temp extremes and desiccation because fungus traps moisture but are sensitive to air pollution

Biosafety Levels - For handling microbes

  • BSL-1: no special precautions

    • microbes not known to cause disease

  • BSL-2: lab coat, gloves, eye protection

    • microbes can be associated with human disease especially those who are immunocompromised

  • BSL-3 Biosafety cabinets to prevent airborne transmission

    • microbes that do cause human disease when encountered

    • treatable and/or have vaccines

  • BSL-4 : sealed, negative pressure

    • exhaust air is filtered twice

    • deadly organisms for which we have no treatment or vaccine

Metabolism of Microbes

  • Metabolism - all chemical reactions and physical working of a cell

  • Two types of chemical reaction

    • anabolism - building process

      • 2 small substrates → 1 large molecules = make bonds E used

    • Catabolism - degradative process

      • 1 large molecule → 2 small substrats = bonds broken , E released

    • RXNS are cyclic and self regulating

Summary of Enzyme Function

  • Enzymes are biological catalysts:

    • Increase the rate of chemical reactions by lowering the activation energy.

  • The enzyme is not permanently altered in the reaction

  • Physically promote a reaction = serve as a physical site upon which the reactants (or substrates) can be positioned for various rxns

  • Since enzymes are not a part of the products, it is not used up by the rxn

    • ENZYMES CAN BE USED OVER AND OVER, are not changed and eventually wear out

Enzyme structure

  • Enzymes can be simple or conjugated

  • simple enzymes - protein only

  • conjugated enzymes - protein + nonprotein molecule

  • Non protein molecules are called cofactors

    • cofactors are either organic molecules which can be called coenzymes

    • coenzymes or inorg elements such as metal ions (cofactor)

Enzyme - substrate interactions (when cofactor is inorganic)

  • For a reaction to take place → temporary union between enzyme and substrate occurs

  • once the enzyme-substrate complex has formed

    • appropriate reactions occur on the substrate

    • often with the aid of a cofactor

    • a product(s) is/are formed and released

  • Function: “better fit” between enzyme and substrate

  • The general function of a coenzyme is to remove a functional group (amino groups, hydrogen atoms) from substrate 1 and add the fucntional group to substrate 2

  • Coenzyme function

    • serves as a transient carrier of the functional group between substrate 1 and substrate 2

    • increases rate of catalysis

Enzymes sensitivity

  • when enzymes are subjected to changes in environmental conditions = Temp or pH extremes = Unfolded/degraded/denatured

    • low temps inhibit catalysis = by lowering kinetic energy

    • high temperatures, certain chemicals and low and high pH = denature enzymes