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Chapter 6

Microbial Nutrition and Growth

Objectives

Describe the uses of carbon, oxygen, nitrogen, trace elements, and vitamins in microbial growth and reproduction.

Compare the four main categories of organisms based upon their carbon and energy sources.

Distinguish among anaerobes, aerobes, aerotolerant anaerobes, facultative anaerobes, microaerophiles, and capnophiles.

Explain how oxygen can be fatal to organisms by discussing singlet oxygen, superoxide radicals, peroxide anion, and hydroxyl radical and describe how organisms protect themselves from toxic forms of oxygen.

Describe how quorum sensing can lead to formation of a biofilm.

Describe methods for collecting clinical specimens from the skin and the respiratory, reproductive, and urinary tracts.

Describe four methods by which microorganisms can be isolated for culture.

Describe six types of general culture media available for bacterial culture.

Discuss the use of special culture methods including animal, cell, low-oxygen, and enrichment cultures.

Contrast refrigeration, deep freezing, and lyophilization as methods for preserving microorganisms.

Describe logarithmic growth.

Explain what is meant by the generation time of bacteria.

Draw and label a bacterial growth curve.

Describe what occurs at each phase of a population's growth.

Contrast direct and indirect methods of measuring bacterial growth.

Metabolism Results in Reproduction

Microbial growth – an increase in a population of microbes rather than an increase in size of a cell

Result of microbial growth is a discrete colony – an aggregation of cells arising from single parent cell

Reproduction results in growth

Growth Requirements

Organisms use a variety of nutrients for their energy needs and to build organic molecules and cellular structures

Most common nutrients – those containing necessary elements such as carbon, hydrogen, nitrogen, oxygen, phosphorus and sulfur (CHNOPS)

Microbes obtain nutrients from variety of sources

Nutrients: Chemical and Energy Requirements

Sources of Carbon, Energy, and Electrons

Oxygen Requirements

Nitrogen Requirements

Other Chemical Requirements

Sources of Carbon, Energy, and Electrons

Organisms categorized into two groups based on source of carbon

Those using an inorganic carbon source (carbon dioxide) are autotrophs

Those catabolizing reduced organic molecules (proteins, carbohydrates, amino acids, and fatty acids) are heterotrophs

Sources of Carbon, Energy, and Electrons (continued)

Organisms categorized into two groups based on whether they use chemicals or light as source of energy

Those that acquire energy from reactions involving inorganic and organic chemicals are chemotrophs

Those that use light as their energy source are phototrophs

Oxygen Requirements

Oxygen is essential for obligate aerobes (final electron acceptor in ETC)

Oxygen is deadly for obligate anaerobes

How can this be true?

Neither gaseous O₂ nor oxygen covalently bound in compounds is poisonous

The forms of oxygen that are toxic are excellent oxidizing agents

Resulting chain of oxidations causes irreparable damage to cells by oxidizing compounds such as proteins and lipids

Four toxic forms of oxygen

Singlet oxygen – molecular oxygen with electrons boosted to higher energy state

Occurs during photosynthesis so phototropic organisms have carotenoids that remove the excess energy of singlet oxygen

Superoxide radicals – some form during incomplete reduction of oxygen in aerobic and anaerobic respiration

So reactive that aerobes produce superoxide dismutases to detoxify them

Anaerobes lack superoxide dismutase and die as a result of oxidizing reactions of superoxide radicals formed in presence of oxygen

Four toxic forms of oxygen (continued)

Peroxide anion – formed during reactions catalyzed by superoxide dismutase and other reactions

Aerobes contain either catalase or peroxidase to detoxify peroxide anion

Obligate anaerobes either lack both enzymes or have only a small amount of each

Four toxic forms of oxygen (continued)

Hydroxyl radical – results from ionizing radiation and from incomplete reduction of hydrogen peroxide

The most reactive of the four toxic forms of oxygen

Not a threat to aerobes due to action of catalase and peroxidase

Aerobes also use antioxidants such as vitamins C and E to protect against toxic oxygen products

Classification of Organisms Based on Oxygen Requirements

Aerobes – undergo aerobic respiration

Anaerobes – do not use aerobic metabolism

Facultative Anaerobes – can maintain life via fermentation or anaerobic respiration or by aerobic respiration

Aerotolerant anaerobes – do not use aerobic metabolism but have some enzymes that detoxify oxygen’s poisonous forms

Microaerophiles – aerobes that require oxygen levels from 2-10% and have a limited ability to detoxify hydrogen peroxide and superoxide radicals

Nitrogen Requirements

Anabolism often ceases due to insufficient nitrogen needed for proteins and nucleotides

Nitrogen acquired from organic and inorganic nutrients; also, all cells recycle nitrogen from amino acids and nucleotides

The reduction of nitrogen gas to ammonia (nitrogen fixation) by certain bacteria is essential to life on Earth because nitrogen is made available in a usable form

Other Chemical Requirements

Phosphorus required for phospholipid membranes, DNA, RNA, ATP, and some proteins

Sulfur is a component of sulfur-containing amino acids, disulfide bonds critical to tertiary structure of proteins, and in vitamins (thiamin and biotin)

Trace elements – usually found in sufficient quantities in tap water

Growth factors – organic chemicals that cannot be synthesized by certain organisms (vitamins, certain amino acids, purines, pyrimidines, cholesterol, NADH, and heme)

Physical Requirements for Growth

Temperature

pH

Osmolarity

Pressure

Temperature

Effect of temperature on proteins

Effect of temperature on lipid-containing membranes of cells and organelles

If too low, membranes become rigid and fragile

If too high, membranes become too fluid and cannot contain the cell or organelle

pH

Organisms sensitive to changes in acidity because H⁺ and OH^- interfere with H bonding in proteins and nucleic acids

Most bacteria and protozoa grow best in a narrow range around neutral pH (6.5-7.5) – these organisms are called neutrophiles

Other bacteria and fungi are acidophiles – grow best in acidic habitats

Acidic waste products can help preserve foods by preventing further microbial growth

Alkalinophiles live in alkaline soils and water up to pH 11.5

Physical Effects of Water

Microbes require water to dissolve enzymes and nutrients required in metabolism

Water is important reactant in many metabolic reactions

Most cells die in absence of water

Some have cell walls that retain water

Endospores and cysts can cease most metabolic activity for years

Two physical effects of water

Osmotic Pressure

Hydrostatic Pressure

Osmotic Pressure

Is the pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane; related to concentration of dissolved molecules and ions in a solution

Hypotonic solutions have lower solute concentrations; cells placed in these solutions will swell and burst

Osmotic Pressure

Hypertonic solutions have greater solute concentrations; cells placed in these solutions will undergo plasmolysis (shriveling of cytoplasm)

This effect helps preserve some foods

Restricts organisms to certain environments

Obligate halophiles – grow in up to 30% salt

Facultative halophiles – can tolerate high salt concentrations

Hydrostatic Pressure

Water exerts pressure in proportion to its depth

For every addition of depth (32ft, 10M), water pressure increases 1 atm

Organisms that live under extreme pressure are barophiles

Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape

Ecological Associations

Organisms live in association with different species

Antagonistic relationships

Synergistic relationships

Symbiotic relationships

Biofilms

Complex relationships among numerous individuals

Form on surfaces often as a result of quorum sensing

Culturing Microorganisms

Inoculum introduced into medium (broth or solid)

Environmental specimens

Clinical specimens

Stored specimens

Culture – refers to act of cultivating microorganisms or the microorganisms that are cultivated

Obtaining Pure Cultures

Cultures composed of cells arising from a single progenitor

Progenitor is termed a CFU

Aseptic technique is used to prevent contamination of sterile substances or objects

Two common isolation techniques

Streak Plates

Pour Plates

Culture Media

Majority of prokaryotes have never been grown in culture medium

Six types of general culture media

Defined media

Complex media

Selective media

Differential media

Anaerobic media

Transport media

Special Media Techniques

Techniques developed for culturing microorganisms

Animal and cell culture

Low-oxygen culture

Enrichment culture

Preserving Cultures

Refrigeration

Deep-freezing

Lyophilization

Measuring Microbial Growth

Direct Methods

Viable Plate Counts

Membrane Filtration

Microscopic counts

Electronic Counters

Most Probable Number

Measuring Microbial Growth

Indirect Methods

Metabolic Activity

Dry Weight

Turbidity

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