Source : Prescott et al Microbiology, Microbiology by Pelczar, Brock Et al

Microbiology

Microbial Nutrition

• Purpose

To obtain energy and construct new cellular components

• Nutrient Requirement

The major elements: C, O, H, N, S, P

The minor elements: K, Ca, Mg, Fe

The trace elements: Mn, Zn, Co, Mo, Ni, Cu

Factors affecting growth: Nutritional Factors

Nutrients – Chemicals taken in and used by organisms for energy,

metabolism and growth

Water (Hydrogen and Oxygen)

Carbon

Nitrogen

Sulfur

Phosphorus

Trace Elements

Growth Factors

Macronutrients - Required in large amounts

Carbon

Needed for synthesis of cellular material and energy source

Nitrogen

Needed for protein synthesis, nucleic acids, ATP

Sulfur

Needed to synthesize amino acids and vitamins (thiamine, biotin)

Phosphorus

Needed to synthesize nucleic acids, ATP, phospholipids

Factors affecting growth: Nutritional Factors

Factors affecting growth: Nutritional Factors

Trace Elements required in trace amounts

involved in enzyme function and protein structure

Examples: Zn, Cu, Fe

Present in tap water and distilled

Growth factors Organic compounds that cannot be synthesized by bacteria

Bacteria are “fastidious” (require relatively large amounts of growth

factors in the media. Can be used to test samples for presence of growth

factors )

Examples: amino acids, purines, pyrimidines, vitamins

Micronutrients

Sources of Essential Nutrients

• Carbon – obtain in organic form, or reduce CO2

• Nitrogen – Fix N2 or obtain as NO3-- NO2

-, or NH3

• Oxygen – Atmospheric or dissolved in water

• Hydrogen – Minerals, water, organic compounds

• Phosphorous – Mineral deposits

• Sulfur – Minerals, H2S

• Metal Ions - Minerals

Make it, or eat it?

• Some bacteria are remarkable, being able to make all the organic

compounds needed from a single C source like glucose.

• For others:

– Vitamins, amino acids, blood, etc. added to a culture medium are

called growth factors.

– Bacteria that require a medium with various growth factors or

other components and are hard to grow are referred to as

fastidious.

Nutrient Requirements

• Prototrophs vs. Auxotrophs– Prototroph

– A species or genetic strain of microbe capable ofgrowing on a minimal medium consisting a simplecarbohydrate or CO2 carbon source, with inorganicsources of all other nutrient requirements

– Auxotroph

– A species or genetic strain requiring one or morecomplex organic nutrients (such as amino acids,nucleotide bases, or enzymatic cofactors) for growth

Organisms are categorized into two groups:

Autotrophs

Those using an inorganic carbon source (carbon dioxide)

Heterotrophs

Those catabolizing organic molecules i.e. reduced,

preformed organic molecules (proteins, carbohydrates,

amino acids, and fatty acids)

Carbon sources

Autotrophs Phytosynthetic bacteria:

Few purple sulphur (e.g., Chromatium) bacteria possess pigments, such as, purple pigment, the bacteriopurpurin, and green

pigment, the bacterial chloroyhyll etc. Bacterioviridin occurs hi green sulphur bacteria, e.g., Chlorobium. Such bacteria

synthesize their carbohydrate food in presence of sunlight by photosynthesis and are known as chlorophyll bacteria.

2H2S + CO2 → (CH2O)2 + 2S + H2O

Chemosynthetic bacteria:

These bacteria get their energy for food synthesis from the oxidation of certain inorganic chemicals. Light energy is not used. The

energy obtained from the chemical reactions is exothermic. The Chemosynthetic bacteria are of the following types:

(a) Sulphomonas (Sulphur bacteria): These bacteria get their energy by oxidation of hydrogen sulphide into H2SO4, e.g.,

Thiobacillus, Beggiatoa.

CO2 + 2H2S → 2S + H2O + CH2O + Energy

3CO2 + 2S + 8H2O → 2 H2S04 + 2(CH0) + 3H2O + Energy

(b) Hydromonas (Hydrogen bacteria): These convert hydrogen into water, e.g., Bacillus pantotrophus.

H2 + ½O2 ® → H2O + Energy

(c) Ferromonas (Iron bacteria): These bacteria get their energy by oxidation of ferrous compounds into ferric forms,. e.g.,

Leptothrix.

2Fe(HCO3)2 + H2O + O → 2Fe (OH)3 + 4CO2 + Energy

4FeCO3 + O2 + 6 H2O → 4Fe(OH)3 + 4CO2 + Energy

(d) Methanomonas (Methane bacteria): These bacteria get their energy by oxidation of methane into water and carbon dioxide.

(e) Nitrosomonas (Nitrifying bacteria): These bacteria get their energy by oxidation of ammonia and nitrogen compounds into

nitrates. Nitrosomonas oxidises NH3 to nitrites.

NH3 + ½O2 ® → H2O + HNO2 + Energy

Nitrobacter converts nitrites to nitrates.

NO2 + ½O2 → NO2 + Energy

HeterotrophsSaprophytic bacteria:

These bacteria obtain their food from the dead organic decaying substances such as leaves, fruits,

vegetables, meat, animal faeces, leather, humus etc. They secrete enzymes to digest the food

and absorb it. The breakdown of carbohydrates is fermentation and of proteins the putrefaction.

The former produces alcohols, acetic and other organic acids by fermentation of carbohydrates.

Putrefaction decomposes proteins into ammonia, methane, H2S, carbonic acids. The enzymes

secreted break down the complex compounds into simpler soluble compounds, which are easily

absorbed. Examples are Bacillus acidi lacti, Acetobacter etc.

Parasitic bacteria:

These bacteria obtain their food from the tissues of living organisms, the hosts. They may be

harmless or may cause serious diseases. The disease-producing bacteria are pathogenic which

cause various diseases in plants and animals. Examples are Bacillus typhosus, B. anthracis, B.

tetani. B. diplheriae, B. tuberculosis, B. pneumoniae, Vibrio cholerae, Pseudomonas citri etc.

Symbiotic bacteria:

These bacteria live in close association with other organisms as symbionts. They are beneficial to

the organisms. The common examples are the nitrogen-fixing bacteria, e.g., Bacillus radicicola,

B. azotobacter, Rhizobium, Ctostridium etc. Rhizobium spp.,B. radicicola and B. azotobacter

live inside the roots of leguminous plants and form bacteria nodules for fixation of nitrogen from

the air.

Organisms are categorized into two groups:

Chemotrophs

Acquire energy from redox reactions (oxidation

of chemical compounds) involving inorganic and

organic chemicals

Phototrophs

use light as their energy source

Energy sources

Groups of organisms based on carbon and energy source

Figure 6.1

Electron sources

Lithotrophs

use reduced inorganic substances as their electron

source.

Organotrophs

extract electrons from organic compounds.

Nutritional classes based on primary sources of carbon,

energy and electrons:

• Phtotolithotrophic autotrophs or photoautotrophs or photolithoautotrophs

Source of energy – light energy

Source of electrons – Inorganic hydrogen/ electron

Carbon source - CO2

Example: Algae, purple and green sulfur bacteria and cyanobacteria.

• Photoorganotrophic heterotrophy or photoorganoheterotrophy

Source of energy – light energy

Source of electrons – organic hydrogen/ electron

Carbon source –organic carbon sources (CO2 may also be used)

Example: Purple and green nonsulfur bacteria (common inhabitants of

lakes and streams)

• Chemolithotrophic autotrophs or chemolithoautotrophy

Source of energy – Chemical energy source (inorganic)

Source of electrons – Inorganic hydrogen/ electron donor

Carbon source - CO2

Example: Sulfur-oxidizing bacteria, hydrogen bacteria, nitrifying

bacteria, iron-oxidizing bacteria.

• Chemoorganotrophic heterotrophs or chemoorganoheterotrophy

Source of energy – Chemical energy source (organic)

Source of electrons – Inorganic hydrogen/ electron donor

Carbon source – organic carbon source

Example: Protozoan, fungi, most non-photosynthetic bacteria

(including most pathogens)

Microbial Growth

Metabolism Results in Reproduction

Reproduction results in Growth

•What is microbial growth?

– an increase in a population of microbes (rather

than an increase in size of an individual)

•Result of microbial growth?

– a discrete colony – an aggregation of cells arising

from single parent cell

Mathematics of Population Growth

Mathematics of Population Growth

Number of generations (n) = (log Nt – log No) / log 2

Growth Rate Constant (k) = n/t

It is expressed in units of generations per hours (h-1)

Generation time (g) = 1/k; it is expressed in units of hours (h).

Nt = No + 2n

Exponential Growth by Binary Fission

1. DNA replication

2. Cell elongation

3. Septum formation

4. Septum completion

leads to separation or

further division

5. Process repeats

Generation time (g= t/n)

Duration of each division

Determined by type of

bacteria

Example: E. coli (20 min)

Bacterial Growth Curve

The Population Growth Curve

In laboratory studies, populations typically display a predictable pattern over time – growth curve.

Stages in the normal growth curve:

1.Lag phase – “flat” period of adjustment, enlargement; little growth

2.Exponential growth phase – a period of maximum growth will continueas long as cells have adequate nutrients and a favorable environment

3.Stationary phase – rate of cell growth equals rate of cell death caused bydepleted nutrients and O2, excretion of organic acids and pollutants

4.Death phase – as limiting factors intensify, cells die exponentially in theirown wastes

Stationary Phase

What

–metabolically active cells stop reproducing

– reproductive rate is balanced by death rate

• Why

– nutrient limitation

– limited oxygen availability

– toxic waste accumulation

– critical population density reached

• Starvation Response

–Morphological change

– Decrease in cell size

– Production of starvation proteins

Diauxic growth

• Growth in two phases

• Utilize one carbon source

first

• Utilize the second one until

the first one depleted

• Resulted from inducible

enzyme synthesis

Environmental Effects on Bacterial Growth

• Temperature

• Oxygen

• pH

• Osmotic pressure

TemperatureCardinal temperatures

• Minimum Temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow.

• Optimum Temperature: Temperature at which growth rate is the fastest.

• Maximum Temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow.

Classification of Microorganisms by Temperature

Requirements

Temperature Classes of Organisms

• Psychrophiles ( 00C-200C)– Cold temperature optima

– Most extreme representatives inhabit permanently cold environments

• Mesophiles ( 200C – 450C)– Midrange temperature optima

– Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes

• Thermophiles ( 500C- 800C)– Growth temperature optima between 45ºC and 80ºC

• Hyperthermophiles– Optima greater than 800C

– These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC

Temperature

Psychrotrophs and Mesophiles

– “Growth” is generally used to refer to the acquisition of biomass

leading to cell division, or reproduction

– Many microbes can survive under conditions in which they

cannot grow

– The suffix “-phile” is often used to describe conditions

permitting growth, whereas the term “tolerant” describes

conditions in which the organisms survive, but don’t necessarily

grow

– For example, a “thermophilic bacterium” grows under

conditions of elevated temperature, while a “thermotolerant

bacterium” survives elevated temperature, but grows at a lower

temperature

Growth vs. Tolerance

34

Preserving Bacteria Cultures:

• Refrigeration:

– Storage for short periods of time

• Deep-freezing:

– -50° to -95°C

– Preserves cultures for years

• Lyophilization (freeze-drying):

– Frozen (-54° to -72°C) and dehydrated in a

vacuum

– Can last decades

Use of Temperature to Preserve Microbes

Oxygen Requirements

Oxygen sources Found as gaseous O2 or covalently bound in compounds

Essential for aerobic respiration

Oxygen is the final electron acceptor

• Deadly for some types of bacteria (anaerobes)

Toxic forms of oxygen are highly reactive

are excellent oxidizing agents

results in irreparable damage to cells by oxidizing compounds such

as proteins and lipids

Classification of organisms based on O2 utilization

• Obligate (strict) aerobes require O2 in order to grow, Ex. Bacillus,

Pseudomonas

• Obligate (strict) anaerobes cannot survive in O2 , Ex. Clostridium sp.

Facultative anaerobes grow better in O2,Ex. E. coli, Staphylococcus

• Aerotolerant organisms don’t care about O2 ,Ex. Lactobacillus sp.

• Microaerophiles require low levels of O2

Capnophile – requires higher CO2 tension (3-10%) than normally found in

the atmosphere, Ex. Neisseria, Brucella, S. pneumoniae

38

– “Obligate” (or “strict”) means that a given condition is required

for growth

– “Facultative” means that the organism can grow under the

condition, but doesn’t require it

• The term “facultative” is often applied to sub-optimal condition

– For example, an obligate thermophile requires elevated

temperatures for growth, while a facultative thermophile may

grow in either elevated temperatures or lower temperatures

Obligate (strict) vs. facultative

OxygenToxicity

Hydrogen

peroxide

Superoxide

Hydroxyl radical (OH)

•Result of ionizing radiation & incomplete reduction of hydrogen peroxide;

extremely reactive but danger averted in aerobes because of catalase &

peroxidase

41

Oxygen Toxicity

Effects of pH

•Classification of Microbes based on pH– Organisms sensitive to changes in acidity

– H+ and OH– interfere with H bonding

– Acidophiles – prefer below 7

– Neutrophiles – prefer 7

– Alkalinophiles – prefer above 7

– Most bacteria grow between pH 6.5 and 7.5

– Molds and yeasts grow between pH 5 and 6

Physical Effects of Water

Microbes require water

to dissolve enzymes and nutrients required in metabolism; to react

in many metabolic reactions

Some microbes have cell walls that retain water

Endospores and cysts stop most metabolic activity to survive in a

dry environment for years

Two physical effects of water

Osmotic pressure

Hydrostatic pressure

Osmotic Pressure

Osmotic pressure

The pressure exerted on the semipermeable membrane by a

solution containing solutes, which cannot move across the

membrane.

Osmosis

Diffusion of water across a semipermeable membrane driven by

unequal concentration of solutes across the membrane.

Osmotic Pressure

HYPERTONICISOTONICPhysiologic Saline

Osmotic Variations in the Environment

– Isotonic

– External concentration of solutes is equal to cell’s

internal environment

– Diffusion of water equal in both directions

– No net change in cell volume

– Hypotonic

– External concentration of solutes is lower than cell’s

internal environment

– Cells swell and burst

– Hypertonic

– Environment has higher solute concentration than

cell’s internal environment

– Cells shrivel (crenate)

– Halophiles tolerate higher salt concentrations

Hydrostatic Pressure

Water exerts pressure in proportion to its depth

For every addition of depth, 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

Culture MediaMEDIA

• Nutrient preparation for microbial growth

• Must provide all chemical requirements

• Physical state (Broth-liquid, Agar-Solid)

AGAR

– Used as solidifying agent for culture media (Typically

1.5-2.0%)

– Composed of complex polysaccharides

– Advantages of agar vs gelatin:

– Generally not metabolized by microbes

– Liquefies at 100°C

– Solidifies ~40°C

Fanny Hesse used agar from seaweed(Red Algae) in her jams and jellies,

which she learned from a neighbor who had lived in Java (Indonesia).

Types of Media Used Defined medium : precise amounts of highly purified chemicals

Complex medium (or undefined) : highly nutritious substances.

Basic Nutrient

Designed to grow broad-spectrum microbes

Enriched

Add enrichment to encourage growth of microbes

Blood, growth factors, serum

Selective

Suppress unwanted microbes and encourage desired microbes to grow

Salt, dyes, alcohol

Differential

To distinguish colonies of different microbes from one another

Dyes, pH indicators

Reduced (anaerobic) media

Contain chemicals (thioglycollate) that combine O2, Used for anaerobic

cultures

Chemically Defined vs Complex Media

Selective medium

MacConkey agar as a selective and differential medium

Anaerobic Culture Methods

Gas Pak Jar Glove Box

Capnophiles require high CO2

• Candle jar

(3-10% CO2)

• CO2-packet

Planktonic vs Sessile Bacteria

Robert Koch

•All lab tests use “pure cultures” of

suspended cells called planktonic

bacteria since they float around in

liquid.

•In fact, pure cultures are virtually

absent in nature.

•Most microbes exist as sessile

bacteria– attached to a surface –

and they live in communities called

biofilms.

Biofilms

An organized, layered system of microbes attached to a surface

Biofilms form when microbes adhere to a surface that is moist and

contains organic matter

– Complex relationships among numerous microorganisms

– Develop an extracellular matrix

– Adheres cells to one another

– Allows attachment to a substrate

– Sequesters nutrients

– May protect individuals in the biofilm

– Form on surfaces often as a result of quorum sensing

– Many microorganisms more harmful as part of a biofilm

How does a biofilm develop?

1. Planktonic cells attach to surface

2. Cells multiply ;Produce glycocalyx

3. Slime layer entraps nutrients, cells, microbes

4. Dynamic pillar-like layers form

How do biofilms communicate?

•Cell to cell

communication- send and receive

chemical signaling

molecules

•Quorum sensing- accumulation of

signaling molecules

- enables a cell to

sense the cell density

Where are Biofilms Found?

Biofilms Found in Health Care

Dental caries

Contact lenses

Lungs of Cystic Fibrosis patients

Indwelling medical devices Endotracheal tube

Mechanical heart valves

Pacemakers

Urinary catheters

IV connectors

Prosthetic joints

Biofilm on a contact lens

Staphylococcus biofilm

on inner surface of

IV connector

Medical Importance of Biofilms

Are 1000X more resistant to antimicrobial agents than

planktonic cells

Easily transfer genes to express new and sometimes

more virulent phenotypes

Are more resistant to host defense mechanisms

80% of nosocomial infections are biofilm associated

(NIH)

20% of patients with biofilm-related septicemia die

Quorum Sensing• A mechanism by which members of a bacterial population can

behave cooperatively, altering their patterns of gene expression

(transcription) in response to the density of the population

• In this way, the entire population can respond in a manner most

strategically practical depending on how sparse or dense the

population is.

Mechanism:

• As the bacteria in the population grow, they secrete a quorum signaling

molecule into the environment (for example, in many gram-negative

bacteria the signal is an acyl homoserine lactone, HSL)

• When the quorum signal reaches a high enough concentration, it

triggers specific receptor proteins that usually act as transcriptional

inducers, turning on quorum-sensitive genes