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Title: PROKARYOTES

1
PROKARYOTES
NUTRITION
ECOLOGY AND GROWTH
2
BACTERIAL UBIQUITY

Prokaryotes are found almost everywhere
From deep oceans to volcanoes
From polar regions to equator
From the Great Salt Lake to freshwater streams
Individual species differ and grow in a limited
set of environments
May grow at temperatures near boiling but not at
moderate temperatures
May grow in the Great Salt Lake but not in
freshwater streams

3
BACTERIAL UBIQUITY

Growth is dependent upon many factors
Host organism
Temperature
pH
Available nutrients
Etc.

4
CULTURING BACTERIA

Microorganisms grow in mixed populations in
nature
Joint contributions to numerous processes
Microorganisms are grown in pure culture in the
laboratory
Requires isolation from a mixed culture
Facilitates study of functions of a particular
species

5
CULTURING BACTERIA

Only 0.1 of prokaryotes can be grown in pure
culture
Most environmental microbes are difficult to
study
Growth requirements difficult to determine
Most medically important bacteria can be grown in
pure culture
Why do you suppose this is the case

6
CULTURING BACTERIA

Various techniques of culturing microorganisms
Equipment must be sterile
Free of microorganisms
Aseptic technique employed
Minimize chance of inadvertent introduction of
microorganisms
Various types of media
Contain necessary nutrients
Solid or liquid

7
MEDIA

Liquid media
Nutrients required for growth
Different requirements for different species
Cannot be used to isolate pure cultures
Solid media
Liquid media plus solidifying agent agar
Allows isolation of pure cultures
Containers
Petri dish (plates) tubes (deeps slants)
Not airtight (allows gas exchange)
Excludes airborne microorganisms

8
AGAR

Resistant to bacterial degradation
Survives high-temperature treatment
Can be sterilized in autoclave
Liquefies at high temperatures
Can be poured into convenient containers
Remains liquid until below 45oC
Once solidified remains solid over a wide range
of temperatures
Melts at 95oC
Translucent
Colonies are visibly apparent

9
PURE CULTURE

Bacteria are separated and placed on solid medium
Various types of dilutions and plating techniques
employed
Individual bacteria divide to form visible
colonies
Visible at gt 1 million cells
Genetically identical
Clone
Each clone is a pure culture

Streak-Plate Isolation
10
STOCK CULTURES

After obtaining a pure culture it is maintained
as a stock culture
Maintained as an inoculum for later study
Various means of storage
Agar slant
Frozen in glycerol solution
Glycerol prevents damage to cells from ice
crystals
Lyophilized (freeze-dried)

11
BACTERIAL GROWTH

Defines as an increase in cell number
Division by binary fission
Simpler than mitotic division
Cell size doubles
Cell components doubled
Cell divides
Repeat
Growth is exponential
1248 etc.

12
BACTERIAL GROWTH

Doubling time / generation time
Time required for population to double in number
Variable dependent upon
Species
Growth conditions (nutrients temp etc.)
Escherichia coli optimally 20 minutes
Mycobacterium tuberculosis 12 24 hours
Longer under suboptimal conditions

E. coli dividing
13
EXPONENTIAL GROWTH

How to get rich quick
On the first day of the month put a penny in a
jar
On the second day double this amount (2 cents)
On the third day double this amount (4 cents)
Etc.

Day 1 0.01
Day 5 0.16
Day 10 5.12
Day 15 163.84
Day 20 5242.88
Day 25 167772.16
Day 28 1342177.28
Day 30 5368709.12

14
EXPONENTIAL GROWTH

Bacteria have the ability to increase
exponentially
Exponential growth increases numbers incredibly
quickly
Exponential growth is not always possible
Growth can be limited by various factors
Environmental factors
Nutrient availability

15
ENVIRONMENTAL FACTORS

Various environmental factors affect prokaryotic
growth
Temperature requirements
O2 requirements
pH requirements
Water availability
Interactions with other organisms

16
TEMPERATURE REQUIREMENTS

All species can grow within a particular range of
temperatures
Enzymes denatured above this range
Optimal temperature within this range
Divided into groups based on temperature
optimum
How does temperature affect the rate of chemical
reactions
How would the enzyme DNA polymerase differ
between mesophiles and thermophiles etc.

17
TEMPERATURE REQUIREMENTS

Psychrophiles (-5ºC 15ºC)
Psychrotrophs prefer gt 15ºC but tolerate lower
e.g. Listeria monocytogenes (food poisoning)
Mesophiles (25ºC 45ºC)
e.g. Escherichia coli
Most other common bacteria most human pathogens
Thermophiles (45ºC 70ºC)
Thermus aquaticus from thermal springs
Hyperthermophiles (70ºC 110ºC)
Members of Archaea
Many members of Archaea are extremophiles
Pyrolobus fumarii isolated from a hydrothermal
vent has max growth temp of 113ºC

18
TEMPERATURE AND DISEASE

Leprosy (Hansens disease)
Caused by Mycobacterium leprae
Typically affects body extremities
Ears hands feet fingers
Syphilis
Caused by Treponema pallidum
Lesions generally appear on genitalia lips
tongue throat
Early treatments involved induction of fever by
deliberate infection with malaria parasite
Antibiotics are a kinder gentler treatment
What can you determine regarding the temperature
requirements of these bacteria

19
OXYGEN REQUIREMENTS

O2 can be useful
e.g. aerobic cellular respiration
O2 can be damaging
Readily converted into toxic compounds
esp. superoxide (O2-) and hydrogen peroxide
(H2O2)
Enzymes detoxify these toxic compounds
Superoxide dismutase O2- H2O2
Catalase H2O2 H2O O2
Not all bacteria possess these enzymes
Other enzymes exist that detoxify these compounds

20
OXYGEN REQUIREMENTS

O2 levels vary widely in different environments
Earths atmosphere is 20 O2
O2 is absent from some environments
e.g. swamps beneath soil surface human
intestines etc.
Bacteria differ in their requirements for O2
e.g. Some bacteria absolutely require O2 for
cellular respiration
Obligate aerobes
Typically possess enzymes detoxifying O2- and
H2O2
Bacteria differ in their tolerance of O2
e.g. Some bacteria cannot tolerate O2
Obligate anaerobes
Typically lack enzymes detoxifying O2- and H2O2

21
OXYGEN REQUIREMENTS

O2 requirements determined using a shake tube
(Could also use thioglycollate broth)
Sterile tube of nutrient agar boiled
Agar melted O2 driven off
Cooled to 50oC bacteria added
Agar hardens
Incubate note areas of growth
What enzymes do you think each of these bacteria
possess
Why do facultative anaerobes grow better when O2
is present

22
pH REQUIREMENTS

All species can grow within a particular range of
pH values
Optimal pH within this range
Divided into groups based on pH optimum
Acidophiles (optimal pH lt 5.5)
Neutrophiles (optimal pH near neutral)
Alkalophiles (optimal pH gt 8.5)
Internal pH constant and typically near neutral
Often maintained by ion pumps

23
WATER AVAILABILITY

Water is absolutely required for growth
Constitutes 70 of cell
May be present but osmotically unavailable
High solute environment removes H2O from cell

Solar Evaporation Pond
(Red color due to pigments of halophilic
organisms)
24
WATER AVAILABILITY

Microbes differ in their ability to live in high
salt environments
Two main ways to deal with high osmolarity in the
environment
Actively pump ions into cell
e.g. K
Produce small solute molecules to increase
internal osmolarity
e.g. proline

25
WATER AVAILABILITY

Osmotolerant
Organisms able to tolerate salt concentrations up
to 10 but not requiring high salt
concentrations
a.k.a. facultative halophiles
Halophiles
a.k.a. obligate halophiles
Organisms requiring high levels of NaCl to grow
3 9 minimum NaCl required
Frequently spoilage organisms in high-salt or
high-sugar preserved foods
Examples of such foods
Many extreme halophiles belong to domain Archaea

26
MICROBIAL ASSOCIATIONS

Microbes do not grow in pure culture in nature
Live in shared habitats and interact with other
organisms
Symbiosis
Close partnership between organisms
Three main types
Mutualism
Commensalism
Parasitism
Non-symbiotic relationships
Synergism
Antagonism

27
MUTUALISM

Association between organisms in which both
benefit
Lichen consist of a fungus and an alga (or
cyanobacterium)
What does the fungus gain
What does the photosynthetic partner gain
Protozoa in a termites gut hydrolyze cellulose
What does the termite gain
What does the protozoan gain

28
COMMENSALISM

Association between two organisms in which one
partner benefits and the other is unaffected
e.g. Satellitism
One bacterium produces a growth factor required
by the second bacterial species
Small colonies of the second species are able to
grow near colonies of the first
See figure 7.11 p. 206 Talaro Talaro

29
PARASITISM

Association between two organisms in which one
partner benefits and the other is harmed

Oral syphilis lesions
Bacteriophage infection
Tobacco mosaic virus
Ergot fungus on rye
Chlamydia attached to oviduct mucosa
30
NON-SYMBIOTIC ASSOCIATIONS

Synergism
Interrelationship between two or more free-living
organisms of benefit to all
Relationship not necessary for survival
Similar to mutualism
Antagonism
Association between free-living species arising
from competition
One organism secretes a substance that inhibits
or destroys other organisms
e.g. antibiotics

31
MICROBIAL NUTRITION

Process by which chemical substances (nutrients)
are acquired from the environment and used in
cellular activities
Nutritional requirements
Source of elements
Source of energy
Substances required by an organisms are termed
essential nutrients

32
MICROBIAL NUTRITION

Energy source
Phototroph
Derives energy from sunlight
Chemotroph
Derives energy from chemicals

Carbon source
Autotroph
Obtains carbon as CO2
Heterotroph
Obtains carbon in organic forms

33
NUTRIENTS

Macronutrients
Required in relatively large amounts
Principle roles in cell structure and metabolism
e.g. energy source
e.g. carbohydrates proteins etc.
Micronutrients
a.k.a. trace elements
Enzyme cofactors etc.
e.g. Mg Zn Ni etc.
Requirements differ between species

Organic nutrients
Contain both C and H
e.g. carbohydrates lipids nucleic acids
proteins CH4 etc.
Not required by all microorganisms
Inorganic nutrients
Lack either C or H
e.g. metals salts gases water
Natural reservoirs are mineral deposits water
air
Required by all microorganisms

34
CELLULAR CHEMISTRY

Cytoplasmic composition of Escherichia coli
Aside from the 70 H2O
97 organic molecules (mainly proteins)
What are the four major classes of biological
macromolecules
96 composed of six elements (CHNOPS)
5000 different compounds
Nutritional requirements very minimal
Water glucose and a few salts
No growth factors required (more on these later)

35
REQUIRED ELEMENTS

Major elements
Elements comprising cell constituents
e.g. C H N O P S etc.
Must be supplied in a usable form
e.g. CO2 is usable by some (not all) organisms
e.g. N2 is usable by very few organisms
Trace elements
Required in minute amounts
e.g. Co Zn Cu Mb Mn etc.
Typically enzyme cofactors

36
GROWTH FACTORS

Some bacteria cannot synthesize some of their
cell constituents
e.g. certain amino acids vitamins etc.
A supply of these compounds is required for
growth
These required compounds are termed growth
factors

Not all bacteria require growth factors
Growth factor requirements differ for different
bacteria
e.g. E. coli requires no growth factors
e.g. species of Neisseria require 40 growth
factors
7 vitamins 20 amino acids etc.
Bacteria requiring many growth factors are termed
fastidious

37
NUTRIENT SOURCES

Ultimately derived from an inorganic reservoir
Source of nutrients
Replenished by organisms
Nutrient cycling is a critically important
ability of microorganisms

38
CARBON

Heterotroph
Requires carbon in an organic form
e.g. proteins carbohydrates etc.
Nutritionally dependent upon other life forms
Not all organic carbon us usable by all organisms
Did you ever eat a pine tree
Autotroph
Uses CO2 as its carbon source
Converts CO2 into organic carbon
Not nutritionally dependent upon other life forms

39
NITROGEN

Important component of proteins DNA RNA
Primary nitrogen source for heterotrophs
Main inorganic reservoir is atmospheric gas (N2)
79 of Earths atmosphere
N2 can be used by a small number of
microorganisms
Nitrogen fixation
N2 NH3

Rhizobium on clover root
40
NITROGEN

Decomposers convert organic nitrogen into NH3
Ammonification
NH3 can be converted to other inorganic forms of
nitrogen by various microorganisms
NO3- NO2- NH3
Many organisms can use one or more of these
inorganic forms of nitrogen

41
OXYGEN

Required for aerobic cellular respiration
Major component of various macromolecules
Even anaerobes require oxygen in some form
Main inorganic reservoirs
Free oxygen (O2) constitutes 20 of Earths
atmosphere
Oxygen is also present in many inorganic salts
e.g. sulfates phosphates nitrates etc.

42
HYDROGEN

Major element in all organic molecules
esp. carbohydrates proteins etc.
Also important element in many inorganic
compounds
e.g. H2O many salts
Important element in certain gases
H2S CH4 H2
These gases both used and produced by microbes

43
PHOSPHORUS

Important component of certain molecules
DNA RNA ATP phospholipids etc.
Main inorganic source is phosphate (PO43-)
Present in rocks ad other mineral deposits
Generally very scarce in environment
Scarcity of phosphorus is typically limiting
factor for growth
Overabundance in water leads to eutrophication

44
SULFUR

Essential component of some vitamins and amino
acids
Mineral form widely distributed in environment
Hydrogen sulfide gas (H2S)
Elemental sulfur (S)
Sulfides (e.g. FeS)
Sulfate (SO42-)
Various forms usable by various microorganisms

Acid drainage from a mine
Bacteria oxidize metal sulfides producing
sulfuric acid (low pH)

45
MEASURING GROWTH

Direct cell counts
Counts all cells alive or dead
Viable cell counts
Counts only cells able to multiply
Measuring biomass
Measures turbidity total weight or nitrogen
Measuring cell products
Byproducts of metabolism

46
DIRECT CELL COUNTS

Direct microscopic count
Bacteria placed in counting chamber
Holds known volume of liquid
Counted under microscope
Requires gt107 cells/ml
Cell-counting instruments
Coulter counter
Counts cells as they pass through a small hole
single file
Cells interrupt an electrical current
Flow cytometer
Similar to Coulter counter
Measures passing cells by scattering of light
from a laser

47
VIABLE CELL COUNTS

Plate counts
Prepare serial dilutions
Known volume added to plate
Pour plate or spread plate
Incubate count colonies
Isolated cell colony
Colony-forming unit
Requires gt100 cells/ml
Membrane filtration
Used when cell concentration is low
Known volume of liquid passed through filter
Bacteria retained by filter plated

48
VIABLE CELL COUNTS

Most Probable Number (MPN)
Prepare serial dilutions
Incubate tubes
Note growth
Compare results to MPN table
Gives statistical estimate of cell
concentration
Frequently used to quantify fecal coliforms

49
MEASURING BIOMASS

Measuring turbidity
Spectrophotometer measures light transmittance
through specimen
Requires gt107 cells/ml

Measuring total weight
Wet weight
Dry weight
Measuring N content
Treat cells with H2SO4
Nitrogen converted to NH3
Assay NH3
Compute biomass
Cells are typically 14 NH3
Cannot differentiate between living and dead
cells

50
MEASURING CELL PRODUCTS

Acid production
Acids often produced as a result of the breakdown
of sugars
Detected by pH indicators
Gas production
Visually detected in Durham tubes
Detected by pH indicators
CO2 slightly reduces pH
CO2 H2O H2CO3 H HCO3-
ATP production
Add firefly enzyme luciferase
Fluorescence requires ATP hydrolysis
Fluorescence quantified

51
BACTERIAL GROWTH

Bacteria have the ability to increase
exponentially
Exponential growth increases numbers incredibly
quickly
Exponential growth is not always possible
Growth can be limited by various factors
Environmental factors
Nutrient availability

52
GROWTH CURVES

Populations typically a predictable pattern of
growth over time
Growth curve
Four distinct phases
Lag phase
Log (exponential) phase
Stationary phase
Death phase

53
LAG PHASE

No cell division
Preparation for cell division
Synthesis of macromolecules
Length of phase depends on conditions
e.g. age of cells richness of medium storage
temperature etc.

54
LOG PHASE

Exponential growth
Cell division exceeds cell death
Shortest generation time measured here
More susceptible to antibiotics and other
chemicals

55
STATIONARY PHASE

Resources depleted or toxins accumulate
Cell division cell death
Total number of viable cells remains relatively
constant
Cells more resistant to antibiotics and other
chemicals

56
DEATH PHASE

Cell death exceeds cell division
Cells die at a fairly constant rate
Slope generally less steep than growth phase

57
COLONY GROWTH

Growth in colonies follows a growth curve
Different parts of the colony are at different
points in the growth curve
Resources depleted sooner in center of colony
Reach stationary phase sooner
Exponential growth continues at edges

58
MIXED MICROBIAL COMMUNITIES

In nature species live in close association with
other species
e.g. mouth intestines soil water
Interactions between species can optimize
environment for other species
e.g. aerobes use up O2 reducing O2 levels for
anaerobes
e.g. metabolic wastes may provide nutrients for
another species
Difficult to reproduce in laboratory
Generally hard to grow these species

59
BIOFILMS

Bacteria attached to surfaces encased in
polysaccharide
Slippery rocks in streams
Slime in kitchen drain
Scum in toilet bowls
Plaque on teeth
Etc.

Dental plaque a biofilm
60
BIOFILM FORMATION

Bacteria adhere to surface
Loose glycocalyx produced
Other species may attach to glycocalyx and grow
Cells form characteristic architecture with open
channels for nutrients and waste
Cell-cell communication important in structure

Latex bead moving through biofilm channel
(time-lapse sequence)

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