Extremophiles

Life on the edge

Life at High Temperatures, Thomas M. Brock

Extremophiles

Images from NASA, http://pds.jpl.nasa.gov/planets/

Goals
 Overview of Extremophiles
– Review some biology
– Give some applications
– Motivate you to study Microbiology!!
Introduction to Extremophiles
 What are they?
– Microbes living where nothing else can
 How do they survive?
– Extremozymes
 Why are they are interesting?
» Extremes fascinate us
 Life on other planets
» Practical applications are interesting
– Interdisciplinary lessons
» Genetic Prospecting
Extremo -
phile

 Definition - Lover of extremes

 History
– Suspected about 30 years ago
– Known and studied for about 20 years
 Temperature extremes
– boiling or freezing, 1000C to -10C (212F to
30F)
 Chemical extremes
– vinegar or ammonia (<5 pH or >9 pH)
– highly salty, up to ten times sea water
 How we sterilize & preserve foods today
Extreme Temperatures
 Thermophiles - High temperature
– Thermal vents and hot springs
– May go hand in hand with chemical extremes
 Psychrophiles - Low temperature
– Arctic and Antarctic
» 1/2 of Earth’s surface is oceans between 10C & 40C
» Deep sea –10C to 40C
» Most rely on photosynthesis
Thermophiles

Obsidian Pool,
Yellowstone National
Park
Hydrothermal Vents
Psychrophiles
Chemical Extremes
 Acidophiles - Acidic
– Again thermal vents and some hot springs
 Alkaliphiles - Alkaline
– Soda lakes in Africa and western U.S.
 Halophiles - Highly Salty
– Natural salt lakes and manmade pools
– Sometimes occurs with extreme alkalinity
Acidophiles

pH 0-1 of waters
at Iron Mountain
Alkaliphile

e.g. Mono Lake

alkaline soda lake, pH 9
salinity 8%
Halophiles

solar salterns
Owens Lake,
Great Salt Lake
coastal splash zones
Dead Sea
 Survival
 Temperature extremes
– Every part of microbe must function at extreme
» “Tough” enzymes for Thermophiles
» “Efficient” enzymes for Psychrophiles
– Many enzymes from these microbes are
interesting

Life at High Temperatures, Thomas M. Brock

Survival
 Chemical extremes
– Interior of cell is “normal”
– Exterior protects the cell
» Acidophiles and Alkaliphiles sometimes excrete protective
substances and enzymes
» Acidophiles often lack cell wall
» Some moderate halophiles have high concentrations of a
solute inside to avoid “pickling”
– Some enzymes from these microbes are interesting
What are enzymes?
 Definition - a protein that catalyses (speeds
up) chemical reactions without being
changed
What are enzymes?
 Enzymes are specific
– Lock and key analogy

Enzyme
Product B

Substrate A Product C
What are enzymes?
 Activation energy
– Enzymes allow reactions with lower energy

Without Enzyme

With Enzyme
Energy

Time
What are enzymes?
 Enzymes are just a protein
– They can be destroyed by
» Heat, acid, base
– They can be inhibited by
» Cold, salt
 Try doing this with an egg white or milk
– Protein is a major component of both
Practical Applications
 Extremozymes
» Enzyme from Extremophile
 Industry & Medicine
 What if you want an enzyme to work
– In a hot factory?
– Tank of cold solution?
– Acidic pond?
– Sewage (ammonia)?
– Highly salty solution?
One solution
 Pay a genetic engineer to design a “super”
enzymes...
– Heat resistant enzymes
– Survive low temperatures
– Able to resist acid, alkali and/or salt
 This could take years and lots of money
Extremophiles got there first
 Nature has already given us the solutions to
these problems
– Extremophiles have the enzymes that work in
extreme conditions

Endolithic algae from Antarctica; Hot springs in Yellowstone National Park,

© 1998 Reston Communications, www.reston.com/astro/extreme.html
 Thermophiles
 Most interesting practical applications so
far
– Many industrial processes involve high heat
– 450C (113F) is a problem for most enzymes
– First Extremophile found 30 years ago

Life at High Temperatures, Thomas M. Brock

 PCR - Polymerase
Chain Reaction
Life at High Temperatures, Thomas M. Brock

 Allows amplification of small sample of

DNA using high temperature process
– Technique is about 10 years old
– DNA fingerprints - samples from crime scene
– Genetic Screening - swab from the mouth
– Medical Diagnosis - a few virus particles from
blood
 Thermus aquaticus or Taq
 Psychrophiles
 Efficient enzymes to work in the cold
– Enzymes to work on foods that need to be
refrigerated
– Perfumes - most don’t tolerate high
temperatures
– Cold-wash detergents

Algal mats on an Antarctic lake bottom,

© 1998 Reston Communications, www.reston.com/astro/extreme.html
 Acidophiles
 Enzymes used to increase
efficiency of animal feeds
– enzymes help animals extract
nutrients from feed
» more efficient and less
expensive

Life at High Temperatures, Thomas M. Brock

Alkaliphiles
 “Stonewashed” pants
– Alkaliphilic enzymes soften fabric and release
some of the dyes, giving worn look and feel
 Detergents
– Enzymes to dissolve proteins or fats
– Alkaliphilic enzymes can work with detergents
Halophiles
 What is a halophile?
 Diversity of Halophilic Organisms
 Osmoregulation
 “Compatible Solute” Strategy
 “Salt-in” Strategy
 Interesting Facts and Applications
What is a halophile?
 The word halophile means “salt loving”.
 A halophile is an organism that can grow in higher
salt concentrations than the norm.
 Based on optimal saline environments halophilic
organisms can be grouped into three categories:
extreme halophiles, moderate halophiles, and
slightly halophilic or halotolerant organisms.
 Some extreme halophiles can live in solutions of
35 % salt. This is extreme compared to seawater
which is only 3% salt.
Diversity of Halophilic
Organisms
 Halophiles are a broad group that can be
found in all three domains of life.
 They are found in salt marshes,
subterranean salt deposits, dry soils, salted
meats, hypersaline seas, and salt
evaporation pools.
Unusual Habitats
 The bacterium pseudomonas was found
living on a desert plant in the Negev Desert.
The plant secretes salt through salt glands
on its leaves.
 Bacillus was found in the nasal cavities of
desert iguanas. These iguanas have salt
glands in their nasal cavities that secrete
KCl brine during osmotic stress.
Osmoregulation
 Living in high salinity poses a serious stress
that halophiles have overcome through
special processes or adaptations.
 The stress lies in the microbes ability to
maintain an internal osmotic potential that
equals their external environment.
 Osmosis is the process in which water
moves from an area of high concentration to
an area of low concentration.
Osmoregulation
 In order for cells to maintain their water
they must have an osmotic potential equal
to their external environment.
 As salinity increases in the environment its
osmotic potential decreases.
 If you placed a non halophilic microbe in a
solution with a high amount of dissolved
salts the cell’s water will move into the
solution causing the cell to plasmolyze.
Osmoregulation
 Halophiles have adapted to life at high
salinity in many different ways.
 One way is through the modification of
their external cell walls. They tend to have
negatively charged proteins on the outside
of their cell walls that stabilize it by binding
to positively charged sodium ions in their
external environments. If salt
concentrations decline their cell walls may
become unstable and break down.
“Compatible Solute” Strategy
 There are two strategies that halophiles have
evolved to deal with high salt environments.
 In the “compatible solute” strategy cells maintain
low concentrations of salt in their cytoplasm by
balancing osmotic potential with organic,
compatible solutes.
 They do this by the synthesis or uptake of
compatible solutes.
“Compatible Solute” Strategy
 Compatible solutes include polyols such as
glycerol, sugars and their derivatives, amino
acids and their derivatives, and quaternary
amines such as glycine betaine.
 Energetically this is an expensive process.
 Autotrophs use between 30 to 90 molecules
of ATP to synthesize one molecule of the
compatible solutes. Heterotrophs use
between 23 to 79 ATP.
“ Compatible Solute” Strategy
 Energy is also expended in pumping out
salts that dissolve into the cell.
 The uptake of available compatible solutes
in the environment is an adaptation they
have evolved to reduce the energy cost of
living in high salt concentrations.
“Salt-in” Strategy
 Cells can have internal concentrations that
are osmotically equivalent to their external
environment.
 This “salt-in” strategy is primarily used by
aerobic, extremely halophilic archaea and
anaerobic bacteria.
 They maintain osmotically equivalent
internal concentrations by accumulating
high concentrations of potassium chloride.
“Salt-in” Strategy
 Potassium ions enter the cell passively via a
uniport system. Sodium ions are pumped
out. Chloride enters the cell against the
membrane potential via cotransport with
sodium ions.
 For every three molecules of potassium
chloride accumulated, two ATP are
hydrolyzed making this strategy more
energy efficient than the “compatible
solute” strategy.
“Salt-in” Strategy
 To use this strategy all enzymes and
structural cell components must be adapted
to high salt concentrations to ensure proper
cell function.
Halobacterium: an example of an
extreme halophile
 Halobacterium are members of the
halophile group in the domain archaea.
They are widely researched for their
extreme halophilism and unique structure.
 They require salt concentrations between
15% to 35% sodium chloride to live.
 They use the “salt-in” strategy.
 They produce ATP by respiration or by
bacteriorhodopsin.
Halobacterium
 They may also have halorhodopsin that
pumps chloride into the cell instead of
pumping protons out.
 The Red Sea was named after
halobacterium that turns the water red
during massive blooms.
Facts
 The term “red herring” comes from the foul
smell of salted meats that were spoiled by
halobacterium.
 There have been considerable problems
with halophiles colonizing leather during
the salt curing process.
Applications
 Current applications using halophiles
include:
– the extraction of carotene from carotene rich
halobacteria and halophilic algae that can then
be used as food additives or as food-coloring
agents.
– the use of halophilic organisms in the
fermentation of soy sauce and Thai fish sauce.
Applications
 Many possible applications using
halophiles are being explored such as:
– increasing crude oil extraction
– genetically engineering halophilic enzyme
encoding DNA into crops to allow for salt
tolerance
– treatment of waste water
Conclusions
 Halophiles are salt tolerant organisms.
 They are widespread and found in all three
domains.
 The “salt-in” strategy uses less energy but
requires intracellular adaptations. Only a
few prokaryotes use it.
 All other halophiles use the “compatible
solute” strategy that is energy expensive but
does not require special adaptations.
 Genetic prospecting
 What is it?
– Think of a hunt for the genetic gold

Pr. Patrick Forterre, Extremophiles Laboratory of IGM at Orsay, France http://www-archbac.u-psud.fr/

Summary
 Now you know something about
Extremophiles
– Where they live & how they survive
 They are interesting because
– They have enzymes that work in unusual
conditions
– The practical applications are interesting