Extremophiles

Life on edge

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Life at High Temperatures, Thomas M. Brock

 Extraterrestrial microbial
life-does it exist?
Extremophiles

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

Lecture Aims

 What are Extremophiles- an introduction

 Strategies for growth & survival
 Biotechnology
Introduction to Extremophiles
 What are Extremophiles
 Live where nothing else can
 How do they survive?
 Extremozymes (more details later)
 Why are they are interesting?
 Extremes fascinate us
 Life on other planets
 Life at boiling temperatures
 Practical applications are interesting
 Interdisciplinary lessons
 Genetic Prospecting
Extremophile
 Definition - Lover of extremities
 History
 First suspected in 1950’s
 Extensively studied since 1970’s
 Temperature extremes
 Boiling or freezing, 1000C to -10C
 Chemical extremes
 Vinegar or ammonia (<5 pH or >9 pH)
 Highly saline, up to x10 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 1-40C
 Deep sea –10C to 40C
 Most rely on photosynthesis
 Hydrothermal Vents- Black
smokers at 350 oC

Thermophiles

Obsidian Pool,
Yellowstone National Park
Psychrophiles
Chemical Extremes
 Acidophiles - Acidic
 Again some thermal vents & hot springs
 Alkaliphiles - Alkaline
 Soda lakes in Africa and Western U.S.
 Halophiles - Highly saline
 Natural salt lakes and manmade pools
 Sometimes occurs with extreme alkalinity
Acidophiles

pH 0-1 of waters
at Iron Mountain
 Alkaliphiles

Mono Lake- alkaline

soda lake, pH 9 &
salinity 8%
 Solar salterns Owens Lake Great Salt Lake coastal
splash zones
Dead Sea

Halophiles
 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 concs 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
Energy

With Enzyme

Time
What are enzymes?
 Enzymes are just a protein
 They can be destroyed by
 Heat, acid, base
 They can be inhibited by
 Cold, salt
 Heat an egg white or add vinegar to milk
 Protein is a major component of both-
denatures
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 saline 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 P

© 1998 Reston Communications, www.reston.com/astro/extreme.html
 Thermophiles
 Most interesting, with practical applications
Many industrial processes involve high heat
 450C (113F) is a problem for most enzymes
 First Extremophile found in 1972

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 &
feel
 Detergents
 Enzymes dissolve proteins or fats
 Detergents do not inhibit alkaliphilic enzymes
Halophiles
 What is a halophile?
 Diversity of Halophilic Organisms
 Adptation Strategies
 Osmoregulation-“Compatible Solute” Strategy
 “Salt-in” Strategy
 Interesting Facts and Applications
 What is a halophile?
 Halophile = “salt loving; can grow in higher salt
concentrations
 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
25 % salt; seawater = 2% salt
Diversity of Halophilic Organisms
 Halophiles are a broad group &t can be
found in all three domains of life.
 Found in salt marshes, subterranean salt
deposits, dry soils, salted meats,
hypersaline seas, and salt evaporation
ponds.
Unusual Habitats
 A Pseudomonas species lives on a desert
plant in the Negev Desert- the plant
leaves secretes salt through salt glands.
 A Bacillus species is found in the nasal
cavities of desert iguanas- iguanas nasal
cavities have salt glands which secrete
KCl brine during osmotic stress.
Osmoregulation
 Halophiles 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.
 Structural modification of external cell
walls- posses negatively charged proteins
on the outside which bind to positively
charged sodium ions in their external
environments & stabilizes the cell wall
break down.
 “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- glycerol, sugars and their
derivatives, amino acids and their derivatives &
quaternary amines such as glycine betaine.
 Energetically synthesizing solutes is an expensive
process.
 Autotrophs use between 30 to 90 molecules of ATP to
synthesize one molecule of compatible solute.
 Heterotrophs use between 23 to 79 ATP.
“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 uniporter. 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 extreme halophile
 Halobacterium are members of domain
archaea.
 Widely researched for their extreme
halophilism and unique structure.
 Require salt concentrations between 15% to
saturation to live.
 Use the “salt-in” strategy.
 Produce ATP by respiration or by
bacteriorhodopsin.
Halobacterium
 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
 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
 Other possible applications being explored:
 Increasing crude oil extraction (MEOR)
 Genetically engineering halophilic enzymes
encoding DNA into crops to allow for salt
tolerance
 Treatment of waste water (petroleum)
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
 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