1. Extremophiles
Life on edge
Life at High Temperatures, Thomas M. Brock

2. Extraterrestrial microbial life-does it exist?
Extremophiles
Extraterrestrial microbial life-does it exist?
Images from NASA,

3. Lecture Aims What are Extremophiles- an introduction
Strategies for growth & survival
Biotechnology
Info on me, in NZ on volcanic hot springs and than here on subsurface GAB
***Any Questions, go ahead and ask **
1) You need to understand them before you can teach
2) A great deal of Info on web sites & new text books due to interest in astrobiology & who we are etc
3) These are fascinating creatures and I enjoyed putting presentation together

4. 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
2) Review of enzymes, then extremozymes
Why is this the second talk??? A natural lead in to BioTech/Computing
3) - Interesting to scientists, and are interesting to us too.
- Life on other planets
- DNA fingerprinting, stonewashed jeans, and oil extraction
- Interdisciplinary lessons

5. 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

6. 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
Who lives in the temperature extremes?
Photosynthesis???

7. Thermophiles Obsidian Pool, Yellowstone National Park
Hydrothermal Vents- Black smokers at 350 oC
Thermophiles
Obsidian Pool,
Yellowstone National Park

8. Psychrophiles

9. Chemical Extremes Acidophiles - Acidic Alkaliphiles - Alkaline
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
Can see that there is some overlap Thermophile/Acidophile and Halophile/Alkaliphile
White Sands???

10. Acidophiles
pH 0-1 of waters
at Iron Mountain

11. Alkaliphiles
Mono Lake- alkaline
soda lake, pH 9 &
salinity 8%

12. Great Salt Lake coastal
Solar salterns Owens Lake
Great Salt Lake coastal
splash zones
Dead Sea
Halophiles

13. 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

14. Survival Chemical extremes Different from temperature
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
Different from temperature
Halophiles - Osmosis
3) Because interior tends to be normal...

15. What are enzymes?
Definition - a protein that catalyses (speeds up) chemical reactions without being changed

16. What are enzymes? Enzymes are specific Lock and key analogy Enzyme
Product B
Substrate A
Product C

17. What are enzymes? Activation energy
Enzymes allow reactions with lower energy
Energy
Time
Without Enzyme
With Enzyme

18. 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
Wrapping up review of enzymes, they have limitations
Remember definition, a protein that catalyses chemical reactions without being changed

19. 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?
Remember last slide on enzymes, each of these situations destroy or inhibit an enzyme

20. 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
2) leads to genetic prospecting, but first

21. 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,

22. 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

23. 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

24. 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,

25. 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

26. Alkaliphiles “Stonewashed” pants Detergents
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
Here is a wild application that caught my eye…
Enzyme needs to work in soapy (alkaline) solution

27. Halophiles What is a halophile? Diversity of Halophilic Organisms
Adptation Strategies
Osmoregulation-“Compatible Solute” Strategy
“Salt-in” Strategy
Interesting Facts and Applications

28. 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

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. “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.

35. “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.

36. “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.

37. “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.

38. 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.

39. 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.

40. 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.

41. 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.

42. 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)

43. 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.

44. Genetic prospecting
What is it?
Think of a hunt for the genetic gold

45. 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