Microbiology of High Temperature Environments

1. Microbiology of High Temperature Environments Microbiology of High Temperature Environments Microbiology of High Temperature Environments AmeerBallout Rui Saito Britta Voss Steve Thorsen Ameer Ballout Rui Saito Britta Voss Steve Thorsen Ameer Ballout Rui Saito Britta Voss Steve Thorsen

2. Where Are High-Temperature Environments? Hot Springs • Yellowstone National Park • Japan • Russia • Iceland • New Zealand Deep-Sea Hydrothermal vents • Found at mid-ocean ridges (Mid-Atlantic Ridge, East Pacific Rise)

3. History of Hot Springs Microbiology

4. History: Pre 19th Century • Believed aquatic plants and animals cannot live at temperatures above 37˚C • 1862 – Ferdinand Cohn first observed organisms living in hot springs in Sicily, Italy • 1875 – Felix Hoppe-Seyler recorded algae in hot springs in Naples, Italy at temperatures above 60˚C

5. History 1900 to Present • 1903 – William Setchell observed algae in hot springs at ~ 75˚C and 89˚C in Yellowstone National Park • 1978 – Thomas Brock found cyanobacteria in hot springs at 75-77˚C, and observed growth at temperatures up to 95.5˚C • Early 1990s –Thermus aquaticus discovered, utilized this organism’s thermotolerant polymerase (“Taq”) in PCR reactions • Recently – discovery of archaea, bacteria and viruses in hot springs continue Time-lapse plot of PCR

6. Terminology • Psychrophiles grow at 0-15˚C, e.g. microorganisms found in polar regions • Mesophiles grow around 25-40˚C, with optimum temperature often ~37˚C • Facultative thermophiles have maximum growth temperatures of 50-65˚C, but also grow at temperatures below 30˚C • Obligative thermophiles have maximum growth temperatures of 65-70˚C, and will not grow below 40˚C • Extreme thermophiles grow between 40-70˚C with an optimal growth temperature of about 65˚C • Hyperthermophiles grow over 90˚C with a range of optimal temperatures between 80-115˚C

7. What it Takes To Live at High Temperatures • Almost all eukaryotes cannot live above 68°C • Bacteria and archaea are usually the dominant inhabitants of hot springs (by numbers and biomass)

8. Problems Caused by Heat Proteins denature Cell membranes and genetic material denatures as well Thermophile solutions Bacteria have more saturated fatty acids in their membranes Archaea have ether (rather than ester) linkages in cell membranes Synthesize proteins that stabilize DNA conformation

9. General Hot Springs Ecology • Photosynthetic microorganisms dominate, ranging from 102 to 108/ml, found mostly in euphotic surface layers or on rock surfaces • Heterotrophic microorganisms are also found, but in lower concentrations (101 to 106/ml), and consume DOM from terrestrial and biological sources (dead photosynthetic organisms)

10. Life in Hot Springs • Thermophiles: optimum growth temp: > 45°C • The upper temp. limit of eukaryotes: 68°C • Three dominant organisms in hot springs • Archaea • Bacteria • Viruses Blue Lagoon, Iceland: Humans utilize hot springs as well.

11. Archaea Most archaea grow at temperatures of 80-85ºC • Methanogens (Methanobacterium thermophilicum) • 2 distinct lineges present? in Yellowstone hot springs (small-subunit rRNA analysis) • A little is known; research continues. A microbiologist collecting water sample at Yellowstone hot spring.

12. Bacteria I • Cyanobacteria (Synechococcus) • Thermophile photoynthetic autotrophs • Grow up to 74ºC. • “Algal mat” (Yellowstone) • Many distinct species living in vertically stratified mats based on 16S rRNA phylogenetic studies Algal mat in Yellowstone

13. Bacteria II • Heterotrophic bacteria (thermophiles) • Chemolithotrophs • H2S & sulfur oxidizers (e.g. Thiobacilus thiooxidans, Sulfolobus acidocaldarius: found in highly acidic hot springs) • Sulfate reducers (e.g. Desulfovibrio thermophilus) • Methane oxidizers (e.g. Methylococcus capsulatus)

14. Viruses • Absence of eukaryotes… but viruses are there • Thermophilic bacteriophages observed living at up to 92ºC: extreme thermophiles • Predators of bacteria • “Microbial loop” in hot springs Electron micrographs of viruses found in Little Hot Creek, OR (Breibar et al. 2004)

15. Microbial loop in hot springs Bacteria DOM Viruses

16. Biogeochemical cycles in hot springs • Carbon cycle: cyanobacteria, methanogens, methanotrophs • Sulfur cycle: sulfide hot springs: sulfur oxidizers, sulfate reducers

17. Carbon cycle Organic compounds Thermophilic cyanobacteria (Photosynthesis) Thermophilic heterotrophic bacteria (Respiration) CO2 Thermophilic methanotrophic bacteria Thermophilic methanogens (Obligative anaerobic archaea) CH4

18. Sulfur cycle Thermophilic sulfide & sulfur oxidizers (Thiobacilus, Sulfolobus) H2S SO42- Thermophilic sulfate reducers (e.g. Desulfovibrio thermophilus)

19. Applications • Great source of thermotolerant enzymes: polymerases, protenases, amylases, xylanases • Valuable tools for agricultural, medical, and industrial markets • New species of microorganisms will be found as research continues

20. Hydrothermal Vent Communities

21. A brief history • 1960s: geologists’ theory of plate tectonics predicts the existence of submarine “hot spots” • 1976: scientists in the Galapagos Rift report anomalous seawater temperatures (among other data) indicating hydrothermal activity • 1977: hydrothermal vents are directly observed by scientists in Alvin • 2005: Australian mining company Neptune Resources granted the right to explore hydrothermal vent sulfide deposits… • the UW supports many research projects in this area • Studies on the origins of life—early earth was much hotter, more acidic than today

22. Alvin

23. Alvin: deep submergence vehicle • Holds 3 people: 2 scientists and 1 pilot • Can explore depths up to 4500m • Remotely-operated vehicles can reach >6500m • Same vessel used to explore the Titanic • http://www.youtube.com/watch?v=HHlGlWyJ34I

24. How deep-sea vents form • At sea-floor spreading centers (e.g. mid-ocean ridges), 1200°C magma pours out of cracks in the lithosphere • Surrounding water is heated to 300-400°C • Minerals in sediment precipitate when hot fluid meets cold sea water (2°C) • Accumulation of minerals builds chimneys, which serve as substrates for microorganisms that can metabolize sulfur and sulfide compounds • Extreme pressure (>250 atm) keeps water from boiling • Chimneys grow (and collapse) at very high rates

26. Sea-floor spreading

27. Where vents are found

28. A unique environment to support life • NO SUNLIGHT—primary producers must utilize chemical energy (from SO42-, H2S, etc.) to fix CO2 • Extreme heat—water nearby vents where microorganisms thrive is 60-150°C • Literature argues over the hottest environment that can support life • 115°C is generally accepted as the upper limit, some claim as high as 150°C • Extreme pressure—many of the adaptations that protect against heat also protect against pressure • Highly acidic—plume pH ~2.8

29. Diversity of Microbes around hydrothermal vents • Conditions • Temperature • Superthermophiles (>115˚C) • Hyperthermophiles (80-115˚C) • O2 requirements • Anaerobic • Aerobic

30. Diversity of microbes around hydrothermal vents • Metabolic Pathways • Autotrophs • Chemolithotrophs • Nitrifying bacteria • Sulfur-oxidizing bacteria • Heterotrophs • Chemoorganotrophs

31. Diversity of microbes • Heterotrophs • an organism requiring organic compounds for its principal source of food. (C obtained from organic compounds) • Autotroph • Make their own food they extract carbon from carbon dioxide in the environment around them, and use it to build more complex, organic molecules. (C obtained from CO2)

32. Chemoorganotrophy • The most common form of bacterial metabolism • Requires organic compounds • Growth usually occurs aerobically at the expense of O2 as the terminal e- acceptor. • In the absence of O2 growth may continue either by fermentation or by the reduction of alternate e- acceptors, including NO3-, NO2-, Mn4+, Fe3+, SO4-, and CO2. • In most marine environments SO4- is found in relatively high concentrations and in the absence of O2 is the preferred e- acceptor.

33. Chemolithotrophy • Energy is obtained from the oxidation of inorganic compounds • Most chemolithotrophs are also autotrophic • There are two major objectives to chemolithotrophy: the generation of energy (ATP) and the generation of reducing power (NADH).

34. S-Oxidizers • Sulfur oxidation involves the oxidation of sulfur compounds such as sulfide (S2-), inorganic sulfur (S0) and thiosulfate (S2O22-) • A classic example of a sulfur oxidizing bacterium is Beggiatoa • Sulfur oxidizing organisms generate reducing power for carbon dioxide fixation via the calvin cycle using reverse electron flow

35. Sulfur Oxidizers cont’d • compounds are converted to sulfite (SO32-) and subsequently converted to sulfate by the enzyme sulfite oxidase • energy released is transferred to the electron transport chain for ATP and NADH production. • In addition to aerobic sulfur oxidation, some organisms like Thiobacillus denitrificans use nitrate (NO3-) as a terminal e- acceptor (anaerobic) CO2 + 2H2S -----> CH2O + 2S + H2O 2CO2 + H2S + 2H2O -------> 2CH2O + H2SO4

36. Sulfur Cycle

37. Nitrifying Bacteria • Nitrification is the process by which ammonia (NH3) is converted to nitrate (NO3-). • Nitrification is actually the net result of two distinct processes: oxidation of ammonia to nitrite (NO2-) and oxidation of nitrite to nitrate by the nitrite-oxidizing bacteria • Both of these processes are extremely poor energetically  very slow growth rates

38. Nitrifying Bacteria cont’d • Ammonia oxidation occurs by the stepwise oxidation of ammonia to hydroxylamine • The enzyme ammonia monooxygenase in the cytoplasm • The oxidation of hydroxylamine to nitrite by the enzyme hydroxylamine oxidoreductase in the periplasm • In both ammonia and nitrite oxidation O2 is required, meaning that both nitrosifying and nitrite-oxidizing bacteria are aerobes

39. Nitrogen Cycle

40. Symbiotic Relationships • Giant tubeworms with trophosomes (live in gut and within tissues), aid in supplying nutrients. • Live in gills of shrimp and limpets. The symbiotic relationship between them is still poorly understood. • Plays a significant role in sulfide detoxification (protect hosts)

41. Summary

42. References Karl, D. M. (ed.) (1995). The Microbiology of Deep-Sea Hydrothermal Vents. CRC Press, Boca Raton. Breitbart, M., L. Wegley, S. Leeds, T. Schoenfeld, and F. Rohwert (2004). Phage community dynamics in hot springs. Applied and Environmental Microbiology 70:1633-1640. Brock, T. D (1978). Thermophilic microorganisms and life at high temperatures. Spring-Verlag, New York. http://www.oceanexplorer.noaa.gov http://www.mbari.org/molecular/vents.html Jjemba, P. K. (2004). Environmental microbiology: principles and applications. Science Publishers, Enfield, New Hampshire. Lacap, D. C., G. J. D. Smith, K. Warren-Rhodes, and S. B. Pointing (2005). Canadian Journal of Microbiology 51: 583-589. Maier, R. M., I. I. Pepper, and C. P. Gerba (2000). Environmental Microbiology. Academic Press, San Diego, California. Miller, S. R. and R. W. Castenholz (2000). Evolution of thermotolerance in hot spring Cyanobacteria of the genus Synechococcus. Applied and Environmental Microbiology 66:4222-4229. Sigee, D. C. (2004). Freshwater microbiology. John Willey and Sons, Chichester, United Kingdom. Varnam, A. H (2000). Environmental Microbiology. ASM Press, Washington, DC. Ward, D. M. and R. W. Castenholz (2000). Cyanobacteria in geothermal habitats. B. A. Whitton and M. Potts (eds), The Ecology of Cyanobacteria: 37-59. Kluwer Academic Publishers. Waters, J. F. (1994). Deep-Sea Vents: Living Worlds Without Sun. Cobblehill Books, Dutton. Wright, D. J. (1996). Rumblings on the Ocean Floor: GIS Supports Deep-Sea Research. Geol. Info. Sys. 6(1): 22-29.