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Home , Xerophile

Extremophiles: defining the envelope for the search for in the universe

Lynn J. Rothschild NASA/Ames Research Center & Stanford University Moffett Field, CA 94035 USA [email protected] Overview

• What is an ? • Why do (and did) we study them? • Environmental extremes • How do they do it? • Examples of extreme ecosystems • Space: new categories of extreme environments What is an “extremophil e”? “extremus”, the superlative of exter, being on the outside plus “philos”, love equals Extremophile

Coined by Bob MacElroy, NASA Ames Research Center. Macelroy (1974) Some comments on the evolution of . Biosystems 6:74­75. Philosophical issues

• Physical, chemical, biological(?) extremes? • What is “extreme”? Simply in the eye of the beholder? Evolutionary definition? Objective definition? Caveat: it is important to talk about extremes for certain organisms - e.g., people • Must extremophiles love extreme conditions? • Must extremophile apply to all life stages? At all times? and frogs. Environmenta l extremes Categories of extremophiles Environment Type Definition Examples Temperature Hyperthermophil growth >80°C -113°, strain e 121°? Growth 60-80°C Synechococcus lividis Growth 15-60°C Mesophile humans Growth <15°C Radiation Psychrophile Psychrobacter, insects D. radiodurans Pressure Weight loving Barophile Pressure loving Shewanella viable at 1600 MPa Desiccation Cryptobiotic; Salinity Xerophile anhydrobiotic Haloarcula, Dunaliella pH Salt loving (2-5 M NaCl) Spirulina, Bacillus firmus Alkaliophile pH >9 OF4 (10.5); 12.8??

Oxygen tension Low pH loving Cyanidium, Ferroplasma

Anaerobe Cannot tolerate O2 jannaschii Miroaerophil Clostridium Chemical extremes Aerophile Homo sapiens Cyanidium caldarium Vacuum high CO2, arsenic, Electricity mercury electric eels Which taxa contain extremophiles ? Taxonomic Distribution of Extremophiles

…but these are just a few examples

Courtesy of Pace lab, 2004 Why study extremop hiles? Why study extremophiles? • Food preservation. • Basic research; model organisms for basic research • Biological warfare. • Biotech potential. • Future use in space. • Biodiversity on Earth. . Origin of life? • Mechanisms of survival: divergence or convergence? Apparent convergence by LGT? • Limits for life in the universe. Life elsewhere Richard Proctor. 1870. Other Worlds Than Ours possibly first to connect study of life in extreme environments and life on other planets. “If we range over the earth, from the arctic regions to the torrid zone, we find that none of the peculiarities which mark the several regions of our globe suffice to banish life from its surface.” (ch. 1) NASA Ames and the history of extremophile research • Ken Souza: We were working with ”extremophiles" well before the term became popular. In the 60's and earlier, people worked with halo, thermo, psychro, alkalo and acido -- philes. Ames dedicated a branch of about 25 civil servant researchers and technicians to biological adaptation (which utilized "extremophiles" to identify and understand the mechanisms life used to survive and reproduce in such harsh environments.) In the early 60's Young and his staff created a ”Mars simulator" and were looking at in freeze/thaw cycles trying to select those most suited to survive putative martian conditions. I spent some years on as did Macelroy as potential models of venution life. I also did some work with alkalophiles as potential models of jovian life (putative aerosol water layer). Hochstein, lanyi and others worked with as putative models for martian life. • Don Devincinzi: I suspect that he (Dick Young) and Chuck Klein started NASA's work on extreme environments at Ames in the mid-60s. • Sherwood Chang: Organisms that survived or thrived under ammoniated conditions were also of interest (remember the so-called primitive Earth's ammonia, methane atmosphere of Urey and Miller), particularly a rather strange bug that I recall was called "kakabeckia" or some such thing. • Bonnie Dalton: “At that time, (1963) we were all looking toward a Mars lander and the need to understand life (if it was there). Of course any life form would have to be of a nature adaptable to very extreme organisms. Our extremeophiles were the halophilic living in the SF bay.” • “Ames was the Center for this work and thus grew the concept of Astrobiology.” Examples of extreme parameter s Temperature: what difference does it make? . Solubility of gasses goes down as temperature goes up (impetus for colonization of the land?). . Organisms have upper temperature limits. Chlorophyll, proteins and nucleic acids denature at high temperatures. . Enzymes have optimal temperatures for activity; slow down at low temperature . Low temperature water freezes. Breaks membranes, increases solute concentration, etc. Temperature limits for life* 150

sulfur dependant archaea methane-producing archaea 100 Pompei worm heterotrophic 70?Pompei worm bacteria cyanobacteri anoxygenic

s a photosynthetic

50 e fung l fung alga

i protozo h i e p mosses a o s e

m vascular plants insects ostrocods 0 fish fung alga protozo bacteri archaea i e a a Himalayan midge and….?

-50 * However many organisms, including seeds and spores, can survive at much lower and higher temperatures. Synechococcus Life at high temperature

°C 65 ~ s, xu fe o C or ° hl 5 C 7

Source, > 95°C

The rmo rube crin r ~8 is 3°C Octopus Spring, Yellowstone National Park, 4 July 1999 History of high temperature extremophile research - Tom Brock, Karl Stetter • Brock’s first visit to YNP was July 1964, and was surprised by organisms in run-off channels. Stopped again to sample later in the summer. Then started to do work in YNP to get field experience before field trip to Iceland. Studies in photosynthesis, chlorophyll, RNA, DNA and protein. • 1965 tried to culture at high temperature using spring water as culture medium and springs as incubators (didn’t work.) Noticed pink, gelatinous mat at Pool A (Octopus Spring.) High temp at the time was considered 60°C. • Fall 1965 wrote NSF proposal, which was funded, and serious YNP work started 1966. Hudson Freeze cultured YT-1, later known aquaticus. • Near end of summer of 1967, starting using immersion slide technique. String in source of Octopus - first evidence for life growing in source. Later than summer showed life in boiling water. • Stetter’s original interest was looking at polymerases in Woese’s newly-proposed Archaea. History of Deep Sea Extremophile Research - Alvin

In 1977, Alvin traveled through the Panama Canal for the first time. Geology work in the Galapagos Rift was completed during February and March. The major discovery of an abundance of exotic animal life on and in the immediate proximity of warm water vents prompted theories about the generation of life. Since no light can penetrate through the deep waters, scientists concluded that the animal chemistry here is based on chemosynthesis, not photosynthesis. Since then, Alvin has located more than 24 hydrothermal sites in the Atlantic and Pacific Oceans. It also has allowed researchers to find and record about 300 new species of animals, including bacteria, foot-long clams and mussels, tiny shrimp, arthropods, and red-tipped tube worms that can grow up to 10 ft long in some vents. History of research

• Food preservation: As old a method as drying. Many Arctic communities preserved food in holes in the ice. • Snow algae - Ferdinand Cohn? • Early work on psychrophiles from the marine environment (Certes in the 1880’s and Conn in the early 20th century). ZoBell and his students particularly Dick Morita studied these organisms and were the first to get really deep-sea samples from the 1957 Galathea expeditions to ocean trenches. They were the first to study psychrophiles and barophiles. • Antarctic research, especially in prep for Mars Lake Hoare Dry Valleys Antarctica

under Lake Hoare preparing to dive under Lake Hoare

mat layers lift-off microbial mat heather pH limits for life sedges Natronobacterium sphagnum Bacillus firmus algae ephydrid flies OF4 Spirulin fung a i protist Archae Synechococc s a us rotifer Sulfolobus carp s haloarchea

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 pH Zygogonium sp.

Zygogonium is a genus of filamentous green algae. This species is acidophilic. What has driven pH work? • Food preservation. • Biotech. Recent example. Kenya Wildlife Service (KWS) planned to launch a multi-million dollar legal claim against Genencor and Procter & Gamble, alleging that a microbial cellulase enzyme illegally obtained from a soda lake in the country was used as an ingredient in the latter company's Tide laundry detergent. Kenyan dispute illuminates bioprospecting difficulties. Nature Biotechnology 2004. http://www.nature.com/news/2004/041108/pf/nbt1104-1337_pf.html • Biochemistry. • Life on Venus. Sechbach and others in 1960s. History of Halophile Research

• Noted red waters in biblical times, as did Darwin in South America. But halophiles first noted as cause because of food preservation issues. • Preparation for Viking. • Evaporites - endoevaporites; panspermia and Mars. • Biotechnology. Salinity

• Halophiles: 2-5 M salt • Include Archaea and a . • Dunaliella salina is used in biotech industry. Produces glycerol and - carotene. • The bacterial halophiles have been fown in space. • Have internal osmotica (e.g., K+, glycine betaine). Desiccation - food preservation

• On of the oldest methods of food preservation. In ancient times the sun and wind would have naturally dried foods. Evidence shows that Middle East and oriental cultures actively dried foods as early as 12,000 B.C. in the hot sun. • Later cultures left more evidence and each would have methods and materials to refect their food supplies ﾑ fish, wild game, domestic animals, etc.Vegetables and fruits were also dried from the earliest times. • The Romans were particularly fond of any dried fruit they could make. In the Middle Ages purposely built still houses were created to dry fruits, vegetables and herbs in areas that did not have enough strong sunlight for drying. A fire was used to create the heat needed to dry foods and in some cases smoking them as well. • Includes meats (esp. pork), fruits (e.g., raisins) and cereal grains (e.g., rice, wheat). Desiccation • Can be correlated with salinity tolerance. • Cell growth at normal temperatures usually requires water potential, aw (defined as pH2O [liquid solution] / pH2O [pure liquid water], where p is the vapor pressure of the respective liquid) of >0.9 for most bacteria and Evaporite, Baja >0.86 for most fungi. California Sur • Lowest value known for growth of a bacterium at normal temperatures is aw = 0.76 for Halobacterium. • Possibly a few organisms, e.g. lichens in the Negev Desert, can survive on water vapor rather than liquid water. • Don’t repair cell damage during desiccation, so must be good at repair upon rehydration.

Inca ruin, 15,000 ft, Bolivian altiplano Radiation: Food Preservation

• The story of goes back to 1957 when it was found in a can of ground meat that spoiled despite having been sterilized by radiation. Scientists were shocked. How could it withstand such damaging radiation? • But, even earlier, one or two years after the discovery of radioactivity, 1879, and X-rays, 1895, these phenomena were were found to be lethal to microbes. The component of light which was lethal was UV. This was published in 1903. Thus the ideas which gave rise to ultra-violet sterilization, 60Co radiation and particle accelerated sterilization were known by the end of the 19th and beginning of the 20th centuries. http://www.genomenewsnetwork.org/articles/2004/10/15/radiodurans.php Radiation

• Some forms of radiation have been a constant for organisms over geological time, whereas others vary seasonally and diurnally. Exposure may depend on ecology. • Man-made radiation can result in novel exposures or exposure levels. • Some radiation is blocked by the Earth’s atmosphere, and thus is newly relevant with respect to interplanetary travel or to an potential extraterrestrial biota. The Solar Spectrum

UVC UVB UVA visible 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 6 1 2 3 4 5

DNADNA proteinprotein

rays x-rays UV infrared microwaves radio waves

2 1 0 - 9 - 8 - 7 - 6 - 5 - 4 - 3 - 2 - 1 0 1 2 3 4 5 - 1 - 1 - 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 wavelength (m) Growth rate of yeast under solar radiation

30000000

25000000 OP3

l 20000000 Mylar m / s l l e

c 15000000 UVT 10000000

5000000

0 0 6 12 18 24 Time (hr)

•Parent strain, S. cerevisiae obtained from Research Genetics. Yeast grown in 50% YEPD, full solar radiation. Jackie Garget, 22 March 2000. UVB had most effect. High oxygen

. Oxygen is the one environmental extreme that we consider “NORMAL” . I will argue that this is one of the WORST environmental extremes. . Conclusion: WE are extremophiles too. Aerobic metabolism: a critical evolutionary innovation • Aerobic metabolism is more efficient than anaerobic • Without it, no oxygenic photosynthesis • Without it, the origin of large animals is unlikely Chemistry of oxygen

The relatively inert O2 can be reduced to a number of different reactive oxygen species, including the superoxide anion

¯ (O2 ), hydrogen peroxide (H2O2) and the hydroxyl radical (OH), and to H2O.

O2  O2¯  H2O2  · OH  H2O How do they do it? pH

+ . pH is as –log10[H ]. At pH 0, [H+] = 1 M. . Easiest is to keep internal pH near neutral. . : maintain neutral pH. Strong proton pump or low proton membrane permeability? . : internal pH 2+ units below medium. Need effective proton transport system. Serious problem for membrane-bound ATP synthase system in bacteria. . Thus, to get extremozyme, must isolate them from cell wall or underlying cell membrane. Mechanisms of Low Temperature destruction

1 2 3

cell cell cell

ice extracellular water ice

1. Cell in extracellular water containing solutes. 2. Ice formation removes water and concentrates the solution in the remaining liquid water. 3. The increase in osmotic concentration outside the cells draws water out of the cells. (redrawn from Wharton, Life at the Limits) Ways to deal with UV damage...

AvoidAvoid damagedamage production of UV-absorbing pigments (i.e., scytonemin, MAAs, phycoerythrin) production of quenchers, antioxidants, living under UV- enzymes absorbing material to neutralize (water, dead radicals cells...) RepairRepair damagedamage Select ecosyste ms Examples of extreme ecosystems

• Geysers, vents • Ice, polar regions • Subsurface • High altitude • High salt • High oxygen • Mine drainage • Nuclear reactors • Soda Lakes • Atmosphere Biota associated with geysers

• Biota depends on pH, temperature (distance from source), chemical composition. • Ex: When the correct combination of temp (~40°C) and pH (~2.5- 3.5) exists, Cyanidium caldarium is found.

Left, Nymph Creek; upper right, micrograph of Nymph mat, bottom right, Iron Spring Radon bubbles

•Contain radon gas, radium Paralana Spring, source

In spite of high levels of radon, radium and uranium, not to mention a high UV fux, there is a lush microbial community growing in the source of Paralana Spring. High Altitude: Bolivian Altiplano

• Low oxygen • High UV • Desiccation • Often winds and cold temperatures New Zealand radiation: Environment of early •PAR earth •UV atmosphere: •oxygen/ozone •carbon dioxide

rotation of Earth impactors

water: •temperature •radiation (PAR, UV) •chemistry (pH, DIC, DO....) The Atmosphere. Aerobiology

• Major challenges include: desiccation, temperature, ultraviolet radiation, trace gases in the atmosphere & “open air effect” • Pasteur early work. • Military involvement. • Today - preservation & disease spread. Space: a new category of extreme environment Why is life beyond earth difficult? √ Differences in atmospheric composition √ Altered gravity √ Space vacuum √ Temperature extremes √ Nutrient sources (e.g., organic carbon, nitrogen) √ Different radiation regime (solar and cosmic) BioPan exposure to space. to exposure weeks two survive could prokaryotes two that showed Mancinelli rocket. Foton Russian the aboard orbit earth in • exposure to space. to exposure weeks two survive could prokaryotes two that showed Mancinelli rocket. Foton Russian the aboard orbit earth in m 1 m 1 ESA’s fown facility exposure space biological shaped pan ESA’s fown facility exposure space biological shaped pan   Extremophiles beyond Earth

Multiple Mars possibilities

spacecraft Titan? meteors comets ? "biozone” in Venusian clouds European ice & ocean Extant life on Venus? Dirk Schulze- Makuch, David Grinspoon

• Life started in Venusian ocean (or Mars or Earth) • As conditions deteriorated on the surface, organisms adapted to life in clouds. • On Venus, clouds are at 31 miles (50 kilometers) altitude - potential "biozone" . Water droplets present, temperature a pleasant 158°F. Below that height it is too hot, and any higher there’s too much UV radiation and cosmic rays. Possibly use sulfur compounds as UV shield. • Rationale: atmosphere relatively low in CO, but do find H2S and SO2 together. Also find carbonyl sulphide, which is difficult to make except by life. Mars as an extreme environment • Temperature: nippy. • Radiation: Mars is 1.5 AU, so overall solar radiation is

43% of Earth. CO2 atmosphere protects surface below 190 nm. Other forms of radiation? • Oxidants: Realized presence of oxidants after Viking. • Liquid water? Past, periodic, hydrothermal activity? • Note that atmosphere is not extreme Earth analogs for Mars: Temperature Earth analogs for Mars: Radiation • Subsurface communities • Self-shading - mats • • Endoevaporites

Rothschild, L.J. and Giver, L.J. (2003) Photosynthesis below the surface in a cryptic microbial mat. Intl. J. Astrobiology 1: 295-304. Earth analogs for Mars: water Polar caps. Liquid water? Hydrothermal systems. Fluid inclusions, Sea Ice Earth analogs for Mars: Endoevaporites

Rothschild, L.J. (1990) Earth analogs for Martian life. Microbes in evaporites, a new model system for life on Mars. Icarus 88: 246-260. Rothschild, L.J., L.J. Giver, M.R. White & R.L. Mancinelli (1994) Metabolic activity of in evaporites. J. Phycol. 30: 431-438. Why Europa? Liquid water, charged particle-induced chemistry, volcanic activity(?)

iron/nickel core surface ice, (600±150 km radius) several to 10 km thick

water (ice or liquid, ~100 ±25 km)

rock shell

Radius of Europa: 1565 km, a Thin, disrupted, ice crust. little smaller than our Moon’s Images collected in 1996 by radius. Galileo. Concluding thoughts. • Extremophiles are intimately connected with the fact that we are based on organic carbon in liquid water. • The story of extremophile research is many stories that have come together in the field of astrobiology. • The envelope for life is far beyond what we could have imagined; thus, the habitats for life have been expanded. • Some adaptations to life in extreme environments are biochemically simple or convergent, suggesting that it could happen elsewhere. • The reasonable man adapts himself to the world; the unreasonable one persists in trying to adapt the world to himself. Therefore, all progress depends on the unreasonable man. George Bernard Shaw A good start in the literature

• Rothschild, L.J. & R.L. Mancinelli (2001) Life in extreme environments. Nature (London) 409: 1092-1101. • Rothschild, L.J. and Giver, L.J. (2003) Photosynthesis below the surface in a cryptic microbial mat. Intl. J. Astrobiology 1: 295-304. • Rothschild, L.J. & A. Lister (eds.) Evolution on Planet Earth: The Impact of the Physical Environment. Academic Press. 2003. 456 pp. • Human adaptation: Frances Ashcroft (2001) Life at the Extremes. The Science of Survival. Flamingo Press, London. 326 pp.