IN THE icy expanse of the Arctic Ocean, a strange beast glides through the endless tunnels that honeycomb the floating sea ice. Propelled by a whip-like tail, it thrives at temperatures that would kill a human in minutes.
As winter approaches, the mercury drops and the sea ice hardens. Those tunnels of water close up and almost disappear. Temperatures plummet below -20 °C. But the whip-tailed beast, a bacterium called Colwellia 34H, remains alive and well, sealed in the ice in bubbles of briny liquid not much larger than its own single-celled body.
Colwellia used to be seen as a freak of nature, the hardiest of all cold-loving bugs. But biologists are starting to realise that it is not at all unusual. Wherever they look – in permafrost, icebergs, glaciers or ice caps – they find die-hard life forms whose appetite for enduring the cold simply astonishes.
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Take the recent discovery that deep in the ice sheets of Antarctica and Greenland, at temperatures as low as -40 °C, bacteria buried kilometres down survive for hundreds of thousands of years, hunkered beneath a liquid film of life-giving water as little as three molecules thick. These bacteria aren’t even cold specialists. They ended up here by accident, but somehow they are surviving. Even more amazingly, Colwellia has shown signs of life at the temperature of liquid nitrogen, which would solidify your finger within a minute if you dipped it in.
“It’s mind-boggling,” says Dirk Schulze-Makuch, an astrobiologist at Washington State University in Pullman. “It could extend the [temperature] range for possible life quite a bit further down.” Not just on Earth: these are some of the surest signs to date that maybe we are not alone in the solar system, that life might be eking out an almost immortal existence on icy worlds such as Mars or Jupiter’s moon Europa.
Over the past 30 years biologists have become comfortable with the idea that life can thrive at extremes of temperature, pressure and pH, but most of the action has been at the upper limits – the scorching springs of Yellowstone park, for instance, or boiling vents at mid-ocean ridges.
Compared with these extremes, cold-loving species, known as psychrophiles, have always looked like wimps. Before the latest discoveries, the record for the coldest temperature at which bacteria were known to metabolise was -20 °C, and microbiologists estimated that life’s “absolute zero” was not much lower. All of this put a damper on the prospects for life elsewhere in our solar system, since many potential habitats are on the wrong side of the lower limit, never rising above -40 °C and often plunging much lower.
“Look at the study of life in extreme environments,” says John Priscu of Montana State University in Bozeman, “and almost everything in the last 30 years has been hot springs. People never even thought of ice as a habitat.” That, he says, is changing.
Speculation about life in extremely cold environments has long hinged on the principle that life needs water. Water provides a medium for nutrients to diffuse into cells and wastes to drift out, as well as providing a solvent for the metabolic reactions essential to life. That water must be liquid – encroaching ice crystals make mincemeat of living cells.
However, where there is ice, vestigial bits of water often remain, even as temperatures plummet. Icy microenvironments like the one where Colwellia spends the winter are more common than we thought.
In the sea ice where Colwellia lives, ice crystals exclude salt as they grow, concentrating it in liquid veins trapped between ice crystals. The salt drives down the freezing point of the water by up to 50 °C, providing Colwellia with a liquid home. We have long known about these liquid veins, but only in the past few years have they been appreciated as potential habitats for life.
Another haven is provided by nanofilms. These thin films of liquid water coalesce on the surface of mineral grains trapped in ice, because water molecules cling to the electrically charged mineral surface in a way that prevents them orienting into ice crystals.
Physical features of ice such as these all help life along, but bacteria need their own survival tricks too. The recent publication of Colwellia‘s genome sequence, the first for a psychrophile, show that the bacteria themselves engineer their environments (Proceedings of the National Academy of Sciences, vol 102, p 10913). Many of DZɱ’s genes encode proteins that are secreted into its surroundings, or that manufacture other molecules for secretion. “This bacterium is very interactive with its environment,” says Jody Deming, the marine microbiologist at the University of Washington in Seattle who led the genome sequencing team. “It’s investing a lot of genetic information in releasing things into its surroundings.”
As the weather gets colder, Colwellia starts oozing a gloopy concoction called exopolymer. This mixture of stringy, starch-like molecules absorbs water and forms gels inside the ice veins. As temperatures drop, expanding ice crystals suck water back out of the gel, leaving the remaining water molecules isolated from one another. “These pockets of water molecules are so small that ice nuclei can’t grow,” says Christopher Krembs, a biological oceanographer at the University of Washington. The result is a glassy state that is still essentially liquid, but resists freezing. No one knows how powerful the exopolymer’s effect is, but xanthan gum, a similar substance used in the food industry, can prevent ice crystallisation at -200 °C. Swaddled in exopolymer, Colwellia is shielded from encroaching ice crystals that would damage it, and from concentrated salts that would dehydrate it.
DZɱ’s protective coating certainly does the trick. At -7 °C the bacterium zips around as quickly as our gut bacterium Escherichia coli swims at body temperature. At -14 °C it can still grow and divide, and in sea ice at -20 °C it continues respiring (Applied Environmental Microbiology, vol 70, p 550).
Last year, Deming’s colleague Karen Junge brought Colwellia into the lab to test its cold tolerance more precisely. To get a measure of its metabolic rate, she incubated it at various temperatures between 13 °C and -20 °C to see how quickly it incorporated a radioactively labelled amino acid, leucine, into newly manufactured protein. As a control, she also incubated Colwellia at -80 °C. At that temperature even Colwellia, she assumed, would be dormant or probably dead.
She was wrong. Colwellia built radioactive leucine into new protein at all temperatures, including -80 °C. So Junge tried harsher conditions. She plunged a tube of bacteria into liquid nitrogen, -196 °C, and left it for 24 hours. When she read the results, she could hardly believe it. Colwellia had again incorporated a small amount of the leucine into protein.
There had to be a simple explanation. Maybe Colwellia had metabolised the leucine in the few seconds before its temperature plummeted below -80 °C. So Junge incubated the bacteria in liquid nitrogen for 1, 2, 4 or 8 hours. Again, she was amazed. The longer the bacteria were incubated, the more labelled protein they accumulated.
Another possibility was that radioactive leucine was being incorporated into proteins through some purely chemical process unrelated to life. To find out, Junge substituted the bacteria with E. coli or freshly killed Colwellia bacteria. Almost no leucine found its way into the protein. The same was true if she used live Colwellia with a toxin called azide, which blocks respiration. “That means the reaction needs energy,” she says, “so it again points to a biological process.”
However many times she repeated the experiment, she got the same results. “I didn’t believe this for many, many months,” she says.
Life in liquid nitrogen
Junge’s results were finally published in June, in the journal Cryobiology (vol 52, p 417). “I would like to believe it,” says Priscu, who has extensive experience working with Antarctic bacteria, “but I think there will be more disbelievers than believers.” He would like to see more evidence to back the extraordinary claim that Colwellia is metabolising at -196 °C: the incorporation of nucleotides into DNA, say, or fatty acids being built into membranes. He would also like to know which proteins the leucine was incorporated into.
“I don’t have a particular problem with seeing metabolism at very low temperatures,” says Roy Daniel, an enzymologist at University of Waikato in New Zealand. “I’m not quite sure about -100, but at -50 or -60 probably.” He has looked at the problem from a different angle: measuring whether purified enzymes can function at very low temperatures. Working with freeze-resistant solvents such as methanol, Daniel sees low but measurable levels of activity as far down as -100 °C.
Junge herself is still struggling to interpret the results. The amount of labelled leucine incorporated into proteins at -196 °C was very small – just 100 to 300 molecules per cell in 24 hours. That means only a tiny proportion of each cell’s 20,000 protein-assembling factories, or ribosomes, were active.
One possible explanation she has yet to rule out is that some leucine might have entered the cells and docked with ribosomes before deep freezing, leaving a simpler biological “finishing off” process to occur at -196 °C. She says that if leucine is already at the right position on the ribosome before the temperature falls, only one conformational change needs to take place to add it to a protein. That could mean that after plunging to -196 °C, each ribosome adds just one more amino acid to the protein it is already building, and then grinds to a halt.
Whether that level of metabolic activity constitutes “life” or is merely its death rattle is still not clear, but such discoveries have stimulated interest in looking for life in Earth’s ultra-cold niches. Junge is looking at cloud droplets, which can capture marine bacteria and carry them high into the atmosphere, where temperatures drop to -50 °C. Priscu is focusing on Don Juan Pond in west Antarctica, which stays unfrozen throughout the winter, even below -50 °C, because it is 18 times as salty as seawater. Preliminary results suggest that something lurks in Don Juan. At -2 °C, samples of lake water slowly converted radioactive leucine into carbon dioxide. Priscu is running more tests. “If life is in there,” he says, “it’s barely getting along, not exactly cranking.”
That still throws up some intriguing possibilities. Buford Price, a physicist at the University of California in Berkeley, has lately turned his attention to bacteria locked kilometres deep in the ice sheets of Greenland and Antarctica. He has calculated the fundamental balance sheet of survival for such bacteria, the minimum metabolic rate that a microbe must maintain in order to repair spontaneous damage to DNA and proteins as soon as it happens. According to his figures, a cell that sits tight and does nothing except repair damage requires just one millionth the metabolic rate of a cell that is actively growing and dividing. What’s more, even though a cell’s ability to repair damage slows down with decreasing temperature, so does the rate of molecular damage it must repair. “There is no evidence of a minimum temperature for metabolism,” concludes Price.
Price then turned his attention to real microbes trapped in ice or permafrost at various temperatures. The actual metabolic rates he measured in the trapped bugs “amazingly agree with what we’d expect if all the energy was used to repair damage,” he says. What’s more, a cell surviving this way might live indefinitely, limited only by food.
Consider the bacteria found in 2003, in 200,000-year-old ice extracted from 3 kilometres down in the Greenland ice sheet. These bugs live at -9 °C, says Price, clinging to the surfaces of microscopic clay grains and gradually consuming iron (Astrobiology, vol 6, p 69). The only liquid is a water nanofilm three molecules thick that covers the surface of the clay grain and the bacterium. The cell cannot move and its only food source is the grain it’s attached to. Price calculated that one clay grain could provide a cell with enough food to last a million years. Surprisingly, these bugs aren’t cold specialists, but rather innocent bystanders that were scooped up by glaciers from underlying soil.
“We think in terms of years,” says Schulze-Makuch. “But who can say, even if reactions are very slow, that the lifetime of an organism could not be a million years?” There is some evidence that bugs can live that long. If bacteria 60 metres down in Siberia turn out to be as old as the permafrost they were found in, they clock in at a youthful 3 million years old.
“If bacteria in Siberia turn out to be as old as the permafrost they were found in, they clock in at a youthful 3 million years old”
Extreme cold tolerance appears to be a capacity that is shared by many bacteria. Whether bacteria could ever grow and divide at such temperatures is not known, but there can be little doubt that they are alive.
Extraterrestrial ice bugs
This combination of extreme cold tolerance and extreme longevity has important implications for the search for extraterrestrial life. For one thing, if Earthling bacteria can perform basic metabolic housekeeping in liquid nitrogen, it bumps up the odds of life existing elsewhere in the solar system. Consider the Martian polar ice caps, which may vary between -120 °C and -40 °C. Or Jupiter’s moon Europa, which may harbour an ocean of liquid water below its icy crust. Theoretical models put that ocean’s temperature anywhere between -10 °C and -90 °C. Even Saturn’s moon Titan, with rivers that are probably liquid ethane or methane, now looks vaguely within life’s striking range at -180 °C.
“Even Saturn’s moon Titan, with rivers that are probably liquid ethane or methane, now looks vaguely within life’s striking range at -180 °C”
What is more, Priscu and his colleague Steven Jepsen say liquid nanofilms may be found on Mars in the “dirty ice” that is widely thought to be buried just below the planet’s surface. Accounting for atmospheric pressure and temperature in the upper metre of Martian crust, Priscu and Jepson calculated that dust grains buried in the ice could support thin films of water down to -80 °C.
Extreme longevity also increases the chances of finding life on other worlds. Life may have evolved on early Mars, but if it is still there it would have had to have survived a couple of billion years of frigid purgatory. Sounds unlikely, but Price says it may just be possible. “If bacteria can live a million years at -9 °C in Greenland,” he says, “then they could comfortably survive a billion years at -40 °C on Mars.” If nutrients occasionally percolate up through liquid veins, it could extend a Martian bug’s lifespan almost indefinitely. “If life ever arose on Mars, I think some of it is still there,” says Price.
Back in 2003, researchers at NASA’s Goddard Space Flight Center in Greenbelt, Maryland, discovered that methane gas was seeping from Mars’s surface into its atmosphere. Could it be the waste products of bacteria hunkered down in survival mode? Last December, Price reported that if such microbes were evenly distributed below the surface in a 10-metre layer of permafrost, only one cell per cubic centimetre would be needed to produce the observed quantities of methane (Proceedings of the National Academy of Sciences, vol 102, p 18292). “One per cubic centimetre is a very reassuring number,” he says. “We have instruments that could detect that many.”
Optimism about life surviving on Mars is also causing concern that future missions could contaminate the Red Planet with Earthlings before we even have a chance to search for Martians. “There are a lot of cold-tolerant bacteria from Earth that I think would run in these conditions,” says Priscu. A prime candidate would be a bug called Deinococcus radiodurans. Its legendary radiation tolerance would help it survive the trip, and it has been found in snow at Vostok Station in Antarctica, where winter temperatures plunge below -70 °C and where the coldest ever natural temperature on Earth, -89.4 °C, was recorded in 1983. Those concerns prompted the US National Research Council to issue recommendations last year on preventing contamination of Mars.
Confirmation that we are not alone in the solar system would be one of the most amazing discoveries of all time. But how would we feel if our neighbours turned out to be a bunch of billion-year-old bugs whose idea of excitement is to fix a bit of molecular damage every millennium or so? It might not get pulses racing, but consider the possibilities. Life on Earth started out small, and took billions of years to evolve. The sun will eventually expand and warm the outer planets. Who knows what the future might hold for our icebound alien cousins?
Freezing eden
We malign ice as inhospitable, but could it have been the cosy womb in which life arose? Pierre-Alain Monnard, a chemist at Harvard University, believes so.
Scientists probing the origin of life have always been puzzled by how small biomolecules in the primordial soup could have assembled into the long chains that were presumably necessary to form the first functioning enzymes and genes. Most RNA enzymes, for example, would need to be at least 70 bases long, but in experiments scientists have trouble getting anywhere near.
Ice could be the answer. Ice crystals could have crowded RNA monomers into microscopic veins of liquid, concentrating them up to 500 times. “It forces the molecules into a certain organisation,” says Monnard.
In experiments performed at -18 °C for up to 38 days, without any template for the molecules to assemble on, Monnard gets better yields of RNA chains – up to 30 bases long – than in similar experiments at room temperature.
Meanwhile Hauke Trinks of the Technical University of Hamburg-Harburg, Germany, grew RNA molecules in artificial sea ice incubated for a year between -7 °C and -24 °C. Unlike Monnard, Trinks seeded his original mixture with small RNA chains as a template for the monomers to arrange on, an advantage that the first life on Earth would not have had. He claims to have grown new RNA chains up to 400 monomers long.
Life on Earth may well have arisen in the cold, since the sun was once less bright than it is today. Dirk Schulze-Makuch, an astrobiologist at Washington State University in Pullman, finds the ice scenario attractive. “Most people look at the origin of life at hydrothermal vents,” he says, “but the organic molecules fall apart very easily at high temperatures.” In the cold, they might have lasted long enough to assemble into something more interesting.
