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RUSSELL VREELAND couldn’t quite believe his eyes. A few weeks earlier, he had
drilled into a crystal from a 250-million-year-old salt bed and extracted a
droplet of brine, which he transferred to a tube of nutrient broth. Now, as he
stared at the tube, the broth’s cloudy appearance could mean only one thing:
bacteria were growing happily—bacteria that had lain dormant for aeons.
These microbes were a quarter of a billion years old, making them the oldest
living things on the planet.

When Vreeland and his colleague William Rosenzweig, microbiologists at West
Chester University in Pennsylvania, reported their find last October, reactions
ranged from amazement to disbelief. Some welcomed the news as evidence that
bacteria can live far longer than anyone had imagined. Others were simply
incredulous. Tomas Lindahl of the Imperial Cancer Research Fund in London, whose
research has shown that DNA breaks down in mere thousands of years, says
Vreeland’s microbe must be a contaminant and that wondering how it might have
survived is a waste of time. “It’s like discussing Jurassic Park,” he
says.

As such strong reactions suggest, a great deal is at stake. No one can yet be
certain that Vreeland’s bug is as old as he and Rosenzweig claim. But if it is,
it’s unlikely to be the only time traveller. Our world may throng with ancient
bacteria that have escaped from their imprisonment in salt, clay or amber and
got on with their lives in the present. And it might not be just our world. If
organisms can survive so long, living relics of an ancient ecosystem could even
be waiting for us in the Martian permafrost.

This isn’t the first time this kind of controversy has flared up. Claims of
reviving ancient microbes go back to the 1920s, when microbiologists extracted
bacteria from coal seams at least a million years old. But critics pointed out
that coal is porous and could have allowed bacteria in much more recently. Many
reports of finding preserved bacteria since then have faced similar
objections.

Suspicions have also been raised about Vreeland’s report. Although the salt
deposit dates back to the Permian period, critics claim that water could have
seeped through at any time, bringing modern bacteria with it. Others argue that
when you bring a sample into the laboratory, it’s impossible to avoid
contamination.

But Vreeland’s team had these criticisms in mind from the very start. They
began by selecting a salt bed that had remained undisturbed since its formation.
Salt beds are the remains of ancient oceans where a pinched-off coastal inlet
repeatedly evaporated and re-flooded for millennia before drying up altogether,
leaving behind all its salt. Microbes in the water would have become trapped in
the salty sediment accumulating at the bottom as the water evaporated.

The Permian Salado Formation, a salt bed near Carlsbad, New Mexico, was laid
down 251 million years ago. The deposit can be dated by the decay of radioactive
isotopes and the type of invertebrate fossils found in the salt. A series of
tunnels and air shafts dug into this particular salt bed for the storage of
nuclear waste has given geologists the chance to check it out for signs of any
disturbance since it was formed. Dennis Powers, a geologist from west Texas,
took Vreeland 600 metres down an air shaft. All the overlying layers of sediment
were intact here, indicating that the deposits were unaltered. Dangling in a
metal cage, they pulled dozens of salt crystals out of the wall, and Vreeland
took them back to the lab in Pennsylvania.

There, researchers selected 53 crystals that had no detectable cracks that
might have admitted modern bacteria. They sterilised them in concentrated sodium
hydroxide followed by concentrated hydrochloric acid—a combination which
kills just about anything, including the type of bacterium that ultimately grew.
They sterilised their drill bits and syringes with steam and heat, and drilled
the crystals in cabinets that had been irradiated with ultraviolet light, washed
with disinfectant and filled with sterile air. They placed open Petri dishes of
nutritive medium all around the work area. Bacterial growth on any of these
dishes in the days following the experiment would have meant that some
contaminant had survived the sterilisation, but nothing grew.

In all, the scientists transferred 66 droplets of trapped salt water into
separate sterile test tubes of nutrient solution. The researchers put these in
an incubator and waited. After many weeks, signs of cloudy bacterial growth
began to appear in four of the tubes.

Vreeland was cautiously excited, but he still wanted to prove these were not
simply miraculous survivors of the sterilisation treatment. So he and his
colleagues intentionally contaminated their tools with the bacteria they had
grown and then sterilised them again. Only when they failed to culture any
bacteria from these tools did they claim success.

So far, Vreeland has identified only one of the four strains, a species in
the genus Bacillus dubbed 2-9-3. Part of the secret of 2-9-3’s
longevity may be its ability to form spores. When nutrients are scarce, bacilli
can toughen themselves up into a hardy state that resists heat, desiccation and
ultraviolet light. Vreeland says 2-9-3 probably weathered the aeons as a spore,
because the 2-microlitre droplet of brine it came from couldn’t have contained
enough energy for it to grow and divide.

Despite the meticulous care the researchers took, many scientists harbour
doubts about the age of 2-9-3. Among them is David Nickle, who researches the
genetic evolution of viruses at the University of Washington in Seattle. He says
Vreeland’s paper caught his eye because the DNA sequence of one of the
bacterium’s genes was too similar to those of modern bacteria. “It struck me
that his 2-9-3 wasn’t behaving like a fossil,” he says.

The gene in question codes for 16S ribosomal RNA. This gene is commonly used
to make genetic comparisons because it is an essential component of the cell and
changes only slowly over evolutionary time. Nickle says that 2-9-3 couldn’t be
as old as is claimed, because the gene has changed too little. It is 99 per cent
identical to its closest modern relative, the Dead Sea bacterium Bacillus
marismortui, and differs only slightly more from other modern salt-loving
bacteria. The difference would have to be more like 5 or 10 per cent to be
consistent with an evolutionary separation of 250 million years, says
Nickle.

While Vreeland agrees that genes tend to change over time, he argues that
predictable change will not happen to every gene in every case. Related
organisms living in similar environments at different times might have similar
genes. “I don’t see biology as a clock-oriented thing,” he says.

And he has an even more compelling defence against the charge that 2-9-3 is
too similar to modern bacteria. The entire world, he argues, could be littered
with living fossils—species of bacteria which at some stage in their
evolution lay dormant for an extended period until some chance event brought a
fresh supply of nutrients and triggered them to start dividing again.

Old salts

It may not be as far-fetched as it sounds. Over the past 2000 years,
humankind has dug millions of tonnes of salt out of the ground and spread it far
and wide. It has been used to de-ice roads, preserve food and cure hides. Roman
soldiers were sometimes paid in salt, a practice which gives us the word
“salary”. Natural processes, too, continually bring salt to the surface. Unless
Vreeland happened upon the only salt crystals on Earth to contain microbes, the
modern world is probably rife with ancient bacteria, and B. marismortui
could be one of them. If Vreeland is right, dating bacteria will be extremely
difficult.

None of this satisfies critics such as Lindahl or Svante Pääbo at
the Max Planck Institute for Evolutionary Anthropology in Leipzig. They say the
DNA simply can’t survive that long. Pääbo has extracted DNA from
Neanderthal remains and frozen mammoths, and he has measured the rate at which
the bonds in the phosphate backbone of DNA break down. “If there is water
present, as there would be in a living cell, then the longest the DNA would last
is maybe 50,000 or 100,000 years,” he says.

But such arguments don’t trouble Raul Cano of California Polytechnic State
University in San Luis Obispo, who in 1995 claimed to have isolated a bacillus
from 25-million-year-old amber. He thinks people are missing the point: “Life is
more than just DNA,” he says. Spores, in particular, are loaded with protective
mechanisms. Since water is so damaging, the bacteria partially dehydrate
themselves. Then they coat their DNA with small proteins that stiffen the helix
and fend off chemical attack. When a spore eventually germinates, it immediately
mobilises a host of specialised enzymes that repair any DNA damage that might
have occurred.

Deep in the salt beds, spores are even better protected. The high salt
concentration may help stabilise biological materials, Vreeland says. UV light,
a real DNA killer, cannot penetrate. And there appears to be no oxygen in the
deposits, preventing oxidative damage and the formation of harmful free
radicals.

Pääbo agrees that factors such as the lower temperature and higher
salt concentration in the salt bed could extend the life of the DNA. And the
tricks spores use to protect their DNA may extend their lives by as much as
three or four times. But a thousand-fold extension seems utterly improbable, he
says.

Such calculations have not deterred some scientists from trying to work out
how ancient microbes might actually survive. Richard Morita, a microbiologist
retired from Oregon State University in Corvallis, for example, points out that
bacteria are excellent survivors. Many live in places almost devoid of
nutrients: granite outcrops, estuaries, desert sand, coal beds, frozen tundra
and Arctic ice. Below the root layer, soil is very low in nutrients, yet
microbiologists continually discover new forms of bacteria living in mud, clay
and sediments hundreds of metres underground. To survive in these conditions,
bacterial cells shut down all non-essential metabolism and divide rarely or not
at all.

Morita believes bacteria can endure these conditions, and possibly survive
for vast lengths of time, by consuming hydrogen. “Hydrogen is everywhere, from
the mantle upward,” he says. Chemical reactions in the Earth’s crust produce a
constant supply, and hydrogen permeates through everything, rock, salt, even
bacterial cell walls. The first bacteria probably consumed hydrogen, and many
bacteria, including bacilli, are still capable of using it today. Morita argues
that bacteria trapped underground for millions of years may consume just enough
hydrogen to repair DNA damage and reverse any degradation of proteins.

Vreeland says he is not convinced that any metabolism could have occurred in
the tiny brine inclusions he found. Metabolism produces waste products that are
toxic at high concentrations, and even a tiny amount of activity would produce
far too much waste over the course of a quarter billion years. He prefers the
hypothesis that spores can manage just fine without consuming energy.

It may prove impossible to test Morita’s hypothesis, because the repair
reactions he is proposing are so slow that they would take centuries to produce
a measurable effect. But if ancient microbes are discovered that are not in
their spore state, it may be the only way to explain their longevity.

At what point does longevity become immortality? If a bacterial spore can
survive a quarter of a billion years, why shouldn’t it live forever? One
possible limit might be damage from natural radiation. The biggest threat facing
spores in salt beds is the small amount of radioactive potassium-40 present.
This releases a high-energy electron called a beta particle, which can slam into
DNA and break the phosphate backbone. The half-life of potassium-40 is 1.25
billion years, which makes it a minor threat in the short term. But over the
aeons, the damage could add up.

“If you have a trapped bacterium in a dormant state you have a sitting
target,” says physicist Anthony Nicastro of West Chester University. “The
question is, are there enough bullets to damage its genetic material?”

Nicastro estimates that over the course of 250 million years, a bacterial
chromosome would receive hundreds of single-strand breaks in its DNA. But that
sort of damage is easily repaired. To get a lethal double-strand break, a second
beta particle would have to hit the opposite strand within five base pairs of
the first break. That’s a very unlikely event. Nicastro calculates that at least
1 per cent of cells would suffer no double-strand breaks at all.

“When you put all that together you go well into the multibillion-year
category [for bacterial survival],” says Vreeland. “If that same spore were in a
situation where no radioactivity could get to it, I could see the organism being
immortal.” It’s a hypothetical immortality, though, since the chances are that
no such place exists.

It’s still great news for proponents of panspermia—the notion that life
could have spread from planet to planet on meteorites. “If organisms can really
remain dormant for 250 million years, that’s much more than required for going
from planet to planet,” says astro- biologist Chris McKay at NASA’s Ames
Research Center in Moffett Field, California. The Martian fragment known as
meteorite ALH84001, in which scientists said they found signs of extinct
microbial life in 1996 and again this February, spent a mere 15 million years in
space before falling to Earth near the South Pole. Other Martian meteorites have
taken even less time to get here—mere hundreds of thousands of
years—although none has been found to contain evidence of life.

Parts of Mars were almost certainly once covered with water, and planetary
scientists are still hoping to find the fossils of ancient life buried beneath
the polar ice caps or in the sediments of dried-up lake beds. But if Vreeland is
right, the Red Planet may contain more than just fossils. “This has very
important implications for the possibility of organisms surviving a long time on
Mars,” McKay says. “Maybe they are still alive in the subsurface.”

Vreeland is now examining more salt crystals. If he and others working
independently can find other bacteria trapped within such crystals, it will go a
long way toward silencing the sceptics, says Cano. The Detroit Salt Company has
offered to cut Vreeland a large block of salt from its Michigan mine for him to
drill into in his lab. The salt in that deposit was laid down a little more than
400 million years ago. There are also Precambrian salt deposits well over a
billion years old in western Canada and the Australian outback, but prospecting
in such remote locations is still well in the future, Vreeland says.

If the Detroit expedition is successful, Vreeland will be looking at
organisms one-tenth as old as life itself here on Earth. If anything will turn
out to be immortal, surely these are our best bet.