Glimpses of the Deep Biosphere

We are The Noisy Upstairs Neighbors to a Vast and Unknown World 

Maciej Ceglowski 

 

 

It may be that we shall find a simple general rule to apply: that microbial life exists in all the locations where microbes can survive, that would mean all the locations that have a chemical energy supply and that are at a temperature below the maximum one to which microbes can adapt. There would be no locations on the Earth that have been protected from “infection” for the long periods of geologic time.

—Thomas Gold, The Deep Hot Biosphere, 1992

If you’re reading this on one of our several fine continents, then the ground beneath your feet for four kilometers or more is full of fractured rock home to a starving, sleeping population of microbes. If you’re on an island or on the Pacific rim, then somewhere below you is a layer cake of microscopic wildlife that has accumulated over millions of years, and in the coolest parts of the crust (subducting tectonic plate edges) may extend down a dozen kilometers or more.

These microbes at these great depths are demonstrably alive, but is this living? In her wonderful book on the topic, microbiologist Karen Lloyd coins the term aeonophile to describe microbiota that live on timescales closer to the geological than the diurnal. Microbes in 101 million year old sediment have been discovered in a dormant but living state, repairing the inevitable radiological damage to their DNA and metabolizing just enough to fight off the depredations of entropy. When a food source appears, their day-to-day speeds up again, sometimes even to the point of reproduction. Otherwise they’re just there, vibing, making a mockery of all of us up top who need fresh air and sunlight and a constant stream of digital entertainment.

We have yet to drill a hole so deep that we don’t find a microbial population at the bottom of it1. In terms of biomass, this hidden biosphere is equivalent in size to every plant, animal, insect and microbe on the surface and in the oceans of the Earth. But we know almost nothing about it. Most of our knowledge comes from opportunistic drilling in deep mines in South Africa and Canada, and from marine sediment cores brought up by the two scientific ships capable of doing such work, one of which is being scrapped.2

So it’s a weird situation. Even weirder is that it took us so long to discover this world, with much of the initial progress being made only because a particularly ornery civil servant (Frank Wobber) at the Department of Energy in the 1980s decided that, since the government was digging deep holes to store radioactive waste, it should first take a good look at what was already living in them. His arbitrary and immensely fruitful decision kick-started an entire field of study.

Cleanroom drilling

One reason it took so long to find the deep biosphere is because it is difficult to convince yourself you’re not just studying whatever microfauna came down for the ride during the drilling process. Sulfate-reducing bacteria had been observed in crude oil as early as the 1920s, but lab techniques for contamination control and for studying such exotic beasts didn’t really exist until the 1980s.

Drilling is inherently dirty work, involving hot pumping mud and sweat-soaked muscle men working in conditions that are both homoerotic and intensely septic. This creates a tension with the needs of microbiologists, who yearn for quiet, pristine cores that have not been contaminated with microbes from the surface or from the drilling process. Compounding the difficulty is the fact that microbe counts in deep cores are orders of magnitude lower than in soil samples from the surface. We are not drilling into deposits of rich Greek yogurt here. Particularly nutrient-poor sections of deep core may have just a few tens or hundreds of living microbes per cubic centimeter, so even a mild degree of contamination risks swamping the signal.

The rough procedure is this: as cores come up, they are bagged in inert gas and sliced into sections. Scientists on the scene go to work on these segments in a glove box, trimming off the ends and paring off the outer layers the way you might peel a leek. These trimmed hearts of core are rinsed with alcohol (sometimes flamed!), individually bagged, and put on ice. Within a few hours, they are shipped to secondary laboratories for analysis. There the core sections are often trimmed again, with the goal of giving a central chunk that has not come into contact with any foreign matter.

The physical aspect of this work is challenging. Scientists are trying to follow cleanroom protocols right next to a running drill rig, sometimes working kilometers underground in the stifling heat of a gold mine. Their work is time-sensitive, and has to take place on a schedule dictated by the drilling crew. And many of the organisms being pulled up in those cores are primadonnas who will faint or die if they are exposed to even small amounts of oxygen.

Microbiologists adopt multiple strategies for contamination control. One simple approach is to add a visual tracer (pink dye) to drilling mud, to give a quick signal that any of it has reached the heart of a sample core. If you are working in the glovebox and see that your core has a pink center, like a filet mignon, you can save yourself precious time preparing it and move on.3

A more sophisticated family of tracers are chemicals called perfluorocarbons (PFCs), a cousin of the teflon on your frying pan. PFCs are some of the most chemically boring compounds ever discovered, reacting with nothing and no one. But because they light up electron capture detectors like a searchlight, they can be measured at the parts-per-trillion level even with the kind of rough-and-ready equipment that can be operated on an oil rig. This extremely low detectability threshold is helped by the fact that they do not exist in nature. A further convenience is that PFCs are volatile. To check whether any drilling fluid got into your pared-down sample, it’s enough to put the sample in a plastic vial and test the air for miniscule concentrations of the stuff.

A PFC assay tells you that fluid reached a certain part of your rock core, but it doesn’t tell you whether the pathway it followed was big enough for microbes to physically pass through. So a second control involves placing a packet of fluorescent microspheres at the head of the core. These little beads are roughly the size of a microbe (0.5 µm) and swirl around during the drilling process, ideally squeezing into every nook and crevice a cell could conceivably pass through. You crush a bit of your sample and look at it under fluorescent light in a microscope for any hint of a microsphere.

Together the combination of microspheres, PFC tracers, and ribosomal DNA fingerprinting of found organisms gives a good quantitative control of contamination. The techniques are precise enough that researchers have been able to identify patches of sterile rock, along with native cell counts as low as a few hundred cells per cubic centimeter while keeping intruders below one part in a few thousand.

How deep does life go?

Extremophiles by their nature enjoy a challenge, so asserting absolute limits on the depth of life is asking for trouble. We have yet to drill deep enough to find a portion of the Earth’s crust that does not harbor life, and as Gold points out in the provocative paper I cited at the outset, it is possible that chemical processes contiguous with life might extend into higher temperature ranges (hundreds of degrees C) in the deep crust of the Earth, where there is plenty of available chemical energy to drive something like metabolism, even if it is not wrapped in lipids and proteins and all the trappings of the modern microbe. Finding complex processes that look like biochemistry in very hot rock would give us a big clue about the genesis of colder, low-pressure forms of life like ourselves.

But if we’re talking about recognizable microbes, little bags of DNA in aqueous solution, then we should expect to hit physical limits set by both temperature and pressure. Temperature underground goes up with depth at a speed called the geothermal gradient. In most places, this is about 25˚C per kilometer. Under continental crust, that runs into the known 122˚C upper limit for microbial life at a depth of four kilometers, and the 150˚C conjectured upper limit another kilometer down.

The deepest places on Earth that are still cool enough for microbes to survive are probably the underside of the Siberian craton (frosty, radiologically depleted and abnormally thick) and the edges of subducting ocean plates, which take time to heat up as they plunge back into the mantle. In both those places, it might be possible to find livable temperatures extending twenty or more kilometers underground.

Pressure also puts a limit on life. At pressures above about 2 GPa (about the weight of 80 km of rock), any liquid water present will freeze into a high-temperature form of ice. This exotic hot ice has been found as inclusions in natural diamonds coughed up from the deep mantle, suggesting that pockets of free water may exist at extraordinary depth, perhaps equal in volume to the Earth’s oceans. But water turning into rock places obvious stress on a cell, and we can consider this a hard limit for all known organisms.

High pressure also smushes down the fissures and pores in rock that microbes call home. The conventional minimal size of microbial cells is 0.2 µm, but it’s not clear how deep crystalline rock has to get before fissures of that size. And it’s worth remembering that underground is the place we first discovered particularly tiny microbes (CPR and DPANN) that are much smaller than what had been considered a universal size limit. A large part of the reason they evaded detection for so long was because everyone was using 0.2 µm mesh as a low-pass filter for microbes, confident that necessary molecular machinery for life couldn’t fit into a smaller package. In microbiology, the surest way to not find something is to not look for it, a lesson worth keeping front and center as we further explore the deep biome.

The upshot is that the Earth is inhabited and inhabitable for at least four kilometers down, and possibly down to tens of kilometers or more.

So who all lives down there?

Sedimentarians

The first broad category of intraterrestrials comprises regular, law-abiding microbes that get buried in ocean floor sediment. How much of this sediment there is varies greatly with location. Close to land and at river mouths, there can be absolute mountains of the stuff. The bottom of the Gulf of Mexico, for example, sits under 18 kilometers of what used to be the Rocky Mountains, eroded and carried down over the aeons by the Mississippi river system.

In parts of the ocean far from land, productivity is limited by the availability of iron (which has to arrive as wind-borne dust carried from continents). With few plankton and other primary producers to provide the bodies for it, sediment in nutrient-impoverished areas of the remote ocean might accumulate at a rate of a millimeter per thousand years.

Sediment by its nature is rich in biological matter, and for the first few centimeters there’s often oxygen to go with the food. The living is good. But when the oxygen runs out, the population balance in the sediment changes character. Anaerobes that had been holding their breath in seawater start to come into their own. Within the first few centimeters of seafloor muck there’s a transition from deep-sea like populations of microfauna to a fully anaerobic ecosystem of lucky colonizers.

The good times continue until deeper in the sediment, when all the readily available food has been eaten. Microbes that once frolicked and played now begin to live in slow motion, expending the minimal amount of energy needed to keep entropy at bay and maintain their DNA and proteins in a viable state. Sex is rare—the cell division time for microbes in sediment goes into the thousands and tens of thousands of years, with evidence that it may extend to millions of years. Organisms expend astonishingly little energy, but still live.

And sometimes food shows up! As the sediment layers sink deeper, they also get warmer, and the higher temperatures begin to cook previously inedible fractions of the organic sediment into a nutritious substrate. Life in the deep is full of these ups and downs—long millennia of hunger, punctuated with brief moments when heating or some nearby cataclysm makes food available.

In writing about the far future, Freeman Dyson once speculated about whether a sufficiently advanced civilization could defeat the entropic heat death of the universe by going into successively longer stretches of hibernation, punctuated by periods of activity. Provided the universe always got colder, and that the periods of stasis got longer, Dyson showed that a finite amount of energy could still translate into an infinite length of subjective experience. It was a vision of practical immortality through sustained cold napping that is strikingly similar to the life microbes have built for themselves underground. The ocean floor conveyor sets a natural time limit to seafloor life, but some of the communities living in deep rock have now been out of contact with the rest of creation for the best part of two billion years, a sort of natural time capsule of slow-living immortals.

For most microbes (like the rest of us), the end does finally come. There comes a point when the sediment gets deep enough that the microbes are cooked to death. But a few lucky survivors find themselves squirted back to the seafloor by a cold seep or mud volcano, the tallest of which can return material from tens of kilometers below the ocean floor. The ocean floor is covered with spores from thermophilic bacteria from these precincts that found themselves suddenly ejected from a warm, happy home kilometers below the surface into a frigid sea. Since they can’t grow in such cold conditions, they serve as a kind of natural tracer, a reminder that a vast unknown world is up to something down there.

Rock Lickers

Under the continents, the picture is a little different. There is neither the permanently accumulating layer cake of sediment, nor the conveyor-belt like movement of ocean crust away from mid-ocean ridges. Instead, there is mostly still rock and the slow passage through it of gases and water. Microbes live in fractures and fissures that also serve as conduits for groundwater, some of which has provably been out of contact with the surface for a billion years or more. It’s not a totally sessile lifestyle--if a nearby province of rock gets sterilized by volcanic activity, microbes will colonize it at a rate of several centimeters per millennium. But their life is utterly disconnected from anything we get up to on the surface.

One ubiquitous food source that is present in deep rock but not a key player in ocean sediments is hydrogen. Much of this forms chemically, through the reaction of hot rocks with water in a process called serpentinization. Some even forms mechanically, through the physical scraping of stone against stone. But a significant fraction is the product of radiolysis: decay products from naturally occurring radioactive elements in continental crust (primarily uranium, thorium, and potassium) cleave apart water molecules, leading to a steady supply of molecular hydrogen deep in crustal rocks.4

One of the great finds in intraterrestrial biology has been an organism called Desulforudis audaxviatohyr, a sulfate-reducing hydrogen eater found as the sole microbial inhabitant in fracture water in a South African gold mine in 2008. Coming across a single-species ecosystem was surprising enough, but digging around in the microbe’s genome has revealed a lifestyle completely decoupled from anything we might call the biosphere. Desulforudis is a strong, independent organism that likes to dine on hydrogen ultimately derived from radiation, making this organism completely independent of any other form of life. It is capable of synthesizing its own lipids and proteins from molecular nitrogen and inorganic carbon in the rocks around it. Its genome carries a set of intriguing additional capabilities, including genes coding for a flagellum (useful for chasing nutrients when they are plentiful), gas vesicles (for drifting along in a current), and CRISPR genes that form a rudimentary immune protection against viruses that are also apparently alive and doing well in the deep biosphere. The organism seems best adapted for life at 60°C, a far higher temperature than the South African and Swedish rocks it has so far been discovered in and has no chemical defenses at all against oxygen, suggesting a full and unapologetic commitment to underground living.

 

 

This stuff is suggestive! Desulforudis audaxviator has not gone through its existence as a James Dean like loner—it acquired all those cool genes by horizontal gene transfer from fellow microbes, presumably also living in the deep underground. But its level of disconnection from the biosphere is food for thought. Hydrogen eating in particular is an ancient and venerable lifestyle that seems to have evolved at least three times independently, suggesting that maybe life had a deep origin, with many of the fancier later metabolisms (ours included) bolted on as an afterthought.

Patterns of deep life

In looking at intraterrestrial organisms, certain patterns come up again and again. The most striking one is that deep life is slow-living, operating at metabolic rates almost indistinguishable from death. The organisms have to be doing something, because otherwise their DNA would get torn up by background radiation, and the amino acids forming their proteins would lose their natural left-handedness over time and devolve into a racemic mixture. But they are consuming almost no energy doing it, part of what makes noticing them (let alone trying to culture them) such a challenge.

Another pattern is metabolic opportunism. Up on the surface, multicellular organisms are expected to choose a metabolism and commit to it the way Japanese salarymen used to commit to a lifetime job. You and I and all the people we know burn glucose with oxygen as an electron acceptor, and will do so with our final breath. The plants around us use sunlight to run this reaction in reverse, using carbon dioxide as an electron acceptor and regenerating oxygen. Methanogens in our bellies will spend their entire lives making methane.

Down under, things get complicated. Microbes change metabolism like you might change allegiance to a sports team, adapting to new surroundings and peer pressure by putting on a new set of colors when there is an advantage to doing so. In the absence of oxygen, almost everyone underground is an anaerobe, but that category still covers a lot of metabolic ground. Some microbes eat carbon dioxide to make methane. Others eat methane and make carbon dioxide. Some pursue suboptimal but kinetically faster metabolic strategies to kneecap their competition. Sulfates and iron are a popular metabolic substrate for everyone, and easy to find in a world of rocks. Name any plausible redox reaction that can generate a little free energy, and you are likely to find a microbe experimenting with it underground.

A final deep pattern is commensalism. Like people, microbes benefit from living in communities with a division of labor, and a lot of discoveries to the contrary (like the putative single-species ecosystem I described above) may be an artifact of our technical limitations. We’re good at finding organisms in groundwater, but still struggle with microbes that attach physically to rock, the way almost all intraterrestrials do. And here it’s worth pointing out that underground communities extend past the bacterial. Nematode worms have been found grazing on microbial mats several kilometers underground, as well as tiny gnats trying to lay eggs on the worms. More bizarrely, there are metazoans (multi-celled animals) that are able to live in anoxic conditions in sealed-off deep caves. And there is growing evidence that those caves themselves, including vast underground systems like Carlsbad caverns, are themselves the product of microbial activity, with sulfide-oxidizing bacteria generating sulfuric acid that then carves out great underground cathedrals with complex internal ecosystems of their own. The interactions between species underground are an area where our ignorance is almost total.

Consequences

The existence of a large and unsuspected deep biosphere has important consequences for space exploration, which I’d like to talk about in increasing order of nuttiness.