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Oliver Strimpel: This is Geology Bites with Oliver Strimpel. The fossil record goes back through the Phanerozoic Eon, about 540 million years, and even earlier into the Ediacaran Period. But while fossils provide incontrovertible evidence of ancient life, the fossils themselves are certainly not alive. In fossils, the original organic matter belonging to the fossilized life form has been replaced by inorganic materials cast into the shape formerly occupied by the life form. However, in some situations the original organic material does survive. For example, original spores as old as 350 million years have been identified using their original organic material, and DNA can survive for as much as a million years. But in 2019, bacteria that had been buried for a hundred million years with barely any access to nutrients were not only identified, but shown to be alive. Had they been in suspended animation for all that time, or were they managing to eke out a living using much less energy than was previously thought to be necessary?

Steve D'Hondt is a professor of oceanography at the University of Rhode Island. He studies life beneath the seafloor and was on the team that discovered the bacterial cells living in hundred-million-year-old sediment. Steve D'Hondt, welcome to Geology Bites.

Steve D'Hondt: Thank you very much, Oliver. It's a pleasure to join you here today.

Oliver Strimpel: Where were the hundred-million-year-old sediments containing these ancient bacteria?

Steve D'Hondt: The sediment was in the middle of the South Pacific Gyre. It's this extensive area of abyssal clay that carpets the middle of the South Pacific Ocean.

Oliver Strimpel: And why did you focus on that area?

Steve D'Hondt: We focused on that area because the sediment accumulates very slowly. In some places it accumulates as slowly as a few centimeters per million years.

Oliver Strimpel: And why was it important to study an area that had such very low deposition rates?

Steve D'Hondt: My colleagues and I wanted to study the microbes that had the lowest access to energy anywhere in the ocean, and we wanted to do so in an environment where we could core them with relatively little contamination. So that meant relatively easily penetrated sediment, relatively close to the seafloor. And that clay is very low in organic matter. It has a tiny amount of organic matter, and the amount of organic matter is relatively constant for tens of millions of years in the sediment, so it's not eaten very quickly.

Oliver Strimpel: So these sediments are below about 6 kilometers of water. How does one go about recovering sediments from that depth?

Steve D'Hondt: Well, we're fortunate to have access to the JOIDES Resolution, which is a drill ship operated on behalf of the International Ocean Discovery Program, a multinational collaboration to core the sediment and rock of the world ocean.

Oliver Strimpel: I'm fascinated by how you actually go about doing that. Do you have a rigid drill that goes down that enormous distance?

Steve D'Hondt: It's a rigid drill from my perspective. It's drill pipe, and when we stand next to it, if we lean on it, it's not going to bend. But when you have six kilometers of pipe out, it's a little bit more like soft spaghetti.

Oliver Strimpel: So it dangles from the ship, and then when it hits the bottom, it starts spinning to drill a core?

Steve D'Hondt: It dangles from the ship. There's a driller, and the driller controls the rate of spin. This drill ship is somewhat like oil and gas drilling vessels, but the rig is set up very differently. With an oil or gas vessel, they have a really wide outer pipe to prevent blowouts. We don't use that outer pipe. We use an inner pipe, and we make sure that we drill where there's no chance of oil or gas. That allows us to work in much deeper water, because we don't have the weight of that big outer pipe.

Oliver Strimpel: And how far below the surface did you have to go to get these bacteria that we're talking about here?

Steve D'Hondt: This sediment is really thin out there, because the sedimentation rate is really slow. So even where the basement is 100 million years old, we might have only 70 meters of sediment. This is a really different situation from what people usually think of when they think of drilling the deep sea. Other expeditions have gone out and drilled as much as two and a half kilometers beneath the seafloor. But our target wasn't depth; our target was age. So we chose this region, where we knew the sediment accumulates very, very slowly, and we could get hundred-million-year-old sediment roughly 70 meters beneath the seafloor.

Oliver Strimpel: Wow! So you only have to drill a few tens of meters. Really not very much at all.

Steve D'Hondt: Exactly.

Oliver Strimpel: So when you got down to those bacteria, to the 100-million-year age, how many bacteria were actually present in the core samples, and how does that compare roughly to the bacterial population on the seafloor surface?

Steve D'Hondt: We have roughly 1,000 cells per cubic centimeter of sediment in the old sediment. At the seafloor in that region, we might have more like a million cells per cubic centimeter, or 10 million cells per cubic centimeter. And in environments on continental shelves, we might have a billion cells or 10 billion cells per cubic centimeter. So at some level, a thousand individuals is a lot, right? You can imagine a room full of a thousand people. But compared to what you would have living at the seafloor, even at the same site, almost all the cells that were at the seafloor are gone.

Oliver Strimpel: So this is a pretty unlikely environment to see something that could survive for this length of time. But to understand why it was unlikely, before answering that question, it would be good if you could explain what it is that a bacterium normally needs in order to be alive and well.

Steve D'Hondt: Well, bacteria need nutrients and they need energy. Microbiologists who work in labs often think about nutrients, but when they discuss them, they're often lumping in the sources of energy. In my mind, I separate these things, because I think a little bit more like an oceanographer. Classical oceanographic nutrients are elements like phosphorus and nitrogen and sulfur and iron. And then we need a source of energy. Normally, when people think about bacteria, they think of bacteria eating organic matter, whether they're attacking our bodies or attacking something in the world around us. In that case, they're getting their nutrients and their energy from the same place, in a way: they're getting the nutrients from the organic matter that they eat, and they're getting energy from oxidizing the organic matter, just like we do. And if you think about that for a second, you realize that the energy really doesn't come from the food. It comes from the oxidation of the food, so it comes from the combination of the oxidant and the food source. In the broader world of bacteria, there are many, many categories of bacteria that use oxidants in the world around them, like oxygen or nitrate or sulfate, and then use reduced compounds in the world around them, like methane or hydrogen or organic matter, and they get their energy from combining those two things.

Oliver Strimpel: So you need both things: you need the nutrients, which would be the reduced matter, and you need the oxygen supply, but you need to have the energy-generating reaction that then keeps the organism alive, right?

Steve D'Hondt: For the energy, you need something reduced and you need something oxidized. For you and me and our listeners, the thing that we use to oxidize is oxygen itself, and the reduced matter that we eat is our food.

Oliver Strimpel: OK. So what was extremely scarce, then, in this environment for these bacteria?

Steve D'Hondt: What's extremely scarce in this environment is food. One of the things we discovered on this expedition is that so little organic matter is consumed in the sediment that oxygen is present all the way from the seafloor down to, and into, the basement below. So even where the sediment is 100 meters thick, there's oxygen in the sediment all the way down.

Oliver Strimpel: How does it get there?

Steve D'Hondt: It diffuses in from the ocean above. It takes a long time for oxygen to make it 100 meters into sediment; it's probably a few hundred thousand years. So the process of organic oxidation in this sediment is incredibly slow. Very little organic matter is eaten in any cubic centimeter, or even in any vertical 70 meters of sediment.

Oliver Strimpel: But nonetheless, they did have oxygen, but they had virtually no nutrients. So what was previously known about the ability of bacteria to survive under these sorts of conditions?

Steve D'Hondt: People have found bacteria in marine sediment up to around 35 million years old. And I should mention that people have pulled bacteria out of gold mines in Canada and in South Africa, where the rock is more than a billion years old. Those bacteria in the gold mines are living in the water in the fractures of the rock, and they've been isolated from the surface world for probably most of that billion years. But it's not a very analogous situation to the sediment that we're talking about, because the bacteria can move around in the fractures, and their oxidants and their reductants can move through the water in the fractures. We'd previously found microbes living in sediment from the Eocene, a little over 34 million years ago, and we were trying to push back deeper in time here. But the thing we were really interested in wasn't finding the oldest living bacterial communities. It was finding bacteria that were challenged by so little access to energy. Until this expedition, people really had not looked at bacteria from environments that had been exposed to oxygen for tens of millions of years, where there was too little organic matter around for them to use up all the oxygen.

Oliver Strimpel: So let's get back to the experiment that you did. What was done to the sediments that were taken out of the core once you got them back to the lab?

Steve D'Hondt: With the experiment that you and I are talking about today, our colleague Yuki Morono took little pieces of the sediment using sterile sampling. He'd set one piece aside so we could use DNA sequencing to determine what was in it, and then splits of that same little piece he would put in different vials with different food sources. In some he would put an amino acid mixture; in others he would put ammonium as a reduced molecule; he put carbon dioxide in some, which could be a source of carbon but not a source of reduced material; and he put acetate in some. So, a variety of very simple organic molecules, carbon-bearing molecules and nitrogen-bearing molecules, and then he waited to see what would grow.

What he found was that after a few weeks, he had growth in all of the vials where reduced material was added, where there was ammonia added, or acetate, or amino acids. The growth can be measured in terms of the number of cells per cubic centimeter, or milliliter, of fluid in the vial. He went from 1,000 cells per cubic centimeter in the sediment to having something like 100,000 cells per cubic centimeter of culture. So after a few weeks, essentially, these organisms, this community that had been trapped for 100 million years, woke up and said, wow, food. And then they reproduced. And that didn't happen in the samples where he just added oxygen. It didn't happen in the samples where he just added CO2, and it didn't happen in the samples where he did not add any oxygen. He did some control samples without oxygen to see what would happen, and they didn't have growth.

Oliver Strimpel: So this confirms that it was the nutrients they were really lacking, not the oxygen. In a sense, it's the food that was really lacking.

Steve D'Hondt: Yes. So from this result, we can say that some bacteria from the sample had survived a hundred million years of food starvation, and they started to behave like normal bacteria.

Oliver Strimpel: Does this mean they were alive, i.e., dividing or repairing, albeit at an extremely slow rate, all along? Or were they essentially frozen or hibernating, and just started living again when they had access to a supply of food?

Steve D'Hondt: This is a really good question, because we're faced with a conundrum here. These microbes, on average, had so little access to energy that they really had barely enough energy to keep repairing molecules as they broke, just like you or I might get a muscle tear if we strain ourselves. So they had enough energy to repair their equivalent of a muscle tear, but they didn't have enough energy, formally, to reproduce as we know it. If they weren't reproducing, though, this meant that the individual cells had survived something like 100 million years. So we're left with two ridiculous alternatives: one, that individual cells have lived for 100 million years, or two, that the population has turned over during that hundred million years, but at much, much slower rates, on much less energy, than our current understanding allows. And I think that the answer probably lies somewhere in between: these cells are probably living much longer than the bacteria in the world around us, but they're probably reproducing over those hundred million years.

There have actually been some recent studies by a group at the Technical University of Munich looking at microbes from parts of the North Atlantic. They've shown that at least some bacteria multiply over the first few million years of burial. That's not quite the same thing as showing that something's been multiplying over a full hundred million years, and it's not the same thing as showing that every lineage that survives is multiplying. But they've been able to show that at least a couple of lineages do multiply in the subseafloor environment. So that provides, I think, reasonable evidence that at least some of them can reproduce on much less energy than we've previously believed realistic.

Oliver Strimpel: So you said that there was a paucity of nutrients, which was the limiting factor down there in those sediments. But if they were at least eking out a very sparse living, but nonetheless dividing, does that mean they had access to some form of nutrients, some reducing agent that could just keep the processes ticking over?

Steve D'Hondt: That's another really good question, and they really have access to two sources of energy, if you will. The first source is that the organic matter is being oxidized at very low rates. We can tell that because, as we go down in the sediment, we find slightly higher nitrate concentrations and slightly lower oxygen concentrations, and we can tell from the ratio of those things that oxygen is being used to oxidize marine organic matter at very, very slow rates. The other source of energy, in the form of reduced compounds and oxidized compounds, is the natural radioactive splitting of water.

In all natural environments, there is some concentration of naturally radioactive material. Even in our bodies, we have radioactive potassium and radioactive carbon, for example, and deep-sea sediment has radioactive uranium, radioactive thorium and radioactive potassium in it. As those radioactive elements in the minerals decay, they release radiation that strikes the water around them in the sediment, and striking that water breaks it up, and then it can recombine into hydrogen and various oxidized chemicals. Hydrogen peroxide is one. If you've got reduced metal around, the oxidized products of the radioactive breakage will oxidize the metal, and then they can use that as an oxidant. We've done some basic calculations that suggest that in this sediment, where organic matter is so scarce, there may be 70 times as much energy available from radioactive splitting of water as from the oxidation of organic matter.

Oliver Strimpel: Wow, that's amazing. But if that is the primary source of the nutrients, and that presumably is pervasive throughout the crust and the mantle, in fact, would this kind of survival mechanism be viable at arbitrary depth?

Steve D'Hondt: This kind of survival mechanism should work in any mineral environment where there's water. It's the survival mechanism that the people who study the gold mines believe supports the microbes in their fractures. It's the mechanism that we believe supports life in deep sediment. And, as you can surmise, it's a mechanism that might support life on Mars or Europa or extrasolar planets.

Oliver Strimpel: Are the bacteria that survived, those thousand per cubic centimeter versus the million or 10 million that you would get on the seafloor surface, a special subset of the population? A kind of extremophile, specially adapted to living under such energy-poor conditions?

Steve D'Hondt: They're extremophiles in the sense that they've survived burial for 100 million years, but they're not typically thought of as extremophiles, because they're part of the standard community at the seafloor. And so this leads us to this curious thought that, although they dominate subseafloor sediment, they may not be very happy there. They're a normal part of the seafloor community, but unlike most of the seafloor community, they can survive burial for up to 100 million years or more. So we would not normally think of them as extremophiles. If we weren't collecting them from the sediment beneath the seafloor, we would think of them as just part of a normal marine community.

Oliver Strimpel: Wow. So the same bacteria that manage to survive under these extreme conditions are also viable in normal conditions.

Steve D'Hondt: Yeah.

Oliver Strimpel: But if this ancient population represented the bacterial population of 100 million years ago, wouldn't we expect it to be different from a modern-day population that has been evolving at a normal pace continuously since that time? I think it's reasonable to expect that you'll have some mutation that would separate this individual cell from that individual cell over a hundred million years. The subseafloor populations are probably not accumulating mutations very fast, and the populations in the surface world, like at the seafloor, have got a lot of mutations.

Steve D'Hondt: I'll say two things with regard to this. The first is that there's undoubtedly been some mutation in both: a lot of mutation in the surface world, and perhaps some mutation in the subsurface world, since they've been separated. But the richness of mutations, if you will, in the surface-world populations is already so great that it wouldn't be very easy to pick out the differences. We do know that the mutation rates are low enough that we can still assign these microbes to the same categories of bacteria, so we know which family and which genus they belong to. And if we wandered around the world exhaustively sequencing everything, we'd probably find lineages that we would call the same species. In fact, in other studies my lab has done on shorter time scales, looking at things over a few million years, we actually find that, to a first approximation, what we see in the subseafloor is not just a subset of a seafloor community, but a subset of the local seafloor community. What I mean by that is that if we see something that we might call a separate species in the subseafloor at some site, in million-year-old sediment or three-million-year-old sediment, we typically have the same species at the seafloor.

But it's important to bear in mind that definitions of things like species and genus for bacteria and archaea, which are other very simple one-celled organisms, are numerical. In the world around us, we think, oh, horses and zebras are different species because they can't have fertile offspring. But bacteria don't reproduce by having sex with each other; they just divide. So our sexual definition of a species doesn't apply to bacteria. What microbiologists do instead is say, oh, if it's 97 percent similar, or more, to some other thing in its gene content, we're going to call it the same species. What that basically means is that for the genes that are used for identification, which are like a barcode, we take a chunk of the genome, always the same chunk, over and over again. If that chunk of the genome is, say, 400 bases long, then there have got to be more than 12 mutations separating it from another one for us to call them different species, because we have that three percent we can play with. So I've wandered a little bit in this explanation, but the basic point is that you can accommodate mutation and still call it the same thing, and that's basically what we do. We see that as we go deeper and deeper in the sediment, we're still calling them the same thing, but they can be separated by some number of mutations over time.

Oliver Strimpel: But just from a naive point of view, it seems a bit surprising that over 100 million years, a population that's been subject to this very extreme selection pressure, if you like, to survive with virtually no food, should still be represented in the population that's on the seafloor surface, which has had none of that pressure and could have evolved quite happily in other directions.

Steve D'Hondt: You know, one of the curious things about selection is that it applies differently to different genes. Ultimately, what you select against is the organism, right? The actual living thing, not the individual gene. But this is something, again, that Bill Orsi's lab in Munich has looked at recently. What Bill finds is that, again over just a few million years, which is the age of the sediment he's looked at in the North Atlantic, you can see mutations accumulate in the genomes of the organisms that live beneath the seafloor, but the mutations are preferentially accumulating in genes associated with functions that the bacteria probably aren't using. So the genes for motility, for example, for swimming, are accumulating mutations, and the genes for reproduction are not; they're remaining more stable.

One thing worth bearing in mind is that only something like one in five thousand mutations is positive, right? Most mutations are negative or neutral. And so essentially what Bill is seeing accumulate are mutations that are basically neutral, because these things aren't swimming anymore. If you had a mutation in the gene sequences that are used for reproduction, almost all of the time that would be negative, and the offspring just wouldn't survive. So there's a lot of pressure to keep their core genes from changing very much. And then, once that pressure is released from genes that are used for functions the microbe no longer uses, we start to see the mutations accumulate. It would be fun to look at evolution in the context of a hundred-million-year-old community and see if what Bill sees over a few million years just carries on. We haven't really tested the cells that Yuki brought back to life to see if they can swim, for example.

Oliver Strimpel: Are there any special constraints, then, on the nature of the geological environment that enables such extremely long-lived bacterial colonies to survive? I mean, obviously, they won't survive passing into a subduction zone, for example.

Steve D'Hondt: One can imagine a number of constraints. The usual constraints that people talk about when they think about these natural environments revolve around temperature: if it's too hot, everything dies. So when people talk about extremophiles, there's a lot of focus on temperature. One could imagine more subtle constraints. For example, the sediment that these microbes were removed from has been continually exposed to oxygen for 100 million years or more. It's conceivable that anaerobic bacteria, bacteria that can't live in the presence of oxygen, are driven to extinction in environments like that, and in fact Yuki's control experiments suggest that that's the case. He wasn't able to grow anything in the vials without oxygen.

And in the case of subduction, which you mentioned, it's an enticing thought. The combination of temperature and pressure in subduction zones is extraordinary from a surface-world perspective. But if you think about the temperature, you can also get deviations from a long-term equilibrium: the core of the subducting slab can be cooler than the edges as it penetrates into the mantle. So you could imagine a temporary refugium of even a few million years in the core of the sinking slab.

Oliver Strimpel: That's an amazing thought: tens of kilometers down, in the middle of a slab.

Steve D'Hondt: It just depends on when you break through the temperature threshold. The known temperature limit to life right now is about 120 degrees Celsius. Under normal circumstances, the average geotherm will take you down, you know, about 20 degrees per kilometer, so that's six kilometers down. But where the geotherm is cooler, it can be deeper, as, for example, in a subduction zone.

Oliver Strimpel: So if bacteria can survive for 100 million years, is there really any upper limit as to how long they might live?

Steve D'Hondt: From a practical perspective, I really don't think so. The bacteria in our digestive systems are living a few hours before they divide. The bacteria in this sediment have been living in isolation for 100 million years, and so, however slowly, they're dividing. They're dividing a lot more slowly than the bacteria in our digestive systems, orders of magnitude more slowly. And the community has survived a hundred million years. So to survive a billion years on Mars, or even a billion years on Earth, that's only one more order of magnitude. If we took an extreme example and thought of the community in our digestive systems as surviving the duration of our lifetimes, if each of us lives 100 years, that is the tiniest fraction of 100 million, right? The communities in the sediment have lived a million times longer. And so to pick up one more order of magnitude doesn't seem so striking.

Oliver Strimpel: Gosh. So you can really imagine communities living for of the order of the age of the planet, or even the age of the universe, if you're up to about, what, 13 and a half billion years.

Steve D'Hondt: If you could retain the environment for that long, you could in principle retain the community. What we see on Earth, of course, is that stuff that approaches four billion years old has been massively heated and pressed and probably lost its bacteria a long time ago. But if you could somehow take a piece of ancient Greenland when it was young and hold it aside from the rock cycle, the community might reside there for four billion years.

Oliver Strimpel: You touched on the space aspect when you mentioned the possibility of such communities perhaps living below the surface of Mars. And in fact, we had a podcast on the Perseverance rover, which is looking for just that right now. But what about the implications for life, and perhaps bacteria, traveling from one planet to another?

Steve D'Hondt: There are experiments and a lot of calculations that show that bacteria can survive relatively small-body impacts, right? So if you have a chunk of Mars that gets knocked out into space by a big impact... You probably know that when you get a really big impact, the center of the target is vaporized. Then, as you move farther from the center, it liquefies, and as you move farther still from the center, it's just mechanically disrupted, and that mechanically disrupted stuff flies out of the crater area during the rebound. Stuff that's mechanically disrupted never reaches the temperatures necessary to kill life. And people have shown with vertical gun experiments at NASA that you can essentially take a bacterium-impregnated piece of sandstone, fire it at a target, and it will survive. So you certainly have the possibility of moving life around on these time scales.

Oliver Strimpel: So perhaps potential support for the panspermia hypothesis for the origin of life?

Steve D'Hondt: It is potential support for panspermia. I find panspermia hypotheses interesting, because from my perspective, the simplest explanation for the origin of life on Earth is that it originated here. Most of the people who talk about panspermia in the Earth-Mars context talk about it coming here from Mars. I think it may be likelier that it's gone to Mars from here.

Oliver Strimpel: Steve D'Hondt, thank you very much.

Steve D'Hondt: Thank you very much, Oliver. It's been a pleasure to speak with you today.

Oliver Strimpel: For more about Geology Bites, as well as pictures and illustrations that support this podcast, you can go to geologybites.com.