This article and interview is a guest post by Lucie Ciccone (LC), a student in Rob Dunn’s course on the Future of Life. Lucie is a sophomore at North Carolina State University where she is studying zoology. She plans to pursue a career in conservation biology and is interested in ecology, our cultural and historical relationship to the environment, and our changing urban landscapes. She enjoys thinking about time and making cartoons. 

Karen Lloyd is a deep subsurface microbiologist, which means that she studies the life that exists thousands of feet beneath our feet. This is life that we didn’t even know existed until about 20 years ago, when exploration of sediment and rock taken from beneath the ocean floor revealed that our understanding of microbiotic life on Earth had, quite literally, barely scratched the surface. What we previously thought was lifeless rock has been revealed to harbor a multitude of species as different from each other as they are to life on the surface. I wanted to know more about these mysterious microbes, what drew Karen Lloyd to study them, and her thoughts on what it means to study the unknown. 

LC: What drew you to studying life that you can’t see, living in environments you can’t visit? What was your first exposure to extreme microbial life? 

KL: I took a summer science course at North Carolina Central University when I was in high school, and I learned that microbes are like tiny invisible things that are running all of Earth’s processes “behind the scenes.” I was fascinated to discover that microbes can live in places that seemed impossible, and I was hooked. So I knew I wanted to be a microbiologist then, but I got a little sidetracked in college because I liked chemistry so much. So I ended up majoring in biochemistry, which ended up working out well since you can view all of microbes like a bunch of catalysts, lowering activation energies and removing kinetic or thermodynamic barriers to speed up Earth’s chemical reactions. So, I still feel like I’m a chemist, even though I’m really a microbiologist. 

To answer the question of when I first saw extreme life, we’d have to define extreme life. I think that the thin crust of microbes living on the hot surface of a rock in the desert are extreme. But if you mean, life that’s so extreme it lives in places that would kill us immediately if we went there, I guess that’s when I first dove in a submarine called the Johnson SeaLink to methane seeps in the Gulf of Mexico. I was freezing in that little titanium submarine, sitting all day about half a mile under water. But I didn’t want to take my eyes off the porthole, even though it was at an awkward angle relative to where I was sitting, because outside was a teeming mass of happy worms whose ancestors had lost their guts millions of years ago. Instead of a stomach or a mouth, they have a sack of microbes inside them that help them eat the chemicals bubbling up from deep inside the Earth. 

LC: That sounds like it would be crazy to see, and moving on a number of levels.

LC: How is studying microbes from the subsurface biosphere of the ocean in the lab complicated by the fact that they are from the deep ocean?

KL: Well, they’re expensive to get, both in time and money. And, in some cases, they’re impossible to get. I would LOVE to have samples all the way through Earth’s crust—straight through to the mantle—from all over the world, but we don’t have the capability to drill that deep everywhere. In fact, it’s never been done, although there are good attempts happening in places where Earth’s crust is the thinnest. But the hardest part is probably making sure the samples are not too contaminated. We solve that problem in many different ways, but it is always a problem.

LC: Can you describe the scene when you pull a sample up, say, from the bottom of the Baltic? What does it look like? What does it feel like?

KL: The look and feel of a core all depends on how you’re sampling, and what type of sediments you’re coring into. I’ve done gravity coring through methane seeps in the Gulf of Mexico that was pretty exciting. A gravity core is a long 18 to 30 foot pipe that’s about 5 inches in diameter. You load it up with about 2 tons of weight and a big metal top-section that makes it fly straight through the water, like the feathers on an arrow. You slowly lower the whole heavy thing off a winch on the A-frame on the stern of the boat. When you think you’re in a good position, you drop it, and let it slam into the seafloor, then you winch it back up to the back deck of the ship, and catch it with hooks and lines to make sure it doesn’t swing around and hurt anyone. This task is done by scientists and the ship’s crew who are wearing uninflated life vests, hard hats, and steel toed boots. And, yes, the women do this job alongside the men. If you’ve hit sediments that don’t have too much methane in them, the core comes up gushing water, but you just use the A frame to lie the core on its side, slide the core inside its plastic core liner out the end, and carry it inside the ship’s labs to cut it in half and begin sampling it. The mud can be all kinds of colors, depending on where you are. I’ve seen bright red, tan, gray, black, brown, seafoam green, and even brilliant blue. Sometimes there will be frozen solid bits of methane hydrate inside that fizz and pop as they dissipate. When that happens everyone moves really fast to sample and document it before it’s gone. Sometimes, if you sample a high-methane sediment, the mud shoots out of the top of the gravity corer as you bring it up to the ship. Not only does it make a huge mess of the back deck, you have to just watch helplessly as your precious samples spray everywhere.

LC: What is the deepest that people have been able to drill and to find life?

KL: The deepest samples have come from gold mines in South Africa that were already drilled by mining operations. The deepest marine sediments that have been drilled and examined for biology are somewhere 2.5 km. (Inagaki et al., 2015)

LC: The microbes that you study are very different from what people typically think of as being life. Could you describe some characteristics that make these microbes very different from other life we have encountered before? 

KL: Most of the life we’re concerned with on a day-to-day basis uses the same set of things to eat and breathe. We breathe oxygen and we eat sugars, fats, and proteins. But microbes can breathe rocks—even many different kinds of rocks. They can even breathe radioactive uranium. And they can eat oil that we spill out of tankers, or even plastics which are normally thought of as non-biodegradable. Given the right conditions, the right microbes, and enough time, they’ll slurp up every plastic bottle we’ve dumped into the oceans. The unfortunate thing for us is that question of time. If we can figure out how to get them to work faster on this problem, they can really help us out.

LC: How does this breathing take place? What processes or mechanisms allow the microbes to do this?

KL: Breathing just means that you have an enzyme that can add an electron to a chemical in such a way that energy is released, and results in a gradient across a cellular membrane. Microbes have enzymes that are chemically tuned to work with chemicals other than oxygen to do this.

LC: Earlier you mentioned time. If I understand correctly, early attempts to grow these microbes in a lab were stymied due to the microbes seeming to be inactive or dormant. But researchers realized that this apparent inactivity is really just an illusion caused by their slow metabolism—and that they can in fact live for thousands of years. This contrasts sharply with many of the microbes we are most familiar with as humans like mold or bacterial infections. What allows them to live as long as they do?

KL: We’re not sure yet! So far, we’re still working on feeling certain that they even can live this long. But the consensus is that they do. Finding out “how” is harder because much of what we learn about molecular biology is only possible with cells that grow. We’ve inferred that they use enzymatic transcriptional regulators (Bird et al., 2019), pack their cells with high proportions of osmolytes (Bird et al., 2019) or small protective proteins (Buongiorno et al., 2020), and switch to using more highly-degraded food sources (Steen et al., 2019). There’s probably a lot of other things that make this possible—and we’re working on finding out.

LC: I am fascinated by the realization that these organisms are, potentially, immortal, as well as by the idea that we can miss things—crucial things—about the world around us because of the time scale within which we operate. How is our understanding of life limited by our own perception of time/time scales? 

KL: They can’t literally be immortal, because they weren’t all plopped down right where they are when Earth formed 4.5 billion years ago. They just seem immortal, relative to our lifespans. I think it’s always difficult to conceptualize quantities of anything (size, age, weight) that are vastly outside our experience. Trying to think about microbes that live for millions of years is like trying to think about how far the farthest galaxies are from Earth. Possible, but only if you really free your mind.

LC: Besides timescale, these microbes are also intriguing in their independence from light. I grew up thinking that the sun was essential to life, but for these microbes that isn’t true. How has being independent of the sun influenced the physiology of these microbes? How does reliance on the sun limit the lifespan of terrestrial or surface organisms?

KL: Being independent from the sun determines everything about these microbes—how they get their energy, where they can live, how they interact with each other and their ecological niche. I have no idea how the sun limits the lifespan of surface organisms (or whether it does so at all). But I would love to know!

LC: Could you speak some more to how these organisms are able to function without sunlight? Do they experience a circadian rhythm?

KL: Since many of them haven’t been grown in a lab, it’s hard to know if they’ve got circadian rhythms. They do have parts of the clock genes, but they do not have the full set of genes known to drive timing in circadian rhythms.

LC: I guess what I’m also getting at here is: how do we think like a microbe? What does the world look like from their perspective? Do they interact with each other at all?

KL: Another interesting question! One thing to realize is that small life does not experience viscosity like we do. A swimming microbe can’t just swim up to a food particle. If they try to do it, they just push that particle away. So, our basic interactions with the matter around us would feel different if we were that small. Many scientists think that many of the microbes on Earth team up with each other to help each other out. So, your experience as a microbe would likely involve having your fate tied closely to another microbe’s on whom you depend to help both of you eat your food.

LC: Whoa, that is fascinating! I feel like it is easy to take for granted what it means to move…and it is interesting to be reminded that our experience of movement is dependent on our size and is very much not universal. I also wouldn’t have imagined such intimate collaboration between microbes. 

LC: Could you describe how subsurface microbes find each other and how this partnership works?

KL: We really don’t know a lot about this. But, since there’s not a lot of energy available, they probably don’t do much swimming. People hypothesize that there are syntrophic relationships, where they rely on each other [for metabolic processes]. 

LC: Learning about the existence of this vast, unexplored biome beneath earth’s surface changes (at least for me) how one views Earth as a life-sustaining planet, with these ancient rock-consuming organisms making the planet itself seem to be alive. How long have microbial communities existed in these environments? For how much longer could they exist? 

KL: The oldest known fossil life is from about 3.8 billion years ago. Life could have existed on Earth even earlier, but we don’t have a lot of rocks from that time. These microbes could theoretically continue to survive on Earth long after the sun burns out. The thing they probably couldn’t survive is if Earth is so old that all the chemical reactions have been completed and all the geothermal gradients have dissipated. That would kill them all, for sure.

LC: There is something really beautiful about that. It gets me thinking about how all life is just a set of chemical reactions occurring down an energy gradient—and at its most essential, that is all life needs to survive. 

LC: I’m curious, how did microbes get to be thousands of meters below the surface of the earth to begin with? 

KL: Life may have started in the subsurface. So the question might be, how did we get to the surface and do the audacious thing of actually surviving there with this harsh UV radiation. 

LC: In talking about these microbes’ ability to survive on these simple (well, perhaps not so simple) conditions, I am curious about their relationship with the upper world. Through climate change and habitat destruction (among other things), surface biomes are changing relatively rapidly—and many species on land and in the oceans are having to adapt to these new conditions. 

LC: How are subsurface microbes affected by, or involved in, these processes? How removed are ocean subsurface microbes from the going-ons up above? 

KL: We’re just learning about how the surface and subsurface biomes interact. Subsurface microbes will certainly be a part of the long-term adjustment of the Earth to the evolution of humans. But it might be possible to get them involved even sooner, if we can pump our excess carbon down to them and have them use it as building blocks for their bodies, or help precipitate it as rock.

LC: That is so cool! How fast would these microbes be able to consume and metabolize carbon in such a scenario? I know that carbon sequestration is already being used in places as a method of reducing atmospheric CO2. Might some of these efforts already be inadvertently feeding microbes?

KL:  I think the speed would depend on the size and dimensions of pore spaces, the saturation level of carbon, calcium, or magnesium in the pore water, and the availability of electron acceptors and donors to drive the microbial energetics. So, in a nutshell, there’s no universal answer for this, and I wouldn’t even dare to guess!

LC: These microbes challenge many of our assumptions about life and what its requirements are: in regard to energy, time, and environment. How do these microbes change our understanding of life? How might they provide insight into the life that might be present elsewhere in the universe? 

KL: The discovery of abundant life at deep-sea hydrothermal vents in the 1970’s expanded our understanding of the range of temperatures and pressures capable of supporting life. Every new limit that’s broken continues to push that even further. I think the more we learn about life in the subsurface, the more we’ll be able to expand our search for life outside our planet.

LC: I now want to turn away from the microbes themselves and ask about how they might (or might not) be emblematic of the increasingly apparent limits to our knowledge of the life we coexist with. Your work shows us yet another (very drastic) example of the limits of human understanding of life on earth. And yet, throughout history, people have repeatedly been confident about the limits of knowledge, and of having reached “all there is to discover”. 

LC: Humans seem simultaneously fascinated by the unknown and set in believing the limits of discovery. Why do you think this is the case? 

KL: I don’t know. That’s sort of a philosophical, sociological, or psychological question. I’m not aware of any scientist throughout history who has declared that we’ve reached the limits of discovery. I certainly wouldn’t ever say that!

LC: I’m glad. I was thinking more of old naturalists and physicists, such as Linnaeus and Albert Michaelson, who when they were alive thought discovery of new species or laws of physics was close to being exhausted (Dobson et al., 2008; Bradshaw 2001). Perhaps another way to phrase this is:

LC: How do you see these microbes changing our cultural awareness of ourselves in regard to the environments of which we are a part? 

KL:  Again, this is a bit out of my area of expertise. But, I, personally, love knowing that we’re less alone on this Earth than we previously thought. Our planet has even more varied life than we ever knew before. Somehow that feels cozy to me – maybe to other people too?

LC: Yes, for sure—I feel the coziness too. 

LC: Do you identify with any of the microbes you study? Do any of them remind you of you? 

KL: What an interesting question! I don’t normally think of identifying with the microbes, like you would if you worked on something more charismatic like puppies or frogs. I guess I really like their resourcefulness. If there is a chemical reaction that falls in the energetic sweet spot—exergonic, but kinetically stalled—they will find a way to capture the energy from that reaction as they catalyze it. I’m the same way. I really like to derive happiness from whatever opportunity is available, even if I have to evolve a little to do so. 

Glossary

Enzymatic Transcriptional Regulators: Enzymes are specific types of proteins that regulate many cellular functions. Enzymatic transcriptional regulators are enzymes that help with gene transcription, which is the process in which a cell’s genetic material is read by the cell and used to build new proteins and cell structures, as well as to carry out cell activities. 

Osmolytes: These are molecules that help an organism’s cells maintain structural balance with their surroundings through aiding with water absorption and release. They can also be involved in stabilizing proteins so that they are able to function longer.

Highly-degraded food sources: Organic (carbon-based) molecules that have been broken down into less complex molecules from their original form.

Clock genes: Genes that regulate daily biological cycles.

Viscosity: The friction between molecules in a liquid. This can refer to both the molecules of a liquid itself, and other things (such as microbes) within a body of liquid.

Syntropy: This is when multiple microorganisms work together to metabolize nutrients. By splitting the work up, it is easier to accomplish under low energy conditions.

Pore spaces: Openings within rock and sediment that contain air and/or water.

Electron acceptors and donors: In chemical reactions, electrons are transferred between different molecules. Molecules that gain electrons through this transfer are known as electron acceptors, and those that lose electrons are electron donors. This electron transfer can result in the release of energy, and it is this energy that powers metabolic function within organisms.

Works Cited

• Bird JT, Tague ED, Zinke L, Schmidt JM, Steen AD, Reese B, Marshall IPG, Webster G, Weightman A, Castro HG, Campagna SR, Lloyd KG. 2019. Uncultured Microbial Phyla Suggest Mechanisms for Multi-Thousand-Year Subsistence in Baltic Sea Sediments. mBio. 10(2): e02376-18. DOI: 10.1128/mBio.02376-18
• Bradshaw AM. 2001. Physics from the inside. Nature. 412: 121-122. https://doi.org/10.1038/35084119
• Buongiorno J, Sipes K, Wasmund K, Loy A, Lloyd KG. 2020. Woeseiales transcriptional response to shallow burial in Arctic fjord surface sediment. PLoS One. 15(8): e0234839. https://doi.org/10.1371/journal.pone.0234839 
• Dobson A, Lafferty KD, Kuris AM, Hechinger RF, Jetz W. 2008. Homage to Linnaeus: How many parasites? How many hosts? PNAS. 105 (Supplement 1) 11482-11489. https://doi.org/10.1073/pnas.0803232105  
• Inagaki F, Hinrichs KU, Kubo Y, Bowles MW, Heuer VB, Hong WL, Hoshino T, Ijiri A, Imachi H, Ito M, Kaneko M, Lever MA, Lin YS, Methé BA, Morita S, Morono Y, Tanikawa W, Bihan M, Bowden SA, Elvert M, Glombitza C, Gross D, Harrington GJ, Hori T, Li K, Limmer D, Liu CH, Murayama M, Ohkouchi N, Ono S, Park YS, Phillips SC,  Prieto-Mollar X, Purkey M, Riedinger N, Sanada Y, Sauvage J, Snyder G, Susilawati R, Takano Y, Tasumi E, Terada T,  Tomaru H, Trembath-Reichert E, Wang DT, Yamada Y. 2015. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor. Science. 349(6246): 420-424. DOI: 10.1126/science.aaa6882
• Morris BEL, Henneberger R, Huber H, Moissl-Eichinger C. 2013. Microbial syntrophy: interaction for the common good. FEMS Microbiology Reviews. 37(3): 384-406. https://doi.org/10.1111/1574-6976.12019 
• Steen AD, Kevorkian RT, Bird JT, Dombrowski N, Baker BJ, Hagen SM, Mulligan KH, Schmidt JM, Webber AT, Royalty ™, Alperin MJ. 2019. Kinetics and Identities of Extracellular Peptidases in Subsurface Sediments of the White Oak River Estuary, North Carolina. Applied and Environmental Microbiology. 85(19): e00102-19. https://doi.org/10.1128/AEM.00102-19