Keynote: Slow, energy-efficient, and mysterious life deep within Earth's crust

by Karen G Lloyd

The keynote presentation by Karen G Lloyd at RubyConf 2019 explores the fascinating biology of microbial life deep within Earth's crust, primarily focusing on marine sediments. The talk highlights how the vast majority of lives on Earth exist within these deep subsurface environments, suggesting that to truly understand life on our planet, we need to investigate its hidden aspects. Lloyd emphasizes that approximately a third of the microbes on Earth could be found buried beneath the ocean floor, creating an immense and largely unexplored ecosystem.

Key points discussed include:

- The significant amount of Earth's surface covered by oceans and the importance of marine sediments for studying microbial life.

- The method of sampling deep-sea sediments using specialized drilling ships, such as the JOIDES Resolution, and the challenges associated with cultivating these microbes in laboratory settings.

- The concept of 'microbial dark matter,' which refers to the many unknown and uncultured microbes that exist in these environments.

- Research findings that reveal these microbes exhibit incredibly slow growth rates, with some remaining dormant for centuries, which poses questions about their survival strategies.

- The exploration of specific case studies, such as Lloyd's work in the Baltic Sea and sampling in the Central American volcanic arc, demonstrating the adaptability of microbes to extreme conditions.

- A discussion on how these microbes utilize limited resources and may exhibit collaborative behaviors that challenge traditional biological assumptions.

- The implications of these findings for our understanding of life not just on Earth but potentially on other planets, such as Mars and its subsurface environments.

In conclusion, Lloyd underscores the need to rethink our assumptions regarding microbial life, especially those that live in extreme environments, and invites further exploration into microbial ecosystems that might exist beyond our planet. The takeaway from the presentation emphasizes the secrets held by life in the deep Earth and the potential for discovering new forms of life in seemingly inhospitable conditions. The study of these microbes not only deepens our understanding of biology but also expands the possibilities of life beyond Earth.

00:00:12.190 So, I'm going to talk about biology and life in the deep Earth. This image from my title slide is actually from the volcanic arc of the Andes. My Argentine and Chilean colleagues invited us down to study this, but really, most of my talk will focus on the oceans. Hopefully, by the time I'm done, you’ll see how it connects back to the volcanoes.

00:00:18.020 What planet is this? It’s refreshing to flip the Earth around and check out the Pacific Ocean every now and then. We spend so much time looking at Earth from the ‘front side’ that it's easy to forget that there’s a lot of ocean and very little land.

00:00:24.590 New Zealand creeps in, hard to get it out, but it’s a nice reminder that we probably all know that 74% of the Earth is covered by oceans. Well, it really is true, and looking at it from the back can help remind you of that. Since most of the Earth is ocean, if you want to know what’s inside Earth and how life interacts with it, you're really talking about marine sediments. That’s where the majority of the crust is habitable by microbes.

00:01:07.250 We’ve done some estimates, and there are probably 3 times 10 to the 20 living microbial cells buried in the sea floor. I know numbers that big are really hard to conceptualize, so to put it in perspective, that’s about a third of the microbes on the planet buried underneath the sea floor, which is roughly ten thousand times more than the number of stars in the universe.

00:01:14.180 This represents an incredibly vast ecosystem. So if you want to study life on Earth, you have to look inside the Earth, because that’s where the majority of it is.

00:01:20.360 But how do we go about learning about these deep subsurface microbes? The big thing is that we go out into nature and collect samples. Occasionally, people will ask me if I'm just sitting at my computer in the lab while someone else, a kind of adventurer, goes out to collect samples for me. No, that’s not how it works. I certainly could be doing other fields of science, but I do this one because I get to go to cool places.

00:01:49.379 We collect samples and measure all the chemicals around these microbes. We try to measure as many things as possible to discern what they are eating and how life works in these strange environments. The most important thing we typically do in the laboratory is chemically extract their DNA. We pull out their DNA and other biomolecules directly from the natural sample because we use this to infer what they are doing.

00:02:16.250 We can also bring these samples home to try to grow things in our laboratories. The work that I’m telling you about now comes from oceanic sediments. We take a ship, the JOIDES Resolution, which you can see has a big drill stack on top of it. This is a long series where we have to load up pipes that get taken down to the seafloor.

00:02:29.459 There’s a little explosion at the seafloor, and those pipes get shoved down into the sediment. This advanced piston coring method can bring back hundreds of meters of Earth sediments, which is very exciting and fun work. So, we go out on ships and collect these samples. If you want to start thinking about what kind of microbes are out there, one thing to know as background is that we already know quite a bit about microbes elsewhere on Earth.

00:02:55.859 We know for sure that there are many microbes on this planet. I found a picture on the internet that I thought was very beautiful. Someone left their handprint on a petri dish, which shows the microbes present on their hand. It may seem disgusting at first, but if you think about it, that person probably did not suffer from an infection due to all those microbes.

00:03:11.970 Most of the microbes on the planet do not kill us. While we spend a lot of time in microbiology studying pathogens and diseases, for the most part, microbes on Earth are not pathogens; they don’t kill us. I do want to point out that while we have only been on this Earth for a few million years, microbes have been here for literally billions of years. They may just not have figured out how to kill us yet.

00:03:40.659 So, when we collected our first samples that we could be sure were not contaminated, we found really good samples from the deep subsurface environment off the coast of Peru. I was lucky enough to have a lot of collaborators for this work. No scientist ever works alone! If someone tells you that a single person discovered something, it’s usually a collaborative effort involving many others.

00:04:06.990 This was my first project as a PhD student, and it was an exciting time. We discovered significant findings. I will show you the data that we found, but I won’t bore you with microbial names or phylogenetic trees. Instead, I’ll visually represent each microbe with a box.

00:04:25.590 For each time we obtained a strain or a DNA sequence from a microbe that was similar to something that someone had discovered before, I’ll show it with a darker blue box. If it’s a lighter box, it indicates a completely new type of microbe that was previously undiscovered. We were amazed to find almost all the boxes were white or light colored. This finding opened the door to a previously hidden world full of interesting life.

00:04:43.970 As microbiologists, when you see a result like this and notice that everything appears to be new, it’s time to start culturing things and getting them to grow in laboratories. Luckily, we had some of the best culturing labs in the world on the ship.

00:05:01.800 They took our samples back to their labs and began growing the microbes. Every time they got a microbe that matched with what we found through our DNA sequencing, I put that result directly underneath the corresponding box so they matched up. If the matches were new, I placed them to the right, and they were colored accordingly.

00:05:22.830 This is what we found: unfortunately, they managed to grow nothing from our samples. There was no overlap between the microbes we found and the ones they could cultivate. Moreover, everything that they cultured was very similar to the kinds of microbes we already knew about, indicating we had a significant challenge.

00:05:43.750 I don’t want to malign those labs; they know what they're doing. The issue was simply that these microbes were living in ways that we were not able to adequately replicate in the lab setting. Many people believe it’s about pressure, and while experiments under pressure have been conducted, they alone do not solve the issue.

00:06:05.300 This has become a pervasive problem recognized worldwide. A lot of people refer to these mysteries as microbial dark matter, borrowing the term from astronomy. Just as most of the matter in the universe is dark matter, which we know exists due to its gravitational effects but cannot see, most of the microbes in this Earth are similarly elusive. This microbial dark matter phenomenon means we have to rethink our basic assumptions about microbiology.

00:06:44.750 We usually teach that microbes grow in cultures, like in test tubes, and we have a general conception of their behaviors. However, these strange microbes challenge the frameworks we have operated within. It is crucial to recognize these assumptions because they dictate our understanding of what life looks like in different environments.

00:07:10.330 Let’s consider the microbes’ perspective: what is it like to be buried in the seafloor? This is my schematic representation, showing seawater overlying sediments where microbes are buried. The process is slow, but sometimes they can get kicked up. On average, the microbes get buried deeper and deeper.

00:07:40.470 The best thing to breathe is oxygen, a fabulous energy source. The fact that we can be these large multicellular organisms spewing out heat everywhere is remarkably inefficient, but it’s because oxygen incredibly energizes our metabolic processes. As this viable source gets depleted, what’s left to breathe becomes increasingly less efficient.

00:08:01.220 Eventually, you’re left with carbon dioxide, which is a wild concept since it is strange to think of carbon dioxide as a metabolic requirement. However, these microbes can utilize it — but it means the energy they can harness becomes more and more limited as they are buried. Additionally, the carbon that they use to create biomass is not replenished, so as they go deeper, their available energy diminishes.

00:08:42.460 As a consequence, these microbes must rely on the remnants of what was laid down in the sediments, often thousands of years previously. This all results in a dramatic decrease in energy availability. Our predictions indicate that the number of microbial cells in these sediments should decrease accordingly, and we have measured this effect quite accurately across multiple sites.

00:09:03.600 This chart illustrates our data compilation from various locations worldwide, with the y-axis representing the total number of cells. As you descend into the marine mud, the cell count decreases significantly. This pattern is consistent in all oceans around the world. However, we are interested in the growth rates of these microbial populations.

00:09:33.360 On one hand, we can see that populations are shrinking with depth. But we want to determine how quickly these microbes grow. Just like observing a patch of grass getting smaller every summer, that doesn’t mean the grass blades didn't grow; it implies that the total population size has changed.

00:10:04.980 So, we can analyze burial rates in marine sediments and see how they wax over 5,000 years. We've found that at the surface, cell generation times are already in the months, which is extraordinarily slow compared to, say, E. coli, which doubles every 30 minutes. In deeper sediments, we have seen cell duplication times stretching to 50 to 70 years.

00:10:38.450 These microbes may not divide for decades. This may sound surprising, yet in my field, it is accepted that these living cells could be hundreds of thousands of years old. My graduate student was shocked when he first learned this concept, and he coined a term I love: 'It’s not a biome; it’s a die-home.' That’s brilliant in its simplicity.

00:11:15.930 At this point in the talk, some of you might wonder why I am so invested in studying what appears to be an entire ecosystem on the brink of dying. Some might wonder why I devote my entire professional life to studying this matter. However, I'll tell you that I find it utterly fascinating to explore how a cell can exist for so long. What does it mean for a cell to survive over such extended periods?

00:11:45.430 This mode of biology is fundamentally different from everything I have ever encountered. I am curious about how evolutionary pressures manifest over such long timescales. When we remove the conventional rules of biology, we open ourselves up to unexpected phenomena. For instance, in Darwinian evolution, rapid growth seems like the best strategy.

00:12:18.500 While we usually consider competition in ecology in terms of increasing population size, we recognize that growth alone does not define success. In ecology, we refer to these as R-strategists, who grow rapidly, and K-strategists, who take their time to increase their population. Being a K-strategist means you conserve resources; you’re steady and patient.

00:12:55.370 We can extrapolate this concept to deep marine sediments where microbes exist in a zero growth state. Now, what does it look like for them? I’d argue that perhaps the goal in this unique environment is merely to survive and persist over time.

00:13:37.530 I had conducted a project in the Baltic Sea, located between Sweden, Finland, Latvia, and Estonia, with an incredible group of students. We wanted to explore how bacteria and archaea could survive in near-zero growth states for 8,000 years, located 50 meters deep within Baltic Sea sediments. The technique we employed was to isolate individual microbial genomes from this heterogeneous mix, which was quite a challenge.

00:14:20.650 To achieve this, we used flow cytometry. This effectively involves creating tiny droplets of water, each containing one microbial cell, and analyzing them using lasers to confirm what they are. After isolating a single cell, we managed to crack it open and, using enzymes, amplified its genome.

00:14:49.500 This was then sent to a sequencing machine to get the DNA sequences, which were fragmented and reconstructed to create a complete genome. We analyzed the genes present, which act like a menu displaying what the microbes can do.

00:15:22.630 Upon examining the genomes obtained from these deep subsurface organisms, we looked for abundant interesting features. Something that immediately stood out were various toxin-antitoxin systems. We realized that none of these microbes belonged to phyla that had ever been cultured before.

00:15:47.590 This is when I introduced the concept of toxin-antitoxin systems. Evolution has led to many cells producing a toxin that essentially kills them, while also producing the antidote. This peculiar mechanism acts as a safeguard, slowing down metabolism and allowing cells to persist even in harsh conditions.

00:16:25.970 This phenomenon is also observed in Mycobacterium tuberculosis, where this mechanism allows it to endure antibiotic attacks. The cells slow their growth and enter a low-energy state, which they maintain by recycling internal resources.

00:17:06.800 Now, while we have identified numerous systems, I'll focus on just one because explaining all of them would take all day. We believe that one characteristic these microbes possess in the deep subsurface is the ability to utilize toxin systems to slow their growth.

00:17:44.380 This doesn’t suffer them, specific food sources must be identified. To determine what they consume while existing in nutrient-scarce conditions, we studied metabolites — the tiny molecules that reflect cellular metabolic processes.

00:18:24.420 My colleagues have successfully performed this analysis on known organisms, creating visual representations to summarize the metabolic pathways at play. We decided to apply the same analysis to our sediment samples. Initially hesitant, I was convinced by my colleague Hector's suggestion to examine our deep-sea mud. Reluctantly, I agreed to explore the metabolites in the sediments.

00:19:03.670 The results were surprising; instead of the expected thousands of molecules, we obtained only 20 molecules from our analysis — which is substantially better than the anticipated absence of results! I went over the obtained molecules to investigate their functions.

00:19:38.490 One molecule that caught our attention was called alanine. It’s nitrogen-rich, and intriguingly, it was present in nearly every sediment core we examined. This molecule is a product of decomposition and could represent a food source for these microbes.

00:20:05.840 Given that we had access to a wealth of genes, we sought to determine if these microbes were actively consuming alanine. We examined various groups of microbes present in our samples, comparing them to different enzymes important for alanine metabolism.

00:20:26.660 Interestingly, one clade of microbes seemed particularly adept at processing alanine. This discovery led me to contemplate why these microbes wouldn’t simply proliferate like other organisms that possess a unique food source.

00:21:01.549 To answer this, I collaborated with colleagues at Texas A&M and USC, where they worked on meta-transcriptomes, essentially analyzing the transcriptional activity of genes in our microbial community. They successfully managed to extract active genes with remarkable results despite the challenges of working with crude sea sediment.

00:21:44.460 Among the genes expressed, one stood out: an exporter of amino acids. This was intriguing because amino acids are energy-rich, representing a fundamental food source, yet it appeared that the microorganisms were not only consuming them but also actively releasing them.

00:22:28.080 In contrast to typical apocalyptic narratives where resources are hoarded for survival, these microbes seem to adopt an alternative strategy by sharing their nutrient wealth with others in their community. Perhaps this selfless act is a calculated risk to avoid triggering competitive growth.

00:23:10.650 Based on our findings, we hypothesize that by allowing amino acid concentrations to rise too high within their cells, they would promote division and competition with themselves. Consequently, these microbes might be purposely releasing amino acids to maintain a stable equilibrium.

00:23:55.210 Interestingly, this sharing behavior could also sustain other organisms in the community, even if this isn’t done for altruistic reasons. It raises intriguing questions about adaptation and coexistence in microbial communities living in extreme environments.

00:24:34.360 As I’ve discussed adaptations for thriving in low nutrient environments, a critical consideration emerges regarding how natural selection allows microbes to persist over such immense time spans. With many of these organisms operating on geological timescales, we must consider geological factors during their lifecycle.

00:25:16.110 To illustrate, oceanic tectonic plates undergo processes like subduction over thousands of years. As oceanic plates are pushed beneath continental plates, this could potentially allow the return of dormant microbes to the surface.

00:25:59.490 In my new project, we've sampled various sites in conjunction with my Costa Rican colleagues along the volcanic arc of Central America in Panama and Costa Rica to explore these connections further. The collaborative efforts span deep into tropical jungles, where we collect samples in muddy environments.

00:26:44.640 This particular environment is rich in microbial life, abundant with the deep subsurface microbes that are utilizing the interfaces created by the oxygen present in surface environments. As a result, we've been diving deep, sampling environments which may be the most dangerous places we've encountered. One instance included sampling inside the crater of Poás Volcano.

00:27:29.680 My Costa Rican colleagues were familiar with the crater's characteristics but we approached with caution, since volcanic activity frequently and suddenly surfaces here. The scary part is that what appears to be a volcanic lake is laden with acidic properties, with steep walls composed of loose debris.

00:28:01.880 To reach it, we hiked and gathered samples while dodging the risks of falling into the lake filled with caustic water heated by volcanic gas. Thankfully, we avoided dangers during our sampling, but just a month and a half later, a massive eruption occurred at the same site.

00:28:59.250 Nature is indifferent to the scientists gathering samples, and we must always be cautious and calculated in our endeavors. Post-eruption, it’s incredible to consider that microbes can thrive in seemingly inhospitable environments. They are used to living in such extremes, which challenges our understanding of what constitutes a viable ecosystem.

00:29:47.170 Pointing our perspectives outward, I want to stress that life need not be limited to Earth. When thinking about life on other planets, we must acknowledge the importance of subsurface environments. Mars, for instance, may not seem conducive to life due to surface conditions, but any subsurface could potentially harbor thriving ecosystems.

00:30:37.670 Similarly, moons like Europa or Io might have incredible hidden life, and it’s crucial to broaden our horizons beyond Earth when seeking life in our universe. I'd like to end by expressing my gratitude to my funders, as their support helps sustain critical scientific exploration.

00:31:17.250 This is my building at the University of Tennessee in Knoxville, located a few hours east of here. A slightly different version of the glittery Nashville, but I appreciate the opportunity to speak with you today and share this journey with you. Thank you!