Deep Sea Life: Studying Subseafloor Microbes

Part 1: Microbes, Fluids, and Rocks

00:00:07.21 Hi.
00:00:08.21 My name is Julie Huber, and I'm an oceanographer at Woods Hole Oceanographic Institution.
00:00:14.16 My background is in marine science, microbiology, and molecular biology.
00:00:19.18 And what I want to do today is tell you a story about how I've woven those three fields together,
00:00:25.09 studying life in the deep ocean.
00:00:27.19 But to start out, in Part I of this talk, I just want to give you a really broad introduction
00:00:31.28 to microbes and how they operate in the deep ocean.
00:00:35.12 So, first, I need to convince you that microbes are important and they basically run our planet.
00:00:41.28 So, I like to think of Planet Earth as also Planet Microbe.
00:00:47.09 Microbes are so-called "simple" organisms but in fact they can do some pretty complex things.
00:00:54.20 Shown here is a very basic Tree of Life, where we have the two domains of life
00:01:00.08 that belong to microbes: bacteria and archaea.
00:01:03.03 Now, microbes have no true nucleus.
00:01:05.12 They have a single chromosome.
00:01:07.00 They don't have any membrane-bound organelles.
00:01:09.06 And they divide by simple binary fission.
00:01:12.03 However, given how simple they are, they take up a huge diversity of life on our planet.
00:01:18.02 This is a much more complicated Tree of Life that's been put together using the power of
00:01:23.17 DNA sequencing.
00:01:25.11 And just to orient you on this tree, you're down here, in the eukaryotes at the very tip.
00:01:31.08 The rest of the tree, in the bacterial and archaeal domains, are taken up by microbes.
00:01:37.02 So, they fill up a huge phylogenetic space on our planet.
00:01:42.18 These microbes are not only diverse, but there are a huge amount of them on the planet.
00:01:47.17 So, there's upwards of a hundred million microbial cells in a single gram of soil.
00:01:52.17 And there's more biomass, or more carbon, trapped inside these microbes
00:01:56.04 than plants and animals combined.
00:01:58.14 And if you were able to in fact count every microbial cell on Earth, you would see
00:02:03.02 that there are more microbes than stars in the universe.
00:02:06.24 These microbes come in a lot of shapes and forms and colors, many of which are illustrated here.
00:02:13.16 And they serve a huge range of functions on our planet.
00:02:17.06 Some of these are probably familiar to you, such as the microbes that provide our drugs,
00:02:22.12 over here, in the form of antibiotics.
00:02:24.13 They also help us make food, things like cheese and beer, which we all enjoy.
00:02:29.16 They also help plants grow, for example, by fixing nitrogen in the soil.
00:02:34.03 And of course, the oxygen that we breathe on our planet, many microbes,
00:02:38.08 in the world's oceans in particular, make that oxygen through photosynthesis.
00:02:42.24 They're also really important here, in the human body, right?
00:02:46.10 We are covered with microbes, and they fill our insides, and they keep us healthy in most situations.
00:02:52.01 And in the oceans, they also play a really critical role in associations with other animals,
00:02:57.10 such as coral reefs, and also, recently, with the Deepwater Horizon oil spill.
00:03:02.27 It was actually microbes that were primed to eat oil that were the first responders.
00:03:09.01 These were the microbes in the ocean that ate a lot of the carbon and the hydrocarbons
00:03:13.17 even before scientists and engineers were able to fix that problem.
00:03:18.09 So, we have a lot of things on our planet to thank microbes for.
00:03:22.22 And what I want to do now is show you how they operate broadly in the ocean,
00:03:27.01 and then move into the deep sea.
00:03:28.17 So, I already called our Planet Microbe, but it's also obviously Planet Ocean.
00:03:33.03 70% of the Earth's surface is covered in seawater.
00:03:36.23 And in the oceans, microbes do some pretty amazing things.
00:03:40.15 This is another way of looking at the ocean.
00:03:43.21 This is basically a map showing the topography of the seafloor and the continents.
00:03:48.28 And what I really want you to notice here is these large swaths of blue.
00:03:52.23 This isn't seawater.
00:03:53.26 This is actually the bottom of the ocean.
00:03:56.12 And there's a huge range of features on the bottom of the ocean, which I'm gonna be
00:04:00.01 talking about today.
00:04:01.10 And the microbes that are living in the deep sea.
00:04:04.15 So, before I get deep, I wanna make sure you understand how microbes operate in the surface ocean.
00:04:11.07 And this is a really complicated look at how they work in the surface ocean.
00:04:15.24 But the very basics are that there are microbes in the surface ocean that are using sunlight
00:04:21.05 to fix carbon, over here, and they produce oxygen, and this moves a bunch of carbon
00:04:27.03 into the microbial food web and higher life forms.
00:04:30.00 We can really simplify this diagram, though, and look at those key players again.
00:04:34.05 So again, we have photosynthetic primary producers up here.
00:04:37.20 And that carbon, also called dissolved organic matter, it feeds a lot of different organisms.
00:04:42.28 But the first step is it goes into heterotrophic microbes, organisms that eat that organic carbon.
00:04:50.05 We then have the secondary consumers, which in this case are grazers.
00:04:54.07 Things like copepods or zooplankton.
00:04:57.06 And this feeds into sort of our classic food chain, higher life forms like fish and crabs
00:05:02.24 and things like that.
00:05:04.13 One important component of this microbial food web are these viruses, shown over here.
00:05:10.02 And I'm not going to be talking about viruses today.
00:05:12.27 We mostly think about them in causing the flu and things like that, but viruses
00:05:17.07 are actually also very important in cycling that carbon, and also moving genes around
00:05:22.06 between microbes in the ocean.
00:05:23.27 So, in the deep ocean, what we need to do is flip this whole picture upside down.
00:05:29.20 So now, instead, what we have for our primary producers are microbes that are harnessing energy
00:05:36.04 from the Earth's crust, down here, and fixing carbon.
00:05:39.20 So, these primary producers are not using sunlight.
00:05:42.25 Instead, they're using chemical energy, and I'll be talking more about that as we move on.
00:05:48.11 But we have a lot of the key players... the same key players as we do in the surface ocean.
00:05:51.27 So, we have this carbon being produced.
00:05:54.24 We have heterotrophic organisms consuming it.
00:05:57.19 We have these grazers.
00:05:59.10 But what of course is most interesting about these systems is the higher life forms
00:06:03.06 that are supported by these microbes look very different.
00:06:06.08 Importantly, we do also have viruses in the deep ocean, although we know very little about them.
00:06:11.22 So, what do these ecosystems look like?
00:06:13.16 Well, this is one example, perhaps the most well-known example, of a hydrothermal system
00:06:19.15 being supported by these microbes in the Galapagos spreading ridge.
00:06:23.25 This is where hydrothermal vents were first discovered in 1977.
00:06:28.04 And what you're looking at are these giant tubeworms that have sort of a fake gut
00:06:33.13 that is packed full of bacteria.
00:06:35.15 And what those bacteria are doing is harnessing chemical energy from the fluids leaking
00:06:40.26 out of the seafloor.
00:06:41.26 They're fixing carbon.
00:06:43.21 And they are producing the carbon that then feeds the worm and the other animals
00:06:48.02 you can see in this movie, like there's some squat lobsters, these are limpets and other organisms.
00:06:53.24 And so we're supporting an entire ecosystem in a very different way than what we have
00:06:58.15 in the surface ocean.
00:07:00.01 These types of ecosystems are called chemosynthetic ecosystems.
00:07:04.02 As opposed to the surface world that we all live in, which is really driven by photosynthetic organisms.
00:07:10.09 So, the fact that these ecosystems exist was a huge surprise to science when they
00:07:15.17 were first discovered in 1977.
00:07:17.14 They were not predicted.
00:07:18.26 There was no hypothesis that giant tubeworms would be living at underwater volcanoes.
00:07:24.00 And the fact that it exists really has to do with this long-term interaction
00:07:28.14 between Earth and life on our planet, for the billions of years of Earth's history, and in particular,
00:07:33.23 the last 3.5 billion since life first originated on this planet.
00:07:38.04 And what happens is the rocks that make up the surface of our... the bottom of our ocean,
00:07:44.20 they interact with seawater and create a variety of energy sources that can support life.
00:07:50.05 And I'm gonna spend the next couple parts of my talk only talking about one potential example.
00:07:55.26 But I do want you to appreciate that there's actually a huge range of environments on our planet
00:08:00.05 that can use this water-rock reaction to support life.
00:08:03.17 Now, I just wanna go into that a little bit to help you understand the broad differences
00:08:10.14 based on these water-rock reactions.
00:08:12.16 So, shown here is a highly schematized version of oceanic crust, right here.
00:08:17.25 And at the very top are these rocks called basalt or mafic rocks.
00:08:22.14 And if you've ever seen videos of... like the Big Island of Hawaii erupting, for example,
00:08:26.26 that's the type of rock that's being erupted there.
00:08:29.17 When basalt reacts with seawater at high temperature, you get things like hydrogen sulfide and iron,
00:08:35.12 which microbes really love to eat.
00:08:37.21 And as a result you get ecosystems like the one I just showed you the movie of,
00:08:41.24 at the Galapagos spreading ridge.
00:08:44.07 In the last 15 years, however, oceanographers have discovered a variety of different types
00:08:49.14 of habitats with different types of water-rock reactions occurring.
00:08:53.12 So here, if we go to deeply buried rock, which sometimes gets thrust up onto the seafloor,
00:08:58.26 we get rocks that are called ultramafic.
00:09:01.15 And when these rocks react with seawater, you get very different food sources for microbes.
00:09:06.05 You get things like hydrogen, methane, and even simple organic compounds.
00:09:11.08 And in turn, these ecosystems -- this is one example called the Lost City hydrothermal vent field --
00:09:16.24 they look different, the microbes are different, and the animal communities
00:09:20.04 are thus different.
00:09:21.09 So, there's this very, you know, deep connection between the types of rocks present,
00:09:26.10 the types of microbes, and then the other life forms that you can get.
00:09:30.00 So, as I mentioned, I'm only gonna be talking about one ecosystem in detail today.
00:09:35.16 But I really want you to appreciate that there's a huge range of water-rock reactions on our planet
00:09:41.01 that can sustain life.
00:09:42.20 And they include everything from mid-ocean ridges, where plates are spreading apart;
00:09:47.17 to volcanic arcs, which are very common, in particular in the western Pacific;
00:09:51.19 to even really cold environments, where fluids are just eking out of the seafloor.
00:09:57.10 So, I've been studying these ecosystems because I'm especially interested,
00:10:03.24 not only in the life at the seafloor but also life beneath the seafloor.
00:10:08.22 And this is a relatively new field of study.
00:10:11.12 But I really like this cartoon that was in an article in Nature a number of years ago,
00:10:16.05 kind of illustrating very simply the habitat I'm interested in studying.
00:10:20.12 So, this is seawater.
00:10:22.23 And beneath all that seawater, as I already mentioned, is the seafloor.
00:10:26.08 And you have marine sediments.
00:10:28.04 And then, beneath that, the rock.
00:10:29.28 And within those sediments and rocks are microbes.
00:10:33.02 And I'm especially interested in those microbes in these rocky environments.
00:10:37.10 And the way I study them depends a lot on what the question is, but in particular
00:10:42.10 go to places where fluids are naturally leaking out of the seafloor, such as at volcanic systems,
00:10:47.24 and sample those fluids to infer what's happening beneath the seafloor.
00:10:53.15 This is a really exciting area of research, I think, because it's very exploratory in nature,
00:10:58.27 and we don't know that much.
00:11:00.20 But the general idea is that seawater is circulating not only through the ocean but also
00:11:07.17 through oceanic crust.
00:11:08.28 And so this is very similar to aquifers that we have on land,
00:11:12.22 where groundwater is moving through some matrix.
00:11:15.15 And in this case, it's seawater moving through the rocky ocean matrix.
00:11:20.05 And that's illustrated in this cartoon, where there are areas of the seafloor where seawater
00:11:24.22 enters the crust and travels for some period of time.
00:11:28.21 In some cases, we have no idea how long.
00:11:30.23 In other cases, we've constrained it to a few hundred years, or a few days.
00:11:36.19 But along this pathway, the seawater gets modified.
00:11:40.22 And the seawater can change in its chemistry.
00:11:43.15 But it's bringing microbes and viruses and nutrients with it.
00:11:46.26 And eventually, it has to pop back out somewhere.
00:11:49.06 So, in this case, at points of hydrothermal discharge.
00:11:53.02 So, in all cases, and what I'm going to be talking about today, is finding those points
00:11:57.26 where it's popping out.
00:11:59.19 And basically capturing those fluids, and trying to understand what's happening
00:12:03.23 beneath the seafloor.
00:12:05.02 So, as you can imagine, this is a pretty challenging thing to do since I've never walked
00:12:10.21 beneath the seafloor.
00:12:11.21 I'm always looking at the seafloor, trying to figure out what's going on beneath it.
00:12:16.08 So, to do this, I often have to go to sea.
00:12:20.06 And this is one example of a ship that we stay on for weeks at a time.
00:12:24.17 This is the RV Atlantis, which is operated by Woods Hole Oceanographic.
00:12:28.28 And the ship is fully equipped to take a bunch of scientists, engineers, and ship's crew.
00:12:35.04 And they basically take care of you while you're doing your science.
00:12:38.25 And once we're on the ship, we also need a way to then get to the bottom of the ocean.
00:12:44.22 Most of the systems that I study are a mile or more beneath the surface of the ocean,
00:12:49.28 so we need vehicles and instrumentation capable of handling the deep pressures...
00:12:55.05 the great pressures and depths of the ocean.
00:12:57.08 So, shown here are a couple examples of how I study the deep ocean.
00:13:02.00 Up here is a remotely operated vehicle called Jason.
00:13:05.08 So, this is a vehicle that's tethered back to the ship, so no people actually go
00:13:10.19 to the seafloor in it.
00:13:12.16 And we gain... we get... because of that cable, we get video, and we can interact
00:13:17.17 with the vehicle the entire time to collect our samples.
00:13:20.07 Shown next to that is the human-occupied submersible called Alvin.
00:13:26.05 This is the vehicle that first discovered hydrothermal systems.
00:13:30.03 And two scientists and one pilot go down.
00:13:32.21 You basically launch after breakfast; you come back at dinner.
00:13:36.08 And it is not attached to the ship in any way.
00:13:39.00 So, the people on the ship don't really know what's going on, but you get to
00:13:42.18 go to the seafloor and do your studies that way.
00:13:45.19 There's a couple other examples here.
00:13:48.03 This is a vehicle called Nereus, which unfortunately imploded a couple years ago and was lost.
00:13:54.20 But Nereus had the ability to go to the greatest depths of the oceans, up to 11,000 meters.
00:14:01.26 And finally, shown here is something I'm gonna be talking about in Parts II and III,
00:14:06.18 which is, instead of sending ships and vehicles, oceanography is trying to move toward
00:14:11.26 more observational science using infrastructure that's constantly in the environment.
00:14:17.05 This is obviously really challenging in the deep ocean.
00:14:19.24 But what this is showing is a number of cables that have been put on the seafloor,
00:14:24.17 basically plugged in to land, so we can constantly monitor and take samples more frequently
00:14:30.20 than when we have the opportunity to go on these ships.
00:14:33.25 So, this is a shot of me diving in Alvin, which is, as I said, one way that we do our work.
00:14:41.15 It was definitely one of the coolest experiences of my career, to travel, you know,
00:14:45.20 thousands of meters -- in this case, 2,200 meters -- to the seafloor to study these hydrothermal systems.
00:14:52.06 If we're using a remotely operated vehicle, we have a control station up on the ship.
00:14:56.25 And this is an example of what I see sitting in that room, where we have a group of pilots
00:15:01.01 sitting at the front and engineers who are operating the vehicles.
00:15:04.19 And then scientists interacting with them to get our samples, to navigate on the seafloor,
00:15:10.10 and things like that.
00:15:12.22 Once our samples are back, in my lab we use a huge suite of tools that draw on the training
00:15:19.00 that I've had in microbiology, molecular biology, and ocean sciences.
00:15:23.28 And just a few examples are shown here, ranging from chemistry to trying to grow these microbes
00:15:29.16 in the lab, which is especially challenging given the pressure and extreme conditions.
00:15:34.06 We do a lot of engineering on the side to try to get our samples back.
00:15:38.15 And what I'll be talking about Parts II and III is kind of combining genomics and stable isotopes
00:15:45.06 to understand how we can go from DNA, down here, all the way up to
00:15:50.24 how this ecosystem functions.
00:15:52.02 So, if you stay tuned, in the next part of the talk I'll start focusing in on some of these methods,
00:15:57.07 and how we use them to study sub-seafloor life.

Part 2: Subseafloor Life at Axial Seamount

00:00:07.19 Hi.
00:00:08.19 My name is Julie Huber, and I'm a scientist at the Woods Hole Oceanographic Institution.
00:00:13.07 And in Part II of my talk, I want to tell you about subseafloor at Axial Seamount.
00:00:19.13 I wrapped up Part I by telling you about the amazing reactions created between water and rock
00:00:26.17 that create energy sources to support microbial life in our ocean.
00:00:31.09 And I also told you that there are many types of water-rock reactions on our planet
00:00:35.10 that can sustain life.
00:00:37.01 What I want to do in this part, as well as Part III, is really focus in on one of
00:00:41.17 these types of systems, and tell you a story about how the science of understanding subseafloor life
00:00:46.17 has evolved over the last 20 years of my career.
00:00:49.27 So, I'm gonna take you to a mid-ocean ridge, which is where oceanic plates are spreading apart,
00:00:55.04 new ocean crust is being formed, and underwater volcanoes occur.
00:01:00.12 So, at these underwater volcanoes, as I discussed in Part III... or, Part I, sorry...
00:01:07.00 I collect these crustal fluids that are leaking out of the seafloor to try to infer
00:01:12.15 what's happening beneath the seafloor.
00:01:14.03 Now, these crustal fluids are a mix of hydrothermal vent fluid and seawater that are usually
00:01:20.22 below about 100 or 120 degrees Celsius.
00:01:24.03 These fluids are the largest source of new carbon into the vent ecosystem,
00:01:29.15 meaning the main source of primary production into the deep ocean in these systems.
00:01:34.06 And they contain a lot of biomass.
00:01:35.22 They contain about 5-10 times more microbial cells than you have in the surrounding ocean.
00:01:41.20 And one of the really neat things is if I collect this fluid up here, which is
00:01:45.17 about 20 degrees or 30 degrees Celsius, let's say, I can actually culture microbes that grow
00:01:51.02 at much higher temperatures, up to 90 degrees Celsius.
00:01:55.02 They don't like oxygen, and they're eating things like sulfur and hydrogen.
00:01:58.28 And that implies that they live in a habitat that's warmer, that is without oxygen, beneath the seafloor.
00:02:04.24 So, by collecting these fluids, I'm inferring what's happening down below.
00:02:09.01 So, I want to take you to one place on the seafloor, where... which is actually
00:02:13.16 where I started my graduate work in 1998.
00:02:16.11 And that is Axial Seamount, which is located about 300 miles off the shore of Oregon,
00:02:22.17 along the Juan de Fuca Ridge.
00:02:24.18 And this is kind of the closest underwater volcano to the continental United States.
00:02:29.12 And this is shown here in a blow-up, where, again, we have two plates spreading apart,
00:02:34.20 and the Juan de Fuca plate is the one that is subducting underneath the United States,
00:02:38.15 and is a reason we have this string of volcanoes from Washington down to Northern California.
00:02:44.16 And so, Axial Seamount is located right on that spreading axis.
00:02:48.20 And if we zoom in a step further, you can see the... sort of the outline of this volcanic caldera,
00:02:54.27 over here, where there is a lot of active venting of these fluids.
00:03:00.15 And in fact, this is a very active volcano.
00:03:03.10 And it's erupted a number of times in the last 20 years.
00:03:06.23 And I'll show you a video that in just a few minutes.
00:03:09.14 So, at Axial, as well as other systems I study around the globe,
00:03:14.01 there's some very basic questions I'm interested in.
00:03:16.15 And I've listed a bunch of them here, although I'm really only gonna talk about
00:03:21.04 the first two over the next two parts of this talk.
00:03:23.27 So, because this is such exploratory research, in the beginning we were asking questions like,
00:03:28.17 who is there?, what microbes are present?, how does the community change over time?
00:03:33.13 More recently, we've been moving into, what do they do?,
00:03:36.15 and how do they cycle important nutrients like carbon, nitrogen, iron and sulfur?
00:03:40.19 But there's a whole slew of other questions that my research addresses.
00:03:45.10 Is there a microbial loop?
00:03:47.06 That's one of the things I introduced in Part I.
00:03:49.24 Are there viruses and grazers?
00:03:51.12 How deep does this habitat go beneath the seafloor?
00:03:54.21 What's its global distribution?
00:03:56.17 How are these microbes contributing to carbon fixation in the deep ocean?
00:04:00.14 And really, how connected are these different habitats, both beneath the seafloor and in the ocean?
00:04:06.11 And when we think really big picture about Earth history, you know, what can we learn
00:04:10.12 about Earth's past and future from the deep ocean.
00:04:14.15 So today, I'm really gonna talk, here in Part II, about the first question.
00:04:19.20 And I want to tell a little story about how my research has evolved, as DNA sequencing
00:04:25.08 has evolved as well, in answering what seems like a really simple question of, who is there?
00:04:30.13 So, when I started my graduate career, environmental microbiology had discovered cloning and sequencing.
00:04:38.09 And we were using this method to help identify the microbes in an environment
00:04:43.12 without having to culture them in the laboratory.
00:04:46.10 Only about 1-2% of microbes on our planet have actually been grown in somebody's lab.
00:04:50.28 And so the way that we are very commonly going out into the environment to study these organisms
00:04:55.25 was by extracting their DNA, carrying out a polymerase chain reaction,
00:05:01.04 really targeting a specific region of the ribosomal RNA gene that's a great phylogenetic marker
00:05:07.01 for these organisms.
00:05:08.04 So basically, telling us who they are and who they're related to.
00:05:11.28 And then taking those PCR products, and going through a cloning and sequencing exercise,
00:05:16.26 which is now of course very, very common in molecular biology.
00:05:21.04 But was pretty revolutionary for marine microbiology.
00:05:24.19 What happened shortly after I finished my PhD, however, is that DNA sequencing changed.
00:05:30.15 And these next-generation approaches were developed.
00:05:33.26 And during my postdoc, the first of these new sequencers came out.
00:05:37.28 And so I was able to apply some of those techniques to ask this question of, who is there?,
00:05:42.24 in a very different way.
00:05:44.03 So, this next-generation sequencing approach has really reduced the cost and
00:05:48.23 increased the returns for sequencing across every field, but particularly in marine microbiology.
00:05:55.25 And during my postdoc at the Marine Biological Lab, we helped develop the application
00:06:00.27 of using these types of sequencers to profile microbial communities.
00:06:05.12 And so, now, we do the same PCR steps, but instead of the laborious and kind of
00:06:11.03 slow cloning and sequencing, we go straight into one of these sequencers,
00:06:15.17 instead of having to do those cloning steps.
00:06:18.10 So, the machine that we developed this approach on the first time was called the 454.
00:06:23.02 And there's now many others including Illumina and others.
00:06:26.14 So, how did that change my view of who is there at Axial Seamount?
00:06:30.28 Well, what it did is it greatly expanded the diversity of life that I was able to detect
00:06:36.04 in this environment.
00:06:37.06 This was actually originally part of the census of marine life that was carried out
00:06:42.10 by the Sloan Foundation.
00:06:44.02 And we were... went to a lot of different habitats in the ocean to try to describe
00:06:48.14 the diversity of microbes present.
00:06:50.20 And this is just one example of two different vents at Axial Seamount,
00:06:54.15 where all the different colors represent different microbes that we were able to detect.
00:07:00.03 And just to emphasize how much this changed what I knew about Axial, I'm putting up here
00:07:05.21 a figure from the paper I published in 2003, during my PhD, where I was able to sequence
00:07:12.13 --- you can see these numbers for these clones -- you know, upwards of, like, 150 clones
00:07:17.18 from some of these venting fluids.
00:07:19.28 And shown on the x-axis is the number of clones, and on the Y is the estimated number of phylotypes,
00:07:26.11 or different types of microbes in this environment.
00:07:29.05 And you can see the slope of this line is incredibly steep.
00:07:32.03 There's a lot more diversity out there that we weren't able to capture with this method.
00:07:37.06 And there's maybe 10 or 15 different colors shown below.
00:07:40.04 Well, in 2007, after developing this approach, the numbers have changed a lot.
00:07:45.20 So again, here's two different vents at Axial Seamount.
00:07:48.20 You can see there are many, many more colors.
00:07:51.12 And it's really the numbers on the axes that are important here.
00:07:54.00 So, on the x, we are going up to 750,000 sequences that we were able to collect.
00:08:00.01 And in the y are estimates of how many different microbes are present.
00:08:04.08 Interestingly, though, this line still isn't flattening out, which again emphasizes
00:08:09.12 how much diversity within microbes we have on our planet.
00:08:12.24 So, the ability to apply these types of new methods to my own research really expanded
00:08:18.20 my own view of the types of microbes living in this environment.
00:08:23.02 And the big question when I saw this data was, why are there so many different microbes,
00:08:27.15 and what are they all doing in the environment?
00:08:30.08 And so that's really where my research began to move, in trying to understand
00:08:34.16 the functional significance of all of that diversity?
00:08:37.27 So, when I'm talking about function in these types of systems, it's really,
00:08:42.19 how are they gaining energy?
00:08:44.28 And how are they cycling different nutrients and contributing to
00:08:48.25 different geochemical cycles in the ocean?
00:08:52.01 And so, today I'm mostly gonna be talking about the bottom part of this
00:08:56.02 pretty complicated drawing.
00:08:58.03 Just to take a step back, what I'm showing here are things like hydrogen, CO2, and methane.
00:09:03.22 And at the bottom, here, these are all the primary producers in this ecosystem.
00:09:07.18 We talked about those autotrophs in Part I that form the base of the microbial food web.
00:09:12.22 And these organisms can use things like iron, sulfur, hydrogen, and nitrogen to generate energy.
00:09:18.14 I've also drawn in here some of these viruses or phage, which I mentioned in Part I as well,
00:09:23.09 to indicate that we know they're there, but we're not sure what they're doing.
00:09:27.15 But I think it's important to keep in mind.
00:09:30.00 And then this carbon, of course, gets moved up to the secondary consumers,
00:09:34.16 the heterotrophic bacteria.
00:09:36.22 And of course this is all connected.
00:09:38.05 So, what I really want to know about these microbes at Axial and the questions I'm going
00:09:43.11 to be addressing here are, who are the active subseafloor autotrophs at Axial?,
00:09:48.11 how do they gain energy?,
00:09:50.11 and how does their activity vary over space, time, and geochemical gradients?
00:09:54.08 So, to answer these questions, I'm gonna talk about two different approaches.
00:09:58.18 The first, here in Part II, is shotgun sequencing using both metagenomics and metatranscriptomics,
00:10:05.06 which I'll describe more in the next couple slides.
00:10:07.24 And in Part III of the talk, I'll be focusing on stable isotope experiments that we've done
00:10:12.22 that kind of combine the power of DNA sequencing with stable isotopes.
00:10:19.06 And we're gonna be using three "model systems".
00:10:21.13 And in this case, there are three different vents at Axial Seamount where I had
00:10:26.15 a large amount of historical data, so I could ask these questions in a fairly focused way
00:10:32.06 rather than a more exploratory way, as I did for the first part of my career.
00:10:35.23 And so the three events... you're gonna hear these names a lot are Anemone, Marker 33,
00:10:41.15 and Marker 113.
00:10:42.18 So, these three vents are located in different parts of Axial Seamount.
00:10:47.08 This is the same image I showed earlier, showing the caldera.
00:10:51.28 And they are only, at most, two kilometers apart, this distance across the volcano.
00:10:58.02 But we chose them because we... again, we had historical data, and we had also
00:11:02.12 characterized them geochemically.
00:11:03.24 So, if we really want to figure out what microbes are doing in the environment,
00:11:07.24 we also need to know what energy sources are available to them.
00:11:11.16 So, what's shown here are those three vents, and a variety of different features,
00:11:17.23 environmental characteristics, ranging from temperature, pH,
00:11:22.05 and a number of nutrients and things that,
00:11:24.15 you know, microbes really like to eat.
00:11:26.13 So, you can see these fluids range all in the mid-20 degrees Celsius.
00:11:30.13 They have slightly acidic pHs.
00:11:33.03 And I've highlighted, here, three things that I really wanted you to pay attention to.
00:11:37.10 So, the first, in blue, is nitrate.
00:11:40.26 And the Anemone vent has a lot of nitrate in it.
00:11:43.24 And if you draw your eye down, in green, it also has the most oxygen.
00:11:48.04 So, this event in particular is rich in electron acceptors.
00:11:53.03 And if you go to the other end of that nitrate column and look at marker 113,
00:11:57.15 the nitrate is much lower.
00:11:59.21 And if you pop down there again to oxygen, you can see the oxygen is also lower.
00:12:03.22 So, we're going from a more oxidizing to a more reducing environment.
00:12:09.25 And the vent in the middle sort of falls in between.
00:12:13.04 And if you look at the hydrogen concentrations, you can see that same trend, where we have
00:12:17.12 a lot of hydrogen available at Anemone, and very little at marker 113.
00:12:22.17 So, it's important to keep this chemistry in mind as we interpret our microbial data.
00:12:28.28 So, one interesting thing that happened while we were conducting this study, which was
00:12:34.12 over a three-year time period, is in the middle of our last two sampling periods Axial erupted again.
00:12:41.14 And so the video you're seeing now is seafloor footage taken by the ROV ROPOS,
00:12:45.26 by the University of Washington,
00:12:48.22 showing what the volcano looked like shortly after one of these deep-sea eruptions.
00:12:52.15 And this is... new lava has been erupted onto the seafloor.
00:12:56.13 And all this white stuff that we see is mainly composed of microbes and sulfur.
00:13:02.09 And when these eruptions occur... this is that material being sucked into a sampling container
00:13:06.20 on the remotely operated vehicle... when these eruptions occur,
00:13:11.02 you get a huge influx of heat and energy into the environment.
00:13:14.09 And there's a bunch of microbes that take advantage of that hydrogen sulfide in particular.
00:13:18.26 And they oxidize it.
00:13:20.04 And it creates sort of these... what looks like snow.
00:13:22.23 And we call these snowblower events, in fact.
00:13:26.25 And so you can think about this sort of like a deep-sea phytoplankton bloom,
00:13:31.11 except instead of phytoplankton it's these very deeply buried microbial communities.
00:13:36.09 But this happened in the middle of our sampling period, which began in 2013.
00:13:41.13 We sampled again in 2014.
00:13:43.23 And then in early 2015, before our sampling season, Axial erupted again.
00:13:48.25 So, once we get material like this, like new fluids coming out of the seafloor back into the lab,
00:13:55.02 we carried out a number of DNA and RNA sequencing approaches.
00:13:59.15 And I just want to review exactly what it was that we did.
00:14:03.28 So, we extracted DNA.
00:14:06.06 And instead of doing a PCR this time, we just sequenced it all.
00:14:10.17 And this is called metagenomics.
00:14:11.18 So, if we were doing this from an individual, we would extract DNA from their blood
00:14:17.19 and then stick it all back together.
00:14:19.04 However, we have a mixed microbial community -- we have everything that's in that sample --
00:14:23.21 so it's actually really hard to stick it all back together again.
00:14:27.28 But what can we do with that type of data?
00:14:30.24 Well, basically what we're doing is we're accessing the genomes of these microbes
00:14:34.28 that we don't have in culture.
00:14:36.04 So, we're identifying who the key players are.
00:14:39.03 We're looking at their functional potential, their metabolism.
00:14:42.19 And we can also look at some really neat things like how genes are moving around
00:14:46.07 and some of the evolutionary dynamics.
00:14:48.14 So, what did we find when we did the sequencing of metagenomes from Axial?
00:14:54.16 I'm gonna be showing a number of plots like this, which I recognize are a little bit overwhelming.
00:14:59.07 But basically, we have the three events and the three years over here.
00:15:04.27 And the different colors represent, in this example, the different types of microbes
00:15:09.07 we found.
00:15:10.13 That first column is background seawater, as well as a plume above the volcano
00:15:15.28 after that large eruption.
00:15:18.04 And what I hope you can see is that the microbes that you find in background seawater,
00:15:23.23 all the way over here, are still found in many of these vent fluids.
00:15:27.18 However, there's also a lot of organisms
00:15:30.12 -- such as these epsilonproteobacteria, or this yellow group in the archaea over here --
00:15:35.00 that are not found in background seawater.
00:15:36.20 So, these microbes are coming out of the vent fluid, mixing with background seawater,
00:15:41.27 and that's what we're collecting.
00:15:43.21 Hopefully, what you can also see is that not all the vents look the same from this perspective.
00:15:47.16 So, there's a lot of blue in the Marker 33 vents, and a lot of orange in the Marker 113 vents.
00:15:54.06 And I'll talk a little bit about what those differences are and who those microbes are
00:15:57.16 carrying it out.
00:15:58.16 But this tells us that the vents have different key players,
00:16:01.19 and they're distinct from background seawater.
00:16:04.15 We can then start digging into the who and getting to what.
00:16:08.11 So, what are they doing?
00:16:09.20 So, what I'm showing here, again, on the upper part of this diagram, are still the same thing:
00:16:14.25 the three different years, the three different vents.
00:16:17.20 But now these colors represent different metabolisms focused on oxygen, nitrogen, methane, hydrogen,
00:16:25.03 and sulfur.
00:16:26.10 And so here, we're looking for known genes that are key to carrying out metabolisms
00:16:31.16 related to those five compounds.
00:16:33.22 And that's what's listed here, the gene name, down the y-axis.
00:16:36.24 Now, what's important to note here is that almost every sample has at least
00:16:42.24 a little bit of color for almost every different gene.
00:16:46.17 And this shows that there's kind of widespread shared gene potential among these different sites.
00:16:52.08 There are some exceptions, which I've highlighted here in yellow.
00:16:55.13 These genes are all related to making methane through a process called methanogenesis,
00:17:01.15 which is carried out within the domain archaea.
00:17:04.18 And you can see that there's a lot of methanogenesis genes over here at Marker 113,
00:17:09.09 and far less in the other samples.
00:17:11.06 So, this is giving us some indication that maybe there are some different metabolisms going on,
00:17:15.26 although there is a lot of shared potential.
00:17:19.19 So, the next thing we did with these samples is instead of extracting DNA, we sampled...
00:17:25.20 we extracted RNA.
00:17:27.18 And RNA allows you to access the gene expression patterns of these uncultured microbes.
00:17:34.06 The methods are very similar, although they're certainly more challenging.
00:17:37.21 But basically, extract RNA, make cDNA, and then directly sequence it again.
00:17:43.00 We don't always try to stick it all back together.
00:17:45.00 We're often using our genomes to then map all those transcripts back.
00:17:50.17 But with this tool, you can identify who are the active microbial players by seeing their transcripts.
00:17:57.09 And what is actually happening when you first capture that sample on the seafloor.
00:18:01.08 And in our case, when we filter the sample on the seafloor, we immediately fix it
00:18:06.12 so that all the message RNA stops, so the cells just stop, you know, metabolizing.
00:18:12.03 And so we really are capturing what's happening at the point of sampling.
00:18:16.09 And then we can start looking into these gene expression and metabolic pathways.
00:18:20.17 So, now what you're looking at is a very similar diagram to what I showed you with
00:18:26.11 the DNA sequences, but instead what we're looking at are the transcripts of these
00:18:30.01 different key metabolisms.
00:18:31.13 So again, the vents on the top.
00:18:33.11 The different colors representing oxygen, nitrogen, methane, hydrogen, and sulfur.
00:18:39.03 And what I hope you can see is that there are fewer dots on this plot, meaning that
00:18:43.25 a lot of the genes that we detected in the DNA pool are actually not turned on
00:18:49.06 at the point of sampling.
00:18:50.26 And just one example, again going back to those methanogenic pathways,
00:18:55.07 we see a lot fewer of the vents are actually carrying out...
00:18:59.17 the organisms at the vents are carrying out methanogenesis.
00:19:02.05 And overall, we see less shared transcription across these different sites.
00:19:06.20 There's another way of looking at this data, which isn't quite so biased.
00:19:10.07 Right?
00:19:11.07 I'm only looking at genes I already know exist in this particular example.
00:19:14.24 But we can also take all of the genes and calculate how different the vents are
00:19:20.02 from one another.
00:19:21.02 And that's what I'm showing over here, with a correlation distance, here, on the x-axis,
00:19:26.17 with each vent.
00:19:27.21 And what you can see is that the vents are overall similar to one another.
00:19:32.20 So, they're more closely related to the vent that they came from than they are to a different one.
00:19:39.00 But also, this correlation distance at the bottom is very small.
00:19:42.02 So overall, the organisms in these vents, and their genes, are very similar.
00:19:48.00 However, when we look at the metatranscriptomes
00:19:51.04 -- again, where we're looking at these gene expression patterns --
00:19:54.12 we can see large differences.
00:19:56.00 Again, down here on the x-axis, looking at that correlation distance.
00:20:00.06 However, the vents are still mostly grouping by which vent we sampled.
00:20:05.01 So, Marker 113 vents are distinct from Marker 33 and down to Anemone.
00:20:10.21 So, these functional profiling techniques allow us to get a handle on what the potential is
00:20:15.14 of these microbial communities versus what they're actually doing when we sample them
00:20:20.04 on the seafloor.
00:20:22.18 Another really neat thing that you can do with this type of DNA sequencing data is
00:20:28.12 try to look at the microbial populations.
00:20:30.22 So, not just these marker genes, but also try to capture the entire genome
00:20:37.05 to figure out how these different organisms are related to one another
00:20:41.08 and what they're doing in the environment.
00:20:43.28 And the way we do this is we take all of the sequencing data, and we stick it back together.
00:20:48.15 But we try to stick it back into individual microbes.
00:20:53.17 And so, in this way, we're calling it a potential microbial genome.
00:20:57.20 Because, in fact, you could have strain variation that computationally you cannot detect.
00:21:04.03 And then what we have is a bin of sequences that we believe all belong to
00:21:08.21 a very specific population of microbes.
00:21:11.23 We can identify it, and then take our RNA data to figure out which ones of those potential genomes
00:21:18.04 are actually active in the environment.
00:21:21.04 So, this is two... these are two examples, where we really focused in on genome bins
00:21:27.22 from some of these high temperature microbial populations that I mentioned.
00:21:31.28 So, the first example I'm showing... this is a group of these bins from Axial
00:21:37.28 that all belong to a group of bacteria called the Aquificales.
00:21:40.26 These are organisms that can carry out the oxidation of hydrogen,
00:21:45.13 and both sulfur and nitrate reduction.
00:21:48.01 And what you can see -- this is a heat map of how abundant they were at different vents --
00:21:52.17 is that these Anemone vents had a lot of expression of these different populations
00:21:57.17 of Aquificales, and then a very different group was present at Marker 33,
00:22:02.16 and almost none of them at Marker 113.
00:22:05.07 So again, even though we had these very broad functional similarities, when we dig down
00:22:10.05 to that population level, it's in fact very different groups and populations
00:22:15.01 that are active in the system.
00:22:17.21 And this is another example.
00:22:18.25 You've heard me talk quite a bit about these methanogens.
00:22:21.12 These are archaea, in this example belonging to both Methanococcus and Methanothermococcus.
00:22:27.22 And these are organisms that are important primary producers also.
00:22:31.15 They basically use hydrogen and CO2 to make methane.
00:22:35.01 And similar to what we saw in our... in our transcripts, we see that these genes are
00:22:40.24 really turned on big-time in these organisms at Marker 113, and to a lesser extent at Marker 33,
00:22:48.10 and not at all at the Anemone vent.
00:22:50.11 So, we have very vent-specific subseafloor populations that are active.
00:22:55.12 They're living beneath the seafloor.
00:22:57.02 And they're carrying out different functions based on these types of measurements.
00:23:01.15 So interestingly, even though we're in this one place on the seafloor
00:23:06.01 -- it's just a few kilometers wide --
00:23:09.02 we see that even just within Axial Seamount, all the vents are not
00:23:13.17 hosting the same populations, especially when we get beneath the seafloor into these warmer anoxic zones,
00:23:19.00 which I introduced at the beginning of my talk.
00:23:21.26 So, we certainly, as I showed, have a large seawater influence on this system,
00:23:27.00 with seawater cycling through and bringing a lot of common microbes
00:23:31.09 into all of the vents that we're sampling.
00:23:34.18 However, when we get deeper, we see some of these vent endemic epsilonproteobacteria,
00:23:38.07 as well as these high temperature populations that I just referred to.
00:23:42.05 So, these organisms are not present everywhere.
00:23:45.24 And something is making it so each vent develops its own population.
00:23:50.21 And I'll be talking a little bit more about that in Part III.
00:23:54.06 So, just to wrap up this section, I wanted to thank the funding agencies for this work.
00:24:00.09 Way back from 1998, when I started my PhD, to present day, where I've been funded
00:24:06.03 mostly by NSF and the Moore Foundation.
00:24:08.08 And of course, a great group of people that made this work possible.
00:24:11.05 Thank you.

Part 3: Combining Stable Isotopes and Sequencing to Understand Subseafloor Life

00:00:07.20 Hi.
00:00:08.20 My name is Julie Huber, and I'm a scientist at Woods Hole Oceanographic Institution.
00:00:12.27 And in Part III of my talk, I want to bring together a lot of the information from the
00:00:17.06 first two talks, and combine it with a new method we developed in my lab
00:00:21.08 to study subseafloor life at Axial Seamount.
00:00:24.06 So, I ended Part II basically telling you that, through our genomic tools, we determined
00:00:29.25 that not all hydrothermal systems host the same populations, especially in the warm anoxic zone
00:00:36.04 beneath the seafloor.
00:00:38.21 And what I wanna talk to you about now is how we've really been probing that subseafloor signal
00:00:44.06 using something called RNA stable isotope probing.
00:00:47.20 And the goal of this is really to focus in on the autotrophs living beneath the seafloor
00:00:52.27 without this swamping signature of deep-seawater mixing through the crust.
00:00:57.22 So, RNA stable isotope probing is a relatively new method.
00:01:02.03 But it basically brings together the power of stable isotopes with molecular biology.
00:01:07.02 And the idea is you do an experiment where you put in a labeled substrate
00:01:11.23 -- whether carbon, nitrogen, oxygen, or, for some, others --
00:01:16.23 in our case in a bottle.
00:01:18.16 And you let an experiment run.
00:01:20.08 And then at the end of the experiment, you gather and extract the RNA,
00:01:24.06 you physically separate it, and you can look at which organisms have taken up that stable isotope.
00:01:30.06 And this allows you to identify the active microbes during your experiment.
00:01:35.07 You can look at carbon flow or interactions between different organisms.
00:01:39.23 Or in our case especially, really looking at what processes those organisms were carrying out
00:01:44.08 when they took up that label.
00:01:47.13 So, at Axial, what we did is basically collected this vent fluid, and then set up a series
00:01:54.02 of bottle experiments.
00:01:55.24 And in half of them we put a control of bicarbonate, because remember, again,
00:02:00.26 we're interested in who the active autotrophs are.
00:02:04.03 And so we're looking at the organisms that have the ability to take up CO2.
00:02:08.06 And we have both an experimental control with the light isotope and a 13C experiment
00:02:14.19 with the heavy isotope.
00:02:16.19 We then take those bottles, we add some hydrogen gas to we create the reducing conditions
00:02:22.02 beneath the seafloor, and then we incubate them at a variety of temperatures that we think exist
00:02:26.21 beneath the seafloor based on our genomic data.
00:02:30.03 We then let these experiments run for anywhere from about 9 to 36 hours.
00:02:35.22 And this actually took a number of years to work out the right timeframe for carrying them out,
00:02:40.00 because you want to catch the label when it's in the organisms that first took it up,
00:02:44.18 not look at secondary consumers, unless that's your question, which in our case it was not.
00:02:50.03 So, at the end of the experiment, we filter all of the vent fluid, we extract the total RNA,
00:02:54.01 and we put it in a density gradient in an ultracentrifuge,
00:02:57.28 and we spin it at very high speeds for a couple of days.
00:03:01.25 And what that allows is for the RNA to distribute itself based on the density,
00:03:06.04 and you can separate that light isotope from the heavy isotope.
00:03:10.19 You then precipitate RNA from each of those different density fractions, and you can make
00:03:16.01 a plot like what's shown here, with buoyant density on the x-axis and RNA concentration on the y.
00:03:23.05 We can take a zoom in on that, and talk a little bit more about what we're seeing here.
00:03:27.26 So again, on the x, buoyant density, and on the y, RNA concentration.
00:03:33.14 And what I'm showing here in orange is the heavy isotope, and in the green is the light isotope.
00:03:39.11 And you can see that this heavy isotope is a higher density.
00:03:43.09 And what that represents are the organisms that took up the 13C label in our experiment.
00:03:49.24 And what I'm mostly gonna be focusing on today is really the active autotrophs
00:03:54.19 and what we're learning about them in this part of the plot.
00:03:58.07 However, it's important to know that we in fact did a lot of different sequencing of this RNA
00:04:03.14 to convince ourselves that the experiment worked.
00:04:07.27 And so we sequenced both the light fraction, which was enriched, as well as its corresponding control,
00:04:14.02 and we did that with both experiments.
00:04:15.28 And that was to convince ourselves that what we're seeing in this heavy 13C peak
00:04:20.13 are actually the organisms that took up the label in the experiment.
00:04:24.06 So again, I'm only gonna be showing this red fraction, which is those active autotrophs
00:04:29.04 in the experiment.
00:04:30.13 So, here we have an experiment that we carried out at Marker 113, at three different temperatures:
00:04:37.14 up at 30 degrees, 55, and also 80 degrees Celsius.
00:04:42.08 Again, representing different habitats that we think exist beneath the seafloor.
00:04:46.24 You can see that the 80-degree experiments aren't quite as lovely as the other two,
00:04:51.03 and that has to do with these high temperature organisms having a higher GC content in their DNA,
00:04:55.22 which often makes these experiments a little bit... can be a little bit messy.
00:05:00.19 And so, again, in all three of these experiments, though, we do have good separation
00:05:05.05 between this lighter isotope and the heavy isotope.
00:05:07.19 And what I'm gonna be showing you in the next slide is just the sequences
00:05:10.24 from this active autotroph peak in all cases.
00:05:14.14 So, in this plot what I'm showing you is data that I showed you in Part II of the talk,
00:05:20.20 that first plot over here, which is just the taxonomy of all the messenger RNA transcripts
00:05:27.08 that we detected from that in situ filter fluid.
00:05:31.07 And what I want to do is then compare that to the three experimental conditions,
00:05:36.10 just to say, which organisms were active and taking up the label in our experiment?
00:05:42.12 And so, what I hope you can see is that when we compare what was happening
00:05:46.08 at the point of sampling to our three bottles, they're quite different.
00:05:50.19 All the colors are present in that metatranscriptome, representing the diversity of organisms
00:05:55.15 in this environment, and in our experimental conditions we see less colors.
00:06:00.08 And so, basically, fewer organisms took up the label under these different temperature regimes.
00:06:06.11 So, at both 30 and 55 degrees, we have a group of bacteria called the epsilonproteobacteria,
00:06:13.03 which took up the label.
00:06:15.04 In this case, these organisms tend to oxidize sulfur.
00:06:18.22 And here we have organisms that both reduce sulfur and nitrate.
00:06:23.02 We also see some methanogens popping up, here, at 55 degrees.
00:06:27.00 At 80 degrees, we have a different group of methanogens within the archaea.
00:06:30.26 Again, these are high-temperature autotrophs.
00:06:33.02 And interestingly, this purple bar, here, these are actually a heterotrophic archaea.
00:06:39.03 These experiments we probably ran a little bit too long, and so we're starting
00:06:43.05 to see those secondary consumers come up and eat the autotrophs.
00:06:46.13 And of course, if that's something that you're interested in, sort of those trophic cascades,
00:06:50.11 this is a great method to get at it.
00:06:51.26 In this particular example, we weren't, but it's definitely set off some future experiments
00:06:56.04 that we're gonna be doing.
00:06:57.22 So, we can then dig in to those transcripts that we've created from these different experiments,
00:07:04.08 and say, well, how are they fixing carbon?
00:07:07.13 How are they gaining energy?
00:07:08.22 So, in this example, I'm just showing different genes for five different known carbon fixation pathways,
00:07:15.22 which are shown here in the different colors.
00:07:18.20 And then, similar to the first part of my talk, this is the metagenome,
00:07:23.21 representing all the potential,
00:07:26.13 this is the metatranscriptome, right here, representing all the genes that were turned on when we took our sample,
00:07:31.22 and then our three experimental conditions in the bottles.
00:07:34.26 And what you can see is all five of these carbon fixation pathways are present in the metagenome,
00:07:41.01 meaning the potential for it exists in all of the... all of the samples.
00:07:45.10 However, when we look at the metatranscriptome, you can see that only four of the five
00:07:51.04 were turned on at the time of sampling.
00:07:53.05 And then, in our experiments, there is even less diversity.
00:07:56.09 So, at 30 degrees, it was only the reductive TCA cycle, versus down here at 80 degrees,
00:08:02.19 where we have methanogenesis turned on.
00:08:05.19 And so we can really hone in on which organisms, which... are using what pathways under
00:08:11.01 these different thermal regimes.
00:08:13.15 We can also look at how they're gaining energy.
00:08:16.14 And again, this is similar to the diagrams I showed in Part II, where we have the metagenome
00:08:22.04 and the metatranscriptome.
00:08:24.03 And then the three temperature conditions.
00:08:26.04 And again, looking at oxygen, nitrogen, sulfur, hydrogen, and methane.
00:08:30.18 Again, what you can see is in that first column, almost all the bubbles are present.
00:08:35.11 In the second, less are present.
00:08:37.08 You can really tell what's going on in situ.
00:08:39.27 And then within our experiments, even fewer.
00:08:43.06 And again, what this is showing is certain organisms use certain metabolisms under
00:08:47.14 different environmental conditions.
00:08:49.24 And we can take a look at what's actually happening in our bottle versus what happens
00:08:53.28 in the... in the environment, because obviously we have major experimental artifacts here.
00:09:00.09 But by comparing it to the natural sample, we can see, for example, that a lot of organisms
00:09:05.02 are using these genes related to oxygen.
00:09:08.01 But in our bottles, we pretty much got rid of oxygen, and so organisms are switching
00:09:12.10 to anaerobic metabolisms.
00:09:14.20 We see a lot of organisms doing nitrate reduction at these lower temperatures.
00:09:19.20 And as we move into some of the higher temperatures, we see a lot of methanogenesis going on,
00:09:24.17 and this maps really well back to what we see in the environmental samples.
00:09:28.14 So, now we know who's taking up the label, we know how they're fixing that carbon,
00:09:33.05 and we know the energy sources they're using to do it.
00:09:36.27 So, I've just showed you one example from one place on the seafloor.
00:09:42.03 And in work that we still haven't published, we've done these experiments
00:09:46.02 at a variety of vents across Axial, and comparing them.
00:09:49.17 And I just wanna show you one example.
00:09:51.17 This is the data I already showed you from Marker 113,
00:09:54.01 where you have these high-temperature methanogens who took up the label.
00:09:58.15 And if we compare that to the two other vents that I introduced in Part II, Anemone and Marker 33,
00:10:04.16 you can see they look completely different.
00:10:06.16 And so, at this really high temperature range, as we had... as was suggested by our metagenomic data,
00:10:12.09 there are different populations of high-temperature organisms living beneath the seafloor
00:10:16.23 carrying out autotrophy.
00:10:18.11 So, at the Anemone site, it's this group of high-temperature bacteria within the Aquificales.
00:10:23.27 And then at Marker 33, a different group with... which is in the same phylum, and these organisms
00:10:29.27 tend to use hydrogen, sulfur, and nitrate.
00:10:32.28 And you can see that there are a few methanogens coming up at Marker 33 in our experiments,
00:10:37.10 but they really thrive at Marker 113.
00:10:42.07 Another cool thing that we've been able to do is kind of compare these experimental results
00:10:47.08 with some of our genomic data from Part II.
00:10:50.16 And in this case, I'm comparing it to those metagenomic bins that I talked about,
00:10:55.13 where you could really focus in on populations that were... are active in situ, or in the environment.
00:11:01.22 And again, we saw... just without doing any experiments, we saw that methanogen populations
00:11:06.25 were very active at Marker 113.
00:11:09.12 And that's what's shown in our SIP experiments, in our RNA-SIP experiments.
00:11:13.14 And again, two different populations of these Aquificales were present in the other two vents,
00:11:19.10 and that is also reflected in both the SIP and the RNA...
00:11:23.14 and the metagenomic and metatranscriptomic data.
00:11:26.16 So, by having both the omics data, in and of itself, as well as the experimental system,
00:11:32.15 we're really able to constrain who is doing what and under what conditions.
00:11:38.04 What we're doing now is we're trying to take all this data together and put together
00:11:42.11 kind of an ecosystem model for how autotrophy works in Axial.
00:11:48.12 And there are a lot of other components to this model that I haven't brought
00:11:51.13 into the discussion today.
00:11:53.02 This is really just focused on those primary producers.
00:11:55.09 But we've also been looking at their potential rates, we've been looking at the viruses that kill them,
00:12:00.03 and we've been also trying to culture a lot of these microbes in the laboratory
00:12:04.12 so that we have model systems to study and constrain a lot of these environmental parameters.
00:12:09.24 And so, what I'm just showing here are... is the plume above the volcano,
00:12:14.02 all the way over here, and then the three vents, and kind of the dominant metabolisms
00:12:19.02 and organisms carrying them out across this temper... temperature regime.
00:12:23.03 So, we're bringing together both the SIP and the omic data to make these types of models
00:12:27.21 about how life works beneath the seafloor at Axial Seamount.
00:12:31.18 So, to wrap this up, both Part II and Part III, our omics data shows us that we
00:12:38.11 have different taxonomic and functional profiles across these vents.
00:12:42.16 They're quite stable from year to year, despite things like deep-sea eruptions.
00:12:47.12 And we're not sure what maintains that stability.
00:12:50.18 But we think it is definitely linked to the geochemistry, as well as the physical structure,
00:12:55.20 of each of those sites.
00:12:57.19 Our genomic binning data shows that we have very vent-specific populations,
00:13:02.13 especially deeper, in the warmer parts of the subseafloor.
00:13:05.04 And our RNA-SIP data also showed those same differences in those active high temperature groups,
00:13:11.19 as well as different metabolisms at each temperature within a vent.
00:13:16.14 So, even though all we're collecting is maybe a 25-degree fluid coming out of the seafloor,
00:13:22.02 we see a huge range of metabolisms and temperatures that these organisms can exist at
00:13:27.06 beneath the seafloor.
00:13:28.06 So, this combined approach is really helping us to define subseafloor primary production
00:13:33.10 at Axial Seamount.
00:13:35.03 So, this RNA-SIP work and genomic work, it's really kind of a deep dive into details.
00:13:42.06 But since this is Part III, and the end of all parts, I just wanted to remind you of
00:13:46.03 some of the big take aways from this type of work.
00:13:49.13 First is that the discovery of deep-sea ecosystems, the hydrothermal vents in particular,
00:13:54.19 really changed the way we view life on our planet.
00:13:57.07 And it's actually opened up even more possibilities about how we could think about life beyond Earth,
00:14:02.08 potentially on ocean worlds such as Enceladus, which is Saturn's moon,
00:14:06.15 or Europa, which is Jupiter's moon, both of which are known to have an ocean.
00:14:11.23 Another big take away is that this very fundamental reaction between seawaters...
00:14:17.07 seawater and rocks creates diverse energy sources that microbes can use, not only to gain energy
00:14:23.06 but also to support these chemosynthetic ecosystems.
00:14:27.16 And in thinking about all the possibilities, I'm quite certain we have not discovered
00:14:32.28 all the possibilities for the types of energy sources that microbes can extract to support ecosystems.
00:14:41.08 The third point is that the subseafloor is really a global oceanic habitat.
00:14:46.11 It's everywhere there is a seafloor, which is the entire ocean.
00:14:50.12 And the dominant organisms in those environments are really these amazing microbes,
00:14:54.20 who put up with an awful lot to thrive in this environment.
00:14:59.07 And finally, the deep sea is, you know, one of the most unexplored corners of our planet,
00:15:04.14 as is most of the ocean.
00:15:06.21 And even though it's mostly out of sight, it should not be out of mind.
00:15:10.14 So, with that, for Part III, I just wanted to thank the agencies that funded this RNA-SIP work.
00:15:16.16 In particular, the Moore Foundation, NSF, and the Schmidt Ocean Institute.
00:15:20.24 And thank all the great people who worked on this project to develop this technique
00:15:25.09 in a pretty challenging place.
00:15:27.06 And with that, I just wanted to say thank you.

Videos in this Talk

Part 1: Microbes, Fluids, and Rocks
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: Subseafloor Life at Axial Seamount
Audience:
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 3: Combining Stable Isotopes and Sequencing to Understand Subseafloor Life
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Julie Huber

Total Duration: 0:56:36

Recorded: July 2018

All Talks in Microbiology

Talk Overview

A mile or more below the surface of the ocean, microbes dominate the deep sea life. In this seminar, Dr. Julie Huber describes her research to better understand the microbial ecosystem in the rocky crust below the ocean floor. She begins the series by describing how reactions between seawater and the elements in ocean rocks enable chemosynthetic ecosystems to exist in the deep sea. She then introduces us to the tools scientists use to study microbial deep sea life below the ocean floor.

In her second talk, Dr. Huber describes her research, which integrates microbiology, molecular biology, and ocean sciences approaches to characterize the microbial ecosystem below Axial Seamount, an underwater volcano off the coast of Oregon. Dr. Huber outlines how her group used environmental DNA and RNA sequencing techniques to analyze the crustal fluids (mix of ocean water & hydrothermal vent fluid) leaking from underneath the sea floor at three deep-sea vents. Her group determined that the metabolic potential of organisms was similar across vents (as indicated by DNA sequencing) but that there were larger differences in the “activity” of the microbes across vents (as indicated by mRNA profiling). Furthermore, Dr. Huber’s group identified vent-specific subseafloor microbial populations.

In her third talk, Dr. Huber describes how a method known as RNA stable isotope probing (SIP) was used to characterize the metabolically active autotrophic microbes at an underwater vent at Axial Seamount. Dr. Huber’s group found that temperature influences the metabolic pathways, including the carbon fixation pathways, used by different organisms collected at the same vent. In addition, Dr. Huber’s group compared microbial activity across three vents and found that all three have different microbes that are active in a similar temperature environment. These findings suggest that subseafloor microbes prefer some environments over others and use different metabolic pathways in different environmental contexts.

Speaker Bio

Julie Huber

Julie Huber is an Associate Scientist of Marine Chemistry and Geochemistry at the Woods Hole Oceanographic Institution in Woods Hole, MA. Before joining the Oceanographic Institute, Huber was an Associate Scientist at the Marine Biological Laboratory, also in Woods Hole, for 10 years. Her lab studies the microbial life found in the unique, and largely… Continue Reading