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RubyConf 2019 – Keynote – Slow, energy-efficient, and mysterious life deep… by Karen G Lloyd

February 27, 2020

Good morning. So, I am going to talk about
biology stuff in the deep earth. The background picture is in the volcanic arc of the Andes. My
Chilean colleagues invited us down, but my talk will be about oceans. Hopefully you will
see how that connects back to volcanos when I’m done. What planet is this? It’s great
to flip the earth around and check out the Pacific ocean every now and then. You can
almost get a view of earth that has no land in it. New Zealand creeps in. It’s a nice
reminder that we probably all know that percentage of 74%of the earth is oceans. It really is. If
you flip it around, you can remind yourself of that. So, because most of the earth ‑‑
since most of the earth is oceans, if you want to know what’s inside earth, like how
life goes into earth, how deep it goes, then what you’re talking about is marine sediments. That’s
the majority of the crust that is habitable by microbes. There are so many microbial
cells living buried in the sea floor. That’s about a third of the microbes on the planet
are buried under the sea floor. It’s roughly 10,000 times than the number of stars in the
universe. This is just a fast, fast eco‑system. So, if you want to study life on earth, you have
to look inside earth, because that’s where it is. 
So how do we do this? How do we go about learning about the deep subsurface microbes? The
big thing is we go out in nature and get samples. Certainly you’re a scientist sitting at a
computer or at the lab bench and some adventurer goes and gets stuff for you, right? No. That’s
the point. I could do other fields of science, but I do this one because I get to go to cool
places. We get samples and we measure the chemicals around the microbes. We measure
as many things as we can to get at what they are eating and breathing, just how life works
in this strange place. And the big thing we do, usually we do this back in the laboratory
is we chemically extract their DNA and other biomolecules directly from the natural sample.
And this is what we use to infer what they are doing. And we can bring home these samples
and try to grow things in the laboratories.  So the work that I’m going to tell you about
was taken from oceanic sediment. This is the Joides Resolution. This is a long series
of ‑‑ you have to load up pipes. So the pipes get taken down to the sea floor, and
there’s a little explosion that happens at the sea floor, and they get shoved down into
the sediment. So, this advanced piston coring can bring you back hundreds of meters into
the earth sediments coming back on ship. It’s exciting and fun stuff to do. So we go out
on ships and we get this stuff. And if you want to start thinking about what kind of
microbes are out there, one thing that you sort of have to know as background is that
we actually already know quite a bit about the microbes elsewhere on earth. So, one
thing we know for sure is that there are a lot of microbes on this planet. I just found
this picture on the internet, and I thought it was really beautiful. It may seem a little
disgusting, because somebody put their hand down on a petri dish, and these are the microbes
that were on their hand. If you think about that person who made that hand print probably
didn’t die from an infection from the microbes. So most of the microbes on the planet do not
kill us. We spend a lot of time in microbiology studying pathogens and diseases. But for the
most part, the microbes are not pathogens. They do not kill us. I do want to make the point
that we have only been on this earth for a few million years. Microbes have been here
for literally billions of years. So, they maybe just haven’t figured out how to kill
us. I’m going to put a yet there. It could be…
So when we got the first samples that we could be sure were not contaminated that were from
the deep subsurface environment, they were from off the coast of Peru. I didn’t do this
work alone. No scientist ever works alone. If somebody tells you, like, so and so discovered
something, like, no. So, and so and a bunch of other people discovered things. This was
my project as a PhD student. It was a Heady time. I will show you the data that we found,
and I will show you not microbial names or a genetic tree or something technical. I
will show you each microbe by a box. I will put a rectangle like this up every time we
got a DNA sequence out from a microbe that was similar to what somebody discovered before. I
will make a heat map where the light color is totally different, a DNA sequence that
we got that was something that was an entirely new type of microbe that nobody had ever discovered
before. So, this is what we got. We got almost all white boxes or pretty light‑colored
boxes. It was truly amazing as an entry into the field that there is a whole undiscovered
world down there. You look at a result like this and you say everything there is new. It’s
time to start culturing things and get things growing in laboratories. Luckily we had some
of the best culturing labs in the world on this ship. And they took samples back to
their lab and started growing things. I will show you their results, too. Every time they
got a microbe that we found, I will put it directly Underneath the box, and new ones
I will pack to the right of them. And they will be color coded the same way. Ready to
see what they found? Zero! Zero of the things we found they managed to grow. Nothing. No
overlap. And further more, everything that they got is very similar to kind of stuff
we already knew about.  So we realized we had a big problem here and
there was actually another lab that did this as well. And they got a similar result. And
I don’t want to malign these labs. They know what they’re doing. It’s just simply that
these microbes were living in a way that we were not adequately replicating. And I know
a lot of times people say pressure, it’s all because of pressure. And these experiments
were not done under pressure. But that has been done quite a bit, and it’s not the missing
link. You can’t just apply pressure to these things and have them grow perfectly. This
is such a pervasive problem. A lot of people refer to these things as microbial dark matter. I
don’t know if you have heard that word, but it’s borrowing from the astronomers, but most
of the matter in the universe is dark matter that we know exists because of its gravitational
pull, but we don’t know what it is. And most of the microbes on the earth are just weird
stuff that we don’t know what it is. So, we call it microbial dark matter. So the fact
that this stuff exists means that we have to crank back the clock and talk about microbiology
101. We teach this in classes. Microbes grow in cultures like test tubes. And we have
a general conception of what microbes do on the planet. I want to point out that these
weird microbes do this. That’s a framework. It’s not necessarily true. These are basic assumptions
that our whole field has been based on. But we don’t think of them as assumptions because,
well, it’s true. Maybe they’re not true. This stuff is making us question that. I think
it’s important to be able to see your frames and they have allowed us to do that. 
So we want to try to figure out what do they do? Let’s be aware of our frame of thinking.
And let’s think about this from the microbes’ perspective, get in their head. What’s it
like to be buried in the sea floor? This is how I think of things, so this is my schematic
of sea water, overlying sediment, as microbes are buried, it happens slowly. Sometimes
they get kicked back up, and on average, they are buried and buried and buried, slowly,
slowly, slowly. The best things to breathe are breathed up quickly. Oxygen is a fabulous
thing to breathe. The fact that we can be these big multicellular organisms literally
spewing out heat everywhere is so inefficient. We can do that just because we breathe oxygen,
which has so much energy. And your next things to breathe like sulfate or iron or manganese,
they get breathed up next. And then after that’s all gone, the last thing you’ve got
to breathe is carbon di oxide, which is crazy to think of using that as something to breathe,
but these microbes can do that. It means there’s a decreasing oxidation potential. The
things you can get energy from are getting crappier and crappier as you get buried. You
combine this with the fact that the food you eat, the carbon that you use to make your
biomass, where you get your electrons from, that doesn’t get replenished. You have to
eat the leftovers of what got laid down thousands of years previously. So, this adds up to
massively decreasing energy availability as you go down in marine sediments. What we
predict is that the number of microbial cells in the sediments should decrease with this. And
that’s one thing that we actually have measured pretty well around the world. And this is
a compilation of data from all around the world. And this is actually a form of the
plot that I will show more, so try to remember it. Up at the surface, that’s the sea water,
that’s the sea floor. As you go down into marine mud, on the Y axis here, we have the
total number of cells. It’s decreasing. So, pretty much everywhere in all different oceans
around the world, the number of cells are decreasing with depth, which is what we expect. 
But what we want to know is how fast do they grow? Like, you know, on average, these populations
are decreasing. But if you see a patch of grass one summer and the same patch of grass
another summer but the patch got smaller, it doesn’t mean that the grass blades didn’t
grow, they just changed in population size. So, we
can put this on an age model. If you look at the sediment age, we go down to 5,000 years
on the Y axis. This is work that my colleagues did looking at the respiration rates. And
at the surface, we have cell generation times of months. Which is really, really slow. If
you put E. Coli in a test tube, it will double every 30 minutes, but these things are taking
months to double. If you fill in the rest of it, they go up to sometimes 50 years or
70 years. So, these are microbial cells that don’t divide for 50 years, which is weird. 
Biology is not really designed to deal with that sort of problem. Yet, that’s how we
find ourselves. So, we have to go to it. So, they’re not really dividing or making new
cells at all, because they don’t even have to divide in every 50 years. They could just
is be switching out their internal biomolecules and fixing the parts that are broken. This
is a carbon turnover. They don’t have to make a daughter cell. So, an individual cell
could hang out for thousands of years or hundreds of thousands of years. And the thing is, like,
maybe that sounds crazy to all of us, and it is crazy. But it’s not. This is actually
the consensus in my field. We say oh yeah, these cells are probably hundreds of thousands
of years old, living cells.  So what this has ‑‑ what this means is
that my graduate student, as he was learning this stuff, he was shocked as I was to learn
this. So, he came up with a word that I love. So, I’m going to share it with you guys. He says
Karen, it’s not a biome, it’s a die‑ome. I was like that’s so good! That’s great. But
at this point in the talk, I should probably raise the thing that maybe some of you are
thinking, like why am I doing this? Like, this is an entire eco‑system that’s dying,
and I’m devoting my entire professional life to studying this. Not just me, but other
people, too. What’s the point? They’re just dying. Let them die! But I’ll tell you the
reason, and the reason is that I find it utterly fascinating. How does a cell live for that
long? What does that mean? They’re still alive. This is a mode of biology that is
fundamentally different than all other biology that I’ve ever heard about, and I don’t know
how the evolutionary pressures work over such long time scales. When you take the rules
off of biology, suddenly really strange things can emerge. For instance, if you think about
what it takes to get ahead in darwinian evolution, fast growth makes a ton of sense, right? So
when we talk about competition and ecology, we normally talk about the goal is increasing
your population size. So, we have different flavors of how to win a race, right? You
can be the hare, and you can sprint and get there fast, but we know that being a tortoise
is a valid way to win. In ecology, we call this R strategists and K strategists. And
K strategists will wait until the rabbits die off and then they will catch up. But
all of that is based on growth, increasing your population size. What about this group
of microbes who are in not actually increasing at all? So maybe I’m proposing that the goal
in the deep marine sediments is just existing.  So, like, a Zen sort of thing. You know,
you look at a Buddhist monk who is meditating, and he may not look like he’s doing anything,
but he is. He’s making forward progress, even though he’s not racing. If you’re not
into the religious reference, maybe you will get this one. For years, I didn’t understand
the point of this movie, the big Lobowski, Then I realized there is no point. I think
that’s what these microbes are doing. They’re just looking for a white Russian. 
[ Laughter ] So, in this eco‑system where this seems
to be the goal is just kind of hanging out, what does that look like? How do they do
this? So now I’ll tell you some ‑‑ sorry to go from pop reference to technical stuff,
but you can handle it. We did a project in the Baltic sea, which is between Sweden and
Finland and Latvia and Estonia. These are my students who did all the work on it. This
is actually not the same ship. We worked on a drill ship that was retro fitted be I
the Europeans. The Japanese have a ship and the Europeans make their ships as needed. So,
we don’t have a ton of ships for this kind of thing. But we wanted to ask the question,
what are the potential mechanisms that allow bacteria and archaea to persist in near zero
growth state for 8,000 years 50 meters deep into the Baltic sea sediments. I got to tell
you about the technique that we used to get these things. Because the situation in marine
sediments is there’s a big group of microbes there, heterogeneous, they’re tiny, bundled
together. And we needed to pick apart individual genomes, but we can’t culture them. So, we
have to pull out a genome in mixed population in literal muck. Think of dirty, crappy muck. That’s
what we’re working with. So, what we do is we physically pull out a single cell from
this weird, mucky stuff. We do this with flow cytometry, which makes droplets of water. Each
droplet has one cell in it which we pass by two lasers to look at the fraction patterns
as the light scatters off. So, this lets us know roughly the size and shape of what’s
sitting in the water droplet. So, we say that thing looks like a cell. Flick it into
its own well. So, we get a tube that only has one cell in it, which is not much to work
with. Then we have to crack it open. We do this usually with changing the pH, so like
shooting alkaline stuff in, because it’s reversible. And then we copy its genome with an enzyme, and
we make lots and lots of copies of that one cell. Then we send it off to a magical sequencing
machine. It’s all proprietary. I don’t know how they work, but they come back with sequences
of DNA. So, tons of data that we get from this. And we bioinformatically knit it together. This
is a fun area of research, putting together data like this. 
So in this way, we analyze the genes that are present in the microbe. You can imagine
genes are like a menu for what the microbe can do. Okay? 
So when we first got these genomes out of the deep subsurface organisms, we looked for
what’s abundant and what looks interesting. Something that looked interesting right away was a bunch
of toxin‑anti‑toxin systems. I’m showing the data where we have the different microbes
that we caught. None of these things belong to a filum that has ever been grown. And
we have this number of different toxin and anti‑toxin systems. You probably have not
heard of toxin/anti‑toxin systems. Evolution worked out such that we have a lot of cells
that will make a toxin that kills themselves and simultaneously produces the antidote to
it and that’s why they don’t die. Doesn’t that sound like a terrible system? That’s
how biology works. It’s like a governor on your system. You can have this toxin that
slows down your metabolism, makes your cell sick. This is using in mycobacterium tuberculosis. This
is from a published paper showing how many different toxin/anti‑toxin systems are present
in this. Tuberculosis, if you hit it with a bunch of antibiotics that destroy a growing
cell, it just stops growing. It’s like I’m going to persist and hang out until your body
forms a granuloma around it and then you have tuberculosis for your whole life. So, the
mycobacterium tuberculosis uses these systems to get in this low, low zero growth state. So,
we have lots of other things that we have identified that I’m just telling you about
one because it would take all day. We think one of the things that the microbes do is
they’re using toxin systems to slow down their growth rate and be very, very slow. So, that’s
kind of cool. That means that we’ve known that everyone in this community is starving
because the food is 8,000 years old. But that’s okay, because they’re not trying to
grow fast anyway.  But can we identify what they actually eat? What
does one eat when living so long without much food? So we looked at the metabolites. That’s
another technical thing. All the metabolisms that are happening inside cells occur by changing
molecules. So, individual monomeric molecules. So, if you get a view of all the molecules present
in a cell at once, that’s called a metabolome. They get these subway‑looking map things where
you can look at the ‑‑ it’s like pipelines or dials. So, you can look at the upregulation
and downregulation of the different metabolites. This is something that people have done in cultures,
but we are working in sediments. It just seemed like a big mess. It’s already a mess
in a living organism, and we’re going to take this natural sample that’s full of tree leaves
or whatever dead stuff and try to pick apart individual molecules from it. It seemed intractable
to me. How much do I know? The only reason why we ended up doing this work is that guy,
Hector, whenever he saw me was like Karen, you got to put some of your weird deep sea
mud and let’s just look at it and see what the metabolites look like. It just seems
like not a great idea, but fine. We did it. We put all of this stuff in. How did it work
out? Did you get anything? He was like no, it was total crap. It didn’t work at all. Obviously. Nothing
ever works in the deep sea. That’s a truism. We only got out of the thousands, we only got
20 molecules. And 20? That is so much more than zero! I can work with 20! And it was
great.  So I went through, my students and I went
through those 20 molecules and tried to figure out if we could see what they were important
for. And I’ll just tell you about one of them, and that’s this ‑‑ it’s a molecule
that I had never heard of before, it’s called allantoin. What stuck out to us about it
is this is another heat map showing the depth on the X axis here, looking at two different
cores. It goes down quite deep. This is down to 100 meters, almost, into the sea floor.
And we got allantoin present at every depth and another form of it called allantoate. This
is potentially a food substrate, a product of the degradation of everybody else dying. This
is something that would result from that. And it’s a substrate that we never would have
thought ‑‑ we wouldn’t have guessed that they could eat this. So, luckily, we have
all of these genes to look for. Like, are they eating it? Let’s see. 
So we looked across the major groups of microbes that we have. Each column is a different
microbial group, and then the rows are different enzymes that are important for allantoin metabolism. So,
it looked like this one clay had a leg up on everybody else. It was able to eat this
food genetically. That’s what we found. It looked like they had a corner on the market
for allantoin. So, this brings up the question if atribacteria are the best at eating the
food, why don’t they kill everybody else and win the game? You’ve got an inside track
to a food source that nothing else has. So, divide your cells a bunch and take everybody
over and be the king of the thing. So, I’m gonna answer that. 
So we put together its whole metabolism from these genes. This is a hefty reconstruction
of all the genetic machinery that we could put together, which, you know, obviously you’re
not going to go into depth and read all of this stuff, but it looked like they were a
fully functional microbe. And further more, we found that they had all of the genes necessary
to make amino acids, to basically just produce the essential building block for all the proteins
that they need. So, it looked like what they were doing with this food source was producing
some biomass. That’s good. That means they should divide. So, to answer this question
of why they weren’t just growing like crazy, I went to more colleagues at Texas A & M and
USC. And they looked at how many of the genes are turned on in the system. It is amazing
to me that they were able to do this in the muck that is deep under the Baltic sea, but
they were. From this list of RNA, or genes that are turned on and active and doing stuff. The
second most abundant one out of all the genes that could have been there in the cellular
metabolism was this one that I circled in blue. And it’s an exporter of amino acids. Amino
acids are energy rich. But instead of hanging on to this stuff, it looked like, at least
genetically that this organism was actually giving it away to its friends. I don’t know. 
I do not like apocalyptic movies, because I am scared of movies. I have only seen one
of them, it was Zombieland. I liked it. It wasn’t too scary. I learned from woody Harrelson
that the thing to do when your resources are limited is you have got to get guns and keep
people away from your food, keep your Twinkies. That is the apocalyptic story, that narrative that
we tell each other. Resources are slim? Gather them up and keep everybody away. Here we
have this eco‑system that has been dying for 8,000 years and they are literally giving
their amino acids away? That doesn’t make sense at all. If they allow their intracellular
concentrations of amino acids to build up too much, they will have to divide. It will
force their enzymes to go forward, and they’ll make competition from themselves. This is
a hypothesis. We don’t know. We could be totally wrong. We think they are releasing
the amino acids simply because they don’t want to divide. They want to keep in the
zero growth state, because that’s the way to persist. If they hold it, they would grow
too fast, so they have to release it. This has the secondary benefit of feeding everybody
else in the community. I don’t claim altruism on the part of microbes. That’s ridiculous. But
it is an amazing system. Apocalyptic movies are about a recent apocalypse. But these
guys are pros. And they share.  How does this get set up? What I have discussed
are adaptations to doing nothing. But we know that Darwin said that you had to pass
your genes along to the next generation to get them to be selected for. So, how does
natural selection happen to allow a microbe to persist for this long? How did this system
get set up? And I think for that, too, we have to take our heads out of our frame of
reference. For that, we have to remember now these microbes live for thousands of years
or longer. So, now we have to start saying are there geological factors? That’s geological
time that could allow them to come back to the surface. So, now don’t think about writing ‑‑
getting buried down in marine sediments. Now we have to think about the oceanic sediments. Plate
tectonics start to matter when you live a long time. What happens in oceanic tectonic
plates is they get crushed under continental plates, usually. This is a schematic. This
happens offshore. There’s a subducting oceanic plate that get crushed under continents. All
of this stuff gets squeezed and shoved back up. So, it’s possible that these microbes
are brought back to the surface early in subduction zones. And of course they don’t survive going
down to the mantle and mixing with mantle and lava. But all of that is connected. Maybe
this is how this connects to volcanos. Maybe life deep within the earth is connected across
spatial scales and time scales that biologists are restricted from thinking about. 
So, as a new project, I don’t have a lot of results to show you, which is fine, because
I’m almost done. But I will tell you about what we’re doing with this project. I put
a dot up for every time we have a sample. So, we have sampled with my colleagues from costa
Rico, and this is what it looks like. We checked in just a few miles into the jungle,
and all of this stuff right here? This milky stuff? This is me and some of the rest of
the team. We just sort of fan out with our tubes and real cheap‑looking stuff, because
it is cheap. And we sample all of this stuff. And this milky red stuff right here is all microbial. So,
there’s tons of these deep subsurface microbes that are spewing out of the earth and taking
advantage of the fact that there’s a great interface with oxygen at the surface of the
earth. Definitely ‑‑ the second most dangerous site I have ever sampled, inside
the crater of Poas volcano. This is the closest to San Jose. We went because it was pretty
on that day. We had to hike and it’s scribbly rock. It’s not settled stuff. There’s nothing
growing here because the thing blows up every now and then. And this is getting ‑‑
this is basically like a lake full of phreatic water. So, it’s literally battery acid. The
pH, this is me with a pH meter right there. It was .85. It goes down to ‑2. It’s sad. It
is terrifying to be there, this is a picture of me. It’s very steep and scree‑filled. The
ground is hot. Duh.  [ Laughter ]
It’s really hot. If you fall, you fall into battery acid. But you don’t want to fall,
so you put your hands down, but it’s 600 degrees, and you’re like crap. You just walk along
and keep your boots moving. I will say I don’t now how many volcanos I’m going to go
into in the future. A month and a half after we went in, this happened. That’s ‑‑
this is the shoreline I just showed you. This is where I was standing. Right there. It’s
just a reminder that nature does not care about scientists who are sampling it. It
will kill you. And we have to be really, really cautious and not be cowboys out there
and really take mitigated risks. And it also means we now know the microbes that are present
in the place. This is the biggest eruption in 60 years, so we were awfully glad to miss
it. We did all call each other on the phone and were like nobody’s in the volcano, right? There
are microbes that survive this. This is a place where microbes live, which is crazy. To
think that is a valid eco‑system for microbes that are like cool. I’m good with an explosion
every now and then. It really makes you want to extend your thinking of where life is to
outside of earth. So, if you ever think about life outside earth, which I hope that you
do, because it’s awfully myopic to focus just on our planet. It’s hard to imagine life
on other planets within our solar system and planetary bodies, because they lack the thick
atmosphere. It’s hard to live at the surface because you have to deal with magnetic and
uv assault. It’s a rough place to live. But nothing is stopping their subsurfaces from
teeming with life. When I think about life and where it might be in our solar system,
I think about the subsurface. I am not going to think about what’s sitting on the surface
and dealing with the sun’s rays. I think there is stuff in Europa and possibly mars. I
hope you will continue to get out of your frame of references as well and think about
life on other planets. I will end by thanking my funders, because
I think it is amazing that we have a society that values this kind of science. And I am
grateful for that. This is my building at the university of Tennessee in Knoxville,
four hours east of here. It’s lovely, and a slightly dirtier, scruffier version of Nashville. Thank
you.  [ Applause ]

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