We once again welcome two guests to this episode where we talk about using digital PCR and other methods to study microbial life and survivability in extreme environments. Dr. Brandi Kiel Reese and Lydia Hayes-Guastella from the Dauphin Island Sea Lab at the University of South Alabama tell us all about their work collecting and analyzing samples from places remote as Antarctica and the Mariana Trench. They share what working in such unique locations is like, how samples are collected and analyzed, and what they hope to glean from such studies. As always, you’ll also get to know a bit more about them and their personal stories too!
There are very few remaining locations on Earth that are untouched by humans, and those that do remain are in very extreme environments that are difficult to access. However, accessing and studying life in these extreme environments can provide unique insights to the biology of life. Understanding how simple organisms adapt and survive in seemingly unlivable conditions is a unique field of study with the potential to inform and affect the human condition.
We’re joined in this episode by Dr. Brandi Kiel Reese and Lydia Hayes-Guastella from the Dauphin Island Sea Lab at the University of South Alabama. They are both geomicrobiologists that study microbial life in extreme environments like the Mariana Trench and Antarctica. They do an excellent job of painting a picture of how extreme conditions are in these environments and how they manage to collect and preserve samples from such harsh conditions. We learn about the various methods they use to analyze the microbial samples they collect, including the use of digital PCR (dPCR) to detect and quantify transcripts that would otherwise not be detectable given how few cells they’re able to collect.
Brandi and Lydia also share their unpredictable career path journeys, while sharing some insights and learnings from their respective experiences. We learn what they each love about their work and what qualities is takes to be successful at what they do. Once again, we’re reminded of what a small world it is, especially when you’re in a specialty field such as geomicrobiology of extreme environments.
Visit the Absolute Gene-ius page to learn more about the guest, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.
Jordan Ruggieri 00:00
I used to be known for my puns. I was the I was the king of dad jokes. I was known for anytime someone said, "Oh my gosh", I would say "You have a gosh? I've always wanted to know what one of those looks like."
Cassie McCreary 00:13
I love it.
Jordan Ruggieri 00:26
Welcome to Absolute Gene-ius, a new podcast series from Thermo Fisher Scientific. I'm Jordan Ruggeri.
Cassie McCreary 0:32
And I'm Cassie McCreary. And today we're taking a deep dive into the world of microbial ecology with two geniuses, Dr. Brandi Kiel Reese and Lydia Hayes.
Jordan Ruggieri 00:41
Brandi is a senior marine scientist at the Dauphin Island Sea Lab and an Associate Professor of Marine Sciences at the University of South Alabama. Her work explores the microorganisms that survive and thrive in extreme environments like the deep ocean. Lydia is a PhD student at the University of South Alabama and works closely with Brandi on several amazing projects.
Cassie McCreary 01:03
So grab your headphones, a roll of duct tape, a toilet brush, and a cool refreshment as we MacGyver our way through this fascinating conversation.
Jordan Ruggieri 01:12
The toilet brush comes in handy who we promise.
Brandi Kiel Reese, PhD 01:18
My name is Brandi Kiel Reese, and I'm a geomicrobiologist, which just means that I study microbes and microbiology in an Earth setting. And in this case, things that live in the bottom of the ocean things that live in the crust below the sediment of the ocean. And that includes microbes like bacteria, archaea, and even fungi.
Lydia Hayes-Guastella 01:40
Hi, I'm Lydia, and I am a geomicrobiologist in training. I'm a PhD student here at the University of South Alabama. And I am interested in microbial life in extreme environments, what they do, how they survive, and how they interact in the environment.
Jordan Ruggieri 01:59
Amazing, I have to say to that I know you're looking at the Mariana Trench and some of the mud volcanoes there and also had some expeditions to, to Antarctica. But we'll talk about some of that as we go through as well. What questions generally are you trying to answer as you study these microbes?
Lydia Hayes-Guastella 02:17
We have a lot of different research questions that we're addressing. But overall, when we look at extreme environment, like the Mariana, or Antarctica, we're looking at what kind of organisms live there, what they're doing, and how they're able to survive in those extreme conditions.
Brandi Kiel Reese, PhD 02:36
It just in general, we want to understand survivability on this planet, and single cells use unique genetics to survive. And so we want to look at it from a genetic standpoint, but we also don't want to leave out those that we can grow in the lab as well. So, we use culture-independent and culture-dependent approaches.
Jordan Ruggieri 02:55
With such tough conditions, there must be ways that these organisms’ kind of adapt and compete with other organisms that are there? Very low biomass, low oxygen, is this the case? Do you do you see these with these organisms?
Brandi Kiel Reese, PhD 03:10
Well, exactly. Each site is unique in terms of what is considered extreme. Extreme can be, you know, low oxygen, or no oxygen. Or extreme can mean you know, high pressures. It also can mean high or low temperatures. It could also mean nutrients, right? So the lack of carbon, the lack of nitrogen, the lack of, you know, other, you know, micronutrients that are needed for survival. And what we're trying to understand is, you know, some of those responses to survivability. To anthropomorphize a microbe or give it a human characteristic, we can think about this as a game of survivor in which you have microbes that can adapt or evolve to, in response to their environment. In other words, can use the nutrients that are available or use, you know, the environment that's, that's available and can thrive in that situation, other microbes can't. And so those that can evolve, or that can adapt, will, will survive and will even thrive. Another choice, if you will, or another option is that they can just go dormant, they can go be inactive, or it can actually, you know, build a spore, you know, create a spore around that cell. So then they just shut down to just the most basic genes or most basic needs, that are keeping that organism alive. These are kind of zombie organisms just hanging on there in some sort of purgatory. And then the third, you know, choice, if you will, it's to create secondary metabolites that can disable or even lyse, meaning kill, your neighbor. So you can, at that point, use your neighbor for its necromass. It's a little bit like cannibalism, where one cell is eating other cell, but they're very different species, if you will. Through the production of these secondary metabolites, which are antibiotics, they can consume their neighbor and, or even, at least disarm their neighbor so that they are no longer a competition.
Jordan Ruggieri 05:17
That is incredible. I mean, you did mention a little bit of, you know, they don't know if they're alive, they don't know if they're dead. I can just imagine that that that can actually cause a lot of issues when studying these organisms. You don't actually or may have trouble figuring out which ones are doing a better job at living if you have trouble identifying in the first place if they're alive or dead. Is that something that you also run into regularly?
Brandi Kiel Reese, PhD 05:43
Yeah, absolutely. So bacterial cells and archaeal cells, for that matter, really exist on a continuum. Live and dead isn't as clear cut as it is in our eukaryotic world. A cell can exist in various forms of damage states and even in dormant states. So we use several different context clues to try to suss this out. DNA is only one of those and DNA is like your library. We're not reading everything all at once all at the same time, we just know that it's contained in there, that information. And if we take out a book, and we open up a page, you know, that's like our RNA that's being expressed. So, we can start expressing those genes. And we know that if an organism is alive, that it's expressing genes. So it's reading the book. If it is dormant, or if it's dead, the books are put away, the information is still there, but it's not being read actively. Other levels of activity include, you know, proteins, or proteomics or even metabolomics, in which we're gonna go ahead and start reading the page. And so reading the page and, you know, garnering that information, that's like proteomics. Then when we start, maybe we're writing down, and we're going to copy every word of that page, and then we'll have a copy. That's our metabolomics. So we go through these different processes to try to understand not only, you know, who was there, that's the DNA, what are they doing, and then also, what actually happened, you know, and that's our proteins and our metabolome. Putting all those pieces of information together, we can try to help us understand the microbe’s survivability.
Jordan Ruggieri 07:28
Are there complications? So looking at all of these different if you're looking at DNA, you're looking at transcripts with RNA, you're looking at the at the proteins as well? Are there complications with going down drilling in the soil, bringing samples back up, and then having good sample to study from?
Lydia Hayes-Guastella 07:47
Yeah, contamination is definitely an issue, also preservation of samples. We're taking them from so far below the sea floor, when they come up, you have to process them as quickly as possible to get them preserved before, you can actually be done with that sample for the time being before it gets back to the lab to be processed. So, we store everything for DNA and RNA extractions and analysis in a minus 80 freezer onboard, usually on the ship. And you want to get that processed as soon as possible because RNA and DNA does degrade fairly quickly. And also, you want to know that the activity in your sample is being preserved at the state when it was collected rather than having those organisms continue to acclimate to their new environment. If you're taking a sample from really high pressure, and you're bringing it up, you want to preserve it as fast as possible before those cells could either lyse due to less pressure or activity could continue and it might not be the same as it was when you collected it. Contamination is also a large issue when we're dealing with our samples. Because you want to know that the RNA and the DNA that you're collecting from your sample is from that environment and that it's not from ambient sources around you.
Jordan Ruggieri 09:18
Yeah, I was gonna be my next question is, especially with the with the Mariana samples, I mean, you're going so far down, you have to bring that sediment up through all of the different levels of the ocean there? So can imagine the difficulty of trying to eliminate some of the contamination that's coming from just bringing the sample up out of the ocean.
Brandi Kiel Reese, PhD 09:37
The nice thing about what we're doing at the Mariana of course using remotely operated vehicle, Jason.
Jordan Ruggieri 09:44
Oh. Didn't even think about that, that's amazing.
Brandi Kiel Reese, PhD 09:46
Yeah, so they're able to have like your eyes and hands. I don't know, like a drone, an underwater type of drone. And so they filtered a lot of water on the seafloor. In order to collect these samples. We use what we call a CORK, a circulation observation retrofit kit. And this is a sub sea floor observatory. It extends hundreds of meters below the sea floor, into the basement in which there are tubes, like a straw, like a slipper that extends all the way down into the basement comes up onto the seafloor. There's an ROV platform, and just starts, you know, hooking up to those, you know, to that end of that straw, if you will, and then filters on the seafloor, and then all you're bringing up is that filter itself. That is then, you know, closed off.
Jordan Ruggieri 10:38
That's, that's incredible. I had I had falsely in my head, I had this this vision of Lydia just standing on this, you know, two-mile-long drill on a boat and trying to go all the way all the way down to get that. But that's, that's incredible. Another question, if we're looking at things that are so deep down so far away, you know, what, why should we care about these microbes?
Brandi Kiel Reese, PhD 11:01
So microbes are the most diverse entity on our planet, the most diverse organisms on our planet, that are responsible for recycling Earth's nutrients, recycling Earth's carbon. Without microbes, you know, we wouldn't have air to breathe. We wouldn't have food, you know, our food is digested by microbes. I mean, honestly, they touch every aspect of our lives. In trying to understand not only how life has evolved on our own planet, we can also begin to understand what life might look like on other planets, both past life, and also life that might exist currently. Definitely is important from an esoteric standpoint, but also from a practical standpoint of. You know, what is recycled among our nutrients, also how harmful things are mitigated through bacterial respiration, bacterial activity is what we call bioremediation. And also from a basic standpoint, you know, what kind of understanding survivability of microbes in extreme environments has given us things like, you know, the opportunity to seek out new natural products and antimicrobials or antibacterials.
Lydia Hayes-Guastella 12:13
Yeah, why are microbes important? They are also one of the largest biomass categories on the planet. So they're definitely significant. They're very diverse, like Brandi said, and understanding microbes, really helps us understand how life is recycled. Understanding how the limits of life exist. When we look at these microbes in these extreme environments, and how they adapt to survive, or how they are there at all, can really give us insights onto how life formed here on Earth, and potentially, how we might find it in other places. We also get to understand new resources, potentially new antimicrobials that we haven't gotten resistance to yet, which is definitely important for the human aspect of things.
Jordan Ruggieri 13:14
Are there any potential climate impact, impacts your studies that you're conducting as well?
Lydia Hayes-Guastella 13:20
Yeah, definitely. Microbial communities play a large role in nutrient cycling, or biogeochemical cycling, whether that's carbon, nitrogen, sulfur, all of these types of elements need to be recycled. And carbon cycling plays a huge role in what we think of as climate change. And so how CO2 is produced or sequestered through microbial respiration, or biomass, definitely plays a large role in climate change.
Jordan Ruggieri 13:55
So what methods are you using to run some of the genetic analysis? Do you do sequencing? Are you using, you know, qPCR, dPCR? What kind of methods are you employing?
Brandi Kiel Reese, PhD 14:07
We do a combination of all of those from whole sequencing approaches, you know, shotgun sequencing, if you will, next generation sequencing. From there, if we want to look at specific genes or specific transcripts of interest, that might be associated with a particular metabolism or a secondary metabolite, something like that, then we'll go into it further with quantitative PCR or digital PCR. And then also we take, you know, some of those and we try to grow them up in the lab. But of course, we know that you can't grow everything in the lab, we can't replicate the environment exactly. And, you know, you do the best you can, but you're lucky if you get just a few percentage of what's in the environment, you know, growing within your lab. So yes, we take, you know, sequencing both the DNA through metagenomics, the RNA through meta transcriptomics. The quantitative PCR allows us to quantify, you know how active a gene might be maybe through looking at its gene expression doing qPCR on, you know, on the on the transcripts themselves. And we often run into the problem of that we have such low biomass environment that we have to look at it a little bit more specifically. The qPCR is a great tool, if you have 1000 to a million copies already, then it's, it's, it's great, it's in its sweet spot. But if you only if you're starting out with only 1000 cells per gram of sediment, then you're only going to have you know, maybe tens of copies, or just single copies of that particular gene or transcript. So that's where digital PCR is much more useful. And we're able to use that to get very low detection and that technology has enabled us to go further within the analyses.
Jordan Ruggieri 16:02
That's excellent. We love digital PCR here at Absolute Gene-ius, so it's great to hear that you're able to employ it in looking at some of those really rare targets. Do you use digital PCR in particular to you, so you do look at particular genes? Are you trying to quantify genes of interest? Or exactly how do you kind of employ that in terms of the primers and probes that you're developing and, you know, things of that nature?
Lydia Hayes-Guastella 16:29
From project-to-project kind of varies how we use digital PCR. But from my perspective, as a researcher, I'm really interested in specific nutrient cycling pathways. In the low biomass environment, you won't have very many transcripts of particular genes if you only have one cell. We would use digital PCR to follow a particular gene transcript pathway for nitrogen cycling, or sulfur cycling, and look at those specific genes.
Cassie McCreary 17:08
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Jordan Ruggieri 17:20
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Cassie McCreary 17:43
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Jordan Ruggieri 17:56
Let's get back to our conversation.
Cassie McCreary 18:02
Welcome to Cassie's Career Corner, laser beam noisily confetti cannons party horn. Anyway, I digress. So we've talked a little bit about these, like these expeditions and everything and what is the day to day like when you're on these, these trips?
Brandi Kiel Reese, PhD 18:18
The neat thing is that, you know, we can go out for you know, two months at a time or, you know, give or take, you know, a couple of weeks here and there. And you can collect all your samples at once and then get back in the lab and you know, work on them for the rest of the year. So, that's the benefit but that's also the drawback to is that you only have two months to collect all your samples at once, and then you've got the rest of the year to work on them back in the lab. And that if anything goes wrong, you know, while you're out at sea, weather doesn't cooperate, mechanical failures, that's your one chance. So you have to be a little bit of MacGyver and then, you know, at the same time be very flexible. You know, flexible with your environment, flexible with your colleagues. It's a fun experience. You know, I will admit that it's a lot of hard work. You don't have breaks, you're essentially on mentally 24/7 for that period that you're out there. But it's fun. It is just so much fun and it's so peaceful just to see the world from a whole different perspective. And you just feel very, very proud but insignificant at the same time because the ocean is a big place with a lot of power and I, you are a small player, you know, in this big wide world.
Lydia Hayes-Guastella 19:44
My trip to the Mariana we were out there for 27 days on the boat. It was the most exciting and also the longest 27 days of my life, probably. Our sampling was round the clock 24 hours. You just have so much adrenaline because you're in a cool place. And like Brandi said, you really have to learn how to go with the flow. Sometimes you think you have everything planned out and something doesn't work for you, and you have to figure out either how to move on or how to fix it. And you only have what you have on the ship with you. You can't magic a piece of equipment to you to make sure something works. And then also sometimes you find something that you hadn't intended you wanted to sample and then you have to find a way to sample it. Like for instance, we had some really cool biofilm growing on one of our CORKs, which was the straw like structure that Brandi talked about earlier, and we wanted to sample it because it was really cool. But it was on kind of a PVC pipe kind of thing and we had to be careful about our method of sampling affecting the structure. So we sent a toilet brush down, and the ROV collected it and its claws and just scrubbed the side of this pole with the toilet brush, and sent it back up. And like you have to be able to think of innovative things like that.
Brandi Kiel Reese, PhD 21:18
I always prepare my students for the what ifs, the incidentals. So we have, you know, I tell everyone, you know, bring duct tape, bring electrical tape, bring zip ties. When I hire students, I want to hire them for, you know, their creativity and for their adaptability as much as I hire for book knowledge. The book knowledge can come. Common sense and creativity and flexibility, those make a scientist. I think much more than, you know, even what is you know, written in a textbook.
Cassie McCreary 21:55
I kind of can't help but wonder how did we get here? For both of you, this is a pretty like, I would say niche area, right? So like, how did we, how did we get here?
Brandi Kiel Reese, PhD 22:05
You know that, that meme that you sometimes see, the way that we think science goes from point A to point B and the reality of it is that it's a circuitous path that goes through loops and turns and you know, and then you get there. Well, I would venture to say that career trajectories are exactly the same way. For me, I started out as pre-med. And I decided that I didn't want to work on humans. So I took a geology class. And I took the geology class because it offered a field trip to the Grand Canyon, and I thought that would be great. And I just fell in love with geology with the Earth. I've always been a bit of an environmentalist, even back into my elementary school days, you know, save the planet, save the Earth. It wasn't until after college, when I first became a consultant before I even you know, went back to graduate school. I became an environmental consultant, and I was introduced to the world of bioremediation, in other words using microbes to clean up our, our planet to clean up our spills or messes our accidents. And it combined the interesting parts of you know, microbiology from that I enjoyed in the pre-med days, to the soil, the environment that I came to love in my geology, undergraduate degree. Bioremediation was just my first introduction to microbes, not only existing, but also manipulating and changing their environment. And from there, from my consulting days, I met some PhD researchers that were on various projects that I was working on as a staff scientist and a staff geologist. And they actually told me how to go to graduate school, which is not something that I learned or came by naturally. In other words, I applied for graduate school once and I got rejected. And I got rejected because I would just apply just the same way you would as an undergraduate. But I never reached out to an individual researcher and said, "Hey, I like what you do. I want to be in your lab. Do you have room for me?" I reapplied for graduate school, and I reached out to those that had, you know, interests in both microbiomes and biogeochemistry. And then the fit was natural from there, you know, I didn't have a rejection anymore, I had, you know, resounding acceptances. And so I that's where I started to, you know, really explore microbes in the environment. And from there, I just kept moving further and further offshore. Inland lakes to rivers to offshore to onshore to offshore, and then just going deeper and deeper. Until I found myself in extreme environments.
Lydia Hayes-Guastella 25:03
I had a very different experience getting to academia than Brandi did. I definitely thought I was going to be a professional musician. When I grew up,
Cassie McCreary 25:15
Oh. Okay.
Lydia Hayes-Guastella 25:16
I played piano since I was eight, I grew up in a very musical family. My dad played the cello, my mom plays the guitar and she's a vocal teacher. I took every music class I possibly could in high school, and then went to college and pursued my future in music, and then realized that maybe I wanted piano to be a hobby, and not a career. So, then I kind of thought to myself, let's regroup. From there. I went back to community college, and I decided maybe I was interested in human health. So, I took some pre-med prerequisites. And the very first one that I took was a introductory to microbiology class. I just loved it. I realized that microbes were just so intriguing, and all of them are so different and you can learn so much from them. So I went on to my four-year institution and as a microbiology major, and in order to graduate you have to do research experience for that program. And so I reached out to some researchers at the University and found a lab that was doing geomicrobiology and did my first semester there and I stayed there for the last two years of my undergrad, and just loved it. And from there, I was like, “Well, maybe I am interested in grad school for environmental science.” And kind of stepped away during my master's, from microbial research into chemical nutrient cycling in coastal wetlands. From there, I graduated, and I took a break from academia and went to work in industry for a little while. And then I get a phone call, out of the blue, from Brandi and then she's like, "I have this project that I think you'd be really interested in", which happened to be this Mariana project. And that was kind of my squiggly route to getting here.
Brandi Kiel Reese, PhD 27:18
It's small worlds, it really is. So I know her former, you know, undergraduate advisor. Geomicrobiologists in extreme environments, is a very small world. And also when you see talent, like we saw in Lydia, you don't let that go. You know, you don't let that pass you by at least without making sure that their goals and their, that they're where they need to be, you know, and so hence the phone call to Lydia. And I said, "Are you still interested in pursuing, you know, a PhD because we've got this, you know, we got this grant that's funded, and I think it'd be perfect for it." Yeah, you don't let that slip through your fingers.
Cassie McCreary 27:59
That gave me the warm fuzzies. I now want to bring us to our first ever Cassie's Career Corner Challenge. So congratulations, you guys are the first participants.
Brandi Kiel Reese, PhD 28:08
I'm a little nervous.
Cassie McCreary 28:09
Isn't that great? I know. Sum up for us and our listeners in five words or less your field.
Brandi Kiel Reese, PhD 28:14
You're asking a scientist to be short winded.
Lydia Hayes-Guastella 28:19
I have to think about. I'm like over here counting words.
Brandi Kiel Reese, PhD 28:25
Once we think of our words, we can say them all together as if this was recorded right for the first time. Microbial survivability,
Lydia Hayes-Guastella 28:32
Survivability, adaptability,
Brandi Kiel Reese, PhD 28:35
Microbial, … Microbial survivability in extreme environments!
Cassie McCreary 28:38
There.You see, look at that. Teamwork makes the dream work. Well done you. I have no prizes for you but consider yourselves blessed. Okay. I like to do this every episode because we love consistency. I want to hear from both of you please your best or proudest lab moment or research moment, what have you. And also your biggest oops or "oh no".
Lydia Hayes-Guastella 29:00
There were a few very proud moments.
Cassie McCreary 29:03
Okay.
Lydia Hayes-Guastella 29:04
The toilet brush was definitely a proud moment.
Cassie McCreary 29:06
The toilet brush.
Lydia Hayes-Guastella 29:08
Another proud moment would be, we were having some problems with our sampling method for fluids. And I came up with an idea to send a really long tube down into that straw and we got very pristine samples from that. And I was very proud of that idea, because we kind of thought at one point that some of our samples were a wash. Sometimes the simplest solutions are the best solutions. So that one was a really proud moment as well. My biggest oops, I haven't had any that were super detrimental, yet.
Cassie McCreary 29:44
Okay.
Lydia Hayes-Guastella 29:45
Knock on wood.
Cassie McCreary 29:45
Well, that's good. Yeah.
Lydia Hayes-Guastella 29:47
I think probably my biggest oops, was one time I was mixing some really high concentration sodium hydroxide and didn't know that it created a very exothermic reaction when you mix it all together. And luckily, I was doing this under a hood, and I was wearing all my proper PPE, but I mixed it all in and the exothermic reaction bubbled and exploded in the hood, and just shot, high concentration, sodium hydroxide everywhere. Now I know that you add in a little bit at a time, and you mix it very slowly, otherwise, it is bad news bears.
Brandi Kiel Reese, PhD 30:29
The funny thing is that we have that exact same story that we both did that exact same thing. You know, during our, you know, early career stages. We've we joked about that, before we learned the same lesson, not together, obviously, you know, separated by, you know, maybe 15 years, probably, but that I did the same thing with the sodium hydroxide, you know, created a pressurized, you know, cap, you know, of a bubble that underneath it, start to swirl it and, you know, explodes. My proudest moment, I absolutely comes when I graduate students or watch the students accomplish something big that was, you know, very challenging or overcoming obstacles that might be in the lab or their challenges. You know, out at sea or just in general, you know, once they publish, you know, that in of itself can be, you know, a challenge in overcoming, you know, you’re writing fears or your writing, you know, blocks. And, of course, graduating and, you know, hitting those milestone milestones. So, students are my proudest moment overall. Absolutely. Far, you know, beyond discoveries or naming organisms or anything like that.
Jordan Ruggieri 31:49
Brandi and Lydia, thank you so much for joining us here on Absolute Gene-ius. It was an absolute pleasure to have you.
Brandi Kiel Reese, PhD 31:54
Thank you for having us.
Lydia Hayes-Guastella 31:56
Yeah, thanks for having us.
Cassie McCreary 31:58
That was Dr. Brandi Kiel Reese and Lydia Hayes-Guastella from the Dauphin Island Sea Lab at the University of South Alabama. Thank you so much for joining us for today's episode of Absolute Gene-ius. Stay curious and we'll see you next time.
I'm gonna do the whole Aspire ad in that voice. Being a scientist has its perks. Okay. Are we feeling aspirational? Are we ready?
Jordan Ruggieri 32:22
Let's do it.