 Yeah, welcome back to likable science. And we have the defense of a master's thesis today from Andres Salazar-Astrona. Welcome to the show, Andres. Hi, Jay. Thanks for having me here. Sure. So not too long ago, a couple of months I guess it was, you defended yourself in your master's thesis at UH, SOEST, and your committee members and members of the public came around and asked you questions. That's almost, you know, that's really you're getting it from both sides. And because the questions you get from your committee are different than the questions you get from the public that attends and has a whack at you. But what's the difference? You know, if you had to, you know, sort of anticipate the questions, what's the difference between questions from the committee and questions from the public? Well, usually when the Q&A portion begins, for let's say the last 15 minutes that you have allotted for your defense, people are usually looking to understand what you just presented a little bit better or clarify certain things or see really how this connects to other parts of their research, right? For the committee portion that happens after the Q&A, after essentially the public, the public part, public portion of the defense finishes and then they bring you meet in the room alone with your committee and then they just, they ask you questions where, rather than getting the right answer, like precisely the right answer, you really wanna see how your brain is working, what's the path that it's taking to try to understand the question. What are you taking into consideration where your mind is sad, especially based on the research that you undertook? So it's a little more, you don't wanna jump the gun on those. You do wanna sort of consider the whole war day and the context and then try to give the best answer you can. You know, half the battle on these graduate papers, you know, master's thesis and dissertations is you have to write well. And boy, if you hand in something with typos, oh my God, it has to be good English. It has to be cogent. It has to be well-written, like an English student. Am I right? And that's why when you talk to a master's, you know, a master's person or a PhD, they're usually pretty articulate in English. And that's because they had to go around the horn through the mill on writing the paper, am I right? Yeah, in fact, it's a double-edged sword because it really after you, for example, for me, I defended the first week of, the last week of July, I think, for the oral presentation and then I finished writing it in the first week of August, right? It's kind of amusing to look back and think about all of the words that I use now, like that are just kind of central to my thesis, right, to what I wrote, to how I presented that information, right? But it's really hard to kind of let go of it, but that's like very specific terms. So those ideas and how they were laid out, it is a strange feeling to like start using very, yeah, very specific words to the topic at hand, right? And now if I just kind of bring them up in regular conversation, it's strange, yeah. Yeah, well, I imagine that sometime before and sometime after, and even now, you wake up at two o'clock in the morning, going through those same, what do you wanna call it, thought channels about writing it and defending it, am I right? For a while, yes. For I think the first maybe month after I defended, it was pretty much the same. I tried to take a break, but I soon realized that I was doing the same things that I was before, except without the writing, right? I was sitting in the same spot with my computer and have sort of like the same writing setup, the same position, and I don't know, my same, like I see at the same place. And I was like, I'm still just like, still going through these motions, even though I'm not working on it actively, right? I'm still thinking about it. I'm still trying to connect ideas. And yeah, and I try to come back and reread those things. Yeah, it's- It becomes part of you, doesn't it? And if you make a major academic effort that way, write it up, defend it, think about it, it becomes part of you, you know, it's kind of like in the center of your identity, the same thing with the dissertation. It defines you, at least so far, don't you think? Oh yeah, absolutely. And I felt similarly when I was an undergrad and did my senior thesis, right? It is just these large portions of your schooling, really, right? It's a whole year, a whole semester, it's maybe many years, right? But yeah, it becomes part of your subconscious almost. Yeah. You cannot not think about it, especially when you hear about other people's work. And it's like, how does this connect to what I do? Yeah. Right, or what I did, or my video, I did write it. Yeah, it puts you in the scientific community, which is not just that so as, it's a community that's global. It's the whole oceanography community in this case. And that's the way it'll be for you, whatever you do. Anyway, the title of your paper was Effects of Nutrient Supply on Metabolic Rates in the Olig, I practiced this, and now I can't remember it, Oligotrophic Ocean. And I looked it up and this is my perception of what you were researching. Nutrient supply means how much nutrients they're in the water and wherever they come from. And some water has more nutrients than some less. And metabolic rates would actually pertain to the animals in the water and what their metabolism was and how fast their metabolism, their metabolic rates were going, which is important both on a natural selection basis and I guess a survival basis and a thriving basis. You wanna see them have the right. This is my perception, right? You can correct everything I say. You wanna see them thrive. You wanna see them, you know, survive. And the Oligotropic Ocean refers to the ocean that does not have a lot of nutrients, a lot of animal life and plant life. And it would be clear water. It would be like a lake that did not have a lot of nutrients in it. Or in this case, it's the oligotropic ocean and some parts of the ocean do not have a lot of plant life. So you're experimenting with that. And then you start talking about a one month microcosm experiment, which means that you did this in fairly short order. And it says to me, you did it indoors that you didn't go out on the ship or anything. You did it indoors and you did it with a controlled environment and you controlled it all. You had to stand by your experimental equipment like every day and measure everything and so forth. So how close am I? Can you help me define your experiment and your paper better than that? That was pretty close, Jay, especially for just having the title on it and the picture. So yes, the full title of my thesis is the effects of nutrient supply and metabolic rates in the oligotropic ocean, insights from large scale and long-term microcosm incubation experiment. So when I say metabolic rates, I'm really talking about the rates of photosynthesis and the rates of respiration or the microbial community that is in the surface water. So like you were saying, the oligotropic waters are defined as, some people call them the oceanic deserts where they're very far from land. There are not very many inputs of nutrients, right? As for example, rivers supply nutrients to coastal oceans. Right? Hawaii is in the middle of one of the largest of those ecosystems being the North Pacific Jire. So we have been as oceanographers, particularly in the work of Dave Carl and the Hawaiian Ocean Time Series along with a lot of collaborators has been to understand how these really large areas of the ocean behave now, how they behave, how they vary throughout the year, throughout the years, through the seasons, through the decades, right? So trying to... Why do we care? Why do we care? Because these are, like I was saying, about 70% of the surface of the oceans is oligotropic. About half of all of the oxygen that is present in the atmosphere comes from the ocean. So one of the short hands that we like to use is that every other breath you take comes from microbes from the ocean, right? Oceanic plants, unicellular plants that are conducting photosynthesis, right? And this happens not because of the ocean, this happens not because they're more biomass, right? There's more of them, there are trees. In fact, I think it's less than 1% if you compare them by weight, how much biomass there's on land compared to the ocean. But since these areas are so vast, right? They take up a lot of that work. And in particular, this area being so big, the North Pacific, trying to understand how it reacts to disturbances or how just it behaves throughout the seasons is really important for us, particularly thinking about the carbon cycle and how much carbon dioxide is going into the water, how much it's actually staying there, how much it's coming back as carbon dioxide, how much it's sedimenting to the bottom of the ocean, et cetera, et cetera. There's something called eutrophic also. There's many trophics out there. So what kind of trophic did you prefer in terms of creating an environment that's beneficial? Well, there needs to be a balance, right? That is what we really want, not to really manipulate these cycles or these processes, but to sort of take a deeper look and see how they work, see how they behave, see how they deviate from the norm that we have assumed because like I said, they call these oligotrophic waters with very little chlorophyll, right? Very little biomass. So they work at very low rates. But on the chance that they do get a surge of nutrients, for example, of nitrogen, they will change their behavior, right? And that's part of what my research was trying to see. It's part of this larger collaboration with other universities about how, if we had all of these different tanks, right? We had 24 of those, the tanks shown in the image. Those tanks, those are tanks? It looks like little pup tents. Can you explain how the tank works? So the tank is about 30 gallons, right? And we had 24 of them. Each of them was filled with about, I would say maybe 25, close to, actually really close to the 30 gallons of whole surface sea water. That means that it had everything, every little bug, the experiment was called peri-fix and it was led by the John Lab at USC, in particular by Emily Seaman and Emily Townsend, you can see there in the top right. And the main goal I was saying was trying to see what were the effects of supplying nutrients to a natural community, right? A natural oligotropic community who's not used to having all of these nutrients. This happened in August through September of 2021. And on that date on August 9th, about 10,000 liters of the whole sea water were taken from the surface, about 13 miles south of Honolulu Harbor. This then was brought onshore onto the University of Hawaii Marine Center, Pier 35, and allocated into 24 of those tanks that we see. And we also see them here on that diagram on the bottom, right? And each one of these had its own light source and they had their own magnetic stairs so we could like, switch everything in there. But these 24 tanks were given eight different treatments of nutrients. So some of them got nitrogen, some of them got phosphorus, some of them got iron. And then they got all other combinations of those, including one that we call the control which had no additions, right? And in this way- All the other elements, experimental factors for the tanks were the same. In other words, the only thing that you were changing among the 24 tanks was the kind of nutrients it was supplying, that's it. Exactly. And the water was presumably all the same because it was all taken from the same area, right? Exactly. You know, they used to say you are what you eat, but looking at and listening to you, I realized that it's not as simple as you are what you eat. Your metabolic rate determines what you eat. So if I feed a human being, certain kinds of nutrients, certain kinds of foods, I get a completely different result. I can make you fatter, skinny. I can speed up your metabolism or slow it down. And different foods have a different result. And it's the same thing here. It's like you're giving it a different kind of food and then you're measuring how these animals or flora fauna, whatever you're testing, react to those nutrients. So what did you find when I think that the more nutrients you give, the higher the metabolic rate, is that, can I say that as a conclusion? No, you would have to actually qualify a little bit. The effect of each one of those that I mentioned before, nitrogen, phosphorus and iron were completely different even when they were provided together. So for example, here now I have, this is a little bit more technical, but we have the length of the experiment with all of the dates on the X axis, on the Y axis here I have micromoles of oxygen per liter per hour, which is really the rate at which they are photosynthesizing on both the left and on the right. The points that we see are the mean and the standard deviation are in the arrow bars for the three tanks of every treatment. Right, and I also have what I call the totes in a blue cross, which are the measurements from the data that water was collected offshore Honolulu. So what we see here is that on August 9th, the nutrients started being provided and we can see that there's a big separation between the treatments that received nitrogen and the treatments that did not receive nitrogen, which that really shows us that the approximate limiting nutrient, means the nutrient that is really limiting this metabolic activity, the photosynthesis, was nitrogen in these waters, right? For the community, for the microbial community that was present. Now, why did you limit the experiment to one month? Would you have learned more if you did it for six months or a year? And why did you limit it to 24 tanks of that particular size? Would you have learned more if the tanks were bigger or there were more of them? The numbers is relative, right? Depending on how many treatments we have. And here, part of the design that was done by the John lab, we thought the 24 was good, particularly because of the logistics of having to do it, having to build everything. Like I said, this was really spearheaded by the MLAs at the John lab and the rest of them who physically built all of this. They set up the 10th, they did all the sampling, the sub-sampling. And in fact, 30, we called it, like I said in the full title of my thesis, we call it a long-term and large-scale experiment because we usually don't have incubation experiments that are this long. The Hawaii Ocean time series, a large part of its power is the fact that it's been going on since 1988. We have a lot of data to interpret, between seasons, between years, between decades, but for monitoring that is this for a resolution, we're sampling, we're sampling every three days, every other day, and we really wanna see a quick effect. These experiments usually only last a week or two. So by making it a month, we actually did see, we got to see more of how the community reacted. Yeah, talk about reaction. Can we see those charts one more time? So the charts reflect changes, what by the day, or by every couple of days? Yeah, I sampled every, I sampled twice a week, yeah. Okay, and so that means there's some sampling days. Something profound is happening. You can see the changes every time you look at it. It's different. That is quite something. How do you measure though, the metabolic rates of the algae or the fluorophonics you're testing? How do you do that? Is there a little device, a little you stick in the metabolic rate device and then go, it tells you what to put on the chart? I wish, no, it's a little more complicated than that. So what I did was I sampled, like I said, twice a week. So every time I went to sample, I would get one liter out of each of the 24 pericostumes, as they would call it. Then that one liter, I would separate it into three bottles, to three smaller bottles. One of those bottles I would kill immediately with poison, which would maintain all of the gases that are the dissolved gases that are there, the oxygen, CO2, et cetera. The second bottle, I would actually spike it. I would give it 650 microliters of water that is enriched in oxygen 18. So H2O, the O instead of being an atom 16 of oxygen, it was an atom 18 of oxygen. And then that one, I would put it in an incubator that was in the same tent, so the same temperature and the same light source as all of the tanks. But separated, but separated. Yeah, but separated. Then the third bottle would be what I call the dark bottle and that I wouldn't do anything to that one, but I would still put it in an incubator, but with a lid. So it would not receive any light, right? Then those two incubations, I would have to wait for them about six hours for them to, well, the first one that had exposed to the light, it would photosynthesize and it would respire, right? It would produce oxygen and it would consume oxygen. Then the third bottle, the dark bottle, it wasn't really exposed to light. So there was no oxygen production, only oxygen consumption, which would give us a rate of respiration of the community. But the fun thing about the light bottle was that both things are happening, right? I use the oxygen 18 to track the production of oxygen when I see how much O2 that is molecular weight of 34, right? That's 16 plus 18, right? The 16 being the common 18 being the uncommon one. Then I could use that the known fraction and the ratio between those isotopes to see how much was the total gross oxygen production. So the total photosynthetic rate for that. And then comparing that to the time zero bottle, right? The one that I killed and like all of the, before the incubation I poisoned it, so there were no microbes would change the oxygen, et cetera. I can tell from, for those six hours, how much oxygen was produced, how much oxygen was consumed in the light bottle and also in the dark bottle. So it gives me also a way to separate the respiration that the microbial community is undertaking while they are exposed to the light, which is actually something that's very difficult to do with any other method. You killed them? Yes, I have to. You didn't put them back where they came from you. Isn't that cruel? A little bit. I do get attached to my little homies. You're a microscopic animal. Yeah, which I rarely, yeah. Some people call me a microbiologist, but I really only care about their gases, what they're putting out, what they're putting in. So I don't really spend much time on the microscope as I used to. So it strikes me that a couple of things. One is you're going to have different results with different animals and different results with different nutrients. So where does it fit in the halls of science that you have all these results? You can have so many results that it's hard to say what that would teach us in a larger sense. In a larger sense, what is your contribution in this paper, this experiment and in defending it? What is remarkable that the rest of the scientific world can use? I think one of the biggest findings that I have here which corroborates a handful of papers that have come out is that separation of being able to determine the rates of respiration when the microbes are exposed to the light and when they are not exposed to the light. Why? Because like we see here on the left, very similar graph to what I showed with the gross oxygen production right except that this time the same rates but it's for oxygen consumption, right? We see that there's still a separation on both of them for light respiration and dark respiration for the treatments that received nitrogen and the treatments that did not receive nitrogen. It is not as visually striking as it is for the oxygen production for the photosynthesis but it was a statistically significant effect. The difference though is that here, light respiration is a lot higher. Well, it's noticeably higher than the rates that we measured for dark respiration. This in papers that have managed to address this is common, this has been found before but still throughout oceanography, especially in what they call biogeochemistry of the surface ocean, a lot of people use the dark method to determine their respiration estimates to their respiration rates. And that's really important to constrain properly because when you are producing your gross oxygen production, right? Is your gross photosynthesis and then you have your respiration. You need to make do a balance between those two to determine what we call the metabolic state of those waters, right? Are they producing more? Are they consuming more? Is if they are producing more than they're consuming that means that there's a lot of carbon that could be sequester that could be could end up in the oceanic cycle for thousands of years. I wanna ask you about that. I saw on PBS recently a very interesting story about the redwoods, California and there's some guys who are taking clippings, the cuttings off those redwoods and planting them in other climates where they somehow managed to grow. Why redwoods? Because redwoods generate or use more carbon. They sequester more carbon than most other trees and the big redwood can really do a job. And so if you plant a lot of redwoods you're doing something for climate change. And so if you can control the whole carbon process in the oligotropic ocean then maybe you could have an effect on carbon in the world and am I right to think that maybe what you're doing learning about nutrients and different animals and metabolic rates, thriving or not thriving, living or not living could have an effect on the ocean which is a great source of oxygen and sequestering carbon, I think. And so these experiments that you're doing that you have done could lead to what I wanna call it an artificial change in the ocean by giving certain nutrients to certain animals and certain places and letting the ocean change for that. Is there that possibility? I personally don't think so Jay. So a lot of these actually there have been scientists in the past who have suggested, for example, dumping iron in the Southern ocean which they would assume would really activate all of this photosynthesis and all of this molecular activity, all of this carbon sequestration. But like I was saying before what we really want to understand is how these systems behave. How do they behave to already their natural disturbances, right? And these disturbances that are gonna continue to increase with climate change, right? With the change of pressure differentials in the atmosphere and the ocean, changes in temperature, changes in how often nutrient pulses occur in the oligotrophic waters. So before we really want to do any of those things or even consider them we still don't have a good grasp on what like for example fertilizing the oceanic desert would do really. If it would really be effective what kind of scale it would have to happen. And yeah, there's a lot of caveats as to what would happen or what could happen if we were to manipulate at a scale that large. Yeah, it could go in the wrong direction too. Exactly. Yeah. But what you're talking about microbes and after all Seymour stands for the stent of her microbial oceanography research and education with the Ocean Time series going on for 30 years and there must be a lot of information already accumulated in the Ocean Time series about these very points. So why did you select this for your master's thesis? I mean, is it a master's thesis that occurred to you, appealed to you or was this your committee telling you an area that was interesting and that science needed to hear from you about it? It was a little bit of both, right? Well, coming here, I didn't really have a project in mind but my advisor, Sara Ferron, she really developed this method of measuring aerosolation production with a particular mass spectrometer. So this method had already been existed using the isotope of Oxygen-18 since the late 80s and it has sort of been polished since then but she has been using it to measure these metabolic rates in the ocean. So she would take out the mass spectrometer into on the ship and measure them out while they were out there and get the results right away. And working with her, I thought that was really interesting with how we could determine, like I said before, what little gases are coming in and out of these little, what we call them, redox machines, right? Like reduction oxidation machines which are these little cells. And I thought that was fascinating. The deeper that I got into it and the sooner that sort of the deadlines for the thesis were looming, I definitely got a lot of help from my committee and sort of like guiding me as to what could be a great opportunity and luckily, like I said, the assignments collaborations on ocean products and ecology scope which is major funder of Seymour and the Hawaii Times series as well as some other PIs had this project ongoing that it was going to happen and they needed someone to quantify the photosynthesis rates and the respiration rates. So it all really fell into place. It seems to me that a lot of the effort you had to put in was to establish the systems, establish the devices you would use, the size of the tanks, the various experiments that you were conducting both on the nutrients and the animal side. And furthermore, that you had to look around for that. You had to do a fair amount of research to just am I right to establish the experiments, to establish what you were going to use, how are you going to do it, how often are you going to look at it, what data you're going to pull out of it and so forth. And aside from the data itself and the scientific conclusions you make into the scientific community, you have to have what scientific credibility and reliability of the data. So therefore you have to use the right equipment and the right systems to make this credible for science. Am I right? Exactly. And that's why I cannot give enough credit to the Emily's and the John lab because they were the ones that designed these, what they call the Perry costumes, right? The tanks with lighting and the stairs and everything. And they tried them out over in California and as part of the USC in Catalina Island and they did all of these trials and all of this work before any of Perry fix really happened to make sure that it was ready for all collaborators, right? I think we were about 10 or 12 different laboratories, not only in the United States. Were you on the phone to them? Were you emailing them? Yeah, we had meetings, right? Online meetings to sort of go through what everyone was going to do and how the sampling was going to happen. And yeah, the logistics were all handled by. So Andre, right now you're working in the same laboratory and you're doing experiments. How close are those experiments related to the experiment about which you wrote the thesis? The work that I'm doing right now is, it has two parts. One of it is yes, finishing up all of the work that I had done, right? All of the readings, all of the writing, some of the writing that I may want to add if we want to publish these results beyond being a thesis on a scientific journal. But at the same time I have other responsibilities to actually just was out at sea for about 45 days trying to get some samples for my supervisors out in the closer to the equatorial Pacific just in the middle of the ocean. And that didn't really have anything to do with metabolic rates, but I love going to sea so it was a welcome opportunity. So what about the future for you? You taking your masters, the next benchmark is the PhD. What are your thoughts about that? What subjects might you consider? What's the timeline in taking your PhD these days? And will you do it at SOS or somewhere else? So right now I'm not thinking about doing my PhD right away. I've definitely thought about it. When I came here, I thought I would go straight through the PhD and just try to sort of blast it out to some extent. But particularly with COVID I am originally from Chile on South America. So it's been a long time away from home. It's been a long time in school also, right? So I'm thinking of taking a little bit of step back enjoying the work that I do enjoying the science rather than feeling that pressure of writing and producing and graduating that I had felt before. So yeah, taking a little bit of a, take a little bit of pause on the academic side, hopefully continuing on the research side and hopefully still related to ocean biogeochemistry. I think what really fascinates me is how powerful these little microbes are and what they can do in cycling all of these nutrients that have monumental effects. They matter a lot for our, so yeah. And being lucky to be able to look at the world that way. Yeah, and you're pretty lucky coming from Chile too. Chile is so environmental. It's really aware and conscious of environment as so few countries are. And it's beautiful as you go south and it's got all this storyline, all this ocean to look at. And of course it's got astronomy too, doesn't it? Yeah, they do up in the desert. Well, thank you, Andres. Salazar Estrada who joins us to tell us about the defense of his master's thesis at UH. Thank you so much. Thank you, Jay. Aloha.