You still hear me? Can you hear me now? Yes. I can hear you. Okay. Sorry. You're real bit about JP PHD at University of North Carolina at Chapel Hill with Carol Arnott, and apparently, C advises. I thought he just managed them the way we all managed them while we were there. He then went overseas to So University and the University of Southern Smark for Post and now he's back at high since fall of 2023. So getting started there. And we're excited to hear what you have to say and someday meet you in person down here. Awesome. Yeah. Well, thank you so much for the invitation to give a talk. So the title of this talk is squeezing Microbes impacts of experimental pressurization on microbial organic matter remineralization. And before I get started, I'd like to acknowledge my collaborators. So Carly Nasty, who is my PhD advisor Ronni Glue, who is my postdoc advisor, Chad Lloyd, who's a postdoc with Carly Nasty Devangsaf who is a PhD student that I co supervise along with Ronnie Glod Mites Middle Bo at University of Copenhagen, mi Schlemyer who is my PhD student here at Lehigh and was my master's student at University of Southern Denmark. Doug Bartlett, who was my undergraduate supervisor at Scripts and is now a collaborator, and then Stephanie, who is an undergraduate student at UNC Chapel Hill. So and just acknowledge NSF paid for some of this work. So, before I get started, this is actually the new Lehigh Oceans Research Center, so it's a little bit unusual that upstream of the Delaware Bay in this relatively terrestrial and freshwater setting, we have the Lehigh Oceans Research Center. But this is a recent thing, and it's because we now have a nice group of people who do oceanographic work. So I'm just introducing them so that you all are aware that upstream here near the Lehigh River, there's also an Oceans Research Center. We have Santiago Herrera, who is a deep sea coral genomics person. Jill McDermott, who's a hydrothermal vent geochemist, and Mike Leyden, who is an Nigerian biologist. So if you'd like to visit, this is our website. Our outline for today's talk is as follows. I will begin with an introduction of our labs research perspectives, questions, aims, approaches, and projects being that we're new. Not a lot of people know of us in our work, and therefore, it's nice to just introduce all of you to the range of work that we do and and the way that we think about our work. About the questions, so we ask. And then we'll move into the body of the talk, which is thinking about how enzymes degrade organic matter from the surface of the oceans where it's fairly productive and there's relatively high microbial abundances all the way to the bottom of the ocean. And in some examples, actually, talking about enzyme activities in the Hadal regions of the ocean. Regions that are greater than 6,000 meters depth. And then we'll transition into the pressure work that we have started doing and looking into the pressure effects on these different kinds of enzyme activities. We have different methods of measuring them and they have various benefits. So this first talk is going to look at these different enzymes. So peptidases glucosidase citas, alkaline phosphatases, But the second part is going to look into other kinds of enzyme activities that degrade polysacride. So these are higher molecular weight polymers. And this is a method that was developed by Caryl Noss at UNC. And then the third part is expanding out. So thinking not just about the pressure effects on enzyme activities, which is that initial step in organic matter degradation, but also things like bacterial production, right? So the uptake of organic matter to produce biomass, the respiration, so the conversion of organic carbon to carbon dioxide and depletion of oxygen in the process, and then the ratio of these two with regards to the bacterial growth efficiencies. Then I'll wrap up with some future work on our upcoming crews as well as a brief summary on the pressure effects that we have found so far. So our research perspective really considers bacterium relative to its food, right? So heterotrophic bacterium relative to its food particulate organic matter and that they need to eat, but they sort of have this size problem and that what they need to eat is much larger than what they can take into their cell. And so to alleviate this problem, they have devised this way to produce their own utensils, which are these hydrolytic enzymes, which hydrolyze or break down Using water, different types of organic carbon and different kinds of bonds in order to be able to break these down into much smaller size fractions, which we would call dissolved organic matter. And this dissolved organic matter can eventually be incorporated into biomass, right? So this is how microbes produce biomass. They can be respired, as I said previously, converting them from organic carbon to carbon dioxide. Or they can be sequestered. So if they remain untouched in the water column, they can eventually sink down to the bottom of the ocean, perhaps if they remain continuously untouched, they can become sequestered over longer time scales and sediments. And of course, that was a fairly simplistic depiction. In reality, what we have is a very complex microbial community community, and some distinctions that I've put on here are organisms that are particle associated, so they live attached to essentially their food sources, something that I completely resonate with as a human being who lives very close to my food sources, as well as free living microorganisms. They have very different genomic capabilities, very different genome sizes, and so on. And it turns out that they probably also have very different ways of accessing their food. And also, we try to understand whether these different complex microbial communities have consequences on their enzymatic capabilities. So we asked this question of how do rates of these microbial enzyme catalyzed transformations of organic matter vary when we go from the surface of the ocean to the deep oceans. And as I'll talk about in a little bit, this has massive implications on the microbial loop, the biological carbon pump, and the persistence or reactivity of organic matter. I also recently expanded into virus host interactions, recognizing that microbes are certainly hosts to viruses, and the activity of viruses as they infect and lie their cells might affect various community succession, community composition, as well as the biogeochemical cycles that microbes carry out. So we also asked the questions of, to what extent do these biotic interactions. So these virus host interactions influence micritical structures and biogeochemical cycling. So here we're thinking about viral ecology and also with the implications of viral influences on biogeochemistry on ecological stoichiometry. And then the sort of overarching theme in the work that we do is thinking about changes of these interactions and processes as determined by differences in environment. So we ask how sensitive are these aquatic microbial community structures and functions, environmental perturbations and regime shifts. So here we're thinking about disturbance ecology. We're also thinking about the relationship between biodiversity and ecosystem function. So our overall aims are to understand the maintenance, the stability, the sensitivity of microbial biodiversity, the extent of their control on organic matter reactivity and persistence and identifying the influences of environmental change on microbial structure and function, and just very broadly, This is sort of the conceptual diagram of that where we think about the environment that we're investigating. We think about how the environment influences microbes, as well as how this influence carries over into microbial functions. And then also how that function of microbes eventually loops back to the environment and alters it as well as loops back to other microorganisms that are within the community through microbial interactions. We do quite a bit of field work as well as experiments, and we use tools rooted in analytical chemistry, molecular biology, and bioinformatics as do many, microbio ly ecologists and biogeochemists. So the work that we do focuses probably on kind of three domains. So thinking about rivers to coast lines. One of the things that I really love thinking about is this transition from terrestrial and freshwater ecosystems to salt water systems, right, coastal ecosystems, and how microbes have adapted to live in these very different kinds of environments, and what the flux of terriginous material implicate the flux of the Tterriginus, what the consequences of the flux of terrigenous material on coastal ecosystems are. We also do surface to deep sea work as well as polar work. So a lot of the work that I did starting in my PhD and actually now carrying over into my faculty position is entirely in polar regions. And since fall, it was really funny that during my interview here, I was asked, What does your lab look like, you know, what's the size of this and being that we're in a relatively small school, and that I sort of grew up academically in a fairly small research lab. I thought I would have about I don't know, three people in my lab. That was sort of ideal. I like the close interactions. And surprisingly, I got more than that. So we have a great group of undergrads who are very, very motivated and self sufficient. And then we have now, I have two PhD students, Alex Sabo and Nomi Shulmyer. And then I have a co supervised PhD student who's based in Denmark Dibong Savi. So together, these group of folks do a lot of great work together. Just a brief, very brief overview about our ongoing projects. This is one of our polar projects. So it was just recently funded by an internal grant and it is trying to establish this new time series in Svalbard in Newson, which is this old mining town in the Norwegian Arctic and that has been turned into a fairly international Arctic research base. And so this is looking at Kgsfordin right here. And so what we're looking to do is establish the routine monitoring program so that we can look at seasonal variability of various microbial processes and how that links up to other macro ecological processes as well. In terms of the coastal domain of our work, we have a project funded by the Pennsylvania Sea Grant to look at microorganisms on microplastics, you know, with a perspective that microplastics are essentially a man made human made habitat for microorganisms within which there are numerous microbial interactions. And so this is a two year project funded by the Pennsylvania Sea Grant. And then the rest of this talk the pressure part of this talk is actually funded by a collaborative research project that's funded by the NSF. It's called pressure effects on microbial catalyze organic matter degradation in the deep ocean with Carl or Nasise PI and myself is CPI. And this is largely what Noah M Shulmers going to be doing for her PG. So to move to the specific research questions about these microbal enzymes from surface to the deep ocean? I already posed this very first one, this first set of questions. And this is really observational here, largely observational, right? So we do trans latitudinal transects. We also do vertical transects in the water column, and we try to ask how do rates of these enzyme activities vary from surface to the deep oceans? What kind of factors might regulate enzyme production and activities, and what are the environmental correlates of these enzyme activity rates. Then the specific pressure related questions that I'll be asking, which are in blue is, to what extent does increasing hydrostatic pressure affect the functionality of hydrolytic enzymes both from pelagic and ventic communities? Does the effect of these pressure of hydrostatic pressure act primarily on enzymes, right? So if enzymes are freely released, might their catalytic efficiency be affected by hydroctic pressure? Or is this effective pressure primarily based on its effect on microbial cells? So perhaps it could reduce or inhibit the ability of microorganisms to produce new enzymes? And then the last question is, does this effective pressure vary depending on which microbial process we're looking at. So kind of thinking at when we go from surface to the deep ocean where hydrostatic pressure increases, does pressure kind of make certain processes of bottleneck in organic matter remineralization. So we're looking at enzyme activities versus biomass production versus respiration. So we'll focus on this first set of questions based on observational data. So the relevance of enzymes, as I previously said, is with regards to this bigger picture of the biological carbon pump, right? So this biological carbon pump, as many of us know, is this fixation of carbon dioxide in the surface waters and through various pump mechanisms, gravitational, sinking pumps, and so on, this carbon gets transferred down to the bottom of the ocean unless it gets remineralized by various heterotrophic or mixotrophic organisms throughout its descent from surface to the bottom of the oceans. And because a lot of these organic compounds are way too large for microbial in For microbial consumption, these enzymes are necessary for organic compounds to be degraded. So in this way, enzymes truly determine what compounds are used in a way, determine what compounds may be used or what may be sequestered and remain untouched. And from the microbial loop perspective, it connects a lot of these high molecular enzymes connect a lot of these high molecular components of dissolved organic compounds, which is measured at around 660 pedigrams of carbon back to organisms of hydrotrophic levels. So organisms of hydrotrophic levels don't have access to this DOC unless microorganisms are able to bring them to take them into their biomass, and so microbes and their enzymes really play a critical role in this process. And the way that we can measure enzyme activities or organic matter degradation by enzyme activities is based on two different methods. And as a quick background for this is that the method uses substrate additions. So we add substrates that are reminiscent of the kinds of organic matter that are found in nature. So anything that is protein based, carbohydrate based, or their monomers, as well as their oligomers, they are natural constituents of aquatic organic matter, and therefore, they're fair game for evaluating how microbes might have access to these kinds of compounds. So the first way that we can do this is by using a fluorescence based method. This is these compounds are low molecular weight, and you can see what these different kinds of substrates are, right? So alpha glucopynide, a Beta glucopynocide, which is just a very slightly different bond. And then leucine then a bunch of these are different amino acids, right? So leucine, Alanine, alanine, phenylalanine, so on and so forth, bound to each other. And they're bound to a fluorophore that is flogenic. That is, if this fluorophore is bound to the substrate, the fluorescence is quenched. But if the enzyme is in the sample is in the sample where you've added the substrate plus this fluorogenic molecule. If the enzyme is present, then it cleaves this. It hydrolyzes the bond between the fluorophore, as well as the substrate. And then this fluorescently quenched flipor becomes fluorescent. This is essentially an assay based on increases in fluorescence. The second method is based on these polysacrides that have been fluorescently labeled by a different kind of fluorophore. This is not a fluorescently quenched molecule. So it's constantly fluorescing, so we cannot rely on an increase in fluorescence to be able to measure this activity. So this has to actually be based on changes in molecular weight. So we run this on a permeation chromatography, and from there, we're able to back calculate a rate. And so, based on the work that I did as a PhD student, as a postdoc, and then continuing into my faculty position, we actually have a fairly good view of enzymatic activities across various oceans, right? So I have this conceptual diagram here that kind of views the ocean through an enzyme lens. So I've sort of removed the microbes. That's a fairly typical conceptual diagram. But instead, just placed in here all the diverse kinds of hydrolytic enzymes that we've been able to measure. And we've done this work in the Central Arctic Greenland. So we have some polar representatives in polar regions. We have Atlantic the North and South Atlantic largely carried out by Adrian Forkss and myself, as well as Carla Arnaz, other PhD students. And then from the South Pacific to the Bering Sea, we also have unpublished data that's being worked up from the Indian Ocean. And so I'd like to just highlight three key findings from just synthesizing all of these different studies in that we have now a fairly Okay. I mean, it's not super comprehensive, but we at least have some sort of understanding about the latitudinal trends of these enzymes. We also have an understanding of some of the sources of these enzymes as well as the potential of different kinds of microbes to produce these different kinds of enzymes. So I'll go through that in the next few slides. This work sorry, this figure that I'm showing here is a correlation plot that correlates all these different enzyme activity rates, right? So this is based on the fluorescence method, right? So these are low molecular weight substrates, correlated with a wide range of abiotic parameters and and biotic activities. And what I want to highlight here are these five different columns. So anything that is within these green boxes shows fairly positive correlations with these enzyme activities, and anything within this red box, so the temperature. Has differing effects on, sorry, different correlations between temperature as well as these different kinds of enzyme activities. Essentially, what correlates most consistently positively with enzyme activity rates to no surprise are things related to primary production. So fluorescence, particulate organic nitrogen, particulate organic carbon, and chlorophyll A. Whereas temperature can either have a positive correlation between one of the trypsins that we measure or can have a negative response. Or sorry, negative correlation with, for example, Beta glucosidase and leucine aminopeptidase. And this is data ranging from surface for the tropics to polar regions and from surface to the deep sea. And what this essentially tells us, particularly of polar microbial communities is that they're actually not temperature limited. They're probably likely to be fairly carbon limited. This is something that Dave Karishman has also already alluded to in his 2009 paper. Um, But to demonstrate that with enzyme activities, these are the figures in the lower right hand corner of the page of the slide, where I compare the enzyme activity profiles in the Equatorial Pacific versus in the Bering Sea, which is about four degrees Celsius, whereas Equatorial Pacific, I believe was around like, I don't know, 25 degrees Celsius. In any case, what we see here is very high activities of some of these enzyme activities. And this is because we hit we were likely in the middle of a spring phytoplakton bloom in the Bering Sea. And of course, as we know, parts of the Pacific, particularly the subtropical and some parts of the Equatorial Pacific are fairly gotrophic. So here what we really nicely see is that when there's carbon that's awaiting degradation, it looks like enzyme activities can be fairly active despite the fact that temperatures are relatively low. Another key finding here actually stems out of the approach that we take when we measure enzyme activities because typically when people measure enzyme activities, and this is to no fault of their own, you know, these substrates are fairly expensive. But Typically, folks will use a few substrates as proxies for carbon degradation or yeah, related enzymatic activities, nitrogen acquiring enzymatic activities and phosphorus acquiring enzymatic activities. But what we decided to do here was actually measure a wide range of them. So we've got, you know, things that are representative of carbon acquisition, things that are representative of nitrogen acquisition, and a lot of these have not been measured in marine systems, and most commonly measured here are the Beta glucosidase and leucine aminopeptidase, which is in the purple and in the gray. But as is often the case, here shown with the North Pacific subtropical gyre, there's actually other nitrogen likely nitrogen acquisition enzymes that are equally or more active than what is most commonly measured, which is leucine aminopeptidase. And so, And we also find that a lot of these are produced by particle associated and bacteria. So we've done assays where we separated out the contribution of particle associated versus free living bacteria and enzyme production, and particle associated bacteria are just much more active, and they produce a wider range of these enzymes, particularly these endo acting enzymes. These endo acting enzymes are different in their mode of degradation of organic matter in that rather than kind of cleaving externally or at the terminal end of organic matter, It cleaves within these polymers. So one hypothesis for why these endo acting enzymes might be produced by particle associated bacteria is that they actually if you cleave larger polymers mid chain, then it's sort of a more efficient degradation process rather than just gradually starting from the ends and then gradually moving inwards. And then it also raises this fairly important point, which is, if we don't measure a wide range of these enzymes, how much might we be underestimating the activities that we measure, right? If we say that cineminopeptdase is fairly representative of the nitrogen acquiring enzymes, then presumably we'll completely underestimate these activities in nature. And then going from the surface to the bottom ocean to the bottom of the ocean, we also observe several trends. So there's alpha glucosidase, which kind of shows us this fairly low and invariable trend, right? It's not really robust. It's never prominent. It's just kind of there in the background doing its thing and not really changing much with depth. Some of these enzymes, Beta glucosidase, and leucinoptdase are actually increasing. So from the surface of the ocean to the bottom of the ocean, I actually increases. But a lot of these endo acting enzymes. So chymotrypsins, two different kinds of chymotrpsins, and then the trypsins they tend to be highest in the upper part of the ocean, and then they just decrease gradually towards the bottom of the ocean, which is fairly consistent with this idea that if these enzymes are produced largely in response to primary production, they're used for efficient polymer degradation and are produced by particle associated bacteria, then it makes sense that these would be decreasing with death because those other parameters are also decreasing with depth. Such that by the time we get to bottom waters, this is the most typical profile that we observe, high activity of Lucy Mino peptides and not really much else in bottom waters. So it led us to think, Oh, bottom water microbes are fairly inactive. But actually, we did an experiment where we spiked bottom water communities with a representative high molecular weight biomass, so coming from TalacoC or is flow GI, which is a fairly dominant coastal diatom. And what it did was it dramatically increased the activities of these bottom water communities. So essentially, alluding to this idea that Even though it looks like they're enzymatically not doing much, when primed with organic matter, these activities become much more prominent and they start producing the same range of enzymes that are observed in surface waters but isn't currently observed in the bottom waters in the absence of organic matter. And then another really fortuitous finding that we had was relates to an expedition that we carried out in 2012, where we were in the Central arctic and we found a lot of these meloca arctic aggregates. These are sea ice diatoms that grow in the underside of the sea ice, and a lot of them were sinking down to the bottom of the central arctic ocean, which was a highly unusual thing, but we think it's probably related to sea ice melt, and therefore, these sea ice attached diatoms are detaching and sinking down to the bottom of the ocean. Well, once these aggregates settle to the bottom of the ocean and specifically on sediments, what we find is that this is respiration of enzyme activities, we find oxygen concentrations completely depleted. So essentially, deepwater microbal communities exhibit substantial activities when liberated from carbon limitation. And when you contrast the oxygen profiles in sediment cores, without these aggregates, which is what's shown on the right over here, you see that it's relatively oxygen and replete. And this shows very oxygen depleted conditions. So bottom water deepwater microbal communities are not inactive. They're probably just waiting for pulses of food, sort of like a feast and famine kind of and boom and bust type of scenario. We started thinking about these patterns of NzM activities and part of what might be driving them. Part of that is certainly abiotic, but we're also thinking about biotic drivers to some of these patterns. And so we played around with this exercise where we tried to correlate the patterns of enzymatic activities with the patterns of microbial community structure and assembly. And I think this is a bit of a far reach, but perhaps, hopefully, I'll convince you that there's some sort of linkage here. So what we're showing here is going from the deep chlorophyx somewhere in the epipolagic going down to the base or the upper part of the bathyplagic at 1,000 meters, and then comparing the whole it enzyme activities in the blue. And then the particle associated activities, in the pink. We see very different trends as we go from surface to the bottom of the ocean. And what we're plotting here are break curtus dissimilarity. So essentially how different what is the beta diversity of these trends of these rates? How different are they from each other in that if the distance to the group centroid is higher, so this value is higher, then they become a lot more different from each other. So essentially the enzyme profiles that we're measuring at 1,000 meter particles is very different across this swath of the ocean where we measured enzyme activities, whereas in the bulk, or the whole community, which at this depth is largely comprised of free living bacteria. The distance to group centroid is fairly low. So these no matter where you go in this transect, enzyme activity profiles look relatively similar to each other, and therefore, they have fairly low dissimilarities. So this is the trend that we observed with enzyme activities. And so what we are curious about was, well, to what extent could this correlate with certain microbial properties, assembly processes, structure, composition. And we played around with measuring assembly processes. Sorry, with measuring, yeah, with quantifying or estimating assembly processes. So things related to deterministic assembly, things related to stochastic activities. This is the framework done by James Stagan and I believe that he put out this paper in 2012, so quite a bit quite a while back. And What we quantified was the extent to which different microbes are selected for by homogeneous selection, perhaps dispersal limitation, variable selection. And the key takeaway here is that in the surface waters, right where we have fairly low dissimilarities and fairly similar profiles, The particle associated and free living microbes have homogeneous selection, which means that essentially, there's a lot of similarity, and there's a similar set of environmental factors that selecting for these assembly processes, and it's very deterministic. In contrast, when we get to the deep waters, particle associated microbes tend to be more influenced by dispersal limitation, which is not deterministic. It's more stochastic. And essentially it's saying, particles are so far from each other that the microbes start to become very different from each other. And we think that this dispersal limitation among particle associated microbes may be in part the reason why these enzyme activity profiles are also very different from each other. Whereas free living microbes continue to be deterministically assembled based on homogeneous selection, and we think that that might be part of the reason why these enzyme activity profiles exhibit very low dissimilarities, or in other words, they're very similar to each other across this transect where we did the work. So that summarizes the observational part of our work. And I'm going to move into the body of the talk today, which is really about squeezing microbes, right? And it's about the effective pressure on the breakdown and remineralization of organic matter, recognizing that most of the global oceans is under high hydrostatic pressure, right? And this pressure increases from the surface to the bottom of the ocean. And we have this sort of logistical challenge which may or may not be biasing our results, right? So when we take water or sediment from the bottom of the ocean, we decompress them. And when we measure rates, they're typically measured at atmospheric pressures and not at Insitu. Pressures, and we wonder to what extent could this be under or over estimating the rates that we measure from deep sea water and sediments. This might also be responsible for I don't know if you'll remember that Adrian Byrd 2010 paper about the mismatch between carbon supply as well as carbon demand in the ocean. So one hypothesis is, well, we just haven't even taken into consideration, potentially the reductive or yeah, yeah, the reducing effect of pressure hydrostatic pressure on microbial processes. So from that perspective, we may be over estimating rates. We also need to recognize from an ecological standpoint that there's vertical transport of microbal communities and organic matter sinking rate from the surface to the bottom of the ocean. And essentially, these sinking particles act as elevator, and anything that is attached to that that colonizes the particle in the surface water experiences increasing hydrostatic pressure as it sinks to the bottom of the ocean. And because of this, as well as through other mechanisms. Surface originating bacteria and organic matter can reach fairly deep waters and sediments. And so it makes us wonder, is there pressure related inhibition of activities, right? That's kind of most obvious hypothesis for this because it's another abiotic parameter that microbes have to contend with. So we could expect perhaps inhibition of activities is something that may also be helping preserve some of the organic matter during its descent from surface to the bottom waters. And how does this affect different microbial processes. So what we do is we quantify this pressure effect. So when we do these experiments, and again, this is the experimental part of the talk. When we do these experiments, we have to do simultaneous measurements at increased pressures. So this is greater than 0.1 megapascal. 0.1 megapascal is essentially 1 atmosphere that's atmospheric conditions. So anything here in the numerator is a rate measured at higher than atmospheric pressures. And then the denominator here is the rate at atmospheric pressure. So high hydrostatic pressure rate numerator and then decompressed or atmospheric pressure rate in the denominator times 100%. So that if you were to see a pressure effect of greater than 100%, sorry, if you were to see it at equal to 100%, then you'd interpret that as pressure resistant or insensitive, if it's less than that, then that means that the denominator is higher, so the pressure actually reduces or inhibits this process. And if it's greater, then perhaps it's pressure stimulated, or at least that's one interpretation of this. Prior work by Christian Tamburini has actually given us some possible mechanisms for very different kinds of pressure effects. And in this schematic that he presented in his 2013 paper, what's really important to notice are these red microorganisms. These are surface originating prokaryotes, either attached A or free living FL, and then anything in the black Here, or maybe that's actually dark gray green, I don't actually know. But that's free living deep sea prokaryotes. So red surface, black deep sea. And if you look at this schematic, there's several potential outcomes here. So if deep sea microbes, and let's focus right here in the right part of this schematic, if deep sea microbes are decompressed, their rates will be underestimated. And that's because we're taking organisms that are adapted to the deep sea conditions, and when we decompress them, then a lot of them just won't function properly, so we'll underestimate their rates. But if a deep sea community is comprised of both deep sea prokaryotes, as well as surface procaryotes. And there's a lot of these surface originating microbes that live in the deep sea. When we decompress them, then perhaps their rates will be overestimated because that's not quite the rate that they're functioning at in the deep sea and we're relieving them of the stress of high pressure when we decompress them. So we bring them back up to atmospheric conditions, which is what they're used to and adapted to. And then in a case where we've got this convective mixing, or upwelling regions or whatever, where you've got this mix of both. It's also possible to be getting a pressure effect of one because you're essentially mixing the two averages. You're averaging the pressure effects on both of these types of organisms, so that it's possible to be getting a pressure effect of one. So with that background, I'd like to answer some of these questions that we posed with regards to pressure effects. The way that we measure pressure effect is taking these enzyme activities, a lot of which you've already seen from the beginning of the talk and doing these simultaneous incubations in different pressure levels. So going from 0.1 megapascals, and in some instances, we actually push them to 10,000 meters equivalent pressure or 100 megapascals. And we do these incubations at 24 hours. So we do a T zero and 24 hour measurement, and then we calculate the rate, and then we calculate that pressure effect. And we first did this as a proof of concept on this one moderate Paso file photo bacterium for fund of SS nine. This is an islet that resides largely in the freezers of Doug Bartlett's minus eight, sorry, lab. And it's a gammoptobacterium, so you know, fall fairly. But within the same class or whatever gammoptobacterium is now, same general branch as Coli. It's essentially the Coli for high pressure work. And we measured three different kinds of enzyme activities. We have lucen amino peptidase, NAG, which is the substrate for tanase and then phosphatase. So I show you here the pressure effect and the x axis, and then in the y axis, that's just the increasing pressures where we did these incubations. So anything in the gray indicates pressure reduction or pressure inhibition, and anything in the white indicates potential pressure stimulation. So we can see a few different things. We have one organism, It has a range of responses, right? So it's got pressure stimulation, which we observe here, as well as pressure reduction at 75 megapascals, right? Interestingly, the Optimal pressure for this moderate pazopile is 28 megapascals, which is the reason why we did it here. So it's nice to know that when an organism is at its maximum or most optimal pressure, then it will also exhibit optimal enzyme activity rates. So that's a good proof of concept. But then the other thing that I want to highlight here is that the pressure has very different effects across different enzymes. So, for example, when we push it to 75 megapascals, we see almost complete inhibition of leucine amino, aminopaptidase activity and almost complete inhibition of alkaline phosphatase activities, but we don't quite see that with tinase. We see probably around like a 45% reduction here, but not a full inhibition. So this essentially says this approach works. So how does this look like with natural microbial communities? Okay. So we've done this kind of work in various settings, right? So we've got various coastal settings here, the Skaaxtit hus Bay, we have it in deep settings, evolution Trench, Japan Trench, and then slightly slightly less deep settings of Fram Strait as well as the North Atlantic, and those are our maximum depths for all of these sites. And so I'd like to just summarize the work by focusing on sediment enzyme activities. And I show you coastal Tbthial sediment enzyme activity, so anywhere 0-4 kilometers below surface water. Again, same configuration of figures, here is the pressure on the y axis all the way up to 75 megapascals, and then here is the pressure effect for lesinmnopeptdase, Batgccdase and tanase. And what we see is that at 25 megapascal, so in this row, So equivalent to 2,500 meters. All of these different kinds of sediments have an average reduction of lucinominopeptdase by 7%. Beta glucosidase have an average has an average reduction of 0%. And then nag or tinase is also 0%. And a lot of these are coastal sediments. I mean, it's probably from like five meter depth. So we're actually seeing fairly low reduction here. When we push it to 75 megapascals, this is what our average production is. For Lucina is 14% for Beta glucosidase, it's 54% and for nag or itinse it's at 44%. So again, very different pressure responses by these different enzyme activities. And but it's nevertheless showing us that even if we push some of these sediments to pressures that are outside of what we thought their microbes are adapted to, it looks like they can still continue to function. That was a bit of a surprise to me. And when we compare this to a vessel and Hadel sediments, we see really interesting and fairly similar results. So this is a wider range of enzyme activities. And then here what we have is 0.1 megapascals, the situ pressure from which these sediments came, and then 100 megapascal. So we're really pushing them to the depths of the ocean. And at Insitu pressure, so this middle row over here in the green anywhere 43-72 Megapascal, the bissal sediment enzymatic activities are reduced only by about 5%. And the Hadel, enzyme activities are reduced by 12%. And you can think about it from a reduction perspective. We can also think about it from a percent over estimation perspective when you decompress the sample. So if you bring these sedments up to atmospheric pressure and you don't pressurize them and measure activities under pressure, this is how much you would be overestimating likely those insitu activities by. So 5% as well as 12%. And when we pushed them to 100 mega pascals, what we see is that the enzymatic activity rates are reduced by about 19%, and Hadel enzyme activities are reduced by about 19% as well. Possible explanations for these reductions, right? It could be that the fraction of active microbes may increase upon decompression. So this is a scenario where some of those microbes in the deep sea are actually surface originating and when you decompress them, they become a lot more active. Or sorry, the proportion of them become more active, not that each one of them become more active, but There's just a wider range of more active microbes. The second point is actually that specific microbes might increase their cell specific activity when we decompress. Thankfully, there's a paper that came out by Ciomano out of Gerhard Handel's Group that sort of gave us some insights into which of these hypotheses might actually be at play. This is a paper that came out in 2022 in nature Geosciences, where they looked at this question. This is based on bacterial production. And what they're looking at what they've plotted here are cell specific leucine uptake rates. And what I'm showing you here on the y axis is the percentage of total count. Orange is atmospheric pressure, blue is in C two pressure. And you can see that the percentage of active microbes tend to be about the same regardless of where you go for atmospheric and in C two pressure. There might be a little bit of a difference here. But so this kind of rejects the first explanation of a larger fraction might be more active when you decompress them from the deep sea. But their second set of results is, I think what indicates is the more possible hypothesis. So percentage of total uptake percentage, which is showing essentially how active are each of these organisms in terms of their cell specific lucine uptake. And what we can see here is that that's where atmospheric pressure and institute pressure starts to deviate, saying essentially that when we decompress microbes from deep from deep waters, there's a few of them that likely kick up their activity, their cell specific activity. So the same range of microbes may be active, but a few of them are kicking up their activity. They might be their activities might be completely reduced at depth, but once you decompress them, they start to really increase their cell specific activities. And you see much more of this the deeper you go into the oceans, right? So in the bathyplagic 400040003000 $2,000, you see that massive difference between atmospheric versus insitu conditions. And they also tried to identify which of these kinds of microbes might actually be responsible. So essentially, log loosin uptake is shown here for different kinds of microorganism. So I believe this is a SR 11 SR 202, SRF o six, Alton, so on and so forth. And if it's higher at atmospheric pressure, essentially, that would be indicative of this scenario right here, and if it's lower at Is pressure, then it's saying that their activities are stifled at high pressure. And we see that fairly nicely for some of these microorganisms s46 here, both in the mesoplagic as well as in the bathyplagic and the SR 11 here. And interestingly, from the study, they also provided some estimates that taking water samples from the bathyplagic actually might alter the activities of these different organisms, and these are the kind of breakdown in terms of the pressure sensitivity of the microbial community. They sort of put out these numbers, these estimates that about 85% of the microbes in the bathyplagic may just be pasotlernt and they're not actually pazophilic, so they're not actually adapted to high pressure, but that only 5% are truly pasophilic and adapted to pressure, and that 10% are paso sensitive, and those are likely the ones that are coming from the surface and being completely reduced in activity. Next, we try to understand the effect of pressure. And I see I'm almost running out of time here. Next is that I'm showing the self free enzyme activities and the effect of pressure on just the enzymes in the absence of microorganisms that can respond and produce enzymes. And we show this activities for a wide range of enzymes. What we did was we added sargassum and placosa to the water. We stimulated enzyme production, and we filtered out everything except for the enzymes and just measured pressure effects on the enzymes. And what we really see here is that there's a lot of pressure insensitivity. And the implication of this is that sulfury enzymes that are produced in surface waters can continue to function at pressures that are characteristic of haatal environments. And that we saw previously that in the deep sea sediments, there was that relatively high pressure resistance. So we think that that high pressure resistance in deep sea enzyme activities may be due to self free enzymes, and that these sulfury enzymes tend to last long when they're sorted to minerals. So perhaps that's what we're measuring. Okay, because we don't have any more time. I'm going to skip the part on polysacide degradation. I'm also going to skip the part on bacterial production. I guess, I'll have to visit and then go over these data with all of you. And then I'll jump right to what are we going to be doing related to the pressure work on the crews. A lot of this talk has been very heavy, but we are also very interested in the molecular biology of these things. So we're trying to understand how pressure may regulate gene expression. Specifically for hydrolytic enzymes, but in particular, just microbial metabolism as a whole, and how this might be factoring in with regards to surface originating microbial communities, asking the question of as these organisms from the surface sink down, does pressure play a role in inhibiting or maybe even retaining or stimulating some of those activities? We're going to follow up on trying to understand more about the polysachide degradation at increasing pressures, and we're going to try to use redox sensor green to try to quantify the fraction of active microbes. And then we're developing this syringe system. It's relatively simple in its design, where we put these fluorescently labeled polysacides in syringes, and then a motor will trigger these syringes to suck up water in the deep sea and incubate them so that we can actually see these activities at entirely in situ conditions. So just to summarize this, there's a wide range of responses, and it seems that pressure reduction is the most frequent, and we think that the effect is likely on decreasing microbial ability to produce new enzymes. We see that sulfury enzymes tend to show a lot of high resistance to pressure. And even if they're produced in the surface, if they latch on the sinking particles and sink down to the bottom of the ocean, they may still continue to function despite the high pressures at depth. For deep sea sediments, we have this new estimate now of how much depressurization may lead to over estimation of enzyme activity rates. And I didn't talk about this, but just the final result here from the parts that I skipped is that we did experiments where we also added additional carbon and nitrogen rich substrates and we tested the pressure effect of these additions on enzomatic activities, bacterial production, and respiration. It seems like these things change, but when we look at bacterial growth efficiencies, they're actually similar at Insitu versus atmospheric pressures, so that's fairly interesting. And then these are some of the questions that we are hoping to ask in the future. And with that, I would like to thank all of you. There's a lot of people funding agencies, and institutions that I'd like to thank as well for helping or allowing me to do this kind of work and giving me the money to do the work. So I truly appreciate it, and I'm happy to take any questions. Thank you. Thank you Very interesting and actually very topical for things that we're dealing there you can hear it, I want to make sure we've got some class in here. Anyone in the room questions that I want to start with Yeah, I don't know if you're on camera, so I don't know that. Hey. Thank you so much for your talk. I'm actually just interested in your pressure barrels, your pressurized I don't know, barrels do you used to grow. Does that check out? Oh, I'm not on camera. Sorry. But how many of those do you have and also how many cultures or christmas can you do. Like during the study is limited, can you only five or six of them or hundreds of them at a time. Oh, that's a great question. Well, so we had to produce a lot of these pressure vessels because a lot of the work that we did experiments that we do is based on sacrificial timepoint. So once we pressurize them, we no longer to depressurize them so that we can get the samples and measure activities. We no longer want to re pressurize them. So everything has to be a sacrificial timepoint design. Therefore, we had to make a lot. For this upcoming cruise, I believe we have 61 pressure vessels available. Doug Bartlett at Scripts also probably has in the range of hundreds. But they're actually a fairly simple design, and they're not super expensive to create. I think if you were to, you know, work with someone in the workshop, it could be reduced to something less than $1,000. But yes, if I mean, if you're ever interested, I'm happy to send some of these pressure vessels downstream to University of Delaware. Awesome. Thank you. Absolutely. Okay. Well, I'll jump in with the question. So in your very initial correlations, I was really surprised that the cell numbers didn't correlate with the enzyme activities. Yeah. Is that is that from lack of data or do you think I mean, because the three that you had are positively correlated. But I thought for sure like cell counts would be a slam dunk of correlation, and I was kind of surprised that it wasn't in here. So data or is it real? I think it's real. I think what contributes to that decoupling of the correlation is actually self free enzyme activity. So these are the persistent stuff that have already been secreted and are just lying around. And in the time scales where we measure some of these So I think that's probably part of what contributes to that. So I do think that's real. It does correlate. I think with Leucine aminopeptidase. I remember Lucinminopeptidase is one that was a very good indicator, sorry, cell count was a really good indicator or correlate for Leucine aminopeptidase activities, perhaps. That's one of the reasons why people have used Lucinminopeptidase as a kind of all end for nitrogen acquisition enzymes. But. No, I think there's validity to that. I don't think that it's simply I don't think that it's simply a lack of data issue. Anyone on Zoom want to mute and speak up, feel free. Urchmropped off the call. So your card questions are over, Dave Dave e mailed me and said that he would have to leave immediately after the talk and therefore couldn't ask questions. So he'll probably if he has any send e mail over some questions. We will do. All right. No one is unmuting, so I assume that that means they're good with it. So yeah, this is super exciting, D and it's great to keep talking to you. I think a bunch of us here have thoughts. We might be reaching out shortly. So thank you so much for doing this, and we hope to see you in person. Yes, absolutely. Thank you so much for listening and for asking questions. Appreciate it.
John Paul Balmonte - SMSP Spring Seminar Series 2024
From Taylor Link April 26, 2024
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"Squeezing Microbes: Impacts of Experimental Pressurization on Microbial Organic Matter Remineralization"
John Paul Balmonte
Lehigh University
Principal Investigator and Assistant Professor
Zoom Recording ID: 96952447472 UUID: DTcg4gicSxyy/foZQH3oeA== Meeting Time: 2024-04-26 03:19:43pmGMT
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