um well thank you very much for that introduction and it is wonderful to be here okay let's be quiet for Jeff so I I have thrown together a bunch of slides let's see if we can maximize this um that is meant to be a very broad non-exhaustive description of microbial habitats we're going to kind of constrain the physical search parameters Mike focused very nicely on Ice habitats we're going to look a little bit more at Mineral Rock sediment hosted places and then I'm going to kind of finish with a few um examples of these kinds of microhabitats that may or may not be relevant for the types of places we're looking so we're going to start very broad of uh you know what microbial life is it's diversity how we Define it how we think about it how we categorize it so first kind of found life that could not be seen by the unaided eye in the 16th and 17th centuries when the microscope was discovered and then people kind of Drew what they thought they were seeing through there and I love this one of this this mustachioed face we tend to project what we think we are seeing uh in all kinds of when we're looking at the limits of technological ability and this was a first example of that we've since learned that's not exactly what these things look like if we did want to Define what a microbe is it's um you know a microorganism an organism that is microscopic something you need a microscope to see so this is a description based on the tools available and not necessarily actual relationships and ecology so what is life is probably beyond the scope of this discussion um this is the current NASA definition that we can go ahead with in terms of what we need a microscope to see that's kind of a a moving Target as well that's not really a mechanism based definition so this kind of idea of a microbe is a little bit Broad and Squishy and probably not that useful so the preferred way of thinking about microbes and their relationship to each other is through phylogeny or their evolutionary history the ways in which different organisms have evolved through genetic modification over billions of years so the kind of easy cartoon version of this tree of life is that we have the archaea the bacteria the eukaryotes the three domains of life the more recent and more accurate version is a little harder to see on a slide of this size but we have the archaea down here the eukaryotes here and this whole group of the bacteria so um when we talk about microbes we're usually referring to at least in this context bacteria and archaea there are eukaryotes of course that are microscopic but usually that involves a higher degree of complexity a little bit more farther along the scale of evolutionary time and therefore maybe not what we would want to look for first in a astrobiological context so the prokaryotes which are the archaean bacteria usually considered more primitive in their cell organization um metabolically we will see that they are much more advanced but there are kind of some physical differences that we can uh cast umbrellas around to kind of categorize what we're talking about so prokaryotes are usually smaller they don't have the sort of traditional internal organelles their cell walls are different they're kind of physical things we can look for that distinguish the microbes from the macrobes all right so um Mike did a good job of kind of uh talking a little bit about some of these parameters that we are um constrained by I'm going to focus on four of them here of our physical chemical boundaries temperature pH water activity and pressure so um in terms of just kind of figuring out what the the search space is at least on Earth that can maybe be a little expanded when we move beyond Earth but this is what we have to deal with so far so we can think about the parameters that cells might enjoy and um deal with based on these kind of sliding scales so this is in the context of temperature but it could be any of these parameters in the sense that there's one zone where everything is very happy they're actively replicating they have all the energy they need to grow and thrive then there's a temperature at which metabolic reactions can still happen but maybe there's not enough energy maybe they are too burdened by dealing with these hazards to really have a full replicative life cycle but they're metabolically active sort of in stasis in some way then there's the zone at which they could be resuscitated they are not completely dead but they are not actively metabolizing so we can kind of think of these gray areas uh when we're looking for um specifically things that are growing and active and metabolizing is there something that kills them sure yes okay so there there should be a fourth aspect here which is uh point of no return sure yes uh where perhaps especially in the context of ice and freezing they're physically disrupted and cannot really be reassembled good point so with temperature we just heard a little bit about this uh the issue at temperature is that um the real issue is that ice can physically disrupt these cells they can be mechanically damaged the limit at the moment for that is as far as I understand it minus 20 degrees Celsius um and the challenge here isn't necessarily the activity of the enzymes at this minus 10 minus 20 Zone it is the physical disruption caused by ice crystals so there is a way to kind of get around it which is the rate of cooling to turn water into ice and this idea of vitrification so that you can freeze but still have a disordered arrangement of water molecules that allows for life to to continue on and it can still sort of access different things in an ice structure so here we kind of have an example of Water Ice in sort of a vacuum just in its own liquid habitat then in the relationship with a cell membrane so if it is in a liquid form we have our happy membrane we have stuff inside the cell these red and green molecules that a cell might want to metabolize as well as the water molecules when ice crystals form it expands of course and can disrupt the membrane that's no good but if we have vitrification if things cool very quickly and maintain this disordered yet solid state there can still sort of keep the membrane intact and access some of these metabolites in between the ice molecules so I guess the point there it's not just where you end up it is the process therefore the rate of cooling that can also be important um pressure dependence probably yeah I'm not sure but others would probably know more about this um so ways that life can sort of adapt um or molecular tricks they can use so ice binding proteins are one of them this is an example of ice forming in the absence of ice binding proteins and in the present so when there are these ice binding proteins they encourage ice to kind of Stack into these um more sort of two-dimensionally constrained zones which then make for more gaps of liquid so this is a good thing when we want to keep things in a liquid fluid environment in terms of measuring activity at these low temperatures this is a calorimetry experiment where they're looking at sort of the amount of heat coming out of this sample heat being used as a proxy for metabolic activity and these are just some values of the lowest limit at which they saw this little Peak that suggested there is still Active Metabolism so for different types of microbes and eukaryotes um it's all here between about negative 10 minus 25 degrees 26 degrees Celsius and then this is um another example of measuring activity through radio labeled acetate and looking at how much of it is kind of coming out in the CO2 at the end of these metabolic processes and here we're going from a five degrees Celsius down colder colder colder even at minus 20 there was a little bit of activity above background so that is considered sort of our limit of temperature that's a lower bound upper bound which might be relevant if we're thinking uh more about hydrothermal vents at the base of some of these ocean worlds um when you have more pressure you can keep water in its liquid form at higher and higher temperatures and the current upper limits that we're aware of is about 122 degrees Celsius this is an image of of that cell I think this is probably one micron not positive but in terms of cell counts by dappy over a two-day experiment things continue to increase in culture at 122 degrees so that's what we're seeing here all right that's a quick version of the temperature limits now moving on to pH what is the challenge with ph it is um an issue in sort of deactivating functional groups uh that have hydrogens involved so OHS nh3s these can protonate and deprotonate at different levels of acidity this uh destabilizes molecules especially around proteins that can kind of stop these Bridges between amino acids forming tertiary structure falls apart and kind of the hydrophobic Interiors of proteins can all then Clump together it can also disrupt the atpase which is a proton pump that allows for the chemical currency of life to form if this balance of protons either inside or outside is out of whack then it's harder to form ATP so these are the two challenges with ph all kinds of solutions life has come up with from changing the membrane composition to lower the permeability to really keep protons where they're supposed to be DNA repair their specific proteins that can go around and kind of stitch broken DNA chains back together and then these chaperone proteins that can be activated under acidic conditions to refold those proteins right if we have our bridges between amino acids being disrupted by higher or lower acidity there are proteins that can come together and sort of stop that from happening or repair it after it does okay so that's pH um I think the current bounds of known pH activity are like negative 2 to 12 12 and a half something like that so huge range as possible water activity or salinity as it is often less precisely described as kind of how the presence of water is felt or experienced or used by a microbe just because there is water around does not mean it is accessible to the cell so here we can calculate the water activity based on the amount of water that is certainly a component but the activity coefficient the degree to which it is bioaccessible is this other parameter that needs to be figured out essentially it's the idea of bound water that can or cannot be used so different types of salts can be better or worse in terms of grabbing onto water molecules and making it hard for life to get a hold of them that's the idea of these chaotropic salts versus cosmotropic salts um if we move more toward this blue and purple end of the spectrum with our cations and anions we run into more trouble under the same amount of salt so here we have more denaturation of the proteins uh higher solubility of hydrophobic constituents stuff like that so um keeping in mind you know it's not just the the amount of salt but the type of salt is really important as well there are of course adaptations that halophiles have come up with to deal with water activity challenges there are two main approaches one is both of them kind of keep the osmotic balance between interior and exterior intact but they do it in different ways one is with the salt in strategy so you keep a lot of the same salt that's outside but just pack it inside the cell uh this can be a problem with amino acid um structures you often have to kind of reconfigure the types of amino acids in proteins over an evolutionary time scale it's not really a quick transcriptional fix usually the second option is this compatible solute approach which again keeps the osmotic balance the same but it doesn't use salts it use other uses other types of organic molecules to keep that water activity consistent with the exterior uh this is a more common approach and all of these not all of them some of these Halo files uh can be seen and concentrated in sort of salt concentration ponds uh that produce these really beautiful pigments um and a really cool site where this is happening where um is Brian pools on the sea floor and this one in particular in the Mediterranean Sea the discovery brine pool uh is caused by this redist solution of a huge chunk of magnesium chloride that precipitated more than 5 million years ago so this is a different salt than what is accounting for most of the salt and seawater that's sodium chloride here it's magnesium chloride and on our scale of chaotropic salts it is much more in this direction so these brine pools where the water activity is super low is a great could be great spots to think about the limit in terms of water activity um I think Julie can probably inform us uh throughout the week of the latest status of this but as uh at last check uh there were cells in these brine pools whether or not they're metabolically active or what they could be doing perhaps is uh an open question but we can hear more about that later um all right so uh the last thing I'm going to talk about is pressure um often it's the same kind of adaptations that are being used in terms of DNA repair and compatible solutes membrane alterations here are three examples of ways you could change your membrane to deal with higher pressure where you might want to decrease the permeability across your membrane in order to keep stuff that's inside inside keep stuff that's outside outside these things include kind of linking the isoprenoid chain so here we've added a bond here um you can have more unsaturated aspects of the fatty acids it could have more branching all of these are different approaches and um the good thing about studying pieza files on Earth is that the highest pressures that we see um you know in the Mariana Trench for example or even in the subsurface are about the same as sea floors on some of these ocean worlds because the gravitational force of ocean worlds can be smaller if they are smaller um smaller bodies then overall you know they're much thicker oceans but the pressure is about the same and I'm sure many of you know more about that as well but it means that we have a relevant analog on earth that is kind of in the same order of magnitude of pressures that we might expect elsewhere all right so I've just focused on four uh physical chemical challenges there are others uh radiation I guess it's probably a big one I didn't talk about but we can sort of start to constrain this search space Here's a nice chart kind of showing I realize we're not going to actually interpret this at the moment but we have salinity temperature pH pressure this should be a three-dimensional Zone um and based on kind of the types of habitats we have on Earth and that Earth life is known to inhabit we encapsulate most of the types of conditions we might expect on a lot of these ocean worlds and Mars so um the natural laboratory of Earth is sufficient in many ways to test a lot of these physical chemical boundaries at least all right so that is one big category of kind of constraining life's ability to survive and thrive the other is uh where their energy is going to come from um and how much energy there is to do all of these life-sustaining processes so chemical redox energy is kind of the currency of all life that we know here it fuels the reactions that uh you know then turn this into biochemical energy ATP that can you can then spend on various things like repairing biomolecules moving around replicating all of these processes of life um we can often think about this in redox Towers really this is just the difference between the redox potential how strongly different molecules want to hold on to electrons versus how strongly they want to accept electrons um the greater the difference between an electron donor an electron acceptor the more energy you can get out of those couplings this is a kind of traditional zonation that we might expect in sediments and soils on earth based on how much energy you get out of each of these processes so in this case we would have kind of a general carbon molecule as an electron donor oxygen is the most uh the juiciest electron acceptor then you'd have nitrate manganese iron sulfate and CO2 these can be scrambled in all kinds of different ways based on specific concentrations but all else being equal uh this is kind of where things sort out in terms of preference energetically and the great thing about microbes is that they can do so many different types of electron transfer reactions animals all do the same thing in terms of electron transfer to get energy we eat carbon and breathe oxygen microbes can do this is a tiny subset of the list of what they are capable of but for example they can take electrons from hydrogen put them onto sulfate from Iron II onto nitrite all sorts of reactions are possible and that I think is the amazing thing about microbial diversity and the reason we're focusing on the microbes when we think about different kinds of environments so how much energy is possible to be produced how much do we actually need for life to persist those are key questions that we need to calculate and think about before we go hunting in particular locations so we think about this often with Gibbs energy reactions so if we have our metabolism of a plus b going to C plus d The amount of energy that comes out of this reaction is determined by this relationship which is really a log calculation of the ratio of sort of where things are at the moment and where they would be at equilibrium if your abundances of reactants and products is completely off of where they would be at some sort of equilibrium level you will have more energy available to drive biochemical reactions if it's already pretty close to that equilibrium less energy that limits uh is also a moving Target it seems to be that in terms of how much energy is required to sustain life this is a compilation of studies from a while ago at this point it's 2004 showing all kinds of studies and where that energetic limit of life was for mostly cultures and some kind of environmental settings our range as you can tell is about you know minus four to minus 30 kilojoules per mole being produced by these extragonic reactions and that's in kilojoules per mole of the substrate itself so we would want to know that the any environment we're targeting for active life at least could produce something like this kind of energy before spending a lot of time and effort to isolate these micro habitats it's kind of a constraining aspect that is telling us what is necessary if not always sufficient to determine if there's Active Metabolism okay but that's just one part of it that's the amount of energy but the power which is energy per unit time is often a more relevant Factor when thinking about if life is actually around um in a at a at a given time in a given habitat so um it's just what we calculated before this gives energy but times the rate at which it is being produced and people have kind of calculated what the the power limits of life could be this particular study is from sediments uh beneath the South Pacific gyre and they compiled the amount of carbon throughout these sediment deaths they calculated all those Gibbs energies they looked at how many cells there were calculated the power per um per location that was available and this is what they came up with this is zepto watts per cell so that's like 10 to the minus 21 I think um per cell which maybe doesn't make any intuitive sense but it's a number that we can kind of use to constrain where our limits are um and we see that it's you know changes over several orders of magnitude Even In This Very sort of energetically constrained sediments um so the reason power is important is that it's not there's kind of a there are two terms playing on whether a microbe can be viable it's the rate at which it is growing and using energy but also the rate at which destructive forces are acting on the cell so if breakdown rates are super high if it's very high temperature maybe high pressure then you need more energy just to retain your structure keep those that proton gradient intact things like that so the the threat posed by the environment is a key component that the biological energy producing reactions need to push against and compensate for um what are these these budgets of energy being spent on so that can be sorted into all kinds of different metabolic functions taken together the these can be considered the maintenance metabolism of a cell so we'll start with this chart here on the right where based on the cell volume we're looking at the rate of a metabolism of your standard cell and in red we have kind of the business of a cell doing its standard metabolism moving around replicating all of these things uh in Black we have the endogenous metabolism so that's kind of the maintenance energy needed to sustain the intact biomolecules and we can sort of think about this as an like overhead for living and it does scale with size as you can tell but it's less so than the Active Metabolism so at some point there's this uh this trade-off where the maintenance energy exceeds the ability of the cell to to make up for it and to have its Active Metabolism produce enough energy to do maintenance but also do the other business of life this chart frames it in terms of its volume and there's this Zone below which sells um you know you would not be able to do enough Active Metabolism to make up for the maintenance of actually building a cell having those cell membranes replicating The genome and all of those things uh this chart on the left breaks it down into what those specific things are motility ribosome repair proton leakage protein repair um so you know depending on the metabolism and the circumstances these might change but you can sort of calculate the amount of energy to produce and repair different biomolecules and this is a chart kind of compiling a lot of of time so um because you don't always need a lot of energy to survive and because things can sort of be resuscitated if you can stay on the good side of this this maintenance energy um balance you can have really long time scales for individual cells to survive in cultures up here we have turnover time so kind of replication times essentially of days and weeks I think these are I guess it's all done here surface sediments in Marine settings where we're at like a year for organisms to replicate and then an even more energetically constrain zones it can be hundreds of years these are calculated based on turnover a radio label [Music] propagated over calculated over time to get at what you would need to replicate a cell Okay so we've talked a little bit about growth and activity there are some questions about this with Mike's presentation um this is just to say that it can mean a lot of different things and the good part of that is we can look for a lot of different things in terms of from DNA turning into RNA into protein where are we drawing our line in terms of activity what specific markers of this process are we looking for do we mean growth in terms of making new cells or just new biomass or just metabolizing at all um and then activity can mean different things that are we looking for changes in the chemistry that are attributable to life or biomolecules themselves so it's just sort of a placeholder to indicate that we can look for many different things and being clear about which aspects of growth and activity we mean would probably be very useful when thinking about this anymore action-oriented way uh okay so how are we doing it for time a little bit left so I'm gonna now focus a little bit on some specific habitats um ways in which microbes interact with other microbes and with their mineralogical surroundings to increase the diversity and Niche space for life on Earth um and a couple of fun uh symbiotic relationships we can look at uh this is a clump of sulfur cycling cells in salt marshes where one cell is a phototroph oxidizing sulfide in an anoxic setting uh and the other is a sulfate reducer so they can form these very uh pretty large clumps of cells that you know would because one is producing the food of the other they can sustain themselves longer and more abundance than if they were separate that's one example this is another of sulfate reducing bacteria and methanogens in an anaerobic super biofilm so here we're looking at a cross-section here of the cells at the bottom we have these methanotrophs and at the top we have sulfate reducers um here it's unclear exactly how well they're interacting but there's obvious uh sort of physical striations and separation based on their use and affinity for hydrogen a common um metabolite that they both want and finally we have an example of anaerobic methane oxidation where archaeal members are oxidizing methane sulfate reducing bacteria are taking electron equivalents and reducing sulfate um and this kind of operates at an energetic minimum that would probably not be possible if it weren't for both of these being present at the same time and location symbiosis and interactions can be modeled of course so this is a study I will not go into depth on for time um but based on kind of this oxyotrophic relationship where one strain of E coli couldn't make one amino acid another couldn't make a different one they need each other to live um the study was able to color code where things are active and how active they are as a function of space they found in this particular case that the neighborhood size kind of the sphere of influence of a cell in this condition was on the order of 3 or 10 12 microns depending on which of these two strains we're looking at um so I think this is fascinating and really makes the case for spatially resolved analyzes because um you know if we stir everything up and extract genomes and reconstruct them and think that um metabolites from one organism are going to another that might not always be the case uh if that neighborhood size is closer to you know dozens of microns so keeping things as intact as possible to look at these interactions I think is really important another truism of microbial ecology is that microbes love surfaces they love these boundary zones and chemical gradients because that is where the redox transitions will be strongest and it's where more energy will be available I think it's hard to always separate um affinity for surfaces because it's a surface from Affinity from a surface because of its specific chemistry um if we just look at kind of the physical micro geography of a surface there are all kinds of little zones that you think of microbe could be happy with but is it because it's just a physical place to hunker down and be away from predators or is it accessing something in the minerals themselves thinking about this in a more three-dimensional way we can kind of map poor networks with micro CT scanning that's to kind of characterize what these micro habitats could look like one nice example of this is these endoliths from Antarctica where they are inside sandstones and but want to be near the surface for to do phototrophy but want to be away from the absolute outside because of radiation dangers but it's showing that there's like this very clear and intimate relationship with the substrates in a way that's structuring the biology another potentially relevant micro habitat is the um brucite and carbonate chimneys of the lost city hydrothermal event this is a figure showing like the temperature pH and salt conditions of this habitat the available carbon substrates from more reduced electron Rich to more oxidized electron poor and the types of metabolisms that could be happening in these structures and this is a nice sem image down at the bottom of what this chimney structure actually looks like so here we have a Zone that's maybe one of the best analogs for seafloor sort of ocean worlds hydrothermal settings where there's active serpentinization happening lots of hydrogen and small small carbon molecules that are sustaining multiple trophic levels of a habitat and it all starts in direct relationship with the surface itself and the minerals that are there all right um just want to check my time all right should end the quotery quickly so um I'll skip this bit uh there are all kinds of other ways that microbes kind of absorb and form minerals uh which I'd be happy to talk about um but the point is that they both form habitats so that's all these things I just zoomed through in terms of silicas and Clays and carbonates forming on the outside of these microbes but they can also destroy them or degrade them through acid production and this is one example of that where sulfide oxidation is kind of forming these pits in carbonate rocks and actively degrading it so um you know I think typically they're considered kind of Constructors of these micro habitats um but that's not always the case so I guess considering you know the nature of the metabolism the acidity and the nature of the substrate how susceptible it is to that acidity is also key when we're thinking about these micro habitats and the relationships we might find all right I'm going to end with a few questions that were top of mind for me I've kind of previewed these as we went one was how much did the microbes actually control the composition of minerals um and how and why are they doing this is it just kind of a um a byproduct of metabolism in a way that it's not useful it's just like getting rid of a waste product or can it form minerals in a way that can then be sort of energetically or nutrient value value adding propositions next is in relation to these micro habitats and minerals can the microbes access what is inside and within mineral structures this was a nice study showing that water from inside gypsum uh Crystal matrices could be acquired and it would actually restructure the mineral itself so we're seeing very close relationships between microbes and minerals in terms of accessing things in the mineral structure and then finally can we look at this in a non-destructive way um and this is something we will no doubt talk about further but Micro CT scanning could be a great way of starting this the intensity of these scans is related to the X-ray attenuation coefficient which is proportional to atomic number this was a study just looking at the minerals we can definitely separate different minerals based on CT scans but can we do it with microbes one study suggests yes I think there's a lot to explore further in these sorts of data sets but I think it is something we might want to think about with this group so I will stop there that was a very broad quick tour I'm happy to take any questions now or throughout the week of course thanks [Applause]