 It's a pleasure to welcome all of you on behalf of the Board on Life Sciences. I'm Barbara Schall from Washington University in St. Louis and I chair the board. Our goal for this session on biodiversity of microbes is to discuss what is known about the diversity of microbes. We want to identify the scientific challenges that occur in understanding and utilizing the diversity and to identify opportunities for future work. For years, biodiversity in animal and plant populations has been and continues to be a priority for the scientific community. Earlier this year, the national academies along with seven other academies of sciences of the seven countries identified biodiversity as a major global challenge. The earth, as we all know, is inhabited by an enormous number of species of microbes, nearly all of which remain to be discovered. Uncovering the diversity of microbes most certainly will lead to identification of species with unique functions. Functions that contribute to science, to health and medicine, to agriculture and to industries in the bioeconomy. Today, we'll be discussing both issues, understanding recent advances and hurdles to uncovering the diversity and leveraging unique microbial functions for the bioeconomy. The topics are particularly relevant given the release of the recent executive order on advancing biotechnology and biomanufacturing innovation for sustainable, safe and secure American bioeconomy and the bioeconomy research and development section of the CHIPS and Science Act. This should be today an interesting and lively session and with that, it's a pleasure to turn this over to Dr. Kavita Berger who is director of the Board on Life Sciences. Hi, I wanna welcome you all on behalf of the Board on Life Sciences for the National Academies. We are particularly excited by both of these discussions we're gonna have today and wanna encourage you to put in your questions, submit your questions through the Slido which is underneath the video. Please welcome us in, please join us in welcoming Baronda Montgomery who is our board member and will be moderating the session. Good afternoon, it's my pleasure to be here to moderate this panel on biodiversity of microbes, scientific challenges and opportunities. Frequently in discussions of micro to macro levels of biodiversity, we focus on animals and plants, but today we have a real opportunity and pleasure to focus on microbes. Our panelists today will be exploring current advances and opportunities for identifying, analyzing and understanding microbial diversity and function. We have four panelists today and we will start with, I will introduce them all by name and then by one by one, they will be able to tell us their offerings for today followed by questions and answers. So we will be having today Jay Lennon from Indiana University, Victoria Orphin from the California Institute of Technology, Jennifer Martini, University of California Irvine and Jack Gilbert from University of California San Diego. I believe we're gonna start out today with Jay Lennon and so I will turn over the floor to Jay for his comments. Should I use my slides and share or do you wanna use the slides on your end? We have a slide ready to go. Okay, great. Okay, good afternoon everyone. I'm interested in the factors that generate and maintain microbial diversity at scales from test tubes to the globe. And it's only been very recently that we've been able to explore some of those questions in the microbial world in part due to a lot of advances in sequencing technology and computational platforms. If we could advance to the next slide, that'd be good. And so for we mentioned the importance of studying biodiversity in plant and the microbial plant in animal world. And from that, we've been able to go and use traditional techniques to quantify the number of individuals and a plot and speciate those to a high degree of certainty, but it's only been recently that we've been able to generate comparable levels of data in the microbial world. And in part that's a really big challenge just because of the pure abundance of individuals. So when we wanna start making comparisons about whether microbes and macrobes obey the same rules of which we've developed in biological theory, we thought it'd be really important to take into account the vast differences in the abundance of individuals. And so what we did is we, a few years ago, we constructed what we call diversity abundance, scaling relationships to determine whether or not microbes and macrobes obey the same kind of patterns of laws of biodiversity. And when we did that, we found that in fact, microbes, which are studied often these days with molecular tools and macrobes, which might be estimated through binoculars or radio collaring animals, follow universal scaling laws. And from that, and this wasn't necessarily what we sought out to discover was that we could extrapolate or make predictions for the number of microorganisms and the species on a planetary scale. So this is what I'm showing on the left. And through independent means, we were able to estimate that that they're somewhere on the order of 10 to the power 12 microbial taxa or species on the planet. So for people who aren't familiar with current inventories of biodiversity on a planetary scale, this is something that biologists have sought to quantify for decades or longer is that there's currently estimated to be somewhere on the one to 10 million species of plants and animals. So when we start to consider microbes, we're now in orders of magnitude around six orders of magnitude larger. So these numbers are really big. They present us with a lot of challenges in terms of discovery and inventory and the computational ways in which we are gonna move forward with understanding that biodiversity. It also raises a lot of questions about the limits to life on earth. So is it even possible that we could get a trillion species to evolve over three and a half to four billion years of life on earth, given what we know about rates of speciation and extinction? Next slide please. So if you're an ecologist or an evolutionary biologist, there are lots of theories that people have developed to try to understand what allows for the maintenance of biodiversity over such time scales. And one of those theories would probably or many theories would kind of focus on the unique physiological, morphological or behavioral characteristics or traits of organisms that influence the performance or fitness of a group of organisms in a given set of environmental conditions. And so Jennifer Martini, who you'll be hearing from is somebody I've worked on with this idea of trying to think about what are the important or key traits for understanding the maintenance of microbial biodiversity. Next slide please. So in our group, one of the things that we think a lot about is dormancy, which is the ability for individuals to enter a reversible state of reduced metabolic activity. And this is something that's evolved independently, many times throughout the tree of life. So there are viruses that can go into latent forms of status in their reproduction. Bacteria and fungi will form spores. There are insects that go into diapause, worms that can form dower stages. Amphibians will estimate. There are birds that can go into torpor, fish can be quiescent and mammals, of course, due to seasonal variation in temperature or resources can also hibernate. So this is something that's really common. It's evolved again independently. So it's an example of convergent evolution. And it's also really important in the microbial world. And it's received a lot of attention from people who are interested in clinical importance and pathogenesis of microorganisms. So things like mycobacterium, tuberculosis, one third of the world's population is walking around with latent infections. And so we know a lot about in some cases the mechanisms that regulate transitions into an out of dormancy in the microbial world. But less is known about that kind of in the wild. So next slide please. So some of the work that we've been doing is trying to understand what regulates the processes of dormancy in model microbial communities. And so on the left, this is a picture of our model. In many cases, if we were interested in the functioning of a community, whether it be in the host or in the soil or in the ocean, we wanna know something about the distribution abundance and traits of metabolically active individuals. But there are conditions, environmental cues that can cause transitions either into an inactive state or out of it, which is referred to as resuscitation. And there can also be stochastic processes just due to noise in the environment, which falls into our category of bet hedging in an evolutionary context. So one of the things I think that's important to consider is that there's this emergent collection of individuals in the microbial world that are metabolically inactive. And so instead of being purged from the biosphere, they can reside in this inactive state, retaining genetic diversity, allelic diversity, species diversity, functional diversity for long periods of time. He can't stay inactive forever, but there's a lot of evidence from various different ecosystems that these seed banks of this collection or reservoir of inactive individuals is large and also very long lived. There are bacteria that can live in energy limited environments for millions of years. And in addition to serving as this reservoir in time, this seed bank, it also facilitates the movement of organisms in space. And so the last figure that I wanted to show is just some examples from data that we've collected a while back now where we surveyed existing data to get a comprehensive kind of view on how prevalent and what the magnitude of these seed banks are in different ecosystems. And so about 25% of all the bacteria in our guts are metabolically inactive. Oceans and freshwater ecosystems are somewhere on the order of 50%. And in soils where we care about fertility of soil for feeding a planet. And also the production and consumptions of trace gas and the storage of carbon, which is important for climate change, which we'll hear from Jen Martini. All of that, those functions are being carried out by a small fraction of the cells that we find in those ecosystems. So I think this is probably pretty important for the next part of the symposium today or the workshop to be thinking about the bio economy because a lot of these organisms are probably pretty recalcitrant true to domestication and in bringing new organisms into the lab if we're interested in, for example, product discovery, natural products or synthetic biology. So the take-homes are is that there's a lot of, there's a lot of microbial diversity, perhaps more than we ever could have imagined. It's gonna require new computational tools to study. And we also need to think about the seed bank, which I think is important for the maintenance of biodiversity and contemporary and deep time. Thank you. Thank you so much, Jay, for those comments. I'm gonna turn next to Jennifer Martini. You, the floor is yours, thank you. Great, thanks. Are we sharing our own screens or are you running the slides? Sorry. The slides have been shared on our end. If you send your slides in, they can- I did, yeah, okay. So I'm ready for those if you are. Super. Thanks, Jay, for a great introduction. And I'm gonna start with where Jay left off saying how one of the biggest challenges, of course, of understanding microbial communities or microbiomes for short, is their diversity. And so just in this handful of soil, which is one of the main systems I focus on, you'll find billions of cells in this handful. And depending how you count tens of thousands to millions of different species. And so over the last couple of decades, we've gotten better at trying to assess this diversity, but there's still a huge amount of variation from handful of soil to handful of soil. Or if you move into different systems, like the human microbiome from person to person. What we can say, however, if you go to the next slide, is that microbiomes are very sensitive to perturbations. So in the field, in the environment, free living microbiomes in soils, for instance, are sensitive to a variety of different types of perturbations that are associated with the types of global changes we think are going to occur in the future. We seem to have lost the slides. Sorry, I'm pulling it back up. Okay, I'll keep going. I was gonna say that similarly, if you look in other ecosystems, not just in outside sort of natural environment ecosystems, but even in human microbiomes, we also see that changes in diet, in health state and diseases also will affect the composition of microbiome. So if you go to the next slide, that's just showing this very nice meta-analysis study by Eric Elms group, which looked at various different health states, disease states. And the point of the figure on the right is that in almost all cases except some cases where there were very few samples, you could see a change in microbiome composition with a change in disease or health. And so that all of those dots to the right of that axis of 0.5 means that we had a better than even chance of predicting what the disease state was based on composition. But you'll notice that these results are very much sort of yes, no composition changes or not. We're still for the most part, not very good at predicting what the composition changes will be really in any microbiome system. And so I think that to tackle this type, this amount of variation, this diversity that we really need to focus on synthesizing data across labs, across sites and even different types of ecosystems. Of course, this is really challenging. It's challenging for any field to suggest that. But I think that there are a lot of opportunities to do this with microbiomes. And for one thing, it's because of the nature of the data that we collect, the type of omic data, which I'm not gonna get too much into, but is really the same tools and same types of analyses that we use regardless of system. And so we can synthesize this data among microbiome scientists easier than many fields. So if you go to the next slide, I really love this slide. It is data from the Earth Microbiome Project that I imagine Jack will talk about today. And I love it because you can put humans on the same plot as oceans. And you can see that how skin microbiomes, they sit between soil and human gut microbiomes and sort of what you would expect. And so this is the type of compositional data that we can integrate together. So to go a little deeper on like how we can start to predict composition, I'm gonna give an example using phylogenetic approaches that Jay had mentioned. So if you go to the next slide, this is an example where one of my postdocs was just pulling together all the global change experiments out in the field that he could find that measured the change in bacterial diversity and control plots versus those perturbed, for instance, by warming or drought or something like that. And in the next figure, you'll see that if you can gather all that data and put it on the same phylogeny because it really is the same information. And the important point of this result is that each of those dots on this phylogenetic tree, which represents all of bacterial life are significant clusters of taxa that are responding either positively in blue or negatively in orange, in this case to elevated CO2. And so this means that across these different studies that use different types of methods potentially to elevate CO2 and for different lengths of time, you still tend to see, despite all of the diversity and variation across the world in soils, we still see the similar groups that are responding one way or the other. Next slide, please. And we've done this also with human microbiome data. So this was a student of mine, Cynthia Rodriguez, who gathered dietary fiber intervention studies. So different groups had asked patients or asked people to take up or to eat higher amounts of fiber in many different ways. Sometimes those whole foods, other times they just put a pill of some kind of resistant starch. And she asked whether or not then combining again, all of the data and controls and the intervention patients, could we detect significant changes in the same tax across people? Despite that we know that in most of these studies, 80% of the variation is explained by differences between individuals. And yet still you can start to see here that there's clusters in this case, it's blue and red clusters of tax on the left side, they're responding one way or the other to a fiber intervention. And on the right hand side, just for those of you that might be curious, some of the tax that we see respond positively and negatively are ones that we have already knew that consistently come up in people's discussion of these types of studies like Bifidobacterium as positively responding to fiber. So that was a nice affirmation. So if you go to the next slide, the problem though is still even if we can predict how certain composition will change in response to a perturbation, we still are having difficulty translating that composition into functioning. How do we translate composition to functioning? So I'm gonna illustrate kind of the big picture idea here with just a very simple graphic. If the pie charts represent different composition of microbiomes and from left to right, there's a perturbation and it changes if you can move forward. What we really wanna know is whether or not these microbiome compositions perform a rate differently. So if you click forward, sorry, there's a bit of animation here. So if you have a functional rate in soil, perhaps that's decomposition, do those different communities, when you hold all other environmental parameters constant, do they perform some important functional rate the same? If you move forward, they might not be functionally redundant actually, be distinctive and then you see these differences. But you'll notice again, this is just like a yes and no answer. Once again, composition matters, it changes your functional rate. What we need still is some sort of quantitative metric of how composition is going to affect functioning. So what we've been working on in my lab, if you move forward as well as many other people is whether or not we can come to some sort of functional process, functional performance curves, for instance, if you're thinking about global change, we often are interested across a gradient like temperature. If we dial up or down temperature, how is that gonna change some microbial mediated process that we care about? And going back to composition, is this process static, regardless of microbiome composition? Or if you move forward, perhaps you have different curves for different communities. And that's the big question now, is how often do we have those different curves and where and when are we going to have them and we need to think about this, especially as composition may change in the future? One thing I will note is ecosystem modelers have been thinking somewhat about this. I'm working with Jay these days on trying to get ecosystem modelers to think about microbial communities more and not use, maybe one option is not to use a static function all the time, figure out where we need microbial, fully different functions here in some of the models. But one wide open area I would say here is thinking about this in host-associated microbiomes. So if you move forward, we have all these ecosystem processes on the left-hand side that we think about the services they provide to humanity in terms in natural ecosystems like soil fertility, greenhouse gas regulation. But I love this graphic from the Sonnenbergs which talks about microbiota services in humans. And imagine that we start modeling, for instance, the degree of fiber intake of a person and a functional response curve in terms of short chain fatty acid production, which we know is associated with immune function in humans. So next slide please. And nowhere is this more urgent, I would say than in climate change research, the IPCC's new reports, it rarely mentions microbes even though we know they're fundamentally involved in the earth systems responses to climate change. And I would argue that there's a huge unexplored potential not only for improving our current climate models if we integrate microbes and microbial research, but also to uncover mitigation and adaptation strategies as well. So to conclude in the next slide, I'll just wrap up by sort of going from, we were charged with thinking about the challenges and the opportunities here. And I think, as I've mentioned, one challenge is getting a handle on that high diversity and variation of composition. And I think the opportunity is the nature of microbiome data can allow us to synthesize a lot of data across studies. In terms of the challenge of understanding how composition translates into functioning, the opportunity is there's a lot of room for discovery of quantitative functional predictions here. And I think the field has been working really hard towards this. And I think I'm hopeful that we're moving in that direction. And finally, one of my soapboxes is to identify general microbiome principles. And to do that, we need to work across microbiome systems. We need soil people to be talking to human microbiome people and ocean microbiome people. Because ultimately, we need to study these questions of ecology and evolution of microbes in their communities regardless of where they are. Microbes aren't restricted to a particular order or place in the world, our research really shouldn't be either. So thank you very much. Thank you very much for those contributions and those challenges and opportunities I look forward to coming back to in our discussion. So we're gonna move on to our third panelist for today, Victoria Orphan from California Institute of Technology. Thank you, Victoria. We wait for the slides and we can advance the first one. Okay, go ahead and go one more, everybody. So I'm happy to be talking to you all today. And I wanted to start by sort of pulling together some of the concepts that you've heard from both Jay and Jennifer. For those of us that work in the microbial ecology field, this idea of the great plate count anomaly is one that's pretty well ingrained. And that is the very small number of microorganisms that can be successfully cultured out of any one environment, somewhere usually on the order of less than a percent. And that means that the vast majority of diversity that you see in an environmental sample is represented by organisms that we may only know from genomic sequences that are recovered of the ecosystem. And this is really nicely illustrated in this conceptual figure by Laura Hugg from a few years ago that really shows that of the known genera level of diversity that have been cultivated, this is usually less than 20% with over 80% of the phyla class order and genera of diversity across the globe being represented only by uncultured organisms. So if we put this in the context of Jay Lennon's trillions of species, that means that there's hundreds of billions of microbial taxa that we really don't have a good handle on their function. You may know a little bit about their distribution. We can look a little bit about their trends and how they change in response to change. But really this is a big wide open area that we're faced with going forward. Now the fortunate thing is that many of this diversity has been guided by advances in omics techniques, microscopy and different types of micro analytical technologies that are really allowing us to make links between microbial diversity as exists in nature and trying to understand their interactions and functions. So we can go to the next slide. So I wanted to actually instead of focusing on the high level diversity, I wanted to dive into sort of another layer of diversity that exists at the subspecies level. And this is something that some groups have focused on but in large part this gets sort of lost in our broad discovery of diversity in nature. And so I wanted to provide a couple of classic examples. One comes out of the pioneering work of Penny Chisholm's lab looking at this single-celled cyanobacteria called prochlorococcus. And this prochlorococcus at a species level. So that is if we looked at it from the 16S ribosomal RNA, most of these organisms would be greater than 97% similar. And so from that perspective, this is one of the most successful and globally dominant cyanobacteria in the ocean somewhere on the order of three times 10 of the 27 cells. But if we look at a higher level of resolution, we realize is that their global dominance is really split up into a number of very successful strains that are exquisitely tuned to their unique niche in the environment. So whether this be adaptation to light at different depths in the water column, adaptation to temperature regimes at different latitudes in the ocean and as well as being able to utilize different nutrients depending on where they're falling in the water column environment. And a lot of the recognition of these ecotypes came from the success in cultivation of some of these prochlorococcus cells. So they were able to basically look at the physiology of these organisms and then put this into the context of the diversity that they see in the environment using omics types of approaches. And so this for me was highly motivating in trying to unpack the diversity in the type of environments that we study in deep ocean sediments. And so we know the next slide. So in this particular case, we've been studying these archaea that are responsible for oxidizing methane under anaerobic conditions. And they are the dominant organisms that basically sequester methane in ocean sediments. And if you take a little pinch of sediment just like the soil systems that Jen was talking about there's a huge amount of diversity of these methane oxidizing organisms that exist. And you can see this in the right hand side of the picture here. And this diversity exists not only at different genera of these methane oxidizing organisms but also a significant amount of strain level heterogeneity. And so when we started our work on these systems this heterogeneity was really a bane to our existence because it really made using classical metagenomic assembly techniques very difficult. So even though they're the most dominant organism in these environments where methane is fluxing to the sea floor, we had a really hard time assembling the genomes of these organisms because of all of this strain level heterogeneity. And so what was turned out to be an initial methodological challenge has now shifted to real interest in the ecology of what all this heterogeneity means. So the next slide. So in this particular case we were able to unpack the idea of different ecotypes among these Archea by doing physical sorting of individual methane oxidizing Archeal aggregates using flow cytometry. And we were able to sort these into individual wells and then sequence them. And when you do that you start to see even within organisms that have over 99% similarity on their ribosomal RNA genes that there are phylogenetically coherent groupings that have differences in their genome contents. And some of these things are actually very ecologically significant. So what I'm showing you here is three different strain level groupings among one of these methane oxidizing Archeal lineages where the ability for these organisms to fix nitrogen this nitrogenase or NIFP operon varies between these different strains. So we see group two has all have the ability to potentially fix nitrogen. Group three, it's a little bit more of a mix and then group one seems to be lacking it. And so we can take this information based on genome predictions and actually start to test their physiological capabilities directly in the environment by using fluorescence and situ hybridization staining targeting these different groups of organisms coupled with isotope tracers. So in this case, say 15N dinitrogen pulse was provided and we could actually then look to see which of the groups of organisms could take up nitrogen and synthesize new biomass. And so what we see here in the nanosims image at the bottom, the warmer colors are representing 15N incorporation from that dinitrogen gas into biomass. And so we were able to show that indeed not only do they carry the genes for this but also are capable of doing this process directly in the sediment environment. So if we think about this in the context of how do we start to link function to the diversity that we see in nature I think understanding things at the level of strains is actually really important in terms of our overall prediction of ecosystem function. So for instance, if we just look at 16S ribosomal RNA genes all of this heterogeneity in capabilities would be kind of smushed under one umbrella with the assumption of a specific trait. But if it's only 10% of that the strains within that group that are capable of this function this could actually have significant implications in terms of both the identification of rates and assignment to specific groups and also the potential function going forward under different types of climatic changes. So I'm hopeful that the types of methodologies that we're continually develop in the field is going to help us not only resolve these ecotypes but also start to put them into context. So if we go to the next slide I just wanna leave with some parting thoughts that we can hopefully use for our discussion. So really this looking at these strains and ecotypes in many cases, especially for the uncultured majority represent hidden within population diversity but they have not only genomic content differences but also this can translate into really important physiological and ecological differentiation in the environment and we should be paying attention to this characterizing this diversity at the strain level when you don't have culture representatives has been a constant challenge and that's an opportunity for the field going forward in terms of how do we start to unpack that. There are really a number of unanswered questions in the regard and the role of micro diversity and how this plays out in terms of the stability and overall functioning of microbial communities. And then when we think about this going forward should we be considering strain level heterogeneity and incorporating this diversity of whether or not we wanna engineer and or model microbial communities again in the context of changing climate. So thank you. Thank you so much. So our final speaker for this session will be Jack Gilbert from UCSD. The third representing the California contingent. So we're keeping it West Coast on this side. It's very nice to be here. My name's Jack. I'm a microbial ecologist and I'm one of these people that's really diversely distributed myself into many, many different ecosystems. My lab works in soils and oceans, in air, in buildings, in humans, in animals and in space. So we try and spread ourselves as nearly as possible. Next slide please. One thing we were keen to develop back about 10 years ago was an understanding of some of the aspects that Jay and others have brought up regarding the distribution and the diversity of microbial life at scale. So in this one small study, we took a monthly sampling of one body of water for six years. And we read it out about 10,000 observations of microbial diversity per month. And then we took one sample here in December 2007, sequence 10 million reads. So if you order a magnitude more microbial observations, and we found that 99.96% of all of the taxa, all of the bacterial species were present in that one month distributed across all the years. So no matter what month you looked in, no matter where you looked in, all the diversity was there. It was just changing in proportions. Next slide please. And this led us to explore if we could track this diversity of microbial life across the planet. So working with the International Census Marine Microbes, which was led and developed by Julie Huber and Mitch Sogan, we explored whether we could find some of these bacteria we found in, in this case, the English Channel, most important body of water on earth, obviously. And we wanted to see if we could find these bugs in basaltic hydrothermal vents, in cold seeps, in estuaries, in the belithal zone, in mangroves. And see if these microbial organisms popped up and demonstrated that approximately 50% of all bacterial taxa in any marine ecosystem that we were able to observe on planet earth was found in the English Channel, which either makes the English Channel the most important body of water for microbial diversity on planet earth and therefore the most important to potentially preserve. I joke. Or it means that there's a, there's a strong distribution mechanism of microbial life across planet earth. Next slide please. And so this, the idea of everything is everywhere, but the environment selects, led us to launch in 2010, the Earth Microbiome Project, which was a fundamental opportunity to get everybody in the world together to see if we could catalog microbial diversity of the whole planet. So not an ego-driven exercise, but an exercise much like we see in physics, where we get hundreds and hundreds of people together to come behind one research idea. This allowed us to develop a lot of fun, cool new technologies. We really got 6NS RNA sequencing up to scale, got the cost down. We developed strategies for metagenomic sequencing, for analysis that really helped us as researchers to generate data at a scale that was appropriate for these kinds of questions. So again, more than 500 scientists, this is back in 2007, I produced this infographic, 2017, so sorry, it's about five years old. But there's now over 200 to, I think 300 peer-reviewed publications associated with this work. The diversity of microbial life has been catalogued in over 200,000 samples globally. Next slide please. And one of the key things that we observed was again that the world is a big microbial ball, right? A big distribution of microbial life everywhere. So here I've arrayed just 2000 samples in the EMP by how many taxa, in this case phyla, are bacteria, so very, very high level taxonomical observations, how many phyla were observed. And so over on the right, you have really diverse samples. Most of those are soils, sediments, places where you've got a lot of rich heterogeneity and niche space. And then over on the left, we have really hyperselective environments where you find one or two phyla. And the thing we found was that even in these hyperselective environments, all of the taxa we find there are also found in these very diverse environments. So we turn this microbial osmosis, right? It's a bit of a joke. But it means that where you get rich, biodiverse environments, they are shedding bacteria, shedding microbes into environments around the world. And this allows us to try and understand how we can preserve this diversity. So we launched the microceta initiative. Microceta is hard to understand, but it's the micro Rosetta initiative, like the Rosetta Stone. It's to allow us to be able to interpret and understand microbial diversity across the planet by reading off between different levels of omics data, metagenomics, metatranscriptomics, metabolomics, developing these technologies so they can be widely applied and distributed at scale. We in off the back of the microceta initiative, we also launched a new program, next slide please, called the microbiota vault, which is a vault for humanity to preserve microbial diversity. Predominantly in the immediate time, it's focused on collecting samples of human feces and vaginal samples, oral sample skin samples from people around the world to try and capture the human associated microbial diversity. But working with Joe Handelsman and others, myself interested in soil diversity, we're also collecting soils from around the planet so that we can capture the microbial diversity of soils present now and keep them locked away like the seed bank at Svalbard in a secure location that's available to all nations and all scientists is an under sovereign control to allow for the samples to be collected in perpetuity for all time. Next slide please. So to focus back on humans, we understand that microbial diversity is important but the human diversity is also important and for years when we look at poo, we've been just looking at its shape and saying something about our health. Now we're cataloging the diversity in these ways. So using the microbiota vault, we've started to catalog the diversity of the really, really diverse populations, tens of thousands of people around the world and look at how their microbial diversity changes and how it associates with their health. Next slide please. Then we refer to this as a microbiome wide association study. So this is my American gut sample. I'm apparently now American but I have embraced an understanding of how my diversity associates and compares to other people's diversity so that we can understand how things like microbial metabolism shape health, right? So how they release compounds that can be involved in immune regulation, how they can change how our drugs are consumed, how they can alter the chemicals that we release into our gut to affect our health. Next slide. So this is really predicated into really cool work that was done by one of my ex-graduate students, Sean Gibbons, that demonstrated that time is an incredibly important factor. This has been brought up by a couple of people here that we need to monitor things over time in order to understand them effectively. So this little graph in the middle really does show that the number of time points of a microbial community you observe really does influence your ability to identify a biomarker, right? A biomarker that could be reflective of how that ecosystem is changing. If it's a human body, then how the health of that person is changing. If it's an ocean sample, how the ecosystem is responding to climate change, the more time points we have, the better we are at capturing diversity. Next slide, please. And so why is time so important? Next slide, please. Because unlike things like in a human context, I know things like height and weight and age, which are very low dimensional, you know, the numbers are quite small, right? And they're very static. They don't change a lot very quickly. Or things like blood pressure and insulin, which change very rapidly, like hour to hour, but also very low dimensional. Or things like genetics. Our genetics are very high dimensional. We have lots of genes, lots of information in our genome, but it's pretty static. It changes very slowly over our lives. The microbiome is both high dimensional and dynamic. Next slide, please. So that high dimensionality, next slide, please. And that dynamics leads to our ability to capture microbial diversity requiring that temporal analysis, that frequent sampling over time to capture that changing ecosystem. Next slide, please. And so we've developed an automated microbial tracking device. I do, I did co-found this company. I do have a conflict of interest with it, but it's a very cool little device that I really wanted, so that when I went out into the Amazon jungle or into the deserts, or when we're sending samples, collection devices up to the moon, we can collect samples and process them and have them stored in a rigorous and automated fashion. So this is a robotic system built into a box that's battery operated. So we can take all samples from around the world and process them in exactly the same way. This removes all of our shipping, our preservative issues. It reduces the cost. It means that we don't need technicians in the labs anymore. I shouldn't tell them because they'll get upset. But we are literally trying to automate microbial activity analysis at a scale that will allow us as individuals to either analyze our microbiome on a regular basis or as researchers to go out into the field and collect samples in a rigorous and robust fashion regularly. Next slide, please. I think that's it, is it? If that's the only slide, then great. Thank you very much. Thank you so much for that contribution, Jack. So we're gonna move to the discussion portion where we're opening it up for question and answers. You can either populate your question in the chat box. If you're on Slido, those will be carried over. And those of you who are on Zoom and have the capability to raise your hand and unmute, we welcome you to ask questions in that fashion as well. So we're gonna open it up as I'm waiting to see if there are questions in the chat. I had a opening question myself. As I was listening across the four of your contributions, it's clear that there is an importance of looking at specific strains as Victoria offered. But also there's this media coordination across systems and the ecosystem level understanding. How do we prioritize that within our current systems, including our current systems of funding and the ways in which scientists are interacting? I'll interject. One of the key things I thought that was very cool that Victoria brought up is if we want to have translational impact, we're going to need to actually embrace micro-diversity. So for example, we're working with researchers such as Jean-Michel Arnais over at University of Wisconsin on trying to identify micro-variants of biological nitrogen fixers, diazotrophs, that can be used to promote the growth of crops, such as grasses in nitrogen poor or climate change damaged soils. And so we by screening literally tens of thousands of different strains of particular diazotrophic species, we're actually able to identify ones that have random mutations that allow them to produce more nitrogen and provide it to the plant, right? So we don't have to rely on genetic modification which limits the ability to distribute those products into other countries. So, you know, where micro-diversity comes really important is our ability to translate that science into something which could be applicable on a wide scale. Thank you. Okay, let's go to, I see Scott Edwards, your hand is up. Hi, thanks very much. That was really interesting to everyone. Sort of a general question, but one specifically also for Victoria was just the role of technology in sequencing technology, for example, in discovering new microbial diversity. And I was curious, Victoria, how you describe an experiment where you put these facts to sort cells into individual wells, then were you able to sequence those whole genomes from single cells then? Or how did you do that? Yeah, thanks for that question. In our system, as you see in the background here, we have these little glowing dots and each one of those represents a methane oxidizing consortia that consists of many hundreds of cells. So that was the unit that we were sorting out of the environment. So rather than just an individual cell, it was a collective, but each one of those represents usually just two different strains that are co-associated with each other. So it's a very low complexity system. So we were able to use an activity-based tracer. So we were sorting the active fraction of these communities that were coexisting together, put them into individual wells, and then we use an amplification type of technique that boosts the amount of DNA that you have and then you can actually do the sequencing. And so the technology is really, we've been doing this for about seven years, now mostly with the Joint Genome Institute and the technology is just getting better and better that we're able to get really high quality genomes of both partners together and then we're able to sort of pick out these strain level differences that were hidden to us. So if you do a diversity survey of just DNA recovered directly from that environment, you either get a pan genome where things like the nitrogenase gets incorporated in there, but you can't resolve the individual strain. So it's like a big bag of everything, but that's not how evolution and ecology works, right? It's really at the individual level. And so this really gave us new insight into really understanding what's going on potential cooperation and competitions that are occurring in these sediment communities. Thank you. I'm gonna go to Nathan, then I'll go to a question from Slido and then come back to the hands in the room. Great, thank you for this wonderful session. Thanks to all the speakers. And so, yeah, a number of things I think are super interesting here, maybe going along that same direction of, this high throughput monitoring. And you've all made so much progress in that and love even the new robotics that Jack's talking about where you might go to build some of those out. But what I'm really interested in is since so much the microbial world is only available to us by sequencing, kind of how much do you think emerging technologies allow us to get further along in functional space? Like one of the things I'm thinking about, there was this announcement from Metta, I'm sure you all saw it like last week that they've folded now 600,000 microbial proteins that whatever quality that can be done now with AlphaFold, there's these emerging connections between the environmental exposures of the microbiome and the triggering of autoimmune disease or rheumatoid arthritis, seems to be a pretty strong connection from a study male clinic and so forth. I'd just like to get your collective thoughts about kind of what you see as the frontier there, how feasible, what you think the next kind of big leap is for us to be able to interpret, these kind of genomic signals and not just from protein playing as metabolic maps or all kinds of different things that are possible, but just kind of love to get your collective thoughts about that. Yeah, so I mean, I have functions key, right? So akin to what Vicky's doing, we've been exploring ways of capturing bugs and then putting them through phenotype assays so that we can identify more clearly what some of these genes of unknown function and proteins of unknown domain proteins are actually doing in those contexts and then putting them into artificial communities of say 20, 30 different organisms under defined conditions to model those dynamics. So that's working with people like Carsten Zengler who's been trying to build those models for though you work with Nathan. So that kind of relationship of trying to model computationally how these things are interacting and then validate that with experimental output I think is absolutely key to this next phase. And I would say, as much as the ability to culture organisms out of the environment has been really slow going, there has been a lot of innovation and advancement on that front too. And you can almost think of this as like coming full circle. So now we can take the data in terms of gene prediction of function to help do a little bit more customized design of selection media to try to culture these organisms. And I think there's also a shift in thinking away from having a pure culture isolate which is ideal but maybe even having consortia of groups of organisms is still fine and relevant and allows us to study things at a much more detail level than we can do just from going into a soil ecosystem. So I think there's a lot of potential and progress that's being done on that front as well as well as the recognition and funding to support these novel cultivation efforts that I think should continue. So I'm going, are you done? Okay, I didn't know if you were gonna say something else anything. I'm gonna go to a question from Slido which I think is to any of the four panelists. It says in a practical way, what are the biggest challenges for scientists to understanding the connection between the environment, the meta-omic data and diversity? Well, don't make me say it again. For me, it's cataloging all the other variables. So our ability to actually measure things is really limited. Even you take something as simple as temperature actually measuring temperature or pH at the level of the microorganism. I mean, Vicki and Jennifer have done some really, really cool stuff in that space. Jay, some cool stuff in that space, but it's still tricky, right? So the measurement is still difficult for understanding what these microorganisms are experiencing and so how their evolution and their ecology is being shaped by the experience. Yeah, I'll just follow up on that. Also measuring their rates themselves, their processes that they're driving. So we are just so much further ahead on measuring the microbes themselves, their genetic material, even their proteins, things like that. Then we are at measuring simple things like the rate of nitrogen and carbon fluxes through the system, not even at a small scale but even at a plot level scale. It's really hard to do those measurements. It takes a lot of human power. We don't have very good automated systems that are cheaply available to stick out and measure these things. Jay or Victoria? Yeah, I would just build on what Jennifer was just saying, like for me, one of the big challenges is we're very good at taking chemical data sets that exist at different spatial scales than what the microbial community is experiencing but really trying to understand the flux of material through microbes when we do these correlative analyses. I'm not entirely convinced that we're actually looking at the right parameter space in terms of the chemistry and the overall transformations that are going on at rates and at scales that are really difficult to put our hands around. I mean, I think sometimes in soils, we might take a five gram sample and we're just integrating over such large volumes relative to the size of a cell. And that's important, I think, for the issues that Jack was saying, but relating to this question of dormancy in the way I think that was motivated initially was that we don't see responses sometimes to environmental conditions because there's a lot of meta-bond, like heterogeneity among individuals that if we just sequence all of the DNA in a sample, we're not capturing that or a certain signal is getting masked. So I think in the past 10 years, we've developed a lot of new techniques that allow us, for example, like Victoria is showing us how there's a lot of variation in activity among individuals and within molecules that we tend to look at. And I'll just add to that quantification. We're not still not very good at quantifying microbial abundances. Most of our data is proportional. We're getting there, we're developing new technologies predominantly in the health sciences, building on some of the stuff that was done preliminary in the marine sciences, but it's still tricky to get absolute abundances of the microbes, which is key to what Jennifer and Jay is saying regarding understanding their rates. If we don't really understand how they're growing, or what rate they're growing, then understanding how their metabolic activity could be shaping ecosystem properties is always gonna be limited. And that's a major disconnect to say climate science where we can absolutely quantify climate gases. If we can't quantify microbial activity, then we've got a misstep between those two capabilities. David Walts, did you still have a question? I think you were next. No? Okay, thank you. We'll go then to Janet Wesley. Thank you, I've learned a lot. Thank you. Just a microbial geneticist. So my question is, are there lessons to be learned from what you understand about the microbiome to apply to those of us who are interested in microbial consortia, for example, making products for functional things that we would like to do? You know, what can we learn from what you're doing? Yeah. Well, I could start maybe because the system that I study is really a metabolic cooperation of sorts, right? So we have an archaea and a sulfate-reducing bacterial organism that are capable collectively of doing this process of anaerobic oxidation of methane that neither partner could do on its own, right? And so they've become tremendously successful across the globe. It's a really important process that has evolved. And we see convergence in terms of the types of strategies that different genera of these methane oxidizing consortia have used in terms of how they're sharing energy between the two cell types. And so there is, I think, common principles that we can maybe think about. The fact that there's so much diversity that's sustained in these environments, I think also speaks to the idea of stability in communities undergoing change. I'm sure Jen can talk a lot more about that. But I think if we're thinking about synthesis, diversity is gonna be a key piece. And I think that's a general shift in thinking, right? It's not just putting something in E. coli and going. Like it's really starting to talk about smart communities. And what I was trying to get across with bringing up this concept of strains is that maybe we wanna even think about not just having one E. coli and one bacillus and one, but maybe we want to have some of the properties of phenotypes distributed across strains that coexist to each other. And that may even create a more stable or productive system depending on the context. I guess I was asking something more specific. Are there guidelines? Are there rules of the road? Are there things that we should be considering as we design these microbial consortia? Are there things we should avoid? Are there things we should embrace? I mean, we're kind of in a black box of how to go about this. And so are there lessons to be learned from what you've understood about microbial consortia in nature that we can use design principles that we can use to construct artificial ones? I love this question, but I'm afraid I have to... I think it reveals how little we understand even about ecology of larger organisms. I always remind my students how badly we are or how poorly we do at trying to restore even a plant community in Southern California when we put in a new freeway, we have a bare space and they struggle for decades to try to revegetate it. And we're talking about maybe five species they're trying to get to coexist and persist there. And so then you go to something like we wanna build a consortia to give in a pill to a patient. And it doesn't surprise me at all that we can't do that yet. And I think some of the things we've brought up about strain differences and things like that that's gonna be important, but it does reveal how few principles we have in ecology for community coexistence. I mean, we have lots of ideas, but when it comes down to practically speaking, we're not very good at it. That all said, I think there is a huge opportunity to work with people like yourselves that are trying to build these consortia for very simple systems. This is where we could potentially start to learn these principles, right? If we understand the systems very well that we're putting them in and can manipulate them, maybe we can come up with some of those principles that you're asking for, which would make sense. Yeah, and one of the things I would like to add to that is one of the things that we've really uncovered is the fact that inter-kingdom associations are really important. So working in mangrove swamps and restorations there, we've shown it's really important to have fungi and bacteria added into the system. And then also, if you miss out the phage or the viruses, you're missing an entire step of the ecological cycle. So being able to take into consideration that we don't just, as Jennifer says, don't just throw in a bunch of bacteria and hope that everything else is gonna work out fine, right? We have to try and reconstruct the ecological principles of cross-kingdom dynamics that are essential to ecosystem stability. And maybe following up on that question, Jack, there's a question that came in from the question box that was really wanting to look at the interaction between microbes in the pathogenesis sense. So from pathogens, microbes and humans. It says, could you comment on how microbiome composition affects pathogenesis for different pathogens? Yes, that's a massive question with about a hundred different examples, you know? We know very clearly that a diversity in microbial metabolic activity in the gut, for example, alters that local ecosystem to make it more difficult for pathogens to thrive and survive, right? So a very key example is in Salmonella or Clostridiorides difficile infections that can be inhibited by microbial metabolism of bile acids. So we know that that diversity plays a key role in altering pathogenesis. But then also the emergence of what we refer to as a pathobiont, say during somebody who's going to undergo surgery, we're going to pump them for the antibiotics, we're going to flush out their gut with peg, right? And then we're going to cut open their gut. And we're shocked when a pathogen suddenly appears and emerges out of that distraught. You know, it was like what Jennifer was saying about putting a highway through an area and then hoping that the plants will just grow back if they don't, right? So then the toughest bugs tend to thrive in that environment and some of those are the pathogens. So yeah, understanding how to reconstruct an ecosystem to make it inhibitory to pathogens, I think is going to be a key phase of the next stages of health. So there's one additional question, which I think is a great one to ask each of, oh, there's a question, I'll take yours first, Sudeb. Thanks, Bron, my question, first of all, I've learned so much. So thank you for the presentations and for this discussion. The science is really exciting. I've got a more mundane question, which is y'all are sort of sitting at intersections of a whole bunch of different disciplines and sciences. Who are the funding agencies that are providing the resources for y'all? And are there, in addition to the federal sources, are there private foundation sources? And then globally, who are the funders that are interested in this space? I'm just curious as to how that's happening. How are you finding the resources? When we started the National Microbiome Initiative under the Obama Administration, this is like five, six years ago now, it was about the funding rate for NIH and NSF combined was about 400 million a year for microbiome associated research. That's got considerably larger in the last five years. And then you take in DOD, you take in the UK Funding Agencies European Commission who just funded another $22 million microbiome center at Switzerland. There's huge resources in China with I think approximately last year there was almost $800 million spent on microbiome-based research in China alone. It's a good time to be a microbiome scientist. And the foundations, yeah, I mean, there's lots of them. The key thing I think is over-promising. So a lot of promise in the microbiome, it's really hard to realize some of that promise. So that's where most of the funding agencies, like when they dabble, they throw in a billion and then they pull back when they realize the results are still pretty academic. I'll just, oh, can I follow up on that? I was just gonna say, again, to get on my little soapbox about studying microbiomes across systems, I think one of the practical challenges we've been facing is that, yes, there's a lot of funding for microbiome research generally across in the US, across different agencies. There's even an interagency working group of program officers that talk to one another. But they also even recognize that getting funds to study, if you're studying humans to also study soils and to bring those people together is actually a huge challenge. And when we've been trying to work with them on because I, in part of what I've been working on on the service side is running a network of microbiome centers across the country. And just even organizing conference funding for that, I had to write proposals to the Navy, to the NIH, to NSF. And each one, I had to change the proposal because they wanted to see certain things highlighted versus not, even though all of us coming, we're there, coming to talk about how this research needs to span and interconnect. And there's just some barriers there to sort of recognizing the potential to do that. Often using the same tools and approaches, right? Exactly, yeah. So we know we can talk to one another. And yeah, and there are of course exceptions to this and particular calls and so forth, but I would say those are few and far between. I suspect that the nonprofit funding agencies could potentially be nimble enough to provide this kind of environment to allow this sort of cross ecosystem comparisons. But to date, it's really been focused and there's a lot of really impactful research that has come out of things like the Moore Foundation's interest in marine microbiomes, now they're doing symbiosis, the Simons Foundation. So there's been a lot of US-based foundations, but they each pick kind of a topic to go after. And so it's rare to see these kinds of things that Jen is talking about that I think are really important to sort of take things to the next level. Yeah, I didn't realize it was, it's all one thing, even if it's in different niches. So that cross-cutting funding must be, we gotta figure out how to make that happen. So thank you, I appreciate that. Thank you, Nancy. Yeah, thank you. Just harking back to Janet's question about creating consortia and figuring out what the principles might be, are there mechanisms for studying in some of these populations predation? I'm thinking about bedelavibrio and so on. I mean, are there ways you can measure how organisms are actually killing each other as well as, you know, we think about putting a population together by allowing it to grow and grow in the presence of each other, but can you measure predation? We have been doing this, not from the omics perspective, but from actually tracing the flow of things like carbon and nitrogen from an active cell into either a pathogenic microorganism or a phage or potentially a protist that's eating those organisms. And so using stable isotopes and doing these time course experiments gives us insight into the flow and the trophic structure of those communities. So that's how we've approached it in our environment, but these are not high throughput experiments. So you can learn about your particular ecosystem that's still a lot of challenge in figuring out whether or not these principles that you learn on the small scale sort of can be extrapolated up to the global. So there's major spatial scale steps that are sort of missing from, you know, sort of the global processes down to what's going on in your small environment that you have in the lab. Jen, I don't know if you have stuff to add to that, but yeah, I was gonna let you. I mean, I think there's been a lot of simultaneous advances in studying of viruses. So that's just one type of interaction between bacteria and archaea and organisms, you know, that could induce mortality. But I would say that it's sort of interesting that it's an interesting question. I think when we go out and we collect a bunch of sequences from the environment, I think there are many people that might just be a static representation of what's there, but I think many of us understand that there are a lot of interactions going on among those individuals, how to quantify that and make, you know, perhaps dynamical models that might accurately represent how a system's operating. I would say maybe other people have different opinions. I don't think the data that we're talking about today really allows us to get at that. Maybe with the exception of what Victoria's talking about with, you know, tracking isotopically labeled atoms into cells. But yeah, I would say that we're a little bit agnostic or at least unclear about how those interactions are taking place in nature. I mean, people use, sorry, sorry. I've just, just to build on that, from an omics perspective, at least for like phage host relationships now, that understanding things like CRISPR as sort of the sort of adaptive immune system of microbes, you know, they basically have potentially a historic record. And so if you can sequence the phage in a community and relate that, there may be ways now to start to make those kinds of connections. And so there's some exciting work that's coming out of that. And we've been exploring a particular fundamental mechanism of statistical temporal correlation called empirical dynamic modeling, which really only works with quantitative data. So in applying quantitative metagenomic approaches to ocean samples, we're now able to track nonlinear relationships between potential bacterial pathogens that you haven't been able to identify. Of course, then we have to go back in and use Victoria's approaches to actually validate that those are predatory relationships. But it's this first screening approach to say, hey, there's an organism in here that appears to be only spiking shortly after this other organism spikes. And it could be feeding off of it, but it looks very much like a nonlinear pathogenic relationship, sorry, predatory relationship in that system. Kind of like a Volterra, but in a nonlinear fashion. So thank you all. We are close to the end of the time for the panel. So I wanted to synthesize a couple of questions and give each of you a chance to respond to it in addition to thanking each of you again for participating in this conversation today. But there was a question about what each of you see as the kind of pressing scientific or technological challenges, but there was also a question about given the fact that some of you have been involved in the academies before, what advice or guidance you would have to the Board on Life Sciences and the academies in terms of supporting this work. So I just wanted to know in terms of thinking about what you see as the most technical and scientific challenges, what advice you would have for the Board on Life Sciences or the academies moving forward if each of you are willing to respond to that. And thanks again for your participation. Have you always go first? Go first, Jack. Yes, stop them. I have too many opinions. So yeah, so in previous panels and last panels, we've explored the opportunities and this kind of risks off of what Victoria and Jennifer were saying regarding bringing communities together. And the EMP was a grassroots exercise, right? Let's just see who's got samples in their freezers, who's got metadata and just sequence everything using the available technologies. But it did build a community and it did build a load of technologies, right? So if the National Academies can use their convening power to bring together communities around key fundamental concepts and again, use that convening power to make sure that the funding agencies come together to support that in Cross Institute, Cross Federal Organizational Initiatives, that would have a big role. And I think also bringing industry to the table, right? So in nearly everything we put in now, we have our basic science component and then we have related industries who are interested in the outcome of that and so making sure that we can use that convening power to bring together teams at a very large and international scale to tackle, say the UN problems, right? You know, the 15 climate related issues that UN has identified with the key. Victoria? Yeah, I'm gonna probably steal something from Jennifer here but she's been pushing this idea of principles of microbial community structure and function and this still is an outstanding question and one that we don't understand whether or not there is a universal or whether or not there's going to be subtle differences in how we think about structuring. So for instance, between aerobic and anaerobic communities, things that are related to our body that are lower diversity relative to what you'd find in a soil and a sediment. And so really trying to get our handle on this is gonna be instrumental in any sort of ability to go forward and engineer and really truly understand how to harness the power of microbial communities. And so for me, I think not only thinking about like experimentally how to start to unpack that but also thinking about how to work better with modelers and figuring out what the key variables that we need to be measuring either as experimentalists and trying to create a common language. I think would also be a step towards a brighter future in incorporating microorganisms and how we're going about our understanding of natural ecosystems and human bodies. Jennifer? Yeah, great. Thank you for those ideas, Jack and Ray. Those are, I love all of them. I think I'll hit back on one that came up already about measuring processes within communities and maybe bringing together the engineers and the ecologists. Like I always am dreaming about there. There must be an engineer out there that knows what I'm doing with literally duct tape and pantyhose. There's an engineer that can help me with this problem. So I would love to see those communities come together, ecologists and engineers here to get at some of these questions. Again, not so much for me on the omic. It's not the omics that is preventing our discoveries. It's some of these other measurements around them. Thanks. And we'll come full circle back to Jay where we started this panel discussion. Well, I'm working with Jennifer and a colloquium right now with the American Academy of Microbiology who's interested in this topic of climate change. And I think one of the things that will be really interesting, like Victoria was saying is for us to work with microbiologists and people who are generating a lot of large complex data sets to talk with people who are gonna allow us to move up from scales of microns to one kilometer by one kilometer grid scales. I think there's a bit of a disconnect and there's a really interesting and challenging problem there that we need to get a handle on if we wanna use micro information, most abundant and diverse organisms on the planet to understand how our planet's gonna be responding to global change drivers. And so that's not a trivial task, but it's an important one. And I think there's gonna be a lot of work that needs to be done in that realm. Thank you. So again, I just wanted to thank each of you again for a really amazing panel and a great conversation. And I will turn it back over to Kavita at this time. Thank you so much for joining our discussion today. Sorry. Thank you so much for joining our discussion today. We, this has set up the entire discussion so well and we're gonna transition with your indulgence to the next panel, which is focused on leveraging some of the cool and interesting functions that we heard in terms of what microbes can do to really building the buyer economy. And so I'm gonna turn it over to Sudit Parikh who is also one of our partners. Please go ahead. Great. Thank you, Kavita. What, yeah, what a great setup from the previous panel for this one. So the title of this one is Building the Bioeconomy, Harnessing Diversity and Unique Functions of Microbes. You see how it builds on the previous panel. The bioeconomy as envisioned builds on advances in the life sciences, engineering, computer science, data science, material science and every other field to create the processes and the products that address hopefully societal issues in addition to building the economy. Now, many of these efforts seek to replace petroleum-based products with bio-based processes and products and this leads to this concept of the circular bioeconomy. And this illustrates the systemic and continuous cycle of using biomass from biological resources for economic growth and development. Microbes with novel or unique functions feature prominently in this circular bioeconomy and as new microbes are discovered and studied, hopefully new opportunities arise for harnessing their capabilities to develop bio-based and bio-inspired products. And this concept is being advanced through the recent executive order on advancing biotechnology and biomanufacturing innovation for a sustainable, safe and secure American bioeconomy. There is not an acronym in that. During this discussion today, we're gonna have speakers who are gonna discuss the successes so far, the challenges and the opportunities lying ahead for harnessing knowledge about microbial function to design and develop these processes and products that feed into the bioeconomy. I know that there's a lot of excitement around the bioeconomy, so I'm eager to get into this discussion. And today we're gonna look at it from three important perspectives, the government, academia and industry. And so I'm gonna go through and have each of our first three speakers talk to it from those three perspectives and then have our four speakers sort of looking at it and responding to it. So I'm gonna start with, I'll go ahead and name all of our speakers for you and then I'll give a short bio as I go to them. Adam Gus is from the Oak Ridge National Laboratory. Michelle O'Malley is from the University of California, Santa Barbara. David Bresslauer is from Bolt Threads and Janet Westfilling is from the University of Georgia. And we're gonna start today, make sure I get my order right, we're gonna start with Adam today. So let me see if we can get his slides up and just a little bit more of an introduction. Adam is a senior R&D scientist at the Oak Ridge National Laboratory. His research focuses on the development of genetic toolboxes for non-modeled microorganisms and the application of those tools to engineer microbes. So I'll turn it over to Adam. Great, thank you, Sudip. If you can go to the next slide, please. Yeah, so when thinking about producing fuels and chemicals biologically, hopefully from renewable and sustainable feedstocks, it turns out that there's no natural organism that can grow on cheap feedstocks and make industrial fuels and chemicals at the kinds of titers rates and yields that are needed for industrial biotechnology. And so any organism that we want to produce fuels and chemicals is going to need to be engineered. And so the vast majority of metabolic engineering of microbes is done in commonly used model organisms like E. coli or Saccharomyces. And there's a really good reason for this, that there's a really great tool set for genetic manipulations. And there's just a wealth of foundational knowledge about the physiology of these organisms. Next slide. However, the phylogenetic and metabolic diversity of microbes is truly vast. We heard a bit about that in the first session thinking about microbial communities and the diverse functions that they do. And it turns out that E. coli and Saccharomyces represent only the tiniest fraction of this diversity. And so there's just so much more out there. Next slide, please. And so the question is, can we leverage all of this diversity that's out there beyond what is available in E. coli or Saccharomyces? And there are good reasons to want to look at non-model microbes and it has to do largely with complex phenotypes for bioengineering. So there are a lot of traits that are just too challenging to engineer into E. coli, for instance. So if you were to engineer E. coli to grow at pH one, where you may want to bioprocess to occur, that would take so many changes that the resulting organism would just fundamentally not be anything like E. coli anymore. And so perhaps it's a much better approach to start with an organism that actually grows at that pH. And there are a lot of complex phenotypes that are really challenging to engineer. And lignocellulose utilization is one of them. People have been trying to engineer E. coli and Saccharomyces to grow on crystalline cellulose for multiple decades. And it's essentially been completely unsuccessful. And other challenging phenotypes like utilization of gaseous substrates, whether that's CO2 for producing carbon negative fuels and chemicals, syngas and methane and so forth. Organisms have evolved over millions of years to do this really well. And so perhaps we want to leverage of what nature is already providing for us. And so why don't we use these non-model microbes as often as one might guess we should? Well, I think it really comes down to the fact that the genetic tools and the basic physiological knowledge about these organisms is often limited. And it's not easy to translate knowledge from E. coli or Saccharomyces into some of these other organisms. But ultimately one of the biggest barriers is just the development of genetic tools to enable rational engineering. Next slide please. So one thing I really want to emphasize is that things have really changed over the last maybe five or 10 years. And it's becoming fairly straightforward to develop genetic tools very rapidly for new organisms. So we can do things like methyl analysis to understand the host defense systems where an organism protects itself against foreign DNA. And then we can use that information to evade typically restriction systems as the most dominant form of host defense system against foreign DNA. And that allows us to get DNA into the cell without that DNA being degraded. And so that enables transformation of new organisms. And then if you combine that with large collections of genetic parts, origins of replication, select whole markers, reporter genes, promoters and terminators and people are building these libraries that work in diverse groups of organisms. It means that we can very rapidly start developing robust genetic tool sets for different groups of organisms. And then we can start looking at more advanced tools like CRISPR and site-specific recombination and transposition and so forth. And so these tools really allow us to start doing rational engineering of diverse organisms. And so I was asked to talk largely about production of fuels but also industrial chemicals. And so if we want to engineer microbes to make fuels and chemicals, they need to be produced from really cheap feedstocks. We can't afford expensive feedstocks if we want to produce, for instance, fuels where we're ultimately gonna set it on fire. We're gonna burn these fuels. And so ultimately the cost needs to be about 25 cents a pound for the entire process getting a feedstock, getting it to a biorefinery, doing the bioconversion, doing the separations and then transporting it to the infrastructure. All of that needs to be about 25 cents a pound or about $2 per gallon for a fuel. And so we really need cheap substrates, things like cellulosic sugars, lignin, plastics as a potential feedstock, waste gases and so forth. And then we need to take all these feedstocks and put them through an engineered organism to produce the chemicals that we need, solvents, polymer-building blocks and fuels. Next slide, please. So on the feedstock side, there are a lot of needs. We really need access to cheap feedstocks which means that we need better enzymes for breaking down those feedstocks because the feedstocks are pretty much always polymers. And so typically that deconstruction is gonna happen outside the cell and so we need really robust mechanisms for secreting a lot of protein in order to do this biocatalysis. And then the other thing is we need to not be afraid of interfacing with chemical catalysis as mechanisms for breaking down these polymers into soluble components that microbes can eat. Next, please. So thinking about engineering the organism, we really need to be able to control metabolism and this can happen at so many different levels. There's gene content, but also gene expression, protein abundance, enzyme activity, allosteric regulation, localization. Each of these are potential control points for metabolism. And we currently utilize several of these on metabolic controls like gene content and gene expression. Sometimes enzymatic activity through rational engineering and evolution, but things like temporal controlled protein abundance and engineering of allostery are exceptionally rare and points of future research. Next, please. And so the needs on this front are things like the need to better understand flux and how to control it because we generally have a core understanding of the genotype phenotype relationship for so many genes. And then as we think about building more robust organisms, I think we really need to do a better job of mimicking nature in the types of ways that metabolism is controlled while still engineering the organism to do the types of things that we want. Next, please. And then on the downstream side, biology really needs to be engineered thinking about what the next step in separations is going to look like. What pH do we want this to happen at? What temperature do we want this to happen at? How can we enable the costly separations using specialized biology? And so what organisms might enable that type of novel processing? Next slide, please. Okay, so kind of thinking about the future, the challenges and so forth, I think one of the biggest challenges of using non-model organisms is just a fear of the unknown. So many people are trained to work on organisms like E. Coli and Saccharomyces. And so transitioning to organisms that grow at high temperature or need special culturing conditions or only grow anerobically, these can be very intimidating for people and that can be a real barrier to getting people involved in the field and then actually getting technologies deployed. And so if we think about how to potentially solve that, things like internships and training programs, especially for people coming out of college and going out into the workforce. And then we also just don't have all of these tools developed even though the foundation is there. And so maybe we need something like a national user facility for genetic tool development and deployment where people can come to a centralized organization and build out the tools or have someone else with expertise, build out the tools that are needed for very promising organisms. I think we need more sharing of information and of tools and greater investment in public resources, especially for pre-competitive work that can be broadly enabling for numerous industries. I think we need more work at the interface of biology and chemistry. So there's certainly some work going on in the space but biology and chemistry can be really cooperative and can be very synergistic. And so finding processes where chemistry gets the process halfway done and then biology finishes it or the other way around biology gets things halfway done and then chemistry gets it to the final product are all things that we need to encourage even more of in research. As a microbial geneticist, I know that a lot of geneticists are intimidated by some of the advanced analytics that are necessary for things like fluxomics or other things like that. And so just having more accessible analytics is going to be very enabling for the field as well. And also a lot of times the analytical equipment is very expensive if you're thinking about like LCMS and other things like that. And then we really need a large emphasis on anaerobic organisms because there's an old saying that oxygen is really hard to keep out at small scale like at the bench scale but it's really hard to get into solution at large scale. And so it's really expensive to pump oxygen into a bioreactor at like the 100,000 liter scale. And so anaerobes can be very attractive from a bioprocessing perspective and the field that works on anaerobes is somewhat limited. And then also things like products that can be growth coupled so that evolution works for you rather than against you. So these are some of my perspectives and I'd be happy to answer any questions. Great, Adam, thank you so much for the presentation. There's lots of great things to come back to on the challenges for the future. I'm gonna turn now to our second speaker, Michelle O'Malley. And while we get her slides up, I'll give you just a bit of an introduction. Michelle is a professor in the Department of Chemical Engineering at the University of California, Santa Barbara and the Associate Director of UCSB's Bioengineering Program. So Michelle, turn it over to you. Great, thanks so much. And it's very great going after Adam because it set the stock up quite perfectly. Thank you, Adam. We can go to the next slide. I'm gonna start with our favorite emoji as a motivation for looking at anaerobes, which are the organisms that thrive without oxygen that you can think of for a number of biotech purposes. I show this emoji as kind of a starting point for the fact that poop is actually a biotech resource. So a lot of microorganisms contained therein that if we learn what those microorganisms are and some of their metabolic capabilities, it's actually a route to solving a lot of issues that we face not only in sustainability as I'll describe in a minute, but of course also in human health, animal health. Poop is a reporter of everything going on in the digestive tract, for example. So we can go to the next slide. And what I mean by that is that the gut microbiome, for example, is a family tree of very diverse microorganisms that of course work as a community where just like our own communities and societies, there's members that specialize in different functions and there's members that benefit from other members. And this beautiful diversity is actually shown here from a paper in nature of biotechnology that was published several years ago, showing the diversity of the cow rumen. And why that is important is because cows and other large herbivores have actually evolved to thrive on eating crude, non-food parts of plants and crude biomass, lignocellulose. A lot of these really ruddy polymers that we as a society would like to use as feedstocks to make value-added chemicals. And this beautiful diversity is really just waiting to be tapped into from a biotech perspective. We can go to the next slide. And what I mean by that is that I'm inspired by microbial communities such as the anaerobic communities that we see in herbivore poop because those communities are dividing and conquering very difficult tasks. So next. And just like our own societies and microbes have their friends that they benefit from, their enemies that they compete with and the frenemies, you can take that as you will, but microbes can sometimes change the way that they interact with each other and their metabolic capabilities based on who they see. And all of the social dynamics that we see in our own world can be mapped into microbial communities. Next. From our perspective, a lot of how my lab at UCSB has approached this issue is to just use metagenomic tools to understand who is there in these types of communities and then addressing the much deeper question of given who is there, what are those microbial communities and the specific members contributing and what are they doing? Some of the things that might be interesting to know are unique metabolic capabilities, specifically certain parts of the plant polymer that they are breaking off and eating and how that funnels into the products that they are then making. In addition to the enzymes or the bio catalysis methods that they're using to achieve those goals. Next. What I would argue is that this is kind of a grand challenge is how can we learn from microbial communities such as those that we can easily enrich from herbivore poop to design microbial communities such as in a scaled up bioreactor that work for us. This is of course really hard because we don't have the technology yet to really put microbial communities in a large scale bioreactor and know what they're going to do with any reasonable predictive power. So I would argue that this is pretty hard and we're not there yet. But next, I would also argue that within these interesting anaerobic microbial communities there are of course parts that we can borrow from microbes that can advance biotechnology needs. And those parts may be the microbes themselves. So developing them as chassis organisms and using some of the tools that Adam has just nicely laid out in terms of developing genetic interventions to push them harder and do things for us. They may also be borrowing the enzymes, the secretion pathways, the metabolic capabilities and importing them over into well understood, more industrially friendly microorganisms. Next. And so just to kind of set the stage for some of the things that we work on just as kind of a jumping off point, we work on anaerobic communities because they're really, really good at degrading things in nature. In particular, if you peer into the digestive tract of a ruminant like a cow or a goat or sheep, what you're gonna find there is a diverse population of largely bacteria that are working really hard to break down ingested plant matter that our own human digestive tracts cannot break down but cows and other herbivores can. And of course there's a lot going on that I don't have time to go through but I do wanna underscore this beautiful microbial diversity that allows cows to eat. Lignocellulose and grow big and strong, right? And also pointing out that in these communities are not just anaerobic bacteria, there's actually also archaea that are associated with producing methane, protists that very few groups understand or can cultivate and then anaerobic fungi which are there kind of as minority players but are actually key enzyme producers in a very competitive system. So they are producing a lion's share of carbohydrate active enzymes that enables these communities to thrive. Next. And so I'm an engineer but part of what my lab is known for is actually going out into the world and isolating these so-called rare members of the anaerobic microbiomes. And I'm skipping a lot of steps here but essentially how we do it is we take a little donation if you will from zoo animals or farm animals and we grind those up and then we anaerobically separate out and select for microbes that we would like. In this case, microbes that degrade cellulose, we grow them in liquid culture anaerobically. We have a bunch of redox indicators there to assess performance but essentially we're growing them on something that you could think of as wood chips. And below at the bottom of the slide you can see four different strains of the anaerobic fungi that our lab has isolated and named. It's really not important to know what all these names are they're the Latin kind of mishmash but more important is just seeing what these fungi look like. They're actually clamping on to the plant material and literally punching through with these rhizoids in three out of these four cases. And that's really thought to be the place where these enzymes that are degrading so much of this waste material are actually being spat out from. Next. And so these microbes come from a pretty humble place but just by applying simple sequencing tools that we did to these organisms, apologies my cat has just decided to join the presentation. We can find that actually just counting enzymes in these fungi, they have the largest most comprehensive array of biomass degrading enzymes of any sequenced fungi and organisms on earth. And this is a record that still stands much to my surprise. So it just goes to show you what we can learn by peering into these systems that on the surface don't seem like they're so important but if we can develop tools to isolate them we can really learn a lot. Next. And so this is my last slide just to leave you with some perspectives based on and opportunities based on my own experience. To echo a lot of what Adam has said I think that going back to basics I'm a chemical engineer and so I appreciate the problems of scale up and also that most of our biomanufacturing capabilities are built on the action of model microbes. It's really time I think to go back and revisit investment in scalable bioreactors for anaerobes, for filamentous microbes to handle some of these issues that classically the industry is afraid of because the benefit can be huge. Obviously there's a balance there. We don't wanna develop every single chassis organism into a scaled up process because that can't be done but I do think there's a balance that we have to find. For anaerobes the argument is obvious. They have very powerful degradation systems. They also have unique transporters, natural product pathways, a whole bunch of things I didn't mention and they can save a lot of money in a bioprocess because you don't need to mix them and you don't need to add oxygen. And what I'll also add is that when you pour it a lot of these enzymes in this case from anaerobic fungi over other systems they don't often get expressed very well. Some do, some don't. And there are a lot of biofoundries that exist in the United States and beyond that are meant to kind of optimize genes and conon optimization and whatnot to try and kind of get around this issue. But in my view, they're really not set up to tackle non-model hosts. And so I really like this idea of trying to incorporate unusual hosts into a biofoundry type concept to really pinpoint what the bottlenecks are and bio manufacturing and to get around them. I would also argue that unusual microbes like anaerobes they're weird, but they do fill important gaps in our understanding of how biology works that I would argue can funnel into better health outcomes and our ability to produce sustainable chemicals and bio-based energy. What I'll also emphasize here is that we have a lot of there's still a lot we don't know even if we have genetic tools available which I fully argue should happen. Most proteins are still uncharacterized and require basic biochemistry and really have to develop assays to figure out what a protein does. And these fungi up to 75% of the protein encoding genes have no known function. That's huge. And it's really hard to go and unmask the function of all of those genes. And we just really lack high throughput tools to do that. And I would also emphasize that we need a better understanding of how proteins get made and spat out, if you will, is secreted in these systems. Some unusual microbes such as anaerobes actually have very powerful secretion systems so much better than E. coli and yeast. And if we could understand that, that would be huge because we can make more product and it can go out into the media for collection. And also my final note, I, you know, this might be a little controversial but I'm an engineer, but I do a lot of bioprospecting. I think there's always a better chassis organism to start from, not that all chassis need to be scaled up but I do think there's a lot hiding in nature that engineers and bioprocess engineers can learn from. So with that, I'm happy to participate in the discussion later on. Great. Thank you, Michelle. And thanks for those opportunities as well, more fodder for our discussion. I'll turn now to our third speaker who is David Bresslauer. And while the slides get put up, David leads technology innovation at Bolt where he's creating and incubating biomaterials for improved consumer products. David, I'll turn to you. Yeah, thank you so much for having me. And I think this ties in well to the previous two presentations, particularly Michelle's in terms of what is the distinction between what are we trying to scale, what is scaled and what are we trying to research? Oh wow, the font's got way out of whack here. But so apologies for that aesthetic slip up but I will give you some context as to where it's coming from. You know, I was asked to come here and speak from an industry perspective. I obviously can't speak for the industry as a whole but Bolt Threads is sort of a, I'll call it gen three, depending on where you're starting to count, biotech company or industrial biotech company coming off the heels of the amiruses alongside the ginkgos of the world. And now there's already a whole new generation of small startups to who are gonna grow, build and grow the bio economy. And I advise several of them and I've seen a lot of trends in terms of how basic research has translated to commercialization and scale up. And the challenges you face in this industry starting to start a company and where the break points come from working with the public sector and support from the public sector to then needing more. So next slide please. So, oh, those are my marketing team would kill me. Sorry about that. But to give you a brief overview, Bolt Threads makes three materials are two breakout materials. The ones that are really popular right now are Milo which is a leather replacement made from mycelium. So that's growing basically mushroom roots to make leather and a B-silk protein which is a recombinant protein that's a drop in replacement to silicone elastomers. Now the motivation for both of these is more sustainable solutions for the world. The fashion industry is desperately but sort of quietly trying to get away from animals and cows largely because it started as a vegan issue and animal rights issue. And now for them has become a greenhouse gas emission issue and no alternative materials meet the quality of leather. But remember leather is skin. There's a lot of structures that can make the structures that make skin. And so that's all I'll get back to that. Similarly, silicone elastomers are now becoming a huge problem because they're not biodegradable. They bio-accumulate but they make for great personal care products. They're in everything you use on your face and body. And there's a EU mandate to get rid of them all and there are essentially no alternatives. We look to nature and found a recombinant protein that could make the same microstructure. So that's what I'm gonna continue to emphasize from Bolt's perspective as we look at materials and the material science of the bio-economy but a microstructure to replace silicone elastomers. And the previous generations of because it's what made sense at the time really focused on drop-in chemicals, drop-in chemistries and still plenty of those companies around but to replace petroleum based ingredients with a bio-based version. So bio-based nylon, but it's still nylon. It's still not necessarily the biodegradable component. And there's greater demand for a fully integrated circular solution. So we look to how can we mimic the value proposition, the structure, why is leather so great? Why does people so love it so much with a different bio-material? So next slide please. So I put this here to emphasize where Bolt started from because public universities, I was UC San Diego undergrad in bioengineering publicly funded to UCSF PhDs University of Washington UCSF. We are funded, we, you know, it's a feeder program, NSF, SBIR, DOD grant, and the NSF specifically told us we want to make jobs. So I'm proud to say we've made over a hundred jobs, probably employed over 200 people over our lifetime 300 million in venture capital. And what I think you're seeing in the bio-economy now, oh, my face is fuzzy. I'm gonna eliminate the blur. What I think you're seeing in the bio-economy now that's beneficial is particularly with companies like Ginkgo and more contract research organizations, both commercial and public, is it's not gonna take $300 million. It still might take $150 million, but I think the cost is really coming down. And this should be more of an example of where we were in the development of the bio-economy rather than an example of what it's gonna take for everyone. And so if you go to the next slide, just again, because it's pretty, but just to give you an example, this is what it means. So it's like products on the market, consumer materials. These are leather replacements in products, silicone elastomer replacements in products for the industries that are trying to move towards this circular economy. And so next slide, please. Where Boltz really thrives. And again, this is what I'm trying to emphasize here is where we are in this development of the bio-economy is looking at the product, not just the technology. You see a lot of historical developments around industrial biotechnology as we develop the technology. We have a hammer, where can we find a nail? Now as building on all the basic research and the building blocks that have gotten us here, standing on the shoulders of giants, we are now able to say, we have a better idea of what a product. All these startups, what a product needs to be, a lot of the biology is, it's not a solve problem, but it's just much less risky than it used to be. So we can actually identify commercial opportunity and then assess the technical risk rather than just developing a technology and saying, we're gonna hope and pray we find a solution. And next slide. To drive that home on Boltz and again, this is an SEM of Mycelium Network on the left and an SEM of our recombinant protein on the right. You can look to nature and find structures, not just molecules. Again, there's plenty, there's tons of value in finding molecules and co-products and organisms that do the whole thing. In fact, Milo is all the biomass, but we think there's also a particular value in looking at what are the materials that nature makes and how can we make more scalable, more sustainable versions of the materials we use every day. Like cotton's got its own challenges, we've domesticated as much as we can and we're still working on it, but it's a great product. Leather, well, now we're seeing a lot of challenges with leather, but nature and microbes in nature make alternatives that can exhibit similar properties. So next thought, this is my sort of laundry list of food for thought. Thinking also about the material science of microbes, not just their molecular products. I'm also biased in that because that's where my company's success is from. Something to think about here is, in terms of accelerating the bio-economy, we don't have enough wins yet. Ginko went public, impossible went public. Amaris is public and is doing well now, but for the initial investors, I don't know that it panned out particularly well and investors are looking for some proof that there's a there there to keep feeding the beast. There's a lot of momentum in the food space because impossible and beyond what public. Now it's diminishing as beyond stock prices tanking, but that really pushed a lot of money into new foods, new food engineering, alternative materials for food. But they need to see companies that have product in the marketing scale. What the companies of the generations of Amaris did that was so beneficial is they essentially de-risked fermentation. Fermentation went from a every investor was like, I don't believe you can scale this fermentation to, I get fermentation, can you solve the downstream? Now it's all the focus on the risk is on the downstream. Some other things, just food for thought here in terms of how national academies continue to support the industry is the visa process for good talent can be overwhelmingly difficult, particularly for small early stage startups with limited capital with government grant money. You have a hundred thousand dollars. There's only so much you can spend on a lawyer. And if you have to move quickly, relying on the lottery gamble, lack of available and good CMOs. There are some great ones. We have the Department of Energy, ABTDU, the Michigan Biosciences, both uses those extensively. We spend a lot of money, great personnel, great public institutions, but there's a limited number of them and they're very booked. And I know BioMaid is working on this. The industry would also benefit from incentives for local manufacturing. I don't know a single company who wouldn't prefer to just do their fermentation next door or even in the next state, but it's like the most honorable way to be booted out of your CMO is because they're making COVID vaccines. But now everybody's running around the world going, who has space and who can scale? And the downside for the US is then all the expertise from all the associates, the manufacturing techs, the scientists, the engineers developed elsewhere. And so that cycle feeds itself elsewhere. To just jump ahead, I'll say part of my point on this next generation is we're getting much better as a startup industry on thinking about what is the product, not just what is the technology and how do I try to find a market for it or what is the product market fit? I do think there's opportunities for students to be better trained as entrepreneurs and to think really deeply about are they trying to sell their technology? Are they trying to jam their technology into a product? That doesn't mean any of those things are bad, but just understand it more in depth. What does stack risk mean? It's got to develop the technology, got to scale the technology, got to develop the product, got to commercialize the product. That's all multiplicative risk and that's how investors look at it. And I would say, and this goes to the previous two speakers, this moment for companies like Bolt that have a product in the market that is currently operating with a process that's scaled. I get a lot of inbound of people saying, I've developed a new host, will you try it? I don't, unfortunately, I don't have the bandwidth. I do believe in five years, 10 years, we need, if we want the bio economy to be competitive, we need to get cost down, we need anaerobes, we need better, you know, nth generation cellulosic consumption, all those things are necessary, necessary to research, but there is an inertia for people who have an existing process. It's like, maybe a better tool for my existing host, but I can't switch hosts, particularly to a host that hasn't been demonstrated at 3000 plus leaders, to Michelle's point, you know, chemical engineers, bio prospecting, not every chassis needs to be scaled. There's opportunity for new chassis, but really thinking about over what time scale are we trying to solve a problem? A new host, if we want it to actually, if we think it's gonna solve the problem, we have to work on the scale aspect too, not just develop it in the lab and then try to pitch it as a product. And that's where I think some critical thinking on what the solution space is for those different avenues of research would be beneficial. And that is all I have for you and I'm open to any questions. Great, thank you, David. Great food for thought. Looking forward to this discussion. I'm gonna turn now to our cleanup batter, Janet Westfilling. Dr. Jan Westfilling is a professor of genetics in the Franklin College of Arts and Sciences at the University of Georgia. And if the information I have here is correct, you go by Jan. All right, Jan, go ahead. Thank you. So thank you. And it's a pleasure and an honor to be here. And my role here is, as he said, to do a little bit of mop up about where we've been and where we're going. So again, I'm going to focus on harnessing microbial diversity. Next slide, please. So I wanna focus on where we've been because I think a perspective of where, what we've done in this area is important. And then my last slide will focus on where we need to be including persistent and outstanding needs. Next slide, please. So where have we been? So for fuels, for the last 10 to 15 years, work is primarily focused on molecules that provide drop-ins for existing fuels, okay? But the cost of these compounds and the energy we derive from them is not likely going to be justified by their production as fuel molecules. And some examples are terpenes and hydrocarbons. And for hydrocarbons, it's been an even longer investment than 10 to 15 years. Ethanol is a wonderful product. It's a drop in fuel. The world is flush with ethanol, but it's now becoming a substrate for larger chain molecules. If we're gonna get to jet fuel, we're gonna have to either come up with longer chain alcohols or we're gonna have to condense ethanol. That's an incredibly expensive catalysis problem. It involves condensing two carbon units to C14. And at the moment, that's prohibitively expensive. So we really have to look forward to other kinds of fuels and chemicals. Major improvements in ethanol conversion have enabled in advances in chemical catalysis. And I can say that chemical catalysis is now becoming rate limiting and an urgent need in this field to understand chemical catalysis. As Adam said, the combination of biology and chemistry is very powerful and we have some really wonderful biological substrates, but we need to convert them to things that are useful. Greg Beckham at NREL, who is led, basically led in this area, talks about this as the slope of enlightenment. We've gotten some of the basics down and now we can focus on what we need and how we need it, how we need to get there, what will work, what won't work and what we need to invest in in the short-term and long-term. Next slide, please. So for chemicals, the list is so long it would take more time than I have. So I draw your attention to a review article in Nature Catalysis by Sonya Lee. And this is a diagram from the supplemental material of that paper. It's a comprehensive overview of what we've done and how we've gotten there with chemicals from biomaterials, from biosubstrates. And I really draw your attention to this. Even if I could, if I had the expertise to go through this, there's just no time, but I invite you to look at this review article because it's detailed in every way you might imagine. Next slide, please. So for metabolic engineering, while bioinformatic analysis and modeling have been somewhat useful and sometimes it's even predictive, which I would argue is the main purpose of biomodeling and bioanalysis or these modeling exercises, it's now time I think to inject a bit of reality as we look forward. Models in their best form can be predictive of what we need to do metabolically to engineer an organism to do more. But largely their theoretical diagrams generated from these databases are helpful, but they're not evidence that the pathway exists or that it functions. And so it's time for experimental evidence to support these pathways and the analysis of these pathways. And the outcomes of mutations in these pathways are often surprising and they're almost always enlightening, okay? But it's time to get to the experimental part of understanding how physiology in microbes works. The use of systems biology to make non-intuitive predictions for strain improvements is still a major problem, okay? How do we translate those models into something that's useful and that's experimental validation? Next slide, please. So what are our persistent and outstanding needs in this area? Okay, I would argue that E. coli simply does not have the metabolic capacity to do much of what we need, okay? And there are many examples of attempts to engineer pathways into E. coli and they just don't work. And Adam, I think, gave some fantastic examples of what you can't engineer. You can't engineer growth at low pH. You can't engineer in biomass conversion or degradation growth at low pH. And more importantly, and I think very important, you can engineer in a pathway, but you can't make it function at high flux, high, high titers. And that's really the right limiting thing to an industrial process where you have to take a cheap substrate and you have to convert it efficiently and economically into a product of interest, okay? I would argue that the use of non-model microbes isn't just desirable, it's essential. And from what, I understand what David is saying that when you're locked into a process, you can't change that process. But if you're looking at expanding that process or you're starting from scratch with a new process, it's important to look at non-model microbes and what the organism can do for you so that you don't have to do yourself. And that includes economics and downstream processing. High throughput methods for DNA transformation and phenotyping are badly needed. And this really echoes some of the things we heard about microbial diversity and microbiome analysis. Scale-up is also critical. It's virtually impossible to know what is gonna be needed at large scale when you're looking at even a 10 liter bioreactor, never mind a shake flask. Most academic research goes on in small bottles. And I'm just saying I wanna make a plug for anaerobes as well. I'm on microbial geneticists to grew up on streptomyces, E. coli, and bacillus. And I started working on anaerobes about 10 years ago. And if I can do it, anybody can do it. Anaerobic biology is working with anaerobes is not as difficult as some people would have you believe. And so I think that we've heard from many speakers today that anaerobes have a lot of advantages eliminating as, and I think as Adam said so well, eliminating oxygen from a shake flask is hard. Putting oxygen into 100,000 liter from menor is difficult. And so when you get to high, to scaling up these processes, the fact that you can do it anaerobically is a real plus. I'd also like to say that one of the rate limiting factors in the translation of microbiology to industry is workforce development. And this has been touched on a couple of times. We don't need more PhDs. God knows where we would be in biomanufacturing if academic PhDs ran for mentors. But we need real people who know how to run real bioreactors. And that isn't about more PhDs, it's about master's degree students. People who learn these skills in technical colleges or in bioprocessing programs in universities. And I can tell you this, if the current salaries for these jobs rival what I make. And so there is a need for this. There is a way to make these people employed. There are high salaries, good jobs out there to do it. And we just don't have the people trained to do that. Okay. One other issue here that has been touched on in the past is the translation of basic science into an industrial process. It's commonly known as the Valley of Death. You have a great idea that you've developed in an academic lab and how do you convince a company to invest in that and do it. And I think one mechanism that I would put on the table for discussion is an increase in SPIR grants. And this is something the academy can perhaps help with. An increase in SPIR grants that bridge academic institutions and academic investigators with industry. This is a metric driven mechanism that would allow for the bridging of this gap so that it would de-risk the investment of a company and maybe provide some resources to move these things forward. So that's my attempt to summarize and tell you where we've been and where we might be going and I would be happy to participate in the discussion that follows. Thank you. Great. Thank you, Jan. And thank you all for a terrific set of talks. So now we'll open it up to questions from the audience and I'm happy to ask the first but please raise your hands as you're ready as you're ready to engage with this great panel. So the first question I'll ask is really getting straight to that workforce question that I heard across several of your talks. Tell me what's being done so far? So are there reach outs, David, from your company to the community colleges in your area? Are there, you know, in academia is there a move towards the same way the semiconductor industry is trying to build relationships with universities to grow the people who are going to work in fabs? Is that ongoing? Is there, has it not gotten started? Where are we? No, it's a great question and I don't know how this exists more broadly across the country, but at least in the Bay Area there is a Cal State University that does a manufacturing associate feeder system that we've had spotty luck but when the good candidates are phenomenal. I mean, we find that there seems to be a correlation to if you worked at Starbucks, you will be great at managing a wet lab. And I think the only challenge for us then becomes as our manufacturing scales. So we were using these feeder programs when we were doing 100 to 3000 liter but as we needed to go to bigger tanks we moved all of that stuff to where the manufacturing is to where those manufacturing facilities are. And so we're no longer leveraging those burdens. That goes to the point about local manufacturing and then talent. Like we kind of broke the cycle by necessity which would have otherwise been if we were doing all that manufacturing local just growing people out of the feeder program and continuing to employ more people but we have had success in the local one. May I address that? So the University of Georgia has one of the few biomanufacturing training facilities in the country. There are fewer than 10 nationwide that actually do hands-on training for biomanufacturing from small scale to large scale. We have an active outreach program with underrepresented minority campuses, small schools, technical colleges and Biomade has really become a central focus for expanding this nationwide. Okay and I think that they have a great plan in place and they're going to have a call for grants in this area as I understand it in the near future to increase biomanufacturing training. We have a master's degree program but it's hands-on. It's not about, you know, it's hands-on. It's how do you, which button do you push to make the fermenter go? Okay and that's what's really I think needed in this area. If I could add something really quick too is that, you know, being a chemical engineer, I think it's super important. Everybody in chemical engineering is like we need to teach more bio. I would go for them that and say we should teach bio through biomanufacturing. It is the most obvious thing and I've sculpted a course at UCSB around only biomanufacturing that all of the sophomores take and then it really just opens up so much. So I wish we could do that for more of our engineers. Can I just tell you that MIT requires biology of all its undergraduates? Speaking of MIT and Boston, Scott, I'll turn to you for the question. Thanks, Sudeep. Great talks, really very interesting and I'm curious about a comment that I think Adam made at the very beginning about sharing information. And so I'm curious how easy is it to share information, especially internationally? You know, all we hear about is how it's security breaches and not wanting to give information to our fellow scientists in other countries. And so I'm just curious how information sharing is either hampering or facilitating these sorts of innovations. I can start at least. So I think there are a lot of aspects to it. I think the more fundamental the research the easier it is to share. I think it's also something that's been largely ignored by management at most places, but I think that's changing. I think there's becoming a lot more concern about biosecurity and I'm afraid that it's going to get worse, not better in terms of what can be shared. Obviously companies have an interest in holding things close to the vest oftentimes, but for government-funded research, I mean, my opinion is that it should be as close to open source as possible with just things being disseminated as quickly as possible for the benefit of humanity. But yeah, I'm afraid that's going to change over time. Others? Okay. All right, thank you. Thank you, Scott. Turn to Jennifer. Yeah, thanks for those great presentations. I learned a lot. So I was very interested that I think three, if not all four of you mentioned the need for more of a focus on non-model organisms and some bioprospecting. And so as an ecologist, I was wondering how do you think we should go about choosing those non-model organisms? I just offer one suggestion. And that is take a process that is impossible engineer. Okay, and if you look in industry, if you look, so for example, we work on hyperthermophilic anaerobes, okay, but they don't tolerate high solids loadings, okay, for biomass. If you could identify an organism that already did that, okay, even it doesn't have to make anything, it doesn't have to do anything. And if you could just tolerate deconstruction of high solids loading, that would be an enormous contribution to being able to use plant biomass as a substrate. And there aren't any organisms that can do that. So if we start with the problem in mind for organisms, in the incredible diversity I've learned about from you guys, if we could go there and find organisms that can do what is most needed, that would be really great. Yeah, I would add on to what Jenna said too and say that from a practical perspective when we do this type of mining, I mean, as an example, so we look for lignocellulose during an activity, that's number one. But then our second criteria is can we freeze it? Does it wake up again? We've learned the hard way that, you can go crazy characterizing something that's really great, but gosh, it's so fragile you'll lose it. And so that's a very important second step of, okay, screen for a function and then screen for practicality. What can be saved? What can be revived? What can be, doesn't need some sort of special sauce? And that's what we've used as guidance. Just to add from the sort of industry perspective where I see and see people looking and there's I think a lot of people just hoping for the future that these things over some timescale will come out of academia to continue to lower costs is everything from processing difficult to process waste streams, next gen cellulose, which people have been researching for a long time since biofuels and they used to be like, you would have to pitch to investors that one day that solve problem would be solved so that you could get cheap enough later, but we're still not there yet. Then products that are difficult, no existing model microbe can make. That's a big one. And then anything that can continue to reduce costs, simplify supply chains, chain, make organisms or I'll even abstract it, make making your product as feedstock agnostic and feedstock cheap as possible is essentially the dream. If you could just say, I just need to make this protein and everything else is a solved problem. That's kind of the ultimate long-term dream from an industry perspective. Great, thank you, thank you, Jennifer. Nathan, I'll turn to you. Sure, yeah, I kind of have two questions, but I'll ask one of them, so I'm debating which one. So one of the things that was brought up, I think David brought it up at the end was this need to create small-scale bioreactors that are predictive of scale up. And I totally echo that, it's been a huge problem for a long, long time. And I've kind of been sitting here just thinking about, is that a pie in the sky kind of thing or is this something we can do? It's sort of interesting from the standpoint of, I think it was Adam brought up a really interesting point that it's hard to keep out oxygen at small scale, but it's hard to get oxygen in at large scale like that's fundamentally different. The diameter, you're gonna have different gradients of shear stresses. And there's all these kinds of things that are just different at scale. And I'm sort of curious from those who have been thinking about it for a long time, an enumeration of some of the thoughts around, what are the kinds of things it would actually take to get something in a small scale to be predictive of scale up? And because I guess the interesting thing is how many of those are maybe technically feasible and are there some that just, it seems like from the physics of it would be incredibly hard or impossible or I'm just kind of curious on thoughts about that. I can jump in to start off, at least for the more established, I'll use the term domesticated, not really domesticated, but how we think of sort of known processes, organisms, E. coli, Saccharomyces, Pichia, Aspergillus, the usual suspects. I think in standard liquid fermentation, it's become somewhat expected that, so long as you're limiting your variables on the small scale to things that are reasonable at the large scale, so you're not trying to cool to some incredible degree that which is great for your get out heat, but you could never do it at a large scale. As long as you're not pumping sugar faster than you could ever pump syrup, your two liters can be fairly predictive of success. And some people even go down to 250 mils. I've found that even with that predictability, the super large manufacturers, the people who are making in these 100,000 liter fermenters still wanna see you do it several times at 3,000 liter and 10,000 liter, but the data you get from a two liter, as long as you're sophisticated in limiting your process to what could be feasible at scale is generally accepted as this is doable. Now, that is for a subset of liquid fermentation for those microbes. And I see that generally, that's also thinking in terms of industrial biotechnology where you want to go to a much larger scale than pharma where you're doing GMP. How that pans out if you're doing gas fermentation and aerobic fermentation, that I'm not familiar, that's outside my wheelhouse. So if I could contribute to that, so there is an unnamed company in California, not David's company, since it's not bioadvertisement, I won't mention it, but it's one of the few companies in the country that does small scale bioreactors and the ability to do that, I can't overestimate how important it is, but they have now developed shake flasks that you can sample biomass, that you can sample aerobic cultures growing on biomass. Okay, so the advances in this area are substantial, but there just aren't enough of them. You know, there just aren't enough companies that do this, there aren't enough bioreactors, there aren't enough people investing in this. There's a need for software, there's a need for hardware, there's a need for analysis of these bioreactors and how they scale up. That's part of the Valley of Death that comes from translating basic science to an industrial process. To join, like, careers were made in solving the chemical engineering problems to scale up aerobic fermentation. How many people are working on that for others? I have no idea, but like, it took work. Credit, credit where credit's due. It also takes money and an investment in things that matter. Perfect, perfect, thank you. All right, I know we're very short on time, so maybe if I can just hit one person to answer each of Patrick and Nancy's questions. So Patrick, go ahead. I've heard a lot about E. coli and Saccharomyces for recent industry that, you know, like, I guess my view from industry is that there's actually less of those, less E. coli and Saccharomyces than you might expect. There's a lot of filament to spongi and, you know, other, you know, legacy organisms like, you know, S. cryptomyces, Lactococcus, but you know, a lot of different stuff up there. And as a result, there's a lot of esoteric fermentation designs at scale and that offers an interesting challenge for industry when the folks who run those facilities might be interested in producing new products. So I'd be interested from the academic viewpoint in terms of identifying new hosts, whether you feel like you have the right engagement with the industry around how to revitalize existing scale manufacturing plants because that's a significant opportunity for new hosts, in my opinion, because, you know, you've spent the money to build a plant, but you may be interested in producing new products. Can I just say that academics are incredibly ignorant about what industry needs and wants? And that's because there's just not enough communication from small startups to the major players in fermentation technology. There's not enough discussion. There's not enough understanding. I can speak as an academic. Maybe I'm not that great at it. But still, I think SBIR grants, as I said before, that bridge this gap. If you have an academic and an industrial partner involved in these processes, they can talk to each other, develop workshops and go forward with this in a way that really will, I think, be game-changing. And I think that's something the academy could promote. Great. I'll leave that one there and let's fair Nancy for our last question. Thanks, yeah. So I'm going to leave my groups for a second. I just finished being a judge at iGEM, which we could consider a sort of feeder really for the industry end. I was struck anecdotally, but I was struck with the number of teams who chose to develop self-resistance or certainly for detection, but also for some of their synthetic processes and so on. And that seems to be a trend to me anecdotally. And I wondered, you could argue that they do it because they can control maybe the reaction better. They avoid detrimental interactions. If there is something they want to release into an ecosystem, for example, and then finally they avoid the whole GMO issue and their respective countries. But I wonder whether you could just come, one of you could comment on the impact of self-resistance, which are getting quite robust on your thoughts about it. I could chime in real quick. I mean, I think that self-resistance have a lot of power in the ability to do high throughput screening that have not been successfully harness. So I got it part of, how do we end mass proteins of unknown function? If you could set up a cell-free screening system to perhaps do that, that'd be really interesting. But I think cell-free systems, I don't think they're ever gonna be used for scale up. It just doesn't make sense. But for onsite manufacturing, a little bit of something, maybe. I think they have a lot of power there, but not for scale up, that's my view. All right. Certification is a bit of a challenge when the cells are all exploded. Amen. All right. We got to leave that as the last word in this great conversation. You can tell from the level of questions and from the question that I didn't get to online that we are in an area of extreme interest and a lot of energy. So thank you very much to the panelists for great talks and a great discussion. And I'll turn it back over to Kavita. Thank you all again. We hope that you enjoyed both of the discussions today. This is not the end, just the beginning. So please keep a lookout for more on these topics and related topics. And we'd love to engage with you more on them. So this is the close of our formal open discussion. Board members, we have a 10 minute break before we join back in again. Thank you all. And I wish you a very good afternoon. Take care.