 Good morning. Good afternoon and for some good evening. I'm Max Hegblum, Editor-in-Chief of FEMS Microbiology Ecology, and it's my pleasure to welcome you to this webinar on microbes versus metals. FEMS, the Federation of European Microbiological Societies, invest in science. We're using the income from our journals to fund charitable activities and support our community. So providing grants to scientists, organize and support conferences, and sponsor a range of events such as this webinar series, which provides a forum for the presentation and discussion of key research, enabling the flow of ideas to continue despite the cancellation of in-person events. Each month we are highlighting a different topic of microbial ecology. Fortunately, or unfortunately, we have many interesting topics to cover as the pandemic and various states of lockdown and restrictions continue. Also, if you missed our earlier webinars, they are available via the FEMS and OUP websites, so you can look these up. Today, we focus on the topic of microbes and metals. Metals and metalloids are the backbone of modern society, but also central to the lifestyles of many microorganisms. They have developed a highly sophisticated repertoire to traffic, metabolize, counteract and exploit metals, either individually or in various types of communities. From respiration of metallic compounds to adjusting and responding to their extracellular environment to merely surviving in the presence of metal polluted habitats, microbes have found ways to incorporate metals into their physiology and using surprising sets of mechanisms. These microbial processes are also key for the efficient recovery and reuse of critical elements. Our speakers today will highlight some of these fascinating metabolisms and our quest to decipher them. First, we have John Stoltz from the Department of Biological Sciences at Duquesne University in Pittsburgh, who will talk about microbial selenium metabolism. Followed by Lucian Steiku from the Faculty of Biology, University of Warsaw in Poland, on microbial biomaterialization processes. Valentin Syriak, now at the Department of Biology, University of Copenhagen in Denmark, will discuss metal-induced bacterial interactions in river sediment microbiomes. And finally, Ron Ormland, who is a Merida senior scientist from the US Geological Survey in Menlo Park, California, who pioneered arsenic and selenium biochemistry will reflect on these discoveries with some salty and metallic tasting stories from the great basin desert. So this is the lineup for today. After the talks, we will open the session for questions and discussions, so please submit your questions, comments via the Q&A link. So with that, let's get this going in our first speaker, John, selenium and microbes. Okay, great. So Max, I want to thank you and Femmes Microbiology Ecology for not only hosting this webinar, but also for allowing Lucian and I to do this special issue. We've got some really amazing papers and I'm really pleased with how it all turned out, despite the last year being in COVID lockdown, so it's been very special. So I'm going to do a little brief introduction of selenium, which is actually a metalloid. So back in 2011, our last speaker, Ron Ormland and I, we published a book on microbial metal metabolism. And during that time we put together this periodic table and what's really amazing if you look at the different colors, this is like the microbes view of the periodic table. Almost two thirds of the elements in the periodic table are used in some biological process and by some microbe. And that includes some of the basic fundamental structures like, you know, everyone knows carbon, nitrogen, phosphorus and sulfur, etc. That they're used in basic metabolism and structure, but metals are also very important as cofactors and the like. And even more so in things like respiration. And so today our focus is going to be on selenium. And the fact that, you know, I when I first started as an assistant professor did not know that seleno cysteine was the 21st amino acid. And so for those of you who might not know that either, you know, I would like to introduce some of that to you. So, as part of the review and I must thank my former student Michael Wells, he was an amazing student and did an amazing thesis on selenium metabolism. And is the first author on our review for the FEMS special issue. But this brief history of selenium back in 1916, respiration via selenide was established in microcoscus selenicus. And it was the first time that, you know, selenium more or less was was thought of possibly as a source of energy over the years as you can see and I won't go through this in detail. But it became more obvious that there were things not only for resistance because it turns out too much selenium can be a problem it can cause certain diseases, especially in cattle for example but a lack of selenium also leads to health impacts. And more so in micro organisms as it turns out that in many micro organisms selenium is an essential element. And so what, why, what is that reason. And so now we understand that not only their instances in which organisms are resistant to high concentrations but more importantly, that they're actually that selenium is incorporated into certain essential enzymes and features. So that they also can be used for energy and so I'll talk a little bit about that so. As you can see here, seleno proteins, what are those well those are proteins that actually contain selenium and often selenium in the active side of those enzymes. So in 1954 selenium was a requirement for format dehydrogenase and if you do any sort of bioinformatics you often see enzymes annotated as anaerobic dehydrogenase typically selenocysteine containing. And how do we know that well, again, with the work that's been done over the years and we've reviewed in this manuscript. We understand now what seleno proteins are. So, just in brief and it turns out that there are some major differences between selenium metabolism and archaea and selenium metabolism and bacteria summarized in this particular slide. But just to cut to the chase over the years of variety of different researchers have looked at both assimilatory processes and dissimulatory processes using selenium for assimilation we know that there are at least now five genes that are specific to selenium metabolism. There's a selenocysteine synthase cell B is an elongation factor that's involved in actually incorporating the selenocysteine into the protein of interest. So the cell C encodes at the specific tRNA so as I said, so you know, cysteine is the 21st amino acid. Interestingly enough, it's encoded by a stop codon. And so the understanding is how do we know that or how does the cell know well because there's this interesting little hairpin loop in the, both the DNA and of course the expressed MRNA. And it's called the Sequs binding protein because it tells the cell, don't stop, keep going and put a selenocysteine there. Cell D is the selenophosphate synthase and cell U which is a rather recent discovery is involved in another assimilatory pathway in which selenium is not incorporated as selenocysteine, but just as selenium. Getting to Michael's work. One of the things we wanted to know back way back when in Mono Lake, Ron Ormaline's group discovered organisms that actually use selenium for energy generation as a terminal electron receptor in both selenate and selenite. And although Joan Macy's group many years ago had demonstrated selenate reduction for respiration and identified the selenate reductase. We didn't know what the selenite reductase was so in the last few years Michael's been working on this, and he identified a putative selenate selenite reductase. And interestingly enough, the story gets even better because not only do we have the putative enzyme and it belongs to a much larger group of enzymes in the DMSO reductase family of enzymes, but it's related to polysulfide and phylsophate reductases. Again, this is very exciting because Michael then was able to do a really deep dive as we like to say in deep time and looking at the various enzymes within the DMSO family, and it turns out that this clade here with the polysulfide reductase and the selenite reductase is a rather old clade. So that's exciting too because it suggests to us that selenate or selenite respiration is actually pretty ancient. And so if we look at just how the different types are given within the timeframe of geologic time, we can see that again that selenite respiration might indeed be very, very old. So now getting back to our selenocysteine, well it turns out that a lot of these enzymes have selenocysteine at the active site, and it also turns out that there are cysteine homologues. So then that raised another question which came first, cysteine or selenocysteine. And again through Michael's deep dive into the protein record, we basically have come to the conclusion that actually selenocysteine was the key amino acid and then subsequently cysteine replaced that. Lastly, and to move things along, I'm probably going very, very fast, but that's okay, we can have more time for discussion. The true focus of the paper as well is looking at the selenium cycle. So what's really fascinating, people have made connections between the selenium cycle with sulfur, but there are some really, really unique differences. For example, when we get these methylated species of selenium, they're always in the minus two or fully reduced form. So that's kind of unique. And the other thing is is that many environments have a robust selenium cycle in which both selenate and selenite is reduced to elemental selenium. It's still a mystery and maybe Ron will shed some light on this as well because the work that his group has done is basically, you know, how things get oxidized is still a true mystery because you know the at least looking at how the processes are going in the environment. Selenium oxidation is a minor constituent of this cycle, but it has to be because otherwise over the millennia we would have reduced all the selenium. So anyway, in conclusion, selenium is definitely on the map as far as an essential element. It's required in many seleno proteins or all seleno proteins. Seleno cysteine is the 21st amino acid and it's encoded by a stop codon, but the secret element allows the cell to recognize the or at least the protein synthesis machinery that you need to put a seleno cysteine in there. And I really hope it is my hope that that seleno cysteine will make it into the basic biochemistry textbooks because I sure as heck didn't learn about it until I was a professor. So with that, I will, you know, again, thank my lab, thank Michael, thank Ron for years of collaboration and once again, Max and Lucien for putting together this great special issue. Thank you. Well, thank you, John. Really nice introduction. I already have a whole bunch of questions for you, but I will save them until later. And again, also, if you have questions, please submit them via the Q&A link and we'll get to them then at the end of the webinar. So we'll continue with microbial bio mineralization processes and our next speaker is Lucien Stegu from the University of Warsaw. Thank you, Max. Hello everybody. Today we'll be talking about microbial biominerals. It is a fascinating topic because biominerals is a research area that is the boundary between several research directions, for example, microbiology, physics, chemistry, mineralogy. So somehow it's a synthesis between different areas of knowledge that it's truly remarkable. So today I will talk a bit about metals, why they are important for our society. Then I will move to biominerals. We have a case study about biogenic lead sulphide that is the topic of the article that we published in this thematic issue. And then we conclude with the perspectives. So metals, as you see here in blue, practically they dominate the periodic table of the elements. They form the vast majority and also with the metalloids they occupy most of the space. In red here we have the lead that is the metal that will be described in more detail today. So at the beginning of the human history, early humans realized that working with metals is not so productive. So then they discovered metals and we have several ages. In history we have the bronze age when the people discovered this wonderful alloy which is a mixture of copper with antimony and with other metals. Then here on the right you see some artifacts belonging to this time period. Then people worked with Tyrone and they learned how to improve its quality. And also today we live in an age where metals are essential and I will show in the next slides. This is the global metal production in 2019. I chose this year because it's the year before the pandemic when the industry was fully working. You can see here on the left huge amounts of aluminium that were produced and consumed but also copper, zinc, lead. Metals that are highly used in the economical production by the industry. Also we use very important quantities of silver, gold, palladium, platinum, precious metals. So practically this shows that metals are essential for our human society. In the future we have to secure their safe production in order to have good economic growth and also sustainable growth. This brings us to critical raw materials or CRM. These are materials that are economically and strategically important for an economy. But on the other hand they have a higher risk associated to their supplies. So practically this means they are very important but their supply is a bit at risk. Different continents or different associations develop these kind of tables. And I'm citing one from the European community. In 2017 they drafted the third list where there are 27 critical raw materials listed. And as you can see metals and metalloids occupy 63% of this. And here I'm giving the examples in black we have the metals and in green the metalloids. This again shows the tremendous importance of metals for our society. This is an example of the demand for metals. You see here on the left aluminum, zinc, copper there is like an exponential demand with time. But also on rare elements you heard today talking a lot about this in the context of electronics on smartphones or computers. And also their demand is increasing exponentially. On the other hand their supply is limited. So this brings us to the very important aspect which is the conflict between this exponential growth of the human population and on the requirement of these metals versus the finite resources. Because metals, unlike other, for example, energy, they cannot be replenished easily. And here in 1972 it was the first report of the Club of Rome. It's like a think tank and they drafted this report that is called Limits to Growth. And this practically shows that the exponential economic and population growth cannot be met by finite resources. So the solution for this is to ensure efficient recycling so that the metals return in the economy after they are used. With regards to biological systems, metals are also very important. You see again the periodic table and in black some of the metals that are essential for microbes because they act as cofactors in various enzymes that are also used to produce cellular energy. With regards to the relationship of bacteria with metals, it's like a very complex one. Because metals on one hand can be essential because they act as cofactors in enzymes. I'm giving some examples here, copper, iron, molybdenum. On the other hand metals can be used to generate cellular energy or ATP. And other examples are arsenic, iron, selenium. There are more metals that are involved in this. But also metals are toxic and bacteria have developed various strategies to counteract this toxicity. And here I color coded different colors. For example, copper, which is the same time essential but toxic. For iron that is essential but it's also used in anaerobic respiration. So this shows that the bacteria have developed ways to use metals in various ways, either in a beneficial way but also when they are toxic to counteract them. In the case of bi-mineralization, this is a process that is widely employed by living organisms. They produce minerals that are either crystalline or amorphous. And to date there are more than 60 bi-minerals that are described. In the case of bacteria, we can talk about two different aspects. We have biologically induced mineralization, which is commonly extracellular. And we have biologically controlled mineralization. In this case, the microorganism controls the nucleation site, the growth, the morphology and the location of the mineral. So this is a process that is highly regulated at the molecular level. I'm giving some examples of microbial bi-minerals that they are much more. For example, in the case of iron, you know that iron minerals have a complex geochemistry. Some examples of green rust, shebanella is involved in the production of green rust. We have geotite, gallionella ferruginea. On the other hand, we have magnetite, some examples of magnetosterilum that produce these chains to orient in the geomagnetic field. We also have calcium carbonate with different polymorphs, calcite, aragonite, vetherite. Some other examples, truvite, which is a phosphate mineral mixed with corpus, was shown to produce truvite. There are also mono elements. So in this case, there are minerals that contain only one chemical element. We have gold, we have the elemental sulphur, elemental selenium. But also lead sulphide or galena, which will be the topic of the second part of the talk. Here you can see two examples. On the right, we have a gallionella that is producing iron stocks. These beautiful stocks here on the right. And another one, we have biogenic selenium produced extracellularly. And you see that it is nicely deposited at the boundaries of the cell. With regards to the function of microbial bioenolus, this is somehow complicated and a topic that needs much more investigation. Because in contrast with the plants or with animals where the bioenolus have a function that is very well established, in the case of bacteria, the things are not so clear. Because oftentimes, bioenolus don't have an evident function in bacteria. And because they are a byproduct of microbial metabolism or energy generation. And some examples, we have borerite, we have various iron minerals. We have ornament here you can see in the image, the yellow suspension. This is a mineral formed by the sulfutomaculum during the respiration of arsenic. On the other hand, bioenolus are toxic and metals are toxic, so bioenolus are a way to detoxify them. And some examples are galena, lead phosphate, elemental selenium. But also the bright side bioenolus can be beneficial. And some ecological roles that I'm citing here, in the case of magnetotactic bacteria, they synthesize magnetite and gray jite. These two minerals that have magnetic properties and they are used by this bacteria to orient in the geomagnetic field in search of micro-aerophilic environments. On the other hand, some bioenolus are an energy generating function. An example is volutine, which is polyphosphates or elemental sulfur, which functions for some bacteria as an electron donor. Finally, we have a very puzzling example of elemental selenium that results from respiratory processes. That might appear to be an evolutionary data. And currently, we have a publication with Larry Barton on this topic. We hope to have it published in the next weeks. And I encourage you, if you are interested in this topic, to look for it and to read it. Now we move to the case study, the lead sulfide. Lead is a post-transition belonging to group 14. It's a copper. This means that it's copper-loving. It's present with copper deposits. And it's main or it's galena or lead sulfide. You see here on the right an example of galena. As you know, lead is also a major anthropogenic pollutant. For example, today in the United States, I found that they want to exchange all the lead pumps, conduits, water conduits, because of toxicity. Some sources of pollution of lead are ore mining, the production of lead is batteries, and the burning of fossil fuels. So, for this study, we used a strain of Bacillus cereus sensulatus. It's a strain that we named Bacillus albuquerque, ABQ. Albuquerque is the capital of New Mexico, and this strain was isolated by the team of Larry Barton, who worked for many years at the University of New Mexico. And this bacterium has the capacity to degrade the system to hydrogen sulfate, and it degrades the system as a result of lead toxicity. And sulfide reacts with lead, forming lead sulfide, which has a very high stability. You can see here the KSP, the solubility constant product showing that this mineral is very stable. It's very insoluble. And here you can see on the right, we have the control, and we have an incubation with cysteine and lead acetate, leading to the formation of a black lead sulfide. Very interestingly, this strain cannot use other sulfur sources to produce sulfide, and we explored several, we tried the sulfate, we tried the tyrosulfate metionine, and it seems that it's only, it's enzymatic machinery, it's only able to degrade the system. And this is a research direction that we are currently pursuing in Warsaw in collaboration with our friends from abroad. So then in this study, we showed that lead removal is time dependent and is biotic. It's related to this strain. And cysteine degradation occurs intracellularly, but the formation of lead sulfide is extracellular. And moreover, we show that these minerals are crystalline and they have a very narrow size, below 10 nanometers. You see here on the right the contours, the shapes of bacteria, and the black iron like metal fillings, these are the crystal of lead sulfide. Then we dried this pallet and we performed mineralogical analysis. We showed by X-ray diffraction that the mineral is lead sulfide or galena. Then we performed high-resolution transmission electron microscopy. We characterized the crystals. Again, we showed that it's lead sulfide and it's high purity. Here it's a model that we proposed and I invited to read our publication for more details about it. So in the case of the toxicity when bacteria is exposed to lead toxicity, it internalizes cysteine from the extracellular environment. Cysteine is degraded in the cytoplasm to hydrogen sulfide, which is released, and then it reacts with lead cation forming lead sulfide, the black dots. So in this way, lead cannot enter in the cell to produce, to have a toxicity reaction, but it's stopped outside the cell by forming an insoluble mineral. The follow-up of this study we are currently looking at the enzymes that are responsible for cysteine degradation. We are also having an in-depth characterization of various sulfur sources. We are doing this in collaboration with Professor Daniela de Biasse from Rome. We also sequenced the genome of this strain and we plan to perform transphosal mutagenesis to determine the genetic determinants involved in lead metabolism. This is a collaboration we are doing with Dr. Rob van Hout from Belgium. We also look at the insight. We have an insight into the bio-miralization process. We look at other metals like iron, cadmium, cobalt, and nickel that might also be precipitated to understand the complex biogeochemistry of metabolism in this strain. And finally, we plan to do a study on the lead recovery in the form of PBS from industrial reference and we have a candidate, the waste water released from lead acid batteries, which contain a lot of lead. Finally, I would like to acknowledge my research assistant from Warsaw, Paulina Witović, my collaborators from Poland. I also have many collaborators abroad, Larry Barton, Michali Posvai from Hungary, he helped us a lot with the study. Andreas Kapler and Liane Benning in Germany, Eric van Hulipus in France. As I mentioned, Daniela de Biasse and Rob van Hout, but also we have good collaborators in Spain, Mohamed Merun and Encarnacion Ruiz Agudo, also in Oviedo, we have a good collaboration. Also, I would like to thank the National Science Centre in Poland who funds my research. The various funding agencies and friends for organizing this thematic issue and this webinar. Also, I would like to thank the participants of this webinar and Max Hegdom for his support. And with this, I thank you for your attention and I look forward to your questions and comments. Thank you. Well, thank you again. Please put comments into the Q&A box and we'll get to them then at the end of the webinar. So thank you, Lucien. We're going to continue on our metallic tour and our next speaker is Valentin Siriak, now at the University of Copenhagen and we go to river sediment microbiomes and their metal interactions. Hi, everybody. And first of all, I would like to thank you, Max, for the invitation as well as fans. And for the opportunity to present this work that I've been doing during my PhD in the University of Mons in Belgium in the group of proteomics and microbiology. And during this PhD, I was interested in the interaction between metals and bacteria in reverse sediments and more specifically how macular communities adapt to high metal concentrations in the environment. And we were partially interested in sediments because they are known to be, to host very dense and diverse macular communities because they are seen for many compounds, including nutrients. It also means that they absorb a lot of metals and has been shown that up to 99% of metals entering river actually ends up in sediments. And this is because they absorb to play and bind to many compounds such as organic matter, sulfides, carbonates, etc. And this absorption will actually define the bioavailability. So the degree and the rate to which they will be available for living organisms, which also mean that at high concentrations, an increasing bioavailability also leads to toxicity of these metals. During the last century, the prediction of these metals increased sharply. And if lesion problem is that metals is a limited resource, my problem is here is that it comes along with the pollution of the environment and an increasing pollution. As an example, OLAB is working on the dirt river in North France that is located next to a fondry, that is responsible for the contamination of this river, the dirt river. So we were interested in how the macular community of the sediments of this river actually adapts to this metal pollution. And to investigate this, we compared sediments from metaraup and upstream sediments that we sampled in ferrin, upstream the contamination. Previous studies showed that the metal contamination in these metaraup sediments were very high. And up to 30 times more contaminated for some metals such as cadmium, but they also were concentrated in copper, lead and zinc. The same study characterized ferrin and metaraup sediments with metagenomics and metaproteomics, and they showed that diversity in metaraup sediments was very high, actually is high and in the control, when both considering Shannon index and the richness. They also shown that the dominant genera that are found is these sediments are very similar in ferrin and metaraup, and that functionally both mobile communities are also very similar. Here you have metagenomic profile of ferrin and metaraup. And you see also that this study shows some genes involved in metal resistance where enriched in metaraup impacted sediments. So we have two macular communities upstream and downstream and metaraup contamination that are very similar. And the part of this study here was to have a closer look and a more detailed picture of the tachymic profile of these communities to try to explain the adaptation, considering of course the missile contamination, but also keeping in mind that other factors could be involved in the adaptation of these communities, such as the release of wastewater in the river or the farming activities surrounding the river. We had two approaches here. The first one was to sample sediments in situ from in ferrin and metaraup extract DNA and the RNA, and get the 16S RNA and PICN profile from these sediments. We also use cognitive PCR to quantify some plasmids. The second approach was to go back to the control side in ferrin, collect some sediments and monitor them over six months in microcosm. We use four of them as a control and we added lead, cadmium, copper and zinc in four other microcosms to finally reach concentration found in the metaraup sediments. Over the six months, we followed the tachymic profile of the communities every month and also used cognitive PCRs to quantify sediments and plasmids. By combining these two approaches, we expected to decipher the mechanisms involved in the adaptation of these communities and leading to this very high diversity in the metal content in this spot. We then looked at what bacteria were resistance of sensitive to the metal, what could be the potential interaction that they are involved in with the rest of the community, and if plasmids could play a role in this adaptation process. The first step was then to check if this diversity was as high in metaraup as in ferrin. As you can see on the left graph here with other results in C2, you have the richness index on the top graph and channel index on the bottom one for both DNA extract in grey and RNA extract in white and ferrin and metaraup sediments. You can see clearly here that the channel index is as high in metaraup than in ferrin and the richness is even higher in the metal content in the sediments. In microcosm, we quantify these diversity indices over the six months of the experiment and you have in grey here the control microcosm and in dark grey the metaraup impacted microcosm for both the richness on the top and the channel index on the bottom. And you can see clearly that the diversity of these sediments were maintained all over the experiment and that after the six months the diversity in metaraup impacted microcosm was even a bit higher. An important factor in the experiment setup here is that daily we were renewing the supernatant water with fresh water that we sample directly into the river and by doing so we're actually adding new living organisms, living bacteria in the system and we load a coalescence process, a mixing process between the community of the water and the community of all sediments. This is particularly relevant here as we've seen in a previous experiment where the water added to the microcosm was still less before addition. We thought we showed that the channel index was decreased over the six months of incubation and metaraup impacted microcosm. So we believe that here we have a combined effect between metal filtration for metal resistant bacteria but also a bottleneck process due to the microcosm setup. Confirming here that this coalescence process might play a very important role in the adaptation of the community. Then we wanted to see what bacteria were significantly responding to the metal first in situ here and once we have found this significantly impacted bacteria which will hit map with them so each line is a bacteria, an OTU, and each column is a sample and the abundance is represented by the color so a red color represents the highly abundant OTU. We have on the left of the heat map sample collected from the control sediments in Fehan and on the right of the heat map we have sample collected in metaraup and metal impacted sediments for both DNA and RNA extract for each. And thanks to the heat map and the associated cluster we could define groups of bacteria that were actually behaving similarly in these sediments and get a closer look at what bacteria were enriched in metaraup sediments if you consider the group pink and orange here or what bacteria were sensitive to metal and almost absent in metaraup sediments if you consider the yellow and purple group here on the bottom of the heat map. An interesting group is the blue group here, the fourth one that include bacteria that were very present in the RNA samples of the metaraup sediments involving that these bacteria were mostly very active in the sediments and they count a lot of permacryptus, clostridia, but also anterobacteria that are known to be markers of fecal contamination so it indicates that they might come from wastewater or the fields and that these coalescence corp and participate to this coalescence process. We're also interested in what kind of metal resistance this metaraup and which bacteria from the group pink and orange were carrying so we went through the literature for each OTU to see what kind of resistance they could carry and we actually considered two types of resistance, selfish resistance such as metal pumps that actually benefits only the producer of the resistance system but also corroborative system including intracellular and extracellular sequestration, the production of exoperative saccharides that will decrease the bioverbity of the metals and then facilitate the growth of surrounding bacteria and when we went back in this pink and orange here of metal and rich bacteria we found some that could be responsible for this facilitation process because they were known to precipitate metal, sequester them or produce a lot of exoperative saccharides then we did the same exercise for microcosm so here on the heat map you have once again each OTU on the lines and the abundance in the corresponding sample on the left of the heat map you have microcosm, controlled microcosm samples in blue and on the right of the heat map in red you have the metal-impacted microcosm and you can see clearly that we have two groups here, a group A of metal-sensitive bacteria and a group B of metal-enriched bacteria this group B was divided in four depending on the time of occurrence of each OTU and in these B groups we looked for these facilitators bacteria and we found plenty of them for example we found a lot of zeorglia and legionella that are known to produce a lot of exoperative saccharides we found bacteria that were known to precipitate metal and then decrease their bioavailability by producing polyphosphates or sulfates such as dichloroneous anharmonic acid or sulfate reducing bacteria from the delta proteobacteria then we wanted to see how are these bacteria included in the community so we used a dynamic network that actually identified bacteria with simultaneous or delayed occurrence in our microcosm to have time-related succession and on the top graph here you have a network built from the control microcosm and on the right here you have the network from the metal-impacted microcosm the first thing that we see is this high number of positive links between all members of the community and this blue cycle here that's combined or OTU from Aneurinaceae and I've shown you before where a facilitator of bacteria and if you zoom on this network and have a look at the positive link that any kind of facilitator of bacteria have with the community we found that Aneurinaceae but also sulfate reducing bacteria in dark blue here also Zeroglia and dichloroneous had positive interaction with many bacteria but more importantly with keystone species one interesting keystone species is exonsobracter because it's a nitrogen-fixing bacteria and might play an essential role in the nitrogen cycle in these sediments and the interaction with facilitators bacteria could explain the resilience of these micro-community in metal-impacted microcosm finally we wanted to see if some plasmids were enriched in our system on the left of the slide you have the copy numbers of ING-F, ING-I and ING-P plasmids in C2 and if you can see that ING-F and ING-I plasmids were not enriched in metal-impacted sediments they were indeed in for ING-P plasmids and this is not really surprising as ING-P plasmids are broad-ore sponge plasmids so they can transfer easily to a very diverse bunch of bacteria and they're known to carry a lot of antibiotic and metal-assistant genes so we wanted to see if these plasmids were also enriched in our metal-impacted microcosm so on the white graph here you can see the evolution of the relative proportion of this ING-P plasmids through time in the control microcosm in light gray and in the metal-impacted microcosm in dark gray and we're very disappointed to see that the proportion of this plasmid was increasing in the control but this enrichment was impeded by the presence of the metals however this can be explained as both metals and plasmids have a cost, a burden for the bacteria and these commuted costs would impede the acquisition of plasmids to avoid a loss of fitness so if plasmids have a role in the adaptation of our microbial community this could be on the long term where we could see the enrichment of plasmids actually carrying metal-resistant genes so to conclude we have two system here, two microbial communities upstream and downstream metal contamination where the metal-impacted community was very diverse and this diversity that took place over 100 years could be the result of three processes or combined processes, a community coalescence process, bringing bacteria from upstream area of the river to the material of sediments also bringing bacteria from surrounding environments such as wastewater or the fields surrounding fields we have potential facilitators bacteria in our sediments that will decrease the bio-vibration of the metals and then allow all the bacteria, more sensitive bacteria to thrive in the sediments and finally plasmid propagation of these sediments might have played a role on the long term adaptation of these sediments but of course all of these processes must be investigated separately to actually confront them with that I would like to thank the PROTME group from Belgium and my PhD supervisor Fadi Wachier and David Jilo also Augusta who worked with me on this amachicosome experiment I would like to thank Gabriel from the University of Lille and Jonas from the University of Lebsic Samuel and Joseph and Serhan from the University of Copenhagen where I'm doing my post-doc now and finally thank you all for your attention and really quick question at the end of this seminar well thank you Valentin some really interesting community interactions around the metals so we'll come back to the discussion so thank you about that so to wrap up the webinar we have Ron Ormland who's going to take us on a trip to the Great Basin Desert and give us some of the insights of what really went on in elucidating the geomicrobiology of arsenic and selenium so Ron it's a delight to have you here and hear about the history behind the discoveries thank you Max and thank John and Lucien for being involved and for publishing that got selenium retrospective I do have a confession to make in the couple of years since I've retired no longer having a lab or conducting any new research it's not so much fun for me to talk about science anymore because I have nothing new to contribute but what I can't contribute is a perspective sometimes tongue-in-cheek looking back on what we accomplished and the interactions that were involved that sometimes make fun stories so with that in mind how do I get to my slides here so this is the title salty and metallic tasting stories mostly true because this goes back 40 years and I remember everything this grows out of a chapter that I was asked to write and the book edited by Kristen Hearst microbes the foundation stone of the biosphere and it is in the very last section of the book the party calls the adventure stories and with that in mind let me see if I can call this up give me two seconds here so who is this guy on the screen that's Jean Shepard one of the idols for my youth and this was its theme song the Fibon you may not know him but I used to listen to him all the time on the radio and I was in my late 18s and early 20s he's famous for the movie Christmas story which is kind of a cult classic so much for that I always wanted to be able to do that okay I want to read to you at the beginning of this chapter that I wrote the part one the moto basin and a trip to snarl it was during the summer of 1978 when I first caught my first glimpse of Mono Lake as I descended from Taioga Pass elevation 10,000 feet located at the eastern entrance of Yosemite National Park I was tasked with driving a field vehicle from this up to the Sierra Nevada Aquatic Research Lab better known as snarl located about 25 miles south of the Lee Vining and Mono Lake near Mammoth Lakes, California now the vehicle itself was an old surplus U.S. Postal Service Jeep with a clunky manual transmission and a very disconcerting tendency to alternately sway left and right as it meandered over the highway due from overhead the Jeep would display an evident sine wave function as I drove at east across California Central Valley but the time I reached Manteka I learned how to drape my body and chest and arms over the steering wheel as a necessary expedient adaptation to dab in the amplitude of that sine wave so as not to gear into oncoming traffic or drive off that narrow two-lane highway a further test of the efficacy of that became as I ascended old priest grade a series of switchbacks that eventually brought me up to the foothills of Sierra Nevada's gentle western slope in the town of Groveland I continued east along Highway 120 as it slowly rose towards the western entrance to Yosemite after which I stayed on Yosemite 120 rather than descend into Yosemite's stable valley the road gains altitude and emerges out of the forested region and continues along the exposed granite batholith with superlative views of the valley below as well as half-dome exposed mountain peaks and many stop and see vistas of exfoliating granite rushing streams and other resplendent natural wonders such as Tenaya Lake let's see and Twelomy Meadows compared to the very tame Catskill Mountains of my New York youth pictured here five years spent in graduate school in very very flat Florida this was quite an eye-opening treat now in descent upon descending from Tioga Pass I once again experienced driver's anxiety as nature's vista opened before me snow covered nearly vertically ascending mountain peaks on either side of the road with precipitous chasm-like cliff in between also known as glacier-cawed U-shaped valleys this is a thousand foot drop here I sweated nervously as I struggled to keep on the jeep on the road as I switched back and forth over its long descent now halfway down the gradient east and the vista opened up to encompass the mono basin with its surrounding volcanic craters evident that is white mountains of in the distance and with monolake itself resplendent to the foreground that's what appeared to be a vast inland sea I clearly remember thinking what the f is that as I drove down to where Route 120 intersects with Highway 395 and made a right turn and continued south towards Snarl and then this route took me over dead man's summit elevation to 8,000 feet and my convict lake all named after a posse caught up with a group of escaped convicts from Carson City in 1871 and commemorating the ensuing gunfight and war recapture this was the wild west in every sense of the words yet as far as monolake was concerned it was to be another six years before I began working there I first had to cut my teeth on a smaller version located further east, Big Soda Lake, Nevada as well as to experience my first genuine California earthquake while waiting for Snarl's experimental streams later in that fall of 1978 so that's monolake, there's Snarl, I actually made it there and now a few pictures to sum up my talk that's Big Soda Lake located by that land of Nevada it's the volcanic crater that blew up 20,000 years ago John Stolls was there with me once these were the early operations me and Chuck Culvertson in this little tin boat and the LCS were done on shore in some of the local interesting flora that adorned the shores of monolake back then it's gotten better as I understand slowly as we went by our operations improved our equipment improved and we would stay at the Lariat Motel which was another story itself and we've conducted the biological experiments on Big Soda Lake initially focused on methane and its precursors there's a bacterial plate of photosynthetic bacteria evidence at 20 meters in the lake during summer you can see a vague pink tinge, ectothirate ospera and Chad Salkukov is still working on that apparently as an arsenic oxidizing capacity but this is the same thing in monolake, this is a hot spring on Peoaha Island if you go ashore, out of the hot spring you see these beautiful biofilms of ectothirate ospera they're absolutely gorgeous there's photosynthetic bacteria here that oxidize the arsenic 3 coming out of the hot springs, the arsenic 5 in the light but when the light goes away there are also anaerobic arsenate respirers that take the sulfide in the spring as their electron donor and oxidize the arsenate that was formed by the phototrophs back to arsenic 3 and give a full cycle to this process but we look so much at gels and of course this is what these guys look like you take a portion of that biofilm and put them under a microscope you really need to do this, this is mind blowing it's one thing to look at a gel and to do its sequencing of this RNA or DNA of the isolated microorganisms but this is what these guys look like under high power and it blows my mind there are slow movers, there are more immobile there will be long fast jetting forms of all kinds you just tear your hair out if you can get, if you're still in use by this you'll never get bored and we eventually started incorporating moda lake into our repertoire of summer excursions and we would come from its soda lake way out here in Nevada and enter the monobasin and setup operations in Lee Vining in a motel, the Lakeview Lodge we essentially made a laboratory this is that hot spring in Payoa Island this is volcanic gases that you can smell, hydrogen sulfide it's really a neat place it's a shot by Larry Miller looking west from that hot spring over the portion of Payoa Island which is just a raised sediment from a volcanic event some 500 years ago and those are the Sierra Nevada mountains background there in early fall after the first dusting of a snowfall although many people were involved in the work here over the years it was a great place to study arsenic the three of us, the three of me, was Larry Miller and Chuck Colbertson I think this was the last time we got together on the lake back in 1984 and the many folks that were involved in this process in these studies it was a wonderful experience and that's it, thank you well thank you Ron we will open this up to discussion questions on all of the speakers so please join us back again Ron let me start with you immediately here in terms of early work I mean now yes we're all taking it for granted that of course microbes can respire, salinate or arsenate but what was it early on in terms of why go into these toxic elements to start examining the microbes involved with them? well it was all a fluke as I wrote in my chapter where my piece for FEMS it was a pollution prone in the Kestison Wildlife Refusion the San Joaquin Valley in California where the birds, the migrating waterfowl came into this man-made marsh that received agricultural drainage water which was rich in selenium the birds came in, a lot of them died had tyrannogenic effects on their offspring and it was attributed to a selenium pollution effect and John, our branch chief then came down the hall said there's money available if you want to start looking into selenium studies and I was working on methane I was happy to work on methane for the rest of my life but there was a little bit of money there enough for a postdoc and I said what the hey, this is selenium being right under sulfur in the periodic table this is all being done by sulfate-reducing bacteria it's going to be a breeze to do this wrong but we went out we started looking at the methylation of dimethylsilanide because that was related to some work we were doing with dimethylsulfide at the time again as a precursor of methane and then just out of frustration we added selenate to a matter of exetamins and it disappeared and it became elemental selenium and just tearing that apart we stumbled on the fact that bacteria were using the selenate as an electron acceptor and we're reducing it with organic electron donors to make elemental selenium it's very obvious from this beautiful orange color the cultures are beautiful they're wonderful it's almost like looking under the microscope you get seduced by this and we eventually paired up with John Stahls and others who would jump in where we lacked the expertise they were more than eager and happy to complement and work with us and then from selenium we jumped, just by chance I threw in some arsenate into a test tube and the bug that we isolated so theorists were on bonzi eye I came in the next day and it was turbin just the opposite of what you'd expect but Jody had gone out on maternity leave so I was lacking people in the lab but then I need lava men who came in my lab teamed up with Phil Dowdle and Jody came back from maternity leave and we eventually started working with arsenate and a lot more people knew about arsenate and selenium toxicity it blossomed from there and it's been wonderful to follow how this field has continued of course you've been isolating them from these wonderful scenic areas but I know that one of our new arsenate respiring bacteria came from a wastewater treatment plant sewage sludge in oculum so they're everywhere and this is actually a question both to you, John and Ron as well so now selenate and arsenate respires are routinely being isolated and they fall all over the place in terms of different phyla of the bacteria and archaea and John you're alluded to this being a very ancient form of metabolism so are you thinking that these have been maintained throughout evolution in these clades or is the lateral gene transfer later on with the selenate and arsenate reductases moving on do you have evidence for one or the other in terms of what's happening? Yeah I think there are both but clearly from what Michael has shown and going through and now looking into metagenomes as well the ability to metabolize both selenium and arsenic are ancient traits and I would say with Ron one of the most exciting things because I've been working on microbial communities for decades and again alternative sources of energy and even with phototrophs tying in that arsenite can be used as an electron donor in photosynthesis because there you can have the complete cycle and the absence of molecular oxygen free oxygen is not required and so you build total ecosystems with these alternative electron acceptors and you know that to me is very exciting and then of course as an integral I mean I have no idea why seleno system is such a mystery to a lot of people and the fact that that itself goes back a long time at least what the proteins are telling us so you know the answer is the enzymes that are used to metabolize these elements are old and their functions in biology is old. Actually here's a question from the chat about selenocysteine and cysteine in proteins how does that change the function or does it? Well again it's a very interesting question because there are some basic differences and I think Michael is far more tuned to answer that but there is a fundamental difference of how cysteine is and how selenocysteine given the radox conditions of the ancient earth. One of the first experiments we did when we started working with selenate respiration there's a radioicentope of selenium I think it's selenium 75 which is a gamma emitter and my postdoc at the time Nissan Steinberg we said let's work out a radio assay where we add radioactive selenate to the anaerobic sediments let it incubate and then take subsamples with time because the selenate is going to elemental selenium and precipitate we can wash away the selenate and just count the sediments so with a gamma emitter unlike sulfur for example you don't have to work with beta emitters it just goes you put it into a gamma counter as washed mud and what's ever there you're calling elemental selenium and it worked really well so we went out fascinated I don't know about ten different sediment types running from pristine freshwater systems to polluted saline systems and the big surprise was first they all reduced selenate to elemental selenium in a linear fashion with no lag and they all took off their rates were different but they all were poised at 20 net micromolar so they all were tackling the same amount of selenate there was no lag and that's kind of what was a surprise to us because if this is an ancient enzyme the bugs still have the ability to do this and if there's selenium is introduced into their environment where it's not there they can jump on it and take advantage of it and presumably because there was no lag the enzymes are constitutive go think about that so a number of the other genes involved in metal transformations are on plasmids or transposons so you jump around and especially if it's detoxification so here's a question actually from Joanne Santini about the plasmids what about uncharacterized plasmid groups so you have mainly the INC-P group as dominant but are there maybe other things that you might be missing have you thought about that? We kind of find plasmids, we had primers at this position but of course might be a lot of other plasmids and it's unexplored so as I say at the end of the presentation there's a lot of stuff to do with that and another thing that could be done is with metagenomic to go and see what genes encoded by a mobile genetic element and if we have more resistance in matter of sediments encoded by these mobile elements then we could have a very broad picture of the mobility of these genes in the system So yes and more to figure out on this as well Always more Yep and then let's see I'm looking through here on what's coming in for Lucien this is about the lead sulfide is it sitting on the cell wall attached or is there a reaction occurring as the sulfide species are removed from the cell so you said that there's internal production of sulfide that I guess then is released so is there then some kind of a direct formation of the lead on the surface So yes definitely lead sulfide is present extracellularly we did a very fine electron microscopy we did not find any lead sulfide present internally maybe maybe it's attached also because of this of the EPS from the surface that's why it's clusters there because usually we don't see it freely in the extracellular environment but it's attached to the surface of the cell okay so really is it just a very rapid reaction that occurs yes sulfide released up so then let's see this is one about we're talking about microbial systems now actually we've all focused on finally bacteria and archaea and this is one question about fungi and metals anybody want to jump in in terms of what functions are similar or different than in bacteria or archaea well max I mean there's also when you talk about fungi are we talking about yeast or are we talking about you know because there's been a lot done especially with arsenic one of the first resistance systems was worked out for saccharomyces so you know there there's definitely a linkage and even some of the bioremediation technologies are based on fungi because they are very fastidious and are able to mobilize things from rocks and etc no and again the question of respiration is probably not in the fungi with any of these but it's interesting when I was a graduate student a fellow graduate student Michael San Francisco who's now at Texas Tech found fungus growing on chikemic acid and was puzzled of how this could possibly be and then because he was working on nitrogen fixation grew it up in some nitrogen free media and it grew and we're like whoa, this would be amazing to find a nitrogen fixing fungus but in the end it turned out it was just an aspergillus but it was incredibly good at stripping any trace nitrogen out of the air out of the media whatever and it wasn't nitrogen fixation it was able to do it so Joanne Santini is just commenting in here on the oxidation reduction as detox of microbial eukaryotes which is of course there's actually many of the same systems that bacteria use so the question this is one other here about selenium how and why does it become toxic to bacteria does it differ on the oxidation reduction state you know I've never Ron we haven't looked at that specifically with arsenic that they can be very susceptible to arsenite and for obvious reasons but with selenite and elemental selenium whether or not we've never pushed the limits one observation was on this selenium nanospheres that we picked up later we observed them early on when we started isolating these bugs but only got an opportunity to study them a bit further down the road and when we had transmission electron micrographs done we were looking at them at the outside but also internally there were lots and lots of selenium nanospheres so I infer that selenium gets into the cell and once they are the bug wants to get rid of it or if it can't get rid of it it has elemental selenium which is relatively armless but then there were so many of these nanospheres outside the cell it wasn't clear if it was being formed on the membrane where other workers say it's being formed internally and then being exported it would be rather hard on the cell to do that because the volume was immense but I think of them as two processes going on so internally it's precipitation as a detoxification process and externally it's gaining energy back to the Galena question for Lucien one thing we did see with the selenium nanospheres on the outside of the cell when on a scanning electron micrograph we saw a diaphanous veil around them there was something that they were encapsulated in that you could see through and workers in Holland went on further to say there's peptides associated with the biologically formed nanospheres but not obviously when you make them chemically some later work we did with Mitch Herbal was only this selenium nanospheres formed biologically it can undergo further reduction to hydrogen selenide not the artificially produced chemically there's something that bugs are attacking attaching to those peptides and transfer their electrons to the biologically produced ones there's a lot more mysteries at least for Selenium if I can contribute a bit these particles are coated with a biopolymer layer we don't know at this moment if this layer is acquired from the extracellular environment or part of it it's also a contribution from the internal environment of bacteria but we know that it's negatively charged because we did some studies during my PhD in Netherlands and we used various counter charge anions we neutralized these particles and we sedimented this show that in fact this surface charge is negative but it can be counteracted by counterions and it's interesting because this negative charge contributes massively to its colloidal stability we left these particles like for five days in a sedimentation count and less than 10% of it sedimented which is amazing because the chemical system that you try to keep colloidal it will not last so much yeah and it's interesting also in terms of the concentrations that some of these trains can tolerate I think we've grown Selenate respires at 30 millimolar Selenate where one reviewer actually said that's true toxic they can't survive it and said well didn't you look at figure one where they were doing but again it's interesting in terms of really I mean concentrations way higher than they would actually ever encounter in the natural environment so there's Selenate respires Selenite respires Arsenate respires Joanne is asking what about Arsenite respiring bacteria are they out there well Arsenite is able to be used as a donor so you got to think of your what respiration is you have your donor and your acceptor so Arsenite is definitely can be used as a donor I think Joe has always wanted to see elemental Arsenate being precipitated yeah Selenium is being precipitated we've never stumbled upon that somebody someday may but in fact that we have a full Arsenic cycle between Arsenic 3 and Arsenic 5 was enough to keep us busy for the rest of our careers beyond well I think it's never saying never regarding microbial activities if you can gain energy from it somewhere there's probably an organism that has figured it out I would agree we just need to find it yeah I like to call them odd couples it's amazing what microbes are able to couple as far as reactions are concerned to make energy I was wondering with Lucien so you had the Sistine link to what about Selenium Sistine this is a very good observation because my student Paulina will travel next month to Rome to do a research stage with Professor De Biasse and we plan to explore more surface surfaces for example we have Taio Urea, Glutataio what else we have Taurine, Elemental Sulphur but also Selenium Sistine we want to see the full repertoire of this bug and it will be very interesting if it extracts sulphide only from Sistine because this will be totally unusual knowing that sulphide is very important for microbial metabolism yeah yeah I think there's a lesson there too in the sense that because of the work that has been done that arsenic has specific genes Selenium has specific genes even though they are closely related to phosphate and sulphur but they're unique in their own way and there are unique pathways and yes they can sneak in various like arsenic sneaks in through phosphate pathways and Selenium can masquerade a sulphur but there's still unique pathways and enzymes involved for arsenic and Selenium and we can then continue on the periodic table go to antimony and so on exactly so I think we are going to have to wrap this up right I want to thank all of you Ron, John, Lucy and Valentin and everybody here in the audience it's been a really interesting session I've enjoyed every minute of it and again thank you to everyone who submitted questions we didn't get to all of them but we'll send these by email and then the speakers can respond later and of course finally thanks to the FEMS and OUP staff for making these webinars happen and all the work behind the scenes as well so thank you all and we may see you in end of May for our next topic on agriculture and sustainable agriculture and soil microbiomes so keep looking out for posting on a new webinar in late May but thank you again and take care