10:02:19 Hello, everyone.
10:02:20 Thank you for coming back to the microbial metabolism forum.
10:02:26 And we are totally delighted today to have Cornelia welter here.
10:02:31 Canadian is a microbial physiologists and who just got tenure at the University of Michigan. So this is a great event.
10:02:42 And she has been doing really interesting work in microburst see one the tablets and specifically in methanogens, and has been taking on one of the sort of really big last frontiers in microbiome metabolism, which is the anaerobic oxidation of methane.
10:03:03 So, and when sort of Terry Canadian I talked about what to prepare. We thought it would be really useful to provide a perspective how to look at what looks like complicated metabolism.
10:03:22 But it is not, but really look at this from from principles. And I think these principles are those that most of you are interested and they should form the basis of a discussion with Cornelia then, so please again interrupt and ask a Canadian anytime,
10:03:55 Yeah, good evening everyone or good morning.
10:04:10 Yeah at seven 7pm, well I'm reciting things Alfred and Terry for inviting me I'm excited to be here and tell you a little bit about the work that my group and I've been doing on measurable nice and metabolism.
10:04:12 And I want to briefly introduce myself so I hold a master's in biology were focused on microbiology and biochemistry, and I hold a PhD in microbial chemistry.
10:04:23 And after that, as a little bit of postdoc hang and then started a tenure track at Robert University in the Netherlands where I'm currently also still residing and as Alfred said just attained tenure in my group.
10:04:36 We work with micro interactions so we work a lot with me time cycle. I get really excited about yeah anaerobic microbiology and especially anaerobic methane cycling microbes.
10:04:46 But I also have a separate branch in my group where we work on symbiosis between pests insects and microbes, something that I only mentioned now but I'm not going to talk about today naturally.
10:05:00 I have one slide in which I want to kind of explain to you why I became a microbiologist.
10:05:06 And this is if you look at animals or humans, they convert sugars proteins and fats to carbon dioxide, which is all fine.
10:05:15 And then you look at plants and they use light and carbon dioxide and they make sure it's from this.
10:05:20 So from a metabolic point of view, even though they're very complex and exciting and so on. I think microbes are just much more interesting, because they can take all kinds of substrate and make all kinds of products, so they're not as restricted in their
10:05:33 metabolism, as animals and plants are.
10:05:39 And then one last advertisement so yeah the Netherlands are again, great country to live in. I'm German and I moved here and I really like it. And one of the things that open a few years ago is actually the first microbiology Museum, which is artists
10:05:54 micro PA.
10:05:55 And I think they have, if you browse the web page already they have a couple of really nice statements, but also if you ever come to visit the Netherlands, please go there it's really worthwhile.
10:06:05 And something that I really liked how they phrase, what microbiology is when you look from really close the new world is revealed to you more beautiful and spectacular than you could ever have imagined.
10:06:17 And I want you to keep this in mind while we go through the maybe a complicated bio energetics that I'm going to explain to you.
10:06:26 Where is that museum Columbia and Amsterdam. It's actually part of the zoo, so it's it's part of them. Yeah, the normal Zoo and they have a separate building, which is then the microbiome microbiology zoo.
10:06:41 So as I said, I get excited about anaerobic microbes. And I want to briefly introduce this to you, even though you know and other and other tutorials you heard about this already.
10:06:56 And to start this I would like to quote from a paper that was published some 20 years ago, this one here. And it says, The earth is a microbial planet on which micro organisms are recent additions, highly interesting, and extremely complex in ways that
10:07:12 most microbes are not. But in the final analysis, relatively unimportant in a global context.
10:07:20 Coming to anaerobic microbiology, please look at this, a timescale of life on Earth. So, life evolved, some 4 billion years ago.
10:07:31 And then for a very long time, microbial life existed in the absence of oxygen.
10:07:39 And then at a certain point oxygen was introduced, and then only like these last. Yeah, these last two little branches are the evolution of plants and animals.
10:07:50 So looking at anaerobic microbiology anaerobic microbes really had 4 billion years to evolve.
10:07:56 So, that explains some of their complexity. But I also think it's a treasure trove for scientists and also for society for biotech applications.
10:08:14 So to start with the, with the context in which, in which the, the meat and metabolism that I want to talk about operates. I want to show you the anaerobic food chain, and I guess that you've already seen this and better things talk or some of the other
10:08:27 talks in this series.
10:08:30 So in the anaerobic food chain, you.
10:08:33 Yeah, this happens in the absence of oxygen naturally.
10:08:36 You have organic polymers, and then a number of different micro groups, hide some, some microbes that Hydra lies the problem areas you have fermented, you have a seat regions you have Central.
10:08:49 And then at the very end of the anaerobic food chain you have the mythology and mythology and take the waste products of some of the other members of this anaerobic food chain and convert those to meet thing that no big food chain, it's not some exotic
10:09:06 thing that happens only at Yeah, at one or other place. It happens in all kinds of anoxic environments and we have plenty of those on our planets.
10:09:18 So I think you also had a had a talk on on gut microbiology so.
10:09:25 Most, if not, if not all intestinal systems are actually anoxic.
10:09:29 Of course we have anaerobic digesters and like bio gas plants are wastewater treatment plants that are anoxic large parts of our oceans are anoxic, not only the sediments, but also the water column, and actually ocean and oxy is a big event happening
10:09:47 in the context of climate change that. and these anoxic areas are expanding so the anaerobic microbiology beget gets more and more important, especially in the context of climate change.
10:10:01 Then sediments, so when I talk sediments it's kind of the soil around here are marine sediments. after the first few millimeters or maybe the first centimeter.
10:10:11 They're all anoxic, and yeah the anaerobic food chain happens. And then of course we have also a lot of subsurface environments. So when you go further Baloo and below and below.
10:10:23 And, and most, if not all of these subsurface environments are anoxic.
10:10:28 And with this I would like to illustrate the anaerobic food chain happens virtually everywhere, even though we as humans, maybe are used to your toxic environments and think that this is where everything's happening.
10:10:42 Yeah, as I said, I want to talk about methanogens, so about me 10 generating micro organisms, and they at the very end of the anaerobic food chain.
10:10:55 Remember realizing the Yeah, the waste product of organic molecules to me thing.
10:11:04 I would like to start this by giving a comparison of envelopes and our robes and and how I see those.
10:11:11 So, first starting with our robes on the right hand side here.
10:11:14 If you have our herbs that grow with organic molecules, basically that take what is up here in the anaerobic food channel so they combine it with oxygen.
10:11:25 They do metabolism to this and that produce you to, which means all the energy that's that's harbored in the organic molecules, they get for themselves.
10:11:35 They don't have to share with others, because they have a very efficient or yeah the bathroom metabolism, releasing huge amounts of energy.
10:11:44 So the Delta G's associated to our aerobic organic compound degradation are usually very very negative. So there's a huge amount of energy to be that to be gained for the Arabs.
10:11:57 If you look at the envelopes, you see that, basically what the Arabs can do in one step. They do in several steps with several microbial guilds involved here.
10:12:09 And they have to share the energy that is released from from the process here.
10:12:17 And because of that, so they need to cooperate because one group is dependent on the next one and the next one and the next one.
10:12:24 And because the need to cooperate. Yeah, they need to share also the yeah the energy that's released in these processes.
10:12:33 So obviously their metabolism also has the Delta gee that's, that's negative.
10:12:38 However, it's not usually the free energy change associated to anaerobic process or individual microbes anaerobic processes.
10:12:47 Yeah, it's much smaller.
10:12:50 And I would even go as far as saying that many live at the feminine AMEC limit, and I will talk about this a lot and the first part of the tutorial actually.
10:13:04 And the second part I will also talk a little bit about thermodynamic tricks, because no ropes are oftentimes limited and the way how they can generate energy, they need to use very inventive mechanisms in.
10:13:14 Yeah, in which they can, they can keep the energy that they have.
10:13:23 And with us, I'd like to provide you the roadmap that I've prepared for for you today. So, my yeah basically I've structured this in three parts, part one and part two, part of the tutorial.
10:13:35 And the third one is then the research talk.
10:13:38 And the first part, I will talk about seminar nomics of me 10 generation, and what it does tell us and also what it does not tell us.
10:13:47 And the second part, I will talk about them as energetic metabolic pathway map and introducing an anaerobic thermodynamic trick to you.
10:14:01 And then the third Park parts. So I've titled my talk, how to reverse the metabolic pathway and still make a living, or novel discoveries and anaerobic Nathan oxidation.
10:14:11 So for this first part, the thermodynamics of meeting generation and what we can learn or maybe also cannot learn from it. There are a couple of things that I really need you to, to know, and these are what Delta GS so what excellent can enter going to
10:14:25 corrections are what ATP is and how it is connected to the proton motor force, and you need to know a general understanding of what respiration is.
10:14:37 And I'd like to take a few minutes to ask you whether these concepts are clear to everyone. Otherwise I would, I would rather take a few minutes to, to explain those again because then it will be easier for you to actually follow what I'm going to say,
10:14:54 we're going to just briefly summarize in your own words, the important aspects on each, so that we know what is how you look at, at these three areas.
10:15:10 Yeah, yeah, that's fine. Um, so actually for ATP pm fn respiration I have a slide for delta gee I don't have a slide.
10:15:16 So let's start with delta G. So delta geez the gives free energy change.
10:15:22 And there's a certain formula the nerds formula, how you can calculate it, and it basically tells you whether a reaction is x or gone and got an organic.
10:15:34 So, and when a reaction is extra chronic that delta G is smaller than zero, and energy is released from from that particular chemical biochemical process of delta G is positive, then the, the chemical biochemical process cannot happen unless you couple
10:15:49 it to, to an extra clinic process.
10:15:57 Okay, this is, this is a slide summarizing proton motor force, the PMF and respiration and also how ATP is coupled to this.
10:16:06 So, Imagine that what you see here is to sell.
10:16:11 There's the cytoplasm and the center in the Yeah, inside the cell and there's the extracellular space outside
10:16:19 to in order to obtain ATP which is required for all kinds of cellular processes such as growth biosynthesis of amino acids and nucleotides locomotion cell division, so all kinds of things require ATP.
10:16:35 And yeah metallic metabolism, or respiration is used to obtain ATP.
10:16:41 Now you've probably heard about respirator or learned about respiration so and respiration you have a couple of membrane proteins that some of those are accepting electrons from a donor molecule for instance entity Ah, there's Allison transport chains
10:16:55 happening that can be very easy, very complex, and then electrons are finally taken up by an executive acceptor molecule, and an aerobic respiration that's oxygen and an anaerobic processes, this can be all kinds of things such as nitrates organic molecules
10:17:11 sulfates, whatever.
10:17:14 And the course of in the course of respiration are of this respiratory chain protons are translated or sometimes also sodium ions, and this will be become important later.
10:17:25 So protons of sodium ions are translocated.
10:17:29 And they build up the PMF the proton motive force.
10:17:33 And then the proton motor force is used by by an enzyme called ATP synthesis, that is using the proton multiverse. So at trends locates protons from the outside to the inside.
10:17:46 And like this it can it can produce ATP.
10:17:54 Okay now I'd like to start on the mythology mythology methanogens. Yeah, just said that methanogens have an ancient metabolism devolved probably very early.
10:18:05 And sometimes people think if things have been around for very long than they're very simple and not so sure I agree, but it's also not so important.
10:18:14 I brought you here to microscopic images of two of my favorite methanogens that it also talks about in this top here.
10:18:23 One our offensive sign which which form these nice packages from eight cells the Yama, the sinus. And this one is an image of my son of Satan. Another mythology and that forums like long filaments
10:18:40 methanogens grow on a number of different substrates but the substrate spectrum is very small, they only grow on really a handful of very small.
10:18:51 Yeah, small substrate such as acetate methanol methylation means or sulfites or hydrogen and co2.
10:18:59 And what they do is really that they make me think from these compounds.
10:19:05 If you look at the metabolic pathway map and I've made this little cartoon here, illustrating, that's also the metabolic pathway looks yeah maybe relatively simple that takes you to do a number of things to it, and then we think gets out.
10:19:21 And in the course of this metabolic pathway they produce ATP.
10:19:27 So, Montana Genesis is the part of the yeah Miss Anna Genesis is the energy conserving pathway of Miss energetic archaea and, yeah, these, these microbes are also called methanogens
10:19:46 someone's not able to see my slides.
10:19:49 Can you all see my slides.
10:19:54 Yes.
10:19:55 Okay.
10:20:03 Okay, now, now I come to the free energy change, so that gives free energy change delta t associated to Montana Genesis.
10:20:12 And this is a table in which you should only look at the the red boxes and forget about the rest.
10:20:20 So if you look at my center Genesis from hydrogen and see you two minutes associated to a free energy change of minus 130 kilometers per mole.
10:20:30 And it will also talk, so I will talk about this later. Later in this tutorial.
10:20:35 And there's also Montana Genesis from acetate ch three co ah here.
10:20:41 And this is associated to free energy change of minus 36 kilos your promo.
10:20:47 Just as a comparison. If you look at the aerobic respiration of sugar, such as glucose down here.
10:20:58 Then this is associated to a free energy change of minus 2800 kilogram per mole.
10:21:04 So just appreciate how big the differences, as I said, the Arabs they get everything for themselves, whereas Arabs have to share, Share the free energy change with different microbial guilds.
10:21:17 And you could say that that these free energy changes here, minus 30 and minus 100 are really low.
10:21:24 And I want to discuss with you a little bit, and the yeah and the and the part to come, what kind of implications. This has for the metabolism of the methanogens.
10:21:37 And to start this part I would I have three statements here to think about, and I would like to hear your, your opinion about this after maybe you've thought about it for a minute or so, and I will read them first to you.
10:21:51 So the first one is acetate dependent Montana Genesis has the lowest gives free energy change of all most energetic processes, yet it is the most relevant method method energetic process on our planet.
10:22:05 The second one is low gives free energy change of a macro level metabolic process does not equal low growth rates, so does not say anything about the speed of cell division of a micro organism.
10:22:19 And the third one is the gifts free energy change does not usually determine the substrate threshold concentration. So the minimum required concentration, for a microbe to grow.
10:22:33 So if anyone has any ideas about this or any thoughts about this I would like to hear about it now, before I go on and, yeah, it will kind of be convoluted them step by step in the slides to come.
10:22:53 Can you hear me.
10:22:56 Yeah. I mean we had in the previous talks we had a number of discussions about the second statement that you made about therapy being they're actually being some potential connection between.
10:23:14 that and and the rate of growth. So is that a statement, in principle, or is that a statement, or, or like a bigger statement you know what I mean, like, you saying, it does not necessarily mean that or that it never means that.
10:23:27 Yeah, I think there is.
10:23:30 So, if you would plot, all metabolic processes and all growth rates, you would probably see a correlation.
10:23:38 but I'm going to show you one example. Yeah, and I'm not sure where that's the next slide or the one after that it doesn't necessarily mean that, so it's very dangerous to think, only because a metabolic process is associated to a high or low Delta Gee,
10:23:57 that that does say something about the speed of growth. But it is true that in more general terms, you can you can say that there's a correlation between, between the gifts reality change.
10:24:08 And, and the growth rate, but especially in a couple of animals. So, I will show you one example of media metabolism here, but there's also an example from nitrogen metabolism, for instance the animals bacteria.
10:24:19 They yeah they have enormous Delta Geez, if you look at just the energetics and they grow extremely slow.
10:24:27 In those cases, is there a rationalization that can be made like that. Some of the wiki. If you look at the net delta G it looks favorable but if you look at the individual steps of the pathway that there there are bottlenecks and some sort.
10:24:44 So certainly there are bottlenecks but there doesn't, they don't need to be some dynamic.
10:24:50 So I think one of the points that I want to want to come to also during this tutorial is that in some cases thermodynamics can be limiting, but in many other cases it's not the thermodynamics that's actually limiting metabolism.
10:25:02 It's other aspects of the metabolism, that, yeah, that are more limiting to to speed for instance.
10:25:11 And that can be toxicity that can be enzymes that can be all kinds of different things.
10:25:22 So thank you.
10:25:22 Regarding your first statement, the dependent maternal Genesis.
10:25:26 Why is it more most relevant Is it because the concentrations of acetate or some other reason.
10:25:35 It's a little difficult to say really, I think, one of the reasons it's that acetate is a very important final product of the anaerobic food chain, and it's maybe even more important than the hydrogen answer to, which is the the second kind of big mess
10:25:51 energetic substrates.
10:25:53 And another one is that if you think about the environment. Then,
10:25:59 when acetate is there in the, in the absence of other electron acceptor.
10:26:05 Then there's really only dermatologists that can pick it up. Whereas if you have hydrogen and co2 there, there's more competition for it. So, the hydrogen is a very true substrate, as I'm sure that you've, you already know.
10:26:19 So, there's more competition for hydrogen and less competition for acetate in a way.
10:26:30 One more question, clarification. So when we talk about when you're talking about gifts for energy here and you're talking about the standard state for energy or like after correcting for the observed concentrations in environment that you're.
10:26:43 Yeah, I'm talking about standing up gives free energy changes. And actually, I've not included it here, but for most substrates, there's not much of a difference.
10:26:54 Like one.
10:26:57 One substrate where does make a big difference is hydrogen, and I think you've seen that. Also in, probably the central talker so.
10:27:07 But for most other substrates, because it's, It's a neat the relevant concentration.
10:27:12 Environmental concentrations that you talk about, but when you think about the energetics, it's the proportion of oxidized versus reduced species, and most of the times, the oxidized species is then also in low concentrations.
10:27:27 So the actual delta G doesn't change so much.
10:27:31 This statement is I think makes sense to me from the perspective of the net reaction, like if you're considering only the extracellular species but I don't think is accurate for intracellular reactions.
10:27:41 Like if you if you think about, for example, a reaction that has like mixed geometry or something and then you very different concentrations in the cell.
10:27:49 the this you know the cut the delta G and one molar reference concentration, different from $1 million for now but some of the nomics doesn't think about intracellular concentrations to start with a thinks about extra selling the concentrations.
10:28:06 And that has to do with the way how
10:28:10 how delta t is kind of defined because it's it's always against the standard hydrogen electrodes.
10:28:16 So, yeah, maybe it's it's beyond also what I'm going to talk about, but delta is as such you really look at extracellular concentrations and don't consider, like transport phenomena and actual concentrations in the cell, for a start.
10:28:33 I mean I think we should put it aside. But I think the reactions inside the cell so have to obey the second law, right.
10:28:40 Yeah, that's true, that's true but I think my, what the pointer wants to make us that the Delta geez I'm showing here. Overall Delta Geez, of the overall chemical reaction of the overall biochemical reaction.
10:28:53 But yeah, you're right, of course that in the cell energetics also have to work. Yeah, so I mean reactions that that that happened voluntarily have to have a negative delta t.
10:29:04 Also another concentrations that that are encountered. But it's a little dangerous to, to look at an individual Delta geez because if you look at the pathway and actually thought about including this in this talk, but I decided against it.
10:29:17 If you look at the map energetic pathway, then some of the steps have a positive delta G, but they can still happen because there are a couple to all the other steps that have a negative delta t.
10:29:27 Yes, but again this is this is the problem with, considering the standard.
10:29:32 Right. They give you, they are misleading, because they're not living in the standard.
10:29:41 Yep.
10:29:49 You want to come for for for comparisons, often standard states are useful. Even if you go to NC to concentrations, you're pretty close. Yeah, there might be you know depending on the state geometry of the substrates, there might be differences in in
10:30:09 by, you know, an order of magnitude gives you a 5.7 kilowatt hours so depending on where you are in the Delta G value.
10:30:19 There might be some deviations but in the first approximation, God, pretty close. It all depends on where you want to go with the argument and I think Cornelia is saying she's looking at the ecosystem for making predictions and petitioning of energy.
10:30:41 Yeah, I think I think I've understood I just that I guess my, my specific hobbyhorse here is just that, for example, it seems the use of the standard state potential seems very specific to Redux reactions where you're sort of stood geometry is sort of
10:30:55 important, because there's two couples.
10:30:57 Right, so you're always doing like to to to type reactions or something like that, but ok so now you're talking about red X potentials, as opposed to wg and yes they are related by Nancy creation but the standard stage rhetorics potentials inside of a
10:31:15 cell, maybe deviate by plus minus 30 milliwatts but they're not totally off so using the standard state red X potential that you're talking about now is nevertheless useful first step to orange you on the Red Sox scale I think I just, just as a very quick
10:31:38 thought exercise just to illustrate the point that I'm making which is not really go back to the talk I'm sorry but imagine that you have a one to two reaction right like you have a bond session.
10:31:50 And then imagine the transition from one millimeter from one molar to one millimeter standard state.
10:31:56 Right. That's three orders of magnitude difference right so that that's 18 kilojoules promote in the right so that that's a huge difference so I All I'm saying is that the use of the standard state value is defensible I think for this extracellular Redux
10:32:11 perspective, but then the moment you consider another type of reaction and start thinking, but but if you're going from one molar to 1 million mala you're doing this for both the substrates and the product so you're doing this on both sides so yeah actually
10:32:25 ending up in a value that is not too far away and all. What matters is sort of this the chemistry.
10:32:41 No alpha, don't agree with that if you go if you combine your break on molecule up into two when you combine the two into one and you pick up that in from the concentration, I mean it's not like a, like ATP and so right.
10:32:48 So, so if you go. If the substrate concentration, you go from one molar to one minimally. You also go to them for the end product concept.
10:32:57 One minute to $1 million.
10:32:58 So then, in the deviation terms that these, these changes cancel out and what is left is the difference in stick yummy tree, and that's where you have been per sort of order of magnitude 5.7 killer jobs, but that's right if you're breaking a molecule
10:33:16 up into two. Then, then you'd that's not that argument isn't working.
10:33:24 Well, that's what I said it's just a chemistry. No, it's not.
10:33:34 gentlemen Gentlemen, okay this is getting too technical, but I have I have another question. What's on the next slide.
10:33:35 Let's, let's have a look here.
10:33:37 Thank you.
10:33:41 So first I'm coming to this first statement. I want to point out that tema nomics, basically, even though you can calculate a lot of things from it. First of all, it provides you with a yes or no answer.
10:33:55 If delta G smaller than zero metabolic process can yield ATP, and if it's bigger than zero, a metaphorical process cannot yield ATP.
10:34:04 Obviously, as we also seen in these arguments it's not as simple as that always but I think as a first approximation it's useful to keep this in mind.
10:34:16 Of course, a minimal delta G is required in order to make a living.
10:34:23 And sometimes it's said that the Yeah, the minimal amount that is required as something that's used to.
10:34:29 Yeah to to make an ATP but actually the minimal energy quantum has one proton, and I hope to illustrate this to you and one example from a fan of Genesis.
10:34:42 Then the second statement of which we also talked about a little bit that there is a correlation if you plot all metabolic processes and all the Delta G's then more negative delta Geez, those processes tend to be associated high growth rates.
10:34:57 However, the two examples that I'm using and, and my talks today are acidic classic pathogenesis so my fan of Genesis on acetate associated to the minus 36 key which will put them all.
10:35:09 And the microbes growing.
10:35:11 With this, they have a doubling time of route around 24 to 30 hours.
10:35:17 And in my research talk I'm actually talking about anaerobic meats and oxidation with my trades that is associated to it, free energy change of minus 500 killed your promo, which is huge.
10:35:29 And we end the buyer actors that we keep them in the lab, if we keep them really very very happy and the optimal growth conditions. They have a doubling time of around 10 to 14 days.
10:35:40 Just saying that more negative delta G doesn't doesn't necessarily mean faster growth.
10:35:51 And then the third statement that actually no one had a question about or any thoughts that gives free energy change is not directly associated to the subject threshold concentration.
10:36:03 And this is actually what I'm gonna talk about in the next like 10 minutes or so.
10:36:08 If we look at the two general that grow that make me sound from acetate.
10:36:15 Then one of the general one of these, this general mechanical signer gross. Only at concentrations higher than one minimal acetate, whereas my tennis with, whereas my fantasy can grow at like super low concentrations.
10:36:32 And when you look to the environment. This is also where you see these two groups occurring. So within a society grows that high acetate and high acetate environments, whereas methanol Seta growth in low acetate environments.
10:36:49 even though they, their metabolic processes associated to the same delta G.
10:36:56 And, yeah, why is that
10:37:00 so there are a number of contributing factors and I was told that I shouldn't talk too much about about enzymes.
10:37:08 But I wanted to include that enzyme kinetics are very important in determining the substrate threshold concentration of a certain microbe. And what you can see here on the left hand side is to make it obvious men's an equation that maybe or maybe not
10:37:21 you've seen before. What's plotted here on your on this graph is basically they were actually velocity of particular enzyme plotted against the substrate concentration.
10:37:34 And you see that, this, this follows a certain. Yeah, a certain line here.
10:37:42 Where at lower substrate concentration, you have lower reaction velocities and how these curves look like is particular to a specific enzyme so enzymes can that take the same substrates, that are, if you will either enzymes that can look very differently
10:38:00 in terms of how these curves look like. So some a very steep and reach their maximum velocity already at like very low substrate concentrations and other Mr shallow and reach their maximum velocity only at very high substrate concentrations.
10:38:16 So the primary factor determining the substrate threshold is actually enzyme kinetics.
10:38:23 The second one is maybe too easy to even put on a slide but it's of course also which enzymes are.
10:38:30 Yeah, are comprised in a cell. So, only because some enzymes exist that have spectacular enzyme kinetics doesn't mean that all the cells have them.
10:38:41 And then there's a third one that I'm not going to talk about, which is the substrate availability.
10:38:48 And with that, I mean not necessarily how many moles are many moles or whatever, available in the environment.
10:38:56 But for some substrates substrate availability is a little tricky such as for gases that don't solve well in and liquids, such as methane, or member and transport and we've talked about this a little bit already that the concentrations that you find outside
10:39:11 of the cell, don't necessarily mean that you find the same concentration are even enrichment of the substrate inside of the cell.
10:39:23 Coming back to the to methanogens Matheson sign and mathematics data. I want to explain to you how they Yep, how they actually incorporate the acetate molecule into me thing.
10:39:38 So this is the the acetate molecule here on the left hand side for both of us on this side and from us on a Seta.
10:39:45 They do something to the acetate molecule and then they make a tool called a at an esoteric or a is split into methane and see you too. That's important how exactly this works.
10:39:55 What is important is that the esoteric molecule needs to be activated before it can.
10:40:00 Yeah, it can really go into Masonic Genesis,
10:40:05 and what is not interesting is that mythos a signer who grows at high substrate concentrations uses a different method for acetate activation, so different enzymes, as Madonna Satan within us assign a uses a two step, a two step process.
10:40:21 And what is important here is not what the intermediate or so, but that they use one ATP molecule in order to activate the acetate molecule.
10:40:31 In contrast, methanol Seta only use one enzyme, and that's hydrolysis ATP to a MP a Pyro phosphate and the Pyro phosphate is further hydro lies to phosphates.
10:40:44 And what that means is that a sign I use this one HTTP for acetate activation, the one us here.
10:40:51 And my son of Satan uses to ATP for acetate activation down here.
10:40:58 And I think at first instance, you might think that my fantasy data is throwing away energy that it comes actually spare because it uses to ATP for process that Matanuska signer managers with just one.
10:41:13 However, this comes at a trade off. And this trade off is the substrate specificity, so the threshold concentration, which these enzymes can can utilize.
10:41:25 So this one step process down here with formats on a Seta, it has it has specificities that are in the few micro molar range, so they can grow it extremely low concentrations, because this enzyme is very good at capturing tiny amounts of acetate
10:41:43 this enzyme f6 system up here is not so good at that. It starts becoming active at like one minimal acetate, but it only becomes really happy at like five 610 milli molar acetate.
10:41:58 So, even though this process up here for my son as a signer looks much better in terms of energetics, so it only uses one ATP. It actually pays back.
10:42:11 This, this low energy investment with the substrate threshold concentration in that it can only use milli molar concentrations.
10:42:23 Whereas this enzyme down here. Yeah, it uses to ATP for acetate activation exact same exact same same processor, as up here, but then just with one HTTP more.
10:42:34 But what the gains from this is the specificity of the enzyme.
10:42:39 And it goes too far not really really explain how enzyme biochemistry does this.
10:42:45 But what I want to.
10:42:48 Yeah, what I basically want to transport to you with a slide is that you really cannot have it all and metabolic process can either be highly substrate specific or highly efficient.
10:43:00 So you cannot have a very low substrate thresholds, and at the same time, have very low ATP investments.
10:43:09 Let me say specificity here. What kind of.
10:43:13 Is it a family of competing metabolites or just broadly, yeah.
10:43:28 specificity is actually just the, the term we use for how this curve looks like. If you have a highly specific enzyme then it's a very steep curve, and you reach high velocity is already at very low substitute concentrations.
10:43:34 And if an enzyme is not so specific, it has a very shallow curve.
10:43:39 That's nice. It's more like a sensitivity it's not like there's a competing metabolite.
10:43:46 No, it's really about one metabolite this one particular metabolites is a question here in the chat that I want to read. Why can't you have it all. Are you saying this trade of is inherent in the laws of physics, or is it an empirical statement.
10:44:05 I'm not sure I can answer this question really well. If you look, I mean I'm a, I'm a lab rat right so I do, I do a lab experiments and what we see with enzymes, is that
10:44:20 is exactly what I'm just saying that you cannot have both.
10:44:24 And I think it has to do with the way how patella says, In this catalyst in the enzyme works.
10:44:32 So I think it does have a physical basis, but I actually don't exactly know I wouldn't know how to explain that.
10:44:43 It was sort of a related question can you provide some intuition about why an enzyme cannot be both highly specific and high heat up game.
10:44:54 That's probably more complicated.
10:44:59 I, I really can't, it's just what we observe. I mean this is just an example with what I think it's very clear that you have this two step process and the one step process.
10:45:09 And we observe that that's how the enzymes work.
10:45:14 So this specificity that you that you kept track, like micro molar concentrations comes at a cost.
10:45:22 Maybe we can pick up on these questions at the end towards the end of the tutorial,
10:45:29 whether any other questions.
10:45:39 Okay, let's maybe go ahead.
10:45:46 It's a little challenging for me to see the chat and the presentation at the same time, don't worry about your chair.
10:45:52 Okay, you can I let you run the show and I did okay okay I was saying that a minimal delta t is required, equaling the amount of one proton translocated across the set of plasma membrane.
10:46:05 And again, I want to illustrate this with an example from Aesthetic Plastic within Genesis.
10:46:26 So in this slides, please don't take the time to look at the specifics of these respiratory chains that I have that I've pasted here. They only here for illustration, again we have the mechanical signer that the text one up for activation at the math
10:46:29 and as stated that takes to to http for for activation.
10:46:33 stated that takes a to ATP for for activation. And then I'm do something that the true bio energetics specialist will frowned upon. But you can just count the protons and sodium ions that are translocated in the respiratory chain that these pathogens use to, to make
10:46:47 me then from acetate.
10:46:49 And when we do that, we see that there are seven protons, or sodium ions per acetate molecule translocated yeah you know what ATP synthesis ATP synthesis takes protons and sodium ions and makes ATP from those, and the Stoke geometry of ATP synthesis.
10:47:05 Yeah, it's not so easy to say how many protons are sodium ions exactly are used by that but we would say three to four ions per ATP. So if we have seven protons.
10:47:16 That equals about two ATP.
10:47:19 We, we have to retract the ATP that was invested for acetate activation, so we come up with an one ATP net gain for mythos assign them from a parasite it becomes a little more challenging right because we had to ATP invested we still have seven protons
10:47:38 translocated respiratory change chain looks a little different.
10:47:43 So there will be no net gain, which means a metabolic pathway that has zero ATP or zero protons in the end.
10:47:52 And obviously that's impossible, because you can only do metabolism, if you gain something in the end.
10:47:59 There are a number of things. So when we investigated this, there were a number of things that we calculated and and measured in the cells, and one is actually associated to the ATP synthesis here, ATP synthesis.
10:48:14 How many protons, it takes to make an ATP is dependent on on the makeup of the enzyme. So how exactly the the subunit composition is like, but also on the concentrations of ATP ATP and phosphate in the cell.
10:48:32 And we measured some of those, and actually learned that with the, with the actual intracellular concentrations of ATP ATP and phosphate and empower phosphate, we commit three protons per, per ATP molecule.
10:48:50 So it seems that the ATP synthesis of my fantasy is a little bit more efficient than that of nothingness sign on.
10:48:57 And then secondly, we discovered an extra enzyme and the respiratory chain, a Pyro phosphate, which takes part but not all of the Pyro phosphates to to also translate an additional proton, and only by discovering these, these two aspects of their bio
10:49:18 energetics, we come up with one extra proton, that's left over.
10:49:25 And this is really the thermodynamic limit of life, so if you do not manage to kind of balance what you invest in what you get to at least get one proton out, then you cannot live in Montana Seta.
10:49:38 Actually, yes at the very limit of life, because it's, it cannot have more than one proton.
10:49:44 Extra per round of Masonic Genesis.
10:49:48 And I think this is particularly interesting, because if you look at the Delta Geez, then Matthias assign him as an essay to have exactly the same, minus 36 kilojoules them all.
10:49:58 So, the family dynamics would tell you, it's the same, but only when you look at the specifics of their metabolism and really learn how how their metabolism works, how the whole yeah how the whole pathway how much energy it takes to go through the pathway
10:50:17 in terms of ATP investment, and also how the respiratory chain looks like in terms of iron stuck geometries.
10:50:25 Only then you can really learn about this.
10:50:30 So, with this I'd like to finish this first part.
10:50:37 And, yeah, maybe these take home messages are too easy, but I kind of tried to summarize them. So I would, I hope I could show you that thermodynamics is important, but also that thermodynamics is not directly quantitative and the example of, for instance,
10:50:51 the one he gained from a fan assignment and one proton gain of a fantasy to and the same with the same Delta gene, the enzyme kinetics actually determines the substrate thresholds and high substrate affinity and high efficiency I actually mutually exclusive.
10:51:10 And the minimal entity quantum that you need to sustain life is not one ATP but it's one translocated iron, and in most cases that would be one proton or one sodium ion.
10:51:22 And I think this would not be a good, good time to take some more questions, because then the second part I will go on to some Yeah, slightly different aspects.
10:51:34 was a question Cornelia Why can't we imagine a system that would be doing half a proton per reaction on average.
10:51:43 How would you split the proton.
10:51:48 So the question is really how you can, you can't have a proton rights.
10:51:54 You can you can have to wait for two reactions before you.
10:52:03 I mean it. I mean, it depends on the, there is a there is a way I understand it right studies, studies of voltage across the membrane.
10:52:13 So protons want to go. So, I mean, I can imagine that there are fluctuations and you only need to pay the energy costs on average, right, not every single instance.
10:52:28 So, maybe to relate so the voltage, that's, that's on the membrane it's built up by the proton motive force.
10:52:37 So they are directly related or there. One is directly related to the other.
10:52:43 And I think the problem is that the energy cannot be saved in any other way. Because the protons and also sodium ions and some other ions but mainly protons, they make up the proton motor force, this is the way how you can I mean cells like battery right
10:52:57 so they translate ions to the outside that builds up in electrical potential, and that that makes that the cells charged and can actually run ATP synthesis.
10:53:20 And you cannot charge the battery with something less than a proton and you cannot kind of wait for it to yet to be to be kept our soul. So while I while I understand your question like, why can't we just save it and then do it in two rounds.
10:53:27 I'm.
10:53:29 I don't think any metabolic processes actually doing that.
10:53:33 And I wouldn't know how.
10:53:38 Yeah, I'm sorry that I don't really understand why why one has to pay the bill one proton at a time. I mean these things happen at some rate right i mean.
10:53:58 So I
10:53:51 don't really understand where the district ization come comes in.
10:53:57 I think that it's so discreet because the.
10:54:16 How can you say this.
10:54:06 This is all done by enzymes right and enzymes. As such, they have a certain functionality and some enzymes can translate protons. And the question is basically what you're what you're asking is cannot the enzyme, remember that it got some energy in the
10:54:22 first place.
10:54:23 And then it takes another energy quantum later, and only then translates the proton.
10:54:31 But the enzymes cannot, cannot remember that they cannot kind of constantly energy reaction.
10:54:49 Reactions happening that are energetically not necessarily favorable right there are fluctuations. Right, so I mean, I don't understand. It's not like at any point in time, every single molecule event has to be energetically favorable know.
10:54:58 Uh huh.
10:55:01 That's still the cell count the enzymes can't remember it.
10:55:05 So I understand your your your question and that you're saying, okay, but their, their chemical reactions happening that are associated to like less free energy change or to less, whatever, not leading to a proton translocation they can still kind of
10:55:21 change the energetic states, but microbial metabolism is working in a way that it needs to translate one proton at a time.
10:55:30 And when we look at the actual metabolisms that are happening, then this is the minimal energy quantum that they take.
10:55:38 I think Berlin has is launching an answer.
10:55:42 Yeah, Cornelia.
10:55:45 I assume you are going to pick up the subject in your next part now.
10:55:51 But of course, electron bifurcation gives us a chance to add up smaller and taking taking all my all my.
10:56:04 Okay.
10:56:07 Yeah, I will talk about elephant bifurcation exactly
10:56:14 where is did you have a point, do you want to do.
10:56:18 Well I maybe I wanted to ask the question a little bit differently.
10:56:23 So these enzymes act as single molecules other complexes.
10:56:30 Is there any cooperative phenomenon known in.
10:56:35 In this metabolic context.
10:56:40 Right. Right now we're we're we're we're talking so strictly so one enzyme at the time.
10:56:48 I'm not sure I entirely understand your question so let me rephrase. Are you asking how enzymes that translate protons look like.
10:56:58 Well, I guess that this could happen at any point in, in the chain. So both with channels with pumps.
10:57:09 You know, one can imagine
10:57:12 some, you know, complex as a few enzymes.
10:57:18 Acting cooperate know some interaction between an enzyme, and the pump you know that, you know, in order to fractional eyes this the proton we're talking about, one needs some sort of interactions were needs some cooperative behavior.
10:57:52 Yeah, I think I'm, I'm getting where you're what what Eric was getting at, you know, there are fluctuations so if you can get correlated fluctuations, then you can you can do all sorts of things.
10:57:55 Yeah.
10:57:55 Let me answer this, this question then maybe a slightly different way.
10:57:59 So, in, in, in microbial respiration you usually have several different enzymes that contribute to the proto motor force, so that that pump protons sodium ions, and the individual reactions associated to negative delta t values.
10:58:17 But not every reaction that's associated to a negative delta d value contributes to the proton pumping for all kinds of different reasons.
10:58:28 And in some ways, you can imagine that some of this left over delta G is picked up, then by other enzymes that that benefit a little more from other enzymes not translating.
10:58:40 And then as a result they can translate a proton, for example.
10:58:48 Yeah, so then suddenly causes of interaction the source.
10:58:52 Yeah.
10:58:54 I think I was, I was there was a question earlier. And it's important to always look at the energetics of the, of the whole chain of the whole metabolism, because this is the framework in which you operate.
10:59:09 For instance, let me go here and a question that I often received was okay you have a delta G of the classic mechanic genesis of minus 36 killer general promo.
10:59:24 And the amount of energy that's required to make an ATP is also around minus 30 minus 35 kilo little promo. So if you already invest in ATP here. How can you.
10:59:35 How can you still make make energy.
10:59:39 And the thing is that the delta G if you look so choco a to me saying it's like minus 70 kilo jewel promo because the energy that you invested here. You kind of get back an intermediate step.
10:59:53 So it's not so useful to always look at the end of the energies of the individual reactions, because they can can kind of cloud The picture on what's really going on.
11:00:09 Right.
11:00:09 Any other questions.
11:00:11 You know, I had a question on the number of ATP, that you showed it is quite not clear is it to foster it bonds, or is it to HTTPS, because they're different, right.
11:00:29 You're probably talking about, about this one then. Yeah, exactly this one yeah yeah I didn't talk about it further so ATP is idolized to a MP.
11:00:37 And this actually equals to ATP hydrolysis to a dp because you need to invest
11:00:49 only, only when you have the hydrolysis of Pyro phosphate.
11:00:54 Exactly.
11:00:57 joblessness Pyro phosphate and like that you yeah invest basically two ATP here. And I've, I've not talked about it much. I've said that part of the Pyro phosphate is highly realized that the membrane, but actually the majority of the Pyro phosphate is
11:01:12 probably had realized in the cytoplasm not contributing to the proton motive force.
11:01:20 Yeah, that's good. Thank you.
11:01:29 Again muted.
11:01:30 You're muted if you're saying something.
11:01:34 Sorry, is the psyche, it is a proton psychiatry of that Pyro phosphate as really known. Is it one proton or two protons Pyro phosphate
11:01:48 know we didn't determine it.
11:01:52 Now we didn't determine it but they're also soluble powerful spaces and they're also active.
11:01:57 So it's very clear that not all the Pyro phosphates goes to the membrane. Also, and I mean you know that because all the Pyro phosphate, for instance, make your DNA and RNA synthesis part of it needs to be Hydra less than the cytoplasm for DNA synthesis
11:02:13 to proceed in the forward direction.
11:02:16 So, it's a little tricky to just put Pyro phosphates and the respiratory chain, because it implies that all Pyro phosphate can be used for proton translocation, whereas this is actually not the case.
11:02:28 So not all the Pyro phosphates.
11:02:31 That is released here contributes to the proton motor force only a very small fraction. And that's actually why the change stuck geometry of ATP synthesis at the same time as necessary.
11:02:43 We. Yeah, we can't really prove that but in a way how we calculated it.
11:02:48 It is really necessary that this 80% It only takes three ions. Otherwise it's this metabolism cannot, cannot run.
11:03:02 Can you probably should move on.
11:03:04 Okay. Um, I don't know when our when our intro we do a five minutes, coffee break. Usually we haven't, another half an hour and then we'll take a break, okay that's fine.
11:03:18 Yeah.
11:03:25 Friends locator.
11:03:26 If you knock it out or something. Yeah, it's technically Yes, practically there's no genetic system from a fantasy data.
11:03:35 You don't know the genome really yeah yeah no we know the genome but there's just no genetic system.
11:03:43 Okay, I have the luxury we're with the cola I'm just thinking
11:03:47 about technically, you're right, if, if, if this was available we could check that. OK, thanks.
11:03:55 Okay then, I would like to move to the second part, where we'll talk about the math antigenic metabolic pathway map, and an anaerobic feminine and dynamic trick that Bennett already took away.
11:04:06 So that's going to be electron bifurcation.
11:04:09 And I have some required knowledge for this one. And I think someone already asked about Redux potential so I need you to know what delta G and easier prime is, and I need you to have a firm understanding what reduction oxidation is that already occurred
11:04:25 during the during the course so far. I see some nodding. Okay, that's great.
11:04:31 Okay, so let's get started so I I will now talk about authentic Genesis from hydrogen and carbon dioxide, so slightly different than the aesthetic plastic ones, I was talking about before.
11:04:42 And I've simplified this metabolic pathway to look like this.
11:04:47 So you have carbon dioxide is starting material.
11:05:02 And then there are stepwise reductions of the carbon dioxide, all the way down to meeting, and you see the Redux numbers are written above here so you get an impression of your of the Redux state of the carbon.
11:05:06 Um, let me see. Yeah. And of course, in order to reduce this to two molecule unit electrons or electron. Yeah, electron input into this metabolic pathway.
11:05:18 It's not further important for my talk, but I at least want to mention that the carbon that is reduced. It's actually not.
11:05:26 Yeah, diffusing freely in the, in the cell but the carbon needs to be bound to common carriers, and there are a number of different common carriers that methanogens us.
11:05:37 But as I said for the remainder of much pockets not important to, to know our understand how they differ.
11:05:44 What will be important is that, in the course of this, of this pathway.
11:05:50 Different electron donors are used, such as paradox and something that's called f1 a 20, and co. Nz MB.
11:05:58 And I will explain to you. And, yeah, in the next 15 minutes or so, why different Telecom carriers are necessary and how they differ.
11:06:09 So a few key questions that that I would like to answer in, in this part of the tutorial ry a different co factors used and how are they regenerate it has energy conserved.
11:06:22 What is limiting the rate of Genesis, and how can see you to be fixed, without the use of ATP.
11:06:31 So starting with the first the first reaction that's happening in this type of Montana Genesis pathway.
11:06:38 So co2 is is reduced to a formal group here.
11:06:45 When you look at the CEO to CEO Redux couple.
11:06:48 Then they either prime is very negative, it's around minus 500 million volts.
11:06:55 And that means that for as an electron donor to this reaction, you need an electron donor, that also has a very negative.
11:07:02 Yeah Redux potential, and there are not so many Allerton donors around in anaerobic metabolism, that actually fulfill this specification. And one of the few are small proteins called paradox ins that also have an easier prime of about minus 500 million
11:07:24 volts. And I think interestingly, I think most of you have probably encountered no DHS, our donor, which is the molecule that's my agents do not do not really us.
11:07:33 And that has a Redux potential of minus 320 million volts,
11:07:39 that there are some bacteria that actually do this very same reaction. so see you to reduction to this formal group, and they they use NIH for that. But because NIH has a much more positive Redux potential.
11:07:54 They require ATP to actually run this reaction.
11:07:59 So I think it's, yeah, what I want to show you here should appreciate that by the choice of Redux Redux carrier, you can avoid using ATP.
11:08:11 In a reduction reaction. If only the, the easier prime is negative enough, and actually.
11:08:18 Yeah, to be to be completely honest here, the easier prime of this carrier needs to be slightly below.
11:08:27 Below this number here.
11:08:31 In order for this reaction to run in the forward direction.
11:08:38 Then for the, for the two steps that come after here, the, the further reduction of former group to, to the methyl group.
11:08:50 The Redux potential doesn't need to be as negative, so you can use a Redux carrier that's, yeah that has a Redux potential a little higher than paradox and the when you look at the Redux couple here it's actually has minus 360 million volts.
11:09:06 Then I think just one interesting aspect, on this type of mechanic Genesis, is that there is within this pathway happening in the cytoplasm. There is only one reaction.
11:09:19 That's contributing to energy generation. And this is a methyl transfers reaction and this is something that's, yeah, maybe not unique but doesn't occur.
11:09:30 So very often and measurable metabolism, said that. And because this is not a reduction reaction had this is really a matter of transfers for action that this contributes to the, to the PMF.
11:09:42 When we look at the energetics of this particular reaction, which is written down here, you'll see that the method, this, this particular method of transfers for action is associated to a very negative delta t value, which is why this reaction translocated
11:09:56 one to two sodium ions to the outside of the cell.
11:10:00 And this is really the only the only step in this math energetic process that's associated to energy conservation. So, there's two sodium ions that are translated to the outside of the cell.
11:10:13 And this is what the cell gets for reducing once you to molecule to one methane molecule.
11:10:25 Then there's a third electron donor is Cohen's MB.
11:10:29 And it seems that this reaction, yes even less hard from a Redux point of view because the Redux potential of this co enzyme be and yeah this is a particular had di sulfide not so important to remember exactly as minus 120 million volts.
11:10:47 So it's even yeah less negative.
11:10:51 I think in the key questions that I posted. I also said, Okay, what is limiting the, the speed of the reaction of this metabolic pathway.
11:11:02 And that's actually the enzyme down here, which is called methyl Kwanzaa memory duct tape.
11:11:08 It's a, it's a very interesting enzyme that's doing something that's biochemically very hard, which is release of the methane molecule.
11:11:18 And you can. It's also not a very good enzyme, so it's very very slow. And one of the ways in which we see this is that methanogens are actually packed with this enzyme.
11:11:30 So in one in one with antigen we've recently done some proteomics, and we saw that 12% of the, the enzyme of the cell arm ethical ends and Redux case.
11:11:41 So the cells. They really yeah, there will be charged themselves with this enzyme.
11:11:47 In order to make this, this reaction happen at A.
11:11:51 Okay rate. So this is really not a very good enzyme and some researchers say that methyl QNZMM reductive is the most abundant enzyme and the anaerobic world, because it's so bad that mythology, and also anaerobic my fantasy tropes needs lots and lots
11:12:09 lots of enzymes. And then this tutorial I'm not really talking about experiments but but as I said I'm an experimental microbiologist whenever we do anything with managing some authentic tropes Mexico and reductive totally screws up our experiments if
11:12:32 do transcriptome mix all you see is mefloquine reductive and drivers arms and almost nothing else, if you do anything with proteins all you see is math grammar doctors and nothing else. So, yeah, just to indicate that, that also by looking at things like
11:12:38 proteomics at transcriptome things, you can actually say something about what maybe the bottlenecks in the metabolic process are.
11:12:48 So with this I've answered some of the questions that are posed so I shown you different co factors, I showed you how energy is conserved. I indicate what is limiting the right of McKenna Genesis, actually, this crappy enzyme that's, that's very slow.
11:13:03 And I also wanted to show you how you too can be fixed, without the use of ATP. Whereas, Yeah, yeah, without the use of ATP.
11:13:13 And now I want to come to this last part, how are co factors regenerated
11:13:21 so authentic Genesis, starting with co2 is using hydrogen as an electron donor.
11:13:29 And yeah hydrogen is naturally then used to regenerate the core factors. So paradox and effort 20, and the sty selfish here in order to generate the coins and be.
11:13:42 And for some of these cool factors, it's really easy. So when we look at the standard potential of hydrogen, it's around minus 420 million volts, we have F for 20 at a potential of minus 360 million volts.
11:13:54 So everything's fine it's very easy to actually reduce for 20 with hydrogen. And this is exactly what the cells are doing. They use the hydrogen nice.
11:14:05 They oxidize hydrogen and then reduce a 420.
11:14:10 However, I said it's important that paradox is used as a donor because otherwise you would need to use ATP and and managers don't have, they don't have enough money to actually use FTP but they also just don't do it.
11:14:23 If we then take hydrogen as a donor at minus 420 million volts or minus 414 and paradox in essence accept that as minus 500 million volts, you immediately see that is thermodynamics unfavorable.
11:14:37 And this can't really happen.
11:14:41 Yeah, because it's found that someone dynamically unfavorable, you have you have delta G that's positive, it's an organic reaction, it's not going to work.
11:14:49 So this is clearly not possible.
11:14:52 There are a number of ways that have been known for a long time, how cells can overcome this. And one I've, I'm going to show you in the next slide. And this is to do this at the membrane and use a specific enzyme, for instance, a specific hydrogen is
11:15:12 that called ech type. That is that is reducing the paradox and with hydrogen, and it's, it's running this reaction with, with the energy from the proton motive force.
11:15:22 So it's taking protons from the outside to the inside.
11:15:27 And this energy that is then released by the proton in flux is used to drive this reaction forward.
11:15:34 Of course if protons are used for reducing paradox and that cannot be used for ATP synthesis. So the cells really lose ATP so they lose proton motor force and by this day, they they they pay back some of the ATP that they could have generated with this
11:15:51 with these protons here.
11:15:55 However, in Massena Genesis, this is not really an option, because as I've shown you in the in the last couple of slides only wanted to serve your mindset translocated pr, pr method produced.
11:16:07 And if, and they would use one to two protons to actually drive this reaction.
11:16:13 And that would lead to no energy jet gain off the off the entire process. So even though this enzymatic machinery is technically possible. Practically, it can't work because the mathematicians just don't have the.
11:16:27 Yeah, they just don't have the money to spare, they don't have the one to two protons, that they would need in order to run this reaction.
11:16:38 So, then we have to look again at cytoplasm options to how to do that.
11:16:44 And, yeah, I've shown you this image before I've said this is summit and then if the unfavorable it's not possible.
11:16:50 You could also run this by coupling this to ATP hydrolysis, but, again, this is not possible from the mythologies because they don't have as much ATP in order to do that.
11:17:02 So how can actually obtain reduced paradox and.
11:17:07 And this is the feminine electric.
11:17:10 That's called election bifurcation.
11:17:12 So what is happening here.
11:17:15 There's another enzyme that's associated to the hydrogen as.
11:17:18 That's, that's splitting this dice alpha that's also current you're in within a Genesis, to the, to the mono sulfites, and what the electron bifurcation basically does is that the couples in an end organic process and this is this is this one here so
11:17:36 electron transfer from hydrogen to paradox isn't it couples that to an ex organic process, and the economic process is to transfer from hydrogen to the hetero di sulfites.
11:17:49 And you also see that the Redux potential difference between the hydrogen and the hetero dice our fight is much bigger than the Redux potential difference that this electron needs to be kind of pushed up hill, for, for the production to be reduced.
11:18:07 And yeah by.
11:18:10 So by splitting the electrons so by bifurcating the electrons.
11:18:14 The cell can actually obtain reduced her toxin without the needs to either dissipate the proton motor force or add any ATP to this reaction.
11:18:28 So the
11:18:32 that the energy that's that's contained in this are the potential energy that's contained in this Redux reaction here from hydrogen to to the Hydra dice how fight that can be harnessed for for pushing the electrons upheld the paradox and, and like this
11:18:53 but this really purely possible for for electron transfer between, between different electron carriers.
11:19:18 So, to summarize the sole misunderstanding pathway. There are two hydrogen cases that generate the CO factors, the first one very easy.
11:19:28 Transfer transferring electrons from hydrogen to, co factor for 20. And the second one slightly more complicated hydrogen as that's doing electron bifurcation using electron from hydrogen, then they go downhill to the hetero dice alpha that's producing
11:19:44 this last step here, so that Korean zombie and then co enzyme and that's used here are regenerated, and on the other hand, the energy that's released by this Redux reaction can be used by this very particular enzyme to produce reduced paradox and that
11:20:03 then is used in this upper part here.
11:20:07 And like this the cell manages to recycle all the CO factors that it needs.
11:20:13 And it gets out with a net gain of the one to two sodium ions that are translated in this method transfer is reaction.
11:20:22 So to sum up this part.
11:20:27 Call factors, and with us I mean particularly electric carriers, they need to be regenerated so there needs to be a closed loop in the metabolism.
11:20:36 Otherwise the metabolism cannot run at. However, after closing the loop there needs to be a net gain of ATP are translated ions.
11:20:51 The Redux potentials of the CO factors, actually determine the way in which they can be regenerated.
11:20:57 So not every coach there factor can be regenerated in the same way as I've shown you with the hydrogen aces and individual enzymes can be serious bottlenecks for entire pathways.
11:21:09 Even though you wouldn't I may not have thought that necessarily metal detectors is the limiting step, it still is because it's enzyme ology is very hard, whereas the, the family dynamics of that step, or maybe not that hard but just the enzyme ology
11:21:24 of its.
11:21:34 Yeah. And here I'm, I'm happy to take any questions if there any questions on this part.
11:21:41 Looks like we can do the third part.
11:21:46 Okay, Maybe now we can have a few minutes break.
11:21:51 Sure.
11:21:52 Be the, the research talk, oh yeah that's true, that's true. So then why don't we have come back here at 11 3032 West Coast time which is what is it.
11:22:12 Seven 738 30 right. Yeah, it's your time.
11:22:18 Okay. See you in 10 minutes. Yeah.
11:32:13 Okay. Okay, why don't you go ahead with your research talk.
11:32:24 Okay, yeah, I'm glad to. So, I want to talk about novel discoveries and anaerobic Nathan oxidation. So, yeah, I'm actually going to talk about the reverse reaction of what I've talked about in the tutorial.
11:32:42 And to start off, I'd like to introduce you to the fascinating world of media and microbiology. So, Mr microbiology is important and all kinds of different ecosystems.
11:32:53 So meeting is really important in via energy production and here, particularly in bio gas plants, where methane is producing anaerobic digesters, and then can be burned up put into the into the guest grid.
11:33:07 And it's been for electricity production or friends in the gasification of fossil fuels, where coal coal or oil residents that are yeah down in the earth can be classified to me thing that can also either be burned directly for electricity.
11:33:22 I'll be putting into the guest grid. If the concentrations high enough.
11:33:27 Then secondly meeting is very important in the greenhouse effect. So, the, the three guesses to you to me thing and enter all make up the majority of the.
11:33:40 target and climate policies, because it has a relatively short residents time, and the atmosphere, unlike co2 that stays in the atmosphere for for hundreds of years, Mithun has so much shorter residents time off.
11:34:02 Yeah, actually I forgot I think around 20 years. So if we do anything on the methane emissions we can directly impact the radio to forcing and Nathan is producing all kinds of different environments.
11:34:21 I see someone put some, some something in my on my slides here.
11:34:26 Okay, I'm sorry.
11:34:28 Yeah, this is an unfortunate thing of, you know, you can edit. And actually, I'm not sure. Maybe the KTP folks know how to remove these labels. Yeah, yeah, don't worry.
11:34:41 And then last but not least, Nathan is important a number of feedback processes. And one of the feedback processes within is really important is the Arctic amplification.
11:34:53 So, I'm sure all of you are aware that permafrost is melting and actually in the Arctic, where the pump power for us is melting the climate change is this proportionally strong.
11:35:07 And in addition to that, when permafrost is melting, then, then meat then is one of the major greenhouse gases produced so there's of course also see you to produce an attack a little bit of intro, but there's also a lot of meat and produce and I'm sure
11:35:20 most of you have seen the, the pictures from them across lakes where you have these frozen bubbles that you can, yeah if you, if you if you pierce them you can actually ignited anything comes out.
11:35:31 And because Mithun is one of the primary products in permafrost melting.
11:35:39 It actually disproportionately affects this Arctic. This Arctic warming and the permafrost warming.
11:35:47 And my research group.
11:35:49 I am interested in. Yeah, the ones that I've discussed with you in the first part that produce the biogenic method from co2 and number of other small see one compounds.
11:36:01 And on the other hand, also in the military troops.
11:36:05 And it's interesting to notice that the mythology trips oxidized 50 to 80% of the produce methane before it ever reaches the atmosphere.
11:36:15 So we never measure that.
11:36:17 But that also means, as you can imagine that the interplay between methanogens and Mussina troves is really important in determining how much meat and gets admitted into the atmosphere.
11:36:29 So in my research group we try to understand the biochemistry, the physiology and also the interactions between methanogens and droves.
11:36:41 So when we look at the Convert biological conversion of meeting, and particularly at the mythos of sin, there are two big groups of mythology proofs, one are the Arabic percentage shirts and these bacteria and they use oxygen for me Ethan, Ethan oxidation
11:37:05 Yeah.
11:37:06 Work on the methane molecule, but they're also the anaerobic troops, and these are the ones that I'm, I'm most interested in.
11:37:15 There are a number of different anaerobic McKenna troops, that, that are described in literature. And actually, this field of anaerobic Montana trophy it's not, it's not all that old so I think the thirst.
11:37:29 Yeah, real real proof which kinds of micro organisms would perform these reactions came around 20 years ago, when in microbial mats at the bottom of the ocean anaerobic misanthropic archaea were discovered, and that abbreviated enemies that live in central
11:37:47 fi with South introduce us to perform selfie dependent montana montana trophy.
11:37:54 And interestingly, these enemy archaea are very closely related Timothy antigens, and it appears and I'm going to show this to you and one of my next slides that they use some authentic Genesis pathway in the reverse reaction in order to oxidize the methane
11:38:09 molecule tissue to.
11:38:13 Then in the order of how they were discovered. They're also bacteria that perform anaerobic Montana trophy. And then I tried dependent so they they oxidize me thing with my tribe and I'm going to show you, also in one slide how a little bit how they're
11:38:26 doing that.
11:38:27 And then they're the enemy Trudy Montana troops that are also called Madonna parents that perform nitrates dependent McKenna trophy via reverse Montana Genesis, so also these authentic trips are very closely related to to manage ins.
11:38:45 And interestingly, they don't have an obligate material partner, such as the consortium of the self independent Montana troops.
11:38:56 The area they are also called Montana parents and recent in recent years, we've also come to learn that they are able to perform a Santa trophy with oxidizing metals so metal oxide, like Iran and manganese, and I will focus my talk on these as these other
11:39:17 ones that we that we grow in our laps.
11:39:18 And to start I would like to explain to you how we actually first enrich those Montana troops and where they thrive.
11:39:28 So on the left hand side here you see a map of the Netherlands.
11:39:33 As you know, the Netherlands are small country.
11:39:36 They're very densely populated and close to the coast. And basically the Netherlands are one big peatlands, there used to be Pete everywhere.
11:39:45 And of course, much of this Pete was was excavated, which is one of the reasons why the country is really clustered with ditches, with ditches and candles so there's a lot of water around here also of course because we are low in the.
11:40:04 Yeah, they have this common English. Yeah, We don't have a lot of innovations.
11:40:12 Yeah, we've seen other things it's really getting late for me I'm sorry.
11:40:17 Um, yeah so the country. It has a very organic rich sediments, it has a lot of water, and at the same time it's super densely populated. And also there's a lot of agriculture going on.
11:40:29 So, lots of cows, lots of people, lots of water. So there's a lot of nitrates in our groundwater and also the, especially the surface waters, due to agricultural runoff.
11:40:43 And at the same time because there's so much organic loading in in the ditches and the kennels. There's also were very quickly, an oxymoron, and high methane and the sediments.
11:40:56 So you can imagine if you look for an unknown micro organism that consumes nitrate and methane, then the Netherlands are kind of the ideal place to go to.
11:41:06 And there are a number of something sites that we went to a number of times like the 20 Canal the polar and the blue summer Haider, and the microbes that I'm actually talking about, we enriched from the 10th Canal, which looks like this.
11:41:20 And if you've never been to the Netherlands, then. Yeah, I think, 80% of the Netherlands look like this only with less trees, so there are too many trees.
11:41:30 So what did we do we, we added sediment from the tensor canal to bioreactors and you can see such a director here. It's an anoxic bioreactor, that is run with mineral medium that contains nitrate and in this case also nitrates and methane and has biomass
11:41:50 retention so it's a member and director, and the area and medium is kind of pumped through this director.
11:41:58 You set up such a director, you wait for two years and then you find an enrichment culture of our key and bacteria so you see a fluorescence in situ hybridization micro graph here in Wistar key or a colored and pink and bacteria colored and green.
11:42:14 And when we analyze this enrichment culture, we found that this culture would couple meat and oxidation to nitrate and nitride production.
11:42:26 And I'm actually cutting a very long story short, and this this even happened before I really joined the project. So if you take this director, and you add my traits and meet them to it, then you enrich for the green folks here the bacteria, and if you
11:42:41 add nitrates and meat then you enrich for the UK here briefly talking about the bacteria.
11:42:49 It was later discovered that they affiliated with the NC 10 find them. They're called my title Mirabilis, and they use a peculiar way of metabolism to oxidize the meat and molecule, because they use method on oxygen as from youth and activation yet they're
11:43:05 strict on ropes. So they take the nitrates, they reduce it to nitric oxide.
11:43:12 And then they this mutate the nitric oxide to end to an O two, and actually for 15 years we've been looking for this enzyme and we still are not 100% sure which one it is.
11:43:24 And then they use the oxygen that's liberated by this this mutation reaction for activation of the methane molecule with amount of oxygen as that's strictly dependent oxygen.
11:43:36 And then the oxidize this to co2.
11:43:47 I will talk about the archaea. So, These are here. When 9% of parents.
11:44:01 Parents night reduces, and they use the reverse Montana Genesis pathway for from Ethan activation so they use the resurrection of of a really crappy enzyme methyl cleanse and reductive that's that even becomes much worse if you turn the yeah if you let
11:44:05 it run in reverse. So, yeah, so this is really not not going very well, but it's apparently enough for them to to grow at all.
11:44:17 And at the same time, they couple. The couple of this electron generating method oxidation pathway to the reduction of nitrate and I tried, and also a little bit of ammonium.
11:44:29 And basically, this was the point when, when I picked up this, this project.
11:44:34 So we wanted to learn. This was kind of what we knew about them microbial pathway map and we wanted to learn how exactly they actually perform perform the these reactions and how they can make a living from oxidizing meeting with nitrates.
11:44:52 So, to make a start, we. So we wanted to start characterizing this enrichment culture. And just to, to make sure we are on the same page. So, these anaerobic managers are not yet read and pure culture.
11:45:08 We haven't managed us and no one else in the world has managed. We are very fortunate that by now we have enrichment cultures that are enriched up to, like, 50% in Montana paradigms and 50% other bacteria.
11:45:23 And so the question is how can you learn from such a mixed culture, what one particular microbial players doing. And I know that you you've heard a lot of other talks on microbial consortia and and enrichment cultures, but just to make clear we don't
11:45:36 have pure culture so we're, we're very limited and the type of experiments that we can actually perform.
11:45:43 So, initially, we, we took the reactor biomass, we sequence the DNA and RNA to perform your meta genomes and meta transcript tomes we obtained genomes and expression patterns, and by using these data.
11:46:07 So to start, on the left hand side I'm showing you basically what I've shown you the tutorial just the other way around. So, This is reverse Montana Genesis.
11:46:15 It starts with me thing in this case.
11:46:19 And then the meat and molecule is oxidized all the way down to carbon dioxide by the same set of enzymes that also used in math engines, you immediately see that there are a couple of considerations here because first of all, you have the methyl transfers
11:46:36 for action. That was energy generating in the mythologies, and here it's energy dissipating.
11:46:42 So actually, there's energy invested in this reverse Montana Genesis pathway in order to let it run.
11:46:49 So clearly, just reversing that metabolic pathway leads to delta t that's higher than zero, so something else has to happen and a metabolic pathway in order for it to be energy generating.
11:47:02 Obviously, as for now the Redux loops of the CO factors are not closed.
11:47:22 The nitric production part is not dead yet included, and that cleaning needs to be included for yeah for the compensation of the energy investment here, and also some extra energy to be to be conserved.
11:47:20 So these are a couple of driving questions for our research.
11:47:25 And then I would like to add something else because I, I sometimes feel people that working with uncultivated are hardly know microbes, feel a little misunderstood by the greater microbiology community.
11:47:36 And that our genome contains like 50% hypothetical proteins.
11:47:41 So, there are a lot of unknowns there. So when you, when you generate metabolic pathway maps, you have to you have lots of gaps and you actually a lot of the pathways are unknown unknown unknowns.
11:47:55 So not only do we not know the pathway, we might not know the enzymes for that because they are hidden in the in these 50% hypothetical proteins.
11:48:02 So part of our research and that's actually what I'm going to talk about in the second part of this, this research talk is going to be on unraveling some of the functions of unknown unknowns.
11:48:17 But first country to have the driving questions for, for this particular project.
11:48:22 We asked the question is how do the electrons actually reach nitrates and how are called factors recycles in order to understand this complete metabolic pathway.
11:48:35 So here. Here I'm showing you the energy generating parts, coming from reverse Montana Genesis tonight. Right.
11:48:42 And I don't want to talk in too much detail about the different enzymes that are involved.
11:48:47 But maybe to start with one of the core factors, the F 120 and Howard is recycled. So we found some enzymes in encoded encoded in the, in the, in the genome and also transcribed that we know very well from from with antigens, which is an FFPOFQO complex.
11:49:07 And then we also found some other enzymes that we know from nitrate reduction.
11:49:14 For instance, in rgh here on Earth. Ah. And interestingly, when we look at for instance in rgh here, we find that part of. So in RGHRR no natural production basis but usually they, they, yeah they come together with certain member and anchors that we
11:49:32 that we didn't find an hour in our genome, and instead we found a number of hypothetical proteins that made us believe that they form a novel type of nitrate productive with some Hincapie oxidase subunits not age, which is actually part of a different
11:49:49 nature introductory is usually an a protein that's found also in other nitrogen indicators of archaea, that is a nice name or seven, but it's a B type side of Chrome.
11:50:00 We found a risk aside from B complex, that also has additional subunits probably some extra seat upside of crumbs.
11:50:08 And even though we have never measured this are yeah yeah we've been unable to measure this then most likely. Some of these enzymes are associated to proton translocation such as such as the risk side, it can be complex and the Fq are complex, and we
11:50:24 are not so sure but about this in our G h here.
11:50:30 not so sure but about this in our gh here. Okay wanted to talk. So now I've shown you basically how nitrates possibly can be reduced and now I want to come to the core factor recycling because it's important that the Redux loops are closed.
11:50:41 And for the effort 20 I've already indicated that this enzyme here is doing the trick, but also now coming to the Cohens MB and Cohen's mm.
11:50:50 So just like some of these troops contain an enzyme that can potentially do that that's associated to the, to the membrane.
11:51:02 However, when we now look at the energetic so I saw the, the either prime of the Colby co M and the hetero dice alpha this minus 120 million volts, and usually methanogens use the electron carrier I'm a fan of fantasy and the membranes that has an easier
11:51:19 prime of minus 180 million volts.
11:51:23 So clearly when elections should move from here to there energy would have to be invested, which is why we hypothesize that Montana fantasy is not part of the, of the membrane, in anaerobic Montana tropes.
11:51:39 If you look at Mr Quinones however that are electron carriers and most materia.
11:51:45 Then they have a much more positive freedoms potential and the electron transfer from here to there could be energy generated at least you don't have to invest energy.
11:51:54 And when we looked further. So after we've we drew up these hypotheses we actually looked into their genome and found that the clip complete men a queen and biosynthesis pathways presence.
11:52:05 In addition, we also did some did some HPC analysis of the enrichment culture, and it's a little hard to tell because as I said, we only have 50% and 50% other bacteria.
11:52:17 But we, we failed to, to find Montana fantasy in in these enrichment cultures and found lots of different Quinones.
11:52:25 So, so we believe that indeed this anaerobic Masonic chefs use meta Quinones as their as their cytoplasm or as their membrane electron carrier.
11:52:37 In addition, we again come to the problem of the paradox than that, but now the other way around that reduced fat reduction needs to be oxidized, and we looked for paradoxes oxidizing enzymes in the membrane are in the, in the genome, that would be encoded
11:52:52 in the membrane just as the ones, as I've shown you in the, in the previous part, and we couldn't detect those.
11:52:59 So, when we then looked at this complex that intelligence is electron bifurcating, we actually think that it's electron complicating, which means that some of the core coins and bullion coins mm is combined with a paradox and to reduce co-factor effort
11:53:19 20 that can then be recycled at the membrane.
11:53:24 So what I would like to illustrate with this part here is that the use of. So, the use of mechanical factors results of thermodynamic challenges for me archaea that they circumvent with repurposing of the enzymes or even with a change and co factors to
11:53:43 make to make this metabolism work out.
11:53:47 So to sum up what we learned from the message nomics and the meta transcriptome mix, we found that a complete reverse pathogenesis pathways and coded.
11:53:56 We also found a complex set of plasma membrane bound electron transport systems. And that's something that I actually haven't talked about so much but we found an unusually high number of seats outside of Chrome's around like between depending on the
11:54:09 strain that will be we were looking at between 40 and 50, different setups either Chrome's. And just as a comparison the related methanogens those that have side Chrome's have maybe one or two, or maybe five.
11:54:21 So, and these managers have like 15, so we think that seed upside Chrome's have a very important function, and the metabolism of these archaea but we are not sure what they do.
11:54:31 And also I would like to emphasize that McKenna trophy is not just the reversal of methanol Genesis but additional reactions and alterations are required in order for these yeah for this metabolic pathway to work.
11:54:47 And I'm jumping forward with this, because this is what we did in 2015 16.
11:54:53 So we've done genomic reconstructions. And when I say we, I mean, previous PhD and postdoc in my lap.
11:55:10 A number of people currently in my group are working on the physiology and the micro interactions of the Montana pardons with other with other microchips.
11:55:10 We were lucky enough to actually be able to do some biochemistry so enzyme measurements and also complex some analysis, which means that we retrieved all the protein complexes, from the cells and could confirm some of our genomic and transcriptome make
11:55:28 predictions, but actually looking at the yeah protein complexes that form in the cell.
11:55:35 We are now. Also looking towards the bio technological applications of these initiatives such as electricity production and biochemical systems and also the application of anaerobic McKenna trips and wastewater treatment, were uncertain waste streams,
11:55:55 And also, nitrogen as compounds and these onions can maybe be used for doing this. And the remainder of my talk, I would like to talk about this project here in which we look at electricity production by anaerobic Montana tropes.
11:56:10 So that's kind of the applied part but from a fundamental science point of view, we aim to learn something about the extracellular electron transfer.
11:56:20 When I say we, I mean, my PhD lane is currently working on this.
11:56:25 So, what are we actually aiming at, so I said that there is an electron generating reaction here, the reverse mechanic Genesis part. Then there's an electron transport chain that reduces and I tried to I tried, and our main question was whether an electrode
11:56:44 could replace the electron accepting reaction here, and this is particularly interesting, because these trips are also found in and sediments, where they probably do extracellular Elgin transferred to metal oxide.
11:57:01 exactly they, they can do that, and by by setting up such a by electrochemical system. We want also wanted to kind of invest in a kind of experimental setup in which you can actually study these processes.
11:57:21 As I said, there are a lot of, so this is the, the image of the cell, you have the reverse McKenna Genesis and the cytoplasm.
11:57:30 And as I said they have lots of seed upside Chrome's and other researchers have also indicated that probably the setup side of Chrome's are used for the electron transfer from the inside to the outside.
11:57:42 So, our hypothesis is that the seed outside of Chrome's mediate the electron donation to the bio and out.
11:57:52 So the system we are using is such an H cell that maybe you've, you've also seen before it has two chambers. When experiments a chamber here, where the biology happens and one chemical chamber here, where the catalytic reaction just chemically happens.
11:58:07 And these two chambers are separated by a by a semi permeable membrane.
11:58:13 And you can see this big black thing here that's a working electrode to carbon cloth electrodes, that is then colonized by microbes. So we, we add microbes from our enrichment cultures into this into this chamber here.
11:58:30 Then we apply a potential with the potential steps. And we did that in our first tries at zero volts.
11:58:40 We, we add medium to this chamber but the medium is devoid of any electron acceptor so it doesn't contain any nitrates, it only contains some mineral salts, and we bubble.
11:58:52 This of course anaerobic Lee with me things so the idea would be that, at the working electrodes you have, hopefully Masonic shrubs colonizing the carbon cloth material.
11:59:03 They oxidize meet the interests you to deliberate electrons and the elections, you can measure as a current our current density on this electrode here.
11:59:13 And I want to show you some of the results that we've obtained on the electric chemistry and also the microbial community growing on this electrode here.
11:59:24 So, we set up the system, and of course one of the first experiments that you aim to.
11:59:33 Yeah. And so one of the first experiments that you're doing is actually one in which you aim to see that the current that is produced is dependent on the substrate that you add so in our case me thing.
11:59:45 When you know look at this church here you see the current density on the electrode on this excess and the time here.
11:59:56 And at this. The Seretse points, we actually stop the myth and influence you can see a small but significant drop and currents that remains kind of constant and when you then restart the myth and inflow, the current picks up again.
12:00:14 Ss a kind of complimentary experiments. You can you can add more me send to the system.
12:00:23 And then, yeah, when you add more meat, then you should also see more current. And this basically has to do with, with a metal coins and reductive, that is not saturated under the method concentrations that we add in our experimental setup, but if you
12:00:37 had more methane. Yeah you clearly see that that much more current is produced.
12:00:44 And then when you do pressurize the system so you, you get off the over pressure off the meeting from the buyer electrochemical system, you'll see a drop in and current production.
12:00:54 So with these experiments we could see the show that the current that we produce is Smith independence.
12:01:02 Now I want to talk about the macro community that we found at the bio anode, and then our column.
12:01:09 And this was done with metagenomics so we sequence the whole yeah biofilm on the bio and out.
12:01:15 And we also sequence that in our column that we added to the biological, chemical system.
12:01:21 And what you can see here is the abundance of the different microbial microbial species are there different genome set say, both in an Oculus and the biofilm.
12:01:33 And what you see is that, yeah there's a big red bar here, that becomes even bigger in the biofilm, and this is our. Yeah, our main within a parrot and strain.
12:01:42 That's, that's actually enriched at the bio and out so so we get more more within a period ends on the bio and then we have in the UK column.
12:01:51 We also have a number of other Montana Paragon strengths in there so then called number two and number three, but they had much smaller abundances and they also their abundance doesn't change between the inoculation, and the biofilm.
12:02:05 It's important to to mention that we only ran the biochemical systems. So far the results that I've shown here for eight days, and the doubling time of methanol burdens and the ideal conditions is 10 days.
12:02:20 So we didn't really expect that they would grow much because of the timeframe of the experiments.
12:02:26 But still you see an enrichment so a selective colonization of the bio anode.
12:02:41 And so, and then secondly what's maybe not so obvious to see here is that we didn't find any other, like well known candidates for electric active bacteria in there, which gives us a good indication that indeed Montana paradigms is the main the main microbe
12:02:49 that's that's producing current here.
12:02:54 And yeah, this chart is a little complicated to read. So what you can see here are the different, the different genome, the different genomes that we recovered from, from the Bible from the bio nodes.
12:03:09 And there are different things plotted here, the number of seed upside Chrome's because seed upside Chrome's are oftentimes associated to electric activity of micro organisms.
12:03:21 And the abundance of the microbes and then our column and this light blue and the abundance on the biofilm in this dark blue, and just by correlation we are kind of searching for those microbes that are high end seats left side of crumbs, and also high
12:03:35 in the biofilm. And you can see that one.
12:03:39 the biofilm. And you can see that one. One jumps out to you, which is, I'm a fan of burdens, and most importantly for those of you that are that are familiar with electric of micro organisms.
12:03:47 We did find Geobacter in our in our curriculum and also on the biofilm, but really at all point 2% abundance of social very low, and it became even lower on the biofilm, so it died or it did not select if the enrich and not multiply on the biofilm, even
12:04:05 though it has the repertoire to to be electric active.
12:04:11 So also from this analysis we we think that this is our main electric microbe, and currently we are we are setting up transcripts omics experiments to to see which of these, sit upside Chrome's are actually differentially expressed on the biofilm versus
12:04:27 the anarchism.
12:04:31 So to summarize also this parts with our experiment we've given a proof of concept that methanol paradigms can be cultivated in the biological, chemical system and can generate currents.
12:04:42 We saw that the current production is Mithun dependence. We also did some extra experiments that that didn't show you that that indicated for instance that 1313 Nathan is actually converted to touching co2.
12:04:58 And we looked at the microbial community and found that Mr pardons is a major player in this ecosystem.
12:05:05 Yeah and but with this I'd like to close I'd like to thank a number of people that have been involved in these projects and specifically, Elena Steph you set up the biochemical systems, and the number of collaborators that we're working with from Virginia
12:05:18 University and vessels.
12:05:21 And I'd like to thank you for your attention and I'm happy to take any questions.
12:05:30 Thanks so much community yeah this was exciting story.
12:05:47 Um, I actually I had a question because I didn't know the electrochemical experiments. So,
12:05:46 the current density was pretty low, but I'm wondering still the do tight rate. The mo download potential to see what the threshold is when a method oxidation actually starts.
12:06:02 Yeah, so we we actually did that.
12:06:05 We did that in potential scans, with the biochemical system that was running.
12:06:11 And we found that the potential when, when it when it turns, so when the election donate and accepting reaction turn is around minus 200 million volts, which is kind of close to the minus 244 the methods you to couple.
12:06:28 But on the other hand you could always argue is also kind of close to the minus 280 for the organic compounds you to couple.
12:06:35 But we think that there is some indication that indeed. Yeah, so we did the tetra ation and we see that it's it's close to our kind of expected.
12:06:43 Flipping points.
12:06:47 And if you then sort of convert the anode and cathode can you actually produce nothing.
12:06:56 It didn't.
12:07:04 Yeah, I mean, that's what we saw, we saw we saw that it really, it really flips. So then it would mean that indeed we see the series, I mean this getting too technical, well, why don't we do this.
12:07:19 So, I had to say. I've now shown you kind of our first experiments because for for this for this setup we have a complete experimental sets.
12:07:29 Then Corona happens, and we had to stop all this and also the community in our bioreactors changed so Elaine had to kind of wait for a year before she could Restart Now she's restarting these experiments and we see much yeah we see higher current densities
12:07:43 also.
12:07:46 We can either because our, we have a different strain That's better.
12:07:48 But also because we've kind of tweaked a number of the. Yeah, a number of the factors. So now we have higher current densities of like 5050 or so now I think I've shown you something with five and 10.
12:08:02 So we are getting a little bit higher but it's not like with acetate where you get five hundreds. Right.
12:08:11 So please.
12:08:13 Further questions, thoughts,
12:08:17 maybe even going back to some of the discussion points that we had in the in the first part burnout.
12:08:25 I would like to come back to is this electro chemical coupling.
12:08:31 What is really the rate limiting step in that I had expected that the electron transfer from the cells to the electrode surface would be the limiting step but then I was surprised to see that if you increase the methane concentration you get a higher
12:08:55 electron flux.
12:08:56 Yeah.
12:08:58 Would you assume from that, that actually the electrodes are entirely covered, and there's no free space on that and only the electron flow from the side of the methane is really limiting, can you do kind of a stoic geometry of the incoming methane towards
12:09:20 the total electron flux.
12:09:33 And there are several questions in one, and now I'm, I just deleted one of the slides that I had because I thought it was too complicated but now I regret that. So maybe first answering to the right, limitation and the increase of current that we see
12:09:42 Mexico and aquarium and Redux as has a km for me thing of like $12 million or so. It's ridiculous. It's so high that you will only achieve chief saturation of Mexico and productive.
12:09:55 If you go to really high pressures, which means that if you look at the kinetics we're at the very very very low end of of the like we're not even.
12:10:08 We're not even close to km.
12:10:10 And that's why whenever you work with anaerobic myths and truths, if you just improve increase the method pressure, it will always be better.
12:10:17 And that's a particular case because, because of metal kinds of and reductive. So we clearly expected that that that would increase.
12:10:28 By the way, another thing that we are considering is lowering the temperature because lowering the temperature will increase the methane solubility, but at the same time, it will work against enzyme enzyme velocity.
12:10:40 So we are still not sure what will happen when we do that.
12:10:44 Second. Second point that you that you touched upon was colonization of the electrode surface. So we see that the entire electrode surface is is colonized.
12:10:55 There's no free space.
12:10:58 So, but of course it's not only colonized them by Montana paradigms it's colonized by other microbes as well. So it's a little hard to say how we can really increase colonization, and one.
12:11:13 There are two experiments that we are currently running to, to kind of get insight into that.
12:11:15 One is that we are running long term experiments, and we're now at 10 weeks, because then we have some chance that Madonna paradigms actually grows, and does, doesn't just like colonize.
12:11:28 And the second is that we are that we are taking the electrodes, and transferring it. Like we let the biological, chemical system run.
12:11:38 And then we take the electrodes and put it into a new bio electrochemical system without dead cells and trying to kind of dilute the cells that are not electric active and increase those cells that are electrically active.
12:11:54 And I think there was one more thing I want to say but I forgot, because I think you had one more concrete question. Yeah, it was towards historic geometry but I understand if you have to apply very pressures, then of course you cannot really measure
12:12:10 how much methane has been taken up all the chemistry.
12:12:16 We.
12:12:17 And that was kind of surprising to us because what we see is that only
12:12:23 20% of the meeting that are consumed are converted into current and 80%.
12:12:31 Not so.
12:12:52 And there are a number of things that probably happened to these 80% meeting that we don't find back in the current
12:12:42 co2.
12:12:44 We. Yeah, we can't really quantify co2 because it's a co2 buffered system. So we do find co2.
12:12:53 But we cannot directly.
12:12:57 We cannot directly say the sky cometary exactly because of the way the the biological electrochemical system is set up. So that's a that's a constraint that we are kind of working with, but that would be ideal if we knew exactly how much co2 would be
12:13:11 produced, or you didn't experiment with CC 13 mapping, then you could look for the co2 form.
12:13:21 Yeah, we, We did that.
12:13:24 But it's not so straightforward because we cannot retrieve samples.
12:13:30 Throughout the experiment, we can only retrieve guest samples and not liquid samples because otherwise we disrupt the system so much that we can't. Yeah, we can't do that so we would need to kind of sacrifice, one system for every time point.
12:13:43 and like that you, you only have guessed samples and then you don't know how much is really solved.
12:13:49 But the second thing that I wanted to mention is that we think that some of the Nathan is actually liberated as small organic acids or storage compounds.
12:13:58 And there are some indications also in our biochemistry data that they may be produce some.
12:14:03 Yeah. Some other small organic compounds that maybe other microbes can use, but it's very hard to really quantify that
12:14:19 there was a question here around is the doubling time the same for the says on the cotton cloth versus the cells and the reactor.
12:14:29 And is there any biomass formed in the methane, I assume reactor you kind of answered this question in your talk already. Yeah, yeah, it's hard to say.
12:14:41 So I think within our within the timeframe off the bioelectric chemical system as we are running it now. We don't know whether we have selective enrichment of the cells on the electrode, or whether some of them grow as additional constraints that I didn't
12:14:54 really talk about is that usually they have nitrate as a nitrogen source. And now we didn't put any nitrate and, but the only nitrogen source that they get is actually end to end, they can fix and to but and nitrogen fixation is a very energy intensive
12:15:11 process.
12:15:12 So we expect but we don't have really data to show that we expect that the doubling time on the nitrogen fixation conditions will be will be higher than 10 days.
12:15:24 It's essentially a suspension experiment that you have been doing.
12:15:28 Well, technically, if they can grow so there is nothing that should really be limited limiting them, but practically the questions whether they do grow within a time frame of eight days.
12:15:38 And it's very hard to say. Yeah.
12:15:41 Why don't you give them pneumonia.
12:15:43 Because then all other kinds of microbes grow,
12:15:48 we kind of want to, because that's also not a game I mean it's just technicalities really in our buyer as you have a constant flow through of all of everything.
12:16:00 And then the bioelectric chemical system you can't do that because it disturbs the electrode too much and you can't measure, you can really have complete anaerobic conditions and good flow through at the same time.
12:16:14 So it's a it's a little bit.
12:16:19 We actually have the system exactly running like that so we should probably talk offline about that so we have electrochemical systems as a flow through the system strictly strictly animals methanogens.
12:16:31 Yeah, yeah, yeah I'd be curious I know our collaborators have that as well but they work with sludge so and for me.
12:16:39 We do King conscious, yeah.
12:16:41 Yeah, that would that would be nice, but I think it's actually good to take end to end not ammonium as as nitrogen source because it limits further options of bucks growing and the electrodes.
12:16:55 But I want to redirect the discussion a little bit to what we discussed in the before the break is sort of the the small amount of energies of the proton as the unit energetic unit, and how a bifurcation would actually break this ruler, and I'm not sure
12:17:17 if I'm convinced about that. So do you want to elaborate on this a little bit more. Yeah.
12:17:25 Yeah, it's maybe really a point of view how you what you, what you regard as energy so I think electron bifurcation can kind of redirect.
12:17:38 And so it, it takes some of the feminine remix that is needed for from for metabolic process from the member into the cytoplasm so you can actually reroute reroute Redux pathways in the cytoplasm which before I think we didn't really know about.
12:17:54 But you I mean elephant bifurcation doesn't really generate energy. Right, right, right, it doesn't really make ATP, are generating the proton motor force, which is why I'm always a little skeptical whether, so I mean it contributes to saving, because
12:18:10 of this rerouting and the, it can. So, as I explained it can make reduce production, which before we thought would always require ATP or a proton motive force.
12:18:21 So in that way it can reroute, how, how energy is distributed, but it cannot. Yeah, it doesn't contribute to really to really energy conservation.
12:18:35 Basically coupling some extra garlic to an end organic reaction, and biology does this many ways. One is electron bifurcation. The other one is ATP hydrolysis for substrate activation and so, so it's an.
12:18:52 In this and also produces for a toxin, which is important in the next step for energy conservation, but but I see one aspect of electron bifurcation and that, but sort of going back to the question, you know, one proton, is this the unit of biology right
12:19:17 there in the in the audience more discussion points on that.
12:19:23 Yeah I raised the point before so I have to say something about that.
12:19:32 The point is that you could buy electron bifurcation, you can add up.
12:19:38 Reactions with a small energy potential a small electro chemical potential and get them to doubling the electric here.
12:19:51 Let's say it that way, you have two reactions at 60 million volt potential difference.
12:19:58 And through an electron bifurcation you can convert that into one reaction with $120 million.
12:20:16 And that might just be sufficient to plant one proton although, although your original electro chemical potential does not allow it. So, so it's it's me I'm one energy conversion rather than Yeah, yeah, yeah, yeah I see what you're aiming at you're saying.
12:20:27 When we look at thermodynamics we shouldn't just look at the delta G of an individual reaction, but with electron bifurcation or convocation you can combine different, different reactions that in itself, wouldn't be enough.
12:20:41 But then in some, they could yeah i think i can i can see how this all that contributes.
12:20:49 I think I looked on bifurcation is nothing else than a mechanism to conserve Redbox energy that cannot be conserved. By respiratory chains, but you convert this into a reduced reduction, then you can do other things with that.
12:21:03 But going back to the question about, you know, the holy grail of proton. Well, and I don't know if obvious still around here but I think, Cornelius argument is that a proton is actually the energy within electro chemical bringing our protein model force
12:21:22 force of minus 200 milliwatts.
12:21:26 And, and there's something to be said about that. But if you think of empty partners, for example, sodium, proton anti partners that have a steak Yama tree that is different from one to one.
12:21:40 You can conserve energy in a smaller.
12:21:46 That's a slogan motive force that is smaller than let's say minus 15 killer towels, convert this into a let's say sodium gradient and then with an empty pot and just again military convert to sodium.
12:22:03 Low Energy sodium sodium ions into one proton, with an ATP as that has essentially about what acting in the in the field of proton motive force of minus 200 milliwatts.
12:22:20 You can accumulate pennies to get finally.
12:22:24 That's right right right right so we are talking about pennies.
12:22:34 Do you see theoretically that this works but do you know if any metabolism that's doing that exclusive.
12:22:35 Of course.
12:22:38 Well here we have the level of physics talking about what is today, what is possible. Yeah.
12:22:48 The point has been discussed by farrakhan moola log, where really this lower, lower most level of biochemically convertible energy is.
12:23:00 And he extends that to nearly zero.
12:23:04 But there is no real example for that. And so, I don't think it's not realistic to go that way but that you can go that you can
12:23:16 cleave the transported proton energy into a half's appears still about realistic.
12:23:27 Not far below that, not down to pennies.
12:23:33 I think it's, it's possible right like if you don't focus on what the lower limit of the quantum is but you think about it in the differential sense like.
12:23:42 I cannot hear you. Can you speak out for me to open this microphone sorry.
12:23:48 Is this better.
12:23:51 Yeah, okay, I'll be
12:23:54 like if you think about it like like let's think about like I don't know, something that requires nitrate or something like soon no not requiring oxygen but but has, you know has sizable amount of energy but like has to make some compromises sometimes.
12:24:10 I, it's possible I think to to compromise differentially like you can you can express a mixture of 321 and four to 180 percent basis right and then you're using, then there's like a there's a free variable there, like, what is the H plus state to compete
12:24:28 ratio or something like that.
12:24:30 And in that, in that vision I think it is the case that the, that it's not quantization right that there, it's possible to mix the amount of synthesis and then get a free continuous transition in terms of the energy coupling ratios.
12:24:48 This includes sort of a different membrane potential and the proton motive force. Yeah, that's where the difference to chemistry works. So if you lower the proton model force, you can actually collect smaller Delta G's as as a proton, but the unit is
12:25:08 still a proton. Well, the program has less less energy. Yeah, I want to add this because if you.
12:25:15 So indeed if you change the stock geometry of 80% that you need to change the proton motor force to run it.
12:25:21 And at the same time then it becomes more expensive to translate a proton.
12:25:27 So it because it's all interconnected it's not just changing stuff geometries here. So you have to pay it back elsewhere, the membrane is sufficiently charged you can run the membrane is sufficiently charged to run the for the one ATP for three protons
12:25:43 then you can certainly run the one HTTP for corporate comms right
12:25:48 now. Right, so, but it becomes inefficient then but we want to get sort of what is the smallest amount of energy we can harvest. So I think this goes directly to this conversation of what whether there is a physical trade off between the amount of energy
12:26:04 that you are harvesting and some, some rate of the process or something that we've been talking flirting with this entire set of sessions right like maybe there is a trade off that is deeply physical in nature that that affects the rate at which things
12:26:16 go and then there's a reason. There's a reason to compromise on energy field in order to get some benefit in terms of how much protein you need or the speed at which individual proteins can do the reaction.
12:26:31 So we're now back to the right deal trade off. Is that where we're going.
12:26:37 That's where I was headed Yeah. All right.
12:26:47 Yes.
12:26:49 Okay, you're trying to say something. Yeah.
12:26:54 No, sorry. no that was just noise here.
12:26:57 Okay, my microphone was sorry.
12:27:10 Well, um, any other questions for Iconia.
12:27:33 And let's thank you for this great talking this great session, and we will see you next Thursday at the same time. So, thanks for coming by all and see you again next week.
12:27:36 Thanks.
12:27:37 Bye bye.
12:27:38 Thank you. Take care.
12:27:40 Bye. Thank you.