10:00:53 Hello, I'm not saying good morning to everyone because I have no idea where the rest of the classes on this this planet, and thanks for coming back to the forum and Microban metabolism.
10:01:07 Today we have Bernard sink. He has featured speaker and I'm very delighted that we could recruit to give a talk on microbial metabolic interactions and if you think about food would be the best person to talk about this I can think of anybody who's more
10:01:28 more sort of qualified for that is if this really interesting interface between microbial metabolism, understanding like metabolism on the single soul, and population level, but linking this to ecology of organisms and that's something that really isn't
10:01:48 an important link between a pure ecological approach to microbiology and a more metabolic biochemical approach and over the years, Bernard has links substantial contributions and really
10:02:08 paved the way how to connect cellular metabolism to community metabolism and ultimately to explain ecosystems performance and I really like sort of this Dysport spectrum, crossing the different scales.
10:02:25 So I will.
10:02:27 And I said, I think, Terry and I we are both very delighted that kind of agreed to give us his do have some terrific microbial interactions and microbial community metabolism in general.
10:02:41 So thanks for that and take it away, the floor is yours.
10:02:47 Okay, so thank you very much for this invitation and for your introductory words.
10:02:57 I know it's only morning on your side you have your morning coffee.
10:03:01 Here I would be close to my evening beer but tonight I have to wait a little bit with that.
10:03:08 So, in the first part of this evening lecture, I want to first tell you something about microbial interactions in general and especially about sick traffic cooperations and then I will show you with several examples what that means and in which context.
10:03:31 Such a thin traffic associations may be important.
10:03:37 And so the idea is to give you first the concept of interspecies electron transfer which is one of the key issues to deal with and then, as I said, Will I will give you several examples, especially with respect to the energy ethical implications that
10:03:59 such transfer systems carry with them.
10:04:04 And perhaps if I get that far to the end, I will even get down to anaerobic methane oxidation which is a very important process on the global scale and then perhaps come to some conclusions.
10:04:20 And then in the second part, later in the evening evening on my side, I will show you with some examples what all the housing organisms, solve the problems that these associations as I described them before, and will pose to their biochemistry and how
10:04:44 they can deal with the biochemical challenges they have to live with.
10:04:51 So, let's go out into nature into for instance, a lake sediment. How can I get rid of this thing up here.
10:05:07 Doesn't it. Is that also in on your screen appears it frames okay you see the picture, I see your cursor Yeah.
10:05:16 Okay.
10:05:19 So, this year is a scheme how different types of electron acceptor can be used by microbial communities. For instance, in the lake sediment. And
10:05:36 as long if you go into such a lake we ever been late right here in front.
10:05:44 And if you go swimming in summary, you go out there two years. Lake and if you scratch around and the sediment a bit after the few first millimeters and actually Oxygen.
10:05:57 Oxygen is the preferred electron acceptor at the top of such as sediment and if you dig a little deeper, then probably nitrate reduction will be the next process that acts as an electron acceptor system here, then follows Iran and manganese more or less
10:06:19 less in the same zone sulfate reduction to solve find it.
10:06:25 And at the very ends and finally methane formation.
10:06:30 And whereas with oxygen dependent respiration and nitrate dependent respiration. Organic Carbon is completely oxidized to co2 with the other processes seers is typically a process running in more than one step so I run reduction and solid eight reduction
10:06:54 the organisms doing that, do not deal with complex organic poly Merrick material.
10:07:02 This job is first being done by classical fermentation.
10:07:07 And then the fermentation products are further oxidized in bite of the iron and sulfate reduces. So in these cases here we have typically a two step process.
10:07:20 And when it comes to methane formation then we even have a three step process, because the classical fermentation products here, cannot all be used by mythology directly they need a further secondary fermentation to make them accessible to the massage
10:07:42 and at the end. So as you see, degradation of organic matter in anoxic environments, is more complex than we know it in the presence of oxygen appears This is typically a one step process every organ aerobic organism can not everyone, but many of them
10:08:03 can use a poly Merrick substrate degraded entirely to see you too.
10:08:08 They are not really dependent on cooperative, or whatever else of
10:08:17 cooperative linkages, the sequence of these alternative electronic sectors is determined by the Redux potential of the Redux reactions underlying these processes, oxygen has the most positive electro electro potential.
10:08:43 Meaning, reducing oxygen provides. Most of the energy. If the organic substrate here is oxidized to co2 I calculated he an average Redux potential for organic metal with a sugar molecule.
10:09:02 And you see with such a sugar molecule and oxygen we can spend rather high potential difference of more than 1.2 volt.
10:09:14 And with the other electron acceptor so this becomes less and less.
10:09:20 And therefore, the oxygen is the preferred electron acceptor the others follow in this sequence. So, very microbial community available via tries to get out a maximum of energy of the respective Redux processes, and the poor ones are those at the end
10:09:40 here in the deeper part of the settlement, because they only finally have co2 as an electron acceptor.
10:09:48 And with that, they can just get a small energy span here between the oxidation of organic matter. And for instance the reduction of co2 methane. There is not much to gain at that point.
10:10:04 You can see that also organize you should take a sentiment core that see a from our lake.
10:10:27 oxygen and also the nitrate and I run reusing zone, where it becomes black ish. This is where sulfide is foreign from by sulfate reduction, and the sulfide reacts with iron to to form Fes, and that's the black stuff underneath in comes to mess energetics
10:10:49 around.
10:10:51 And there's also satisfied in the background but not that different iron sulfide here, and therefore it's not black anymore more grave.
10:11:01 And with that, you have this nation that does show would be you before, and you find this same sequence of processes wherever you go if you go into a marine sediment, a freshwater sediment, a hot spring and yellow stone or whatever else just, it depends
10:11:22 on the availability of these various alternative electronic sectors, to which extent these things really stretch into the ground it can be a matter of millimeters in such a hot spring, it can be a matter of many meters in the deep sea.
10:11:42 And here in our legs. This is just a some 20 centimeters or so, in which you find all these processes lined up.
10:11:54 At the end, as I said, comes the message witnesses, and this is an old process, or at least has been described long ago by Alessandra of all time.
10:12:03 And he stared up sediment a bit here and captured the gas, the gas bubbles that came up there and then lighted it.
10:12:19 And with that, he, he calls this guy's RBI in Korea Mambili burning air, and he quit show by explosion experiments that this gas was not hydrogen, Hydrogen had been discovered just a few years earlier.
10:12:45 And he could compare mean the explosion experiments that you need a different style geometries to get an optimal explosion with that and we said he could show that this missing.
10:12:50 Or if this swamp gas that he stood up here, had a different heat capacities and hydrogen, and that was at times when of course there was no simple way of analyzing gases, so that was at just at the beginning of the analysis of gases oxygen at been discovered.
10:13:12 Also about a few years earlier here so that's all at the same time.
10:13:19 Now what happens down into sediment and organic matter can be degraded in the presence of oxygen so co2 and hydrogen.
10:13:32 Water sorry.
10:13:35 And this, of course, is a very simple equation that you all know, I used glucose here as a molecule representative of organic matter in general, of course, the world is not all sugars, but sugar molecule is a good representative, and you can calculate
10:13:56 with that.
10:13:58 There are some formulas for biomass and so on, on the market very complicated including all nitrogen phosphorus whatever else. But that makes it very complicated and you cannot really calculate with a thing, because you don't get a real big value or anything
10:14:17 of that so glucose is a good representative of biomass. And if you oxidizer completely with oxygen you get a lot of energy.
10:14:28 If you convert the sugar molecule to methane and co2 at the very end. Then you get far less energy. As we could expect expect, also from our sequence that we talked about before.
10:14:43 chemically speaking this conversion of a sugar to methane and co2 is a dis mutation of carbon, or of this property nation or whatever term you use. So here the carbon is at the Redux state zero.
10:15:02 And here it is, is at its most negative value minus four. And yet, its most positive value.
10:15:09 So it's a complete this mutation of the carbon.
10:15:15 And that's the maximum amount. You can get out of this process.
10:15:21 Actually, all fermentation and you heard a lot about fermentation in the other lectures before.
10:15:29 Nearly all fermentation, are these mutations of organic carbon. So you start with a substrate, most of the sugar or something like that at an intermediate red state.
10:15:43 And you produce products that are partly more oxidized or partly more reduced.
10:15:51 And typically, such as this mutation yields a little energy. And that's what the organisms involved in can put it aside and live on that.
10:16:03 The difference between these two values here, of course, is the energy that is so fast stored in the methane molecule when you now oxidize the methane again with oxygen that oxygen up here, then you end up with co2 again and you get the difference of
10:16:19 the energy between both that's still 85% of the energy that is originally available in the sugar molecule here.
10:16:37 And, of course, that's the reason why we like to produce methane this way and use it as an energy source for, for whatever purposes in biology, of course, also this energy can be used for instance by aerobic methane oxidizing bacteria which oxidizing
10:17:02 Methane's in with oxygen, and can make use of this huge amount of energy.
10:17:11 You have seen this slide basically in a slightly different way before. It's just a reminder that we need energy to form ATP which is the currency of energy in the cell under standard conditions ATP formation requires 32 kilo tools under the conditions
10:17:33 as we find them in a living cell. This is more energy up to 50 kilo two. And if we include heat loss in irreversible reactions steps.
10:17:46 And they add that also give us a whole sequence of a living organism. Living biochemical process, a direction, then ATP formation requires something like 60 to 70 kilos mall.
10:18:03 Now, fortunately, nearly all microbes have ATP a sis membrane bound complex enzyme machineries that convert ATP into a, an electrical chemical gradient across the membrane either by protons or by sodium ions.
10:18:27 And this is an electrical and also a chemical gradient and together both pirates, make up a proton or sodium motive force of nearly 200 million volts.
10:18:41 And with that energy. For instance, ATP can be synthesized or if ATP is idolized, you can get this energy back and you can use it for any kind of Redux processes at the membrane.
10:19:08 The style geometry of these ATP as is differs at different organisms.
10:19:06 It is usually three to four protons also do minds for ATP.
10:19:14 And that cross the membrane per ATP. And with that, this transported proton or transported sodium ion represents the smallest energy quantum that a living cell can explore, to, or exploit to synthesize ATP, or so to make ATP.
10:19:39 You do not necessarily need the full amount of 60 to 70 kilo tool in one reaction, as you saw it in classical fermentation such as lactic acid fermentation also.
10:19:55 Then you have sufficient energy to form to full ATP units directly by substrate level phosphorylation.
10:20:02 But the membrane bound ATP as, and the energy stored in the electro chemical gradient across the membrane that allows to cut this ATP unit into smaller parts, depending on the story geometry of the HTTPS.
10:20:23 And with that, the smallest amount of energy that can biochemically be used by any kind of process comes down to something like 15 to 20 kilo true per mole.
10:20:38 And that's what we need to try and locate the proton or a sodium ion across the memory. With that, to synthesize ATP in small increments.
10:20:53 Let's keep that in mind, we need to add a little a trick.
10:20:58 Now if we come back to our simple equation aerobic oxidation of organic matter.
10:21:04 You know from textbooks that this amount of energy can be used on the optimal conditions to form 38 ATP per mole glucose. And if you divide these by others and you get to something like 75.
10:21:20 kilo true per more ATP.
10:21:23 So that's quite close to that what we saw before 60 to 70 is a little leeway in the overall process, but basically it's a rather efficient energy coupling.
10:21:37 If we apply that same value for the to methane formation. Then we see, there's only five ATP perhaps in the total process. That's it follows less than the Arabs have available.
10:21:54 Now we can play the game and calculate a bit.
10:21:59 If we also 75 kilos rupiah ATP is every calculated here.
10:22:06 If we need only 17 kilojoules for ATP senses it stays a little leeway left.
10:22:13 And if we add that up with a 38 ATP here.
10:22:20 And then we know how much energy is more or less wasted in the overall process meaning how much energy is there that pushes the overall process to the product side.
10:22:33 If we do that, then we can calculate what the corresponding glucose concentration would be when the whole thing comes to a
10:22:44 energetic equilibrium.
10:22:47 This of course is an extremely low concentration.
10:22:52 It is about one molecule in our huge lake constancy in front of our town with 50 billion cubic meters of water in.
10:23:04 Of course, the poor bacterium in our Lake will not see that one molecule anymore.
10:23:11 It has to stop much earlier.
10:23:15 Somewhere in the nano molar range, meaning 10 to the minus nine Mola be, because after that it doesn't see the molecule anymore.
10:23:27 And if we do the same calculation for the anaerobic conversion of glucose to methane and co2.
10:23:35 And we do the same.
10:23:38 Comparison with the energy that goes totally into ATP formation, then the glucose concentration could go down to something like 10 to the minus hundred nanometer.
10:23:56 And that is actually not so far from the concentrations that we can really measure in such segments. It's not easy to measure in these concentration ranges.
10:24:07 Part of the substrates in such a setting are bound to surfaces are the czar up to it. It's not so clear how much is really kinesthetically, relevant, meaning accessible for the organisms, but you can extract such amounts of sugars and other substrates
10:24:28 from sediment you are not really so far off.
10:24:31 So, what does that tell us.
10:24:34 Aerobic processes are never energy, limited, they are kinda ethically limited the equilibrium is so far on the product side that only the availability of the substrate really limits the overall turnover in the anaerobic world.
10:24:55 You come quite often quite close to equilibrium situations, meaning that also the product accumulation will inhibit your respective processes that you look at.
10:25:10 Now, you will see that scheme a few more times. And I think it also, it has shown it earlier already, that's the general scheme that Marvin Bryant designed many years ago.
10:25:26 And that includes the cooperation of the various groups of organisms in the mess energetic process. Not only that we have only five ATP available in the total transformation of the sugar.
10:25:57 This small amount of ATP is also to be shared by many different partners involved involved in this process. There are the classical fermented up here, they convert first Polonius tomato mirrors and then further degrade them to see one compounds here on
10:26:08 on the left, and acetate on the right, and several classical fermentation intermediates build smelling fatty acids alcohols lactate vaccinate.
10:26:21 It's a much longer list including aromatic residues and whatever else, all that goes into this bucket here.
10:26:30 And to make these accessible also to the methanogens at the very end, these have to be fermented again, because the Montana chickens can only use see one compounds and hydrogen here, and acetate on the other side they cannot deal with such more complex
10:26:49 substrate. Therefore, we need a further group of organisms here that take care of these complex substrates, and convert them to these substrates that I'm a fan of that and can my foundations can use.
10:27:04 There is a further arrow in the scheme here the number five. These are the hormones SC touch any bacteria that had been mentioned I think of an iPhone, and they are able to ferment co2 and hydrogen and some other see one compounds to acetate and gain
10:27:22 additional energy from that they somehow can balance out these two pools here to convert excess of this to asset.
10:27:37 Sorry about just just to just to ask the question to be awesome give other people a chance. So, when you make a statement like mythologise can only deal with acetate or what is that is that a just an observation, there are some deep chemical reasons and
10:28:08 what what is mythology, Janet, the process is so complicated that they cannot do anything else, what clarify that.
10:28:21 Well first of all, we have to realize it is like that. And that was actually also the scientific, the development of scientific knowledge. When people didn't do we deal with defined cultures at the beginning so nearly everything that they threw into a
10:28:28 wild mix of organisms ended up finally in methane and co2.
10:28:44 But the further the techniques were developed to purify these rather oxygen sensitive organisms, then it turned out more and more these, the real mythology ends here at the very end happened very small scale of substrate that can deal with.
10:28:55 Perhaps we come back to the question why is that might be like that to the end of this lecture, then we'll pick that up again.
10:29:04 Okay.
10:29:05 Whoops.
10:29:07 Oh, can I just ask. Um Can you repeat it, because you went through this rather quickly. The five groups. So the first one, it just degrades, and the classical fermenting bacteria, such as close to you, or lactobacillus or whatever else that deal with
10:29:32 organic matter and convert them to the classical fermentation product. Then we have the methanogens in closer sense. So, basically two groups, some can use see one compounds and ferment them to methane and co2, and others are specialists in converting
10:29:51 acetate to methane and SEO tools as one organism and even that can do both.
10:29:59 And, but they only take that small range of substrates depicted here. And then we have these strange organisms said, I will talk about in more detail.
10:30:13 Now later.
10:30:14 They use the production of the classical fermentation, and convert them into these two types of substrate. And then we had the home ice to GNC at the very end.
10:30:27 Okay.
10:30:30 I can't resist to ask you.
10:30:34 We don't have some traffic acetate oxidizes in sort of the opposite direction of the homeless citizens
10:30:45 me is that as well. It comes retro chapter. Okay.
10:30:52 OK, so the linkages between the different groups in these these complex processes may be rather tight, or they may be more or less, lose in an early experiment by a user into the lab of rod fools.
10:31:14 They tried to model such cooperations with pure cultures of organisms here they used a hormone seated gigantic bacterium that can convert glucose, two, three molecules of acetate.
10:31:30 And then they combine that with a mythology and that can ferment acetate to methane and co2, and this way you have a two step process that ferments glucose to maintain it co2.
10:31:45 But this is an artificial situation you will never see that in a more complex.
10:31:52 Natural community.
10:31:55 It would not be stable it would, as long as other substrates, enter the system, it will be virtue baited will not stay it's a model in the lab, but it's not a good model for nature.
10:32:09 Now let's come to these secondary fermentation, that have been coined also seen traffic cooperations that are these ones here.
10:32:22 And the first example,
10:32:27 this kind was isolated by Marvin Bryant in Urbana, and long long ago more than 50 years ago.
10:32:37 And that was a cooperation with the two bowling people they were in animal husbandry and animal nutrition in Urbana, and raw food, of course, who was one of the godfathers in anaerobic microbiology of mythologies especially.
10:32:57 And what they found out was that a culture of a putative pure culture of bacteria turned out to be a mix of two different organisms, all these way of bacteria by that time so today we know of course that mess antigens are not bacteria, but Arusha or bacteria
10:33:21 bacteria whatever you call them. So in those days, these were all bacteria.
10:33:27 And what was the background of that.
10:33:29 So this culture converted ethanol, together with co2 acetate and messy.
10:33:39 And as such, this culture. Culture had been isolated by a Russian micro organism, or at least described by Russian or my microbiologist or meri jaan ski.
10:33:52 I come back to him later again.
10:33:54 And this culture had been isolated in a strictest sense by Baraka
10:34:02 Baraka was a chemist he was perhaps not so critical about pure conscious, in those days, and he thought he had a pure culture, but through the work of Marvin brined it turned out as this culture was composed of two organisms the one was the so called
10:34:21 s organism that oxidized ethanol to acetate and to release the excess electrons in that process as molecular hydrogen.
10:34:33 And then the hydrogen was picked up by a mythology that reduces with this hydrogen co2 methane.
10:34:43 And then this is a highly except chronic reactions and it's well known of course from the foundation. This is an in depth chronic process, under standard conditions, and they would never run this way on its own.
10:34:56 But if you combine these two organisms and the management partner here, keeps the hydrogen partial pressure, low, meaning takes all the hydrogen away and maintains a hydrogen positive pressure as low as, for instance 10 to the minus four atmospheres,
10:35:16 then this reaction becomes exec on it as well.
10:35:20 And then both together can do the job that is described up here.
10:35:26 And with that, the term trophy has not really points by that. But that was the first sin traffic assets, meaning to organisms live in a symbiotic association with each other.
10:35:47 And the cooperate by a transfer of a metabolite to make energy metabolism on both sides possible.
10:35:57 This is a simple scheme that I tried to draw for that, you know, or at least by face lectures at the beginning, you know that you can describe usually a microbial metabolism and also our own as a process in two parallel paths in one one substrate is oxidized,
10:36:21 and the electrons, released in that process are being used to reduce a second substrate to a reduced production. You can make ATP typically on the oxidative side, and also on the reductive side.
10:36:38 And now, in a central figure associations, the two processes are being uncoupled, somehow, or at least are being separated and coupled only via the transfer of a electron carrier, in this case here, molecular hydrogen.
10:36:57 So we have an oxidizer date if branch and oxidizing organism now that is combined with a and other organisms that takes these electrons and use that for his own metabolism.
10:37:13 And now we have definitively to get ATP on both sides because they both have to get their energy.
10:37:23 Today we know a whole series of such in the organic fermentation processes that depend on cooperation with a partner organism that is up here was ethanol oxidation we just talked about.
10:37:41 Here it is now for one ethanol molecule before that was for two. So, for one molecule it's roughly 10 kilo to will.
10:37:51 And if we look at oxidation of fatty acids, such, such as beauty rate that has also to be converted to acetate and hydrogen, and now you'll see that is much worse than the situation up here.
10:38:06 If we look at proper Nate oxidation is improper Nate goes again to acetate now three hydrogen molecule that becomes even more in the iconic. And there is also a special case I get back to that of acetate oxidation assume traffic acetate oxidation.
10:38:30 Is that from co2 and hydrogen only and then you get have to invest 95 kilo two.
10:38:36 And it's a branch chain fatty acid down here is benzo aid and aromatic acids that is being formed into degradation of for instance aromatic amino acids.
10:38:49 And all these are such in the organic processes that become possible only in the cooperation with a for instance methodology Nic partners and keeps the hydrogen partial pressure low.
10:39:03 And with that pulls the overall process, towards the product side.
10:39:12 As I said, the term Sintra fee was not really coined by Marvin Brian in his ethanol system, the term appears at it later with the isolation of an organism that oxidizes fatty acids such as beauty rate.
10:39:33 And as we switch right before.
10:39:36 and mom Brian was involved here as well.
10:39:40 And the here for the first time he soon traffic oxidation of beauty rate was described and he also this term appears so homework or most of the work was done in the lab of Norbit Phoenix inverting and in those days, and in his lap as I might show you
10:40:00 little later as and.
10:40:03 Also, another system of into organist make a relationship, had been described at about the same time as that's why this term has been used. At about the same time for not really similar processes will get back to that later.
10:40:24 Now, how can you purify such organisms, or how can you get to any kind of defined cultures with these books if you cannot grow them in pure culture.
10:40:37 Well, the way to do it is basically what we are used to do in our lab for the purification of strict interrupts we usually don't freak them out on a plate, because then it's the risk is very high that they get exposed to small amounts of oxygen and then
10:40:58 they don't grow. And therefore we grow them in the argon will use a dilute argon 1% agha it's a semi solid Agha.
10:41:07 And we dilute the medium with, we have the medium here in the cubes.
10:41:14 At 40 degrees then it's just a liquid.
10:41:17 And then we add our in our curriculum and enrichment college was so into it and then it goes through a delusion series all the way down.
10:41:25 And in the last tubes then you usually see after a while single colonies. And then you can pick them out with their pastor up iPad or so, and they are happy to grow in here and even if such a tube is slightly exposed to oxygen intermediate layer it doesn't
10:41:43 doesn't change much because that oxygen does not really penetrate deep into the tubes. Down here you can still keep them alive and get them in good order the technique had been developed by the funniest lab for photo traffic bacteria, and that's how we
10:42:05 got used to that.
10:42:12 Now, if you do the same delusion series with such as sin tropical culture where two partners need each other.
10:42:16 Then of course, you can do it the same way and after a few weeks you might see good growth in the first and second and third job.
10:42:23 And then somewhere. The delusions here is breaks off.
10:42:29 So there's still 100 or 1000 colonies here. And then there's nothing that tells you there's something wrong, there is a interrelationship between the partners and when they through the delusion process don't meet each other anymore, then somehow they
10:42:49 cannot work anymore and then you don't get growth.
10:42:52 And that's always an indication that this is maybe a sin traffic or whatever, a relationship in the culture that you have to deal with.
10:43:02 Now how to solve that.
10:43:04 In that case, we know that the second organism in the system here is a hydrogen utilizing the furniture.
10:43:12 And we can use such no organisms they are available in pure culture that's no problem.
10:43:18 And we can provide it in the background here as a media component in all these tubes so there is a background blown of this partner organism everywhere available.
10:43:32 And now on that background we can out dilute our enrichment culture of the traffic system. And then we would again, expect to get a normal dilution effect, meaning step by step it gets less colonies and subway and the last ones we get colonies of course
10:43:50 the is I'm a pure culture.
10:43:53 But if we do that twice. Also we have at least a defined co culture.
10:43:59 And in that defined co culture we know one of the partners because we can grow even a pure culture.
10:44:06 And we know the other one well not directly but we can correct her eyes it in comparison to the pure culture of the partner and then find out what the properties of the first organism in the system.
10:44:22 It's a little more messy. It requires a little more f4 to deal with these sin traffic countries.
10:44:31 To be honest, They are also rather slow.
10:44:34 And that means you cannot really produce papers every other week it takes a little longer to get those out.
10:44:42 But it says worthwhile because the competition is not so high in the world. So, not many foods dealing with this, these funny books.
10:44:54 Now what can you do with these things this is just one example of a actually benzo at grading sin traffic culture that we worked with in our lab.
10:45:06 And we isolated that together with a hydrogen utilizing methylation.
10:45:15 Look at it here.
10:45:17 And then we have some here in a dense self sustaining sustaining together, a co culture defined co culture of two partners.
10:45:29 And now we give them enjoyed.
10:45:31 And this is a one hour the scale, the overall process here was one day. That's the advantage if you do the experiments in den cell suspensions you really have turn overs, that are, that provide data over a day that you can work with.
10:45:53 Otherwise if you do real growth experiments, it takes days and weeks until you have results. So, within cell suspension you can play.
10:46:03 And what we measure it here was benzo at gradation. And who is benzo at predation UC acetate accumulating, and he has some hydrogen partial pressure and as you see this hydrogen partial pressure is roughly at 10 Pascal's This is a log, log scale here.
10:46:25 And now, at this point we added an inhibitor against the mythology, there's a specific in the inhibitor Bama fan south on it that blocks the biochemistry of the mythology, and you'll see immediately the hydrogen Patra pressure jumps up, because it's not
10:46:46 being consumed anymore.
10:46:49 And bands are at where they stopped.
10:46:54 They cannot work anymore if the hydrogen partial pressure is so high, and nothing happens over quite a while.
10:47:03 Now we added another hydrogen utilizing organism. In this case, a sulfate reducer.
10:47:12 That is not impeded by this inhibitor used here.
10:47:16 And now sulfate reduces can even push downs a hydrogen posture pressure, a bit lower than the mythology and scan. So we now have a lower hydrogen level than we had it up here.
10:47:30 And now, the benzo at gradation and acetate accumulation happens again. So you can play with such partner system you can add different partners, you can.
10:47:47 By choosing different partners, you can determine the hydrogen partial pressures that shows up in equilibrium of the two organisms cooperating with each other and with that you can elucidate the energetic needs of the organisms that you are working a
10:48:03 And I can go back to the previous slide, I missed the cultural condition, the addition of what is the essential very high bacterial destiny.
10:48:16 Yeah, so it was a co control of these two organisms that has been grown saying the little culture or so, and then we spun them down, and then you have perhaps an oldie or say five four So, meaning you sell mass in a smaller volume perhaps in a 50 milliliter
10:48:39 culture also.
10:48:42 And then of course everything is more condensed, and the cell activity is a measurable cell activity is much higher than in a dilute culture when you start growing that make it.
10:48:58 And then during this period I guess very little happened to the cells themselves.
10:49:09 Green very middle, you would, of course they might even grow in that but you don't see that on the big background of the ability that you have in the beginning, you don't really see the increase too much, thank you very differently from from the region
10:49:25 I work with. How easy is it to measure that hydrogen, or gas in general, the pressure. In this case we measure the hydrogen in the headspace, because the height of the disorder of hydrogen in the liquid phase, and the hydrogen in the headspace are in
10:49:43 equilibrium. So, you know, you can shake the culture to maintain this equilibrium so you know that these two.
10:50:01 The gods face and the liquid phase are roughly in equilibrium with a hydrogen concentration. And you see the measurement itself so we did that with a gas chromatograph, but if you want to get down to these concentrations you need a specific gas chromatograph
10:50:13 for that, in that range so normal hydrogen detecting thermal conductivity detector is they might go down into this range but not much lower.
10:50:26 So you need a special, special equipment for that that's a detector system that works by the reduction of mercury of metallic Mercury, to make you're a bad parent, that's very sensitive.
10:50:42 The system has been developed by the air chemists, that measure the hydrogen here in our air around.
10:50:50 And that allows you to measure all the way down there.
10:50:54 Thank you.
10:50:57 Now let's look into a culture that degrades beauty rate and build rate by the CO culture is converted to methane and co2.
10:51:12 It's a tree culture. So, it consists of such as in traffic fermenting organism, together with a hydrogen utilizing organism, and an acetate degrade.
10:51:25 You see the overall process is x iconic.
10:51:29 And now if we take a concentrations that is more reasonable to a natural situation that we have about 10 micro Mala. The alteration in a reactor. Also, and then of course the energy gain is smaller.
10:51:46 Now, as I said, we have three organisms involved in the overall process the one is the sin traffic the fermenting organisms that converts beauty rate to hydrogen and acetate.
10:52:00 Then there is a hydrogen utilizing the sanitation, that reduces co2 into methane.
10:52:07 And today is in acetate degrading the sanitation, that converts acetate to methane and co2.
10:52:16 This reaction runs twice.
10:52:21 This runs, once this one, four times.
10:52:26 So we have all together two plus one plus four, seven parts reactions.
10:52:35 And all of the bugs, working here they want to get their chance in energy they have a minimum requirement of energy to make their living.
10:52:46 So, let's assume they share this amount of energy here at equal rights, then every partial reaction gets 20 kilo to.
10:53:04 Sorry.
10:53:07 Here it is.
10:53:07 Now, if we know these 20 kilo true then we can also calculate it's always announced the equation back and forth, we can calculate the concentrations of the intermediate in the process that's hydrogen, and that's that's acetate down there.
10:53:25 And it turns out.
10:53:27 Hydrogen is somewhere between 10 to the minus four and 10 to the minus five atmospheres and acetate is in the range of about 50 micron.
10:53:37 And it doesn't matter now, it is a little more or little less because all these concentrations go as activities into the announced equation at as logarithmic values, but we do not have unlimited leeway here, because they all want to get their minimum
10:54:02 amount of about 20 kilo true. And as I said before, this is about the minimum that we need we cannot go much lower.
10:54:12 Otherwise, one of them, doesn't get anything anymore and then he doesn't work anymore.
10:54:17 And that's the problem of all these mass allergenic processes.
10:54:24 They are rather sensitive to perturbations because they need a high degree of stability.
10:54:36 If anything goes wrong, for instance, in such a wastewater anaerobic wastewater react or anything like that. Something toxic comes in through which one of these organism groups here it is seriously impaired.
10:54:53 And then this function for instance, is not maintained anymore. Then hydrogen accumulates this thing cannot work anymore, fatty acids accumulate the whole thing smells horrible.
10:55:06 And then you have to find a way to get rid of that and get it somehow stabilized.
10:55:13 So, we are the leeway for the intermediates here is limited.
10:55:24 And therefore, the whole system is not unlimited least stable.
10:55:31 It needs a certain stability on the long run.
10:55:36 So your question about wastewater treatment plant, I mean they probably use similar right my understanding that they probably use a similar my corporate consortia is that is that true or well they, they do not plan to of course they grow the in the system
10:55:51 it's a fully undefined community in there.
10:55:57 And I should emphasize this is the inner Robic reactor in the wastewater treatment plant. Most of the water treatment goes through would aerobic process ol all the organic material collected there, and also by mechanical processes that goes in into an
10:56:18 anaerobic huge reactor and Thea the final fermentation to maintain and co2 take takes place. And with that they can gain back some energy also for running the first bite of the water treatment.
10:56:33 But this is the save the money making part of the wastewater treatment, and therefore it is important that it also helps to stabilize the slabs that comes out comes out at the very end.
10:56:47 Yeah, so it doesn't smell so bad anymore, you have questions about how stable are these waste treatment.
10:56:54 Right.
10:56:56 systems, if this is the fans, if you feed it continuously with always the same food, then it's basically okay.
10:57:06 But that's the problem of a wastewater treatment system in general you cannot strictly control what comes into your system.
10:57:15 And sometimes by whatever accident some toxic stuff comes in with a waste water.
10:57:22 And that might cause serious travel somewhere.
10:57:25 I see.
10:57:27 So these clouds have not this wastewater treatment. They have not because they have been doing this for a long time right could they see eventually end up with a community that's more stable way every that's resistant to fluctuations in the input of waste.
10:57:43 Yeah, and why if you want to start such a thing, a new thing you go to a friendly other wastewater treatment plant and get a lot of inoculate them for your reactor so you don't inoculate that with the loop anymore.
10:58:05 But with the tank. And so you really need a truck full of sludge to get such a thing going.
10:58:14 And then, keep it and torture it.
10:58:19 No, no.
10:58:21 Baby it all the time until it gets really stable and you can continuously feed it, and then it will pay off on the long run and you have to keep it in a certain detention time not too fast, not too slow, that the whole thing can continue to run.
10:58:41 Thank you very much.
10:58:42 So I have, I have a different stability related the question, and the.
10:58:49 You may have explained that early on and, and I missed.
10:58:52 So, what is.
10:58:55 So the fundamental reason why hydrogen has to be exported and oxidation and reduction part of the process cannot be efficiently run in a single cell.
10:59:14 Why labor.
10:59:14 I didn't say that is and I could give a reason for that, that they operate in two different cells, we get to that later in the lecture again, actually, a little
10:59:28 explanation going a little bit in the direction was also given by flight form and in one of his earlier lectures in a different context.
10:59:39 I get back to that. Oh wait, thank you.
10:59:41 Okay.
10:59:52 So, he is our network again and now we have to ask how important are the relative branches here. So I drew is I was here a little thicker as you see.
10:59:56 And if you're dealing with a world stabilize systems and the main flux of electrons goes through the outer part of this scheme on only a minor part goes through the inner part because also as a classical fermentation here do better in the presence of
11:00:18 mythology that keeps the hydrogen Patra pressure low, and then they will not form so much fatty acids all the mess in between here. Therefore, the main electron flux will go out there.
11:00:32 Of course the central part will never be zero, that are also from the degradation of biomass there are limits and all this stuff so from that also fatty acids, Arise, that have to be degraded so this central pirate big remains important, but it's the
11:00:51 whole system runs at high rate, and at high efficiency then the central part is not so important.
11:00:59 I will show you that with one single simple.
11:01:09 Sorry, but just a backup so to that slide my question is, do you envision this kind of a century as a kind of a flux balanced state where everything is sort of growing in some sort of a steady state I know there's not much biomass growth, but the flux
11:01:20 steady state, or do you imagine sort of a temporal sort of oscillation. In, when something builds out some are going to consume some paper, something comes you know that says, continuous, at least in in such a reactor system it is a continuous process
11:01:36 and if you look into a sentiment also, of course, it is also continued, I will compare that was a little later. Okay, so perhaps then you'll see that.
11:01:46 Now, let's come to the to such a primary fermentation by a Clostridium organism in pure culture as we grow these Clostridium material eco mind anything like that in the lab.
11:02:01 They prefer to use a mix of beauty rate and acetate and co2 and some hydrogen.
11:02:08 And this ratio of beauty rate and acetate is determined by the amount of ATP that they finally get out.
11:02:18 And that is the formation of beauty rate is actually kind of a sacrifice that they do because the hydrogen becomes otherwise inhibitory for the fermentation, they would prefer to oxidize the glucose completely to acetate, and then release all the excess
11:02:42 electrons from glucose oxidation as hydrogen.
11:02:48 But this would yield 216 kilo true, but that is too little energy to form the four ATP that are strictly associated with this pathway to ATP and glycol Genesis and to ATP.
11:03:06 In the release of acetate.
11:03:09 So they cannot afford it.
11:03:12 But if we again keep the hydrogen partial pressure, low, meaning we run the whole thing on coal cultivate this plus radium with a high background of the sanctions.
11:03:25 And they keep the hydrogen partial pressure low, then the overall energetics change can calculate that. Then we get far more energy out. And with that we can pay for the for ATP.
11:03:42 And that means again. Yeah, that's the scheme.
11:03:45 So glucose to pure power of it as a joke or I acetate.
11:04:05 And if the hydrogen parts for pressure is low enough by the partner, then all these electrons basically can be used up by the partner, and then the overall process can run this way.
11:04:20 This has been a model as well developed in pure culture and several years ago we thought perhaps out there in nature.
11:04:33 In the sediment of our lake.
11:04:44 There might be organisms sitting about oxidize sugars and other complex substrates, always in the presence of that the background of my foundation.
11:04:47 So they never need to switch to the Pro Tools the formation of side products, such as bill to rate or anything like that because the hydrogen partial pressure is always low.
11:05:02 Such organisers perhaps would really depend on such partners.
11:05:10 And that means we cannot grow them in pure culture.
11:05:16 And so we did a rather simple experiment we took some segment, out of the lake and now we do the same thing as we did for purification of pure cultures of the central ops before.
11:05:27 Now we don't have an enrichment culture to start with, but we take the sediment from the lake right as it is about three centimeters.
11:05:39 And then we have modernize that a bit shake it's roughly so bacteria are released from surfaces and so on. And then directly out dilute that in this tube series with a high number of messages in the background.
11:05:58 Now, they all find partners in the background.
11:06:02 What was the outcome.
11:06:05 So we have such delusional experiments did set with glucose and also we started so pros.
11:06:12 And depending on whether we provided a foundation in the background or not, minus plus minus plus minus plus.
11:06:22 We get after a long time, different numbers of colonies for the first colonies of course the way out after two three weeks.
11:06:33 And then we waited. We waited two months, actually.
11:06:39 And then in the tubes with the mythology and in the background there.
11:06:50 The additional colonies small colonies showed up in the background that did not develop in the absence of the mythology.
11:06:56 And with that, we got rather high numbers of sugar tea graders, which obviously depended on the cooperation with a Masonic. So what we expected at the beginning.
11:07:09 If you compare that to the these numbers here to the number of total by the total number of bacteria is counted by Davi staining, which gives you the total number, more or less, you see that is roughly the same order of magnitude.
11:07:27 So, this type of sugar the greater that needs the cooperation with the management is actually very numerous there it's far more numerous in the sediment there, then those ones that do not depend on a partner organise.
11:07:47 So, you know, probably from the literature, it is that it is very difficult to cultivate organisms from nature you only get a small fraction of them.
11:08:01 And it is cultivation is nonsense anywhere. Some people say, because you get kind of an artifact there that is entirely meaningless for nature.
11:08:13 Well, I always say yes cultivation is bad if you use the wrong methods, and you should better. Think of the conditions under which these poor buggers live out there.
11:08:30 First of all, of course you need a medium that is more or less comparable to the natural situation. But you should also think of the fact that they are usually living together with friends.
11:08:39 They are not alone, there is you are supposed to have them later on in your culture they don't like that so many of them depend on friends all around and if you provide these friends, or at least some relatives or friends to them, then it's much easier
11:08:55 for them to come up that ask a question.
11:08:58 Yes, I'm just, I'm wondering, does the heart, what's the standard of choose those friends, like those different types of.
11:09:09 Oh, what was the standard of choosing the right partners. It does the does the different logins matter different types.
11:09:22 We have work nearly always with this madness we look at it as this type of massage and that we first microscopically you can recognize it rather easily.
11:09:39 And second, it's this type of management that usually develops in the public at the center of the culture systems with fatty acids and so on that I mentioned before, so you select in these cultures for this type of organism, and that tells us that it
11:10:02 is a rather good partner to play but I don't say it's the only or the best one.
11:10:09 We don't know that, but it is at least an efficient one because it is usually selected, together with these difficult digger native system we are working with.
11:10:21 And therefore it must be a good one.
11:10:25 Thanks.
11:10:45 Can I ask the same question, a different way. I'm so in the natural community, such as those from the continents. Do you find typically one or a few managers, or do you find many.
11:10:41 Of course, if you go, if you really look for diversity and one way to do that is by the way, not to do a classical enrichment in liquid culture as you do by classical microbiology, that always selects only for the fastest one.
11:11:01 If you instead us you are in our kilometer settlement also directly for such a delusion in the Aga, then you get a far more diversity of different organisms out there, because everyone gets a chance.
11:11:22 Otherwise if you pre select from the beginning by several transfers and liquid medium you only get the fastest one and but that's one way to get a greater diversity and of course there are many more.
11:11:36 But I'm just asking sort of roughly.
11:11:40 These managers.
11:11:57 hydrogen specialists for hydrogen, others for methanol or metal residues. Others prefer for me than the acetate utilizing ones, and it depends a bit Some like to be attached to a surface some others like to link directly with fermenting organisms.
11:12:22 So the diversity is much higher than we select by rather poor principles of course. But the diversity is much higher.
11:12:37 Okay, so that's what they look like let's go a little faster.
11:12:43 What these bugs that we isolated this way did was really they do the fermentation of glucose to methane, to acetate, in cooperation with a mythology and to form methane, and they really depend on this cooperation.
11:13:01 If we and again use this in hip inhibitor here Brahma ethane south on eight. Then glucose is not integrated anymore, meaning if we block them advantage and then also that the gradation of the glucose doesn't work anymore.
11:13:18 No.
11:13:21 We have to speed up a bit time talking to Matt.
11:13:24 Let's compare different message energetic environments.
11:13:30 And I compared here from the literature lots of values that I picked.
11:13:44 I compared to you traffic marine sediment, you traffic freshwater sediment, an anaerobic sewage sludge.
11:13:45 and the room and have a cow.
11:13:47 All these are habitats, where methane is being formed.
11:13:54 Now, these places differ by some physical constraints sediments are comparatively cold and sewage sludge digests the most cases is being run at 30 to 40, sometimes degrees.
11:14:12 And the cow, of course has a very defined temperature range of 737 to 39 degree.
11:14:22 One important factor is the time that the organism sat in the sediment, they can sit forever.
11:14:30 There's very little grazing in the anaerobic world.
11:14:35 So that is the microbes that have grown there they don't have to worry to be eat next day.
11:14:44 There is no need to replace the biomass, all the time because there is nearly no grazing only in very rich environment some in the Robic protozoa have been described.
11:14:57 But otherwise, anoxic settlements and so on are not really being graze upon, so you have a lot of time in the sewage sludge that just, you don't have that much time, you cannot run this thing for unlimited times typically it's, yeah.
11:15:29 And the Romanovs a cow, say as a detention time is roughly one day.
11:15:36 So, we find fatty acids in a broad range, it depends very much how this is older literature as well.
11:15:46 It depends how is the samples were taken how, if the settlement was squeezed if there was a chemical extraction involved or whatever. That's the reason why these numbers change to a certain extent.
11:16:01 But you I mainly in the range of a low micro Mola concentration and drop your native military that lower than the ease and hydrogen.
11:16:14 Fortunately, this thing is always in the way here that's expected so sorry.
11:16:20 And the hydrogen is usually in the range of about one Pascal meaning 10 to the minus five atmospheres.
11:16:28 Now compare that to a sewage sludge.
11:16:33 That is warmer.
11:16:35 You have a shorter detention time. And you have considerably higher fatty acid concentrations, and also hydrogen is higher.
11:16:45 So everything is at higher pool.
11:16:49 That means that the, the energy spans between the different intermediates, Maybe roughly comparable to those that we have in this segment, everything with lower numbers, but basically their relationship to each other is about the same.
11:17:13 So, all these processes.
11:17:16 Basically, operate at similar conditions are energetically speaking, but here with higher pools thing in a setup.
11:17:26 And the situation of course is entirely different.
11:17:30 With in the room.
11:17:33 You have high fatty acid concentrations 60 2010 milli molar.
11:17:41 And this is the fatty acids that go to the host. That's what the whole organism resolves and that he lives on the cow eats grass, but it feeds on fatty acids.
11:17:56 And all those ideas and Patra pressure is Raja Hi.
11:18:00 Why is that so because the detention time is so short, these complex cooperations of fatty acid.
11:18:13 Sit traffic is fatty acid degrading SIP trunks and mythology and they have doubling times have several days.
11:18:21 They will never establish in the room.
11:18:27 And of course, the cow is not interested in that either.
11:18:31 The cow doesn't want to eat the grass all the time, and in the room and everything goes to methane and they don't see any of that.
11:18:39 So, the cow wants to have a fast turn over. And with that gets a fatty acids, as intermediates.
11:18:51 So, sorry, that was the sewage sludge digester, that's a cow.
11:18:54 And if we now.
11:18:59 If we now compare our scheme to you see these pictures as well in it somehow is nasty so.
11:19:08 So here's our old scheme. And now if we compare that to the cow room and then we see that our sin traffic systems here are not existing because they cannot establish, they are too slow.
11:19:23 And that's good because the fatty acids and especially also the acetate is supposed to go to the host.
11:19:32 And of course the cow also produces methane, but it also only with those electrons that come over here as hydrogen. It helps to stabilize the primary fermentation, meaning that not that much alcohol and especially not that much lactate is being formed
11:19:53 lactic can cause at sea doors is in the cow and that's also not really what you want. you want to get a maximum of fatty acids acetate, and that goes to the host.
11:20:08 Now here we have another example.
11:20:13 The example of a termite gut. That is also comparable somehow to a cow is much smaller.
11:20:20 And the term I got gets a considerable amount of oxygen that helps. That's a good feeding to Mike.
11:20:28 So, in this case, a considerable pilot of the
11:20:35 total lumen of the gut, which has a volume of about one microliter compared to 150 liter of in the cow room and so little oxygen comes in here that helps in the degradation of wood and the poly sack rights that are being set free this way, are being fermented
11:20:57 inside the term I got here mainly to acetate in this case, although I see Trojans take a very important part here to take the hydrogen that appears here also to acetate, and that acetate is fed to the host organism, only a very small part of the overall
11:21:22 electron flow gaze goes to methane termites make a little methane, but it's not much far less than a cow.
11:21:31 Not only by size but also related to bio half.
11:21:38 Now we come to the question why is it also complicated.
11:21:42 Why is there not simply an advantage in that eat sugars, and then we don't need to deal with all these funny intermediate fair.
11:21:52 I'm not a real artist I have to admit, so this is one of my figures.
11:22:00 And the left scheme here shows something like an aerobic bacteria that feeds on several different types of substrates, as in complex intermediate metabolism it goes through important internal channels, and at the end several other several products might
11:22:18 come out.
11:22:21 The concept of the communities in such a mess energetic system is a different one.
11:22:30 Here we have kind of a modular arrangement. We have different organisms for every single job.
11:22:39 So they are the classical fermenting bacteria and as I was sitting drops that convert just one fatty acid to acetate.
11:22:50 And the other one is therefore proper need, and so on. And then we have some advantages cooperating with him, everyone has just a very limited
11:23:01 number of tasks to play with.
11:23:05 That makes some things easier. You do not need a complicated regulation.
11:23:11 So, you have just one job to do, and either you have the substrate for it. And that goes.
11:23:19 Of course this is a very simplified view all these books, also have to do all their asset military bio synthetic drugs.
11:23:31 And they do that with a very small amount of energy just one third of an ATP.
11:23:39 And so all that metabolism is also to go into the cell.
11:23:43 So that.
11:24:06 I fish for man in one of his early lectures here mentioned the work by young cliff and his co workers who emphasize that every organism such bacterial cell is packed full with enzymes.
11:24:27 And if you have only.
11:24:31 And let's put it that way. Every collection of enzymes inside. Such an organism, allows only a limited amount of enzyme molecules of every single time.
11:24:45 If you have only a small amount of energy the overall process you need a comparatively high flux of substrate through the system, and to guarantee that you need a comparatively high number of enzymes to do that, do the job and keep that flux going.
11:25:06 So if you have only a small energy, small amount of energy available in the overall process. You cannot have a very long reaction chain.
11:25:21 And that might be the reason why we find in the system where little energy is available only smaller operational units, more or less, running the respective process, and then giving their substrate off to the next one.
11:25:41 That is a concept, I don't say it's the truth.
11:25:45 But it is one concept, how this whole thing could operate. And of course, inside the cell you have a limited capacity of housing enzyme molecules, and therefore, you cannot run too many functions.
11:26:06 Slip please not for the energy metabolism where you need a high throughput.
11:26:12 Inside such a cell if the overall energy gain is low.
11:26:18 I see boys it's not convinced, and I ask a quick question.
11:26:23 Yep.
11:26:25 So what's the alternative here the alternative.
11:26:29 On the left, I see that I have one Barack, can you speak up a bit I cannot hear you. Yeah. Let me try.
11:26:36 It's better. Yeah, that's better.
11:26:54 In one cell or that different cells have the same species are doing the thing on the right. In other words, what is the difference between the left and the right is it.
11:26:57 And I have a genome that that's capacity.
11:27:01 Such a bug that would model more or less than, Arabic to them or not or something like that that can use 100 different substrates. But of course, not necessarily all at the same time.
11:27:14 But it can use many substrates, just depending on when they come in.
11:27:21 So the so the question is then like why do we not see Pseudomonas like organisms in the anaerobic setting but but not that like we're not, there's there's no issue.
11:27:31 There's no surprise why the pathways that compartmentalize they're just thermodynamic reason for that.
11:27:35 Right, they can't do all of the pathways at the same time.
11:27:40 No, they cannot do it at the same time but they can switch on one they ever they are needed for an evidence suited substrate is available. Okay, I understand I understand what the what the comparison is about.
11:27:55 Hmm.
11:27:55 I'm I don't say this is a very convincing model at the very end, I tried to explain or at least to understand it myself. And as far as I understand the argumentation of john correct and his people.
11:28:10 He really emphasizes that the limitation of say metabolic capacity inside the cell under conditions of very small energy increments forces the cell. The to run only few reactions in a reaction line.
11:28:33 But there's a feature album.
11:28:36 Well she let the metabolites of a lot of you.
11:28:39 Sorry. Look, it seems like there's a huge problem once you let metabolite escape from you, you, you just don't know where it will go to.
11:28:51 It is passed on to the partner and both together form an operational unit as we added with see is it drops before the.
11:29:04 Hi.
11:29:03 The hydrogen, that's the same traffic Lee fermenting organism releases is worthless for that.
11:29:13 It's a waste.
11:29:15 And only the Misano, for instance that can take that hydrogen and combine it with co2 to make methane, that really can do something with it.
11:29:26 If, of course you could have both functions in one cell by then you would have a longer operational chain in a year.
11:29:36 And the then you need a higher transformative capacity, have many more enzymes in the same system.
11:29:48 But what do you do was deficient last hydrogen can not escape from the system. I don't either metabolize can just give us away.
11:29:56 It can but it's not sufficient.
11:29:59 And so simple diffusion doesn't help life we try to grow these box in a bottle just with nitrogen around, as a hydrogen could escape but the kinetics of that are very slow so that it's not really convincing a partner sitting close by, and consuming the
11:30:19 hydrogen at very low concentration is far more efficient.
11:30:29 Just more pictures to that later.
11:30:33 They're tightly associated just answered to carry.
11:30:36 So these. That's what what he said why those are those in trophies scientific organisms have fun impact clusters. And so, in fact, I saw the title of it, we get through that in the next pictures.
11:30:50 So, if it's tightly associated.
11:30:54 I'm not sure how you divide into one or the whole.
11:30:59 Right. So here we have such a model, there is the organism, our sin graphically fermenting organism that oxidizes fatty acids so something, and releases hydrogen and this mythology and that takes it.
11:31:15 And now, the distance between both determines the efficiency of the flux.
11:31:23 And if for instance we have them all distributed suspended in liquid here, and you can calculate that some medium concentration of such a culture in the lab.
11:31:36 Say, we have a distance of eight micrometres in that here. And if they sit back together then the distance is much shorter.
11:31:45 And with that, the
11:31:49 flux increases 100 fold, because the flux distance has become much broader.
11:31:58 And this is what we also observe. This is from a certain type of anaerobic reactors that have been worked on in a group of selecting and baffling long for many years.
11:32:12 So they form pellets slide fed about a centimeter in diameter. and that is packed biomass, if you look into those here with a scanning electron microscope.
11:32:24 You see, the cell by cell very close together.
11:32:28 And these are efficient systems that rely that makes this overall process happen.
11:32:36 Now there is another problem remaining with that.
11:32:41 Look, etc flock.
11:32:43 In whatever kind of wastewater reactor or also in a sediment, a group of bugs attached to a little sediment particle or whatever. And I just drew them here as rots and
11:33:00 little balls to symbolize two different organisms.
11:33:06 Of course the efficiency of metabolite transfer would be optimal if that would be mixed like that entirely.
11:33:16 Can you have short transfer distances between the two different types.
11:33:22 But both types of cells will grow, and everyone will be an offspring, only to its own kids.
11:33:33 So, after a while you will get nests formed in such an aggregate is the type one and type two separate from each other, and the efficiency of metabolite transfer of course is far less than it is in such then optimally mixed situation.
11:33:54 So here you have an optimization of a metabolic activity, through a maximization of disorder.
11:34:04 So it works best if it is really mixed up.
11:34:09 Not like my working desk comparable to it.
11:34:15 So,
11:34:18 yeah.
11:34:20 So far, we only.
11:34:22 And this is a problem we don't understand yet.
11:34:25 If you take a picture of such aggregates and try to type if I, the different types of organisms that you'll see here today of course these are all pictures, today you can stain them specifically at least by major groups you can say which type of organisms
11:34:46 are they, and you find both, you find the areas where they the partners appear to be perfectly mixed, and you'll find nests like that, which actually should not operate anymore, and how they manage to optimize that and somehow to avoid such structures
11:35:06 have better have a maximum of these mix situations. That is not known and we don't I I don't have a good idea how to test that.
11:35:20 Now, I was said, Is this hydrogen being transported between the organisms and hydrogen is a very good electron carrier because it's easily divisible it doesn't need transport, it goes through membranes and so on.
11:35:37 No problem at all to do that. And, I admit, it is also easy to measure in at low concentrations and the gas face. And that's why we always look for it.
11:35:49 But there are indications that also for me, it
11:35:54 can be used as such an electron carrier between different organisms the Redux potentials of the format co2 pair and of the hydrogen proton pair is nearly identical.
11:36:10 And, and the NZ two conditions under with the concentrations that we can measure
11:36:18 the are roughly equivalent.
11:36:22 And I show that he in this picture. If we compare the Redux potentials that are established by the hydrogen proton peer, and the four made co2, or for me by carbon and pair they are nearly identical, and compare them at different hydrogen parts or pressures
11:36:45 of the corresponding for made concentrations that's the dashed line. You see, they are rather close together, we have in our systems quite often the hydrogen posture pressures in the range of 10 to the minus four to 10 to the minus five atmospheres that's
11:37:03 here in this range. And this corresponds to a Farm Aid concentration of about 10 to the minus five Mola meaning.
11:37:18 10 micro molar, and that's the value we can measure rather well.
11:37:25 And it's the same range so obviously hydrogen and form eight appear to be at the equal energetic value in such system systems, they might be used even simultaneously by certain organisms as electron carrier.
11:37:45 So part of the electrons are released as hydrogen and others go as for me, there were speculations and also papers that claim that in pure culture or define mixed culture systems.
11:38:02 You can even gain energy from farming conversion tool hydrogen, and that by that way, you can couple formulate oxidation with hydrogen dependent masala Genesis and gain energy from the first step.
11:38:23 It works in artificial setups like that, but in nature, we know there are so many bugs says that have enzymes that put fall mate and tied it with into equilibrium with hydrogen.
11:38:39 And it's very difficult to gain energy from that difference if next door and other organism is living who sets about into equilibrium, then there is no way to gain energy from that.
11:38:54 So, speculations on that so far have been based only on some artificial mixers. And I doubt that they are really representative for natural situation in the cultures that we studied hydrogen, and for made appear to run at equivalent values for this.
11:39:19 I'm wondering, I'm actually wondering if this would be a good time to take a 10 minute break for everyone and then resume with the anaerobic methane oxidation.
11:39:33 Okay. Yes, so we can have a break now.
11:39:47 Yeah, so let's say I'm 1150, or whatever. Our you have 50. I will get back. Thanks so much for that. Thanks so much.



11:54:32 Okay. So, there we go. We are on again. Yeah back to business okay now, I have to turn.
11:54:44 Okay, let's see.
11:54:50 oxidation of method.
11:54:52 That is a business that has entertained already at least two generations of microbial biologists geochemists observe that methane is being used disappears in marine sediments and so on.
11:55:20 at depth where oxygen definitively has no access. Aerobic oxidation of methane has been known for many years more than 100 years.
11:55:39 oxygen appeared. Not very reasonable. Nonetheless, the process happens he is the equation.
11:55:45 It even yields 20 kilo two ways that there should be organisms to grow with that people have tried it.
11:55:53 And I've never been successful.
11:55:57 That's Aleksandr who has dealt with that for a while even collected the whole anthology of papers that claimed to have such organisms and then he picked out what they did wrong they had a vitamin solution with ethanol in it and the Bucs grew on the ethanol
11:56:15 didn't care about failure at all, and so on. So,
11:56:22 this question has remained open and became actually the first access to these things came after the discovery off the gas hydrates as hydrates are huge amounts of methane gas that is trapped in frozen water in sediments along the shelves, mainly.
11:56:52 And so add the depth of about 800 to 1000 meters, water depth.
11:57:02 And this huge amount of methane, 10th of a 19 grams.
11:57:09 That is far more than we have resources of coal and oil still available on this world so if that could be tapped our energy needs would be covered for a long time.
11:57:24 However, unfortunately, probably a lot of that methane would go into the atmosphere and that would be dangerous.
11:57:31 Now, what is so special above such methane hydrates in the sediment now yeah that's a picture of these.
11:57:48 So the ice thing is, kind of, so like a sponge and inside is a structure here you see that inside the water structure, methane molecules can hide more or less exactly in Tuesday.
11:58:02 And this way not only the methane is trapped in the eyes but also the methane stabilizes the eyes.
11:58:10 And so the frozen ice stays solid few degrees over zero, even.
11:58:18 And with that a lot of methane can be trapped there at low temperature, close to zero a few degrees above zero. And at low pressure because the methane is is trapped inside these track.
11:58:34 You can burn that, then it looks like that.
11:58:38 and above such structures were such methane eyes had been found in the shelf sediments such little structures were observed. And they were right at a zone where methane that was confusing up from the gas hydrate and sulfate, that was confusing down from
11:59:03 the sea water where they met.
11:59:05 They are these aggregates existed.
11:59:09 And by specific staining with the 16 s rival Soma RNA probes. It could be shown that the outer organisms here the green stained ones, they were sulfate reduce this or at least really, really related to South introduces.
11:59:27 And those ones that are staying right here. They appeared to be at least related to Masonic chance.
11:59:37 Well methanogens are usually such organisms that produce methane, and these ones here, obviously, operate the other way around, they activate methane oxidize it and to release the electrons to the sulfate reduce us around.
11:59:57 Now, this is again a sin traffic situation in the meantime, there are several types of such anaerobic methane oxidizing dies in communities linked to sulfate reductions that have been observed in such shelf segments and let other places.
12:00:16 And so, we are again dealing with such a sin traffic association of methane oxidizing organism, which is actually a marathon engine running in reverse.
12:00:31 And, a fellow Phaedra to Sir, and they both cooperate, and what is not known is here in between what is being transferred between us.
12:00:51 The energetic situation may explain why such systems have never been cultivated because under standard conditions in the lab with a 20 kilo to, you may be able to grow one organism, but not to work under the conditions in the sediment down there.
12:01:07 We have about 100 atmospheres of messy fans that mainly together with a solid eight and solid five
12:01:16 enhances increases the amount of available energy substantially and now this is sufficient to grow to organisms.
12:01:26 And this is actually being done in Bremen in the Max Planck Institute where they have money for that.
12:01:33 They grow, or grooves these things in the lab of fleets middle at high pressure in thick walls steel cylinders at high pressure, then you could multiply these cultures, really grow them and do physiological experiments on that.
12:01:51 What is still open. It's a question, what is being transferred. It is not hydrogen, it is not for me, it's not methanol it's not acetate all that has been tried it all doesn't help.
12:02:04 One model sets a video lab together with partners in Blaine brought up that it is a sulfur cycle between the
12:02:19 inverted mythology and let's put it that way.
12:02:22 And the sulfate reducer, in which poly sulfide is being transferred here as an electron carrier to this part here, part of that is released as sulfide the other part goes back as sulfate.
12:02:42 And it's partly reduced already over here. So, part of the sulfate reduction also happens in this organism but this one helps to keep the whole thing going.
12:02:55 This is not the only hypothesis Tuesday's process.
12:03:01 in the lab of
12:03:06 freaky all fund in.
12:03:10 She's in Pasadena is she, yeah.
12:03:14 They found indications that there is a direct electron transfer via cycle crumbs between the two partner organisms meaning only electrons go over there.
12:03:25 From one organism to the other one goes through a chain of psycho Chrome's as electron carries all this is possible. There are several types of the systems, and the cells, this is still an exciting story to study how these electrons are being transferred
12:03:47 since the overall process is often enormous global importance. If these aggregates of sin traffic methane oxidizing communities wouldn't sit over these
12:04:04 methane hydrates there, then the continuous release of methane from these places into the atmosphere would cause deep trouble to our atmosphere on the long run, so we should be very happy that they are doing their jobs, even if we don't understand yet
12:04:21 how they do it.
12:04:24 Nonetheless, this Saturday cycle operating here for electron transfer also has a background in another type of sin traffic association that was studied in the lab of Norbert pfennig many years ago and that wasn't photo traffic association of to organisms,
12:04:49 a culture that was originally described club soda one as a truly a culture of a green sulfur bacterium that oxidized organic compounds.
12:05:04 And when they check that culture.
12:05:06 That was broad as I'm from another lab. They check it out and found out it's at least two different organisms in the, in the center here and that's again in a purification in such an aka the illusion series serious, that I mentioned before, we have a
12:05:24 lot of green sub sulfur bacteria around here.
12:05:30 And you see the closer these colonies appear to the central radius colony here, the bigger they get so they are being supported by the central colony here, and this one here is the one that oxidizes an organic substrate for instance acetate and reduces
12:05:51 with that.
12:05:54 Sulfur elemental sulfur.
12:05:57 200 hydrogen sulfide, and with the hydrogen sulfide it feeds the green ones around here.
12:06:02 As a greens oxidize the hydrogen sulfide again to sulfur and that comes back, so you have a little Salafis cycle here between hydrogen sulfide and zero at Vaillant sulfur.
12:06:17 And we're sad and electron transfer again between these partners.
12:06:22 As I said that was observed in the lap of Norbert pfennig at about the same time, when the same traffic beautiful rate oxidizing box were isolated by Marvin Brian, Mike making money.
12:06:37 But, Marvin Brian had been in Norbit Phoenix lab at the time. So, the idea is we're really going back and forth is that this linkage between two organisms was kind of a common pattern of cooperation between such organisms.
12:06:56 Yeah okay that's what they did.
12:06:59 Let's keep that leaves off and that we skip this well. So such solid for cycles can also play an important role in the attack on enamoured electron acceptor outside the cell.
12:07:14 So now we do not have another organism sitting here, which takes electrons from solid fi that has been produced here by another organism.
12:07:26 And the transfer of electrons by that from this organism go it doesn't go directly to the iron it goes through a solid cycle. And this way the irony is reduced out here.
12:07:39 It's an SL external electron acceptor. In this case, not an organism, but an insoluble mineral metal oxide sitting.
12:07:52 So basically you have a similar problem.
12:07:55 How to deliver electrons outside the cell.
12:07:59 And for that now several different solutions exists, such as alpha cycle might be a challenge. Another system that has been discussed in this context.
12:08:14 Are you make compounds. These are complex condensed system of aromatic racist us and India you often have quinoa its structure like this one, or an auto quinoa, here also and they can accept and release electrons and they can help to transport electrons
12:08:33 over certain distances. And with that, deliver electrons from one system to the other one.
12:08:54 So, let's stop that at this point and say well you have observed that in anoxic environments interspecies interactions are much more Commons and in the oxygen supplied world.
12:09:09 Maybe, simply because we never looked with Arab so much for cooperative systems, but at least that's the experience that we have.
12:09:20 And beyond hydrogen or for me It also sulfur compounds and some others might also help to subtle electrons between such as we have seen that sugars and other more complex substrates can be fermented in say partially send traffic associations as well,
12:09:40 with math and engineering partners. And we have found that this minimum amount of 20 kilo to roughly is really the lower limit of at which organisms can work under the conditions we look at.
12:09:59 And with that we have an orientation how much energy we really need for metabolism of subtle.
12:10:09 And with that, if I'm allowed, I would switch.
12:10:13 Yeah, that's a nice picture of our university, it's a lake floor.
12:10:19 And I would now switch to the second part of my talk. Let me have a look.
12:10:24 Okay, we'll open it up and it which I now want to show you a bit. How do they biochemically really solve the problem of dealing with these small amounts of energy they have to deal with.
12:10:41 And I will use two to three examples, just depending on how far we get this is not like work from our own lab.
12:10:52 And, you know, the scheme already, and we know that we need about 20 killer tool for our processes now we look at one of these organisms here that Central Africa oxidizes beauty rate to a hydrogen and acetate, and we already know.
12:11:15 It has certain constraints, under which it has to operate the product concentrations cannot accumulate to a high otherwise they cannot work.
12:11:24 How do they do that.
12:11:27 Well the biochemistry of beauty rate oxidation to acetate is nothing unusual that's a beta oxidation. So the initial activation is paid for by the release of acetone or as acetate by an transferees you activate the jewelry to Goodwill Kool Aid and comes
12:11:53 beta oxidation through quote on your go hey three eyedrops a beautiful. See to answer to call a tool acetone or a the one exchanges he is the other one goes by acetone phosphate to acetate, and forms.
12:12:05 One ATP kind of keeps that.
12:12:12 No, because it has only 20 kilo two ways that you cannot make that ATP. Well, it can make it, but it cannot keep it. We are not done yet, because we have to release these electrons here.
12:12:27 And those in the second oxidation steps they are still kind of convenient that's minus 250 million volt, it's close to the level of NADH meaning fruit minus three on the 20.
12:12:41 And as such, they are easy to handle.
12:12:44 But they have to go to a hydrogen at the end and hydrogen is at minus one and 20.
12:12:51 At least understand that conditions.
12:12:54 And this first oxidation step here from be able to literally go to court on your way that's much more positive. And if these electrons are supposed to go to a hydrogen they have to go up hill.
12:13:07 And that's difficult, how to do that. Well, if we.
12:13:27 You have seen a similar scheme before.
12:13:27 Here I compared hydrogen concentrations at two different scales it's just the same numbers but what's different he didn't pass the bar.
12:13:29 And here you have the corresponding Redux potentials of the hydrogen proton pair and shown and on the right side you have right doc systems inside the cell that have to correspond with that.
12:13:45 So at one atmosphere or one bar of hydrogen here we have the Redux potential of minus 414. If we decrease the hydrogen concentration here on this scale, then the corresponding Redux potential goes upwards.
12:14:04 At the conditions that we use to find in our systems tend to the minus four to 10 to the minus five atmospheres here.
12:14:15 We have a corresponding Redux potential of about minus 300 million.
12:14:22 Meaning, electrons at the NADH level can be released as hydrogen, meaning can be used for proton reduction, that is more or less equivalent.
12:14:35 The same applies for the electrons in the second oxidation step three hydroxyl beautiful KHYC to acid turquoise. That's all in the same range. That's why I put the big black, red dots there by the electrons into first oxidation step, they are at minus
12:14:57 150 125 or according to other values in the literature, even more positive here at $30 million.
12:15:06 So, we cannot release those as hydrogen, we would need hydrogen petrol prices as low as 10 to the minus 10 atmospheres. There's no back to do that for you to keep the hydrogen project prices so low.
12:15:22 Miss Anna Genesis gets into equilibrium already here at 10 to the minus six atmosphere.
12:15:29 So nobody does it.
12:15:32 What do they have to do they have to invest energy to shift the Redux potential of the hydrogen of the electrons of from this oxidation step here to more negative value to finally allows them to be released has hide it.
12:15:54 And many years ago we did a rather simple experiment with a cell suspension of a hydrogen oxidizing, or, sorry, future rate oxidizing sin traffic partner together with a hydrogen oxidizing mismanaging.
12:16:13 And this is again a short time. Experiment with a den sale suspension of the two partners here.
12:16:21 And in that case, we grow them together and then we inhibit again specifically the hydrogen utilizes so physiologically, we look only at the first organism, the other one is not dead, but inactivated.
12:16:39 Now what happens if we feed this cell suspension was built rate, they accumulate hydrogen to a certain limit in concentration here.
12:16:52 And then nothing happens anymore the system gets into equilibrium, we can calculate it, it is really the way we would expect it.
12:17:03 Now, if we stop senate earlier in buying, for instance, adding a, an IO port on our for that destroys the proton gradient across the membrane.
12:17:20 Then hydrogen accumulation stops.
12:17:22 And he is the same effect if we use an inhibitor for the membrane bound ATP.
12:17:35 Then again, nothing happens anymore. So that would indicate that we need for hydrogen where these two this limiting amount we would need an existing proton potential, and an operating ATP is.
12:17:48 Now, to do the control will use cotton eight, which is the partly oxidized and fatty acid. That is right behind the difficult step that we talked about before.
12:18:03 They all exchange YLZ called a derivatives in the cell.
12:18:10 So, If we oxidized quote on a substrate, then neither support or no for nor the ATP ACE inhibitor has any effect. They all accumulate hydrogen to roughly the same.
12:18:34 So obviously, a portmanteau a proton gradient and an ATP as needed to make safe, beauty rate oxidation go.
12:18:38 An early concept that we developed for that was that part of the ATP, that we formed by substrate level phosphorylation in acetate release.
12:18:51 Part of that is hydro lies here to establish a proton gradient. And that proton gradient drives the electron transfer from utero call a to quote on your core a towards hydrogen released by proton reduction, and one model could be that that runs through
12:19:15 a Quinones cycle we know that these organisms have a quinoa, and may not quinoa and goo.
12:19:23 And that has a slightly lower Redux potential than ubiquity known minus $84 million.
12:19:32 And that somehow fits into this transfer here and by degradation of the proton gradient this potential difference, could be energized.
12:19:43 Now that was an old model for that.
12:19:47 In the meantime, this organisms then had been sequenced, and we could show by differential expression of proteins, after incubation after grows with ease a beauty rate, always caught on eight.
12:20:03 After the difficult step. And we could find out that in certain bands he aware and used and we're not in used under the conditions, after growth with alternate, and to cut the story short, here is the scheme now in a condensed version, again here we have
12:20:24 our beta oxidation pathway to ask from military to acetate at the end we form an ATP.
12:20:32 The second oxidation step is not really difficult. We said that already. These electrons go via Nat to a multi enzyme complex that allows to transfer these electrons, either to hydrogen, or to form it, and they can alter this complex can also exchange
12:20:57 for made electrons against hydrogen, or backwards, they are energetically equivalent, as I mentioned before, the difficulty electrons were these here in oxidation of beautiful co a to quarterly is a go via flavorings to a membrane bound Anka here that
12:21:18 to a quinoa cycle. And then, to a site of Chrome and further out here. Unfortunately that's always behind this frame up there. how to is a for made dehydrogenase Can you see that.
12:21:35 Okay, well in my scheme here is somehow something over it. Nonetheless, so for me it is ridiculous, or co2 is reduced the form it on the outside of the cells here.
12:21:50 And with that, electrons are being consumed that goes who was a member in via the Quinton cycle and either the consumption of the protons in the formation of profit of farming out there, or also as a queen on cycle itself, we don't know that yet consumes
12:22:11 part of the proton gradient that is established by the ATP as here.
12:22:19 And the partner, I would say I could either use for me directly as electronic area or convert before made after passage into the cell again against hydrogen and use that as electron as source.
12:22:40 Both is possible, perhaps the box could also use hydrogen, and for me it's similar dangerously. Actually, most Masonic that we know can use both hydrogen.
12:22:55 And for me, so it's very common among them as antigens to play with either one as electron donor, and as I said energetically they are both equal.
12:23:07 So this is our present understanding of sin traffic fatty acid D gradations This is the difficult step. And that needs to be supported through a membrane bound system that consumes part of a proton gradient that is formed by Patrick hydrolysis of ATP.
12:23:28 And with that, the, the overall balance for the organisms is just the remainder of one transported proton meaning one third of an ADP on which they do their entire business.
12:23:53 Sorry,
12:23:53 so much about it.
12:23:56 I want to tell you at least one more story that is acetate.
12:24:03 If we can accomplish even though I said one I don't know.
12:24:06 There is, as I showed you with the old scheme.
12:24:14 There are Masonic friends that use acetate directly, and fermented to methane and co2, that is x iconic releases 35 kilo drool. And a realistic conditions in the sediment with lower than one molar acetate concentrations it comes down to about 20 kilo
12:24:33 tool, and they simply have one third of an ATP again to live on.
12:24:38 That's the situation in a normal sediment in most wastewater reactors and so on.
12:24:45 But it was observed by Steve Cinder many years ago, in a thermal fit like wastewater reactor.
12:24:56 Fat acetate was degraded in a two step process, meaning acetate was first oxidized to co2 and hydrogen.
12:25:09 And then the hydrogen and co2 combined again to form.
12:25:14 The first step is end up chronic. The second one is xi chronic if you add them up then of course you have again this amount of energy.
12:25:26 Now, to organisms have to live off that.
12:25:28 You see, for two organisms that gets a little short at enhanced temperature.
12:25:37 It gets better. So, the temperature plays a certain role here so we observed this two step conversion off acetate to methane and co2 preferentially at enhanced temperatures.
12:25:53 And as you see that helps at least to feed organ to organisms from this step, which is difficult to do at standard conditions.
12:26:20 The first organisms that was isolated by the cinder lab in those days.
12:26:11 Unfortunately, did not live very long it survived three publications, and then it disappeared forever.
12:26:21 And it could not be recovered anymore. Steve's in know gave its of silly name, they bounce it back.
12:26:29 They ballsy back. And this is because if you look at the first equation here, what the organism does is a reversal of what we know as humble as the Genesis, or hormonal acetate fermentation normally co2 and hydrogen will be fermented to acetate.
12:26:53 The thing that's just the opposite.
12:26:56 Of course it does so only in the presence of a hydrogen scavenging massage and otherwise that would run under chronic reaction and couldn't do that.
12:27:08 But as Cinder found out with his people, was, you can actually grow these organism in the opposite way.
12:27:18 So you can feed it with hydrogen and co2, then you get the acetate
12:27:25 that you see how close to summer dynamic equilibrium, such an organism operate.
12:27:34 Nobody of us, you couldn't, I couldn't do that with anybody have you just put you under high pressure of water and co2 and you would not start making sugars or anything like that for sure not.
12:27:47 So, This poor buck can do it.
12:27:51 Or I should say could do it because it's lost.
12:27:54 But a few years later and then the group in Japan.
12:28:00 Isolated a similar organism.
12:28:03 And after they described it, we cooperated with them and then we started this organism in more detail we also sequence that. So we could go into more detailed biochemical analysis of it.
12:28:19 We come back to our old scheme. Now that means that in such a thermal situation in Steve sinners reactor now.
12:28:29 The direct path from acetate to methane and co2 doesn't exist or is very thin here only a little bit on the side, there are some affiliate acetate degrading advantages that do it is direct way, but for some reason.
12:28:48 In these systems, he is a do it in a two step by meaning they run the hormone estrogen metabolism backwards and combine it in with hydrogen dependent methanogens.
12:29:05 It is a little strange that you can run a metabolism in two different ways.
12:29:12 And here again, that's the reaction under standard conditions you can set a boundary conditions under which you grow this book. If you run it in this direction meaning as a hormone estrogen, you can go down with the hydrogen concentration and with the
12:29:30 acetate and keeps the acetate concentration limited and under these conditions at 25 degrees, you can just get your 20 kilo tool out being sufficient energy to keep this thing going.
12:29:45 If you want to run it in the opposite direction you need to lower the hydrogen concentration you need higher acetate concentration and it helps if you enhance the temperature, and then you'll get 20 kilo true in the opposite direction.
12:30:05 And we can even show that in culture.
12:30:10 With this organism in this case we grow it with it some affiliate manager and as partner.
12:30:16 And when both are togethers and we get a quantitative conversion of acetate to methane.
12:30:24 That goes roughly by one by one style geometry.
12:30:29 Then, at this point, we exchange. the headspace the methane is all gone.
12:30:35 And we inhibit the Masonic partner.
12:30:39 By Walmart insane South Lake. Always the same stuff.
12:30:44 And now we feed them,
12:30:51 Hydrogen yes the hydrogen.
12:30:55 That goes slowly down, and now they produce acid.
12:31:03 There's still a bit of methane being formed meanings as a partner still had a little bit to chew on.
12:31:09 But mainly hydrogen is converted to acetate by the same cells that grew here before. That's, again, a den cell suspension experiment.
12:31:21 The same cells that grew before by the in the opposite.
12:31:27 We do not know if they grow here,
12:31:32 but at least they can run the metabolism.
12:31:37 And that means that the reaction chain, probably has been inverted more or less entirely.
12:31:44 We do not know exactly what the differences are. I can show you our present understanding of the energy metabolism, of course, changes are needed.
12:31:57 If you want to make ATP, either way.
12:32:02 So what you win in the one direction you would lose in the other direction if you don't change anything.
12:32:17 So, these are is one paper that came out last year by Kayla from our group, and see so now we start down here if we grow this back for instance on hydrogen forum for made it uses the classical harm ICT Jenny pathway through the tetra hydro for late derivatives
12:32:34 forms and esoteric way, add subtle phosphate to acetate, all the way up.
12:32:42 And they even have a chance to do a substrate, no a electron transport phosphorylation on the way from for made to the reduction of methyl in tetra hydro fully to metal tetra hydro fully.
12:32:59 That's the most positive reduction step here was minus 200 million old, so there is some energetic chance to make a little ATP, and they might do so we don't know that yet that they use a, again, a quinoa dependent electron proton extrusion that is then
12:33:28 coupled to ATP synthesis. Now, that same organism grown in the opposite direction.
12:33:37 With acetate and substrates the pathway is the same now runs in the opposite direction.
12:33:43 There is one very important difference now.
12:33:47 The first step from acetate to asset to acquire a is not an ATP dependent reaction.
12:33:56 But it goes via a ferret docs independent reduction to acid LDL hide. And from there to acetone.
12:34:08 So they cheated but at that point. That's a cheap way to activate the fatty acids the equilibrium admittedly is on the power on the side of the acetate and it is possible only because they get low potential electrons here in the oxidation of carbon monoxide
12:34:29 to co2.
12:34:32 There is a Redux potential of minus 520, and the reduction of acetate to acid LD height is at minus 570.
12:34:43 So, the if we combine those.
12:34:48 Then the equilibrium is on the side of free acetate, and we need comparatively high acetate concentration to get this system going in this way.
12:35:03 But the advantage now is the organism.
12:35:09 Get a net ATP here at the end, and they do not need to reinvest this ATP right away at the beginning again, as you would normally expect that to happen with a citizen running backwards.
12:35:28 But of course again they cannot keep this ATP and full they have to invest it here, and
12:35:37 establish a proton gradient which now drives a reverse electron transport again from the electrons of the method advisor for late oxidation as I said they are at minus 200 million world and to get those out to hydrogen or four made at the very end here,
12:36:02 requires an energy input, which, again, is being shifted by a proton gradient.
12:36:11 They can do so only, this is the difficult step in the overall process, and they can do so only because they get the author the activation of the acetate appear kind of for free, and therefore they have ATP available here at the end of which they can
12:36:29 invest the part into this reverse electron transport business, our current model for that, as I said, we are not entirely through it.
12:36:42 Whether really here, a proton pumping reaction is involved it is probable they have such a main aqui known.
12:37:09 What else look for use it for. But at least.
12:37:04 That's a point that needs to be checked in on some details as well, that we have to work on in the future. So, that is an hour. Yep. Sorry. Excuse me.
12:37:10 I'm so you said several times I to drive the organism This way, you need to.
12:37:23 Where's it going to find such a high ethical situation,
12:37:32 You would. Let's start. This way, the central physically coupled acetate and conversion to methane, that goes by this tool with the two organisms together, obviously has a very bad affinity for acetate so it only works at completely comparatively high
12:37:59 acetate concentration, whereas the classical acetate degrading mismanagement, they operate with far lower acetate concentration.
12:38:12 I can show you I think comes right after that.
12:38:16 I don't want to go into all the details but it's just for that reason that I show these pictures from bio gas reactors that we worked with.
12:38:39 And here is one of them. It is run.
12:38:33 Let's say it's the other reason when such to step acetate conversions become obvious is not only at higher temporary trips, but also at high ammonia concentrations, then for some reason the normal acetate cleaving foundations do not work.
12:38:53 And we observed that here so they get a lot of nitrogen in this reaction through chicken manure or anything like that.
12:39:01 And here we actually observe quite high concentrations of acetate in the room in equilibrium in the continuous operation of the system 10 milli molar is already quite a high concentration.
12:39:24 This is a reactor.
12:39:26 This is a technical system a technical reactor. Yeah.
12:39:31 So it's not a natural system if you will so
12:39:36 this is a working natural system, somewhere. Yeah, these bugs were not born in that reactor, you're right. So, but we we hardly find such conditions in the natural system.
12:39:54 We can do things both ways.
12:39:55 can do things both ways. Yeah, it's crazy. And perhaps these bugs even work sometimes in the opposite way and make their living this way and when the conditions change. They must change dramatically if you have seen, then they can gain energy in the opposite
12:40:15 way.
12:40:17 But, I admit we do not understand the background, the natural background of the strategy, because of course these books are older than our reactors are, and they must have a natural source were really conditions are giving it away.
12:40:40 That's such a system could evolve, where they found will will will the first isolated.
12:40:47 We found that it's not is this here is not isolated the bacteria, we worked with was isolated as well in such a reactor from such a reactor system and again you can ask where comes to stuff from originally.
12:41:08 Can I say selfie link reactor, so it is run at higher temperatures they probably took for bad for him in originally not kill him off this reactor. They might have taken some stuff from a hot spring or whatever else I don't know.
12:41:27 And I'm not sure what the conditions have been there,
12:41:32 you know, your reactor is it must be heavily buffered, this is a.
12:41:39 What's up pH, the buffer system is typically by carbonate co2, and said okay, don't worry.
12:41:50 So, that you have about 100 million dollar by carbonate in there. Okay, fine. 10 milli molar fatty acid doesn't really make it right. There was another question.
12:42:05 I just some quick because you kept them so I noticed that the common theme right in your pocket is that minus 20 kilo job enough for one organism and a minus 44 two.
12:42:15 So I'm just curious since you made such explicit such quantitative statement.
12:42:19 Were you able to show the bunch of, you know, conditions where if you change it say the partial pressure of hydrogen and so on so forth you would have predicted that this temperature, the minus, you will get to minus 20 or minus 40 have this have this
12:42:33 types of experiments being done, sort of like you actually can manipulate the the you know the concentration of hydrogen and say, you know, acetate and so on so forth and just to observe, if you make the conditioning that you you know make the free energy
12:42:47 to be minus 20 or minus 40 that indeed as organisms behave the way you predict the they will behave.
12:42:56 We did not really do consequence studies of that kind, on a single system in the sense that we would change the substrate and productive concentrations against each other to produce exactly the boundary conditions under which such a system would still
12:43:20 just work or not work out at all anymore. That could have been a way to approach that question that you asked.
12:43:31 On the other hand, what we see is that in many cases, we get right into this boundary.
12:43:41 Right. range where we assume that this is the boundary for ATP synthesis that all, and just under these conditions, we find the system just keeps running.
12:43:58 It is not really a, an explanation of or say a,
12:44:08 an argument that this is really the limit, we only can say our models that we have for ATP synthesis, for everything that we understand on the physiological side, that this more or less matches the conditions under which life is just still possible.
12:44:34 It does not mean that we understood the system. It only shows that the models that we have might give a good basis for the explanation of that.
12:44:47 I'm sure this is not really convincing, but thank you very much.
12:44:56 Started sort of with some good questions and in the interest of time we have basically 15 minutes left. I was wondering if it would be okay to switch now to questions that come from the audience.
12:45:13 I'm nearly through anyhow I still had this, it's an old story but it's not, not, I'd love to hear it, and I kind of know it but why not be sort of open up the floor for questions and I from the church, there were a number of questions.
12:45:33 And so please come forward and ask another question.
12:45:40 Hello Bernard. If not, you get more lectures.
12:45:45 And if I asked the question in the chat, which I want to read it already I want to ask you.
12:45:53 So, in earlier lecture, I've talked about the rate versus US trade off, where, when you have little flux and energy available, the metabolic pathways will be longer, so that you get a high yield out of a small plots.
12:46:13 And now you have made it equally clear.
12:46:26 Both sounds very, very good both description some very good, that when there is a energy limited environment organisms have shorter pathways because they need less enzymes and whatever else to sell of me to build this enzymes and stuff.
12:46:38 So how does this fit together.
12:46:45 I,
12:46:48 I would agree that it somehow fits together. But I have to admit that I don't see the connection at the moment.
12:46:57 Um, well, I'm thinking, I'm going to say something.
12:47:03 Tell me what do you think,
12:47:06 Well, my the topic I'm working on is the biosphere. So whatever you're saying I think okay well, what does this mean for me.
12:47:14 And the biosphere is an energy limited extremely energy limited environment.
12:47:39 And as an environmentalist, I think okay what kind of species, or what kind of metabolic pathways and elemental flexes, maybe, can I expect to find there.
12:47:34 And from the theory of red versus your trade off I expect to find organisms there that can get the most out of whatever substrate they find with a very long metabolic pathway.
12:47:50 And when I think about your lecture now.
12:47:55 I would rather imagine that I find several different micro organisms there that maybe live in clusters, and each of them has only a very short, and metabolic pathway.
12:48:14 But this is just a conclusion from the experience that people had with this coma mocks strategy that in us, the US systems they add the biosphere is nearly no energy input at all.
12:48:39 Then you would have unlimited time, you could go extremely slowly with your metabolism you need a very small substrate turnover.
12:48:52 So, let's,
12:48:55 let's put that in a little simple view, you need only one molecule of an enzyme, to keep your glycol Genesis running one copy of a model of an enzyme at all.
12:49:17 For every step to keep the whole thing going because it goes very very slowly. And if you need only these few enzyme molecules.
12:49:22 then you can house.
12:49:27 Many more metabolic steps in the cell.
12:49:28 Then, if you are have to need thousand or 10,000 copies of every enzyme.
12:49:37 So the point is the time scale we're thinking about, I think so. And that was also as a result of this quote marks.
12:50:02 craft had predicted years before on his model considerations.
12:50:08 So perhaps in your system there you have a good chance to find a Masonic and that grows on cellulose
12:50:23 little exaggerated but the half a chance really to see long metabolic trains, even if the overall energy game is small.
12:50:40 I want to sort of add a different spin to what what Ben had said, in the sense, I think there's a difference between small energy and small energy flexes, and I could, it's not just semantics It's huge.
12:50:55 So, Bella talked about small energy said sort of PR kind of wanting reaction little energy is available. This is a consequence of a higher energy flux.
12:51:07 So, if you take these anaerobic digesters.
12:51:14 The ruling of the cow and so on and so forth. He actually have a higher flux and with the high flux of substrates, going through the semester, you have a high influx of energy that strives speciation of pathways into organisms with small delta G.
12:51:30 And maybe that's the confusion.
12:51:34 And again, correct me if I'm wrong. That's sort of the place as a contradiction that, well, how can sort of, you know, a high energy flux have small delta Geez, it's, it's different.
12:51:47 It's one is the flux that causes speciation that results in organisms with pathways that are short and therefore I have a small that G.
12:51:59 Well thank you for clarifying that again.
12:52:03 On the other hand, if you have a high energy Spain,
12:52:10 high amount of energy available in a certain reaction.
12:52:15 Then you can run, or at least the system tends to use a high flux to transform a lot of substrates through it depends on. I mean that's the q&a experiments that Terry has been doing and the Julian Adams experiment in Arabic equalize glucose with oxygen
12:52:37 24 ATP, and the high flux conditions, it stops at the level of acetate, and therefore uses the resource inefficiently.
12:52:48 It's glucose limited there's oxygen around and everything so the fact that the organism has on machinery doesn't mean it Jesus said it actually shuts it off, and then it becomes genetically fixed and in the small economy and the logical me their hands.
12:53:06 And that's a summit, and essentially a simple, a very simplified scheme for, I think, how long can they explain the the genetic speciation of anaerobic energetic pathways.
12:53:23 So it is a little a, say, a different kind of pastor effect. Yes, yes, if you Yes, Pastor effect there you limit the glucose flux. As soon as you get sufficient energy to the end if you aerobically the greater sugar.
12:53:44 Whereas if you ferment it to methanol then you need a far higher glucose flux to keep the same amount of energy production for the cell.
12:53:56 And then Tony has nicely shown that a domestic like gonna look like of equilibrium glucose.
12:54:03 If you have first like a little pathway and then the respiratory which is like a lyric plus respiration, that on a per diem basis, the flux of ATP synthesis through it like a linear pathway is twice as high through the respiratory pathway.
12:54:19 I thought this wasn't just an awesome finding and that that nails it.
12:54:27 Thank you all for that. So, let me as we talk about number. It just doesn't make sense to me I mean the the ultimately.
12:54:36 This type of trade off is in the cell is interested in growing so it needs to it wants to take a lot of protein let's say for growth. But then the type of a system, you guys are talking about a much much slower in growth and and and the same amount of
12:54:57 arm and other stuff that they invested in very little so I really don't understand I couldn't get numbers to make sense aware, like, no for Madonna jails and, and the slow growing animals to me at Comic Con I will flow going and you're thinking of them
12:55:19 fast and the spread of many. And this is just as doesn't make sense to me. I'm.
12:55:21 I'm not sure that the prudent explanation is the explanation for these envelopes, they put in my location because you know in our WordPress methanogens it's actually looks different, so I don't think, actually the podium allocation in terms of kind of
12:55:35 what accountability is the explanation. I think it is more the flux that you get a higher flex to show the pathways.
12:55:54 Yeah, but what's so what's the what's the what's the driving force. I mean, you will get this deal flow right.
12:55:53 It's DATPDT. Right. It's a facet of, You know the overall rate of ATP production through a shorter pathway is higher than through a longer pathway, and a competitive high flux conditioning system that's advantageous.
12:56:15 Ultimately has something to do with enzyme efficiency.
12:56:20 Then, I think the end of efficiency as a result of selection of for these conditions.
12:56:25 It doesn't come first. It's a consequence.
12:56:32 But I just cannot think of. Yeah but, what have you, but do you have okay so for for the reason we went through the call I started was I wanted to see really something.
12:56:44 I mean, whether there's something that's really competitive at the scale of the class right what could it be, and in our case, it was really the rebels on the things like that.
12:56:56 But then, in these other cases when you see the was the DATBDT console that's what ultimately does it announced you were to point your finger on the console.
12:57:10 No, but we are looking here and speciation organisms that drive the speciation, the ultimate speciation speciation is that unlike competitive conditions.
12:57:24 It's more advantageous to have to get a higher flex and with a higher flex more ATP it's a way to trade of from 500 differ in the end.
12:57:37 Yeah, but don't you think that somehow interaction between these two pathways has to be important. If everything you know at the moment. all day here so they're pretty linear considerations.
12:57:52 You know, it's so thermodynamics, or it's flux per enzyme.
12:57:57 But it's all additive.
12:58:00 And you think that breaking you know this separation of labor is the question of convexity and or lack thereof. So, therefore, the wall of interaction, the saturation of labor is just in the eye of the beholder and it's it.
12:58:22 This is not in the view of the microbial community it just happened. Instead, the end product of one organism can be used by another one and it would be consumption is beneficial.
12:58:32 So the division of labor i think it's it's pretty entrepreneur morphic and.
12:58:38 And after the fact.
12:58:45 Not Not quite, you could.
12:58:43 Well, you know, we can always accept an explanation that it is because that's the way it is. But if we're trying to rationalize.
12:58:56 Why, doing two parts of the process into separate cellular bags might be advantageous to doing it, both parts of the process in one bag of twice the size.
12:59:11 You think that
12:59:16 the experiment again I sorry I have to switch to another zoom.
12:59:21 But this this you know the Jane Addams experiment is pretty compelling experiment, right, that you get a speciation under high flux conditions, and there is no no theory behind it it's it's ending you develop this level of metabolic interaction
12:59:43 I can you Terry Can you give me that paper or those papers that uncoupling.
12:59:52 It's in the I put it up on my list of things to look into. So again, thanks. I'm sorry I just have a continuous discussion and I'm so sorry that I have to thank you so much, this was this was awesome.
13:00:08 Thank you.
13:00:09 Thank you very much.
13:00:11 Goodbye night. Thank you.
13:00:13 But maybe, maybe,
13:00:17 just some good the okay we already went to crack but maybe can't Can we hear from, from your perspective, you'll be working on this topic, or this time and it gave us this is just a great overview.
13:00:33 What do you think are the what, in your opinion, what are the sort of important question to address in this field.
13:00:46 To understand better the theory behind the strategies of this multi organizational or multi level degradation systems in comparison to Arabic ones.
13:01:05 I cannot give you a clear answer I think this is a good question to ask, and strategies to go there could really be to find ways to run the same processes at quite different.
13:01:27 Transformation rates are substrate turnover rates.
13:01:33 Most of the systems have been studied especially those ones in reactors and so on have always been studied at comparatively fast substrate and over.
13:01:44 And that's how our pictures have been developed.
13:01:49 Whether that is really applicable to a settlement or even the deep sea sediment as we heard before, is quite questionable.
13:01:59 And.
13:02:01 Okay.
13:02:04 Intelligent ideas to develop experimental systems to study organisms that grow in C to a doubling times over 100 years.
13:02:14 So that's very hard to do and you need very good ideas for that sorry I don't have them right now.
13:02:23 Okay, so I think it's very. That's a very, very reassuring product because because I think I can speak for many people, the students, myself included that are here.
13:02:34 This is a sort of interest of the will. This is one of the things that we are interested in, have a control over everything is super organism versus the separated the modular system.
13:02:50 But then the so, so, so you think you're getting that, because you're still looking at a fast, very fast, very fast or reaction.
13:03:00 Compare to much much slower, but the but then. But then, on the headphone my perspective I thought your system is already at a very very slow scale, but I'm so, so, so sounds like there is a is a pen paddled on a scale at least the number that is, like,
13:03:17 be the one is whether it's this is a such kind of a, a big super organism.
13:03:25 Do we find it so so fun. Nobody had well okay there's example the Nightshade I tried system. I think we will have a separate about later on that. Okay, so, so that that's that's one.
13:03:37 But then, yeah, so then probably to understand it will want to put down on scale what means slow running spots, you know, in terms of the separation.
13:03:46 Okay. And this No Well thank you very much for North, and it's already 10 o'clock, over, over your evening.
13:03:56 Yes.