10:01:02 Yeah. So, we are totally excited today to have Sebastian look at as a speaker today and and Sebastian is a microbiologist and the reason
10:01:21 why we invited him because he led a study that made a very important discovery that is highly relevant for the theme of the trade offs radial trade offs that has been going through the course, so far, and where we mainly talked about speciation as a sort
10:01:45 of a one way, but then sort of the references are there conditions where you don't find speciation.
10:01:52 And the other part reason why we're excited to have Sebastian is that we are going away from organic traffic microbes but we're looking into listen traffic microbes which again is an aspect of microbiology that is unique to microbiology, listen trophy
10:02:09 is unique, and also from an environmental relevance in terms of notification. This is a very important process. So semester and really made with the school bus seminar contribution to finding actually organism that is not specie aided, but has an anti
10:02:28 electrification pathway.
10:02:30 And so, as we had talked about in the first 45 minutes are for you to, you know, lay out sort of the backgrounds, that, that you want and then and the second part is sort of the research talk, but what what really worked well with this terrific group
10:02:50 of participants, is to, you know, allow questions provoke questions.
10:02:56 And Terry and I we will help for the fishing out questions from the chat and contributions and feed the men but feel free to, you know, solicit and induce any kind of questions or indexes that that you want.
10:03:13 Thanks again. I'm the best person for staying up and taking your evening, because you're in.
10:03:23 So it's evening for you and, yeah, We're looking forward to hearing from you.
10:03:29 Thank you.
10:03:31 And I'll start sharing my screen
10:03:36 and start the presentation so
10:03:40 for the first 45 minutes, I decided to take a little bit away from from classical microbiology and take you more into my main field of research with, which is environmental microbiology, and we're going to touch a little bit.
10:03:57 Upon respiratory chain and and these things little trophy in the research talk at the beginning, when I when I give a short introduction there. But in the first half, I would really like to introduce a few concepts and methods, because what you will realize
10:04:12 and please interrupt me when something that I say along the way is not clear what I hope that you'll realize is that if you work with environmental micro organisms.
10:04:25 It's, you have to use completely different tools to study them, then you would in the Northern Territory, if you have a pure culture. So, we usually don't have the luxury of pure culture which we can just simply study and exposed to different conditions,
10:04:41 but rather quite often face microorganisms that we maybe can enrich if you're lucky, sometimes current culture at all.
10:04:49 And still we want to find out what these microbes and students so for the first 45 minutes.
10:04:55 I'll give you an overview of methods in local ecology and I'm sure I saw a few familiar names popping up in the attendance list and I'm sure some of you will be probably a little bit bored.
10:05:07 Sorry for that you can also, if all of you say wait a second, we know all that, please let me know there no hurry on to other topics and maybe give a little bit more research than in the second half.
10:05:19 So yeah, let me know when I go too fast. If something is completely unclear because it's unfamiliar new to you, but also let me know at the same time if I go till things you already know quite well.
10:05:32 So, let's get started.
10:05:34 As I said I will talk about talk about methods. In the first part.
10:05:39 So we'll start with a general introduction to cultivation independent methods, introduce some of the classical methods that we still use a lot and sort of that, you know, we're pushing the field and the knowledge we have today in early microbial ecology.
10:05:57 I will then talk quite a bit of a meta genomics so we can use that to study and understand, environmental micro organisms without cultivation.
10:06:06 And after that it will shortly touch a little bit on activity essays, how can we work with environmental samples to infer processes that happen in this environmental sample, and how can we link function to identity in directly in these environmental samples
10:06:21 so without cultivation without obtaining a pure culture.
10:06:24 What are example methods that we can use there and they will only give one example that will then read later also reappear in the research so that's why I chose that method.
10:06:36 So very gentle yeah what is microbial ecology, it's the study of who is there, when, where and why are microorganisms present and and what do they capitalize.
10:06:49 And of course, your hot springs, especially in the Yellowstone part of a prominent examples that probably all of you have heard about the microbiome of the systems, they also were among this the first systems that were really studied with these environmental
10:07:01 methods were culture independent methods.
10:07:05 And we still old dream of going to this beautiful places and study what's going on there.
10:07:09 I have to say, most of the time.
10:07:13 You're snowed once again.
10:07:15 Most of the inhabitants we are working with actually are not quite as beautiful or not as far away, and not as exotic. So one of my, I have to say favorite talented six you waste water, because it's a very nice model system that is not as complex as many
10:07:34 because it's a little bit more controlled. And it's very easy to work with these microorganisms there because you don't have sediment Soul In the background, all the geo chemistry going on. And so it makes it very nice model system to test new methods
10:07:46 And so it makes a very nice model system to test new methods to work with microorganisms and figure out what do they do and you'll be amazed even, followed by an engineered system that, where you think we understand everything in there quite well because
10:08:01 of course we know what's going in and conditions we apply.
10:08:03 Still, most of the microorganisms in there, we don't really know what they do and how they make a living in there so even in these habits there still is a lot to find out to figure out
10:08:14 a problem we face or a challenge, it's not a problem it's a challenge, I would say, we face in microbiology is the huge diversity of micro organisms in the environment.
10:08:23 So if you take one gram of soul.
10:08:26 This one gram of solo contain billions of micro organisms and their estimates vary, but they will be somewhere between 1000 200,000 different species present in this one gram of so.
10:08:40 So if a compared a handful of soul a few grammar so we'll actually contain more micrograms then we have people on earth.
10:08:47 more micrograms than we have people on earth. But of course if you take this gram of microphones you don't see what is in there you don't see this diversity, you have no idea what they do all you see a storage basically.
10:08:55 And so we need other ways to to understand what is going on in the systems. And, yeah, how can we, how can we learn what these micro organs are doing there.
10:09:06 And the classical approach, most of you probably are familiar with this one is plating. So you take any sample you make a souls layer for example you diluted for enough and you played it on top of an alga plate.
10:09:18 Very nice, very powerful. But in reality, if you look at that, this method, you will see that for most habits, less than 1% of the micro organs present in this environment can be cultured.
10:09:32 So 99% of the microcosms in most environments will not grow in this place, and you will never obtain them in culture and things get a little bit better with sophisticated more sophisticated cultivation methods.
10:09:45 Also, liquid cultivation doesn't have to be that complicated gets a little bit better but still approximate 99% of the microphones out there we will not be able to drop your culture in the laboratory.
10:09:55 And this is why we need alternative methods, why we why we can't just use the standard microbiology methods to study what these microorganisms do out there and that's way beyond microbial ecology and these cultivation independent methods are so important.
10:10:11 The other thing why microbial ecology and cultivation independent efforts really important is that.
10:10:23 Just one question what is activated sludge. There was a question. Oh, I'm sorry yeah activated sludge. It's basically the flux you find in wastewater treatment.
10:10:29 So wastewater treatment selects for for flood forming biofilm suspended biofilm forming organisms. And these slots are purely made up of bacteria. And that's why we call it activated so much because it's, it's sediments in US lunch like structure, which
10:10:45 is actually only made up of bacteria and GPS metrics. And this is where all the reactions happen so carbon cycling nitrogen cycle all happens catalyzed by these bacteria and yeah that's that's why it's activated because it's.
10:10:59 It contains the friction of wastewater treatment.
10:11:03 Alright.
10:11:09 So,
10:11:09 one problem if we do cultivation many micrograms have a very short generation time we call a divide so once every 20 minutes approximately in the laboratory because we provide optimal conditions.
10:11:21 This providing optimal conditions also means not only that we have it in the lab for a few years, which means thousands to millions of generation times.
10:11:30 It also means we temperate, it's not challenged by changing environments much anymore. So, well, you might think you study a very valid deputed vault micro organism.
10:11:42 After few hundred generations which actually study might be completely different than the written well type that you won't have isolated from the environment, or maybe another researcher 20 years ago as isolated from the environment.
10:11:54 And so, also this is a reason for us to always go back to the environment and see how do these micronutrients that we are interested in behave in the environment, do they have this bit half the same phenotypes do they show the same day but as we observe
10:12:09 in laboratory culture, especially if a few coaches that have not seen the environment for for many many generations.
10:12:17 Another problem or microbiology, it's not colorful.
10:12:21 We cannot go out there, we cannot go diving and just look at different fish species also coral species, and just distinguish the micro organisms out there based on their phenotype because if you take an environmental enrichment culture where any Michael
10:12:37 enrichment culture put it under a microscope, it will look something like this. Yes, you will be able to distinguish a few phenotypes you will have short rods long rods Hawkeye, but the, the number of shapes that are micro organism can take an adept is
10:12:51 limited. And so, just based on looking at these microorganisms, you will not be able to identify them and you will not be able to predict anything about their function.
10:13:01 And so, also here we need other ways to to study these mixed communities to see who's there, and who does what what are their functions, how might they also interact all these questions we need methods for that go away from just pure observation because
10:13:16 this is something that that doesn't work.
10:13:20 One of the big biggest inventions on the way to microbial ecology was done in the late 70s by called voce who realized that we can use the rival Soma r amp a, so the RNA molecules that are part of the the ribosome, the structure that is responsible for
10:13:42 translation in the cell that we can use that for fellow genetic analysis, and with starting to use the rival Soma RNA to study bacteria to study archaea but also eukaryotic Colombo's actually was the first to realize that bacteria and the key are completely
10:14:02 different domains. So the three domains structure as we understand the tree of life today. Well, today it's starting to change again a little bit. But still, the standing nomenclature of life.
10:14:14 The division three domains, really was not possible without this realization that we can use certain market genes, as fellow genetic markers to distinguish the microbes is to identify the micro organism and to study their evolutionary history, over time.
10:14:31 And so he was really with that study the first one to realize that it or not, that they are as distinct from the bacteria, as they are from the, from the periods.
10:14:50 So I already mentioned, the these rivals Soma are nice. They are part of the ribosome the ribosome is a very large structure consisting of a small step unit and the lights up unit.
10:14:58 In this most of unit we have one RNA molecule. And, quite a few proteins.
10:15:05 This is the 16 s RNA in the large sub unit we have two RNAs to five so very small one and the 23 is also these can and and to a certain extent have been used for philosophy, but most people started to focus on the 16 s for different reasons that I will
10:15:21 come to.
10:15:25 And why is the 16 s such a beautiful phylogenetic marker molecule and so powerful that has different reasons and the first one, and very important one of course is that it's to be cautious and all organisms, so every organism out there, has a sickness,
10:15:39 Gene, because of course, every organism out there needs to do translation and for that reason it's a ribosome.
10:15:46 And with this conserved function of course also comes that the gene has a conserved function in all of us and it's functionally constant, which means that the mutation rate of this gene is very low.
10:16:01 So, it does not change too fast between micro organisms, which also means that we can use it to infer evolution of very distinct micro organisms, and even of micro organisms self bacterial forcier and eukaryotes, we can calculate back, how long ago, have
10:16:22 they diverge into these two different species. The 16 s, and this is one of the main reasons why we used to six minutes and have the five as it's long enough to have enough information.
10:16:30 So contains words it's, it has approximately 1500 30 base pair, which is long enough to distinguish between all species out there. At the same time it's short enough that you can easily sequences.
10:16:43 And this is nowadays, of course, where we can easily and quickly sequence old genomes not that important anymore. But if you go back to the 70s it could take a researcher, a month or longer to sequence one single sickness molecule and so their links was
10:16:56 very important. And that's why people settle on the 16 s because it was long enough to contain enough information, but not too long, like the 23 years which is much longer.
10:17:05 So it could still easily sequence it, and also very very nice point is that it has varying sequence conservation degrees across the are along the gene.
10:17:19 So it has regions, especially at the beginning and at the end, which are highly conserved, they are almost 100% identical in almost all organisms out there.
10:17:26 So we can use these regions to design primers for PCR, so we can amplify it, and we can amplify basically all micro organisms that are present in an environmental sample without knowing who stare because we know these regions are highly conserved.
10:17:39 And so the probability that the premise we design on these regions.
10:17:52 Also binds to new species, it's very high. At the same time, it also has variable regions there indicated here and very small with these v. So we want to nine.
10:17:54 And these regions are highly variable, which of course is necessary to have a good taxonomic resolution on the species level. So these mutates so fast between microorganisms they can use these regions to study speciation to study.
10:18:10 Do we have, how many different general present how many different species of present.
10:18:14 And of course by now, we have huge datasets, which is important if you want to classify organism so if you want to have Taxonomic Information. It doesn't help me if I amplify something from the environment.
10:18:26 And then can place it, because then you can't put a name to it so you need ways to put an entered and that of course is by putting it in large data sets that contain a lot of well characterized and well named organisms and these data sets are available
10:18:41 right now.
10:18:42 This is what I mentioned right now. The degree of conservation along the sickness molecule. So here again see this, the one to be nine the variable regions.
10:18:53 And what you also see nicely study in between, we do have regions that are really highly conserved in almost 100% of the organism so all these regions can be used for, to the same general premise that allow us to target all organisms that might be present
10:19:08 in your sample.
10:19:10 And with this realization.
10:19:12 It was now able to design the so called RNA approach the rival Soma RNA approach to study, an environmental sample the microbial community in an environmental sample without cultivation without knowing anything about the micro organisms that are present
10:19:26 in the system.
10:19:28 All you have to do is you take a sample you extract the complete community DNA of everything that's in there. And then using primers to target these highly conserved regions you do a PCR.
10:19:39 So you amplify the 16 s RNA gene, until you have a few million copies of it.
10:19:45 This, in the old days was then cloned into vectors that could be transformed into a cola. Nowadays, of course we have high throughput sequencing which makes it a lot faster.
10:19:57 See a question come up Is there any chance that there is a lamp post here, some good organism that not amplified by this premise Yes.
10:20:10 That is definitely one of the, One of the big problems of this approach.
10:20:16 That is something we have learned nowadays with meta genomics which is primary independent, that there actually is a huge group of Michael orders that make approximately 15% of the microbial diversity.
10:20:29 That is not targeted by this primers. So, that is that is one of the big drawbacks of these molecular methods that of course your database that you can design the primers on is based on sequences that have been amplified with similar primers and so your
10:20:45 primer is only as good as the database but the database is only as good as the primers until we started to really do high throughput sequencing without primer so meta genomics I'll come to that later.
10:20:55 And that, indeed, led to the realization that we do miss a lot of organisms. Still, 90%, approximately of the audience out there will be targeted by the general primers, and of course we also have multiple primary periods available because we have these
10:21:11 multiple conserved regions that we can target. And so if you combine different primers due to multiple clone libraries, it is possible to target most micrograms out there, but yes no prime is ever perfect so with any PCR based study you will always miss
10:21:25 microorganisms. Very good question very important point.
10:21:31 But back to the roach
10:21:36 Now we can amplify and clone, and this this six new sequences from the environment without any cultivation.
10:21:44 And then sequence them high throughput clone library so Sanger sequencing doesn't matter.
10:21:49 But why are the sequencing we now can really study, who's present. And so we can use the sequences, after the sequencing do comparative analysis calculate follow genetic tree with nice reference data sets that are available and identify which species
10:22:05 is present in this environment and now versus the environment however versus the community, but also of course you can start comparing samples, how do communities change between two different environments.
10:22:18 And this really led to the so called discovery phase of microbial ecology.
10:22:23 So if you look at the mid 80s.
10:22:25 This all the knowledge we had about microbial diversity was cultivation based.
10:22:30 And so back then.
10:22:32 12 approximate 12 filer were known to procure file to kill files were known. And, yeah, that was the diversity that grew nicely the left so that was what could be studied by researchers in the 80s.
10:22:48 And then with the US over the start of the use of this RNA approach suddenly people realized, wait a second, there's much much more out there than we ever knew.
10:22:57 And then we ever suspected. So, in the mid 90s it was one single study that looked at the microbial diversity in hot springs and the Yellowstone Park.
10:23:18 dependent methods so completely missed up to that day. And if you compare the number of failure we nowadays have in the lab drawing in the lab as a culture, and where we only have seconds information from the environment, the ones which we still cannot
10:23:30 call to cultivate at all the whole file and not a single member of the phylum by far out numbers, the number of cultivated file and that we have available.
10:23:39 So, this shows why it's so important to to have these tools to study the microbial diversity without bringing them into the lab without cultivating cultivating them.
10:23:50 Since it's such an.
10:23:55 Yeah, I guess, I, well, challenge a bit I guess you're making the same point here and I'd be.
10:24:01 So, what do we learn by knowing the 16 s okay there are lots of such thing as our name out there, presumably, but there needs to be ultimately wants to know how these things work and not just their names right and so that needs to be some kind of a spillover
10:24:19 beyond 60 s at some functional kind of similarity. And then, yeah, the, you know,
10:24:28 to a certain extent yes this is what I would I mentioned that the last point, how to link. Okay function to identity.
10:24:45 Yeah, but I can I can already give sort of a short preview here, because you can be right the 60 minutes. It does not tell us much about function. Of course, if you identify 16 S that is highly similar so very closely related to something that you have
10:25:05 characterized in the library, then you can infer something about the function. But this always has to be done with care. Because if you don't know what is to cut off.
10:25:08 Is it 99% identity identity that this Michael wasn't still has the same function. Is it 99.9 or is it maybe it's 97% identity that also depends on the genius you're working with.
10:25:19 So sometimes it works quite well to do these comparisons and say I identified something close closely related to a cola, so it will have similar functions or identified an address bar so it will probably be a natural oxidizer.
10:25:32 That works quite well. But of course, everything beyond that.
10:25:35 So, the Define differences can reduce hydrogen can use for mate, you will never seen the sickness molecule.
10:25:43 About behind I mean, even if you have some strange I'd say 99%, similar to the color.
10:25:50 to ask my very different.
10:26:03 Yep. Yes, definitely because of course it 99% identity, it will also be an issue richer, most likely, I think in that, especially example, she gives us a very very closely related right so they, it can even be a different species or the different Gina's.
10:26:18 But of course you also have no idea is this an pathogenic organisms, or is it an environmental harmless organism. That is something at 99% of entity, you will not be able to predict for this.
10:26:28 This example is set for others it's a little bit easier so natural spera these these these natural fires they are a little bit more conserved in their functions.
10:26:38 So, there you can say okay this is also going to be an oxidizer with a very high probability.
10:26:44 But there's more of these additional functions, because we of course, and that is what we will see in the research talk then have a lot of unexpected functions also present within these it fires, and that you cannot predict based on the 16th.
10:26:58 And so there we really need different methods to link then the function to that entity. The 16 S is only a tool to study the diversity, and to see what is out there and compare different environments.
10:27:10 How does the community change, and we act, maybe two changes in the environment, over time, over different conditions whatever you're interested in. So that's, but but but this seems very interesting so.
10:27:24 But the question is, what is the. So the sensitivity one gets in this kind of compositional labels to resolve temporal dynamics or, you know, some systematic changes in response to certain conditions and change in the nitrogen availability for example.
10:27:43 Yeah, yeah, this is exactly what you can use it for.
10:27:47 Because of course it doesn't cost much to sequences Exodus only and so he can have huge sequencing libraries, especially now with high throughput sequencing, you can get millions of sequence, out of one habitats, very cheaply very quickly.
10:28:00 And so you can executive follow if you want to optimize your wastewater treatment plant out operates how it removes contaminants and you test slightly different conditions temperature, or how much nutrients, you load what is the slots retention times
10:28:22 how long is the microbiome as in the system how much, how long is the liquid in the system. Then you can follow the community changes, how do they react.
10:28:26 And when do I maybe also start losing the micro organs where I think at least they are the most important for the system, so they know nitrate oxidizes the no pneumonia oxidizes when do they start to get washed out of the system or out competed by others.
10:28:37 So roughly what sort of sensitivity is achievable can one sense, a response to I don't know 5%, the change in some environmental factor, or one needs hundred percent change sensitive to another level of present or
10:29:02 scale.
10:29:04 That depends very much on the on the verb to look at, because of course if you think of pH value for example pH is very very important to be more or less stable.
10:29:13 And so a very small pH shift of going from pH seven to pH six five or six, it will change the community drastically.
10:29:23 For other also salinity can have very strong effect. Other factors.
10:29:28 If you go from a few milligrams of ammonia to a few 10s of milligrams might not have such drastic effects.
10:29:38 And so, yes of course the ages logarithmic so it's it's it's a very very drastic change there.
10:29:44 But it really depends a lot on the factor you're looking at, and on the community also there. But usually, if you drop the temperature by 10 degrees for example you will see a response in the microbial community and this approach.
10:29:56 If you sequence deep enough, it definitely sensitive enough to to also see this changing community.
10:30:03 Thank you. And that of course also allows you then to a certain extent without even knowing which which Michael is might be responsible to link certain microorganisms to certain functions.
10:30:12 So if you see at a temperature of 30 degrees notification breaks down, and a certain group of Michael Adams disappears you know one of those Michaels most likely will be involved in notification.
10:30:23 It's not a direct proof.
10:30:24 But it gives you a very good indication already who might be responsible. If it compared to systems that are similar in their operational strategies, but one has good education, the other one is bad notification and look at the difference in community.
10:30:39 I also might already get ideas and who's responsible for the process that I'm interested in.
10:30:47 Hi. Can I follow up on bonuses question.
10:30:51 Yes.
10:30:53 You know, what level of information about temporal dynamics or even reason, environmental history of a micro, can you get from this approach.
10:31:04 I appreciate the point that you can tell what's present and what's not in otherwise intractable systems but for example if you were to take a nap tree, and look at it 16 a sequence presumably you content that it's a boring organism that's been sitting
10:31:19 around in the lab for a few hundred generations. What What you mean is if I can compare it to the vault type of this organism to infer how much it has changed in laboratory.
10:31:32 Yes. So, this is. Yeah, this is something that the sexiness it mutates very very slowly.
10:31:41 And so if you bring an organism to culture and then keep it for a few hundred thousand million generations. The sexiness will not muted, so it will stay constant, so it will not tell you anything about the the changes on the genomic level.
10:31:58 And what makes us even more complex is that quite often these changes in behavior in a lab strain or not even necessarily on the genomic level they can also be on the regulatory level.
10:32:11 And then you wouldn't even necessarily see them very well if you sequence the whole genome, but you will definitely not see these changes have a lead strain, by looking at the six years.
10:32:22 That is not what it's what it can be used for know that that answer the question.
10:32:27 Yes, thank you. Yes. Yes.
10:32:30 All right.
10:32:39 Eco type.
10:32:42 So bacterial species.
10:32:45 Of course, the species concept in microbiology that's something that is very hotly debated. Ever since 216 as it was used and still is.
10:32:56 In the earlier days people started to sort of try to compare for available pure cultures, this DNA. DNA hybridization value so how will discuss the DNA of to micro organisms hybridize to each other, which was one of those tablets ways to classify what
10:33:14 is one species what is a different species, try to compare these values to the 16 s similarity, if you sequence only the 16 s of these two organisms.
10:33:25 Based on that, People defined a cut off of 97% identity of to 16 s to be the species level cut off by now, it is realized more and more that first of all these hard cut offs just don't work.
10:33:40 If you have two species that are below 9997 79% or 97% are identical identical you can be sure that they are different species but if they are more highly similar than 97%.
10:33:55 They might be one species but they might also not be also with the larger data sets people refined this cut off to be probably rather around 98.7%. So it's a much higher than previously.
10:34:21 that it's the same species, meaning that it has the same function, this is how I would define equal time. So, two species that are 99% identical can still have different functions in the environment and might be different equities, but the probability
10:34:28 that they have the same equal type is of course, higher, the more similar the species are to each other.
10:34:33 So that's a little bit of the problem with this 16 s or any any approach that looks at one gene only. That of course it has a very limited resolution.
10:34:43 Compared to whole genome sequencing very rarely see which functions are present between these two organisms.
10:34:48 So, it's very very limited approach but of course very powerful and necessary to learn, diversity, but this is really the main purpose of the 16 s to learn about the diversity of different habitats and and and what what changes between habitats and then
10:35:11 can get nice hypotheses of the functions that are present, but these will have to be tested. If they really are present.
10:35:14 So by now we have data sets which contain more than 10,000,006 new sequences, different species so of course this already gives a very good resolution of the microorganisms out there so if you sequence any sample and and stick the sequences you get back
10:35:29 into these databases, the probability that you will find something highly similar that might already have been characterized to certain extent is very high.
10:35:38 So, of course, the bigger the databases, the more characterized openings. We also put in there, the easier it will be to get a good hypothesis about the functions that might be present in your environment.
10:35:49 But still, these are only hypotheses that should be tested with other methods, so they are completely agree.
10:35:54 That is the limitation.
10:35:57 Sometimes, you don't want to see everything that's out there sometimes you work with a certain function.
10:36:02 But at the same time this function might not be conserved in one defined group. So one define taxonomic group of micro organisms. So if you look at not just fixation for example this this the first step in the nitrogen cycle going from gases nitrogen
10:36:19 to ammonia which then can be assimilated into biomass. Of course this is a process that is highly distributed in many different bacteria, even in some appear.
10:36:28 But, if this is the research, your research focus to study night vacation in a certain environment and the diversity of 95 organisms, we can use functional genes instead, because the enzymes the catalyst, these functions, often are highly conserved, and
10:36:44 similar to the 16 s also have regions that are highly conserved between most of the organisms out there. And so instead of amplifying the 16 so I can then turn to these functional jeans, for the different processes.
10:36:56 So this is an example for the nitrogen cycle where for every step so nitrogen fixation ammonia oxidation both steps. So first step ammonia to it mean second step of ammonia extension I had exhilarating tonight right then nitride oxidation to nitrate,
10:37:21 notification via all the intermediate processes, animals, they all contain specific marker genes functional market genes that I can use to study this function. So, just like with the six minutes I can descend primers for conserved regions of these things,
10:37:28 and then use the gene to amplify all organisms that contain and ammonium on oxygen as in the environment I'm studying.
10:37:38 And then it doesn't matter if they are closely related or not, I don't need primers that only target the subgroup, and I don't have to to to sort through this huge amount of sickness data.
10:37:48 If I sequence everything that is in there.
10:37:50 But of course functional genes. They mutates always slightly faster than the 16 s and for that reason of course the primers, quite often are even more difficult to design, so that you really target everything.
10:38:04 And of course, sometimes you also have convergent evolution so you might have two rather unrelated proteins that suddenly involve a very similar function, and these of course you will never target with one premise premises.
10:38:16 So sometimes you need multiple premises to really target the functional marketing you're interested in in different taxonomic roots.
10:38:25 organism have a defined function in the environment.
10:38:42 We already mentioned a lot of drawbacks of the sexiness.
10:38:45 And, there certainly are many biases along this way. So PCR bias primer might not find it starts already earlier DNA extraction, the chemical DNA extraction method might not be as efficient for certain microorganisms, then the as a mechanical DNA extraction,
10:39:05 so every method here as its biases, of course also cloning and sequencing can introduce viruses so
10:39:14 what what you get out at the end, in the sequence information, first of all, you can be sure it does not contain everything that's in the environment.
10:39:28 And secondly, it will not reliably represent the abundance of Michael organs in your sample. So if you want to know who's the most abundant organism, and how exactly does it change.
10:39:34 Upon temporal or conditional changes in abundance. This will not be reliable the standard sickness with this approach it can give you again an idea of who will be the most abundant organism.
10:39:47 But that's it. And since you're looking at the gene and not not organized and not anything that is transcribed. It will also not tell you anything about activity, and Michael origin might be abundant because it's constantly flushed in from from somewhere
10:40:00 else.
10:40:01 But it might not be active in your environment still here of course you will detect it because you just measured the DNA that is there, and amplify the 16 s of everything that is present in your sample.
10:40:14 Luckily, we're at least two to somewhat resolve this problem.
10:40:25 Method wasn't invented in the late 80s early 90s, there was that allowed us to use the 16 s information that we had to visualize micro organisms in the environment.
10:40:34 This is called fluorescence and see diabetes ation.
10:40:37 And it's actually a very very nice but very powerful easy it was still very powerful method, very simplified How it works is, instead of using a PCR primer, you also use and more or less identical short stretch of DNA and all legal nucleotide have somewhere
10:40:53 between 18 and 20 base pair that now has a florist and label attached to one end.
10:41:02 Another way is that otherwise has the same properties as the PCL primer so it can target highly conserved regions staining almost every bacteria, but it can also target these these highly variable region so it can be specific for a single species, you
10:41:12 might be interested in.
10:41:14 And of course in between all levels of taxonomic
10:41:19 have targeting different taxonomic levels are possible so you can go for the genius level the family level, whatever you are interested in and what you do here is you make you preserve the microorganisms, I have the complete method on the next slide,
10:41:34 and then you get these probes into the cells where they can bind to the rebel Soma RNA So, not to the gene in this case but really to the RNA. In the ribosome.
10:41:45 And then, if it's 100% identical. Stay bound. And so this is a molecular beacon to detect these microphones because only those microphones that are half to 100% identical sequence to your prob will at the end before simply labeled.
10:42:02 And so now I can distinguish these microphones and study them in the environment.
10:42:07 So here a little bit more detail on fish on for instance in civilization.
10:42:12 The first step of course is you need to preserve the symbol. So you go to the environment you you take an environmental sample, and you fix it. So, the most common method is secure at power formaldehyde so formalin that kills the sample because you don't
10:42:27 want the sample to change while you bring it to the laboratory.
10:42:31 And it stabilizes this the cell morphology of every organism in there, so that they don't degrade, they, they stay intact in their cell morphology.
10:42:41 Next, they're criminalized. So the membrane gets provable so you can insert or get these only going to click type probes into the, into the cell.
10:42:52 Because of course that's where they need to go to be able to hybridize to the rebels Omar.
10:43:18 environmental samples so in their natural environment still, of course, slightly changed or modified.
10:43:26 Since I have to dry it on a microscope slight, but still it's the closest we can get to really visualizing them international environment. And on the one hand, this now of course also allows me to really in a mixed population of microorganisms to distinguish
10:43:38 different cell types that are present, and put a name on them from approach that good enough.
10:43:46 And on the other hand, also to see how many of which Michael Adams audio, so I can quantify them, and really say this Michael wasn't is very abundant and this Michael is very rare might not be that important.
10:43:58 And furthermore, of course I can also learn there.
10:44:04 I'll come back to the slide in a second about the structure, how do they grow in the environment. So, this is a picture from an activated slush flock so from micrograms how they grow in the notification stage of a face would have a wastewater treatment
10:44:18 plant.
10:44:20 And what we see here and white.
10:44:22 These are natural sparrows natural oxidizes, and now we can really see how do they grow in the flock, how abundant are they.
10:44:30 What is the growth form so we see they don't grow there as planktonic single cells because of course they would also be washed out the system, but they're really formed these nice clusters clusters in the activated slush float everything in green here,
10:44:43 this is a very gentle bacterial profits don't stains every bacteria. So, in this image we don't learn very much about possible interactions with other microphones.
10:44:53 But of course, you could also combine it with a probe now for ammonia oxidizing. So of course notification, two step process we have ammonia oxidizing foundation to match right and then nitrate oxidation to nitrate, which is done here by these natural
10:45:07 spiral. They will rely on ammonia oxidizer in their vicinity to produce the natural things. And so if I use drugs for the known ammonia oxidizing it's very likely these microorganisms here, these, these colonies will light up with those probes.
10:45:21 And then if I do these studies I can I can learn something do certain types of mycotoxins always. co. uk your next to each other, because of course if they always procure in close vicinity each other.
10:45:34 It's a very good sign that they probably will interact and then I can form a hypothesis what could be this interaction between these two organism and and design, a targeted approaches to study is this interaction really what I think it is.
10:45:46 So it's a very powerful tool to give you ideas about these interactions to develop hypotheses. But again, it's not a proof it's only a tool to develop our processes that you can then take further and study further.
10:46:01 And so back to this slide, this sort of close this RNA approach to the so called full cycle RNA approach, because now we can use the sequence information about the only ones that you identified wire sequencing, in the environmental sample.
10:46:15 You can use this to design specific probes for the microbes is very interested in and take it back to the environmental sample and study. How often do these microphones appear so what is their abundance.
10:46:26 And where do they appear, and how do they look how did it go, best that already tell us something, give us an idea of their function in this environment.
10:46:38 Right.
10:46:40 Before I move on to me to genomics.
10:46:43 Any questions still left for these sort of classical cultivation independent method.
10:46:53 But then I'll move on. Just also interrupt me if something pops up in a few minutes.
10:47:01 Now of course nowadays, the sequencing capacities, have immensely changed and increase so now we don't have to rely on sequencing single jeans anymore we can do much more we can sequence whole genomes.
10:47:16 Even whole environments in one go. So this brings us to metronomic The next point.
10:47:22 Just to make sure that we're all on the same page, sure you all have heard that before. So a genome can be defined very simply as a parts list of a single species.
10:47:34 And in microbiology, how do we normally get to such a genome. It's by culturing this organism trying to get into your culture, and then sequence the DNA from this pure culture, because then we of course also know everything the sequence belongs to this
10:47:47 one organism, but as I said, way less than 5% probably way less than 1% of the mechanisms out there can be cultured, and especially in pure culture. It is really difficult to get 99% of the diversity out there into pure culture.
10:48:02 For that reason, we are people develop metagenomics we need tools to study, sort of, complete microbial community that is present in a complex sample without this cultivation step.
10:48:13 And that's exactly what made the genomic status. So media genomics is still a partial list but now if the complete community, not have an organism, but everything that is present in your sample.
10:48:25 There are sometimes a little bit in my opinion confusions about the Terminator genomics, because you can do two different approaches with this environmental RNA that you extract DNA that you extract sorry.
10:48:40 The first one is basically what we discussed so far.
10:48:43 This is you amplify the 16 s, and do high throughput sequencing of that.
10:48:49 Even in the Illumina instruments. This is called meta genomics for some reason that I still don't understand because it has nothing to do with McCutcheon omics meta genomics is defined as taking the community DNA of an environment sample and directly
10:49:05 sequencing, without PCR with our targeting for certain genes, without any of these steps.
10:49:12 So don't confuse admitted genomics is really only the sequencing of everything. As soon as somebody says yeah we amplified the 16 s and then admitted genomics community message nomics whatever it's called.
10:49:24 This is not a genomic stress applicant sequencing, which is the, the high throughput version of the RNA approach that we discussed right now because it does exactly the same just using high throughput methods.
10:49:37 But so what I want to talk about is really this community sequencing of everything that's out there without the use of specific primers to amplify that
10:49:46 I see a question coming by. When you say can't be cultured.
10:49:52 The question of why they can't be cultured i think that's that's still a very highly debated topic, and it also differs from my goals to micro organism.
10:50:14 Ensure that for, I don't know another 123 maybe 5%, you can design a medium. People have also tried to get genomes of uncultured microbes and and then sort of learn from the genome.
10:50:21 What do we have to add to the medium, so this Michael was in Joe's. They are surprisingly few examples where it actually has worked this approach. So the genome and formed cultivation.
10:50:34 bird Yes, and I'm sure that still for for many organizations out there. We just don't know the medium to get them and culture.
10:50:43 Sometimes it's not so much the medium sentence can also just be the conditions that for some reason there's something on our plate that's inhibitory or also even in the, in the bottle we use for cultivation might be something inhibitory, we had, we have
10:50:57 seen in the past that for our natural spiral, for example, rubber stoppers that you commonly use in the lab to close bottles. They apparently contain something that goes into the gas face some chemical that is inhibitory for the microwaves we want to
10:51:12 grow.
10:51:14 And so unless you really carefully bowl these rubber stoppers three times before you use them, the microphones will not grow just because you close the bottle with the wrong rubber stopper.
10:51:23 So, these these tiny effects can all contribute to why am I goals and can't be cultured.
10:51:29 But of course often they might rely on interactions.
10:51:34 Because they they need something other woman's produces, and maybe it's not the not even the chemical itself, maybe it's also something on the regulatory level, which we really don't understand that well yet.
10:51:48 So I think this this what you also put in the question the mono culture is the second point to consider often Michael was might be, you might be able to get them in in culture, by not getting them in mono cultures and pure culture but in having a cool
10:52:02 culture of two or three organism. And that is something that is way more often achievable, then really getting up your culture of one organism.
10:52:11 So it can be culture, this is a little sloppy term can mean many things.
10:52:17 It doesn't necessarily have to mean getting it into monoculture but this is how it's usually used It can't be. It's not possible to obtain a pure culture with the methods we have
10:52:30 very short history of genome sequencing, or sequencing in general.
10:52:43 So sequencing started here in the mid 70s with the development of Sanger sequencing sequencing still is very nice very potent so still used quite a bit today because it's gifts, very high quality and long reads it can sequence up to 801,000 base pair,
10:52:54 which most of the high throughput methods, at least the second generation sequencing methods, can't achieve. So that's why it's under sequencing also still is around.
10:53:04 And we're seeing a sequencing really sort of the sequencing kicked off. So this is what Carlos made use of by sequencing the rebel so my RNAs in the 90s.
10:53:16 The first 16 s based study was performed. So, you know, in the in the 90s. This this discovery phase of the microbial ecology, really kicked off in 95 something that's missing here the first part your genome sequence by Craig Venter.
10:53:30 So this was Mycobacterium published in 1995 and 98.
10:53:37 meters. The Terminator genomic was terms by Joe handles man.
10:53:40 So also then people started to to look into this option of sequencing without purifying without cultivation and from there and really the sequencing went up exponentially.
10:53:51 Till today.
10:53:52 So with this introduction of the second generation sequencing machines alumina which is the most widespread.
10:54:01 Nowadays, but but also selected machines for for for this this were second generation high throughput machines that came to the market. In the early 2002 2010.
10:54:15 And now the development is really going into long read third generation sequencing so Park bio is one example nanopores and also also very prominent one, which allows us to to sequence very long reads, but they still didn't take over the market because
10:54:32 they have the problem of a very high error rate. So this is shortly summarized here, Illumina is set the most prominent second generation sequencing machine on the market.
10:54:45 It has many advantages. It has a very low DNA requirements so tiny amounts of DNA from environmental sample can already be sequenced.
10:54:53 There is depending on the kick you use there is some amplification but not using specific primer so it is more or less, unbiased. In this respect, but it has some biases in genome coverage there are regions that Illumina can't do that if they are very
10:55:12 GC rich for example it's more difficult to sequence them. If it's very repetitive. It introduces many errors. So, it has some systematic biases. And the biggest problem, and I'll come to this point a little more on the next slides, is that the read output.
10:55:29 So the sequencing output is very short, the fragments that it generates are maximally 300 base pair that depends on the chemistry on the machine. Sometimes it can be as short as 100 to 150 based view.
10:55:41 And of course if you think of a microbrew genome that has five megabytes beer.
10:55:46 And you fragmented into all in small pieces of 100 bass player, it's almost impossible to stitch back together.
10:55:53 And this is where this why these these third generation long range sequencing machines are so interesting, even if they still have a very very high error rate so you see it here, both nano poor and bio still have approximately 10% error rate so one and
10:56:15 10 basis is probably miss cold is wrong. That's why it's it's still difficult methods are being developed but it's still difficult to sequence the genome, only with these with these long reads sequencing machines.
10:56:26 And then of course stitch it together more easily. Because you do get these these long reads that can spend up to 100 KB, or even longer.
10:56:35 So what most people do nowadays is combine the two methods, which is really powerful because of course the luminary you get a very high coverage with very high accuracy, but this one reads, but if you then have long reads, that might be high and error
10:56:45 rate, but half this links you need to stitch together the genome. You can use a long reach to get a scaffold for a genome, and then use the short rates to correct the the genome to correct all the Miss called basis by the long way sequencing, techniques,
10:57:05 and so to get a high quality genome. In the end, so that's where the field is moving to right now. But of course these methods also get better and better and better per year.
10:57:14 And so, yeah, hopefully in a few years, we might not need this trick anymore, but can really go directly to whole genome sequencing with this third or by then maybe fourth generation sequencers
10:57:27 the workflow of all this cloud now mainly focuses on this the second generation of the short rate, high throughput sequencing because this is still what is most commonly done out there, and and so it's it's most important to really understand what's going
10:57:40 per se quite simple, you're taking environmental sample that contains some mixture of micro organisms.
10:57:50 You do a DNA extract of everything that is in the sample.
10:57:55 This is them prepared for sequencing of course you can sequence it's directly in most methods.
10:58:01 And then sequence, which gives you the so called sequencing reads, and these are set usually have a rather short read length of somewhere between 103 hundred bass player.
10:58:15 Of course with those sweets, you can't do much because they don't even contain a whole gene. The average virtual gene has a length of 1000 base pair. So on a short fragment of even 300 base pair, you don't even have a complete gene on there so you can't
10:58:26 do much with the streets. For that reason, the first step that is done with the sequencing reads is the so called assembly into context where you try to look for overlapping regions in the sequencing rates.
10:58:39 And as soon as the assembly software, find such an overlap it stitches together these, these reads into longer context, and these contexts, they usually started rather than 500 base pair and can already go depending on the complexity of your sample go
10:58:54 up to large parts of the genome, so it can spend up to one mega base pair, something like that.
10:59:00 But that of course depends a lot on the complexity because the more organized Jeff here, the more difficult it will be to find these overlapping pieces and stitch them back together correctly.
10:59:13 And so, for for simple enrichment. This can really yield you quite quite good genomes.
10:59:18 And now of course, this context or long enough, so they contain whole jeans. And now you can extract it will translate these genes of the proteins and start comparing them against databases, so and to be a database for example, which contains a notation
10:59:35 of all these teams and then of course you can extract the documentation and learn something about the function.
10:59:41 But, as you can imagine what happens in this approaches you take your micro the genome.
10:59:49 And by sequencing it with this short rate, high throughput sequences, you really break it down into tiny tiny tiny tiny frequency frequents. And so even for pure culture, it is hardly ever possible to reassemble them to complete genome.
11:00:01 But the advantage of a pure culture of course is you don't necessarily have to care because you still know that all the pieces you get back are from the same organism.
11:00:10 And so you can say all the functions I find on this on my context belong to my organism and soul jeans I find I can also say that my organs and, at least as the genomic function potential.
11:00:22 To perform this function.
11:00:24 If you work with mixed Mykola communities of course that is much more complex because you have so many more organisms in your sample. And so it will also never be possible to reassemble every single my goals and completely and so you end up with this
11:00:38 mixture of context of all the organisms that are in there.
11:00:42 And you still you find all the functions on there, but in this mixture you still don't know which Michael origin does it belong to.
11:00:52 And so what you can learn at this stage is mainly the functional classification of the whole ecosystem so which functions are present in your ecosystem.
11:01:03 And you can learn about the genetic classification of the organism who's there. So you know who's there, and what can they do, but you can't link that you you don't know who's doing what we just know these functions are president these microphones were
11:01:14 present.
11:01:14 So, you need to be very careful not to operate these results because of course if you sequence a lion and an eagle, you don't have a flying lion.
11:01:22 Even if you have all functions in your content library.
11:01:25 But you need to be able to separate them back apart and say these functions come from the line, these functions come from the eagle.
11:01:33 And if you translate that back to also, how do we understand how an ecosystem works.
11:01:40 You can really stay to to to to understand an ecosystem. Well, you need to understand, not the function of the functions president ecosystem, but you need to understand every single species that is present in the ecosystem, and what they might be doing
11:01:56 in this ecosystem. There was a question can organize.
11:02:00 Be sequence and analyze but this method.
11:02:03 You can do transcriptome it very sequence, only the RNA fraction so that that's, that gives you the picture of what is transcribed in your sample. So which genes are active, so to say, which functions most likely are active.
11:02:18 But of course if you only look at the RNAs you will never find the linking region so you will.
11:02:23 Without good genome data for the same habits.
11:02:29 You will never be able to to to stitch these genomes back together. So transcriptome mix.
11:02:37 Unless it's a very well characterized habitats. Always should be combined with me to genomics so you learn about the microbes into the present, and then you can use the RNA sequencing to learn about the functions that are probably most important for each
11:02:49 of the micro organisms and for the community and total.
11:02:54 What is the use of applicants sequencing.
11:02:55 There we go back to the original discussion we had on 16 s sequencing in general, because of course, what you do here is you do the RNA approach you do you do community sequencing to identify who's there.
11:03:08 And how does the community change between different ecosystems between different conditions between different time points. So what happens over time.
11:03:15 The difference to the RNA approaches we discussed it before is it's much more high throughput, so you have a much higher resolution. You are more limited to the shorter wavelengths so that is a little bit of problem and applicant sequencing, that you
11:03:28 cannot sequence the full six minutes but only a shorter region of up to 600 bass player. If you, if you use overlapping sequencing reads, but that contains enough information to give you a good overview of the community structure of the diversity and
11:03:44 of changes between different communities, but of course it does tell you very very little about the function. So they all the limitations we mentioned earlier for 16 hours sequencing, of course, are the same for applicant sequencing, because usually it
11:03:58 is 600 sequencing. You can also use it for functional teams, but also the of course you have these, these limitations.
11:04:06 Other ways to the infrared entity from assemble context Yes, I'll come to that.
11:04:12 So of course that's what we try to achieve that that was exactly the next point.
11:04:17 So, this is called the bidding process, because of course we need to get from this mixed fruit bowl. So this mix mixture of context that have been assembled from the sequencing reads, we want to extract the signal genomes because we don't want this mixed
11:04:33 part list. We really want to know which microphones are there. And what are their potential functions in the environment. This process is called Benny, which is the most complex sort of metagenomics usually.
11:04:47 So the most the time and and work intensive one.
11:04:51 But this is really necessary to learn not only who's there but also what can each single individual do in my sample.
11:05:01 There are different methods available. In the end, what what bending boils down to is, you look at the sequences, the context you have and you try to identify similarities between different certain groups of context and differences between these groups
11:05:16 of context. So in this example if you look at the mixture of Lego bricks of course there are similarities between the Lego bricks so you can sort them by color by size by length by whatever you want.
11:05:27 There are clues that can help you to sort them back to the micro genomes that were in the original sample.
11:05:36 What can be used for this.
11:05:38 The most common ones are seeing based on sequence composition, because of course microbes. Don't all contain the same DNA, the DNA difference in its primary structure, it can differentiate the content you have very cheesy rich organisms that can go up
11:05:54 to 70% THC content.
11:05:59 Others are very secure, they go down to below 30% gc content, and that I can use I can just simply look at the average GC content of every single contact, and then sort them into preliminary bins, one been for the the ITC one but for the low and one for
11:06:14 the medium juicy context.
11:06:17 is slightly related, but a little bit more powerful often these tetra nucleotide frequencies, they have to do with the coding probability for the code on usage story of a micro organism.
11:06:49 triple triplets some amino acids have six different triplets that encode for the same amino acid.
11:06:49 And this preferred code on differs from organism to organism.
11:06:54 And that of course you can also quantify them over the genes on these contexts.
11:06:59 This will mean that these these triplets will differ. And for some reason Don't ask me why because I don't know it.
11:07:07 It was found to be more sensitive not to look at the triplets but attach a nucleotide so for nucleotides fragments that sort of are quantified counted in a sliding window over the complete context.
11:07:18 And then are also characteristic for the different micro organisms.
11:07:24 Could there be organisms that have a party ITC and and other parts of the gym with a low GC.
11:07:31 Not as far as I'm aware, so the the GC content you really usually is quite uniform over the largest part of the genome. But, of course, the second point eg from worrisome to transfer it's very important that can happen.
11:07:49 There are certain scenes like the 16 s usually has a rather neutral, juicy content. And for that reason, in extreme genomes deviates from the main juicy content of the genome.
11:08:00 And of course, also, if a gene was acquired by recent horizontal gene transfer, it also might be different because it might be obtained from an organism that has a quite different to see content.
11:08:11 Over time, so the longer gene stays in the genome, it will adapt, it will also mutate slowly towards the medium to see content of the organism.
11:08:21 So, if unless the assumptions for his recent.
11:08:25 It will not have this difference anymore, but of course if it wasn't recent yes you have these differences, but usually then only over small fractions and not over large fractions of the team.
11:08:36 Besides, This sequence composition methods.
11:08:41 You can also use abundance of certain sequences in in your sample, and that is a very powerful method that I want to go into a little bit more detail the next slides.
11:08:56 Do we know where the GC. Well it's adaptation that has to do with a quote on user just has to do.
11:09:03 Yeah, it was always thought that has to do with the stability of the DNA that ITC is better in very warm habitats. So for them affiliate organisms. Nowadays it seems that this is actually not the case so much, but it might have still limit to do with
11:09:19 habit of adaptation.
11:09:23 I would say the main reason probably is this this code on usage adaptation that. Then of course, they can also select for for more GC by exactly I can't really give you an answer.
11:09:36 What's what the evolutionary purposes behind this this GC contents. So, yeah, I said the first theory that it has mainly to do with stability.
11:09:46 Because GC binding us more stable than at binding and and for that reason it's beneficial in warmer habitats, that's doesn't seem to be the case but still, it might have advantages under certain conditions.
11:10:01 Before I go here into this abundance based methods. You of course can also use taxonomic classification. So you can use every look at every single gene on this context.
11:10:12 Extract marker genes that are conserved in many organisms or even all organisms and and to try to classify them so compare them to databases classify them and, and then try to identify context present in your, in your mixed library that have the same
11:10:28 taxonomic classification so that can be used in addition to all the other methods.
11:10:34 But I want to maintain focus here on this this abundance because that's something we really use a lot, because it's, you know, gives gives you a very high power to to distinguish different samples.
11:10:45 So once you have to count context, you can of course take this.
11:11:01 The Mr Dominic reads the role sequencing output and map it back against the context. And then you can count for every single nucleotide in this context, how often, was it covered by the sequencing weeks, so how often was this position sequenced in all
11:11:07 your sequencing library, and that will differ depending on mainly on the on the original abundance of your organism in the original sample. So an organism that is very abundant Of course will also be sequenced more often, because they will be more genome
11:11:23 copies going into your sequencer. At the beginning, and so it will have a higher coverage, every nucleotide position will be covered 10 times 20 times 50 times, while others will have a low coverage.
11:11:34 And this you can use by simply plotting.
11:11:39 Some compositional metrics like the GC content against this coverage in your sequence library and suddenly you start to two separate single or single clouds of context that then start to really come from set separate organisms.
11:11:58 So now you can really separate and say okay here, maybe I have to sequence clouds maybe it's one, but they all have a very similar very low GC content all have a very similar coverage.
11:12:09 So let's separate these contexts, from this context or sign up here, and also period can already see, there's one big cloud might be a very large genome might be several organisms, but they're also you you start to be able to distinguish additional clouds
11:12:24 of differences in abundance in GC content. And of course you can do this in multiple dimensions so you can attach it to a frequency here to have even more power to distinguish between these organisms.
11:12:37 So, this is where abundance starts to become powerful, but we can take it also one step further, we can apply tricks to use it even more, because of course if you have not, if you sequence not only one sample.
11:12:49 But if you sequence two samples that are not identical, but come from very similar environments. So, I will have a few examples in the next two slides, it can come from two wastewater treatment systems or two bedrooms in one trade in one place will feed
11:13:02 them system very expect the same organisms to be present, but probably since they are slightly different systems parallel reactors, they will probably be a different abundances what it can do then is to simply plot the abundance in one sequencing library
11:13:21 against the abundance in the second sequencing library.
11:13:24 And this suddenly allows you to really nicely distinguish without any looking at any differences in sequence composition, or at fellow genetic modifications or anything.
11:13:34 It allows you to really distinguish between.
11:13:40 Quite, Quite a lot between different organisms that might be present in your environment. So in this example it's still an enrichment culture quite a simple one so you see now, I start to see or I can be quite certain that there probably are four organisms
11:13:53 present this you might still be a mixed population of different protein bacteria, but at least for the most abundant organisms, it becomes quite clear that I seem to have to have Tina material there.
11:14:02 And one night to spiral that are now can extract, so I can now really extract this set of context and be quite certain that all these come from one natural spiral organism, and really belong to my one organism then.
11:14:20 So my main question, of course.
11:14:23 Yes, thank you very much. So, I agree that the abundance is very powerful and thank you for presenting this lecture, but I would like to ask more about the biases that abundance can give you for example you have a diverse environment that they have different
11:14:36 organs with different vocal position.
11:14:51 And maybe, maybe, for example, access the night has to do with your gut negative or positive can give you some kind of problem there is this occasion or is it solved somehow.
11:14:56 This is actually something I come to after this slide because it's a huge problem.
11:14:59 You always have sequencing biases because of DNA extraction biases because a gram negative might build need different DNA extraction methods from to complete your gram positive, but exactly this bias.
11:15:25 patient for two slides, then I come to exactly that and I'll come back to your question. Okay. Great. Thanks.
11:15:23 Yeah, so, because the question of course is how can I get these differential coverages. So where do I get my two samples from, and there are two ways.
11:15:32 The more, maybe more logical one is you can do short term enrichment of the sample of interest if you are interested and this is the biggest drawback of this approach.
11:15:46 It only works if you know how to keep an organism active and maybe even to enrich it a little bit. But if I say I want to, mainly interested in the 90 fires, President wastewater treatment.
11:15:59 I can, of course, take a sample into the lab.
11:16:03 Start a batch cultures, just a bottle start a slightly more complicated reactor, and then rich on a medium that resembles the wastewater, so the water that goes into this tank, and then maybe add specific substrates like ammonium to enrich for then it
11:16:25 fires and then I let that reactor run for a few days two weeks.
11:16:25 And of course, you will have an enrichment process. So compared to your original sample.
11:16:31 The diversity, over time, will decrease, and the organisms you, you select for you enrich for by adding the specific substrate will increase in abundance.
11:16:44 And for that reason of course, if you look especially at the species diversity.
11:16:50 It will be a lot easier to sort through this meta genome, because the diversity will be lower and your interest will be covered more.
11:17:01 As a result, we'll have coverage in the sequence library will be easy to assemble.
11:17:05 But, and this is exactly where we come to the DNA extraction. Often this is not possible and you also don't want to enrich because you don't want to have this enrichment bias you want to know what's present in the original sample.
11:17:18 And the very simple trick to apply here is to use two different DNA extraction methods, one that might might work slightly better for 10 volt grow negatives.
11:17:28 And another one that works better for everything, or even slightly selects for the ground positives. And then you don't mix this DNA to get a good representation of everything that's there but your sequencing separately.
11:17:40 And then you also map, you read back to these two.
11:17:44 So you co assemble both sequencing rates sequencing libraries.
11:17:48 You sequence everything barcoded so you can sort it apart you know which Sequence read comes from example, but you mix them to assemble them because then you can be sure that in your assembly all organisms will be present that were present in your original
11:18:03 original sample, and then you map.
11:18:06 The reads from the two sequencing libraries separately to these context of the cause employee.
11:18:12 And that of course then also will give you differences in coverage because some organisms will be more highly covered in the next direction, one.
11:18:24 Then and DNA extraction two.
11:18:26 And this is an example shown here from a real life wastewater treatment plant sequencing approach, where there was no pre cultivation, where the researchers, simply took two samples, and well took one sample split it into, and that's two different in
11:18:49 extraction methods. And then, by mapping the coverage in these two samples, you got a very good resolution already separation of these different micro organism.
11:18:58 And then if you look at this, zoom in region here for example you see by adding some Taxonomic Information, you can already distinguish these microorganisms quite well but there's still an overlap, so it's still a bit hard to to to reliably separate these
11:19:15 these context, but now of course you can use additional access. So now you can refine the sequencing library or this this context that here by plotting juicy content, against touch on nucleotide frequency or touch another frequency against abundance and
11:19:41 sample one whichever might be more powerful to separate the context that you still have in this mixed population here. And with these approaches it's really possible from very complex samples very quickly.
11:19:46 To get a pure genomes, or to pure.
11:19:50 So, so this is you probably heard the term before these meta genome, assemble genomes, Max, or they're called.
11:19:55 Because of course they are not close genomes, we still have context there still is a risk of contamination in them. They also never guarantee that they are 100% complete.
11:20:04 But here now you can be quite certain that these contexts, really, to 99%.
11:20:12 We belong to one organism. And so now you can start the annotation process and look at this one organism, what are the functions that are encoded by this one organisms.
11:20:21 With combining these different in extraction methods of course you try to get a good overview and unbiased overview of the community.
11:20:29 But yes, in reality, I'm sure there's still our biases, I think that is one of the biggest problems and local ecology that all these methods we have the beautiful they're powerful, but none of them is completely unbiased.
11:20:41 And so the trick is to know the biases, and then to know methods that might have different biases so you can combine them and test for these biases strategically.
11:20:55 So, Yeah.
11:20:55 Okay,
11:20:59 back to long waits, very short notice by a long wait so nice and powerful because of course, a long way it will give you a bigger piece of the puzzle, so I don't need to puzzle together so many small pieces I have a bigger piece, even though it might
11:21:12 be very high error rate that I can correct it was bigger pieces, make it easier to solve the puzzle to stitch it back together.
11:21:20 This is especially important into in repetitive regions. So, if you imagine the stuff we can start very simply you ever you ever gene that is duplicating the genome, but 100% identical on the nucleotide level, this gene will be longer than the sequencing
11:21:37 reads, And so of course the assembler will not know where to put this gene in the genome, it will automatically break off the assembly there. And so it will break up the assembly into separate context, with the language, I can span the neighboring region
11:21:51 of this gene, and then really place it at the two or three or five places in the genome where it belongs.
11:21:56 And so there are a lot of these these structural variants polymer physics repetitive regions that can break up your genome assembly into small fragments and all these I can nicely spend with the long pieces.
11:22:08 And I said the original problem but can be corrected for by combining different sequencing methods.
11:22:18 Good.
11:22:20 Meta genomics and ongoing faster so slow me down if it's too fast.
11:22:26 But if there are no questions left Vantage nomics. Yeah. So McMakin back to this and biases about the abandoned so I agree, I like very much, your explanation of how we can use it for our benefit.
11:22:39 But in the end, when I want to compare the different abundance of organism is that.
11:22:47 not transmitted genomics meta genomics is also, it is a little bit more label concerning abundances then than the standard PCR based methods, but it's still not quantitative, it can give an indication of the most abundant organism, but I would not really
11:23:05 rely on it. If you want quantitative information on your organisms. I would go to either QPR so quantitative PCR or fish can be used to quantify certain organs or function, but but not Mater genomics, it's also for that to bias do too many reasons like
11:23:23 the DNA extraction.
11:23:25 But you can also have just differences in in sequencing efficiency depending on the DNA.
11:23:36 So, yes, there are publications that try to give you ways to to make it quantitative, so of course you can, for example mix in standards of known quantity.
11:23:48 Either after the dean extraction but also before the dean extraction to show that the DNA extraction efficiency.
11:24:09 I would always say, Take metagenomics.
11:24:14 As an indication of shifts and abundance of of these things have also which organisms are the most abundant always because of course these indications are in there.
11:24:24 But if it's really important to have a good quantification combine it with other methods. So don't forget about it completely but combine it with other methods to to prove that the quantification really has been done.
11:24:39 Thanks, can we agree on that.
11:24:41 Yes, thanks.
11:26:58 Then activity assays. Very shortly. Nobody really coming closer to, how can we link, a function to the micro organisms we detect.
11:27:09 We start very simple.
11:27:11 So the simplest activity essays really are. To show is a certain function present in the environment. So I detected with any methods can be functional gene sequencing can even be based on 16 s similarity to non species, or a complete genome meta genome
11:27:30 of the species I detect a meeting oxidizer present in a certain environment.
11:27:36 And I'm not interested in this nice and oxidizer to to see, is it really also active as meeting oxidizer or was there a mistake, is it actually only flushed in externally and not oxidizing anything in this environment.
11:27:51 So what you can do is you can take the sample fresh from the environment, if it's a soul You make a soul slurry. If it's from a wastewater treatment plant it will already be liquid.
11:28:02 So you can directly stick the sample into a bottle.
11:28:06 And then at the substitute interested in missing. In this case, and then use different methods and myths and of course it's a guest that has converted to see you to a different gas, and both of these gases, I can quantify in the headspace of these bottles
11:28:20 directly. so I can use methods like GC.
11:28:24 So gas chromatography to separate these gases and to specifically detect the different classes, and then follow me think consumption over time.
11:28:32 And also co2 production over time, and also look at this the commentary does it fit the estimates and really stick symmetrically oxidized to co2 so do find as much co2 back as it would expect for complete to meet and oxidation.
11:28:46 Because of course if you don't find it back in the product that should be formed.
11:28:51 Something else might be going on, and it might just absorb to something in your sample and then you have to look at your good controls to make sure. Okay, do I see a biologic direction, or does it just disappear.
11:29:03 But so with this very simple test I can only I can take my environmental sample and directly see is the function, I hypothesize that is present. Also there.
11:29:13 And this of course it doesn't have to be these these bottles.
11:29:18 It can be a lot more sophistic sophisticated than that.
11:29:20 So something we use for example in our lab quite a bit as aspiring actors, which is a. I would say a very sophisticated bottle, because you can control the environment very good you can regulate the pH the temperature, you can have a media constant media
11:29:35 flow through the reactor.
11:29:53 You can make it anoxic hypoxic so very the oxygen concentrations, control the, the gas flow rate all these things, but also there you can measure processes, either in the liquid or in the headspace, but you can also take it into the environment, so you
11:29:56 can have these these gas boxes that you put on top of your environment of interest and then directly measure what is going on in the head space above your above above this field above the grassland how much Nathan is submitted, you can follow that.
11:30:09 You can also check something and then see how does the gas exchange.
11:30:15 From the soul with atmosphere, how does that differ. So there are a lot of different setups in the lab in the environment where you can really directly measure the substrate turnovers and and and product production rates, and a method to make it even
11:30:29 more powerful especially in the lab is the use of isotopes.
11:30:35 As you probably know, most or many elements exist in different forms different isotopes have different nuclear weights here shown for carbon. So, carbon 12, that's the normal carbon, which consists of six protons, six neutrons, but we also have a heavy
11:30:53 heavy carbon naturally present in the environment that consists of six protons because that's important to be a carbon but seven nutrients it has one additional neutron, which does not change its chemical properties.
11:31:15 C 12 carbon, but it is heavier. And this white difference we can use and carbon of course is not the only element that has these isotopes. So, the ones that are most used in environmental microbiology, are the hydrogen atom.
11:31:25 So here we have material nitrogen has a stable isotope oxygen has even to then carbon also sulfur has even three stabilizer tops, stable means, they don't decompose so they're not really active, which also makes it very nice to work with them.
11:31:42 Because since they're not already active we don't need any special protection mechanisms, we can just use them in any normal app, because they are not harmful.
11:31:50 And also indicated here.
11:31:53 These stable isotopes they exist in nature, but they are very rare. So, the heavy carbon for example only exists, like what approximately 1% or more than 1% of the carbon in the environment is this heavy carbon for nitrogen it's even less so less than
11:32:10 0.4% of the nitrogen in the environment is the heavy form of the nitrogen. And so whenever I start measuring these these heavy isotopes. I can measure very very sensitively because the background is so low.
11:32:24 So how can we use that in our exam or in our research.
11:32:30 Again in very simple, very similar setups we can take the environment into the lab in all the different forms possible so any incubation that is possible.
11:32:40 The easiest again as you make a soul slurry and stick it into a small bottle.
11:32:44 Very good then add a defined medium or defined headspace.
11:32:48 Then you add the label substrate, and you follow witness this label go into me as an example earlier you can follow, does the do I detect label to to that contains the heavy co2, or the heavy carbon.
11:33:04 And that helps us to distinguish which processes are taking place in mixed communities because of course your to production is very unspecific because if you still have some organic substrates present in your, in your sample.
11:33:16 These will also be respected by other but he took traffic microorganism. So, you will have a lot of co2 production over time but that does not necessarily prove that it comes from is an oxidation.
11:33:25 If I add labeled anything and detect label to you too I know it has to come from anything. And so it can link it back to the process.
11:33:34 How do we measure that I already mentioned, we use GC to measure the chemical compounds like methane gas chromatography with a label we can combine these guests medical field mass methods with mass spectrometry where we can distinguish very accurately
11:33:52 very fine differences in math. So what happens here is your sample is injected into the GC point or them as potent from the GC where it is, ionized.
11:34:06 So it gets a charge. And these ions, then our accelerated.
11:35:18 And so using this
11:35:21 mass spectrometry to, we can then really quantify how much of a certain compounds we detect is unlabeled is maybe half labeled or come to example where that is important so the nitrogen gas can be important, or double labeled.
11:35:37 So where do we detect the label and then we can quantify the ratio of these different labels states.
11:35:43 And then of course we can use specifically
11:35:49 to.
11:35:52 Yes, there we go.
11:35:54 To look at processes in mixed communities, if we know which direction is going on. So if you go back to the nitrogen cycle.
11:36:05 Something or no organism via very interested in in our lab is animals anaerobic ammonia oxidation. Which oxidizes ammonia will risk nitride as terminal electron acceptor two and two guys.
11:36:18 And if you look over here of course, there are also other actions that could produce and two plus, as you probably know, and two is the most abundant gas and atmosphere so it makes up 78% off the gas and atmosphere.
11:36:31 So if we just look at our books without any label to really quantify reliably how much into is formed by animals in an environmental sample against this huge spectrum is how to possibly at all.
11:36:44 But what we can do is we can add labeled substrate so we add ammonium or also and I tried it doesn't matter.
11:36:53 With a label under the conditions optimal for animals, and then we just follow the label. How fast does it go into the into gas, and how much of the into gas is labeled over time.
11:37:08 And since animal access this very unique biology of combining one nitride and one ammonium into the nitrogen gas. If we only add one of the two in this example then I tried in its label state.
11:37:25 It will form a very very very specific nitrogen comp or D nitrogen component because it will be a half labeled and will always be half labeled. And for that reason we can easily distinguish animals from the notification environment, because of course
11:37:39 the notification only uses nitrate and and I tried to form the nitrogen gas so if you add labeled night right, the end to guess that is phone bill be double labeled.
11:37:49 If you turn it around if you add labeled ammonia.
11:38:00 Then you the notification will form unlabeled, the nitrogen gas well animals will still form half label the nitrogen gas. And so with these tricks. We can even in a mixed community, very specifically quantify how active is a certain micro organism that
11:38:09 we might be interested in. And if we design it smartly the experiment even distinguish between different processes that have the same end product.
11:38:22 Very shortly before we go into the break. It's only two slides.
11:38:26 Yeah, nope, nope, we have no we can prove that a certain function is President environment but of course it's this still there's no proof, who does it.
11:38:37 We have indications know from six minutes from the major genome.
11:38:41 From the activity so we have a genetic potential they have a candidate we have to process that it happens but really, we need to link to it.
11:38:55 There are different methods available.
11:38:50 So especially down here, ramen and and and nano Sims are becoming more and more powerful and popular.
11:38:56 I want to present the simplest one that is out there that is fish more, which is fish, combined with micro auto radiography, which means now for this example and fish was actually one of the few techniques that still uses radioactively labeled substrates
11:39:13 and not the stable isotopes form of the substrate.
11:39:18 We directly measure and detect incorporation of a certain subset into the biomass, and that we can detect an intake biomass and for that reason, use other methods like fish that's why it's a fish more to identify the sales so we can link function to identity
11:39:36 identity on the single cell level incubation or the setup here is also quite simple. Again you take, you can take your environment sample just as before, make us all slurry, and you add your radioactively labeled substrates, for you to Trump's you add
11:39:52 labeled sugar for Chelsea usually use carbon labeled co2, and follow the co2 incorporation under conditions where they might cause and should be active, so has its energy substrate and where it shouldn't be active, where it doesn't have its energy substrate.
11:40:06 After a rather short incubation usually overnight is enough depends on the activity of my goals.
11:40:12 You killed a sample by a PFA fixation formalin fixation just as for standard fish you apply your sample to a microscope slight do a standard fish for your micro organism of interest.
11:40:27 So the micro is very suspect, this is the organism catalyzing the process I'm interested in.
11:40:33 And then you overlay, your bio mass that contains the fish crops already with a photographic emotion, and this is really the same emotion that was used in black and white photography.
11:40:45 The nice thing about this emotion is it contains silver, and if this silver in the molten is hit by the radioactive by the beater race that is emitted from the decay of the carbon.
11:41:00 It will form elemental self as silver, so it will form black granules that can be detected in the microscope. Then you go to a microscope and simply quantify or localize the silver, bronze, and localize the fluorescence, which allows you to link the function,
11:41:18 because of course, only the Michael Odin there was active will have similar grains forming above this biomass and identity, because the fish probe allows you to specifically identify a micro level of interest.
11:41:31 And so now you can link it and can really say organism a was active and an incubation conditions always be was not active on the the incubation conditions so now we can really linked the function, back to a single micro organism.
11:41:44 Alright, think it's time for a break here.
11:41:47 Awesome, thanks so much so, That was a lot of great background.
11:41:53 Um, I suggest we take, let's say until 50 1150 pacific time, so like eight to 10 minutes for a break.
11:42:06 Thanks again Sebastian. Yes.
11:51:06 Alright, on to part two complete notification.
11:51:12 The discovery of comics. I put in. Yeah, after a little bit of a general introduction why I'm actually interested in education and and also a little bit more natural spiral, not so much complete notification yet so rather the canonical knighted oxidizes.
11:51:28 I have material for for different topics I don't think we will cover them all. So you can also just stop you when you say okay my brain is overflowing that's enough now.
11:51:37 The first two are the most important one for today, everything after that it's nice if you still have time for it, otherwise I did provide the slides and both of the last two chapters also published or at least available on by archives now so you can
11:51:56 also have a look there.
11:51:58 But let's get started. As you could infer probably quite well so far I've worked with nitrogen cycle organism.
11:52:06 More specifically, I'm interested in this reaction here notification, which is the Arabic oxidation of ammonia, why and I tried to nitrate.
11:52:16 Why is notification important always find a good to also give some some motivational background way they actually are interested in this Croesus night Trojan removals ammonia removal is one of the key processes in wastewater treatment, because if the
11:52:34 ammonia is not efficiently removed from our wastewater, it leads to eutrophication in receiving water bodies. And in the end, in the sea. So notification is really really important because it is one of the key processes combined for st notification to
11:52:51 convert this ammonia tonight try to nitrate which then can be denied unified into harmless, the nitrogen gas that just goes out of the wastewater goes back into the atmosphere and for that reason is harmless does not harm any of the receiving of water
11:53:19 more modern way is analog so the anaerobic ammonia oxidation.
11:53:16 But also here on unlock sterilized on night right which can only be provided by notifications also a notebook still needs.
11:53:24 Partial notification tonight right it does not need the nitride oxidation anymore, but at least the first step always also still has to be present.
11:53:32 However notification is not always wanted. If you go to agriculture, of course a lot of fertilizer, mainly in the form of ammonia or largely is brought out the fields and ammonia, or ammonium has a very nice feature, since it has a positive surface charge
11:53:48 it binds very well to the negative, Rufus charge of soul particle of the soul metrics and for that reason it's very well retained and available for growth of plans.
11:54:00 If notification sets in the positively charged ammonium is converted to a negatively charged nitrate molecule that is mobilized very easily. So if it rains, It is washed out of the, of the soil.
11:54:14 It is lost. So virtualization efficiency goes down, and of course it again leads to eutrophication of receiving water bodies, which means detrimental for the environment also these coastal minimum zones are mainly caused by too much nitrogen fertilizer
11:54:28 ending up in this ecosystem which is to a large extent do you to notification of activity on the field.
11:54:38 So there we actually want to suppress it and not, you know, not sustained nature. The nature fires are quite diverse everything shown in blue here, or nighttime oxidizer so you can see, we have identified mattered oxidizer Spain.
11:54:55 I'm sorry for that I really don't know what's going on today,
11:55:00 collecting a lot lately word never did this I don't know what constantly crashes today.
11:55:08 This is really annoying.
11:55:10 Maybe it just continue talking, so it's not so boring for you well we are waiting for my PowerPoint to restart.
11:55:18 The vino nitride oxidizes for now and six different file.
11:55:27 Fellow genetic groups.
11:55:28 The one I am most interested in our natural spiral, which I will show you in a minute.
11:55:34 Ammonia exercisers as far as we know, are not quite as diverse, so their fuel ammonia oxidizes.
11:55:45 Yeah that's maybe, maybe an idea of everything else fails to upload it I can also otherwise try to present from the PDF.
11:55:54 That might also be a workaround. If it continues to crash let's give it one more try.
11:56:01 And, otherwise we maybe should really switch
11:56:05 So now just spiraled on here that's the genius I'm most interested in. It's also in many in most environments and also in most applications engineered systems like wastewater.
11:56:17 The, these are the main knighthood oxidizes out there. The only exception is the sea in your marine environment usually noticed by now, is the dominant knighthood oxidizer and those those system.
11:56:31 Ammonia oxidizes we have here and read most of the environmentally important organisms in freshwater systems are within the beta product here and it is a moment's notice as viral in marine systems there's usually snipers a caucus, and the marine systems
11:56:47 especially but also in almost everywhere we have a Mani oxidizing a Kia which are not included in this slide this only shows to bacteria.
11:56:55 So that makes the know pneumonia oxidized a little bit more diverse,
11:57:01 a very short excursion into respiratory chain.
11:57:05 Because nitride oxidizes, yet they have a very special respiratory chain, which is due to the substrate they use. So what we see here is the Redux Teller, going from electro negative to electric positive down here we have the best terminal Eric accepted
11:57:20 is oxygen and appear most electro negative are the best electron donors, because the larger the difference between electron donor and electron acceptor in Redux potential the more energy can be conserved.
11:57:35 During respiration.
11:57:37 The nitrate nitrate couple is here at plus 420 million volts. So it is quite close to oxygen already which means there is not much energy nitrate oxidation anymore.
11:57:48 And you see also Moni oxidation ammonia tonight right it's not that much better it said I think 300 plus 240 million volts.
11:57:56 What that also means especially for natural oxidizes is that the electrons have derived from nitrate can only be fed into certain currency. So into the second sequel, which means that all the complexes interacting with the keynote cool are two electro
11:58:18 to directly accept electrons from natural oxidation. And for that reason, the electrons have to go to the second sequel, where they can be passed to the gym oxidase still so to oxygen.
11:58:26 But for everything. We're a reduced keen on is necessary, or if a reduced NADH is necessary for bio mass production. These electrons have to be pumped up hill.
11:58:39 So energy has to be invested into getting these electrons into the pool, because the the substrate the electron donor is to electric positive.
11:58:50 That means that the core respiratory chain so to serve nighters para or if nitric oxide as a general is very short, because it consists of two enzyme complexes only.
11:59:00 You have the nitric oxide inductees that oxidized and I tried to nitrate. The two electrons that are gained from this reaction or then fed probably biocide to come see directly to term oxidase were oxygens reduced and proton gradient is formed that then
11:59:17 can be used by complex five to convert it into ATP as a energy storage, and of course also this, the protons that are pumped out of the cell by complex for their in this case not only used for ATP generation at complex five.
11:59:35 They also have to be used at complex three and complex one to pump the electrons uphill. So to get extra electrons onto or extra energy onto the electron so they can be fed from the secular and people are pill into the Keenan pool.
11:59:52 And from there, even further uphill onto into the end of the age group.
11:59:57 Not just Barrow Of course it's an accumulated traffic organism which means they are need to alter traffic organism, which means they obtain co2 from the environment, directly by fixing carbon dioxide into biomass, and of course, this, they use the reductive
12:00:13 TC a cycle for that so the reversal of the normal citric acid cycle to form biomass, and of course all the energy that is necessary for this reverse TC a cycle has to come from a pill electron transport from the electrons game by natural cetacean so that
12:00:32 sets the, the respiratory chain quite apart, compared to Trump's which are usually run this all in the forward direction that most of the rest of the changes are actually runs reverse and costs energy that has to be gained here, or it has to be formed
12:00:49 as a protocol to force by this combination interaction of the next hour and complex for.
12:01:00 So, let's take it to the complete notification, where, how they identify these complete and it fires in outer space. So, it has been a puzzle. It has been puzzling researchers for for quite a while, why this metabolic labor and notification is divided
12:01:18 between two functions, different groups of organisms.
12:01:21 So why is ammonia oxidized first Bomani oxidizes to nitride only, which then is excluded, taking up a second organism by a nitrate oxidizer and oxidize to nitrate Why is it not present in one single organism, because of course, in theory, if you just
12:01:36 look at pure thermodynamics of these Redux reactions. They of course is more energy in the complete direction the complete oxidation of ammonia all the way to nitrate.
12:01:47 Then in the two party directions.
12:01:49 What you also see here nitride oxidation really does not yield very much energy, compared to ammonia oxidation, but it still seems to be enough for these oils to be quite competitive.
12:02:00 But still, why is it separated Why do ammonia oxidizing not take ammonia, all the way to nitrate and waste this extra energy.
12:02:08 The, there was a hypothesis posted in this paper in 2006, but you can always drift at and colleagues who proposed that the cost of regulation of internal intermediates and have extra enzymes that are necessary for the last step of nitrate oxidation.
12:02:29 And maybe proteins are acting with this nitrate oxy deputize is higher than the additional gain for ammonia oxidizes if they go all the way. And so that their hypothesis was that canonical ammonia oxidizing actually are adapted for fast growth.
12:02:49 High substrate turnover rates, but low growth heels.
12:03:05 And that a complex organism so complete ammonia oxidizing that takes it all the way to nitrate should be competitive under conditions which select for low growth rates, so we're growth rate is not selecting factor anymore.
12:03:08 But we're high yield might be a factor, which means if it translated it should be an organism that can grow very slowly at very low concentrations of their substrate of ammonia, in this case, so they predicted that you would probably be able to enrich
12:03:24 common mocks organisms at in biofilm like systems where you have basically no cell turnover cell wash out because they can stay in the biofilm forever at very very low ammonia concentrations very suddenly this high yield is more important than fast growth
12:03:44 rates high growth rates. When I joined the lab and 900 but there was a direct already started by Marty Marty Frank Castle.
12:03:52 That heads very similar conditions it did not have exactly these conditions but it's used by a phone from agriculture bio filter that was actually running in the basement of rapid University.
12:04:07 So it was very handy for us because we also use the water directly from this filter as media for this vector to feed it.
12:04:16 And to the, to the, to the medium to this director, we added a set the filtered water directly from the agriculture system, and edit very low concentrations of ammonia nitride nitrate.
12:04:29 We didn't add any carbon but of course there was a little bit of fluctuating carbon coming in the agriculture water. They also was some nitrate coming in with agriculture water, and we kept the conditions hypoxic, so we constantly bubbles, the nitrogen
12:04:43 D nitrogen gas and Oregon car to gas through this reactor, to keep it as anoxic as possible, because Marty he was interested in enriching and cold adapted animals that is adapted to these conditions, and to become back to the activity is explained earlier
12:05:02 as Marty was selecting for and I'm also of course she wanted to know is on amongst also present and so she didn't keep patients with labelled ammonia, and unlabeled nitrate, to see do we find this and unlock signature do we find half labeled the nitrogen
12:05:14 gas forms.
12:05:17 And indeed We did see that in batch essays.
12:05:20 The activity in batch was always much lower than the record itself but still we could see that over time. We had a constant formation of half labeled the nitrogen gas so I know Max was indeed their next marketed fish to see okay, where is the Moxie she
12:05:37 found a peculiar species in the her six NES libraries, so she did fish. And, interestingly, she saw in their success libraries already that there seems to be an actress for present and when she applied fish clubs when I respond, and I'm expecting.
12:05:54 At the same time, she saw that these two always courtyard. In the same flux and proximate Lee at similar abundances so that you always had these these mixed flocks of animals, and natural spiral populations, which of course was unexpected because, first
12:06:12 of all, both organisms rely on night, right, because they needed a substrate so they should be competing for their substrate and one organism is Arabic Niger spiral always needs as far as we know, oxygen is terminal and accepted by animals is Amelie inhibited
12:06:29 by oxygen. So needs anaerobic conditions. So of course we got very intrigued What does this not just for do in this culture.
12:06:37 Because knighted oxidation seemed unlikely, and even more strongly. It spoke against this close interaction of these two organisms, it would then rather indicate that they appear in different floods where they don't directly compete for the same substrate.
12:06:50 So we got interested in what could potentially function of this organism.
12:06:56 For that reason we did meet the genomics we sequenced the the complete DNA from this director, and together with collaboration partners and all of them, we were able to build out even two different atmospheric closely related but still different address
12:07:09 by genomes that are shown here, which of course allowed us to look at their philosophy at first so extract the 16 s RNA from these genomes, to see where are they affiliated so which are the most closely related.
12:07:26 And I just barely don't have to read the tree here no it's very small, but he's shown here is the so called supplements to have nightdress para Moscow vendors down here is the type strain of the supplements to.
12:07:38 And the to natural spiral genomes to be extracted from the meta gene and quite closely related based on 16 s, but clearly did form, separate species.
12:07:47 In this stable sub cluster but in this up lineage to have natural spiral.
12:07:52 But, way more fascinatingly when we took a closer look at the genome, we found that both of these genomes so both species, not just by species wanted to hear.
12:08:03 Do not only contain the market genes for knighthood oxidation so the NXR the nitric oxide doctors, but they also contain the complete over on encoding for the ammonium on oxygen as with some additional, sorry, sub units.
12:08:20 And that actually means auxiliary deputies also that here in one large operation, which, of course, was the first genomic indication that we might have found a common works organism because all genes for all steps of notification were present in the genome.
12:08:36 However, when we looked when we took a closer look at the ammonium on oxygen as it actually did not cluster with the known ammonium oxidizes it clustered in an environment and group containing only environmental sequences.
12:08:51 And the only sequence in the sequence group that was linked to an organism was actually annotated as a p most as a meeting mono oxygen is which is closely related to the monument it's nice but of course it's a completely different functional so that's
12:09:05 missing and not ammonia.
12:09:07 This link was not based on genomics, it was only based on linking QPR data is a quantitative PCR data have a mixed population to each other.
12:09:21 And so we of course at this point we're uncertain is this was this link wrong, and did we know really find them on oxidation as it appeared from the genomic environment, or is this annotation correct and our annotation one.
12:09:36 And it says, do we actually have a meeting or oxidizer and not a complete 95, so of course we needed to prove that this slide here shows this whole family of the modem oxygenation and the feminine oxygen as shown in right here by nobody nobody even have
12:09:53 have two different groups of ammonium oxidizes in different comic species but all of these are natural spiral in green mean, we have the known ammonia oxidizing bacteria so the beetle productive mo here, the GM approval to these are the marine ammonia
12:10:09 oxidizing over here.
12:10:11 Here with a very long branch the Akil ammonium Warnock's nice, and then between, we have a lot of enzymes a different function. So, this group here is a nice and oxidized Nathan mono oxygen as all these enzymes you're missing one oxidizes.
12:10:25 You also have some hydrocarbon one oxygen is here in this group that can go for for Ethan or booting.
12:10:32 So, due to this this this large distance to the characterized ammonium on oxygen aces.
12:10:40 We of course couldn't be certain and needed to prove further that yes we really have an ammonia oxidizing here and novel ammonia oxidizing. The first thing we did is returned back to the activity essence, because mighty remember that she actually did
12:10:54 not only see the half labeled the nitrogen gas but she also saw some indications for ammonia oxidation happening in the system. And if you look back at what happens with the, with the stable isotopes.
12:11:07 Okay, we'll switch to the PDF now.
12:11:49 Okay, you can all see it.
12:11:53 Of course I have to move through a lot of slides here which I probably should have done first.
12:12:39 Okay.
12:12:44 In the presence of an ammonia oxidizer, you would have oxidation of ammonia tonight right so you would get labeled nitride. And then if animals would use these two substrates Of course you would suddenly start forming double labeled the nitrogen gas.
12:13:00 And indeed, when we went back to the, to the data from these activity SS.
12:13:05 So looked at the GCs data. We did see that at the same at a lower rate but with the same trends.
12:13:12 We did not only get the athlete multi nitrogen gas we also got double labeled the nitrogen gas for and in these incubation so we had a first indication that yes apparently ammonia oxidation really is happening in this reactor, and is feeding additional
12:13:25 nitride to animals.
12:13:28 So then we wanted to, to show that, yes, it really is this ammonia oxidation as we assumed.
12:13:35 So we took vitamins from director and heavily oxygenated it because that million activates, and I'm ox. And so that one activity that should be left with ammonia, then of course the money oxidation so the comment box activity.
12:13:48 And indeed, in incubation with ammonia. We did see ammonia consumption and nitrate formation, without intermediate nitrate accumulation here.
12:13:58 If we added inhibitors, well known and amateurs so this is a new to real but scavengers away the copper from the active center of the Mana Mana oxygen as we did see this inhibitor also complete inhibits ammonia oxidation.
12:14:12 So these are the open symbols here ammonia stays untouched.
12:14:16 and no nitrate is formed.
12:14:34 So, that already shows Yes This really is a biological oxidation of ammonia and not something chemical or some strange saturation of questions, it's still an atmosphere and we also wanted to show can they still do canonical natural oxidation. So we also fed nitrate, to these bottles, and you
12:14:37 these bottles, and you see the reactions even faster so and I tried to oxidize faster than ammonia in these cultures and also stick symmetrically converted to nitrate, as expected for an oxidizer, so nobody knew.
12:14:50 Yes, they have the genetic potential for ammonia oxidation for complete notification.
12:14:56 And we could also prove that the function itself is happening in this culture, both in director.
12:15:09 So now I'm just curious what was the doubling time or retention time in the system. So what was the rate, with which this organism, Google, I mean yes flux, but, you know, approximate indefinite because we never move by Omar so director we used was a
12:15:30 so called Sequencing Batch Reactor. Okay, which means it has a settling face every 12 hours. So every 12 hours it stops stirring it stops the gas flow, it stops the media flow so that the bonus settles to the bottom, and then we take out the excess medium
12:15:44 from the top and leave in like half a liter so the biomass at the bottom is untouched. And then once the complete by the medium is removed, we start steering and gas flow and slowly add medium again until it's filled up again.
12:15:58 And for that reason the biomass stays in the system forever so we we are not limited by growth rate at all, because even if it's suspended growth in flux.
12:16:08 It is sort of biofilm grows because it stays in the system. It's not much don't
12:16:16 like to talk about this later. Yeah.
12:16:19 Yeah.
12:16:21 So at this point of course we wanted to to really linked to this natural spiral that we identified, they returned back to fish more what I described earlier so we made incubation adding radio, radio actively labeled by carbonate co2.
12:16:38 Since these of course are automatic organisms fixing carbon from co2. And then that innovations in the presence of ammonia in the presence of ammonia and the inhibitor at you in the presence of nitride, and in the absence of all energy substrate so only
12:16:54 radioactively radioactively labeled bicarbonate present. And we can see very very clearly that in the presence of ammonia, the natural spiral which is shown in pink here is very strongly labeled.
12:17:08 Down here we have some animals, which looks on first glance, a little bit labeled but when you zoom into these images you do see to notice Paris, very small cluster sitting here that are responsible for this label well the animal side of the flock is
12:17:20 not labeled. We do see that at you, that's not 100% inhibit the culture but to 95% activity was gone. As expected, also from our big patients that we did earlier with nature, it had a similar picture also nitride we had a clear very strong level of this
12:17:38 mattress power. So, again, I know most of us was unlabeled and of course as soon as we left out the substrates.
12:17:43 The sales did not have energy for carbon fixation anymore and so carbon fixation stopped, and the cells also were unlabeled.
12:17:51 So nobody could really have the combination of these dis results, go from the hypothesis we have a common amongst organism to showing the activity, and to linking the activity on the single cell level to these organisms.
12:18:09 And that, of course, then was enough to convince the revenues that it really is a common amongst organism. And so, that pro to really prove beyond doubt that you know that this this normal reaction is possible in one organism.
12:18:24 Right. Alpha Do you want to discuss here, or at the very end. At the end, you might touch on this anyway.
12:18:30 Okay, good.
12:18:32 Because then I'll move on to some follow up studies, because of course if you listened very carefully.
12:18:40 I said that these two organisms should compete for the night right with the story I told you so far, they actually still compete, because they compete for ammonium nitrate now, because of course a complete I'm only oxidizer, we'll go from ammonia, taking
12:18:56 away one substrate from animals tonight right but oxidize that on tonight right so still take away the second substrate also from animals. So we still are very puzzled why we have this interaction of these two organism.
12:19:08 And this interaction is stable we have this reactor running for more than 10 years now, in the reactor, and you still find the same flux dominated by comics natural power and dynamics.
12:19:19 So, this is one of the ongoing research projects and the data, I present to you at the end of this part is very very preliminary. I also have to say, it's not proven beyond doubts that might still be be that hypothesis I present to you now or wrong, but
12:19:36 we do have very nice first data that this, that there might actually be a novel pathway in this column looks natural spiral that allows them to coexist with matches with the animals organisms.
12:19:49 What you did for this is, first of all we wanted to have a more controlled system, because of course in the original director.
12:19:56 We did not really know what was supplied to the director because there was carbon in the agriculture water I mean, there's fish in there, these fish guts feed so there is also leftover feed but mainly the pieces of the fish in there so there's an undefined
12:20:09 mixture of carbon substrates, and of course also an undefined mixture of nitrate coming in, very fluctuating concentrations but also some ammonia coming in.
12:20:19 So the first thing we did is the way I'm going.
12:20:27 Reverse here, we're thinking about what else could be happening this reactor. So, if, if this reaction isn't happening because this would not explain our interaction dynamics, but rather competition.
12:20:39 What else could be happened his reaction. And then of course, the easiest The most obvious explanation would be that our comics organism, actually does not perform comics, but only performs canonical ammonia oxidation.
12:20:52 And so stops at night. Right. And with that, since the system is nitrate limited feed some additional substrate to animals, which means that animals can then oxidize more ammonia and both oh you should be happy, very feasible explanation, but does not
12:21:09 explain it all the way then come on look suddenly outcompete canonical ammonia oxidizing Why do we then have a common Mazzotta that can do both steps and not a normal ammonia oxidizing stops at night right at the second process doesn't happen any.
12:21:24 So we came up with a new idea, which would be there is nitrate in the system, and also animals forms a little bit of nitrate during its metabolism.
12:21:35 So maybe our organism can actually turn it can use the nitric oxide production is the enzyme responsible knighted oxidation and during the reverse reaction, which we know from other natural spark that they can turn around, the NXR, this reaction and use
12:21:48 it to reduce nitrate back tonight. Right.
12:21:51 And suddenly we will have a system that is very beneficial for both organisms, because I know max would get more nitrate but not only from the ammonia that is oxidized tonight right but simultaneously from the nitrate.
12:22:06 That is reduced tonight right.
12:22:10 At the same time, comics would be better adapted to the conditions that we have in this reactor, because it's hypoxic so it's oxygen limited. So if we compare it to the canonical reaction of ammonia oxidation and knighted oxidation we have two reactions
12:22:24 that require oxygen to activate ammonia in this reaction, and as turmeric acceptor, and he also accepted. So we need to oxygen molecules for each ammonia molecule to complete your oxidizer to nitrate.
12:22:43 In an oxygen limited system.
12:22:43 We still need 1.5 oxygen per ammonia for canonical ammonia oxidation because it still serves to activate ammonia, and as thermal electron acceptor for for the electrons from a monetization.
12:22:55 But if you go to the nitrate producing ammonia oxidation which is the so called nitride combo coordination reaction, because it forms nitride from two different directions by oxidation and reduction reaction we suddenly only need one oxygen for ammonia,
12:23:12 which means at oxygen limited conditions, the comics organism could be much more active in the system.
12:23:21 Because it doesn't rely on oxygen so much because it replaces oxygen with nitrate as terminal acceptor. And so, comics would suddenly outcompete canonical ammonia oxidizes which cannot switch to nitrate reduction so they always rely on oxygen reductions
12:23:37 General Electric sector.
12:23:38 And of course, it would explain the this interaction we observed with the animals organism.
12:23:44 One of the first things will in parallel to starting a new director and trying to prove it in directors, was the visitor back then, Christopher Lawson from, from medicine University, who was very interested in genome scale modeling and so he tried to
12:24:02 model.
12:24:03 In this example that I showed today very simplified very much simplified. Only the growth rate and the growth yield of the different common mocks we actually reactions that we predicted so complete notification.
12:24:16 Partial modification so canonical ammonium sedation tonight. Right.
12:24:21 And now to a couple points nation, and tried to predict what happens of course on the rest of the chain but more importantly what happens to growth rate and deal.
12:24:30 If you look at the respiratory chain, how does that change in these different scenarios. So what are the differences How can it work on the restoration level.
12:24:45 What we see here the AMA, that's the morning on oxygen is where oxidation of ammonia, with oxygen to activate this ammonia tragic Silliman happens.
12:24:50 This is actually consuming electrons from the keynote today because oxygen needs to be reduced to water. And so, net, this is consuming two electrons accelerating then is oxidized tonight right.
12:25:04 This reaction yields for electrons that are fed directly into the keynote to, to have these electrons have to go back to ammonia, to the ammo.
12:25:12 And from there, there has to the trim oxidase, which uses these electrons to reduce oxygen to form a protocol motive force. And of course, under complete notification conditions, you will have additional electrons from nitride oxidation.
12:25:41 That also feed inside conceivable too complex for the term oxidase, and so you will form a lot of PMF. But of course, it needs a lot of oxygen, here, and here.
12:25:44 And so this, the model. This fits in the model predicted to only happen under ammonia limiting conditions, and not under oxygen limited conditions.
12:25:54 As soon as you add nitrate and limit the oxygen, the model also predicted that the respiratory chain, what's sort of remodel itself. And that suddenly, only the electrons.
12:26:06 The electrons here that enter the sight of drum see pool from hydroxyl I mean oxidation.
12:26:15 Do not go to the terminal oxidase anymore, that this enzyme will be down regulated to not be active anymore, but that these electrons are passed to the NXR, which then runs in reverse and uses these electrons to reduce the nitrate to nitrate.
12:26:33 And so now we have this nitride combo coordination phenotype. That is automatically selected for under oxygen limiting conditions, because it maximizes the oxygen flux flux of the system.
12:26:41 So it allows to oxidize the most ammonia, with a limited amount of oxygen that is present in the system.
12:26:48 Kristen also tried to predict the growth rates, and yield. And of course, this under ammonia limited conditions when we have the common max phenotype, that reaches the maximum rates and yield set 200%, we see in oxygen limiting conditions, but without
12:27:07 nitrate presence so selecting for canonical ammonia oxidation tonight right, the yield is slightly lower.
12:27:17 Then, with knighted oxidation because if you remember the additional energy in knighted oxidation was not that much. So the yield actually doesn't decrease so much, but the growth rate goes down quite a bit
12:27:29 in yellow. That's a natural protection of course this is reduced rate here, because not too much ammonia can be oxidized anymore. However, if you now model.
12:27:49 The natural compounds nation phenotype here on the right so the presence of nitrate and oxygen limiting conditions, the yield is slightly lower.
12:27:52 Then, if oxygen is used to termination accepted for monetization. But suddenly the growth rate goes up again is predicted to be higher. Because, more substrate can be turned over with the same amount of oxygen, even if they're slightly less energy in
12:28:06 each single ammonia that is oxidized with nitrate, instead of oxygens to American acceptor more substrate can be turned over with the same amount of oxygen present, and for that reason, the cells can reach a slightly higher growth rate.
12:28:20 And of course, and this is interesting for the interaction dynamics to nitride productions maximized. So most nitride for is formed in this direction.
12:28:42 So we know wanted to know, can we also turn that question. Yes. Sorry. There's a question about how does the flux distribution get calculated, you maximize the maximizing the, it will see maximizing the biomass flux, I guess, the oxygen.
12:28:51 The oxygen flux I think was was, as far as I remember because Christy, is that these calculations. The oxygen flux was set constant, so many amazing, and he said the model so that the bio mass production was maximized on a set amount of oxygen.
12:29:12 Right, so we have another question I want you to do so. So I've never understood this fully but I, my understanding with the FBA models is that, that the the growth rate is kind of the same variable as the as the flux to biomass or something like this.
12:29:29 And so they're not they're not unless you make like thermodynamic FBA or something that includes concentrations in some way, you're not really independently predicting those two variables, but it you were talking as if you had different you had different
12:29:44 calculations for them that they weren't strictly coupled to each other.
12:29:48 Yes. In, and there don't take them for 100% granted, it's it's it's 90%.
12:29:56 That I'm certain on this. So, here.
12:30:00 Chris really tried to uncoupled the two. And so he really, he did not use the, the complete genomic model for these two organs are used to, sort of, minimize trying to.
12:30:18 How was it predict the.
12:30:23 Well, we didn't. So, these are pure predictions we did not measure anything so for for most of the answers, the canonical natural oxidizes really limited the models by calculating by measuring indirect or how much biomass can be for for more promote nitrate
12:30:40 that is oxidized, he'll be only predicted that. So we, so here the all information really comes purely from thermodynamics, how much energy is theoretically present in these reactions.
12:30:52 I know this doesn't fully answer your question.
12:31:01 But he tried to sort of uncoupled it by looking for the by coupling the yield to the energy that is present in the reaction so in the turnover of one mole of ammonia with a different term Alex and accelerators, and the growth rate based on turn over numbers
12:31:20 of substrate.
12:31:23 And so that takes it a little bit away from the classical FBA modeling.
12:31:28 Can we agree on that.
12:31:29 Yeah. Is it is this like, Is there a blueprint available I can just look for this story not. No, unfortunately not this is really very preliminary still.
12:31:40 So there.
12:31:43 There's a paper now available for for the real FBA of animals, and we have a pre print for canonical naturist party but the combination of these two is unfortunate published, you know.
12:32:15 All right. Well, I did something that he did something clever. And he was sorry, go ahead. No, as a, he was appointed to this model also directly exclusively deals with the concentrations, dealing with.
12:32:15 It deals with mainly thermodynamics and substitute turnover so it is sort of oxygen normalized, and then calculates for for per mole of oxygen, how much ammonia can be turned over.
12:32:30 And that of course can then be used back to calculate the growth rate because it gives you an idea of how much substrate can be turned over, per unit of time or.
12:32:41 Yep, it is presumably modeling directly.
12:32:47 Incorporating concentrations into the model to do the thermodynamics, you need a concentration. Yep. Yeah, yeah. So it's, it's, it should be sort of normalized oxygen one mole of oxygen, how many moles of ammonia can be turned over, per mole of oxygen.
12:33:05 And so yes you have concentrations in there because of course that then gives you the, the template and MX how much energy can be gained from all of oxygen.
12:33:18 Hmm.
12:33:22 Interesting.
12:33:22 So I said we went back to the lab, and we said okay now we want to test for this but to be able to test with this we need a more defined culture.
12:33:32 We cannot have this carbon coming in that might feed the notification and all these did other directions because we really want to make sure that the nitrate, that is reduced is reduced by comic books and not by the big fires and then is fed to animals.
12:33:47 So, we sampled our original director and inoculated a new director, that got a purely synthetic medium where the new big system overtime, or decrease them.
12:33:59 In the case of nitrates, where we knew exactly how much ammonium nitrate nitrate when we could be sure that no organic carbon entered the system. So, the system is purely otter traffic purely relate on co2 fixation.
12:34:13 And again, was oxygen limited, so there will be applied the same settings. As for the original reactor, which should gas out most of the oxygen but there's always some oxygen bleeding in with media, and through the tubing into the system so some oxygen
12:34:27 seems to be available.
12:34:31 We of course the first thing needed to make sure that this interaction still was present, so that we still had flux containing on a box and column box, not just Barbara and nicely we could see that after a few weeks to months of incubation the flux did
12:34:45 not change very much.
12:34:47 So we still had the flux.
12:34:48 With with these two organisms present and apparently still interacting so they still call cured in all the flux and did not separate, and now of course, if we needed again tools to to test is this we actually really happening.
12:35:02 So again, we went to labeling experiments activity experiments where we added labeled substrate labeled ammonia and no not. We didn't add any nights right.
12:35:15 But we added some nitrate.
12:35:18 And that of course will then give you different products for the animals. Because on the one hand, ammonia will be oxidized or two nights right so you will get labeled nitride and animals can use the labeled nitride in combination with a labeled ammonium
12:35:32 form, double labeled the nitrogen gas.
12:35:35 On the other hand, of course, on MX can take the labeled ammonia and use unlabeled nitrate, that is produced from the reduction of unlabeled nitric trait that we added to the director tonight right so the nitric competence nation, which will yield half
12:35:49 label to the nitrogen gas.
12:35:53 And, indeed, first of course, we could see that, yes, the comics itself. So the oxidation part of comics is still active we still get oxidation of labelled ammonia to nitrate, which is then used on a Mac so we could see an increase of the double labeled,
12:36:16 and two in director.
12:36:20 As soon as we add it. So this was with only ammonia present as soon as we added unlabeled match nitrate. We still saw double labeled into production, but at a lower concentration, so let's double able to D nitrogen gas was produced by their hands, if
12:36:39 you look at the half labeled, and to production. We do see that in the presence of both labeled ammonia and unlabeled nitrate, we actually get more production of the nitrogen gas.
12:36:54 Then, in the presence of ammonia only where it actually levels off which makes sense because at some point, there probably will not be much enough nitrate anymore.
12:37:02 Only this very little bit that is still produced by nomics in the system.
12:37:06 And maybe most interesting for our hypothesis is that the rate of into production so half labeled plus double able to total into production of the system was higher in the presence of nitrate and then in the presence of ammonia only, which indicates that
12:37:28 Then in the presence of ammonia only, which indicates that yes, the activity of the complete system. So of comics, and for that reason also for animals in the presence of nitrate is higher is increased, because suddenly comics can be more active can supply
12:37:43 more nitrogen to annex. And for that reason also animals can be more active.
12:37:47 So, we have very good data or very good first indications that this cooperation really is happening that unlocks and common walks in this reactor do not compete for substrate but actually cooperate, informing nights right, which is limiting in the system.
12:38:04 And that got the cogs in the system can actually turn around the nitrate oxidation. The second step of notification to supply extra nitrate to animals by also nitrate reduction.
12:38:16 And so that our common species really performed this night or combo coordination reaction, which would explain this interaction.
12:38:24 However, as I said, the, we have been trying to reproduce these results for the last one and a half years. Usually our actors crash at some point and we were not able to reproduce it so taken with a grain of salt yeah very good indication that that this
12:38:40 is really happening in the system.
12:38:42 But the final proof is still waiting for it, but it's of course a very nice. Yeah, showcase how we can combine modeling measurements thermodynamic predictions, with the tools for us and microbiology, to sort of predict and then test for this novel phenotypes
12:38:58 unfortunately sometimes then the cultures do not always act as nice and easy as we would like them to so it's sometimes a little bit more tedious to prove that this is also really happening.
12:39:11 All right.
12:39:13 Do you want to continue a little longer.
12:39:15 What do you want to have more time for discussion.
12:39:19 Or do you want to have more time for discussion. Exactly. I want to throw in the question. So, the way you started this, the motivation was essentially the young craft, I bought this is that rates drives speciation right.
12:39:35 So if you have a sort of a low flux less come low competitive environment, you will not have metabolic speciation.
12:39:44 So when I am if I understand you correctly.
12:39:48 You're retaining retain to complete the organism it's a complete notification pathway, not because of the rate but because of the oxygen limitation is that right because it's switched to a more sort of so to speak, economic way of oxygen utilization although
12:40:08 I'm still unclear how this works because the sub said you as batch, it is a it is a batch fed reactor right and then.
12:40:23 No. So, just very short two drops and then you can finish the question. It's not Bedford, it's the the sequencing better actors.
12:40:28 They get a very very slow continuous media influence so you just have a break in between for half an hour where you have to settling and the removal of the spent media, but then you have a very slow supply of media, so the media concentration of most
12:40:41 of the substrates indirect of fluid is always close to zero. So it's not match for you dump it all in very full up director completely and ventilated react.
12:40:50 We fill it up slowly so that the substrate that goes in Middle East comes in. Okay, so it is kind of like a cumulus that actually, it is a key milestone, just has to settling face in between us that system, because that was the other question, if you
12:41:03 would have added the substrate in batch How would you sort of prevent the the arising and sort of enrichment of the the the partition pathway.
12:41:18 Yeah, yeah, I think in batch we probably would not have selected for for this interaction, we probably would have gotten, maybe a co culture of canonical ammonia oxidizes and animals or animals would have out competed.
12:41:35 The naturalize altogether depending on how much and I tried his presence.
12:41:38 So I think, sort of our, our, our luck here was that we really kept it substrate limited.
12:41:48 And, of course, under conditions that, in retrospect if this model the particular with the natural compensation is correct if this idea really is correct, then select for for a complete 95 or not because it can come perform complete notification, but
12:42:04 because it can turn around this.
12:42:06 This last step and perform denied if I were at least nitrate producing ammonia oxidation.
12:42:13 And that sort of this reversibility of the NXR, which normally does the second step, that that was the key factor why we obtained this organism under these conditions.
12:42:23 Right, so So what is the common mocks organism get out of this metabolic indirect and except that nitride is consumed.
12:42:34 I think it's quite important that the nitrogen is consumed.
12:42:39 Because of course nitride is toxic to all microorganisms. Also tonight Prospero. So, the nitrate concentration gets too high.
12:42:48 It becomes toxic. And I mean yes the thermodynamic for this reaction are also favorable.
12:42:54 But it could very well imagine that sort of keeping the night right concentration so the end product very low makes sure that the nitrate reduction continues, what we know from canonical nature it oxidizes.
12:43:09 If you have natural spiral Muscovy enters in incubated in the presence of four made which it can use as an alternative electron donor and nitrate.
12:43:20 Even when there's oxygen, it will reduce the nitrate tonight right at the same time as it produces oxygen. Yeah.
12:43:40 So it apparently diverts these electrons to these two different channels and accepted. And then once the nitric concentrate concentration exceeds a certain limit, then it's are suddenly turns around again and oxidizes to match right back to nitrate, well
12:43:45 well it's still as oxidizing for me. And so my guess is that the regulation of this enzyme is not very good, and that the direction of the reaction, depends a lot on the concentration of the of the substrates and the products.
12:44:01 And for that reason to continue to drive this reaction into the reductive direction, you need to remove the natural. And that is what we're in amongst us.
12:44:19 So we leave our networks which would be inactivated that the economics would switch again to complete notification.
12:44:21 And for that you would only need to increase the oxygen concentration.
12:44:28 Now that is something
12:44:29 that is something we do see we now have test reactors where we sort of slowly up the oxygen concentration with leaving everything else constant. And then you do see that at a certain oxygen concentration.
12:44:40 The system crashes and animals completely disappears because suddenly common box is too competitive for the ammonia just oxidizes alternate rate. And so I know marks on the one hand sees too much oxygen.
12:44:52 but even when oxygen is still quite limiting in the culture because it has consumed completed by Karl Marx, suddenly, unlocks doesn't get substrates anymore.
12:45:01 And so it
12:45:07 really sort of a lucky hand to set the conditions right in the beginning that you had this system play out in this in this window.
12:45:14 Yes, indeed.
12:45:18 It's, I mean we do know by now that it's not the only way to enrich for comics, because of course there was the parallel paper to our study from Mickey wellness group in Vienna.
12:45:25 Yeah, and and they did a classical Arabic batch incubation for many years, where they also reach for comic books and they're the trick will he was.
12:45:37 It was a patch incubation so it also had some more or less eternal biomass retention because they they they centrifuge that when they exchange the media, and then they also kept it at very low ammonia concentrations and also enriched for comics and not
12:45:52 canonical ammonia oxidizing.
12:45:56 So, it can be done. Well, you can obtain them also with the expected metabolism let's put it this way.
12:46:05 But you know, for us, definitely was, was a lucky catch that we had these conditions where we selected for the for the interaction. Yeah.
12:46:14 Just want to throw in a question here from the chat room. How fast is the conversion of nitride to nitrogen gas, did you see a higher accumulation of my tried to support the enrichment of this animal reaction.
12:46:30 We never measured and I tried in director. So nice right still apparently was was limiting in the whole system.
12:46:40 And always was instantly consumed by dynamics. And I think this is, as I said earlier important to drive direction. And of course also is. Yeah, helped by this close and direction of the organism but the nitrate is nearly passed on to animals, and animals
12:46:55 can immediately consume it.
12:46:57 So, the, the whole turnover to to end to release seems to be regulated by the speed of nitride production and just immediately consumed so no it never accumulates, which is another great example that the environmentally most relevant compounds are those
12:47:18 that you can measure.
12:47:21 Right. Yeah, yeah, yeah, of course, yeah, we are similar. Similarly, relevant, but but yeah the, the compound that regulates the turnover speed. So the rejection rate is the one you can't measure to
12:47:36 me ask a different question so I'm going to forward for the verification of what you presented to us. It's fascinating.
12:47:46 What about the rest of the community because you are always marketers that the bacteria is there either.
12:47:55 How much the rest of you talking about and if it's a lot.
12:47:59 Do you have any idea how do they survive together.
12:48:04 I think that is still one of the biggest puzzles, we have in all of our actors systems which I'll go first. I'll go back to the, to the meta genomics picture here of the first study even though it was a slightly more complex media.
12:48:22 So, here you do see that even among the abundant organisms there still are quite, quite a number of different organisms present. So there's still some plan to my seat for example which are not the organism.
12:48:36 So this cloud here is the animals organism here we have two other planted seeds that are probably he touched lives, and also here, These, these organisms here are mainly to traffic organisms.
12:48:50 And that is something that we really seen all our actors, even if we only feet ammonium at limiting concentrations at as the only energy substrate.
12:48:59 We have 5060 different mechanisms that persist over many years, these reactors, and we cannot get rid of what they exactly do.
12:49:11 It is something people have been looking into a little bit but it's not really that villainous that a lot will probably be just simply grows on dying biomarkers.
12:49:23 It will also be because of course you always have cell turnover.
12:49:27 It will also be gross on on GPS metrics, so these flock formers of course the flock has a structure that is made of biomolecules sort of extra problematic substance, and other organisms can consume that.
12:49:43 But sometimes it also seems to be a certain direction because we always find exactly the same.
12:49:49 The same final type in similar actors so if you have animals in a system, we always find the biggest fear like blank to my seats in there, and not just random diverse set of, he took trips.
12:50:01 So that seems to be a certain specialization of the, the trough that grows in these systems.
12:50:08 If that is because it's best adapted to consume the animal APS matrix, or if it also feeds something back to another box to make the system more stable and so if there's a selection for it out of those reasons, we really don't know.
12:50:24 Yeah, so, so you think it's more convincing, or maybe cross fading.
12:50:30 Do you have any hands or an indication.
12:50:37 When you look for example for security secretion systems on an experiment in the transcript Thomas for example when you look file expressed as a Christian system that could indicate some kind of prediction.
12:50:52 We didn't do we didn't do much transcript topics on the systems yet we once tried to, to look for a specific cross feeding, if there is a specific molecule that is passed, either between column books and another box to enhance the, the interaction also
12:51:12 then fed to the truth. So we did so miserable omics ones on these reactor cultures where we actually did not identify much, there was a little bit of sex innate in the medium.
12:51:24 So, there was some bleeding of TC a cycle the media is mainly sex innate.
12:51:29 That of course will then be consumed by the seat of jobs, but very low concentration and in general we did not detect much in the X amount of alone.
12:51:39 So, at least, but of course the X amount of long will miss much in the peptide range and then these things that will not see so much.
12:51:48 But of course there's also difficult to distinguish them. Do we see expression of peptide export systems and address borrow because they export them for ups production, or because it might be some cross feeding, for some reason,
12:52:06 great mystery. Thank you. Yeah.
12:52:09 Okay,
12:52:13 so thick to hear.
12:52:20 Now we have the choice between two different topics.
12:52:27 The first one goes a bit more method development, because we developed new methods to specifically detect ammonia oxidizing Mike holders and environment.
12:52:38 The second one is the kinetics characterization of comic books enrichment, or you say we stop. That is your decision.
12:52:48 Terry, what do you think I think we have eight minutes left.
12:52:55 Well, other questions in the audience.
12:53:01 I actually have a question for you, Sebastian, if we don't mind.
12:53:07 I'm thinking about ammonia oxidizing bacteria here, and I know that when they have low oxygen, they tend to shut down their complex for and then they have an increase in natural fire do nice vacation as a way to get rid of extra electrons that build up.
12:53:22 up. So, do you have jeans like near care interests, or it says reversal nitrates and I try it kind of a way to build that electron sink under low oxygen, or is there any thoughts about that or.
12:53:35 Yes.
12:53:38 So the best way to start from the back, the fascinating thing about this night if it notification I always find is that it doesn't seem to be as far as I know, at least, but certainly not the expert on this topic that it doesn't seem to be very effective
12:53:51 so that it really seems to be a survival mechanism, but that they can't grow very long on this metabolism, their money oxidizes now.
12:54:00 There might be very few exceptions but in general, this seems to be the case for natural Sparrow, they all supposed to nitrate oxidizing and the complete ammonia oxidizing that just for the container.
12:54:29 But none of the largest part of the sequence so far contains any and all reductions. There was one indication in one major genome, which has not been physiologically proven yet all the ones will be followed up with incubation definitely can introduce
12:54:32 an intro in Vienna they even showed that the entire production rates of the match sign up in artists of their comic books corresponds to our be autocratic Silliman turnover, and it's definitely not biological of biological origin.
12:54:51 Okay, great. Thank you.
12:54:53 Yeah.
12:54:57 So maybe we can spend the last few minutes, I guess maybe summarizing more about this radio think that that you initially search.
12:55:11 So,
12:55:16 so, so, would you say that the verdict is still out about the radio.
12:55:27 Not it well, not if you compare the deal question for comic books, compared to canonical ammonia oxidizer.
12:55:36 So there I go here to this slight.
12:55:43 This are the results from the buy now pure culture of the, so the only pure culture that's available for comics.
12:55:53 And they could show in in 2017 in a paper, where they measured post the affinity for the substrates.
12:56:04 affinity for the substrate ammonia, then all the canonical ammonia oxidizing bacteria and also higher than even most of the ammonia oxidizing a Kia.
12:56:28 And they also determine the yield and they could really see that this complete notification really allows for a yield compared to both a clear and bacteria which only do the partial the canonical and when you oxidation.
12:56:40 So, I think it's it's it's partly proven that at least the yield is really higher, the growth rate. Also, at least compared to the, to the, EOB that sort of grow at higher concentrations also is a lot lower.
12:56:54 So this really seems to be true, and be a large factor in this selection that they do have the higher yield especially, and then are more competitive under.
12:57:08 But that lets the grocery we are talking about. That was what I was asking you before.
12:57:14 Yes, for our actors we never to two minutes for the regional directors.
12:57:21 For the regional directors. I for this study I try to remember they put it in there. I think it was still in the order of days.
12:57:31 Whatever base.
12:57:32 Yeah. Yeah.
12:57:35 So okay, I think I don't work.
12:57:36 Yes.
12:57:38 Yeah, because I mean that that's the funny thing that some of the comics so the, the one be enriched further, they apparently even are inhibited by ammonia concentrations.
12:57:49 So as soon as you go above 100 micro molar of ammonia.
12:57:54 They reduce the maximum rate again and are not completely limited, but but are to approximate 50% inhibited, which of course shows the more strongly that they will only grow with these highly limited concentrations, which then automatically means that
12:58:07 they probably never reach their maximum growth rate, just because they never see sort of the very high substrate concentration is because that for some reason hits them.
12:58:19 But that's not the case for all comic books, seems to be special to some.
12:58:27 Yeah, It's a fascinating story may ask another question.
12:58:33 Yeah, sure.
12:58:34 So, so do you find these two animal animals and karma, calm almost always enough lock on a biofilm, or. Also, the kind of find them in the liquid phase of the chemistry, let's say, well, that question for our culture's is a little bit difficult to answer,
12:58:59 because of course we select for flux, since we have this SPR setup of our Rector, which has the settling phase where all the liquid, then is removed rather rapidly.
12:59:09 This automatically means that all planktonic bacteria are also removed from the system. And so yes I'm sure some of these comment box, probably more than another box will also go into the liquid face, but then they are washed out of the system immediately.
12:59:29 And I think for that reason we usually don't see them.
12:59:33 We now have a setup that tries to mimic trickling filters of drinking water systems where we have glass beads, as biofilm carriers and then just water trickling over them, so that the water flows through the system by gravity and they we do detect some
12:59:50 common logs in the effluent so yes they continue to go into the liquid face. But if they still are active there or if this is sort of excess biomass dying biomass answer.
13:00:05 Thanks.
13:00:06 Yeah.
13:00:10 Any other questions.
13:00:17 I see a question in the chat from David boats, if ammonia oxidizes can also oxidize anything.
13:00:26 Yes and no it's, I think the same story as the other way around for a refund oxidizes some economic status, of course oxidize ammonia, but can't grow on it.
13:00:38 Because the yeah they can't use the intermediate so they can't use it. I mean, they have detoxification systems but they can't use it for energy conservation so they if they see too much and wanting to actually kill themselves because that excellent mean
13:01:06 Nathan oxidation Ville be capitalized, I'm sure, to a certain extent but I'm also, because we also know that meeting is inhibitory for ammonia oxidation, at least in not in all ammonia oxidizes but in many, but it's probably also happens here that the
13:01:08 downstream systems are not in place so that the cells produce me So know that, then cannot effectively be new be used, and so they might kill themselves nice to know.
13:01:17 And even if they convert me some old, they might form formaldehyde which is even more toxic. And so they probably also will kill themselves in the long run if they see too much missing.
13:01:27 As that from from our culture's we do have the impression that they are quite quickly inhibited by the thing, but we didn't do systematic tests on that yet for comic books.
13:01:42 It was the main open question I still saw on the chat.
13:01:48 Yeah.
13:01:51 Now, we have one more. The number of how much do we understand and the genomes.
13:01:56 There the the number of 30% was passed it depends a lot on which which
13:02:02 which Michael organs you've worked with. So in most of the natural spiral genomes where we don't have many Bell characterized close relatives.
13:02:11 It's in the range of 50% of the genes that still are hypothetical. So, half of the genome we cannot predict a function for, which of course you always makes it difficult to predict executive which functions are absent, because you have no idea what this
13:02:26 this 50% of the genome does.
13:02:29 The 30% is is as far as I remember so true for for August lucky cooler.
13:02:34 So even if all i after years and years and years of research on the coolest still has approximately 30% of uncharacteristic genes where we don't know the functions.
13:02:51 I think those were all there were still in the chat. Well, Sebastian I think we will stop here this was a terrific session so thanks so much, you're covered a lot of ground.
13:03:00 It was great to hear so much microbiology outside of the organic world, so listen throw some pretty cool.
13:03:08 And if you want to, I think, understand really the sophistication of microbial metabolism Lissa troves is a great, great place to look as well.
13:03:20 And so thanks again for this exciting morning or afternoon or evening, whatever.
13:03:26 And what my pleasure. Yes.
13:03:31 Yeah, so thanks so much and I'll see the rest of the class will see them in a week. Okay.
13:03:38 All right. Thanks a lot.