 all the other organizers. It's an amazing place and I think the talks have been very stimulating, so thanks a lot for that. Also thank you everyone for sticking around until this late. Hopefully it will pay off and you'll get something out of the talk. So I come from Universal Warwick where we have heavily invested in synthetic biology and we're also sort of having a set of groups working on trying to engineer ecosystems and if I have time I'll try to talk about that at the end but primarily I'll focus on the theme of the workshop and talk about how we're trying to understand microbial communities. So people have been showing rather static pictures of microbial communities except for the beautiful talk on the zebrafish gut where we really seen how dynamic these systems can be and this is just a sort of a tiny example from my lab where we have this system which is a freshwater sample that's been stable in the lab for over a year now and it seems to respond to light. It has cyanobacterium in there and a bunch of other microbes and we don't understand anything about this system. We're not putting manpower on the system yet but this is something that keeps me going. I think these systems are fascinating the way how they can be stable and complex at the same time and dynamically complex. So this is what we are interested in understanding these kinds of systems and obviously this is not just about the basic science. There is a lot of relevance as we've been hearing for human health but also in terms of biotechnology. So a lot of water remediation facilities are actively using microbes, microbial communities but also there are other processes including agriculture where microbes play key roles, microbial communities play key roles. In terms of the scientific questions we've been hearing them already over the week and I'm not going to repeat them but obviously we are also very much interested in these questions and a few years ago we organized a workshop very much like this and there was a position paper coming out of that with around 50 authors on it where we tried to summarize some of these open questions and the questions are closing quickly with the beautiful work we've been hearing. What I'm going to introduce you today is a system that we've been spending a lot of time with which is so-called anaerobic digestion systems and the nice thing about these systems is that you can talk about a community level function because the way they operate is they simply digest an organic matter all the way down to methane and this is actually a very much used biotechnology actually around the world and heavily used in places like Germany China and India where people will build these huge reactors shoveling organic matter you know food waste agricultural waste etc and the microbial community within these reactors will then degrade that organic waste and produce methane as an end product which you can then use as energy source obviously. So that's the biotechnology side of it the anaerobic digestion process itself obviously occurs wherever you have depletion of oxygen and that could be much more common and obviously most of the marine sediments will count as anaerobic digestion systems but also several micro environments which are oxygen depleted could show could act as an anaerobic digestion system so here I have few examples where people have measured for example the oxygen levels in a biofilm where oxygen is very quickly depleted below the surface of the biofilm in the gut I couldn't find much information but at least in some cases there seems to be oxygen depletion and I mentioned the marine sediments where obviously oxygen can be depleted and it's not just about oxygen but other strong electron acceptors in particular sulfate nitrate which a lot of microbes can use can also get depleted so these are very nice studies from 80s and 90s where people have measured these electron acceptor profiles in marine sediments and as you can see for example sulfate in this case can quickly get depleted and this gives rise to different processes so the other aspect of anaerobic digestion should come back to this slide is that it is relatively well understood at least at the course level so as I mentioned the organic complex organics come into system and usually it's believed that they are degraded first by primary degraders into less complex sugars which can then get fed on by so-called secondary fermenters which will then ultimately lead into formation of these organic acids which can now at the final stage be degraded by methanogens and other organisms like acetylcholastic bacteria but in a sense the understanding is that this system acts as a food letter where we go from complex sugars toward much simpler sugars and each of the step is obviously a redox reaction and therefore the presence of terminal electron acceptors is quite crucial for the function of this system in particular we know that these final stages of degradation of simpler sugars can quickly get limited by the availability of terminal electron acceptors I'll tell you a bit more about that as we go on so we're interested in understanding the communities in these systems how they work how they function and our approach has been sort of this taking the two ends that we've been hearing the top down and bottom up approaches at the what we mean by that is on the top down side we try to look at the natural communities that exist in these anaerobic digestion systems they usually contain hundreds to thousands of species so we're talking about complex systems and we try to use different approaches to study those at that level and on the other hand we had this naive idea of can we create such systems from bottom up with very few number of species maybe up to five species that can still achieve that community function and how will those systems look like so we we got we got very lucky convincing the funders that this was an interesting thing to do and I'm leading this rather big project involving multiple institutions and I'll tell you a bit about that but also we started the sister project that I want to tell you first about which we are quite excited about actually so the idea here was that we can use these functional systems and study them over time using metagenomics okay so as I said this is a biotechnological application so there are bunch of facilities across Europe also in the UK where people do anaerobic digestion in these big reactors so the thought was that we can sample these reactors over time and actually the project scope at the moment is for a year but we're trying to extend it and the sampling we're doing is over every week so I think this is going to be the first of its kind in terms of having temporal metagenomics data from a system where we can also claim to have a function and the function is important because we can also collect functional metadata from these reactors in particular pH temperature sometimes the feed that goes in the composition of that feed and obviously the methane production right so we can then also go back to the metadata and and ask questions around how the metagenomics fits with the metadata and so on this project started about three months ago so we don't have much results yet I'll just give you a taste of what we're going to try and do in this system and what kind of things we can learn and obviously the sequencing is being done in batches so we don't get the data as we go along but rather we have to wait a bit so just to give you a sense of what is going on these reactors seem to be quite different in general obviously they're being fed with different things some of them with urban waste some of them with agricultural waste some of them are at the same site hence they are getting the same food and it seems that this is reflected in their microbiomes right so these three for example are at the same site and they are getting fed by the same things and they're pretty similar at least in this family level this is 16 s data I'm showing you you can also reduce the dimensionality of this data and create these pca plots and this is how the different reactors look like we just plugged in some gut samples in there from human gut microbiome project and they seem quite different from from those but the bottom line is that the reactors are different they are fed differently and it seems that their microbiomes are also different so these 16 s plots are useful to a certain extent what we are more interested is the functions so we've been working also on developing bioinformatics platforms to go from the 16 s data to metabolic profiles and andres in the in the room so she's helping with this aspect where we try to then go from the 16 s to genomes to to metabolic pathways and we try to understand whether there are sort of differences in metabolic capabilities of the different reactors and there seems that there's something going on and we don't yet know whether this then relates to the feeds composition and so on so that's that's what we're trying to understand so in the meantime we've been also trying to get some of these microbiomes into the lab and study them in the lab as complex communities and this is mostly working this in the context of this big project I mentioned led by Angus Buckling at University of Exeter and what he's been doing is to set up lab scale reactors these tiny 500 mil bottles on Arabic bottles and grow these microbiomes in these in these bottles using a synthetic feed and he's done that using again microbiomes from different reactors but pairing them up with slurry from these bioreactors or the slurry that is used to set up these bioreactors originally so the idea was whether there was some sort of optimization going on in the reactors that they somehow are adapted better to the anaerobic digestion and it seems to be the case so the dark data points here are from the reactors and the open ones are from paired slurries in most cases the slurry was what originally the reactors were inoculated with yeah yeah and again these systems are open so it's not that simple but we thought that maybe that's a sort of a pair in loose pairing where we have the original source versus what has been in the reactor for a long time and so this is over time in the lab as these communities get adapted or get fed with the synthetic media and they seem to converge and we can we can also see that actually in their 16s profiles so again here the the squares and the circles are the reactors or the slurry and they seem to all converge to to a certain point in in in the compositional space as you adapt them and then you know what this simply shows is that the communities can be different and over time they can adapt to a certain feed but also because we have this sort of function in our hands which is the methane production we thought we can do fun things with it and one of the fun things that Angus tried is to try to combine communities that have different performances right so as I was showing here earlier there are different communities that perform differently in terms of methane production and you can now say what happens if I mix mix a pool performer and a good performer what's going to be the methane production what's going to be the composition and it turns out that this is the pool performer and the dark here is a good performer in terms of methane production and the mixture seems to overline with the with the good performer over time as you as you keep them and what was interesting to us is that as this is happening in the in the sort of performance in the 16S or the compositional space the the mixture was converging towards the better performer so it seems that somehow the better performing community is taking over as a community this is this is a rather sort of preliminary result we we try to still understand what is going on here but Angus has been doing more experiments around this where they coalesce or mix different numbers of communities and it seems that always the best performing community or the mixture always converges to the best performing community so I don't want to sort of make much out of this because we don't really understand what is going on but I guess one bottom line that we can take from this is that the microbial interactions within the community in these natural communities seem to matter and and seem to sort of give them a sort of a real sense of community right so that it seems that functionally speaking there is something that we can say is a community and this community can go through perturbations as a single unit well no I mean what I'm what I mean is they stick together in the sense of that as you mix them and as you grow them over time then the mixture behaves as in the as the better performing community so they are not clumped or anything that these are mixed environments once we have them in the in these lab reactors okay so but it suggests that the the interactions in the community somehow allow the better performing community to dominate the system is what we are thinking so then what drives those interactions it becomes the question right so what is really happening in these communities and I guess many of us are interested in this question as well as the diversity in these communities how it is maintained and I'll just take a sort of a side shot at Johnny Depp here as he stars in Don Juan de Marco there is one scene where it sets up all these philosophical questions and then he says the answer to all of these questions meaning of life what we are we doing here etc he says is one on the the same and it's love in our case I think the answer is metabolism and I hopefully most most of you will agree with that with that obviously there's there are you know we've been hearing about toxins and bacteria killing each other other things but I think a lot of the interactions are driven by metabolic metabolic processes and therefore we need to understand this right and the cellular metabolism in the microbial case is already quite complicated so we're talking about thousands of reactions and we have these textbook pictures where everything is neat and ordered and pathways everything has a function and a role but as we've been hearing this is more like I increasingly started to think about this as like a spawn that you know you squeeze and everything comes from all sides and that's actually a paradoxical thing right why would metabolism result in so much excretion is not an obvious thing right every cell needs all those metabolic elements so why should they be screaming metabolites out but as we've been hearing they do so and all those excreted metabolites give rise to these different metabolic interactions like crossfeeding, synthrophy, oxotrophy and also in some cases metabolic cycles so why are these things happening and they are happening because metabolism behaves in a certain way and obviously metabolism is the result of evolution so we are now facing an evolutionary question why did metabolism evolve in such a way that we have all these metabolic interactions so this is something we've been thinking a lot about and obviously the evolution is not simply an optimization process as we know it's rather like you're trying to swim in the sea and the environment changes as you do things and as you adapt to the environment and that's the thought that we've been playing with to try to understand cellular metabolism and an emergence of interactions so one of the things that we early on looked at is the possible ways of how crossfeeding can evolve and people have been thinking about this problem quite a while and one of the ideas is that constraints within cellular metabolism both in terms of adaptation to different sugar sources but also limitations in terms of enzyme production or respiration rates can lead to evolution of crossfeeding and we want to test that idea using FPA models and we heard a lot about FPA but if I may quickly introduce this is actually a linear optimization approach where you set certain constraints on metabolism which is set out as stoichiometric reaction sets and then assuming steady state and given those constraints you optimize the system for fluxes and obviously it's been used a lot we tried to put a twist on it by trying to use FPA to model multi-species systems as well as evolution so the idea here is that instead of running a single FPA I run multiple FPAs in a shared environment using what is known as dynamic FPA so we run these stoichiometric linear optimizations for each model for each of these models and then we run a differential equation model to simulate the environment so the models uptake certain metabolites and they perhaps excrete something or not so we then update the environment and then we go back and update the models and I think people have been talking about this so this is the same approach used in comet the twist we added is that for each model we edit a sort of a global constraint where we say the total uptake and excretion fluxes on a model are constrained right so you have a certain amount this could be due to enzymatic constraints or other cellular constraints but it's a sort of an abstract notion that we put in and you can now given that constraint on total uptakes you can distribute it across different uptake processes as you wish so that's actually optimized by the model but the only way you can change these investments into the different substrates is through mutations so that's how we introduce then evolution so this is just a sort of abstract way we can model now a multi-species system in the same environment along with evolution so we have both ecology and evolution potentially so we want to try if this model makes any any sense if it does anything any any sensible predictions so we want to try it on a well-studied case and we told the Lansky experiment where they took the E. coli through batch transfers over many years as an example we can study because in some lineages of this experiment they find actually multiple sort of genotypes arising and we thought perhaps we can we can sort of simulate the system and obviously you can so you can set up an E. coli model and you can set up this experiment in the context of this model so the way this looks like is that you start with a single model so each of these lines is a model now the x-axis is just our subculturing just like in the Lansky experiment and then each model grows based on its sort of biomass flux and then this is the population size associated with each model and the models as they grow they give rise to mutants so some mutants arise and then they get lost again because we do the subculturing and some some mutants persist and they stick around as you see we get a bunch of different genotypes surviving at the end this is all published work so I'm just going through it very quickly but the bottom line is that we end up with two types of models evolving in these simulations so we start with the originally coli model set up in a certain way and what what happens is that this model evolves its glucose uptake rate but as it does so its optimal state becomes one in which it excretes acetate and that acetate excretion if you wish creates another niche if you want to think about it like that into which another genotype can evolve which adjusts its uptake rates such that it it it prefers acetate over glucose so it's it's quite abstract and I think some of the elements of this model or of this results arise because directly because we set the model in a certain way assuming a very strong tradeoff between different aspects of the substrate uptake but still we think it is useful it seems to explain some aspects of the system in the landscape experiment so we teamed up with Dominic Schneider who then went back to the clones in that one lineage where they find these different lineages and looked at their growth on acetate and glucose and we find that it seems that one genotype has improved on acetate and didn't change much on glucose whereas the other one has increased on glucose and and sort of went down on acetate growth so as I said I'm happy to talk to you more about this this this model is available to download if you're interested and we're talking about trying to link it up with comets and so on but what I want to move on is this idea that you know constraints in your cellular system either due to enzyme production or perhaps rates of respiration can shift this metabolism towards fermentation and these products can get secreted and act as basically points of interaction with other bacteria and this is as I said quite common especially when not not just because of cellular constraint but especially when respiratory pathways stop simply because there is no terminal electron acceptors available right so the respiratory pathway is is the part where you dump electrons on the on a terminal electron acceptor usually this is oxygen but not necessarily always so you can use sulfate nitrate but if none of those things are available you can't do this anymore the only way you can get rid of electrons is through fermentation and this is very common in anaerobic digestion as I was saying so to start with there is no oxygen so many bugs will use nitrate or sulfate but those things get depleted as well so you end up with a situation where the only thing you can do is ferment okay and this is and these fermentation products will be taken up by other bacteria in a process called syntrophy which is very similar to crossfeeding with a twist because people will usually assume that there is an inhibition here which I'll tell you more about in a bit so the key thing here to note and start to think about is that these fermentative pathways they're low energy pathways okay if you're respiring if you're us and if you're eating glucose and respiring with oxygen we are talking about around 2000 kilo joules of energy per mole whereas if you're doing respiration respiration on more weaker electron acceptors this energy goes down and if you're thinking about fermentation this energy goes even down further okay so we thought about you know what will be the broader consequences of this because this is something that people usually don't think about people think about E. coli oxygen glucose and if you think about this the low energy what or the energy that is close to zero what it means is that we are closer to the thermodynamic equilibrium right as the bacteria converts some substrate into products it is actually running this reaction chemical reaction towards equilibrium and if you start very far from equilibrium you'll probably never reach it but if you're starting very close to it you might actually reach it right so then the sort of thermodynamic aspect the reaction free energy becomes an important factor perhaps and usually people have just considered substrate uptake kinetics you don't worry about products because you're in high energy regime but there are times that where you should consider this thermodynamics and people have been trying to put thermodynamics in microbial growth models now we started to think about the impact of this on diversity right so and we already talked about exclusion principle so the general view on diversity is that you know if you apply a kinetic growth model and if you have a single substrate you cannot maintain two species on that substrate with that simple growth model obviously you can if you have inhibitory interactions if you if these guys are screaming different things that kill each other etc etc but if you just go with the simplest model of there's one substrate and two species feeding on that substrate you cannot maintain these guys one of them with the better substrate uptake rate will win that competition and will dominate for example the chemostat this is that this is just showing your chemostat model here so that's fine I don't have to go into details of that we thought what would happen here if we now have a thermodynamic view of the world so in particular if we now adjust this growth model with a thermodynamic factor what this factor does is basically it brings in the product concentration into the equation right so and now you can create situations where actually you can grow on the same substrate but both species can coexist now the key thing for this to work is that the two species should consume the substrate in different ways so they should have different byproducts otherwise their energetics will have been the same as well so they will have been the same species but if that is satisfied and also if those two reactions were really close to equilibrium then this thing will work right so we have to be really close to thermodynamic equilibrium such that the fast guy even though it's fast in uptake it actually reaches equilibrium and it cannot grow anymore that leaves still some substrate for the other bug to grow so this is an interesting concept we thought because theoretically I can now play this game and I can set up as many reactions as I want and as many species as I want as long as I'm in this very thermodynamically inhibited regime I can have as many species as I want so this sort of creates an answer to the diversity issue but obviously only in this very highly compromised thermodynamic regime and actually the regime where this works is around minus 100 kilojoules so as soon as because the way this equation is set up because it is sort of exponential you have basically a cliff there and as soon as the thermodynamics are above a certain threshold the kinetic term dominates and the diversity is lost but it turns out that actually this this sort of regime we're talking about applies in the case of many fermentation reactions so many fermentation reactions have standard term gives free energies around minus 100 or below hence we think that this this theory could actually be relevant for especially for anaerobic digestion systems okay so this is just summarizing what I've been saying so cellular constraints can give rise to metabolic overflows which can then give rise to cross feeding at one hand and at the other hand depletion of strong terminal electron acceptors will shift you to a thermodynamically compromised regime where actually diversity wouldn't be such an issue anymore because there wouldn't be much competition going on anymore so how relevant are these things really I think they're very relevant in the case of anaerobic digestion so it's well known as I was saying that the anaerobic digestion systems work like a food letter and you reach a point where you're basically having to ferment these compounds like lactate acetate which are which don't have much energy okay and the other aspect is that the electron acceptors are being depleted by all these people above you so that when you come down here you potentially don't have electron acceptors either so one of the key interactions that's known as syntrophy is actually based on this thermodynamics so you have so-called sulfate reducers and this is why people are interested in sulfate so they will normally respire sulfate but when sulfate runs out they have to ferment and they usually live on small organic acids like lactate and similar compounds and when they ferment they ferment lactate into acetate and hydrogen that reaction will reach thermodynamic equilibrium the only way this guy can continue growing is by lowering the product concentration in this case hydrogen and acetate and that's done by methanogens who are sort of specialized on these so who are among the few species who can eat hydrogen and acetate hence you have this you have to have this interaction otherwise nobody can grow so this is known as syntrophy and this is just giving you the delta G is associated with that so we want to sort of study this system to better understand how that system evolves because it's also interesting that whenever there is sulfate this interaction will go away right so there's this ecological component there's the thermodynamics stuff that we like so we said let's put this system together and it's actually a system that David Stahl had you you wash has been studying a lot so they helped us also a lot when we were first getting set up we just tried to set up a co-culture and we failed miserably at this at the beginning and then finally we run a huge experiment with a large number of co-cultures we say like this has to work and then one of them did work so we got methane production on lactate and we know that methanogens cannot eat lactate in the absence of sulfate so the system is working right we got growth and we got methane we were happy we were doing our thing and then we sort of thought wait a second why did it just work only once right so there was the thought that maybe there's something special with this one case so we took this co-culture that was working isolated back the sulfate reducer and we tried to set the system up again and in this case when we did this every time we tried this with a isolate from the working co-culture we got a successful co-culture right so this strongly indicated that there was a mutation or something specific in that one working co-culture that made the syntrophy work so there is potentially a mutant that's our hands that is sort of syntrophic and we wanted to understand this better and it's quite I find it very exciting it must it might be just me but this system is amazingly close to thermodynamic limits right so when you convert lactate and ferment it into acetate and hydrogen that's your overall reaction but actually the first step is lactate oxidation into pyruvate which under standard condition is a positive delta G right so that's reaction is not allowed by the laws of physics under standard conditions but this bug is somehow doing that and then by then going through this step it also produces acetate and hydrogen now obviously we're talking about standard conditions and in the cell the micro environment might be quite different but we thought that this syntrophic mutant could potentially be linked to this initial step in the in the metabolism and I guess it was beginner's luck we just sort of sequenced the the syntrophic isolate that we have against the wild type and lo and behold we only found two mutations that were consisted across the set so we have the isolates from the sort of the original dysulfabial sulfate reducer culture versus the syntrophic isolates and they had two mutations and one of them was on the lactate permease and the other one was on this the hydrogenase which is allowing this lactate oxidation to happen and it turns out this dehydrogenase is coupled to anti-porter ion porter across the membrane so that we hypothesize that maybe there's something happening here that the organism invests from the membrane potential that is facilitated by the ions into the into making this reaction possible and then once it gets going it can produce hydrogen which then allows the metanogen to feed on and grow and you get the syntrophy working okay so the other interesting thing about this is that when we looked at the wild type version so this sort of non-syntrophic version of this few of the clones there were actually polymorphic in this loci so this is interesting because so this wasn't a mutant this was a polymorphism in the culture is what this suggests and then the question is how is that polymorphism maintained but I think the bottom line here what I'm trying to get at is that the thermodynamic limitation seems to be driving the evolution of the genotypes for this for this bug right and and the polymorphism is interesting is because this either suggests that there is maybe internal syntrophy between different genotypes or perhaps the population just maintains a certain amount of um syntrophic genotypes just in case sulfate runs out and they're ready to create a syntrophic interaction with metanogens so the the second idea is quite interesting so we want to we're trying to go in that direction and this is now our recent work where we're trying to do these things it takes time because these anaerobic systems are notorious to grow but we also extended the system at the same time so we introduced another metanogen that can grow on acetate such that we have sort of a full set here of syntrophic interactions so we have the hydrogen based interaction but now we have also the acetate based interaction and this behaves as you expect in the absence of sulfate methane production increases in the three culture compared to the two cultures but where we want to go with this is the maintenance of these syntrophic interactions in the absence of sorry in the presence of sulfate right because for these communities sulfate must be coming in and going out and this this interaction is so central to the whole community that it's quite interesting to try to understand that question the interaction is important to the community because if this is not working properly acetate accumulates in the system acidifies things and the whole thing collapses and that's a common problem for these bioreactors that you have a 10 ton reactor and then suddenly it doesn't produce methane it just does something etc so we want to understand the sort of robustness around sulfate and that's what we've been trying to do set these co-cultures versus the three culture and then we try to sort of maintain them in the level where multiple subculturings each subculturing being three weeks and and as you can see here if I now introduce sulfate at a level that is enough to respire all of that lactate we start to lose the metanogens so the community breaks down but interest and then if you have no sulfate obviously the community stays it's the only way for them to exist is to do syntrophy but in the in the middle levels of sulfate so this is now stoichiometrically half of the sulfate that you need to respire all of the lactate you still see some maintenance so the metanogens can be sustained in the system when this is happening but interestingly only the hydrogen-based metanogenesis can be sustained and the acetate-based syntrophy is not able to sustain itself we don't understand all that is going on here there is a very good increase in methane production in the absence of sulfate that we don't see in the absence in the presence of halve sulfate we're trying to understand that currently okay so hopefully what we are trying to do is make make sense so we try to bridge these two ends you know we want to look at the natural communities try to understand the different functions that are there how the system changes but at the same time we want to also bring those insights in or merge them with the sort of insights we're trying to get from these minimal systems and then we get carried away and with we say okay now that we're getting some insights can we use those also actually to go back and try to engineer actually new systems right and when I say engineer what I mean is just just putting together ecosystems right can I sort of set the environment can I pick the species put them together and it sort of works and now I'm running out of time so I will not be able to tell you too much about it but the sort of the general idea is you know we can create these different interactions and maybe understand them and then maybe put them together to create more complex systems and always with a certain function in mind so with the AD system what we are trying to do is to extend now this core syntrophes with primary for primary degraders to then degrade a higher sugar and we're aiming for cellulose and this might then be studied on its own but it could also be optimized maybe genetically engineered etc but also we take the approach to other systems that are not necessarily anaerobic so one system that we are quite keen on is this sort of idea of closed ecosystem can we do a microbial version of this using a phototroph like the one I showed in the beginning and we've made some some progress with this where we try to use heterotrophs actually initially to create a mineral cycle working on manganese which we know is crucial for maintaining phototrophs because they use manganese only in their light harvesting systems so we've been trying to set that that kind of mineral cycling with two species that we know can oxidize and reduce manganese and we've been trying to set that up we first try to create a common media so we run a lot of physiology experiments on these two guys and it turns out that actually they do can crest feed so these are schnovella schnovella anodensis and rosaobacter ASWK it's a marine organism and this is a freshwater organism and it turns out that schnovella grows on lactate and overflows acetate which rosaobacter can use and we are able to sort of measure all these things and show that it works and then from there we can go and look into the manganese so if i have just two slides here the other system just to briefly mention it is a is a system where we want to sort of create a microbial system that can support plant on a media that normally doesn't support plant and for this we are using a mycarzill fungi that is shown to colonize plants and and support them and it turns out this this this fungi is oxotrophic for thiamine which can be provided by bachillus subtilis so without thiamine this guy cannot grow but with bachillus subtilis it can and we can quantify this using timelapse microscopy but the latest thing we are finding here which probably might be interesting some is that if we if we try to create so the system works if we spatially separate them on a plate but if we premix them it doesn't work so somehow they're inhibiting each other and their inhibition is is dependent on space or time so if we also temporarily separate them that still works but if we don't temporarily or spatially separate them doesn't work and we don't know why at the moment but we think it's either oxygen depletion or or pH but yeah I'll stop there and I apologize running later a bit but this is what we are trying to do sort of try to learn some of the ecological principles as well as the limitations that drive that shape these communities and then try to use those insights to build functional systems and with that I'll acknowledge the people the the the reactor work I mentioned is this is a sort of a public facing project so all the data is on the web so we put the data on the web as it becomes available and we are very open to collaborate with people if you're interested I mentioned the the workshop with it and obviously I thank everybody in the group Andrea has a poster if you're interested on these functionality analysis and I acknowledge the collaborators and your time thank you