 It's an honor for me to introduce a host of really good speakers for the webinar, Complete Ammonia Oxidizers, a new pathway in the nitrification process. So I'd like to introduce the group, the specialist group to you, IWA Neutron Tremul and Recurry specialist group is, I believe the oldest specialist group of IWA and perhaps one of the largest, we focus on all aspects of nutrient removal as well as recovery, the microbial processes, the technologies associated with such removal and recovery, and also the cutting edge and fundamental research in that area and applied research in that area. We have approximately 50% of our members of the specialist group from academia and about the remaining 50% from practice. We host conferences every year. We have typically done that, even during COVID, we've hosted virtual events and there was a virtual event that was just held recently in Poland. Our events are typically, they move from different regions between the Americas, the European region, Europe, Africa, and then Asia and Australia. So typically our events go between those three different regions of the world from a longitudinal perspective. And we also have just started hosting webinars. This is actually our third webinar and we hope to continue to host webinars on, I would say either a monthly or a bimonthly, every other month basis and on topics of both removal and recovery. Next slide, please. Our IWA's flagship event is the IWA World Water Congress and exhibition. This is going to be held in Copenhagen this year between 11th and 15th of September, 2022. I believe the early bird registrations are still available. So I would encourage you to register for this event. I think it's going to be, I know it's going to be a great event for us to get together and meet after such a long COVID break. So register at www.worldwatercongress.org and hope to see you there. Next slide, please. This webinar is recorded and it will be made available on demand. So it's going to be on the IWA website and you're going to also have separately the presentation slides. The speakers are responsible for securing copyrights for any of this work that will be presented and they are not the legal copyright holder. The opinions, hypotheses, conclusions or recommendations contained in the presentations and other materials are the sole responsibility of the speakers and do not necessarily reflect IWA opinion. Next slide, please. This is an important slide for those that want to interact and I strongly encourage everyone to interact and make this a webinar where we can connect with each other, at least on the Q&A box. So if you have questions, please send those questions to the panelists. And the way I would do it is I would say, for example, use the ampersand, add Sebastian or add panelists and then ask the question. Or if you have comments, please do that as well. So please send us your questions, comments to the panelists and we'll either respond during the webinar itself or in the panel portion at the end of the webinar live format. There's also a chat box. The chat box is really for more what I call it general purpose questions that are directed to the IWA staff. If you have some trouble with getting in or if you have some issues with the webinar itself. But really for general requests and any other administrative activities. But really focus on the Q&A box for asking questions and if you would like that question responded to live, also make that known to us. But please do send us questions. Next slide. I'm Sudeer Murthy. I'm the chair of the Neutron-Dremel and Recovery Specialist Group. I'm located in the United States in the Washington DC Metro. I've had mostly experiences and practice, both in consulting, 16 years at a water utility serving Washington DC, and now much more so as an entrepreneur. We have Sebastian Luker, who will be the first speaker. He's going to be discussing the discovery of complete nitrification. So it's an introduction to this concept. The physiology of Coma Mox is Holger Dimes. The model development by Yasek Makinia. Yasek is also the secretary of the Neutron-Dremel and Recovery Specialist Group. He was strongly involved in the development of the webinar. Jianhua actually was really responsible for organizing this webinar. So thank you, Jianhua. He's going to be talking about the applications. And then finally, we go to the Q&A panel at the end, where I'll lead the panel. But really, I expect it to be very interactive amongst the panelists. And then final remarks and conclusions by Jianhua as well. And with that, I'd like to get to the webinar itself. Next slide. The learning objectives are to learn more about the discovery, physiology, kinetics, and metabolic properties of these organisms, and specifically in nitrospira, to enhance their understanding of the role of a nitrospira in biological nitrogen removal processes and opportunities at wastewater treatment plants. And then to explore the potential applications of nitrospira in treatment and removal of contaminants, including ammonia. Next slide. If you have any thoughts on this webinar, please, please tag at IWA HQ on social media. And let us know why you feel Comamox is important for nitrification. How does it affect your life as perhaps an academic or a practitioner? What is the main contribution to, say, sustainable development goals or to the 2030 agenda? And most importantly, don't forget to include hashtags of IWA and Comamox in your thoughts. Next slide. And with that, I'd like to welcome and introduce Sebastian Luker. He's with Rabat University in the Netherlands, and he's going to lead it off and help us discover this process. Sebastian? Yes, thanks a lot, Satir. And I would also like to start with thanking all the organizers for putting together this nice program today, especially for giving me also the chance to tell a little bit about the history of, well, by now, history of how we discovered Comamox, so to give a more general introduction, just still waiting and getting control of the slides here, which always takes a little while to be with me. Well, I am waiting on this yet. There we go. So as Satir said, I will take you back to how we actually discovered complete nitrification, how we discovered Comamox. But I also want to add some slides then, if they start moving, not only on the discovery of Comamox nitrospera, but also on some novel molecular tools that we developed over the last years on how to detect these Comamox bacteria directly in the environment without cultivation, because of course, that is still time-consuming and complex topic for nitrospera. It's really not easy to get these organisms in pure culture, unfortunately. Very short introduction, I'm sure all of you are very well aware of the nitrogen cycle. So of course, today we speak about nitrification. And I'm sure also most of you are still aware that many textbooks still say ammonia oxidation is a two-step process catalyzed first by ammonia oxidizing microorganisms, then secondly, by nitrite oxidizing microorganisms, sorry. And something I want to point out here, because it will appear in the first half of the talk, also is that of course, we also have anaerobic ammonia oxidation by the Anomox organisms that convert ammonia with nitrite as thermal electron acceptor to denitrogen gas. But the main focus obviously today will be on nitrification. It's, Comamox has been around in the literature for a while. So there was this paper in 2006 by the group of Jan Ulrich Greft, who already predicted that in principle, complete nitrification should exist, should be possible, and they hypothesized that the canonical ammonia oxidizers, as we know them, will be selected for under rather high substrate concentrations. So conditions that select for high growth rates, but low yields. And that Comamox, it should be possible to enrich for them under conditions selecting for low growth rates. So low substrate concentrations and long biomass retention systems like biofilms and high yield on the other hand. So meaning getting most energy out of the limited substrate that is available, low ammonia concentrations in this case. By now, of course, you all know we discovered complete nitrification in sub-nitro-spiro organisms. This was published at the end of 2015. And something worth mentioning here is that this was not only discovered in my group in the Netherlands. In parallel, the same finding was made in Vienna by the group of Holger Dimes, our second speaker today, and Michael Wagner. But also almost at the same time, there were two additional publications from a group in the US and a group in Denmark that also found metagenomic evidence of the existence of Comamox bacteria. Both of these were looking at drinking water treatment systems, which also are quite heavily dominated, as we know by now, by Comamox nitro-spiro. How did we here in the Netherlands find Comamox? For us, it all started with this bioreactor. This was a bioreactor being circulated with a biofilm from a biofilter attached to a recirculating aquaculture system that we have here at the university in the basement. And this bioreactor was then fed regularly with water derived from the same aquaculture system that we filtered and added low concentrations of ammonia of nitride and nitrate. But in principle, the system was nitride limited. We didn't add any external carbon. But of course, since it's water from our aquaculture system, there's always some carbon present. And we kept the reactor hypoxic. So we bubbled it constantly with organ and CO2 to remove oxygen as efficiently as possible. And so the conditions we applied here actually should be optimal for enriching anaerobic ammonia oxidizers, Anamox bacteria. And this is also the first thing we tested for. And if you work with Anamox, there's a very nice test you can apply because due to this combination or combining of ammonia and nitride to denitrogen gas, you can use labelled nitrogen compounds to detect if Anamox is present. Because what happens if you add labelled ammonium and unlabeled nitride to your culture, Anamox will form half labelled denitrogen gas. And this is very specific. It's really a signature for Anamox in this case. And with that essay, we put directly in the headspace of the reactor or in batch cultures measure if Anamox is active. And as you can see here over time, we do have a formation of the half labelled denitrogen gas, indicating that yes, indeed, Anamox is enriched, is present in the culture as we expected. However, you can also infer more from this labelled data because of course, if you have simultaneously ammonia oxidation going on, you will have formation of labelled nitride. And if Anamox then combines this labelled nitride with the labelled ammonia that is still present, you will have the formation of double labelled denitrogen gas. And of course, this is only possible in such a system if ammonia first is oxidized because otherwise the second labelled cannot get into the nitride pool. And indeed, when we looked at the measurement data we had from this bioreactor, we could see that there was a constant formation of also double labelled denitrogen gas, showing that ammonia oxidation apparently was present. We of course, at that point, got interested in the community because in principle, we thought it was a completely unarobic system. And so we got curious, okay, which ammonia oxidizer is present? And to our surprise, when we did fish on this culture, we did not find any known ammonia oxidizer. The only nitrify we found in this culture was nitrospera. And these nitrospera cells were always co-localizing with Anamox in the same flux, as you can see here. So they're always co-occurring together, which usually indicates also there might be some interaction. And of course, that puzzled us quite a bit because if we look at sort of the interactions and competitions between these nitrogen cycle organisms, yes, ammonia oxidizers, they should interact with Anamox, but nitrodoxidizers, they should actually compete for nitride. They should compete for the same substrate with Anamox. And so what would they do there? On the other hand, also, of course, also nitrodoxidizers need oxygen as thermal egg-noxceptors. So how can they survive in this reactor? So to find out more about the genomic potential of this community, especially of the nitrospera, we applied metagenomics to the bioreactor community. And together with collaboration partners in Denmark, we were able to extract two high quality nitrospera bins from this metagenome. And when we started to analyze those, we were very surprised to not only find the expected nitrodoxyl ductase gene, so the genes for nitrodoxidation, but also that these bins contained all the genes required for ammonia oxidation. So the ammonia monoxygenase and the hydroxylamine, the dehydrogenase, all these genes were present in both of these genomes, indicating that these organisms have the genomic potential for comomics, for complete nitrification in one organism. When we did phylogeny of this ammo, because of course we were a little bit surprised, okay, why did we not never see this ammo before? We saw that actually this ammonia monoxygenase clusters quite outside of the known and described ammonia monoxygenases of the beta-proteobacterial ammonia oxidizers, and belongs to a group that up to that time point was actually considered to contain methane monoxygenases. And never, so a group that never was linked to ammonia oxidation before. So with that evidence, of course, we were very intrigued, but also felt it's still necessary to prove that these nitrous bars really do ammonia oxidation, not methane oxidation. So the first thing we did was we took the biomass out of the bioreactor into batch incubations that we oxygenated happily. The nice thing, of course, when you work with anamox and you add oxygen, anamox is immediately deactivated. So it's inactive, it doesn't convert ammonia anymore. So the only organism that should still be active is the commomox nitrospera in this case. And indeed, when you add ammonia without inhibitor, we could see that ammonia was disappearing in the culture. Nitrite never accumulated, never was measurable, but nitrate was formed stochometrically as expected. And of course, we also did the test with nitrite to see if they can also still perform sort of the canonical nitrate oxidizer reaction. And indeed, also nitrite was oxidized and converted stochometrically to nitrate in these cultures. So the culture, but of course, this is only an enrichment culture, was clearly able to oxidize ammonia all the way to nitrate if oxygen was present. But we still wanted to link it to this nitrospera cell to really prove on a single cell level that these nitrospera are responsible for ammonia oxidation. For this, we went to a method called fishmoor. So fish combined with microautoradiography, which is very powerful here because you can incubate the sample in the presence of radioactive substrates. We used radioactive bicarbonate to test for carbon fixation, which of course, since nitrospera is an autotrophic organism, is indicative for activity in these organisms. And so if you then incubate your cells in the presence of the radioactive bicarbonate and the energy substrates or ammonium, you will have incorporation of the radioactivity into the cells. And this you can detect on a fish light by overlaying your sample with a photoemulsion that reacts with the radioactivity to form silver grains, which you then can detect in the microscope as black docks. And you can combine this method with standard fluorescence and sea hybridization to have a phylogenetic stain of the microorganisms you're interested in. So you use specific probe for nitrospera in this case to see where are they and does the fish signal overlap with the mar signal. And indeed what we saw is that in the presence of ammonia and of course, also in the presence of nitrite, we have a heavy label of nitrospera cells labeled in magenta here. And if you look here, hardly any label of anamox. In this flock, it looks a little bit as if anamox is labeled also, but there are actually our nitrospera cells sitting right here, which are labeled. If we add an inhibitor, ATU, an inhibitor for ammonia oxidation, we see there's very little label left. There's some residual activity, but hardly any. And if we add no substrate, there's no label. So this labeling is really ammonia dependent. So at this point, we could really prove that we have nitrospera species that are able of complete notification of oxidizing ammonia all the way to nitrate. And that apparently some interaction of anamox and comalmox is also possible, but this is still a question we're very interested in because we still haven't completely figured out what is going on in this reactor. And if comalmox here actually performs canonical ammonia oxidation, stopping it nitrite, or also if alternative metabolisms are happening in these cells, but this is still ongoing research at our department. And here I want to move on to the second part. So some in-sito detection tools of these ammonia oxidizers, especially comalmox organisms. Why is that so important? If you look at this very, very simplified 16S phylogenetic tree, it becomes obvious that the comalmox nitrospera, shown in red, cannot reliably be distinguished from nitrite oxidizing nitrospera. So the 16S can really not be used to infer, do I have comalmox organisms present in my sample or do I have canonical nitrite oxidizers present? So we need some other tools to detect them. One of them, of course, that is obvious to be used for this approach is the AMOA, because the AMOA is, first of all, very distinct from the AMOA of other ammonia oxidizers. And of course, it's a signature of comalmox nitrospera because normal nitrospera don't have it. So the first approach we used was the design of primers that target specifically this AMOA of, so the A subunit of the ammonium and oxygenase of comalmox nitrospera. This was in a very nice collaboration again with Michael Wagner-Tolga-Deyes in Vienna. And here it was possible, don't try to read this tree, this is not important. But what I want to show you is, it was possible to develop two primersets for the two known clades of comalmox. So clade B and clade A, which is shown here, that target the clades very efficiently and allowed us to screen a whole range of different habitats to also show that comalmox indeed is very widespread in nature. However, one problem with these PCR-based approaches is that, yes, we can retrieve a lot of AMOA sequences from the environment, we can show the diversity of comalmox also in these habitats, but it's not possible to link it back to a phylo type. So there's no way you can link these AMOA sequence that you retrieve to the 16S sequences that you might have from the same sample. And of course, one way to do that is metagenomics, but metagenomics can be very time-consuming and of course for complex samples also very tedious. So we developed a more direct approach that allows us to, in situ, label all bacteria that contain an ammonium monoxygenase within so-called activity-based labeling protocol. This was also published last year. Officially, the protocol was adapted from a similar protocol published in 2016 by Benadol. And what was used there is Benadol used octadiene, which is sort of an equivalent of a very well-known inhibitor of the bacterial ammonium monoxygenase octane. The only difference is instead of one alkyne group, it has two alkyne groups, which means that if you incubate your sample with these octadiene, that the octadiene binds to the ammonium monoxygenase, covalently stays bound in the ammonium monoxygenase, but one alkyne group sticks out of the enzyme. And this we can use for the so-called click reaction to specifically couple marker molecule, usually a fluorescent marker molecule to the labeled ammonium monoxygenase and then combine that with fluorescent microscopy to detect which cells were labeled. And the very nice thing is this approach, we can also combine with fish fluorescent zeta hybridization. And so we can combine here a functional marker or functional labeling technique for all cells that are ammonium oxidizing, so contained in ammo. And we have phylogenetic markers of fish growth that can be specific for either ammonium oxidizers or in our case, nitrospera in general. And as we can see in this enrichment culture, we can really nicely distinguish ammonium oxidizing microorganisms that are mainly labeled green. Comamox nitrospera that are in this pinkish white because they have an overlay of the nitrospera probe and the ammo label, but also of canonical nitrospera, they do not contain an ammo because they of course are only labeled with the fish probes, but not with the ammo stain. And the very nice thing about this technique, we can not only use it to visualize the microorganisms, we can also use it in combination with fluorescently activated cell sorting to sort these labeled samples specifically, or these labeled cells specifically out of the mixed community and then do downstream applications like metagenomics. So we have an approach here to do targeted metagenomics of activity defined subpopulation in this community, ammonium oxidizers. And we did this in a proof of principle study first on enrichment. And you can see here nicely that we have a huge enrichment of the ammonium oxidizers presence in nitrosomonas and Comamox nitrospera compared to the untreated biomass. And it even was possible to apply this method to a full-scale wastewater treatment system where ammonium oxidizers mates less than 0.03% of the total reads in the metagenome. So it was in the native metagenome, it was not possible to retrieve a high quality bin of any nitrosomonas here. But after the sorting it was able, we were able to enrich for the nitrosomonas in here more than 50-fold. Well, more than 188-fold even, which meant at the end we were able to put together a very high quality Mac of this nitrosomonas that never would have been possible otherwise. There are still some strange biases in this method. So we also highly enriched some Compitibacteracea Macs which clearly do not contain an ammonia or lithium monoxygenase. So there we don't quite know what's going on yet. The method is still sort of under testing to find out what's going on in these cells. But even with that, it is a very powerful method to enrich these ammonia monoxygenase containing cells in a targeted manner for downstream applications like metagenomics or also just simply for detecting in your sample directly if Comamox nitrospera or other ammonium oxidizers are present. With this, I want to end here. Of course, thank my whole group here at the Drapad University in Nijmegen. Also thank my collaboration partners at the University of Vienna and Alberg University. And yes, thank all of you for your attention. And with that, I'll hand back to Soet here. Thank you, Sebastian. Excellent introduction to not only the reactions of Comamox but also how to detect these organisms and then use these organisms for the profiling. The next speaker is Holger Dimes from University of Vienna in Austria. He's in the division of microbial ecology. And Holger will discuss and describe the physiology of Comamox and also the key features of what he calls the green microbe. Thank you, Holger for presenting. Yeah, thank you Soet here for the nice introduction. And at first I would also like to thank the IWA and the organizers of the webinar for the kind invitation to give a presentation here. And of course I am very happy to contribute. Well, as Sebastian has already mentioned during his talk, Comamox bacteria now I am trying to go to the next slide. But for some reason this is not working. So I restart as Sebastian has already mentioned in his talk, Comamox was discovered several times and about the same time. And I am always amazed about this I must say because after more than a century of nitrification research, when Comamox was hypothesized often but never found that it was finally discovered several times by different groups in about the same period. Yeah, I'm always puzzled about this. This is great. And in our case, it was a nitrifying enrichment culture that a collaboration partner from Moscow, Elena Libedeva brought to us in Vienna. And she got it from about one kilometer deep exploration well in the town of Oshiga in the Caucasus. And it was a moderately thermophilic nitrifying biofilm that felt happy at 50 centigrade. And we found that it had the phenotype of complete nitrification. So ammonium was completely consumed. Nitride temporarily accumulated but then was also completely converted to nitrate without significant total analysis. And fluorescence in Ceto hybridization analysis revealed that it was a binary culture consisting of a nitrospyra organism and a unknown beta-proteobacterium. And of course, we're interested now in this beta-proteobacterium mainly because we assume nitrospyra must be the nitride oxidizer and the beta-proteobacterium and novel ammonia oxidizer. Then we had sequence the metagenome of the binary culture. We were totally surprised. First of all, both genomes could be closed. It was straightforward to sequence that with the high coverage. And in the closed genome of the nitrospyra strain, we found all the genes which are required for a complete nitrification. Not only the NXR, the nitride oxyleridactase, which was expected in nitrospyra, but also ammonia monoxygenase and hydroxylamin dehydrogenase plus accessory proteins that are known to be required for ammonia oxidation. The beta-proteobacterium that was also present in the enrichment didn't contain any nitrification genes and also grew on organic media without any sign of nitrification of its pure culture. Sometime later, it was possible to isolate the nitrospyra strain in the pure culture and we named it nitrospyra inopinata. Inopinata means surprising or against the established opinion which always was that nitrification is a split process of two different organisms. So that was the ComaMox organism and using this pure culture, we then set out to study the features of the ComaMox in more detail because once ComaMox had been discovered by us and by others, of course, there was a number of pressing questions. For instance, what is the importance of ComaMox in agriculture and fertilized soils, in natural ecosystems and water treatment plants? Would there be specific applications for ComaMox and engineered systems? Of course, also what about a greenhouse gas, especially the nitrous oxide emissions by ComaMox and finally what about the biochemistry of ComaMox? Does complete nitrification work in a similar way as nitrification by the previously known canonical nitrifiers? Here we have a number of questions for, yeah, I think decades of follow-up research and today I want to give you an overview of what we have, some main findings we made regarding this pure culture nitrospera inopinata. Here you see a cell cartoon which is based on the annotation of the complete genome of nitrospera inopinata. I will not go into every detail here. You'll see a big number of transport proteins that bring important cofactors and substrates into the cell or export, for example, toxic compounds. Here I have summarized some main features. Of course, it contains the complete ammonia and nitriloxidation pathways. It also has a urea transporter in the UVAs and based on that nitrospera inopinata can very well go on the urea as a source of ammonia and carbon dioxide. It fixes carbon dioxide by using the reductive tricaboxidic acid cycle, which is present in an oxidant tolerant version in this organism, but that is also present in all other known nitrospera species. So nothing special on this side. Interestingly, nitrospera inopinata is unable to go on nitrite as the only substrate. It needs ammonia. Nitrite alone is oxidized for a little while to nitrate, but then the activity stops because inopinata cannot assimilate nitrogen from nitrite and thus it would not be able to go on nitrite. Interestingly, however, it is potentially capable of respiratory amonification. The genome encodes periplasmic cytochrome C, nitrite reductase or nerve. And with that, it would be able to reduce nitrite to ammonia if it has got an external electron donor. That is not a nitrification pathway, obviously, but it is a potential alternative lifestyle of this bacterium. Another interesting aspect is that nitrospera inopinata can oxidize formates, although it has no known formate oxidizing enzyme in the genome. However, it doesn't show growth on formate and it also has a pretty poor affinity for formate. So it is an interesting side note that this formate oxidation is possible, but that is likely an unspecific reaction in the metabolism. And finally, it is able to form glycogen and polyphosphate as a storage compound. So that is a quick overview of nitrospera inopinata and its core and alternative metabolisms we know at the present. Now, in order to address more of the question of ComaMox importance, we need to know more about the kinetics of complete nitrification by ComaMox. And with the pure culture, we are in the happy situation that we can use a tool like microesterometry, which works best with pure cultures. In this case, we have the small glass chambers that can contain a few milliliters of concentrated pure culture. And then they have openings in the lid and through such an opening, an oxygen micro sensor can be inserted. So here you'll see the tiny sensor tip. And then there is an additional port and through that port, a substrate like ammonium can be offered. And once ammonium has been offered, you can record in real time the utilization of oxygen. So the organism starts to respire, starts to nitrify, consumes oxygen and this can be recorded in real time. And then we know the stoichiometry of complete nitrification. So two molecules of both two are used per oxidized ammonia two nitrate. And based on that stoichiometry and based on the oxygen consumption curve, we can calculate the kinetic curve of the complete nitrification that is a straightforward thing to do. And here we have such a result for a micro of a microstereometry experiment. Here we see on the x-axis, the total ammonia and ammonium concentration on the y-axis, the ammonium oxidation rate that was calculated from the microstereometry experiment. We see this plot is a typical Michaelis-Menton curve. It is important to note here that this Michaelis-Menton kinetics was not quantified from isolated enzymes, but this is a whole cell experiment. So the value we get here for affinity is not the classical KM affinity constant, but it is the KM for whole cells and we call that the apparent KM. And from such a kinetic experiment we can derive, including the replicates of course, that the whole cell affinity of comamox is very high. The KM value is very low. Please remember, a high affinity means a low KM value and that is only 63 nanomolars of ammonia. And on the next slide, you will see a comparison of comamox affinity to the affinity of other ammonium oxidizers for some reason going forward. This slide is not working again. Now not even with a switch down here. Now it worked with a keyboard, interesting. Okay, so here we have the next slide where we see a comparison of the affinities. On the left side of this plot we see ammonia oxidizing archaea, AOA. Then we have nitrospera with nitrospera in opinata currently is the only representative in pure culture. And here we see ammonia oxidizing bacteria. And what we see here is please note that this KM y-axis is a logarithmic axis. So apparently small differences are in reality very large. And we see that nitrospera in opinata has a very low KM value, which is lower than the values of all known or tested ammonia oxidizing bacteria so far and of terrestrial ammonia oxidizing archaea and only some marine AOA strains have a lower KM value meaning a higher affinity for ammonia. So nitrospera in opinata must be highly competitive at a very low ammonia concentrations, more competitive than terrestrial AOB and AOA. But that's not the only interesting feature because Coma Mox also turned out to have a higher yield than the other nitrifiers. Yield means the milligram of biomass here as a proxy we use total protein produced per mole ammonia oxidized until you see nitrospera in opinata yield is higher than that of AOA and AOB. At the same time the growth rate is lower. Actually the maximal growth rate is slower than that of the other nitrifiers especially compared to AOB like nitrosomonas-europia which can grow much faster. From that we conclude that Coma Mox is a so-called yield strategist. I will come back to this point a little later because it seems to be optimized for having a high yield but a slow growth rate. Then based on the kinetic curves you see that there is a relatively small window of opportunity for Coma Mox that can outcomplete other ammonia oxidizers. Here we see the red curve is again the kinetic curve of nitrospera in opinata. The blue curve is nitrosophiric agensis which is the terrestrial ammonia oxidizing archaeon and the black one is a terrestrial ammonia oxidizing bacterium nitrosophiral species. And we see that the affinity of Coma Mox is better than the affinity of all the others but there's a point when the ammonium concentration gets high enough that the other organisms show their higher rate of turnover that means they can grow faster. And in this area they may already be able to outcomplete Coma Mox. So Coma Mox would only be competitive here at very low substrate concentrations under extremely oligotrophic conditions. Actually that's an interesting point but it's not the whole story because now the next important issue comes into play which is the feature of Coma Mox to be a yield strategist. Just imagine please if organisms live a planktonic lifestyle like on the left side here it is important for them to capture substrate as quickly as possible because the neighbor is likely a different species and capturing substrate fast means just get it and don't give it for the neighbor for the competitor. So a planktonic lifestyle selects for organisms which are fast they can have a high affinity if the substrate concentration is low but at the same time they must be fast enough. In contrast imagine lifestyle in a biofilm. There we have cell aggregates and the diffusion limitation into the EPS and the biofilm so substrates even if the ambient concentration is high the substrate influx is limited. So in the biofilm we have a slow influx of substrate and that means the organisms also must have a high affinity in order to capture that little substrate but at the same time they can afford to be slow because if they leave substrate for the neighbors the neighbors likely their own clone will aggregate so they support the neighbors and having a high yield means the organism can form a lot of biomass from little substrate which is a highly economic metabolism and that also helps the neighbor because every substrate not used is available to the neighbor themselves. So a yield strategy in ComaMox selects for life in biofilms which we often find in wastewater treatment systems and this is of course very important for engineer applications and it's not a big surprise that actually a nitrous pyra is a ComaMox because nitrous pyra also the nitride oxidizers are very well biofilm forming organisms as you can see in this picture that form big cell aggregates in biofilms. So the high affinity also means that nitrogen removal by ComaMox by nitrification is efficient but before we can make general conclusions here we need more kinetic data because so far only two ComaMox strains have been kinetically characterized in addition to nitrous pyra in opinata nitrous pyra kräftii that was analyzed by Sebastian Lücker's group in Nijmegen this is an enrichment culture and it's turned out that both of them have a very high affinity for ammonia but we see already differences for nitride because nitrous pyra in opinata has a very poor affinity for nitride whereas kräftii has an affinity that is comparable to the high affinity of the nitride oxidizing nitrous pyra in the canonical nitride oxidizing nitrous pyra species indicating that in order to get a more valid more general view of ComaMox kinetics we do definitely need much more kinetic data from different ComaMox organisms. Okay, in the last part of my talk I will briefly address another important issue which is greenhouse gas emission. Nitrous oxide is the third most abundant greenhouse gas in the atmosphere and also the dominant ozone-depleting substance in the atmosphere nowadays and roughly 50% of nitrous oxide emissions are from anthropogenic sources mostly from agricultural soils and smaller part from wastewater treatment plants and biologically speaking the main sources of nitrous oxide are denitrification, nitrification and abiotic processes which are partly linked to nitrification and denitrification directly. And here we have a simplified overview of how nitrous oxide can be formed in the context of nitrification. So the green arrows here are the normal nitrification process from ammonia via hydroxylamine, nitric oxide as known intermediates and then nitrite is formed and finally nitrate. However, when oxygen is depleted many nitrifiers are able to do the so-called nitrifier denitrification where they actually reduce nitrate back to nitrite to nitric oxide and finally to nitrous oxide which then is emitted into the atmosphere. Under oxygen depletion it's also possible that hydroxylamine accumulates in their metabolism but hydroxylamine is toxic and must then be detoxified and one of these detoxification pathways also directly leads to nitrous oxide. And finally, if hydroxylamine or NO leave the cell and enter the surrounding environment there may be abiotic inorganic chemical conversions of these compounds into nitrous oxide. So we have multiple pathways here leading in nitrifying organisms to nitrous oxide as a byproduct, a greenhouse gas, which is of course something we would ideally like to avoid at least in agriculture and in technical systems. Now what about NO production by ComaMux? We found also in a micro spirometry experiment where we observed oxygen use and NO production with different micro sensors. That's why the organism is actively respiring oxygen. Yeah, this is the dotted curve here. And it produces some NO but this NO is quickly consumed again and even when oxygen is gone, this NO, there's no visible net NO production anymore. This is a big contrast to other ammonia oxidizers like the AOB Nitrosomonas Europea which starts to make a lot of NO by nitrifying denitrification especially under hypoxic conditions. All the AOA Nitrosocera DNNs which also produces NO by ComaMux briefly during nitrification but then makes more NO under hypoxic conditions later on. This is not the case in ComaMux and with N2O we also have a very interesting situation here. There's no net N2O production by ComaMux during nitrification and only about 30 minutes after hypoxia set in, you see some little N2O produced which is much less than the N2O production of AOB. You see here much higher activity leading to N2O and also less than in the AOA Nitrosocera DNNs in this case. So we apparently have less greenhouse gas production and the source of the N2O in ComaMux can be determined by looking at the distribution of the natural distribution of 15N in the N2O molecule. Here we have an alpha in the data nitrogen atom in N2O and one can measure the natural distribution of 15N at these different positions and calculate the so-called site preference which is the difference of the delta 15N values at the two positions. And it is known that the heterotrophic denitrification and nitrified denitrification have a site preference of zero per mil whereas inorganic in the hydroxylamine conversion to N2O nitrous oxide usually has values around 30 per mil and this is also the case in ComaMux. From this we conclude that the N2O source in ComaMux is not enzymatically catalyzed but this is inorganic conversion of hydroxylamine to N2O. And then when we look at the actual N2O yields of ComaMux we see that under ammonia limited or oxygen limited conditions they always make very low amounts of N2O per oxidized ammonia whereas other nitrifiers especially AOB make much more especially AOB and the hypoxic conditions make about 10 times more into O than ComaMux and AOA are in a comparable range. So they are also quite beneficial in terms of small greenhouse gas emissions but AOA do hardly occur in place for the treatment plants for example. So ComaMux would be an organism that should be like a green microbe should be from kinetic viewpoints and from greenhouse gas emission viewpoints that are beneficial in engineered systems. Yeah, at the end just a brief outlook in physiology we need biomass for doing these experiments also to study the structures of the ComaMux enzymes like ammonia monoxygenase for example. This has always been a huge bottleneck because biomass production is difficult these organisms grow very slowly and we recently developed a protocol together with collaboration partners here in Vienna and in Hungary to cultivate nitrospera enopinata at a 200 liter scale and that means we can get immense amounts of biomass compared to previous times and here we see Chris and Johanna from our group with a bottle of highly concentrated nitrospera enopinata and this pink color are the cytochromes that are very abundant and this will be the basis for future ComaMux research in our group where we aim to learn more about the physiology and the biochemistry of this fascinating organism. At the end, I would like to thank everyone who has contributed to our studies in Vienna, in Alborg of course also at the Videogradzky Institute with Elena Lebedeva who brought us the primary enrichment and also at Rappau University in Nijmegen with Naiket Mansivastia Luka who are good outstanding collaboration partners and thank you very much for your attention. Thank you so much Holger. Outstanding presentation and introduction to the physiology and the states of different organisms as well as ComaMux. Our next speaker is Yasek Makinia. I've known obviously Yasek for many, many years maybe well over 15 as he's been involved with a nutrient removal and recovery specialist group for quite some time. Yasek will help us develop the models and the model development for the ComaMux process based on some of the work I assume being done by Holger and Sebastian and others. So Yasek, why don't you take it from there? Thank you Sudir for a kind introduction. Good afternoon or good morning, good evening depending where you are. It's my great pleasure to attend this webinar and present our research on modeling ComaMux process. So let's test the slide movement. I cannot move my slide. Yes, so the main points of my presentations are conceptualization of the ComaMux model with three possible scenarios then comparison of kinetic parameters in general for nitrous pyra and specifically for ComaMux bacteria. I have seen some questions about actually kinetics of nitrous pyra already in the Q&A box. Then integration of ComaMux into an extended activated sludge model including two step nitrification and heterotrophic denitrification. Then something about the impact of initial biomass concentrations and kinetic parameters of nitrifiers on model predictions and then assessment of the ComaMux contribution to the nitrogen conversions. Next slide please. Yes, but before I come to my main topic of presentation I would like to start with two golden rules of modeling. The first says that no model is perfect, some are useful and the second a model should be as simple as possible and only as complex as needed. It means that a perfect model does not exist and it's always a simplification of reality and the extent of simplification depends on the intended use. So the best model would be the simplest model that could still help understand the system behavior. The next slide please. Yes, so the first fundamental question that comes is can we integrate ComaMux in the state of the art activated sludge models known for more than 30 years? And the single answer is yes because in those models actually nitrification is modeled as a one step process as a direct oxidation of ammonia to nitrate. Next slide please. But we are in the novel nitrogen removal processes based on nitrate accumulation. We were more interested in two step nitrification models and we found in the literature almost 40 such models in recent 30 years and ComaMux could be integrated with those models actually in three ways as shown here. In model one, we have a direct oxidation of ammonia to nitrate. Then in model two, we have a sequential oxidation of ammonia via nitrite and basically ComaMux bacteria play the same role as two groups of canonical nitrifiers. And in model three, we have parallel oxidation of ammonia and nitrate to nitrate. So we implemented those models in a simulation platform GPS6 using a special utility called model developer. Next slide please. So we should also remember that NOB are not a one group as it was already mentioned here. So on one side, on one hand, we have our strategies represented by nitrobacter and case strategies represented by nitrospira and the advantage of the dominance of case strategies is under a low substrate concentrations as it is shown in the graph below. And those conditions are typical for mainstream bioreactors. Next slide. So if we look at the range of kinetic parameters for nitrospira, we have found them in recent publications. So indeed those ranges confirm that nitrospira can be considered as a case strategist. But when we look at the ComaMux bacteria, the data of course are hardly available, are very limited, but we can see quite a lot of similarities except of course for the affinity constant for ammonia, which does not exist for canonical NOB and very high possibility of very high affinity constant for nitride. Next slide please. So in our study, we have run several washout experiments under laboratory conditions, decreasing the solitary retention time from four days to one day under different temperatures using different nitrogen sources, only ammonia or only nitriding the feed. And the dissolved oxygen concentration was kept at a relatively low level at 0.6 milligrams per liter. Next slide please. So in the preliminary study, we compare those three model concepts and the simulation results were pretty similar for those three models. In terms of the nitrogen species, ammonia, nitrate, nitride, and also biomass concentration. But the difference comes inside. The next slide please. So we use the Sankey graphs to show the nitrogen conversion pathways for different microbial groups mediated those processes. And as you can see the role of ComaMux would change depending on the model concept which is used. The next slide please. During our studies, we observed that there was a high heterotrophic activity in our system. So that's why we extended our model with heterotrophic denitrification on soluble microbial products as there was no organic carbon in the feed. And for the decay of biomass, we use the death regeneration concept applied in the activated sludge model number one. So the next slide please. We used a typical modeling procedure with a couple of steps. This is less important this next slide. And again, we obtained pretty good results in terms of predictions. Also for the experiments with nitrite in the feed. Also for nitrogen species and biomass concentrations. The next slide. But the unique feature of our study was that we also compared the ratios of different microbial groups including NOB to AOB and nitrifiers to heterotrophs. And in the experiments with ammonia, you can see that the NOB to AOB ratios were pretty stable in the course of experiments with the values below one, which is quite typical for mainstream bioreactors, while the ratios of nitrifiers to heterotrophs were very low and even decreasing at the end of experiments. Next slide. For ComaMox, we use the relative abundance approach, which might be questioned, but from the modeling point of view, it provides valuable information. So we could model this parameter. And it's quite interesting that in the experiments with NO2, which is shown on the right side, we observed some activity, at least we observed some activity of ComaMox and it was confirmed also by the model as the model without considering the ComaMox growth worse than the simulation results. The next slide, please. So in terms of the importance for calibration, the ComaMox vaccine growth rate and ComaMox biomass concentration are less important for model calibration than other nitrifiers, especially AOB. Next slide, please. And again, building synchrographs for nitrogen conversions at different stages of the experiments at the beginning in the middle phase and in the end of the experiment, we can see rearrangements of the relative contributions of the different groups of bacteria for canonical NOB and ComaMox at the steady decreasing trend was observed for AOB increase in the middle phase and then decreasing at the end while for heterotroph, denitrifying heterotrophs a steady increasing relative contribution was observed. Next slide, please. So in summary, we can say that integration of ComaMox in two-step nitrification models is not very difficult. The problems come when we start modeling N2O and this multi-step nitrification. The model involving both ammonia and nitrite conversions would be recommended as most more flexible than others. And this nitritation step could be switched off easily There are few challenges. The growth of ComaMox bacteria on nitrite and preferable substrate ammonia versus nitrite. And the initial concentrations of ComaMox bacteria should get some more attention. Also we learned very little about kinetics and stoichiometric parameters, especially in mixed cultures. And from our study, it seems that the role of ComaMox in nitrogen conversions should not be neglected, but it requires further investigation as the content of nitrospirals pretty low in the biomass from the sludge samples. Okay, the next slide. This is a related publication to our study. The next slide. Acknowledgements for the project support. Next slide. So with this, I would like to thank you for your attention creating from Gdańsk University of Technology. And if you wanted to visit this beautiful building, we organized another IWA conference at the end of October. This will be a specialty conference on agro-waste. Thank you. Thank you so much, Essek, for your presentation on the modeling of ComaMox and its possible relevance to our applications in water. Our next speaker, and we're running a little late, so I'll go a little bit faster. Our next speaker is Jianhua Guo. He's going to be speaking about urea-based ComaMox and especially the nitrospira and their potential applications. Jianhua. Thank you for your kind introduction, Sadeer. It's also my great honor to present what we have been doing in ComaMox related research. So today, I particularly focus on urea-based ComaMox, nitrospira, and their potential applications. I'm not sure I can move or not. Yes, please, next page. Oh, you can transfer to the controller to me, like Isabella, I can try to control the slides. As we know, usually for the two-step phone nitrification is driven by like, you know, AOB or AOA together with NOB. So actually, we also have a very, very simple, like organic nitrogen source in the all the earth, so is, which is the urea. So they just have one carbon and the two nitrogen. So it's very simple, like organic nitrogen form. So usually, like, you know, like, you know, that many heterotrophic bacteria can convert urea into the ammonia through the ammonification. But now our question is, are there any organisms who are able to convert urea into the nitrate independently? If so, who are they? And who are they? And how about the pathway? So this is the question we want to address. So actually, so recently, we have very, very, like, interest in phenomena lab, actually also is an extant, so based on our collaboration with Tsinghua University. So we run the one membrane biorector, is we call them NBR, like with work volume, 12 liters. For these reactors, we feed, like, you know, the source of pretty urea, like this rare waste water collected from toilet. So actually, we just want to, like, convert all ammonia into nitrate. So not our aim is not to enrich comal. So that's why we control the DO, maintenance DO to the full PPM. And for the infant, we'll feed, like, you know, around 118 to, like, 215, like, total nitrogen. So in that feeding, depending on the story time, so we have urea concentration around 10 to, like, 15 PPM. So we run these reactors. So let's have a look at the performance. So actually, so initially that we have ammonia left, you know, our reactor effluent, but after one-handed operation. So all ammonia can, all the organic nitrogen, ammonia, or urea feeding on the reactor will be completely converted into nitrate. So majority will become nitrate. So we don't have a nitrate combination. So ammonia concentration in the effluent also is a very, very low level. It's around, like, an earth, one PPM, or even lower. So then, we run the 60-SRI gene sequencing. We want to understand, so do we have AOB or which AOB or NOB are doing for nitrification? So actually we found a very, like, interesting phenomena. So you can see this is the red trigger is AOB nitrosolamos. So at the beginning, we have 1% relative abundance of AOB. But after 115 days operation, all NB disappeared. So correspondingly, for the nitrous sparer, like, you know, this is like a blue circle. So abundance increase from the 5% up to the 13% after 300 days operation. So this is very strange because we have full nitrification, but we don't have a normal or typical AOB. So that's why this trigger us to ask, is that possible we enrich almost nitrous sparer? So then we can do the ammonia oxidation into the nitrate eventually. So we did a few validation experiments. So first experiment is a QP cell. So we're using like a reported primer to target abundance of MOA gene carried by the comos nitrous sparer AOB and AOA. So based on the like a QP cell data we show here, you can say AOA MOA gene abundance is very, really low. And for the comos, the copy number of the MOA gene, actually three order of the magnitude high, there is AOB like MOA genes. That means probably we have the enriched comos nitrous sparer instead of like a typical AOB or AOB in our system. This is true or not, we further demonstrate based on the metagenomic sequencing. So we collect samples from the day 101, 118 or 216 and even the more than 300. We have a three DNA samples. We run the metagenomic sequence. So after the DNA sampling and the BNIN, in total we require the three like comos nitrous sparer bins. So I just show the pink color here. All three recover the genome actually belong or classed with the clade A comos nitrous sparer. So this is the bank one, the gray bank one is all like a clade A comos nitrous sparer. So blue color shows like a clade B comos nitrous sparer. So we also compare the other genome with other typical comos nitrous sparer in terms of an amino acid identity. So we found all three recover the comos nitrous sparer for the AAI percentage is less 85%. That means, so luckily we have enread some new comos nitrous sparer. So it's totally different with already reported in the literature. So we also quantify like calculate the right to abundance beta metagenomic sequence data. So we found at the beginning we have the first BN1 comos at the rate up to the 15%. We also have like a normal NOB but with increased operation time. So NOB abundance, typical NOB abundance like a decrease and decrease. We have more and more like comos nitrous sparer B1 and we also have B2 and B3 popping up in our system. So then we did the gene annotation and the construct like a genome structure feature of this comos, three comos. Very interesting. We also found these three comos nitrous sparer. They carry the URT and URE gene. So they also carry definitely they carry the AMO, AMO-A-B-C-H-A-O and IXR gene. So that means this comos nitrous sparer you reached in the MBR feeding with URE. Actually they are able to transport URE from outside into the inside of the cell. Then URE will be further degraded into the ammonia through the ammonification pathway. After that ammonia will be sequentially octahed into the nitride, then to the nitrate. So that means this comos nitrous sparer they have a super capability. They can even utilize URE. So is that true or not? We did a like a small bad test. We collected biomass from that big reactor and just a dozen 14% or 14 ppm URE are used in the infant. So we monitor the URE conversion and the ammonia and the nitrate production. So you can say once we feed the URE, we'll be octahed into the nitrate. At the beginning we also have a little bit ammonia accumulation. After that both ammonia and the URE will be totally converted in your nitrate in the end. So as I mentioned before, for this 3G loan, so actually they carried all the relative genes like regarding URE utilization pathway genes including URE, URT, A, B, C, B, E subunits. Also they have a URE, A, B, C, B, F, J subunit gene in that 3G loans. That means this comos nitrous sparer they can do the URE conversion into ammonia then can convert ammonia into nitrate eventually. But this paper has been published on the Izmi communication. If you're interested, you can check out the details in the later stage. So now you will be challenging me, okay, this phenomenon is unique or is universal. Can you prove this phenomenon be further reproduced or not? So after that we run the two more reactors. We restart the new two new reactors. So still the MBR reactor. The work volume is two liter. So all conditions are same except the feeding nitrate source are different. For the reactor one is also our control reactor. We feed the 118 ppm ammonia, just ammonia only. But for the reactor two, we feed 100 ppm in URE. So all the conditions DO is the same, four and five. We didn't control DO to the low level. It's a high DO condition for the HRT is three days. So we slowly feed the URE ammonia to the reactor one and the reactor two. So what happened? This is a reactor performance. You can say no matter is a ammonia and the URE feeding reactor. So the performance is roughly same. So after one month or 15 days, so all ammonia reactor reached state to state. So all ammonia will be converted into the nitrate in reactor one. In reactor two, all feed like URE will be oxidized into the nitrate as well. So we also compare the ammonia in effluent. So after like day 15, the ammonia concentration extremely low for both reactor. So around the majority is less one ppm, even though they're all put one like a milligram per liter for ammonia concentrate, residue ammonia concentration. So then we run like a KPSer again. So for the reactor one, so feed with the ammonia, you can say we have a mixture of AOB and AOA and even the COMOs nitrospera. So AOB like it's in terms of MOA gene copy numbers, AOB looks dominate in our system one, but in system two feed with UREA. So you can say COMOs MOA gene is much more dominate over other two organisms. They have more abundant MOA gene carried by the COMOs. That means probably once again, we reach COMOs nitrospera in our URE feed rectors. So for the middle bar chart is based on sequence. So again, for the reactor one, we have AOB, typical AOB. We also have a nitrate vector as another typical NOB. So but for the reactor B, so after 115th operation, all the typical AOB disappeared. So iteratively just nitrospera left, but based on sequence, we can't distinguish the COMOs nitrospera or not. So then we import like the fish and the metadromes. So now I just show the fish dot image here. So this is a reactor two like a fish image. So you can say this white color is the COMOs like a nitrospera. So it's much more dominate compared to the typical NOB, this blue color shoots here. So we have a very abundant COMOs nitrospera in reach again in our URE feed reactor. But for the ammonia feeding reactor, we have a mixture of AOB, COMOs and AOA. So now we also collect the data from the DNA sequencing and RNA sequencing. We are wrong in metadromes sequencing or metadromes and metatransforms data analysis. The project is ongoing. But once again, we use the artificial wastewater. Why is ammonia? Why is the urea? So we enrich like a COMOs nitrospera again. So now last question I wanna address. So how could we apply COMOs nitrospera in our wastewater or water treatment system? Do we have any potential application scenario or situation? So as mentioned by the holder, actually COMOs nitrospera, they are green like a marketplace. They have multiple advantages. So here are at least a few. The first one, they generate less until like emission. Second one, because they have a high affinity towards ammonia. That means we can import COMOs to remove ammonia into very, very extremely low level. The third one, because AMO enzyme, they have a co-metabolic pathway. That means together with ammonia, they even can degrade like an organic marketplace. So that's why I think in the future, probably we can explore the application of COMOs. But on the other hand, we do have challenges or barriers to apply the COMOs in our water system. So what are the challenges we are facing? So first one, they are grown very, very slowly. As mentioned, they are key strategy organisms. The second one is still not clear how to enrich COMOs activity. The third one, so which scenario we can have to apply COMOs nitrospera. So here, I will just share my rough idea like what we are doing now. So we are thinking, so can we use another membrane, different membrane reactor? We call the membrane aerated biofilm reactor. So why we want to use this system? So because we can deliver oxygen through the holding fiber membrane, then biofilm will build up on the surface of the holding fiber. So once we have oxygen diffusion or permeate from inner side to outside membrane, so then biofilm will are swollen or eating oxygen as soon as possible. So then we have a high gas transfer efficiency. So then we can save aeration consumption. The secondly, like we can decouple HRT and SRT because nitrospera, COMOs nitrospera is a slow growing bacteria. So if we can have very, very long SRT, so potentially we can enrich or we can keep our COMOs nitrospera in our biofilm system very well. So then we can address first challenge. Okay, so now how can we apply COMOs? What we want to propose because based on our preliminary funding, so is that possible we can apply COMOs to treat the urea with water, treat it like, you know, the source of separated urea because we have a high urea in that like a source separated with water. We also have a high concentration micropretent like due to the urea discharge. So if we can run the MABR system, if we can enrich COMOs nitrospera in our biofilm lectures, in that case, probably we can achieve two goals. One is we can achieve very high, very low concentration ammonia in the effluent. So secondly, simultaneously we can degrade it or we can convert like a micropretent in some no toxic compound. So in that case, we can address the second change. We find a scenario, we can apply urea-based, urea-based like COMOs to treat our with water. So what we are doing now, we start up like such experiment. So for the time being, we just re-enriched the AOB and the typical AOB and NOB in our system because we feed only ammonia, we didn't feed urea, we didn't feed urea. So we also supply one very, very typical antibiotics, CFX. So we run the MABR system. So this is the sickness data. You can see we have a nitrous solomons, we have nitrous spera. So it's an AOB, NOB plus COMOs enriching system. So we feed ammonia and antibiotics, like based on this figure, you can say we have very good four-nitrification performance and if we feed 100 micrograms like CFX in infant, so we can achieve a more than 16% antibiotic removal efficiency. So we also did a batch test to confirm this is still the COMO tablet for the pathway by the AOB. So because if we only supply the antibiotics, the removal efficiency or rate is extremely low. That means in rich naturophanic sludge, they are not able to convert, like consume the CFX antibiotics without ammonia. If you applied nitride and antibiotics, the rate also is very, very slow. But if you supply both ammonia and antibiotics, the antibiotic removal efficiency is much higher, rate is much higher. That means AOB or COMOs, they do have a co-metabolical pathway to degrade microplutin from wastewater. So with this, I would acknowledge my team members from university Queensland, I also would like to thank my collaborators from Tsinghua and Robert University, Lehmeheng, Sebastian, Mark Yatien and one of the collaborators from UTS University of South Ethnology in China, Zheng Shuang. So thank you very much for your listening. I'm waiting to address any questions you have. Thank you. Thank you, Jinghua. I would like for all of the presenters to show their video and maybe what we can do because we have only about five or six minutes, what I'd like to do is for, you know, I'll start with Sebastian, but to have each of you perhaps respond to any Q and A that would be better done verbally than having done using a text message. So please start starting with Sebastian, give each of you two minutes to respond, maybe the question and then a response to it. Yes, thanks. I think there's not one specific question I would like to answer. It's more something that appeared in different questions. How can we select for common mocks during enrichment and then also in full-scale systems? We just, I try to answer it also, it's probably not that easy and straightforward and this is something we're still trying to understand because of course one factor to select for common mocks is low ammonia concentrations. Yes, definitely true, but there are additional factors because of course you also have AOA that have high affinities and you also have at least in full-scale systems in nature even if you might not have them in culture, you have AOB that also have high affinities for ammonia. So oxygen seems to go into it, but also there are different reports in some systems, oxygen has not an influence on the abundance of common mocks in others common mocks is enriched, especially on the low oxygen concentrations. Definitely the biomass retention has an influence. So you need biofilm systems in longer retention times that is definitely an effect or a factor that plays into it. But for everything else, we're still investigating it. So as you know I showed urea can help to select. Maybe pH can sometimes but that is something we haven't really seen. But if you look at paper and soil, you see that it's like the acidic pH is clay to become mocks. For example, sometimes it's dominant. So I think that the main message here is unfortunately I would love to give an easy answer to, this is how you enrich common mocks, but it's definitely not that easy because there seem to be multiple factors that sort of play into it. Thank you, great response Sebastian. Holger, how about you? Yeah, along the same lines I have seen a number of questions about the interactions and competition between common mocks and canonical nitrifyers. And yeah, here I can also only mainly say we are still working on this, but common mocks are apparently primarily biofilm organisms. They are very, very adapted to a situation in a biofilm where the substrate influx is slow. Here they can really outcompete other nitrifyers, even those which have a high affinity as well for the substrates because common mocks as a yield strategist has a clear advantage in such a biofilm system. Thinking about granules that may also be an interesting context. So because here we also have a relatively thick layers of biomass with limited substrate influx. And based on these considerations, it might be possible to develop systems that enrich for common mocks over canonical nitrifyers, but still I believe that in a complex system it will certainly always be a complex community containing both canonical nitrifyers and common mocks. And of course, the art will then be to support the activity of common mocks in order to reduce, for example, the greenhouse gas emissions, but more in agricultural soils perhaps than in wastewater treatment plants. Because the end to all issue is much bigger in agriculture than in wastewater treatment as far as I know. And Holger, when you talk about yield strategist, did you measure yield as in both the number of organisms that are yielded or more as in terms of mass? In terms of mass using total protein as a proxy, because actually weighting the biomass for the little amounts of biomass we have in common mocks and other nitrifyers is very difficult. And so the yield, is that really related to the opportunity of it going all the way from ammonia to nitrate, the energy available for that reaction increases the potential for the yield? I think it does, but that is probably or certainly not the only factor here. And the yield is also not a constant. It will certainly change with environmental conditions. For example, if there's so much oxygen that stress response becomes more and more important this will also use a lot of energy and reduce the yield of the organism. And the CO2 fixation pathway is also important. Here nitrospera have a good starting position because the RTCA cycle they use is energetically cheaper than the Kelvin cycle that is used by AOB and nitrovector, for example. Very interesting, thank you. Jacek, can you reflect a little bit on some of the strategy questions that were asked in the Q&A and how you would apply perhaps in terms of relevance to modeling? Yes, there were a couple of questions concerning kinetic parameters. And as my colleagues already answered it's still under investigation. So basically we need to distinguish between pure cultures and mixed cultures because a lot of more processes are going in mixed cultures. And those interrelationships are more difficult to follow. With regard to the maximum growth rate it seems at least from our experiments that it's not so low because we run those experiments at very low SRTs. So we are decreasing very aggressively SRT from four days to one day and those bacteria could still be present at the end of the experiment. So definitely we need to verify the maximum growth rate of those. Bacteria concerning the half saturation coefficients and the preference of the substrate looks like it is the ammonia is preferred and still the use of nitrite is under investigation because there are some studies showing that bacteria can grow, some studies that not. So this should be also clarified and models could help with this. Thank you, Jasek. And finally, Jianhua what would you reflect on some of the process engineering opportunities? I know UQ and you have specifically been working a lot on process engineering and certainly you looked at URIA, what are the other opportunities you see? And what I found that was interesting with Sebastian's presentation was the co-use of Anamox and Comomox, what would you think are all of those opportunities for these water related processes? Yeah, thanks for your question. So I need to make apologize for all these questions in the chat box. I can't have time to type my question but I try to make a very short summary. Actually, like, you know, there are some like a new study, very recent study, try to explore the application to cover both Comomox with animal bacteria to achieve autotrophic nitrogen removal from wastewater. Like some study published on paper is a very early state study but based on this data, they said, okay, until generation emission is quite low compared to PNA based on AUB and animals because Comomox generates an unto emission. This is one advantage. Second one, so because Comomox they have a high affinity towards ammonia even like oxygen probably we can, it's much more easier to suppress the typical NOB if we combine like Comomox and animals. In that case, if we have no NOB in our system, nitrogen generation in our PNA system could be less compared to like AUB and animal system. So this is another advantage. Third one, so no matter it's AUB or AOA or even Comomox, I mentioned that they have a co-metabolic pathway to degrade the macro pollutants like pharmaceutical or personal care products. So then if we can combine like Comomox, animals potentially we can also like remove a macro pollutant from our wastewater or from our toilet like a U-ray like system. So now I would promote like because in terms of application I think for the U-ray-based Comomox that could more relevant for the de-centralized the treatment process run at full-scale like large-scale or like centralized system because usually for the full-scale large-scale like in our wastewater treatment plant ammonia concentration is low, is very high. So probably this is like the AUB and typical NOB rather than Comomox. But if we can control ammonia in a very low level in the biofirm system for some small scale or decentralized system so probably we can employ the Comomox together with animals even use the MABR because they can save energy for the aeration. So we can achieve like a sustainable like natural removal from wastewater. Thank you so much. Great big picture prospectors, Jeanne-Ola. Isabella, do we have time for Jeanne-Ola to conclude with a few of his remarks at the end? Yeah, yes. Maybe Jeanne-Ola, if you could spend two or three minutes until the, you know. Yeah, thank, I would make a short like acknowledgement. So first I would thank like the IWA and particularly for the NRR Spanish group like support and coordinate such great like a regular or other like Latin circle or NRR community. I very appreciate. Secondly, I would appreciate like all the speakers for your great talk today. And I very appreciate your time to make such big effort to make such event happen. So third, last but not least, I would thank every like audience from globally like I understand that we do have some audience sitting like in Asian country they are very, very late already. I very appreciate that your patient like to attend that seminar. So I hope, so this such webinar could be a trigger for the future study. So we are looking forward more and more like a fascinating basic size, but also more and more like opportunity for to explore like application of commons in the future. Thank you very much. I'm not sure that we should talk about what professional like late works on our Isabella. Go ahead quickly, very quickly. Yeah, we do have like, you know them, what professional like, you know the registration like probably like we can post on the website. If you're interested, if you're young, what professional please take that registration as possible. We offer very good discount now. Yeah. Isabella, do you have anything else to add on the YWP discounts and the membership in IWA? Yes, so here we are putting the screen again, the share presentation just a second. I think it's on the final slide. And while it's on the final slide, I would also like to thank the IWA headquarters. It's just outstanding amount of effort and close into putting out a webinar and Isabella certainly has been hugely important to all of this and making it all happen. Thank you so much. This is something I'm mentioning is what professional like you can take the photo for this PPT like using that code, you can get a 20% discount for what professional it works. Well, thank you. Thank you, Jinwa and thank you all of the panelists and speakers, it's really enlightening to bring all of these different concepts into one panel. So thank you and hope everyone has a good day. Goodbye, everyone.