 Well, thank you for this opportunity to pay tribute to Dave Stenzo. It really is an honor for me to talk about such a great man. Next slide, please. What I'd like to do is to tell you about my history with Dave Stenzo, how I got to know him, how he worked together, and leading up to the last time I communicated with him and his passion on the subject that we are talking about. We recently had a tribute to Dave Stenzo, of which I've taken one slide here. In this tribute, we made note of his tremendous achievement over all these years. And he got his PhD from Cornell University in 1971. And next slide, please. I also met him in 1972 for the first time. And that was early into his career at a pilot plant. He was working for Angkor at the time. And Angkor decided to take up the pilot plant from the Institute in South Africa. Now, I traveled to Salt Lake City. Dave picked me up at the airport. Took me to his house, which set the patent for the next 40 years. Later, we traveled to the USA to promote the concept and finally we designed the first plant in Calmetta, Florida. And later, we had many opportunities to work together, including defining the first book on being an arm with Dr. Randall as editor, 10 years on the New York City Advisory Committee courses, and so forth. Next slide, please. The same year that I met Dave Stenzo, I discovered biological phosphorus removal in this pilot plant in South Africa. And what I found was this release of phosphorus. But what was really happening was there was a dead zone connected to this second zone. Next slide. And this is a diagram of what that pilot plant looked like. And there was a dead zone, but it was connected to the second and not to the third. And that served as a mentor. And the results was extremely good. It was just a four-stage plan for nitrogen removal. But the past was coming in in 1910, and the Grand Polytechnic was carrying out a lesson from the Grand Polytechnic for the next slide, please. That led then to these configurations that was where we put the anaerobic zone in France. And on the right-hand side, you see a plant that was built on the site. The pilot plant, but we didn't nearly achieve the same result as we did in the pilot plant. Next slide, please. That led to this construction of a number of large scale plants in the U.S. in South Africa. And eventually, next slide, please, the uncle in Salt Lake City decided to take up the plant for patent. I traveled to the Lake City as I said, worked with Dave Central. And we designed to build this first plant in our Metro Florida to achieve the total nitrogen in the three-milligram per liter. And that would be a 21-milligram per liter. And that led to a working relationship that lasted for more than 50 years. Next slide, please. And Dave then, uncle also had the license for the carousel plant. And Dave successfully married the water flow in the carousel to form what you see in this slide here. And we're extremely good with the raw materials of nitrogen removal. The simultaneous modification, denatification, that was taking place in the carousel was augmented by the adenopsis plant. And this plant here on the right-hand side produced effluent total nitrogen of less than 1.7-milligram per liter of ideal period. Another one of Dave Central's legacy. Next slide, please. In the meantime, we discovered that we really need volatile fatty acids. And when I designed the two plants in the rich Colombia, I decided that to ferment primary sludge and to augment the volatile fatty acids. But since that was going into the anaerobic zone, we bypassed most of the flow from the primary effluent to the anoxic zone all this time I was discussing it with Dave. Next slide, please. And that led to the design of this plant, where we fermentate from the primary sludge that's going into the anaerobic zone, most of the primary effluent bypassing, until they just shut off the flow to the primary effluent flow to the anaerobic zone. And that led to basically a side stream fermenter, because now we had the turn after it started going back into the anaerobic zone and we fed the fermentate from the primary sludge in there. This plant performed extremely well. Next slide. And let us do the concept that where we decided, well, let's go ahead with this idea of side stream fermentation, which can take on various forms. In one form, it can lead to a simply switching off anaerobic mixtures in the anaerobic zone and get the sludge petal on the element on the floor. And I worked with Dave on a plant like that too. And the other option is to take some of that sludge out of the anaerobic zone and ferment it and put it back in the anaerobic zone. In the meantime, Nam, in North Carolina, patented the process in which he thought some of the return activated sludge and fermented that and put it back in the system. And in Denver, Colorado, we did exactly that, but added some fermentate from the primary sludge. And that really gave extremely good results in the new MBD plant. Next slide, please. This is an example of a plant where in this particular plant, what you see on the right-hand side is the anoxic zone that was added and the anaerobic zone. And this plant worked extremely well because there was a lot of plant, volatile fatty acids in the end, but then they added nitrate to the collection system for odor control. And in that process, they destroyed the volatile fatty acid coming in and the plant stopped removing pastures. So what we did was to switch off a mixer in the anaerobic zone and that led to the effluent from the primary, from the secondary platter part being around 0.1 millicentry pastures. And so far, we have seen great improvement in SPI when applying to precise implementation. I am showing this plant because I want to go on to other of David's sense of discovery. Next slide, please. Dave's Denzel was very interested in granular formation in mainstream plants. And he produced this paper with Professor Marie Brinkler. And the next slide, please. And that plant that I've just shown the Anderson plant, what they found on this slide that I've taken from their paper, it shows the SPI 30 over the SPI 13. That is one of the reasons you have actually got granules instead of actually sludge. And so what do you see the Anderson plant that I've shown producing a tremendous amount of granules? And so I was working with Dave to see what was the reason for this granule formation. Here you see another plant where I work with Dave Sinckel and Tom Coleman. It is a plant in Kashmir, Washington. And you can see on this left-hand side picture, you can actually see the granules that formed. But the granules that formed in this case had filaments growing out of the granules. And my theory is that what happened here is as we went into the flood flow, aeration basin, we had high energy, we sheared off those filaments. And then at the end of the basin, we had selective wasting. We wasted from the top. And when we wasted from the top, we used the plant to form a few granules. On the right-hand side, you see the granules that was formed in that plant that I previously mentioned in Anderson. And this picture is taken by Dave Sinckel. Thank you. Next slide, please. And lastly, I designed this plant in British Columbia. I mean, sorry, in Kansas. And what we have is a on the left-hand side picture here is an L-shaped anaerobic zone. And the side-stream fermenter, where we put 10% of the mixed liquor, fermented it and returned it to the inlet. And then that was followed by an oxyclone and the aeration zone of the carousel. Again, one of the Dave Sinckel magazines. What you see on the middle picture here is the, what it looked like in the carousel system. And what you really see here is the granules with a layer of clear liquid on top. And this was a remarkable performance of this plant that you see on the right-hand side, where the effluent auto phosphorus was below 0.1 milligram to lead to mostly 0.04 milligram to lead to. But it also reduced very clear effluent. And on the right-hand side, you see the granules that was formed in this slide. We basically had fewer granules. And the total suspended solids was like 6 milligrams per liter. And what really saddens me so is that this is the last discussion I had with Dave a week before he died. We were still talking about this granule formation. And Dave, to me, was sounding more next slide. And so in celebrating his legacy next slide, this legacy, I want to say, he was one of the giants at the time. And his work will live on for many generations here. I was privileged to have known him and worked with him most of his professional life. And to me, he was like a sounding board. And I still find myself saying, here I should discuss this with Dave. And his latest work on granulation I had been very well seen in this webinar. Thank you very much. It's really an honor to have the opportunity to participate in this webinar in remembrance of Dave Stensel. What we'd like to do is to share a little bit of the work that we have done as a team to explore the opportunity for denitrifying polyphosphate accumulating organisms, for optimizing carbon usage for both denitrification and phosphorus removal. Really, the focus in this project is on resource efficient, low carbon nitrogen and phosphorus removal. Next slide, please. To give you a bit of background on this project, we're going to be telling you a bit about the work that we did as part of work project NTRY-13R-16, understanding the impacts of low energy and low carbon nitrogen removal technologies on biopea nutrient recovery processes. We had a very large project team, including two universities, about seven utilities, and four engineering consulting firms as well. And with this excellent team of partners, we addressed three different research tracks. We focused on integrating resource sufficient nitrogen removal processes with biological phosphorus removal through the activity of DPIOs that's our focus for today. But we also focused on testing the limits of A-stage high rate biological phosphorus removal, followed by a second stage of B-stage shortcut nitrogen removal, for example, nitrogen animox processes. And in a third track, we focused on implementation of extractive resource recovery of phosphorus from water resource recovery facilities, performing both biological phosphorus and chemical phosphorus removal. And again, we're going to be focusing on this first track, focusing on DPIOs. Next slide, please. But the overall goal of this project, the primary outcome that we targeted was successful full scale implementation, broadly speaking, of carbon and energy efficient nitrogen removal with biological phosphorus removal in water resource recovery facilities with stringent nitrogen and phosphorus criteria across a variety of different geographic locations and process configurations. Next slide, please. So what I would like to tell you about a bit first is some of the work that we did at lab scale, and then we'll progress to some of the work at piloted full scale as well. But starting with some of the lab scale work and what we learned really a bit more on the fundamental side about denitrifying polyphosphate accumulating organism enriched bioprocesses. We did this work in my lab here at Northwestern over a number of years in collaboration with Karthik Chandran and Krista Barbadillo and many other project partners with the overall objective of quantifying kinetics of phosphorus and nitrogen transformations by denitrifying a cumulambacter PAO clades in the presence of different electron acceptors, including nitrite and nitrous oxide. In electron donors, we focus specifically on a couple of different VFAs, acetate and propionate. We also did quite a bit of work to assess propensity and mechanism for nitrous oxide production by DPIOs. I won't have time to go into detail about that today. And we also characterize metabolic pathways and the molecular microbial ecology of this consortium as well. And again, I won't have time to go into detail about that today. Little bit of nuts and bolts about our experimentation. We used a lab-scale reactor of a picture on screen here of this. We operated this for over three years. It's a 12-liter sequencing batch reactor that we cycled between anaerobic and aerobic phases. And the key thing I wanna point out is that these types of enrichment cultures have been used in the literature previously, but the vast majority have focused on anaerobic aerobic cycling, so aerobic phosphorus accumulation or in a limited number of cases, denitrifying polyphosphate with nitrate as the electron acceptor. The unique aspect of this study was that we used nitrite as the electron acceptor, and that's important because that fits into our conception of nitrite as this key intermediate in shortcut nitrogen removal processes. So we're sort of mimicking influence from an upstream nitritation reactor. If you could go to the next slide, please. So what did we learn from this system? Just at a high level, were we actually able to select for our desired functional group, these polyphosphate accumulating organisms, this key microbial class for removal and recovery of phosphorus. We're just looking at a bit of results from DNA sequencing, 16S RNA amplicon sequencing to look at who's present in this reactor. What we found is that over time, very rapidly we selected for on the right hand side of the screen, you can see this orange bar that increases in abundance. That's candidata succumilabacter. So indeed, candidata succumilabacter was enriched from about 2% up to about 50% of the community in about three months. But of course we're interested in the activity, what these accumulabacter are doing. So if you could go to the next slide, please. What we found was that very consistently if we evaluated the activity of this system, we saw a very robust phosphorus uptake under anoxic conditions with nitrite as the electron acceptor. We're just looking at two representative cycles. We could look at hundreds of additional cycles that look relatively similar, but we're just looking at some representative cycles here on the left with propionates as the feed and on the right with acetate as the feed. And in both cases, we're looking at time within the cycle on the X axis and then concentrations of a number of substrates and products on the Y axis. Most important line to track here is the orange one that's phosphate. And you can see that under anaerobic conditions that is released as we would expect in a biological phosphorus removal reactor. And then under anoxic conditions, we see then reuptake of that phosphate with both propionate and acetate as feed. Now, if we look at kinetics of this process and we compare the kinetics of anoxic phosphorus uptake and aerobic phosphorus uptake, the rates are approximately the same. And so the key takeaway broadly speaking from this slide and from this body of work is that we showed that a dedicated anoxic phase in the presence of nitrites, which again is the end product of nitritation key blend to short cut nitrogen removal with an extended SRT, we had about a 20 day SRT in the system, is sufficient to select for a specialized consortia of high rates denitrifying polyphosphate accumulating organisms. We also showed that these organisms had a strong kinetic preference from nitrite over nitrate. Not showing that on screen and some additional data. And we also finally showed that the anoxic phosphorus uptakes were not significantly different in the presence of propionate versus acetate. So not a clear preference for different BFAs in the system. Next slide, please. I won't go into great detail about our molecular microbiology results, but I did just wanna mention that we found what I think is some interesting diversity or microdiversity in PAOs that were present in the system and that may be important in practice as well, that remains to be shown in full scale systems. Using a metagenomic, genome-enabled metagenomics approach we were able to recover near complete genomes for three types of acenolabacter. The first is type 1A, which has been reported in literature previously. The second was more interesting, that's a type 1C. This is the first reported genome for this particular clade of acenolabacter. And the most intriguing one in my view was the discovery of a potentially novel clade of acenolabacter that really hasn't been observed or reported in literature before. This is a clade 1F acenolabacter that enriched over time in a reactor and that we think is putatively adapted to high rate denitrifying phosphorus uptake. So I'll leave our molecular microbiology results at that and I wanna emphasize these are promising and interesting results at lab scale and a highly enriched condition. But of course in this project we're interested in implementation and practice. And so we also wanted to look at pilot and full scale systems as well. And I'll turn the floor over to my partner, Krista Barbadillo, to tell you a bit more about that work. Thank you, George. As George mentioned earlier, we had the benefit on this project of a very broad and distributed project team that included work by a number of utilities at eight plants and testing 12 different configurations. Much of this was at full scale. Some of it was supported by pilot scale experiments. The work also covered a broad number of configurations including several BNR configurations, a plant with an oxidation ditch, an SBR, an integrated fixed film activated sludge shove system plant and also several plants that were operating with hydro cyclones for external physical selection for denser settling particles. All of these experiments that were conducted by the utility research teams were specifically focused on their own utilities goals and addressing issues or planning work that was being done for future decisions and the future projects. However, there was a commonality between all of the tests in that everyone was quite interested in low energy, low carbon, nitrogen and phosphorus removal and taking advantage of different mechanisms to optimize performance and reduce the use of resources. Next slide, please. So the first step really in taking some of the results from the different utilities and from these different experiments and to try to look for some trends in the results and in performance was to take a look at the effluent phosphorus removal performance statistics. So from each experiment or each utility, the effluent phosphorus results during their periods of testing were compiled and 50th percentile, 91.7 percentile and 99 percentile statistics were calculated. And so what we're showing here really is the 50th percentile as a general indicator of typical performance. And then we're looking at the ratio of 91.7 to 50 as kind of a maximum month type of indicator and the ratio of 99.7 to 50 as a maximum day type of indicator. And the reason for this was just to provide a little bit of insight into variability of performance. And a few themes emerged. One is that a number of plants that operated at reduced DO and at low DO did achieve good phosphorus removal performance at the lower DO concentrations. And so this included the Cary plant in Chicago, the O'Brien SBR plant that was also Chicago, the Ibumal plant in Denmark, work at HRSD and then also the James DeLorio plant in Colorado. At the Cary plant in Chicago, they were also able as they did this at full scale in isolated trains, they were also able to characterize and track actual blower performance and energy usage and did find that they got the benefit that they were seeking of reduced energy costs at the lower DO, but did show slightly more variation in their phosphorus removal performance as shown on the graph here. Another interesting result was at the Rock Creek plant in Oregon operated by Clean Water Services and they tested side by side A2O configuration and sidestream EBPR configuration. And what they found actually was that both configurations really provide a very good performance kind of on an average or median basis, but that there was more variability in the A2O system compared to the sidestream EBPR system. Next slide please. Another interesting result shown through in the phosphorus uptake activity tests that were performed under anoxic conditions. So whenever the utilities were able to conduct these assays under anoxic conditions, so looking at phosphorus uptake in the presence, most of them in the presence of nitrate as the electron acceptor and then to compare that to the rate of phosphorus uptake under aerobic or oxic conditions. Under two of the tests, the anoxic activity tests were able to be done both using nitrate and nitrite. And what was interesting here is these configurations are very different. One was the O'Brien SBR, the other was the Ibimal Full Scale Oxidation Ditch. But what was interesting here is that the difference in the uptake rates were not very different between nitrate or nitrite as the electron acceptor. Although the difference in the uptake rates, anoxic uptake rates between the two configurations was in fact quite different with the results that Ibimal being about double those in the O'Brien pilot. Next slide please. So additional comparison of the anoxic phosphorus uptake rate activity results were compiled based on nitrate for all the facilities that performed this work. And so what we're showing here actually is a graph comparing the anaerobic SRT, the anoxic SRT and then a ratio of the anoxic phosphorus uptake as a percent of the aerobic phosphorus uptake for each of the plants that participated. And what was noted was perhaps a general trend toward the anoxic-to-oxic phosphorus uptake percentage or ratio being a bit higher in the systems that either had a longer anaerobic SRT or systems that had hydrocyclones. And so on that, when I point out the James River plant which really had very, very little anaerobic contact time but it did have hydrocyclones and had a rather high ratio of anoxic phosphorus uptake to the aerobic as observed. Next slide please. Showing a bit more detail of the results from the team at the Rock Creek plant in Oregon. As I mentioned earlier, they performed side-by-side testing of A2O and sidestream EVPR configurations. Both treatment trains were operating in A2O mode and an initial set of phosphorus uptake tests were conducted. Then the two systems were isolated. The conventional configuration maintained operation in A2O, the two basins were isolated and the second basin was operated with a sidestream EVPR type of configuration which included RAS only in the first anaerobic zone. And over a period of about six months it was noticed that the anoxic uptake rates in the A2O system, if anything started to drop while the anoxic uptake rates and the ratio as percentage of aerated uptake rates was increasing in the system that was operating in the sidestream EVPR configuration. And so these basins were operated identically and are of the same basin design. And so the main difference between the two configurations was the fact that the mass fraction, the anaerobic mass fraction or SRRT was higher in the sidestream EVPR configuration which showed the higher anoxic uptake rates. Next slide please. And so moving on to a bit more discussion about the plants operating with hydrocyclones, several of these also seem to have some of the higher anoxic phosphorus uptake values. And honestly, these plants had quite a few differences between each other in terms of configuration and operation but except that they all had hydrocyclones. And so for example, the James River treatment plant that I mentioned earlier is really an IFAS primarily oriented toward nitrification and nitrogen removal with a very short anaerobic contact time as the streams come together. In addition to that at the time during the testing, the zones with the media, the biofilm media were quite highly aerated and the suspended growth SRT is fairly short. So that's one configuration. The Ivy Mall plant in Denmark has high anaerobic and anoxic fractions and operated an oxidation ditch with alternating aeration at lower DOs. The James DeLorio plant in Colorado was operating with low DO and ABN control. So it's so advanced controls to try to optimize aeration and nutrient removal performance. They also had hydrocyclones. Another observation that we made is that at the Denver plant, at the Robert Hyde plant, they were able to run a full scale isolated demonstration train in an AO mode and then change that operation over to AO with hydrocyclones. And after this switch was made, an increase in PAO relative abundance was observed in the train that was operating with hydrocyclones. So another interesting result. And so with that, I'll turn it over to my colleague, Kartik Chandran to talk more about the microbial ecology results. Thank you very much, Chris and George. All right, I'd like to start also by acknowledging that it's really an honor for us all to present our work in honor again of Professor David Stensfield. Dave was really a close friend to me and a really strong supporter and mentor. And yes, I'm happy to share some of our work here. So as Chris and George have already mentioned, this project has been really a remarkable confidence of different directions, different stakeholders, different partners from across sectors, academia, utilities, practitioners and so on. So what I'm here to share today is other results of a more deeper molecular characterization of the full scale treatment plants that Chris just presented. And this also ties into some of the more fundamental work that George had presented previously. And so here first we are showing the results of a very detailed interrogation of different full scale treatment plants that Chris had mentioned. So to compliment what Chris mentioned in terms of what the plants did and that is to monitor performance and kinetics through batch experiments, the overall efficiency of conversion, the kinetics. What we did was we went into the black box of these systems to try and answer three questions. What kind of organisms are there? So who is there and how many to an extent? And then more importantly, what it is that they are capable of doing. So this is the potential activity. And then under the conditions that these plants were being operated, what were these different groups of organisms actually doing? And so that is the progression that I'm going to take you through today. And on this slide, what we are presenting are answers to the first question, in these different plants. So if you look at the schematic, if you look at the graph on the X-axis, we have the different configurations summarized so later and on the Y-axis, we have the relative abundance based on the results of the technique that is referred to as metagenomics. So essentially what we are doing is we are going in, taking out all the DNA and sequencing all the DNA with one objective here, more to follow on the next slide. One objective here is to figure out what kind of organisms there are, who is there and how many. And as you can see across, if you just take a step back and take a look at the overall profile here, what we are able to see is that the fingerprint of the different organisms or the different groups, the different genera based on metagenomic sequencing is nearly identical. So irrespective of how these different treatment plants were configured or operated, we did observe a similar overall microbial community structure. So who is there, how many and so on. And this is especially the case when we talk about the dominant nitrogen and phosphorous cycling organisms, which is really the focus of this project. So again, irrespective of how these plants were configured or run, the overall microbial structure, the fingerprint of the structure is nearly identical. The dominant nitrogen cycling organisms here we found across the board, in terms of ammonia oxidizing bacteria were related to nitrosomonic species. And for nitride oxidation, the dominant groups were related to nitrospyrus species. And then for the phosphorous cycling organisms, we have candidatus acumenibacterus species, again, representing the bulk of the DNA signatures. Next slide please. So now we go deeper. It's, if you want to understand not just who is there, how many, but what it is that they are capable of doing. So the potential function. We look at the same DNA pool that we extracted. And now the question is, what kind of different reactions, what kind of different metabolic pathways do these groups of organisms possess? Not what they're doing, that's coming up on the next slide. What it is that they can do, potentially do, what is it that they possess? So on the left-hand circle, I'm showing you the different reactions of the nitrogen cycle. Most of the nitrogen cycle reactions. And on the right-hand circle, we have some of the key reactions representing phosphorous uptake on the bottom half of the circle. And then, generally speaking, phosphorous release on the right-hand, on the top portion of the circle. So if we focus on the nitrogen cycle, the principal reactions that we are considering, based on what we found on the last slide, who is there? So we're talking about conventional ammonia to nitrite oxidation, nitrite to nitrate oxidation. So that's what is represented by these different pathways. So the first step of ammonia oxidation catalyzed by ammonia, mono-oxygenase, AMO, and then, and so on and so forth. So that's what we track from ammonia to nitrite to nitrate. And then on the denitrification side, we are tracking nitrate to nitrite, nitrite to nitric oxide, nitrous oxide and nitrogen gas. The way to read all of these schematics is as follows. We are looking for a fingerprint of each of these genes as represented by the intensity of the blue color. And so, for instance, if you just look at that AMO rectangle, again, we are tracking all the different plants, we find AMO present in all of these plants. And that is to be expected. If plant is nitrifying or even capable of nitrifying, we expect the signature and that's what we are seeing. And we are not seeing really any plants where any of the big steps are missing in the nitrogen cycle. Again, no surprises, but it's good to actually confirm this. On the phosphorus side, it's a similar story. We're looking at four genes here, PPK, PST, PAP and PPX. Again, we can talk about functions in a bit more detail, but essentially, broadly speaking, generally speaking, we have divided them into parts where it's associated generally with uptake and release of phosphorus. Again, the bottom line is as follows. For all the different treatment plants that we consider, the signatures are there. So the potential is there. There's nothing really missing from that perspective. And so that's what we say when we summarize complete pathways for nitrogen and phosphorus cycling for present across the board at all facilities, despite differences in how these plants are configured or operated. Next slide, please. And now this is the more exciting result as well. It's not just who's there, how many, but it's actually what it is they're actually doing. So this takes some explaining to do now. So if you just take a look at the schematic, what we're trying to do is we'll see the biomass is all there, but the biomass is actually circling round and round the different zones of any given wastewater treatment plant. And these could be anaerobic, anoxic or aerobic. And so the way to, one way to summarize this information is by looking at the expression of these genes. So now we are talking about RNA, messenger RNA specifically. We're looking at the expression of these genes as the biomass goes from one zone to another. The way we have expressed this is as follows. If a gene, if the expression of the gene, a given gene or the messenger RNA corresponding to a given gene increases as we go into an aerobic zone or an aerated zone, or alternately, if the expression of a given gene or a pathway is higher in the aerobic zone compared to an anaerobic zone or aerobic anoxic zone, that signal will be represented in a red, through a red arrow, if you can look at the arrows. And then vice versa for the green arrows. So let's take another look at what it is that we're trying to explain here. So for nitrification, so for nitrification and looking at ammonia to nitrite, nitrite to nitrate, what do we expect in terms of the activity of these organisms, expression of these pathways as these organisms are in an aerobic zone? We expect an increase in the aerobic zone relative to a decrease. That's exactly what we're finding if you track on the left hand circle, the red arrow. So we are seeing that the nitrification related pathways are expressed at a higher level in the aerobic zone compared to the anoxic zone. And that's exactly what we expect. And this is now relating not just to who is there, how many are there, it's actually what are these organisms doing? And we are doing... Thank you, Chris. Thank you, George. And Kartik for sharing one for work We're gonna move on to the next panelist. It's Dr. Yu-Mei Li, who is the chair and the professor at the Tongji University. Professor Li, please. Okay. So it's my honor to be here to talk about my work on EPPR and the phosphorous recovery. Here, I want to introduce a sidestream process designed for nutrient removal and phosphorous recovery. Next slide, please. So here we use a synthetic domestic waste water and we set the SRT from 25 days to 50 days and also for the peer recovery rate from 40% to 65%. And we find that at a higher peer recovery, we can have a higher SRT to maintain the tea content in sludge around the 5% so that to keep the EPPR activities. Next slide, please. We also use the real waste water and to have the similar results. Here, we can see that this purple color is the recovered phosphorous. And here the stage one and the stage two, they are the AAO process. And then we shift to the sidestream process with the different anoxic tank volumes. And we can get a very good phosphorous recovery and also keep the peer content in sludge around the 4%. In the supernatant, we can have a phosphorous phosphate around in the range of 30 to 70 milligram per liter. Next slide, please. So here you can see as a peer recovery process is combined with the AAO process and we actually dispatch a part of returned sludge to the SBR tank in the sidestream. And then that's there for the anaerobic fermentation for a few hours and then phosphorous can be released. So then in the next tank, we can let the phosphorous recovered as HAP hydroxyapatite. And after the peer is recovered, the supernatant will be returned to the anaerobic tank. Then in another tank after the sludge in the this SBR sidestream SBR tank, the sludge will be returned to the anoxic tank. So here we recovered phosphorous and also the part of sludge, the carbon in the sludge can be released as VFA. And therefore the sludge back to the anoxic tank to provide the sludge fuller for PHA. Okay, next slide, please. So we hope that using this sidestream peer recovery process to recover about 60% of phosphorous from the influence. And also we hope that the phosphorous concentration in the sidestream supernatant higher than 15 milligram per liter so that it's easy to be recovered. And this sidestream need to combine with a EDPR process so that we can have many P in the supernatant. But usually if we recover phosphorous in the sidestream process and maybe there's some negative effects, the extraction of phosphorous from the sludge may cause the lower P content in activated sludge and maybe also lead to the depletion of the polyp in biomass and thus may damage the EDPR process. Next slide, please. So we correlate to peer recovery rate with SRT and also the P content in sludge. So we have this equation, just link SRT and R is the peer recovery rate and here PS is the peer content in sludge. So here, these two figures show that if we want to maintain constant P content in the sludge and recover a higher phosphorous from the influence, we need to have a higher SRT. And also the right color show that the higher the influence phosphorous concentration and the higher phosphorous recovery rate we can get at a constant SRT. We're using experiments to have this equation validated. Next slide, please. Next slide. Ah, yes, here. And also we can see that from the AO to the AO SPSPR process with the increase of SRT, we get reduction of the excess sludge. So also the sludge yield, when the SRT increased to 50 days, it resulted in 58% of sludge reduction. So this process, that means this process also reduce the sludge production. Next slide, please. We also have a deep down the pilot scale operation in which the little influence you influence the phosphorous is lower than the lab scale experiment. And we can have around more than 40% of phosphorous recovery. And also in the sidestream, we have released the phosphorous concentration around 50 milligram per liter. Also the peak content in the sludge is still around the 5%. So it can keep the EPPR activity. And okay, next slide. So here is the phosphorous removal efficiency and the total nitrogen efficiency. We can see that the total phosphorous efficiency is not influenced by the peak recovery. And also for the total nitrogen removal compared with the AO process, the sidestream process even have a higher total nitrogen removal efficiency, especially at the higher SRT and higher phosphorous recovery rate. Next slide, please. So from the stoichiometric and kinetics of the system, we can see that the phosphorous release rate is not actually influenced very much by the peak recovery and there's an extended SRT. When the SRT increased from 25 days to 30 days, all the AO SPSPR process have a good phosphorous release rate. Next slide, please. And also we can see that when the systems switched from the AO process to the sidestream process, we can have a much better denitrifying peak removal. And from the stoichiometric, we can see that the maximum anoxic peak uptake rate actually increased much better compared with the AO process. And also the denitrifying rate increased. And with the higher the sidestream biomass, we have a higher ratio of anoxic peak uptake to the AO uptake. Next slide. Okay, so here is a take home message is that based on the phosphorous mass balance equation, if we extend the SRT of the sidestream process it can increase the potential of phosphorous recovery and reduce the impact of phosphorous recovery on the peak content of the activated sludge and keep the EBPR activities. And the second is denitrifying peak uptake was improved in the sidestream process. And thus we have a higher total nitrogen removal. The microbial community structure was changed significantly compared with the AO process. And the important functional bacteria, for example, the nitrifyers and denitrifyers, PAO, especially BPAO, they are all enriched in the AO FBF process. The challenge of the process is that it is more complicated compared with the AO process. And therefore more sophisticated control of the process is required. Okay, that's all. Thanks for your attendance. Next slide, yeah. So next slide have a look at the microbial community analysis. So from the oaths and the kills and the ACEs indexes we can see that the microbial richness of the sidestream process increased significantly compared with the AO process. And also the diversity, the microbial diversity increased. Next, please. So if we focus on the PAOs in the sidestream process we find that the relative abundance of candidate acumen factor was not affected by the both forest recovery and the extended SRT. And even though the acumen factor in the sidestream process actually increased significantly compared with the AO process. Other PAOs such as the tetrospherol and dichloroamonas also presented and especially that dichloroamonas is much enriched in the sidestream process compared with the AO process. Next slide, please. So if we look at the nitrogen metabolism pathways and we find that from the abundance of the KEGG models subject to the nitrogen metabolism pathway we find that actually the denitrifying pathway it is strengthened after the system switched from the AO process to the sidestream process. And particularly so also the data show that the sidestream process could improve the denitrifying P-optic by promoting the growth of candidate acumen factors. Next slide, please. Great, thanks, April. I'll try and be a little quick. I know we're a little behind on time. To introduce what I wanna share about I'll show you the schematic of a pilot system we ran at HRSD for about three years. And of course the ideas that helped build and inspire the system, we stand on the shoulders of giants like Dave. This system is an AB process. So we do carbon capture and removal and then nitrogen removal in the B stage. So for the nitrogen removal step we do intermittent aeration with AVN control to get a mixture of NOx and ammonia that we can then send to a downstream anamox polishing process. The way to do anamox here, ideally, or what we intended is the intermittent aeration and aggressive SRT control can hopefully lead to NOB out selection or in the anamox reactor itself we can add supplemental carbon to do PDNA, partial denitrification anamox. The big drawback to a system like this is that you can't do BiOP because you've removed the readily available carbon and especially the VFA and the influence in your A stage and the A stage SRT is too short to do BiOP. So to try and add BiOP to the system we added a side stream BiOP reactor that's this SBPR here and we added a fermenter for some of the carbon we've captured in the A stage. We take some of that was and we ferment it to get a VFA rich fermentate that we add into this side stream reactor. So, you know, big picture this is capturing the carbon out of the process so you can have a much smaller nutrient removal piece the B stage and then we can put that carbon back where we want it. So just big picture this system works and it works well and it's quite stable as long as you add enough VFA from that fermented A stage WAS you can get really low effluent OPs. So here in these figures you see the VFA to P or the C to P ratios and this is what we added in terms of fermentate and the effluent OP on the Y axis. So you can see above a certain amount of VFA or soluble COD we get very consistent effluent OPs under 0.5 and even as we increase the amount of carbon we're adding to very high levels we don't see any deterioration of the biop performance. That leads into the next point which is that that additional carbon appears to be very useful for nitrogen removal as well. So these are endogenous denitrification batch tests we did. So we take a sludge sample from the end of the aerobic zone so there shouldn't be any soluble carbon around and we don't add any carbon either and then we measure a denitrification rate. So the Y axis here is that denitrification rates and the X axis is that VFA to P ratio. The big takeaway is that these rates are much, much higher than you would expect from endogenous denitrification which maybe is one to 2.5 milligrams of NOx per liter per hour and here we're seeing 2.5 to 5 to up to 10 even and you can see this rate increases as we add more carbon. So what that means is there's significant carbon storage going on in the system and that benefits us with a bunch of extra TIN removal and it's not at the expense of the biop we're doing. That carbon storage can also lead to partial denitrification. So this is a period of about five or six months of operation where we have this happening and here's just a profile in time from the system and you can see in the gray we have the air that's turning on and off because it's intermittently aerated and there's nitrite accumulating while the air is turned off. So we're getting nitrite accumulation in the system which is great for the Animox downstream and it's not from the out selection it's from partial denitrification and we see that in this profile. We also did some additional work in our group and this is not showing up on here. So we did some additional metagenomic work and we did these batch tests where we measured both PHA and glycogen that's shown in the orange and green here in this batch test where nitrite's accumulating and that's only with internally stored carbon no exogenous carbons. And we did single cell Raman micro spectroscopy on those samples and we found that there's three OTUs really present in this system and I'm sorry three OPUs and in OPU one this major cluster we found that in those cells PHA decreased over time as nitrite was accumulating and there was no glycogen in any of those OPUs. So that led us to conclude that PHA was driving this internal carbon storage and partial denitrification and not glycogen and those OPUs or that OPU one likely related to the OTUs for either an acenotobacter or promomona to say. We also saw that that partial denitrification really drove a high TIN removal in the anamox process downstream. So again, we had designed the system to do NOB out selection to get the nitrite or we add carbon in the anamox reactor to do partial denitrification anamox. Here what we were able to do is do the partial denitrification step in the B stage, in the activated sludge portion that we have a bunch of ammonia and nitrite coming out of there and that anamox just knocks out the rest of the nitrogen. So very quickly this figure is showing nitrite accumulation ratio as a percentage in this black line and then the nitrogen removal percentage in the B stage that's in blue and the NBVR in red. So you can see, we're doing 50% nitrogen removal in the B stage and then another 30% nitrogen removal with no extra carbon added in the NBVR just because of all that nitrogen there. So it's an incredibly efficient process and it wasn't exactly how we designed it but if you can drive partial denite with this internal carbon storage is a huge benefit. I'll leave you with this last figure which is just a very broad sketch of carbon. It's a sand key diagram showing carbon flows in a typical A2O and in our pilot process. And the broadest thing I'll say about this is that the AB process, these sidestream processes is focused on internal carbon storage. We're really unlocking tools to take carbon and instead of dealing with it in the influent, remove it and put it where we want it in the most useful places. And it's huge for Bio-P, for nitrogen removal and for mainstream animox. So thank you. Thank you, Caster. Our next panelist is Stephanie Klaus. She's a process engineer working at HRSD and she's also leading several R&D project including the partial denitrification full-scale demonstration. Stephanie, go ahead. Thanks, April. Thank you. Thank you very much, Professor Lee. Our next panelist is Caster McCoolen and he's currently working with Hampson Road Sanitary District and also I'm fortunate to be involved in associate with him as a advisor, as him as a PhD candidate right now at Cornell University. Caster, go ahead. Thank you. So we've observed this phenomenon in some of our other plants, especially the VIP plant here. This is showing how low the methanol doses below expectation at our VIP plant. And we basically have stopped this methanol entirely here because we have this internally stored carbon in our second anoxic zone. So, and we're seeing nitrate accumulation there. So it would be great if we could just, you know, drop in some moving media or drop in some fixed media modules and do partial denitrification animox in, you know, some of our other facilities which really saves on carbon and we can also let this bleed through some ammonia from the aerobic zone. So that's energy and caustic savings as well. So that's it for my presentation. Thank you, Stephanie. All right, so I'm gonna be focusing on full scale plant today. We're gonna be talking about James River treatment plant. So while we were doing the work that Caster just presented on the pilot, simultaneously, we were noticing full scale at many of our different plants, this same phenomenon. In our plants that had a second anoxic zone, we were noticing we didn't have to dose as much carbon as we thought we would have to, you know, below stoichiometric requirements. So we were really investigating this further. We found that it was internally stored carbon that gets carried through the process. So at James River, Chris presented this plant earlier, at some point, we added an anaerobic zone here. So it's an A2O currently. So we have anaerobic, anoxic, and then anaerobic, IFAS zone. Then there's a very small second anoxic zone that we traditionally have not added external carbon and a very small, even smaller, re-aeration zone. So we need to meet some stricter nitrogen limits here in the future because we are adding a water reuse system on the tail end of this plant. So our plans for doing this are either, we're probably gonna do both, but the options are to add IFAS in a second anoxic zone here, which is really small. And we want to do partial denitrification, animox. So we'll have animox here as attached growth in the second anoxic zone and as a polishing step as well. We probably could have just picked one of these options, but because we're still piloting and testing, we're going with both to be conservative. Regardless of where you're doing the partial denitrification animox, it's really important to control the ammonia versus NOx aeration control. So we need the ammonia and NOx to be approximately equal going into this zone to meet animox stoichiometry. So in order to study both of these options, we have a pilot. We've been operating for a couple of years now, both IFAS option and MBBR option. So MBBR receives secondary clarifier effluent, IFAS receives the mix liquor from the main plant. What we found was that mainstream animox took a startup, took about three months. This was good news. We've grown animox from scratch in about four or five startups now. So we're not concerned about growing animox from scratch. And once animox is there, it's not the limiting factor. And we're able to meet low effluent TIN limits as long as we meet that upstream ammonia versus NOx ratio. We had more experience with the MBBR at the time, but we found that IFAS is also a viable option. And this is exciting news for us because we have nine medium-sized plants, some of which provide more at HRSD, which have a second anoxic zone where we could potentially just add animox and do partial denitrification animox in that second zone. And lastly, we found that IFAS can take advantage of internally stored carbon. So using internally stored carbon to do that first step of taking nitrate to nitrite and then animox taking ammonia and nitrite to nitrogen gas. So here's an example. This is the, on the blue is the MBBR. The gray is IFAS phase one where we were adding less carbon trying to meet a lower effluent limit. I mean, sorry, a higher effluent limit. And then the green was phase two where we were trying to push that a little bit. And I think the main takeaway here is just in gray. You can see this COD to N around one is really low considering for methanol, we might expect this ratio to be 4.8 for full denit and maybe half of that for partial. So we're really under that expectation there. And you can see that there's really not too much difference between the effluent TIN remove between phase one and phase two. You're getting maybe an additional one milligram per liter in phase two, but we're really able to take advantage in the gray of this internally stored carbon. And why that's interesting is that it's really getting carried, remember this is getting carried through the whole aerobic process. So this carbon is getting stored in the anaerobic zone, getting carried through the aerobic zone and then being used for partial denit in the second anoxic. And here's just some example of the rates. The top is a nitrate removal rate. The bottom is nitrite accumulation rate where we see sort of this preference for partial denitrification. The endogenous rate is really in quotes, endogenous just meant that we didn't add external carbon. So this is really above just what we think of from decay. This has to be from some internally stored carbon and we have other tests to prove that it is from internally stored carbon that are shown here. But yeah, this is just with glycerol and methanol. And at the time for part of this experiment we were adding glycerol on the main plant, but some of this time we were not. But you can see that glycerol gives the probably the best nitrite accumulation rate in on the bottom graph, but that in the dark blue, you can see that we still get like some good nitrite accumulation even without adding any external carbon at all. So this is great news. We want to take advantage of this. Now we're doing, we did the pilot. Now we're doing a full scale demonstration on two of the nine tanks. So here are the nine aeration tanks. We're converting two trains to fit in this second small second anoxic zone where the HRT is about 20 minutes. We're going to fit in two demonstrations, one fixed media and one moving media. So we can grow anamox there and do partial denitrification anamox with methanol as the carbon source. The time to decide between the two while we're doing the demonstration, moving media is more tried and true and it's really a better choice for wet weather management. I mean, sorry, it's the more challenging for wet weather management because we have to add screens, which increases head loss. So we need to have a wet weather bypass around the moving media zone. It would be nice if we could do the fixed media. This is what the modules are going to look like here on the top and we're using a fabric. The fixed media is great for retrofit because we can just drop this in any second anoxic zone, grow anamox on it and do partial denitrification anamox. But traditionally this, you know, there aren't a lot of anoxic applications of fixed media options. And also you have to be able to control, it's harder to control the biofilm thickness on fixed media instead of plastic moving media. I want to touch upon quickly a concept that at least actually motivated that the study related to the science dream EBPR is actually the requirement of carbon ratio to achieve a stable EBPR. It's well recognized that in practice that we require certain level or minimum carbon to P ratio for us to achieve a stable EBPR. Many studies explored the fundamental reason on one of them is related to the carbon computation among PAO's GAO's as well as others. And we know in most ways what you remember that at least in Europe and the States most treatment plan having a range of 10 to 40 and in that range is unavoidably you're always going to have PAO-GAO coexistence. So therefore carbon computation cannot be avoid. So under this consideration and understanding how can we better control the carbon to make it maximized to achieve the goal we want? It's actually the key, particularly in response to the EBPR side we know that the low-influency to P often lead to unstable and fluctuation. And also if you want to implement any process such as AB stage you want to capture carbon or want to capture carbon to optimize downstream shortcut and remove anemox you have to address the carbon needs for the biop process. So I think of the thinking how we can better do this it's a warranted. And one of the particular direction our group have been involved in working with many in one of the work funded project with many agencies including into looking how potentially the design and operation strategy related to the side stream EBPR can help achieve along that direction. So this particular configuration actually is not really new. It was practiced in Denmark and Europe for a while with at the time the motivation actually to enhance the nitrification. And we actually looked from a little bit of different angle try to see how this particular strategy and design can be better leveraged to enhance and improve the stability of EBPR without external carbon addition. And we have learned through this effort that this particular configuration by simply taking a small portion of the rest through a side stream or semi or first half of the fermentation to enhance VFA production but with minimized mass antigens actually provide a suite of advantages including influence the carbon fluctuation independent P removal therefore is more stable more controlled and efficient on your work zone as well as more flexible implementation. So this particular study was done across 12 facilities including some facility were able to pilot the side stream some using conventional and so three years monitoring of the data we learned that indeed plant implemented a variety of side stream configuration led to more stable performance. Of course, you also have to consider the permit and other facts potentially impacting but nevertheless, the statistic indeed show that the side stream implant patient improved the overall performance. And of course with more mechanistic that combines genomics and modeling we also get some insight to learn that the particular strategy will allow to have a higher diversity more rich micro community and our study as well as previous panel that George and others also show that the population potential selection of a specific organism associated with this strategy. So it means that this particular change of an operation indeed leads to fundamental changes in the ecology and the system. And I want to touch upon a little bit that the key things related to how potentially saturated BPR can be further leveraged to maximize the carbon usage. So particularly related to carbon we know that for side stream that with in situ carbon production not only increased amount that available to a biop process but it's actually provide a more complex carbon mixture that has intrinsic implications on the PAO GAO computation, kinetics as well as the carbon diversion or carbon distribution among different populations. And we learned that this process actually can optimize the carbon utilization directing carbon to more favorable over GAO to PAO over GAO. And also through better and more controlled condition in the side stream in relative to the traditional A2O design it also allow us to have more flexibility to divert the carbon as the case demonstrated by Caster in the AB stage and the P&A or PD&A process combined with a side stream. So overall I think that there are a number of parameters and aspects related to this particular concept not necessarily to this like I mentioned this particular side stream but it could be along this way of thinking how can we put in more strategies to make the internal carbon use more efficient therefore reducing overall carbon footprint. I think that there are suites of evidence including those presented earlier today as well as those I've not get a chance to present today but in the literature that how potential scientists in the PR can enable more flexible and improve the carbon utilization. And one example mentioned earlier is the AB stage that stream plus the shortcut presented earlier. And also here I borrowed the study done by Metro of Chicago and provided kindly by Cindy Chin. They also showed that by implementing side stream with a very unfavorable low carbon condition which was not possible to sustain by LP but now with side stream in BPR now they can do the essence that actually reduced the carbon demand and the carbon loading requirement. And also I think evidence presented today as well as other study also showed that the side stream BPR potentially also enhance internal carbon driven denitrification therefore also that's done through multiple directly of course by having the side stream you already allow more influence to carbon directly goes through the anoxic zone. So therefore intrinsic that strategy already already would allow more carbon go to denitrification but in addition to that I think that there's more room to be explored on how the side stream potentially encourage more population that has stored more higher statistic amount of carbon than conventional system allow and how we can better use that. One of particular evidence I just want to share one here really quick is through the strategy to look at the statistically intracellular cellular level carbon polymer carbon storage shift the change we can find that actually side stream system actually really allow statistically significant improve and increase of internal carbon storage in not only PAOGO as well as unknown and unrecognized organisms. So at the very last just touch upon one slide go back to the DPAO concept the previous several panelists touch upon on the role of DPAO. I think concept wise DPAO that would help us allow us to better use the internal carbon for both NNP removal purposes but I want to touch upon just one point is that we have to carefully look at how much contribution DPAO makes versus other organisms. In this particular study it's a lab skill study with different recycle ratio to allow different amount of nitrate to be recycled back to anoxazone which led to a different level of enrichment of DPAO versus non-DPAO based what so far we know and turn out that the DPAO can be easily enriched by just increased nitrate recycle ratio as expected. And but more importantly I think the key point I want to touch upon at an end through sumo modeling as well as computation we can see that under three different nitrate recycle ratio when the system have a different level of DPAO versus non-DPAO you can see that the overall contribution to denitrification by DPAO is somewhere in the range from five to 10%. That means majority majority of the denitrification are carried out by conventional heterotropic denitrifier as well as other internal carbon driven denitrifier therefore lead a lot of room for us to explore further. So with that I want to thank you all the students who contributed to the information I presented here today including a PhD student Nick Guangyu visiting scholar Dr. Wang who was professor now in Xi'an University Technology and the previous student Nareen and postdoc and the one rule right now is a consulting form and as well as all the participating agencies and the facility to make the study possible. Thank you. So with that I think we got to translate into the panel discussion and the Q&A section. So now I'm going to moving to the final panelist that's me. And hopefully I can wrap up some of the key points presented by a number of panelists at today's talk. So in response to overall incentive to lower carbon footprint and energy in BNR biological process related wastewater or other biological process I think effort has been taken either directly greenhouse gas emission reductions through various strategies as well as indirect reduction mostly it's through the operation innovation or operation optimization along the line of less chemical usage, less sludge reduction as well as overall more efficient use of carbon. So look at some of the efforts been going on recently I kind of put together this summary table list of some of the efforts or certain directions. So shortcut nitritation or nitrate shunt effort have been going on for a while. I have a lot of wealth of the information and the literature in that area. We recognize there's still some challenges remaining particularly how do we better use this concept at the low strengths wastewater and how can we better control the potential emission of greenhouse emission gases. And efforts also been put forward including several panelists today presented along the direction of partial nitrification combined with anamox or partial denitrification combined with downstream anamox. I think the PNA has been quite demonstrated at both pilot and the full-scale but mostly most successful stories are associated in generally with high-strength wastewater. How do we apply that in low-strength full-scale plan I think still there are works ongoing. And particularly in recent years a number of groups have demonstrated that they can successfully achieve partial denitrification with downstream anamox including HRC and several other groups around the world. And I think fundamentals along this line are still being explored to better understand that the biochemistry and the mechanism of ecology behind what is the really controlling factor to allow us to achieve a reliable and a stable PDNA but one element I particularly want to point out is that one of the main challenging to achieve a PNA is actually that it will be out-selection and how can we maintain that the stability? And PDNA so far based on the evidence seem to allow us a little bit more room and flexibility to operate or strategize the process to allow PNA to occur. So if we can allow a system and design strategy such that the PNA and the PDNA can occur in the background in response to those uncertainty or variation fluctuation that typically lead to the challenge related to PNA and allow that in the background provided the nitride accumulation and to enable downstream anamox I think that that's a new thought and new trend need to be further evaluated and showing promises. And another direction is how can we actually better combine phosphorus removal processes with shortcut nitrogen? Efforts in shortcut nitrogen removal process as I mentioned earlier has been successful but mostly how does that take into account of the phosphorus removal has been challenging because they have a conflicting or sometimes opposite requirement regarding to carbon regarding to SRT. So for example, most short cut nitrogen processes doesn't want carbon but as to EBPR actually require a reliable amount of carbon and SRT requirement also difference and other operations. How do we understand those and develop the strategy to allow both? That's another direction people have been looking into particularly how do we complement that in the full scale? And I think a wonderful example showed earlier by one of the panelists at SRT is to combine actually AB stage to capture carbon upstream and then with B stage to do intermittent aeration enable the shortcut nitrogen with downstream anabox and also enable biop with the sidestream is just one example along this direction. And here for the remaining part I want to touch upon a little bit how can we better use the concept of a sidestream enhanced biological peel removal? The concept of it is actually much wider than the specific or currently one or a few implementation strategy and how to achieve but key is really how do we expand the concept to use further allow us to maximize internal carbon usage to further enhance internal carbon driven deny as previous panels as presented and also at the same time improve performance. And I think that there are still rooms remaining for us to better understanding the mechanism actually particularly the link between internal carbon storage internal carbon join denitrification as well as how does a potential sidestream EB paratype of the strategy allow us actually in reach more organisms such as DPOs or PHA or other intracelular carbon accumulating organs to allow us to enhance that to benefits. George, can you hear the question? April, I think George had to leave for his to teach a class. Oh, sorry. So he's not on. Yeah. Yeah. And I'm not able to answer. I don't have anything to add to this question myself. Kartik, are you still on? Would you like to make a few comments related to this? And we collect all questions for any panelists today. So I'm going to start with several questions we have collected so far in the Q&A box. I'm going to direct them to the right panelists. The first question is, this is directed to George Wells from Northwestern University. What do you think about bio mineralization of PNN as a resource recovery from wastewater treatment? I think probably that can be directly to me. So question is that in the side stream or enhanced biop with fermentation, the fermentation side reactor is only actually carried out to the first half of fermentation basically from hydrolysis to fermentation to generate VFA. The SRT typically is controlled below the threshold where the sandages can thrive. So indeed, this process is mostly generating carbon sources. And where does the source come from? It's from those none utilized or particulate or other refractory organic carbon steel residual in the rest or as well as partial for the degradation or decay of the biomass itself. And the answer is yes. The VFA provided in the side stream does directly go to the PAO, which are evidenced by increased the PAO activity as well as other molecular evidence to show indeed the statistic increase in internal PHA in response to the VFA production and the reaction time in the PAOs. The next question is N-O-H also on how DAO possible? I actually do not fully understand this question. Is cardiac still on? I think cardiac is gone as well. Oh, sorry. OK, so we're going to move on to the next question in the Q&A box. So what results in enhanced biop removal when formatted SAS is added back to the ASP? Does the VFA provide additional carbon source for the PAOs? So we're going to move on to next question. Why incomplete nitrification could affect the EBPR? Kaster, do you want to make a few comments on this? The question is raised by Peter Ubeck. If you are there, would you please repeat your question to the panelists? No, I guess he's no longer there. So Yung Mai, I think you're on mute. You may, yeah. The question is, what's the HRT for your site stream pre-release reactor? And whether it was continuously mixed or intermittently mixed? And I think this is a broad question that could be answered by other panelists as well. So Yung Mai, what is the typical HRT for such reactors? I think from the evidence we see so far in operation and practice, it's in a range of between 6 to 38, sometimes even 45 hours. And from a mechanistic perspective, we learned that there is a so-called optimum HRT if your optimizing parameter is to maximize the internal PHA, implying more P removal potential. And we found that transit threshold of HRT varies from plant to plant. And once we are preserved, typically for United States plant, the optimized range is typically between 18 hours to 24. And so now I answer this question. OK, now I can answer this question. For the HRT, for the site stream, it's around 24 hours. And it is actually a SBR module, so we operate it in intermittent modules, not a continuous one. Just for the site stream. But for the mainstream, it's a continuous one. And April, you mentioned that the site stream could be done on part of the RAS. Typically, can you give a sense whether you would do it on the entire RAS, part of the RAS, and how much of the RAS would be subject to this 18 to 24 hour HRT? And how would you go about doing it from a design perspective? Thank you, Sudhir. I think that's a really, really good question. I don't think we have a full answer, but I can share with what we learned so far. Question one is, what is the optimized percentage of RAS should be pulled through the site stream? That's the first question. I think so far from practice or from optimization energy or volume, footprint reduction perspective, we want as small as possible. As small as possible to enable this working. So the lowest possible has been diamonds that are working is only 5%. So that means you need a really, really small portion for RAS. That means your footprint of the site stream reactor can be very small. That means it can be actually smaller than traditional A2O anaerobic zone, allow the same or even better impact. But in practice, people normally put 15%, 25% as more common. And I think that's a question for myself to really have a better understanding. From the mechanistic perspective, I think that the question is, can you allow sufficient amount of enrichment selecting factor to occur in a site stream is the key? So really how much means how much carbon additionally do you really need to use? And what is the balance of the biopea organism you need to enrich? I think that's overall plant carbon balance optimization calculation. And what is really the threshold of that? That's a separate question for Stephanie. Well, in the results we showed, we had full nitrification. It was just followed by a partial denitrification. Broadly speaking, the less NOx you produce and you're recycling to an anaerobic zone, the less carbon you waste denitrifying in that zone if you need the carbon for biopea though. There are some studies, I think that have discussed nitrite inhibition of PAO activity as well. We did not see that up to nitrite concentrations of seven or eight milligrams per liter. Thank you, Caster. Yeah, those are good points. Okay. I have another question coming in. What is the HRT for the site stream P release reactor? It is continuous to mixed or intermittently. This is directly to panelist professor Lee. 10% of the rest. So 10% of the rest and... On the mix later. Yeah. So it can get also some reasonable drastic impact. So dear, can you speak a closer to the microphone? We're losing a little bit. No, it's like a... Can you try one more time? I don't know. Go ahead. I was wondering was a reasonable drastic thing as well as a combination of maybe 10, 15% couldn't really make it for a very compact fermentation tank as opposed... Thanks, April. I could just add that in the final... Go ahead, James. That couldn't have been more than 5%. And it was working extremely well. So we see anything from 5% to 15%. But mostly what we have seen with those plants is like 10%. I think that's all the questions we have so far collected in the QIA box. So if no... Can we open floor more to anybody ask a question before we move on to the next item on agenda? Sorry, Sudha, all of a sudden your voice become really vague and echoed. So we couldn't really hear you well. Sorry, yeah, apologize for that. I'm gonna move on to the last question in the QIA box. Q, in addition, what happens in the sidestream reactor such fermentation to VFA P release or as additional VFA added? I think I can help perhaps answer this one. James, you're welcome to add anything you would like. So far what happens in the sidestream reactor can be summarized as the following aspect. One, as I mentioned earlier, you control the HRT and SRT such that you're maximizing your internal first half of the pathway of VFA production, minimized miscellaneous. And then while the carbon is being released as a mixture of acetate-appropriated and the other fermentation product, you do have simultaneous carbon uptake, PHA formation and the P release. We see the data for that. And over time we found, even without any additional VFA addition, just rely on 100% of internal VFA production. Over time, as long as up to 72 hours we have done in our lab, you take a sample during that 72 hour, you do the uptake release testing to measure the EBPR activity. You found that activity actually over time increases, which is consistent with the continuous PHA accumulation in the PAOs to accumulate for that potential for P removal. And for some cases, VFA can be added as one of the case presented today. That's only if your HRT or SRT design for that set stream is too small, not sufficient to produce that enough, then you can add additional VFA either from west fermentation from A stage or primary slide, but in essence from the mechanism as well as both lab and the full-scale demonstration, we really do not need that. We can run successful sites through EBPR with no external VFA addition. But if you're limited to footprint, you don't have enough sufficient HRT SRT, you can rely on that as a supplement. So James, you want to add more? No, I think you've covered it very well. Can you try again, Sudhir? Sudhir, do you want to try again to adjust? Yeah, I tried to get my speaker working. Oh, it's working now. Is it working? It's working now. So in one of your earlier slides, April, I think you try to summarize the direction of some of the work that's being done in storage that really combines the work done in the work study that Chris, George and Karthik talked about, but also some of the more recent work being done by Kester, Stephanie and yourself. And certainly Yong Mai out in China. And the question is, one, how do you maximize storage and what are the organisms that are responsible for storage? Relatively, you know, the PAOs versus GEOs to get anamox going, especially partial denitrification and anamox, it would appear that you need biofilm processes to somehow mix with suspended growth processes in some reasonable way. So what, perhaps if the panelists could respond is the vision overall. Is it to look at maybe a little bit more of the GEO type organisms as a beneficial resource for storage, especially if we want to improve partial denitrification because it would appear that PAOs may not be enough. And how would you actually combine all of this in some meaningful way? We cannot hear you, Sudhir, somehow. Yeah, Sudhir, it sounds like a connection issue like bandwidth and maybe you turn your video off. So Bryce, would you like to make a few comments representing Dave as a former students before I move on behalf of Pat to give her tribute? Yes, yes. Thank you, hello, everyone. My name's Bryce Figdor. I was actually Dave's last PhD student and graduated from the University of Washington in 2017. And I had a slightly longer statement prepared, but running out of time here, so I'll just kind of get to the synopsis that Dave showed not only my life and career, but those of so many others. He taught, guided and inspired his students and colleagues and anyone that touched him with immense kindness and generosity. He approached students and colleagues from a position of equality rather than authority despite his towering achievements in the industry. And I'll miss Dave greatly, but we'll be forever grateful for his mentorship and friendship. And although I was Dave's last PhD student, upon reflection, this really isn't the case from a broader point of view that Dave's legacy of learning, exploring and teaching will continue to be passed on to others by everyone fortunate to call Dave a friend and colleague, thus creating a legacy of students for generations benefiting from Dave's knowledge, passion and humanity. So thank you for the time to present these words and I'll pass it on to April to present some statements from Dave's family. Thank you, Bryce, for speaking that on behalf of all the students prescribed, I couldn't agree more. So at the last, on behalf of Pat Stenzo, who is Dave Stenzo's wife, who gave me this tribute, just on her behalf to read this. I'm still feeling too emotional about Dave's unexpected passing to speak publicly about him. Sorry, I think I understand totally how Pat feels. And but these are my thoughts about him as a person. Dave loved being a father and a grandfather. He made a point of attending his grandkids concert, games, science fairs. He loved to be in a husband and was a loving generous guy. He's also loved his work and believed strongly in doing what he was doing to contribute to society. I recently found at least a hundred photos of wastewater treatment plants on my iPad that he had to take over the years. I have no idea where they were taken, but it was so typical of him to want to record those as well as to treat me as tourist plants whenever we traveled. But on the other hand, he participated in a great many garden tours with me willingly. Dave enjoyed his years as a teacher and mentor at the University of Washington. Many of his connection with graduate students endured through the years. I've heard such warm comments from things that he's passing. I only wish he could have known how much he was revered and loved by them. Our house became a gathering place during those years where they could enjoy dinner, wine and a conversation without a closing hour. Dave also loved good food and wine. I know that. He became quite a red wine connoisseur over the years. Our trip to Italy definitely contribute to that. He became quite a good cook in his retirement years too. I think he considered cooking a little chemistry experiment. So he enjoyed combining different ingredients instead of following a recipe to the better. Sounds like Dave. Chili was his specialty, the hotter the better. Probably the most delightful quality Dave possessed was his sense of humor. His take on life always surprised me. I could never anticipate what could come out of his mouth. His unique perspective always kept my life with him interesting. Speaking for Dave's family and myself, we miss him every day, but we have such fond memory of our lives with him. Thank you. As Dave wanted for Dave's students and it was also extremely fortunate for me to be connected with Dave through all the years throughout my career, just as James, just a few days before he's passing, we were still talking, joking and discuss wastewater. And he's such a kind person. And I believe everyone who have any fortunate opportunity to be connected with Dave would forever remember him. Thank you so much, everyone. Thank you, April, for organizing this webinar, in tribute of Dave. Thank you for all the panelists and for everyone effort and help. Thank you for the attendance. Have a great day.