Hello and welcome everyone. My name is Dane Menke. I am the digital marketing manager here at Regenesis and LandScience. Before we get started, I have just a few administrative items to cover. Since we’re trying to keep this under an hour, today’s presentation will be conducted with the audience audio settings on mute. This will minimize unwanted background noise from the large number of participants joining us today.

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We are recording this webinar and a link to the recording will be emailed to you once it is available. In order to continue to sponsor events that are of value and worthy of your time, we will be sending out a brief survey following the webinar to get your feedback. Today’s webinar will discuss leveraging molecular data and colloidal remediation technologies for better site management.

With that, I’d like to introduce our presenters for today.

We are pleased to have with us Dora Taggart, Chief Executive Officer of Microbial Insights. Since joining Microbial Insights in 2001, Dora Taggart has advanced the field of molecular biological tools, including pioneering work in qPCR analysis and stable isotope probing. She continues to propel Microbial Insights forward by collaborating with leading researchers in academia and federal agencies to keep microbial insights at the forefront of the industry. As CEO, she has become a global ambassador for molecular biological tools, regularly presenting at environmental and corrosion conferences, leading hundreds of technical workshops worldwide, and co-authoring guidance documents with groups like the Interstate Technology Regulatory Council EMD team to support the appropriate use and interpretation of molecular biological tools.

We’re also pleased to have with us today Dr. John Freim, Director of Materials Science at Regenesis. Dr. Freim is well known throughout the environmental industry for his work in developing breakthrough in-situ chemical reduction technologies, successfully employed throughout the environmental remediation industry. He has over 35 years of experience in materials processing. In his role with Regenesis, he is responsible for the manufacture of colloidal remediation technologies, including plume-stop colloidal activated carbon and S-Micro ZVI.

All right. That concludes our introduction. So now I will hand things over to Doris Haggart to get us started.

Thank you so much, Dane. Thank you, everyone. And I really appreciate the opportunity to speak. Always enjoy doing these webinars with Regenesis. So today I’m going to be talking to you, as Dane was saying, about molecular tools. But first, just wanted to give a brief introduction to microbial insights for anyone who may not familiar with our company. So Microbial Insights is an environmental biotechnology laboratory. We’ve been in business for over 32 years now. And we focus only on doing molecular analysis. So that’s going to be the focus of my talk today.

We’ll talk about the different molecular tools that are used in the environmental restoration industry and focus on those and how those can be really useful to you in site interpretation and in data assessment. We are ISO certified and it’s our goal to make sure that the data that is produced from these tools are something that can really be applied in site assessment. So being sure that the QAQC is something that you can trust.

And we work with great companies like Regenesis who offer these wonderful products and cultures, but we don’t sell any of those products or cultures ourselves. So we are an independent company that is separate from anyone else so that we can collaborate with groups like Regenesis to be able to offer you independent, unbiased analysis. And we’re also not owned by any consulting firms or anything else, so we’re very independent in what we do.

But we do try to be innovative. So we’re looking at new pathways and new identification of microorganisms so that we can always continue to be on the cutting edge that when universities and researchers identify these new microorganisms and new processes that are so important to us in environmental restoration that we can make these assays commercially available.

And so we do this around the world. We have over 4 ,500 customers around the globe, and we do keep a database that I’ll mention briefly at the end of my talk today that has over 150 ,000 samples. So it can help you add some context to the data as you’re looking at different analytics that are done, like QPCR analysis that Dane mentioned earlier.

So, we’ve seen a real revolution in microbiology, and so most people are pretty familiar with quantitative PCR. It’s become kind of a household name over the past five years in particular, but we’ve also seen a real uptake in publications about PCR, as you can see in the slide here, where the annual publication accounts have really grown dramatically over the past two decades in sequencing. NGS is next generation sequencing.

So a ton of publications about new organisms that were identifying new genes that are being isolated. A lot of publications about stable isotope probing and compound specific isotope analysis. So we’re seeing that these tools are regularly used in site assessment all over the world. And so we know a lot of information about how to apply the data at this point. Another thing that has come a long way over the last two decades during my career is something called a bio trap sampler.

And I just wanna briefly introduce that to you here because I’m gonna be talking about that in the case study with the product that we tested from Regenesis here today. So this is a passive microbial sampling tool that is used in a monitoring well so that we can colonize organisms that are active in the subsurface. And these were developed by the University of Tulsa and by DuPont and they are composed of biosep beads. And bioset beads are the real key to this because they’re composed of patterned activated carbon and Nomex.

Nomex is what a fire retardant suit is made from and the pack gives us the ability to absorb compounds that we’re interested in from the environment so the chemicals or the contaminants that are there in the subsurface then microbes can colonize and start to form biofilms and grow and you can see that picture to the right hand side of my slide where you can see the cross section of the bead you can see how porous that is.

So biofilms, little microorganisms, can form inside those beads, and the surface area is really high in each of these beads. So one gram of beads is the same surface area as a football field.

So a massive amount of space for organisms to form biofilms, grow, utilize those contaminants that we’re interested in, and then we can add different amendments, like I’m going to talk about today, to these biosep bead samplers, and then we can see how the microorganisms change over time. So, there are two different types of biotraps that are primarily available. There’s standard ones that are just plain powdered activated carbon and Nomex that are put down in the well so we can look at the normal community that’s there.

Or there’s something called an in situ microcosm study that’s kind of similar to a bench scale study that you’ve probably heard about, but we’re doing them in situ. And that’s what I’m going to show you in the case study in just a few minutes. And so, these are where we can add those different amendments and test them in situ. So, we’ve been doing this technology for over 25 years now. These were initially identified by Dr. White, the founder of our company, at a conference and they were developed for a completely different application, but he thought how great they could be in environmental restoration.

And so, he talked to the patent person for this and licensed this technology for our company. So, you can see this on the left-hand side of the slide, this is the original bio trap that was there, and we use them in monitoring wells all the time to be able to look at microbial populations.

Then over the years, we learned that we can load different contaminants that we’re interested in, and I’m not gonna get into this a lot today, but if you’re interested, you can always follow up with me offline about something called stable isotope probing, where we can put a heavy-weighted compound of interest, like a heavy-weighted benzene, and we can track that degradation into the microorganisms, or we can use them in our in situ microcosm studies so that we can test different amendments like I’m gonna show you.

Another revolution that’s happened is the ability to use something similar to a bio trap called a MEN trap for abiotic sampling. So in the past, when we wanted to understand abiotic interactions in the subsurface, we had to go out and collect and drill a sample and look at those sediments or that material that was being collected from that drilling. And that’s very expensive.

So we worked with Arcadis on this and it is a patented technology to modify our bio trap samplers into this device called a MEN trap that you can see here on the left-hand side of your slide. And what it is instead of the beads that are inside that we were just talking about, we have these mesh bags that contain either material from the aquifer that there, or we can put different sands or whatever composition of material that we want to put inside there.

We’re going to sew these into this mesh housing, deploy them inside our slotted PVC units that we use for the microbial sampling, and then as you can see in that middle picture there, this is what it looks like when we retrieve them. You can see that black precipitate that’s formed showing that iron sulfide formation. So it’s a way that we can mimic the processes that happening on those surface materials and be able to retrieve it very easily in situ and then test it to better understand and document abiotic degradation.

So let’s get to the meat of what we came here to talk about today. This is an in situ microcosm case study. So this is an old manufacturing site, very common to what we see locations around the world. The focus is on the source area. They have a mixture of TCE, 1 ,1 TCA, and they are seeing a little bit of daughter products that are being formed. They do have total CVOC concentrations that are up to 25 milligrams per liter.

And what’s a little bit unique about this site, and we do see this fairly commonly, is that they have a good bit of sulfate. So sulfate can range in this location up to 790 milligrams per liter. So, the site manager decided he wanted to figure out what to do for this and trying to think about the best treatment strategy.

So, looking at the lithology of the site, he could see that when he did a cross section that you can see displayed here, that he had two main parts of the formation. There was a silty clay underlain by a fractured limestone, so he wanted to look at both that upper area with the silty clay, and that’s a monitoring well that was installed MW16 that we’re gonna look at in our study today.

And then a deeper area, MW29B, that’s our limestone area. And these are gonna have differing geochemical characteristics because of the clay versus the limestone. But when he was thinking about it, looking at this high sulfate, he was thinking how it tends to lend to a bio plus biogeochemical type approach.

So what he wanted to do was to take some of the products that are available and test them in the in-situ microcosms to figure out a treatment strategy. So this particular project manager calls these a way that he can fail on a small scale so he can test different amendments in a pretty reasonable fashion so he can decide which amendment he thinks is going to work best in this aquifer. So what he did was he chose two different wells at the different depths that 16 that’s the shallow clay area and then 29B that’s that deeper limestone area. So he put in situ microcosms in there.

And before I get to it, I just want to be sure that we’re all on the same page, because a lot of times we talk about biotic degradation pathways, and we see those slides a million times at conferences when we travel. But we often forget abiotic degradation, which I think can be a very, very important part of site assessment. So with biotic reductive dechlorination, when we’re thinking about TCE, if you’ve to any remediation conference in the last decade you’ve heard of, dehalococcoides.

So dehalococcoides is the wonderful organism that can do complete reductive dechlorination from TCE through cis-TCE to vinyl chloride to ethene very efficiently, so you get complete reductive dechlorination. So knowing we have dehalococcoides is very important to us in our site assessment from the biological side. From the abiotic side, when TCE is transformed with iron sulfides, as you can see in that lower picture here, we get acetylene, ethene, a little bit of ethane, and some carbon compounds that are formed.

Well, since we can produce ethene from biotic reactions, it’s hard to know if we’ve got abiotic transformations, and acetylene, if you’ve tried to measure it in the environment, can be really difficult to capture and measure acetylene because it can be transformed very quickly, so we may not capture that end product. So understanding abiotic degradation is sometimes a little more complex to document and be able to prove that it occurred.

So from the chlorinated ethane side, for 1 ,1 TCA, we know with microorganisms like Dehalobacter and Dehalogenomonas, you get transformation to 1 ,1 DCA and then to chloroethane. From the abiotic side, we’re going to get with iron sulfide formation, some 1 ,1 DCA, ethene and 2-butene that are being produced, but it’s a very low product recovery. And so again, it’s kind of hard to document that abiotic side.

So the great thing with an in situ microcosm is we can custom make these in a lot of different configurations so we can answer whatever question that we want to address in the subsurface. So kind of like when you’d set up a bunch of bottles in a bench scale study, you sacrifice those one at a time from the bench at different time points So we can see how those are responding, what the organisms, what the chemical contaminants are doing. We’re trying to do this in situ. And the way that we do it is with these 15 inch slotted PVCs that you can see here in the center picture.

And the key to this is they have baffles on each end. Those baffles prevent that circular groundwater reaction that happens so that we’re not getting crosstalk between the units. So then we can put multiple of these units down in a well and we can test different treatment strategies. So as you see in the left-hand side, we’ll take iHooks and connect the different control unit to the treatment units, and then we can see what’s happening naturally compared to different treatments that we wanna add.

So then inside each unit, we’re gonna put different types of suppliers and collection devices so that we can test the different things that we’re interested in. So we can have supplier sponges or baggies. So whatever regensis product that you want to test, we can load them in different ways into these sponges or into baggies to be able to deliver them in the unit.

Then we’re going to put a passive diffusion bag that you see that second picture down there to look at the contaminants of concern. We’re also going to put the bio trap sampler to look at the microbial populations. And then we’re going to put a biochemistry vial that has a passive membrane on top. So we can look at how sulfate changes, how VFAs are changing, and then like methane, ethane, ethane type situations.

So in this particular case study, this is how the train looked that was put into the well that you can see here on the right hand side. So let’s first look at the red circle here of our control unit. We had passive diffusion bags for our compounds of interest. We had bio traps to look at the DNA, and then we had some geochemistry bios so that we could look at the sulfate concentrations and how they were changing as we performed the study.

The middle unit, we had a biostimulant product that was added onto the supplier sponges on both sides of the unit. We also had, again, the same middle components of a passive diffusion bag, a bio-trap sampler, and that geochemistry vial. And one thing that was unique in this is we took one of the Regenesis bio-augmentation cultures that they sell, and that was loaded onto the bio trap that was here in this location.

And so we can do that commonly to test this in situ to see how these cultures are going to survive and what impact they’re going to have on the contaminants. So in that bottom unit, it was the same thing as the middle unit. The only difference was it was a different product that was added into the sponges so we could see which one performed the best.

Due to time today, because I don’t want to focus solely on this little in situ microcosm study, I want to show you kind of the whole thing that happened at this site because it was really exciting. I’m just going to summarize what we found in the data. It ended up being that that middle unit, the Regenesis product that was added there, was the one that won in the study across all points that we measured.

We saw the biggest drop in the VOC concentrations within that unit. We also did something called compound specific isotope analysis, where we look at the fractionation changes in the TCE and the TCA within the unit. And we saw the most enrichment of those two compounds in that middle unit. We also saw a decrease in sulfate, so sulfate was getting consumed in this process. We saw not only ethane, but we were actually able to capture acetylene production in that little voivile. So that was an exciting, unexpected surprise that happened in this study. So we were seeing not only biotic degradation, but abiotic degradation as well, and we saw an increase in BFAs.

And one little aside for you guys that are familiar with the microbiology on these types of studies, we typically see a lot of dehalobacter, especially with chlorinated but we really didn’t see a lot of the halobacter in this particular site. It was low in all the units.

Okay. So what they did was they decided to take that amendment in that culture and go full scale with it. And so you can see the plot of the contaminants over time here. You see the injection one that happened and an immediate decrease in all of the compounds. They did have a little bit of rebound that happened in a second injection that occurred, but then quickly everything was gone.

So that what they did was they had decided to put this in MW-29B and just do it in this source area, but it went away so quickly and they had such good success from it. They were very shocked at how fast it went away and they ended up having so much success that their source area treatment actually ended up cleaning up the whole source area from this one location they injected in. So they expected it to just be a pilot study that they would expand into something bigger, but this bio- biogeochemical approach, this electron donor, and this bio-augmentation ended up being so successful that they got total source area remediation.

So it was, it’s one of my favorite case studies because it’s nice when you have an unexpected action like that and it was such a good cost savings for what happened in that total site assessment. Very exciting results and I think it showed that the in situ microcosms definitely helped point us in the right direction to be able to select the right amendments and to be able to move forward in a good fashion at this site and to be able to get both the biotic and abiotic degradation to occur in these locations.

So that brings me kind of to moving forward in the next steps of what I wanted to discuss today before we wrap up. The microbial revolution that I have seen in my career has just been super exciting. So I’ve been doing this for 23 years now and when I started, when we looked at microorganisms and we tried to understand organisms in the subsurface, we were counting them on plates and counting them on plates is really difficult if you’ve ever tried to do that and it’s not very accurate.

You can trend data but that’s about the best you can do. Then we moved to doing PCR and so we would do plus plus plus to tell you if you had some the halococcoides, moderate amounts of the halococcoides or the band of the halococcoides was bright. And that’s about the length of what we could tell you. Then we quickly moved to quantitative PCR.

And then for those of you that are familiar with it to quant array where we can do a lot of qPCR on a sample in a single analysis, looking at the halococcoides and co metabolic pathways and aerobic pathways for degradation all at the same time in a single test. And that was exciting, But I’m super excited to share with you today that we’ve actually been able to move more to moving into the field with doing this type of testing.

And so I wanted to share some results for that as we end our talk today. So QPCR, as you guys know, this is not meant to be an eye chart, but it is something that I wanted to show you just how much we’ve grown over this past 20 years. QPCR targets and applicability are very broad. So whatever the contaminant is, whether it’s chlorinated compounds like we talked about in this case study today, or if it’s petroleum hydrocarbons, or corrosion in industrial processes, whatever it is, we know a lot about organisms and pathways.

And we’re learning about others like new contaminants like PFAS. And, you know, we know things about 1 ,4-dioxane, but we’re always learning all the time. So we do have a lot of abilities to help you guys figure out which QPCR test can be the best to help you have success in your project. And we found in working with one of the oil and gas companies, they say that if they spend 10 to $15 ,000 in testing, they can save anywhere from 300 to $700 ,000 in savings on their site assessment because they can respond more quickly and pivot well and make better decisions more cost-effectively.

So I do think there’s a large value and applying molecular tools like this in your site. And now that we can do it in the field, that makes it even easier for these remote locations. So one of the concerns that we’ve always had is if you’re out at a remote location, say a platform for oil and gas, or if you’re in the North Slope of Alaska, or you’re in some remote area where you just can’t get to a FedEx to ship samples, what do we do to preserve the DNA? Well, recently, Microbial Insights came out with this patent pending kit for DNA extraction. So you filter the water, just like you guys are used to doing or the microbial sampling, and then you extract it in the field.

Any person can do this. It doesn’t have to be a molecular biologist or an advanced degree. It can be any field tech. The procedure is very, very simple, easy to walk through. You can do it in the field. You can do it in your hotel room. It takes about 45 minutes to do six samples. And this way, you can ensure your sample integrity right there in the field. When we’ve got that DNA extracted, it’s very stable. You don’t have to ship it that same day.

You can hold it, ship it at the end of the week. We have a study that we’re doing in Qatar right now that they’re holding it in a freezer, and they’re going to send it to us at the end of six months when they’ve completed this whole study. So it gives you ways and flexibility in your project assessment. So if you want to send the samples to us for extraction, you can do it, but now if you want to do it yourself in the field, this is available.

And not only that, but you can also do the QPCR yourself in the field. And you know, the big value of QPCR is having assays that you trust. So these are the same assays that we use in the lab, but we just lyophilize them into tubes that can be used in the field. And I’m going to show you in just a minute the lab results and how validated it is against the lab data.

These systems were developed for the Department of Defense. And then during COVID, they got tested kind of everywhere because we needed those in kind of extreme environments for COVID testing. So these little units that you see are about the size of a speaker and they come with a cell phone. And so you don’t have to download apps or do anything. It has everything you need in this package.

So once you extract the DNA in the field, You take an aliquot of the DNA, put it in the tube, put it into this little unit, you run it for an hour, you have your QPCR results. Very simple to use. It’s ideal for emergency response. I used it at the East Palestine train derailment when I went up to do sample collection there. It was a great tool. You get reports back very quickly. It’s a great screening tool if you’re trying to choose which wells you want to sample from.

And again, just gives you flexibility in what you want to do. And it’s available for chlorinated compounds like the site we just talked about, for petroleum hydrocarbons, and then for the oil and gas industry in particular, or industrial processes for corrosion-based testing as well. So let me show you the comparison data before we close out today. So these are some samples that were done with the bio extract kit in the field actually out on the bed of a truck in the summer, compared to samples that we brought back to the lab.

And you can see from the total bacterial counts here that it’s very reproducible. And we find that for pretty much every target that we’ve tested so far. So very excited to have this tool capable. And as I finished this presentation today, I just want to remind you, the value of qPCR is in knowing what those numbers mean.

So whether you do it in the field or you send it to us in the lab, adding context to your data is really what’s going to help you understand be able to do a really good assessment. So use the microbial insights database. It has over 25 years of qPCR data from over 250 genes and organisms that we have. There’s over 150 ,000 field samples in that database, and it continues to grow every day. It’s from all 50 states here in the U.S. and more than 50 countries on all seven continents around the world.

So you can go into that database and go, is this number of genes that I have for vinyl chloride oxidation, is that a high number or is it low? And every gene is a little bit different. So being able to add that context can really give you some value in your interpretation. So I would encourage you to use that to strengthen the assessment that you have from these types of tools. So it’s our goal to help you generate actionable data.

And I hope that’s what this presentation showed you today, whether you’re trying to figure out which amendment that you want to use, you’re trying to use it in the field and prove that you did such a great job with this amendment in the application, or if things aren’t going right and you wanna know what’s off that you need to pivot and be able to make a better assessment moving forward. I think molecular tools give you the ability to do that.

And I’m gonna encourage you to use the database, help add some context, and then start to trend your data over time so you can get some real value in the interpretation of that data. So wherever you are in the world, these samples and these tools are being used. And now that we have these field extraction kits, it makes it really simple, but we do have labs all over the world. So wherever you’re collecting samples, reach out to us. We’d be happy to help you.

And so I know I gave you kind of a lot of information and I’m happy to take questions at the end. I will turn it over to Dr. John Freim so he can give you some more information right now.

All right, thank you, Dora, for the informative presentation on my part of the talk. I’m gonna describe using ZVI to promote effective end-of-situ remediation. So when we do our remediation projects using ZVI, there’s a limited number of instances where we actually do ZVI alone. In most cases, we’ve actually learned that it’s better to combine ZVI with other amendments.

So in this talk, I’m gonna give you two specific examples. One will be source zones, where we found that it’s better usually to combine ZVI with emulsion products to do mass removal.

And then the second situation that I’ll describe are dissolved plumes, where we found that is usually beneficial to combine our ZVI product with plume stop colloidal activated carbon in a PRB permeable reactive barrier configuration to mitigate down gradient migration of the plume.

So what is our iron product? At Regenesis, our iron product is branded as S-Micro ZVI, kind of an acronym or shortened version of sulfidated micro ZVI. And its key feature is that it’s sulfidated.

And what does sulfidated mean? Well, what we do is we deposit a thin layer of reduced iron sulfide onto the surface of a zero-valent iron particle. So we start with iron, we process it down to about two to three microns, then we use a proprietary process to electrochemically deposit iron sulfide onto the surface. This is called a core shell configuration. The interior iron is basically kind of the engine, supplies the electrons that drive remediation reactions and the thin surface layer gives it its beneficial properties.

So what are the beneficial properties? One is accelerated reactivity with chlorinated solvents. This feature has probably been known for at least 10 years and it’s really remarkable how much faster we can remove TCE, for example, using sulfated iron in comparison to bare iron. And to prove that we’ve done a lot of in-house studies. On the right is an example of one. This is a closed-bottle microcosm study where we took a small amount of sulfated iron, about four grams per liter, and then added to a bottle with 150 micromolars of TCE.

Periodically, about every day, we remove sample from the bottle, measure the composition, and that way we can determine the degradation rates and the kinetics and the amount of daughter products that are produced. So you look at this plot on the right, you can see that with this small amount of sulfated iron, after only about 14 days, we removed all of the TCE from the bottle.

It’s a nice exponential decay, which is indicative of pseudo first order rate kinetics. And if you back out the numbers, the half-life for this is about three days. If you compare that to bare iron, if we’ve done studies with it, this dose is gonna be a half-life of about 100 days.

So the reactivity is about 30 to 40 times that of bare iron. And this has been corroborated by other groups and universities as well. Another thing that’s beneficial is that your DCE yield is relatively small. If you look on the plot, the yellow triangles, we see about a 10% DCE molar yield. And this is attributable to the fact that we’re using beta elimination as our reaction pathway instead of the sequential dehalogenation that’s used in biological degradation processes.

One caveat is that this TCE does react with iron but the kinetics are inherently slower than TCE and this results has been corroborated by other research groups as well. I think what’s even more interesting that that’s come to fruition more recently is the fact that S-Micro-ZVI or sulfidated iron is going to give you a much longer longevity of persistence in the ground.

When you apply ZVI into remediation site there’s essentially two things that can One of them is beneficial. That’s to react with the contaminants and reduce them to ethane, ethane, or whatever your terminal reaction product is. But at the same time, iron is a powerful reductant. It can also reduce water to molecular hydrogen. This is called hydrolysis.

And I think the first really good paper on this was about eight years ago by Fan et al at Oregon Health and Science University, published in the S &T. And they showed that sulfated iron, almost all the electrons go to the chlorination with bare iron, almost all the electrons go to water. This is really important because iron is a consumable as it reacts, it goes away.

And if you’re boiling it most off the water, it’s going to really severely impact your longevity or persistence in the ground. And once you have something in the ground, you generally want it last for a long time. We’ve verified this, there’s a on our website, a case study availability, where we ran a column with S-micro ZVI for four years and maintained flow reactivity with TCE. So that’s kind of the basics on our products.

And I’ll tell you how we use this in practice and kind of the theory behind how it works. So when we have a storage zone, when we’re trying to do mass removal, we almost always co-apply a mixture of several products. One of them is obviously S-micro ZVI, but we also add an oil water emulsion, such as 3DME or EVO, the chlorine microbes, nutrients, pH modifiers, vitamin B12, etc. This is essentially equivalent to what you’d use on a straight ERD site, but you’re also adding SCBI to enhance the degradation rate in the daughter products.

So why do we use emulsions? Well, they have a really, really cool property that works extremely well in DNAPL sites. So as you know, the DNAPL such as PCE is very hydrophobic. That’s why it’s DNAPL. It doesn’t dissolve into water. If it does, it does very, very slowly. But the droplets in our organic emulsions are also hydrophobic, such as soybean oil. If you go back to your college chemistry class, I still remember the teacher telling you that like dissolves like.

And we use that to our benefit upon contact, the PCE droplets or the DCE droplets actually partition and dissolve into your oil droplets on your emulsion. And get it out of the DNAPL phase, then eventually these droplets will biodegrade and release the contaminant into the aqueous phase where the degradation events occur. So if you ever have a DNAPL site and you try to do a remediation program, I strongly recommend using some sort of emulsion product.

So why are we adding iron? You don’t need to add iron to do this partitioning and dissolution process. Well, we’ve realized, other people have realized over the years, is that biological events do work, but they have a couple of shortcomings.

One is that they’re inherently slow, you know, you talk months, years to achieve closure, and also you end up with inherently, you know, a one-to-one stoichiometric conversion to daughter products, such as cis and vinyl.

So we tested this theory in a column study in our lab, where first of all, we did a biological only approach, where we had a column pumped effluent up through the column with 15 micromolars TCE with sodium lactate as their donor, the halococortis and nutrients, and we measure the concentration of our contaminants in effluent.

This column has about a one week residence time of your solution within the column. And the results are kind of what you’d expect. You see on this plot on the right, it works pretty well with TCE. After about eight weeks, column becomes acclimated. I think your microbial communities start to flourish, and you don’t have much TCE that leaves the column. But what you do see is pretty substantial increases in daughter products.

You have quite a bit of DCE and vinyl, and after 10 weeks in this column, after it’s acclimated, we really only have about a 20-molar percent conversion to ethene. So it does a really good job on TCE, But although it will happen eventually, it’s much slower to get rid of the daughters.

This slide shows the results of this exact same column setup when we actually placed iron S-Micro ZVI into the column before the start of the test. And you can see the results are quite different. There’s no TCE eluted at any time during the experiment. That’s the blue line at the bottom of the x-axis. And although we do have some daughters, they’re lower in concentration and they’re not as persistent as we see in the biological only column. And after eight weeks once the columns become acclimated, we have a clean solution coming out of the column.

So this pretty much clearly shows, and this has been shown also in field results, that adding iron to a donor greatly accelerates the reaction speed and also reduces the amount daughters that you’ll encounter. Bottom line, you get faster remediation. So now I’m going to segue over to the situation where we’re going to do plume treatment instead of source treatment.

And in this case, instead of using S-Micro-ZVI in an organic donor, we’re going to use S-Micro-ZVI in our PlumeStop colloidalactivated carbon. So this figure here kind of depicts what a PRB looks like. On the right-hand side where it’s red that’s that depicts your contaminated water. You in place your amendments in the ground with a DPT rig usually and that’s the gray area in the middle of this figure.

Water advects naturally from right to left and as it passes through the barrier contaminants are removed and you get clean water down gradient. So we found and I’ll explain this a little bit on the next couple of slides, that using S-Micro-ZVI and PlumeStop together gives us the best results. It’s kind of a two-phase approach. The PlumeStop will absorb the contaminants, and while they’re absorbed onto the surface in the PlumeStop, S-Micro-ZVI will destroy the contaminants via abiotic pathways.

So we have a model in-house called PlumeForce, which is developed by Jeremy Bernstein in England. It’s a really powerful model, And what this will do is kind of predict how a burial will perform with different amendments, different groundwater flow, et cetera.

So I’ll explain what this plot is here. The cross hash area is a 10-foot wide barrier that depicts where we apply our amendments. The groundwater is flowing from left to right. So our influence, we have 4 ,500 micrograms per liter TCE and 500 micrograms per liter CIS TCE.

And as we go from left to right, we can see where the contaminants are removed in the barrier and how much. This has six grams per liter S-Micro ZVI in the effective porosity. You can see it works really well with TCE. The red line, it goes away pretty much in the front half of the barrier. It’s converted mostly to ethane. It doesn’t work quite so good with SIFs.

This predicts after four years, you’re going to equilibrate with approximately the same amount of cis coming out of your barrier as you have going into the barrier. And this is because, as I explained earlier, the kinetics with cysts DCE are inherently slow, and there’s just not enough residence time within the barrier to remove that before it advects that ingredient.

So this can be solved by combining S-Micro ZVI and PlumeStop together. This is exactly the same situation we had on the previous slide, but the only difference is that we’ve also added six grams per liter plume stop to the porosity, and you can see that the results are much, much better. As it was the case before, the TCE is generally removed and destroyed in the front half of the barrier.

We also get a complete removal of cis-DCE, and as I explained earlier, we can explain us away by the fact that when the groundwater encounters the plume stop, its eviction rate is greatly retarded by an order of magnitude or greater.

So it provides more time within the barrier, and even though it’s slower reacting, the residence time is great enough that you’re not going to see any downgrading eviction. So these materials work really, really well together in barriers with chlorinated solvents. So I can give you some examples of how we put these concepts to practice. This is a figure of a job that was done a few years ago. The source area is in the red there at the bottom.

And this is an old site. This had been around for 30 years or even longer. This was addressed at the bottom using a mixture of excavation and enhanced bio using iron and 3D media together and downgrading it to intercept the plume and prevent contaminant from migrating offsite.

We applied a series of five PRBs and those are depicted the injection points by the green triangles. You notice there’s also monitoring wells, you know, about 10 to 15 feet downgrading of each of these barriers to measure the performance.

And the goal here is source mitigation, but more importantly, wanted to mitigate offsite migration of the contaminants to the neighboring property. So we did a lot of monitoring on this site. We’ll just focus on the upper left-hand plot right now. This is the shallow area downgrading of the first barrier. The little cartoon of the DPT rig on November 30th is where we applied the amendments, the and the plume stopped together.

And you can see as expected, we had a near immediate and mostly complete removal of all our contaminants and the daughter products both. On average across the site, we had a more than 95% reduction of the contaminants within a year downgrading of each barrier. What’s also interesting is that we measured ethane and each one of these barriers had pretty large spikes of ethane. That’s depicted by the upper line in the upper left-hand corner, showing you that a lot of that was produced.

That’s more or less proof that we’re actually seeing degradation in the barrier. Some people question this in the past. Well, PlumeStop just absorbs the contaminants but doesn’t promote degradation. Well, this is pretty clear evidence that when we add iron to the system, and we get a combination of both and really excellent results downgrading.

I would say that in the past six or seven years since S-Micro ZVI has been on the market, we’ve done well over 200 sites with the enhanced bio approach that I described earlier for the source on mass removal in over 100 PRB sites. So thank you for your time. If you have any questions, we’d be happy to answer them and thank you for your time.