Demonstrating Contaminant Biodegradation in Conjunction with PlumeStop® Liquid Activated Carbon™

So, today I’m gonna be talking to you about the PlumeStop product of these colloidal activated Remediation technologies that we have. And specifically, I’m gonna be focusing on some bench and field-scale studies that we did together with Regenesis, that were designed to evaluate whether biodegradation can occur in conjunction with these colloidal activated carbon applications. So we get not just the sorption of those contaminants to these carbon molecules, but we’re also seeing contaminant destruction. So I’m gonna walk you through a couple of bench scale studies, and then more importantly, what happened when we took this to the field. What are we seeing here? How do we monitor effectively to make good management decisions?

So, as Dane mentioned, I’m the president at Microbial Insights. For those of you that aren’t familiar with Microbial Insights, we’re an Environmental Laboratory. And what our specialty is, is looking not just at the VS-C concentrations, we don’t do the standard chemical and geochemical analysis, we focus on using molecular tools to help us understand these bacterial processes so that we can to look at concentrations of contaminant degraders at different functional genes that can be important in these degradation processes. And so, when we apply a product like PlumeStop and we get the compounds being adsorbed to this activated carbon, what do we see from there? Do we see these contaminants biodegrade? So that’s really what I want to address in this presentation today.

So, I’m certainly not an expert on the product, people at Regenesis can answer those types of questions. But I am more than happy to talk about the microbiology and what we’re seeing at these sites, and the best way to kind of collect and monitor as you’re moving forward.

So, let me move to my next slide here. So, just to be sure everyone is on the same page, when we’re talking about these Colloidal Activated Carbon products, we’re referring to the size of the carbon molecule. So the activated carbon here, we’re talking about is about a nanometer to a micrometer in size. So very small molecules. And these colloidal particles are dispersed through some type of medium so that we can get good distribution of these particles into the environment. And the real key to this is these activated carbon particles allow for adsorption of contaminants to these colloids so we can get a really quick lowering of our aqueous phase concentration because we get this adsorption to these particles.

So then, what’s really interesting is can we use that in our remediation practice, and use it with different electron donors or acceptors to really biodegrade and get contaminant destruction? And so, I think, I wanna point out to you before I get into the presentation that I’m gonna show you some data from chlorinated sites just to keep a simplified presentation today, but this can also be used…and we have used it, at petroleum hydrocarbon sites. So it’s got a variety of applications that I think you’re gonna see coming out over the next few years with this product. So whether we’re using it combined with an electron donor or an acceptor, we can use these same types of tools to help us understand and do better site management.

So when we talk about these products, and we talk about PlumeStop, this is a product that is a stabilized form of these colloidal activated carbons. And it distributes really well in the subsurface. So Regenesis a lot of work with this and they get really good distribution. And then we see rapid reduction of that dissolved phase contaminant concentration in the subsurface. And then what we’re interested in seeing is how we can simultaneously promote biodegradation, so we get a lot of benefit from adding this product.

So, this diagram here that I’m showing represents what we would typically see in our subsurface. And there’s two areas that I wanna point out. We have two main areas of that we’re concerned about. We have our high permeability zones and our lower permeability zones. So when we have our contaminants that happen, they’re transported through these high permeability zones, and over time, those contaminants start to diffuse into our lower permeability zones.

So, when we add our PlumeStop to the site, these colloids are going to be distributed in those high permeability seams. And we know that remediating contaminants in those high permeability seams is much easier, right? Because we can get things in contact with each other and get transport easily through these areas. But it’s much more too difficult for us to remediate those in those lower permeability zones. So with the PlumeStop, the advantage that we get is not only reduction of those high permeability zones, so we also get the ability to capture some of the back diffusion that happens in those lower permeability zones.

So Regenesis, as I mentioned, has done a lot of work on this product over the last few years, and they see good distribution, they get good contaminant adsorption to these activated carbon molecules. And then where MI came into the study is we wanted to use these molecular biological tools to help us look at what happens with these compounds. Once they’re bound to these activated carbon molecules, are they still bioavailable to our microorganisms? Can we show that we’ve got some evidence of biodegradation? So what we’re doing with Regenesis in these is looking at some bench scale studies, and proving efficacy of this, and then translating that to where it’s most important in the field. What can we do in the field to get degradation?

So, Microbial Insights’ role in this was to look at different concentrations of bacteria present at the site, how to do the best sample collection. And those are some of the things that I wanna present to you today because with these tools…for instance, on these chlorinated sites, we can quantify concentrations of our halorespiring bacteria, that we can use that to help us demonstrate the potential for biodegradation following sorption. And that immediate decrease in our contaminate concentrations, we can then understand how bioavailable they are. Can we still maintain these high dehalococcoides concentrations and these other degraders? Are they still present at high concentrations even when our aqueous concentrations are low? So do we have evidence of that biodegradation? And then if we get that in the lab, then we can translate that to the field.

So, in order to answer these questions, Regenesis set up a couple of bench scale studies and then some field applications that I’m gonna walk you through today. And again, I’m gonna be focusing more on the microbiology and what I think is going on there. So for the bench scale studies that I’m gonna be covering, there were two experiments that were conducted. There was a PCE Microcosm and a Tank Study. In the Microcosm study, PCE was continuously added to some control and treatment microcosms. And then in the Tank Study that I’m gonna show you, there were some tanks that were set up which explored the back diffusion, and the effects that we get on our microbial populations from this. And then based on the results of the bench studies, this is then taken to the field. And I’m gonna show you two separate case studies that were selected for different reasons, but they were identical treatments that are applied. And I wanna show you how we looked at the contaminant destruction as well as understanding those halorespiring bacterial populations, and how those changed at these sites.

So let’s start with our bench scale studies. For the first one, this was some microcosms that were set up, and the PCE Microcosm Study was done to simulate the injection of these carbon molecules into an early stage plant where we have high aqueous concentrations of contaminants in those high permeability zones, so we can monitor to see if we get adsorption to the PlumeStop material, and then what happens if microorganisms are present. And then the second study took this a step further and there were tanks that were set up with different conditions, so we could simulate and understand better what’s happening with that back diffusion. So more reminiscent of what we would see from a late stage plume. And then we can take and look at these molecular biological tools, look at all the lines of evidence that we have, and try to understand what’s important for monitoring as we move forward.

So, let’s get into some data from these. Here I’m showing you the results of the PCE Microcosm Study. So our PCE concentration here is our total PCE mass. So it’s our aqueous PCE plus the adsorbed PCE mass on the Y axis that’s plotted here. And we’re looking at it over time. And we have spikes of PCE that were happened. So if you follow the blue line and the green line, that’s our sterile control and then the same sterile control. The only difference is it has PlumeStop at it. We see that we have increasing amounts of PCE that’s being detected…total PCE in both of these as would be expected since we have no bacterial populations, right? We’re just getting sorption of this compound in the one with the PlumeStop.

But look at the difference when we compare that to one where we have dehalococcoides added as a biological. Our biotic microcosm, we see very rapid reduction of our PCE mass, it’s really gone quickly after the spike and we’re presuming that this is due to biodegradation. As they looked at daughter products, they saw the same thing, very rapid degradation as soon as it’s added into these microcosms.

So, that first little study was just to make sure is that still bioavailable to the organisms? It looks like it is. And we have high concentrations in our source area. Looks like this is gonna be effective to not only absorb that contaminant but also to get some biological degradation.

So let’s look at the results from that tank study. Here I’m showing you the concentrations of dehalococcoides and the functional genes associated with dehalococcoides. Looking at our TCE-reductase gene and our vinyl chloride-reductase gene, and there were four different treatments that I’m showing you here. The first is our blue bars. That’s an unamended control. The second is Plume…a tank that had PlumeStop only in it that was added, and a third tank that received a culture of dehalococcoides and lactate but no PlumeStop. So this is the third bar, that green bar that you’re looking at is similar to what we would see with a typical bio-augmentation process. And then our fourth tank, the only difference in that tank was that PlumeStop was added, in addition to the dehalococcoides and the electron donor. So PlumeStopp plus bio-augmentation in that purple.

So if we start with the control and the PlumeStop only, this red dash line here is important. This is our limit of detection for these assays. We can say that we don’t have any dehalococcoides or those vinyl chloride and TCE-reductase genes that are being detected in our samples in those tanks, and that makes sense. We don’t have any microbiology that was added, any organisms that were added. But when we see dehalococcoides added, like the traditional microcosm study, look at this, we have concentrations of dehalococcoides that are around 10 to the 3 cells per mill and around 10 to the 2 of our vinyl chloride-reductase genes. And then the only difference in that fourth tank was that PlumeStop was present. And when PlumeStop is present, we see an order of magnitude greater for our dehalococcoides and our vinyl chloride-reductase genes in that particular tank.

So if we look at the Microbial Insights database where we’ve looked at hundreds of thousands of samples from around the world, we know that getting a concentration of 10 to the 4 cells per milliliter of dehalococcoides is really important to get useful rates reductive dechlorination . So if we take that information, this tank that has the PlumeStop, we hit that mark. So we should be seeing very efficient reductive dechlorination in these types of situations. And we see an order of magnitude greater in that tank with the PlumeStop. So very promising results from this study. Very exciting, when I got this data to see that.

So then moving forward to these field studies, we could have some confidence that this is really having a positive impact on our microbial population. So let’s dive into these field studies now. In order to confirm that these bench scale studies were applicable to the field, people at Regenesis set up some field validations sites. And I’m gonna show you the microbial results from these. We’re gonna focus on two here today. A former dry cleaner in California and then also a former manufacturing plant.

So with any field study, we always wanna look at multiple lines of evidence so we can assess that site. As you know, the first line of evidence we typically look at is our chemistry at the site. Do we have concentrations of our contaminant that are decreasing over time? Are we seeing daughter products form? That would be a generally what I would consider the first line of evidence. Then we’ll look at our second line of evidence here looking at our geochemistry. And we’ll try to understand…as an indirect indication of what’s going on, are we getting reducing enough conditions, if we’re talking about a chlorinated site? Do we see enough electron donor that’s available for our microorganisms to thrive? Are we seeing fermentation with our volatile fatty acids? Are we seeing proof that we’re getting hydrogen production for these organisms?

And then what about competing electron acceptors like sulfate? Are we stimulating our sulfate-reducing bacteria? So that would be our second line of evidence to kind of get some inferential information on what’s happening with our microbial populations. And then the 3rd line of evidence…and I think what you’re gonna see, this is a really important line of evidence when we’re talking about injecting PlumeStop at the site is to look at the microorganisms and cells. And we can use tools like qPCR and QuantArray to help us quantify different microorganisms that we’re interested in.

So I wanna walk through this for just a moment because I think these tools are very important and I know that these tools are commonly accepted. We use them as sites around the world. Most of you are probably familiar with it, but just to be sure everyone is on the same page and that we understand why we pick these tools, I wanna cover them just really briefly here before we get into the data. When we talk about quantitative PCR, quantitative is the key word there. There are very few tools that are out there that we can truly quantify and accurately quantify the number of microorganisms that we have or the number of a particular gene of interest that we have in our sample. qPCR is a well-vetted technology, it’s been used for almost 15 years at sites around the world. We know we can trust the data from this.

And there are two main types of genes that we use. We use a taxonomic identification. So if we’re talking about a chlorinated solvent site, we wanna to know do I have dehalococcoides there? Do I have dehalobacter there? We’re gonna look at the 16S to tell us who an organism is and to more importantly, quantify the amount of that organism. Do we have enough to efficiently remediate our sites? We can also look at functional genes of interest for chlorinated sites. We’re gonna look at a lot of Reductase Genes. Do we have enough of the vinyl chloride to get efficient vinyl chloride ethane conversion for petroleum hydrocarbon sites? We’re really gonna focus on that functional aspect and quantify, do we have enough of these aerobic and anaerobic genes to help us get efficient remediation of these petroleum hydrocarbons once they’re adsorbed to our PlumeStop material?

So we can get a lot of great data from the qPCR. And I’m gonna talk at the end of the webinar today a little bit about the sample collection because it’s a little bit different when we’re applying a material like PlumeStop. In my recommendation from what we found over time, in these studies, that you’re gonna collect typically a groundwater sample or a soil sample and then we’re gonna extract the DNA. And with these qPCR approaches, we’re gonna go in and individually run assays that tell us the concentration of these organisms compared to known standards. So we get individual numbers. So, qPCR is great, well-vetted technology.

The other thing we can do is something called QuantArray. And I wanna mention this because I think for some of these sites, that could be very very important especially when we have mixed contaminants that we’re interested in. We’re gonna collect our samples the same way and I wanna to be sure we don’t confuse this with the MicroArray because these are two very different technologies. With the QuantArray, unlike a MicroArray, we’re quantifying. So we know the quantity of our microorganisms. The other thing that we’re doing is like the tubes, we have little individual holes on our slides here. So the amplification happens within each of this little SubArray holes, these through holes. So you don’t get interaction and cross-reactions, and things that can happen like we have with MicroArrays. So it’s a different technology. We get quantitative numbers, we have individual assays that we’re seeing individualized results. But it gives us the ability to take qPCR to another step. So rather than getting that one individual number, now we can look at 20 to 30 different organisms and genes that we’re interested in, all at the same time. So kind of like qPCR on steroids.

So just quickly before we move on, if it’s Reductive Dechlorination and we’re doing QuantArray, I really recommend this especially if you have a mixed site where you’ve got ethenes and ethanes, or maybe you have chloroform that’s present because we can look at all of these organisms at one time. We can also look to see if sulfate-reducing bacteria might out-compete our dehalococcoides for the available hydrogen so we can have some lags that happen. We can get a lot of information with this type of tool.

The other thing that we commonly miss at sites that I’m happy to see people looking at more are Co-Metabolic processes. So, even in places where we’re trying to do anaerobic degradation, we can still have pockets of oxygen that’s available. And even microphilic amounts of oxygen. We can still have oxygen in methane, get methanotrophs that are present at our site. So they are co-metabolizing some of these compounds that we’re interested in. And then as many of you know, some of your sites have chlorinated mixed with BTEX. We can have these genes that are being expressed for BTEX degradation and we’re getting a side benefit, and getting Co-metabolic degradation of our chlorinated compounds.

So with the QuantArray, this type of qPCR technology, we’re getting a lot of information that we can use in our site assessment to do a better job. And we can see how the sorption of to these PlumeStop really stimulates these microbial populations and make these compounds bioavailable to the microorganisms.

And so today, when I dive into it, I’m gonna be showing you again just two chlorinated sites. So we can use this for petroleum hydrocarbons as well, so I just wanted to mention that before we move forward. Because you know, with petroleum hydrocarbons, we often have a lot of mixtures, right? We might have toluene, and benzene that’s remaining or naphthalene that’s present at our locations. We know a lot about the degradation processes for these different organisms. For instance, with toluene that I’m showing here, we know five aerobic pathways and one anaerobic pathway this compound can biodegrade. With benzene, we’re learning more and more about the anaerobic degradation pathways. There’s three pathways, we have genes for one of them that have been identified. Currently this carboxylase gene and this coenzyme A reductase gene that we can look at for anaerobic benzene degradation.

So if we have a petroleum slide, I would recommend this QuantArray-Petro to give us a better overview of our microbial community on that PlumeStop. We can look at aerobic BTEX and PAHs. I’ve listed quite a few of the genes that are here. We can also look at that anaerobic degradation, and sometimes we see these processes kind of happening hand at hand in our environment.

So, I just wanted to touch on that briefly. In case any of you that are listening today has some petroleum sites and you’re thinking about using this product, that’s the technology you can use for your microbial monitoring.

Okay, so as I promised, let’s get to this case study. And we’ll end the webinar today looking at a couple of case studies and then talking about the sampling. So the first case study that I wanna walk you through is a former dry cleaner site. And Regenesis chose this as a test site for a number of reasons. They had a long history of the ground water monitoring data all the way back to 2001. Baseline conditions at the site were generally aerobic and dehalococcoides wasn’t seen, it was below detection. There was no evidence of daughter product formation prior to this study, so it presented a little bit of a challenge with the redox conditions. But with no evidence of prior degradation, it also gives us the potential to see these great impacts when we add the PlumeStop and bio-augmentation to this particular site. Finally, it’s a sandy aquifer. We got a pretty good flow. The groundwater velocity here is about 10 meters per year. So the site is representative of a site where there’s a need to stop the contaminant migration, which is a common reason we would want to apply this particular product at our sites, right?

So here, I’m showing you on the first graph our contaminant concentrations as a function of time, just before and after the PlumeStop injection. So you can see this red line here. Our PCE concentrations were a little over 500 micrograms per liter. And the injection consisted of…I’m sorry of PlumeStop, dehalococcoides, and HRC for electron donor. And what do we see after injection? Look at our PCE concentrations. They drop to almost non-detect very quickly after our injection. So we’re getting adsorption of our PCE onto the PlumeStop material. So now what we’re interested is what’s happening with the biodegradation.

So let’s add in our dehalococcoides here, these blue bars. We can see prior to the injection we don’t really have any dehalococcoides that’s present. But after injection of the PlumeStop and bio-augmentation with dehalococcoides, we see very high concentrations of dehalococcoides. Look at this 10 to the 4 mark where we wanna be. And that was maintained for a period of about nine months even with removal of that PCE from the aqueous phase. So we’ve removed it from the aqueous phase, it’s now bound to that PlumeStop. We still see very high concentration of dehalococcoides that’s present at the site.

So very positive results here. And when we look after a period of time, we see our available have added to this graph our total organic carbon concentrations. We can see that by 15 months, our TOC has decreased to less than 10 milligrams per liter, and our dehalococcoides population start to decrease by orders of magnitude. I didn’t show it here but we also saw vinyl chloride-reductase genes that had been around 10 to the 3 cells per milliliter, are now dropping down to 10 to the 1 cells per milliliter. So at this point, dehalococcoides concentrations are likely decreasing because the electron donor availability is becoming more limited.

So the first thing I want you to notice here on this last plot that I want to show you is we’re zooming in because I wanna show you something really that I think is interesting here. So we’ve re-scaled the Y-axis so we can take a closer look at the daughter product concentration. And we can see what happened. So as we see electron donor was completely consumed. Unfortunately, we don’t have our qPCR data for this period. But I think it’s pretty safe to assume concentrations continued to decline. And the results make sense because there was no electron donor to support dehalococcoides, and we saw redox conditions at the site become less favorable. We saw a decrease in ferrous iron, an increase in sulfate. So once the dehalococcoides had decreased but look at this, we start to see a little bit of vinyl chloride, now granite, it’s a very low concentration of vinyl chloride, but again, it’s giving us additional evidence that’s convincing that reductive dechlorination of PCE was happening on our PlumeStop material. Once dehalococcoides is not there and biodegrading it very efficiently where we’re not detecting it, then we start to see a little bit of accumulation of that vinyl chloride.

So, the result of this first case study we see that the PlumeStop combined with HRC and bio-augmentation resulted in not only effective adsorption but we also saw some biodegradation. Dehalococcoides is an obligate halorespiring bacteria, and there’s no way we’re gonna maintain very high concentrations of this organism like we’re seeing here for nine months if they’re not active and utilized in the compound. So the compounds adsorbed the PCE and daughter products that are adsorbed to our PlumeStop must have been bio-available for these organisms to grow and thrive like they were doing.

And now, if we’d had an additional electron donor injection, vinyl chloride concentrations probably would never have been detected at that site. And that leads me to a really important point that I think we’ve learned in this. Microbial monitoring, whether you do the qPCR or the QuantArray like I described for you earlier, it’s really important to have this line of evidence because we may not have our first line of evidence that we rely on a lot looking for those daughter products because they’re so efficiently degraded once they’re adsorbed to these compounds.

So, based on the results that we’ve seen from this site and a few other sites, we may not see daughter products to form very effectively especially in aqueous solution that we could use as part of our line of evidence and what’s happening at the site.

Okay, so let’s move on to this last case study, and then we’ll talk a little bit about sampling before we close out. As you’ve just seen, we showed you a case study where PlumeStop was very effective in treating a source area. Now I’d like to discuss another application of using this as a barrier. So, this site was a former manufacturing facility that had been using degreasers since the 1950s. The site was underlined by a coastal plain sediment. There was sandy clay with sand stringers, and groundwater flow is about nine meters per year. And as you can see here in this purplish blue color, we have a dissolved Plume that extends quite a ways down gradient. So the PlumeStop was applied in this red box that we see here, and here are some of the monitoring wells that we used to understand what was happening in that injection. And so again, we had PlumeStop, electron donor, and dehalococcoides culture that were applied here.

So here’s the results from that. And I know it’s a little tough to see on the plot but our total VOC concentrations are this dark red line here. And we can see that our concentrations of our VOCs decreased rapidly post application of the PlumeStop. And look at our concentrations of organisms. We’re talking about the dehalococcoides, dehalobacters, dehalogenimonas and that vinyl chloride and TCE-reductive genes found in dehalococcoides and they increased to substantial concentrations. And look at this, they’re maintained even 300 days post-application.

So again, very positive results that even though our VOCs are getting adsorbed very quickly to this PlumeStop material, it’s still bioavailable for these microorganisms. So we see effective adsorption. Again, concentrations decrease very rapidly. Again, we weren’t really seeing daughter products that were being detected in our aqueous phase. And that dehalococcoides is present and very high concentrations. High enough that we should be getting efficient reductive dechlorination that remain there for over 300 days.

So as I sum up the things that we learned in these studies that we did with Regenesis, PlumeStop really leads to very rapid decreases in our aqueous COC concentrations. We saw this in the microcosms and the tank studies, and more importantly, in the field studies down to below detection limits. And then we are interested in the biodegradation aspect. And we did see that as long as there was enough electron donor that were made available, there were high concentrations of obligate halorespiring bacteria, like the dehalococcoides that were maintained, even though the contaminants resorbed to the PlumeStop and removed from aqueous phase. Again, Dehalococcoides is an obligate halorespiring organism, and that the only way that we’re gonna see our dehalococcoides concentrations remain on the order of 10 to the 4 cells per milliliter as we saw on that last study for over 300 days is if they’re actively performing reductive dechlorination.

Now, in that first case study, there was also chemical evidence for reductive dechlorination when we saw the donor deplete and dehalococcoides concentrations go down, we did see some very low concentrations of that daughter product of vinyl chloride being produced which brings me to my final takeaway on this. With any type of colloidal activated carbon products like PlumeStop, there’s a good chance we may not see daughter products like our first line of evidence, so we need to combine that with convincing second and third lines of evidence to make sure that we understand what’s happening at the site. And I think that’s gonna be the case whether it’s a chlorinated or a petroleum site is that we need to keep an eye on our geochemistry and look at ways that we can monitor these microbial populations as we move forward.

So as I close out the webinar today, one of the last things I wanted to do, a common question that I get from people when they’re calling in is you know, “How do I sample for this?” It’s very similar to what we would do in sampling for any type of project. If we’re talking about reductive dechlorination, the most critical sampling points are definitely within our injection grid. In the source area, if you see changes in your contaminate concentration by an order of magnitude, if there’s differences in your geochemistry at the site, those are important points. The hydrogeology is different, you might wanna collect samples there. There are some secondary locations that you might wanna do depending on the size of the Plume or if there is a receptor that’s of importance. But certainly this area, and maybe just down gradient from the injection grid would be really important for us to sample.

Now, when we’re talking about petroleum hydrocarbons. Those same points are important but we also wanna add in a background location because for petroleum hydrocarbon degradation, the organisms that can do this degradation have a lot of metabolic capabilities. So they can be doing different processes. And what we’re interested in is are they enhanced because they’re biodegrading our petroleum hydrocarbons that are present, right? So if we have a background sample, we can see if concentrations of organisms are elevated in our source area and down gradient locations where they’ve got selective pressure with these petroleum hydrocarbons being present. And that can give us some additional evidence and proof that the organisms are being stimulated, and utilizing this contaminant of interest.

Another thing, maybe if you’re using this as a bio-barrier approach…like we just talked about in that last case study. I would recommend adding some additional sampling points down gradient where that bio barrier is, kind of across the barrier and then just outside the barrier. So really thinking about what questions you’re trying to address. Make sure that your sampling strategy fits with that.

Now, we’ve done a lot of testing with Regenesis. We looked at collecting soil samples, we looked at collecting groundwater samples from a number of sites. And what we found was with the groundwater sampling, we had the best results. And we can use one-liter bottles of the water or the Bio-Flo filters. For those of you that aren’t familiar with these, these are just membranes that are connected. They’re sterile plastic-enclosed membranes that are connected to peristaltic or other groundwater sampling pumps, as long as it’s a low-flow pump. You pump the water through, send this filter back to the lab for analysis, the microbial community is condensed on this filter. Now, the key difference in this is when we’re doing the groundwater sampling, we wanna sample in zones where we think the PlumeStop was dispersed. We want that turbidity that you see from the PlumeStop being applied. Don’t worry if the filter clogs, if it clogs early, that’s fine. But where we saw the best benefit and really seeing the change in the microbial community from the PlumeStop was in areas that we could get that turbidity where the PlumeStop was.

And you could do it from soil samples as well as ground water, but the key with the soil samples is making sure you know where the distribution of your PlumeStop because what we found in that was it was a little tricky to see it in the soil samples once we got out there in the field. So it may be hard to collect at that precise point, and if you miss that, you may get kind of more of what your background soil looks like than what’s happening on the surface of that PlumeStop. And that’s really what we wanna to see here. We wanna see if the degradation is happening on that PlumeStop material. So my recommendation from what we’ve seen and the lessons learned kind of philosophy of doing this is collect groundwater samples where you’ve got some turbidity, where you see the PlumeStop. And those are gonna give you your best representation of what we think is going on with our microbial communities there.

So hopefully, you found this to be informative and got some information out of it. I am very excited again about this product. I think it’s something that’s really interesting and I’m happy to see it in the repertoire of what we could use at site. So I would be happy to take questions that you guys have, and still I’ll answer anything from the microbiological side that I can.

Dane: Okay great, thank you so much, Dora. That is gonna conclude the formal section of our presentation. And as Dora said, we’d like to now shift into the question and answer portion.

Before we do this, just a few reminders. First, you will receive a follow-up email with a brief survey. We do appreciate your feedback. So please, take a minute to let us know how we did. You will also receive a link to the webinar recording as soon as it is available.

All right, so let’s circle back to the questions now.

First question. You covered doing qPCR as the molecular tool, would you recommend other molecular testing on these samples?

Dora: Sure, so with a PlumeStop application, we’re seeing this thing used for more and more contaminants. So I talked about qPCR because those are well-vetted technologies when we’re talking about chlorinated sites or petroleum hydrocarbon sites. But what if you have, for instance, one for dioxane that’s present and you’re using that? There are other tools that you can use. You can use qPCR for some different genes there or if it’s contaminants that we don’t understand as well, we can and we don’t maybe have targets specifically developed for, you can do things like next generation sequencing of these samples to look at the total microbial community. So pretty much anything that you can do from a typical monitoring standpoint, we can do the same thing on this PlumeStop material but I would say, after qPCR and Quant Array, the next most interesting data would probably be to look at the next generation sequencing to see how the microbial communities change as they are growing and utilizing the compounds that are adsorbed to the PlumeStop.

Dane: All right. So next question here. Where do you recommend that we collect the sample? Should it be a soil or a groundwater?

Dora: Okay. And yeah, as I mentioned before, we can do either but my recommendation from the lessons learned is getting it in those turbid groundwater samples like I showed in that last slide. That that may be the best place from what we’ve seen, to really show what’s happening on the surface of that material.

Dane: Okay. All right, we’re getting some more questions coming in here. Next question is, as a microbiologist, were you surprised by the results? At the very least, it seems counterintuitive that an adsorbed contaminant would be bioavailable?

Dora: Yeah, and you know, that’s a question that people had for quite a while. We have a tool called a Bio-Trap Sampler that was developed by the University of Tulsa and Dupont. And it’s powder-activated carbon and Nomex. So it’s kind of similar in that we see that we can adsorb compounds that we’re interested in like a 13-Carbon compound. For instance, a 13-Carbon benzene. And it was bioavailable to the organisms. We would see that 13-Carbon could be traced into the biomass of microorganisms. So, even though the compounds were bound very tightly to that activated carbon and we didn’t see it lost into the aqueous phase, surrounding the Bio-Traps, we were still able to see the microorganisms utilize that 13-carbon and incorporate it into their biomass. So proof positive, it was bioavailable.

So based on my experience with Bio-Traps Samplers over the past 15 years, I wasn’t necessarily surprised. I think it was a great idea to use this technology. And this particle size, I was curious to see how the experience of the bacterial particles molecules would be to the small particle size, but I wasn’t surprised that it was still bioavailable to them.

Dane: All right okay. So our next question here is, would you recommend doing RNA analysis to better demonstrate activity?

Dora: You know, it’s a great question. So, RNA and DNA analysis is something that we’ve looked at and debated over the past decade. It really depends on the contaminant that you have at the site. Which one I would recommend? If we’re talking about a compound like a chlorinated ethane, like our PCE, for example, dehalococcoides can only use those chlorinated ethane compounds. So we don’t have to worry. If they’re present in high concentrations and their DNA is being detected in a higher concentration, they were in the near term utilizing those compounds of interest.

Now, on the other hand, for a petroleum site where you could have organisms that are doing a lot of different metabolic capabilities, the RNA comes in handy because we need to know, are they expressing that particular gene that we’re interested in? For instance, toluene dioxygenase. They have the capability to biodegrade toluene with it. Do we see the organisms actively expressing that gene of interest or RNA comes in very handy there? So I would say it really depends on the compound and that’s something that feel free to contact us and we can kind of advise you based on what our experience is with those particular compounds.

Dane: All right. Yeah thanks, Dora. We’re still getting some more questions here. So our next question is are you counting only the cells suspended in groundwater or are you also counting cells adhered to the PlumeStop?

Dora: That’s a good question. With our extraction process, we should be getting both. That would be the goal. And what we’re doing in the extraction is if we collect those turbid samples where PlumeStop is present, we should be removing those from the PlumeStop as well as anything that’s free floating in the water. So, ideally, we’re getting both of them.

Dane: Okay, all right. Let’s see next question here is how often would you think you would need to sample microbiological elements?

Dora: Again, it depends on the site itself. Sites that are new, that you’re trying to characterize and understand, typically quarterly monitoring is what we see that people will do. And you’re not gonna do every well on the site. You’re gonna do a selection, kind of like I showed in that diagram. Once the site becomes more characterized and you know it and you’re kind of more in a monitoring phase and not making so many changes, then you may move to semi-annual or annual sampling.

Dane: All right, so let’s see here. Next question is is there an optimal or minimum concentration of electron donor to maintain the DHC population?

Dora: You know, I don’t know that there is a cut-off. Off the top of my head, I’m not sure what that concentration would be. I think you would wanna to see you know, some decent TOC values. If you see them start to rapidly decline, I would look at that in combination with your qPCR results because if you see your dehalococcoides concentrations going down, then we would be concerned that something has changed in that environment. So it could be TOC and that’s an easy solution then to go out and you know, add some additional electron donor. But off the top of my head, I don’t know a magic number that I would say is a cut-off. I think it would be kind of site-specific, and we’d wanna to make sure that you do have…you know, from what we’ve seen in some sites that we’ve worked on, I would say you know, 50 to 100 is kind of where you’d wanna be with that number in concentration.

Dane: Okay, all right great. So let’s see here, a couple more questions we have. This one is have you validated your DNA extraction method? Some soil bacteria such as…I’m sorry I might not pronounce this right. Mycobacterium are very difficult to lice and do not release their DNA using the conventional commercial kits.

Dora: Yeah, so we’ve been doing DNA extraction for 20 years here at Microbial Insights so we’ve been through the process and comparing different extraction methods. I would say in comparative studies where we’ve been involved, we have a better lising and extraction methods than any other lab that we were compared with. And we were consistently able to recover the right amount of bacterium from some round robin-type studies that were performed. I don’t know that we can lice every single bacteria. You’re right, some of them like cryptosporidium can have really hard outer shells that could be more difficult to lice. In those cases, we may underestimate how those populations but for the organisms that we’re talking about here and chlorinated and petroleum hydrocarbon plumes, we see very good recovery of the DNA from those types of microorganisms. So I don’t think there’ll be any concern there.

Dane: All right, okay. So you have a little bit more time here, so we have a few more questions. This one is what could cause a decrease in DHC concentration after it is injected at a chlorinated site?

Dora: There are several things that you’d want to take a look at. Dehalococcoides is very sensitive to oxygen. Make sure of that because if you add a culture and you don’t prep the environment for it where you have electron donor that’s making it reducing enough, if there’s any oxygen you can limit the reductive dechlorination capabilities of dehalococcoides so that would be very important. The other thing that I see commonly at sites is looking at your sulfate concentrations because when you add an electron donor, you’re stimulating any of the anaerobic organisms that can use that donor, right? So if we see sulfate concentrations are very high at location, we might have…those sulfate-reducing bacteria really gets stimulated after that donor application, so we add dehalococcoides and they kind of lag a little bit and don’t do as well. But once sulfate reducers start to stabilize out, then we see dehalococcoides can come back and start to compete and utilize that. So there may be temporary lags that happen in those types of situations.

The other thing I would look at is looking at you know, the PH of your site to make sure that it isn’t down like say a three level. I have seen some locations where they’ve injected dehalococcoides and PH was super low. And we’ve had some problems there. So just looking at kind of your site parameters as a whole, I think, off the top of my head, those would probably be my top three recommendations.

Dane: Okay, thank you very much. A couple more questions here. This one is, is your anaerobic BTEX degradation based on denitrification?

Dora: Yes, the genes that have been identified so far, the carboxylation gene has been shown under more denitrifying conditions. And there’s research that’s been done for anaerobic benzene degradation trying to understand the other pathways. We don’t have genes isolated yet that we can target for those, but I foresee that coming and a better understanding, you know under more redox limited conditions, seeing some more information come out about that and literature in the future.

Dane: All right, great. Let’s see here. Next question is, is it hard to extract the microbial DNA from activated carbon?

Dora: No, it is not hard at all to remove it from what we’ve seen. We’ve worked with Bio-Trap Samplers. And as long as you have a good process for how you’re handling those samples, we find that we can very effectively remove the microbial communities from those. And we’ve loaded certain concentrations onto activated carbon and we get very efficient removal with our extraction process.

Dane: All right, okay. So next question is…looks like this person is asking, essentially, won’t these microbiological tools be informative only with respect to activated carbon? Or he’s asking, are these microbiological tools informative for remediation products other than activated carbon?

Dora: Yeah, these tools are used for any of the products. And we did a webinar with Regenesis previously using these same type of tools that we just add say, HRC to a site or if you add an electron etc. like an oxygen to the site, we would use the same types of tools to monitor those microbial communities. So it’s not specific to activated carbon, these tools are ubiquitous. Our gas can be used in multiple applications.

But I was just focusing on that here because it’s a great way for us to show what’s happening on that adsorbed PlumeStop material. We also commonly use these tools for monitored natural attenuation. So, if you have a site where you’re just doing MNA, this is a great line of evidence that you can use to show that the microbial populations, that Mother Nature is taking care of it. That we’re doing it and that things are fine. You know, maybe it’s a polishing tool at the end after you’ve done a treatment at a location. This is a great tool to continue on through that MNA to closure kind of period that you have there.

Dane: Okay great. All right, so looks like this one is probably gonna be the last question here. And it is can you calculate the biodegradation rate based on the bacterial concentration?

Dora: I wish. That’s something that we’re working on. So Microbial and Insights has been involved in a lot of research over the years, with John Wilson and Frank Loeffler’s group, kind of looking at some of these questions. We aren’t there yet with the answer. There’s some additional research that’s being done right now that might help to elucidate it. And there’s some work that John Wilson is doing looking at co-metabolism and its rates of degradation that we see correlate with concentrations of bacteria for co-metabolic degradation. So we’re getting there. We’re closer but unfortunately, we can’t just look at a concentration of bacteria and know a rate from that at this point. Hopefully, I’ll be able to tell you a different answer to that in the next five years. But for now, no. I wish we could.

Dane: All right. Okay, great. Thank you very much, Dora, that’s gonna be the end of our chat questions. If we did not get your question, someone will make an effort to follow up with you. To learn more about molecular biological tools from Microbial Insights, visit microbe.com. And if you need assistance with a remediation solution from Regenesis, please visit regenesis.com to find your local technical representative and they will be happy to speak with you.

Thanks again, very much, to our presenter Dora Taggart, and thanks to everyone who could join us. Have a great day.

Dora: All right, thank you, Dane.