How to Select and Use Molecular Biological Tools (MBTs)

If you have petroleum sites and you’re looking at adding an electron acceptor such as an oxygen-releasing compound or a sulfate, we can look at both aerobic and anaerobic degradation processes for these petroleum hydrocarbons to help you understand what’s happening. And even if you’re doing things like in situ chemical oxidation or in situ chemical reduction to clean up your site, there’s gonna be residual compounds like oxygen or sulfate that becomes bio-available after the main contaminant has been remediated. And so then we can start to use that oxygen and sulfate, and microorganisms will use them to do continued cleanup at that site. So it’s, kind of, a one step. You get a dual benefit from it. And we’re gonna talk about that with some of the products that they have available today. So each of these products have complimentary molecular analysis that we can use to really evaluate that effectiveness, and what that does is it gives you the ability to perform cleanup in the most cost-effective manner that you can.

Now, today I’m gonna focus on two newer products from REGENESIS. I’m gonna talk about PlumeStop and Aqua-ZVI. And we’re gonna look at how we can use some molecular tools to monitor their performance in the field. Now, there was a couple months ago that Matt Burns with WSP did a phenomenal webinar also talking about these two products. And he gave some case studies that demonstrated practical strategies of how to use these products in the field and have real successes for it. So I think Matt’s webinar from a few months ago really complements what I’m gonna show you today. If you didn’t see that webinar, I’d encourage you to go to the REGENESIS website and watch that when you have time.

So as I’ve been moving forward in summer watching kids and grandkids play sports, it made me think about the importance of reaching our goals. Site management is pretty analogous to the perspective of managing a sports team. So in the presentation today, I’m gonna use sports as the analogy for our site remediation. So if we’re a team manager, one of the things that we want to know is who are our players? Who do we have that are available at our site? Similarly, in remediation, we want to know which microorganisms are present. Do we have the right microbial processes so if we add one of these treatment products we can really count on them to work well in the field? As we know, each player has his own strengths and weaknesses, and as a team manager, it’s our responsibility to choose these so that we get the best play we can that complements the strengths of our players. So if we understand those microorganisms, we can really optimize that and do that the best for our site.

The other thing that we need to know in our sites is where are we playing? Knowing the site in detail prior to beginning a treatment strategy really allows us to have a home field advantage each and every time. Then, we can look at our game plan. Now our playbook has grown dramatically over the last 20 years that I’ve been doing this type of work. I’ve seen a lot of new products come out. REGENESIS has done a great job of addressing a lot of concerns that we have in remediation in providing these great products. So our game plans and the playbook that we have is ever-growing. So how do we manage these and select the best play for us? So with enough site knowledge, we can begin thinking through different treatment strategies, how they might work in our specific environment, and then if we implement those treatments, we want to continue monitoring frequently so we can make sure they’re working as they were expected. If the plays aren’t working, we want to modify them quickly so that we can get cost-effective remediation.

Finally, the last piece of the puzzle that we need before reaching our end goal is to determine who are star players are. Who’s the really important key component in our lineup? Do we have the right microorganism for that biotic process that we’re trying to stimulate so we can really count on these microorganisms to bring us home and reach that target end goal that we have? What about our abiotic processes? Do we have the right microorganisms there that are generating the products that we need for this? And we’ll talk about this throughout the presentation today.

So we’re gonna start by looking at one of the newer products that REGENESIS offers called Aqua-ZVI. Now, with any site cleanup, it’s good to understand our chemical and geochemical condition. And it’s important to understand the microbial populations, as their presence and interaction drives most of the processes that happen in our subsurface. So let’s take a look at this with some site data.

Now, our first case study is gonna be a bio plus biogeochemical pilot test at a chlorinated solvent source area. In this situation, we know who the main players are gonna be. We want to stimulate reductive de-chlorination of the site. We know which microorganisms are important. But in order to implement our players effectively, we need to keep a few things in mind to give us that home field advantage.

One, we have to put the data in context. And this is what I was just talking to you about with the Microbial Insights database. We can take this data that we get from our site and start to learn, “Is that a high concentration? What does that mean for our site? Do we have enough to get effective remediation?” You know, if we look at raw numbers of any analysis, it doesn’t really give us much. We have to put that into context. And it takes several pieces of data looked at as a whole to really understand the full context of the site.

So next we’ll ask what the data is actually telling us about that site. If we can start to learn this information, we can then really make effective decisions. Understanding the site through this data lets us peek into the black box of our subsurface. And it gives us a lot more confidence when it comes to choosing the play. And then if we need to make a modification to our play, it helps us to understand what our next move should be. So as you’ll see throughout this presentation today, these steps are not only useful prior to implementing a treatment strategy, but they’re really critical for monitoring whether the strategies are working in our intended manner.

Now, we’ve identified our main players. So let’s take a look at our course. Let’s see where we’re actually playing. So this is a former manufacturing facility where chlorinated ethenes and ethanes have been released. The origin of the release was from aboveground solvent tanks in the area highlighted in the red circle here. The ground water flow is toward the southeast, but the focus of the pilot study was to determine the most efficient way of cleaning up that source area. Now, one thing I want you to note. From the geochemical data, we knew that there were naturally high concentrations of sulfate present. So the site manager was smart to use this sulfate to his advantage. And by using a bio plus a biogeochemical approach, he realized the sulfate could be reduce to sulfide by sulfate-reducing bacteria, and then form iron sulfides in situ in the presence of BDI.

Now, I know that these boxes are pretty small. I’m not asking you to read these. I’m not trying to give you an eye exam today. But I wanted to highlight the geology of the site and kinda show you a cross-section. Some of the shallow monitoring wells lie in this silty clay overburden, while the deeper wells are found in the fractured limestone. So in order to truly figure out for the best place for this site, we need to know how both of these environments are gonna respond to different treatment strategies. So in order to do this, two wells were chosen to perform some preliminary analysis. There was a shallow well, MW-16, here in the silty clay and a deep well, MW-29B, that’s in the fractured limestone.

Now, I know saying this, if we say that we’ll figure out our play, that’s often easier said than done. Coaches of sports teams will study the strengths and weaknesses of the opposing team watching videos for hours on end before they make a play so they can best determine the most impactful play that they want to use. The same applies to us in remediation. The more we study and understand our site, we can determine how it’s gonna react and the better chance we are gonna have of hitting our target more quickly.

Now, before I move on, I’m gonna go ahead and spoil the ending for you. So here’s the spoiler alert. This source area was cleaned up through the pilot study alone, but the presentation I’m giving you today isn’t about whether the treatment strategy worked. I really want to talk to you about how we actually got there. So let’s take a look from the very beginning to see how the best plays were chosen for this site. So in order to test different treatment strategies, the site manager used the tool that Microbial Insights offers called an in situ microcosm. These are an enhancement to our passive sampling tool called a Bio-Trap sampler. They consist of slotted PVC that you can see here on the right-hand side of your screen with different components to help us test different amendments in the field prior to implementation.

Now, the project manager at this site has a saying that I really like that I wanted to share with you. He says he uses these so if he fails, it’s on a small scale as a screening tool before he moves forward to field implementation. So this is a really cost-effective way to evaluate different screening options and figure out your best play.

Now, one of the most important components to these units are the baffles that you see on each end. It’s kinda hard to see them in the picture here, but these little black pieces are baffles. And what they do is they prevent vertical circulation within the wells and allow us to section off a portion of the monitoring well so we can test different treatment strategies.

So in this situation, our top unit… There were three units that were deployed connected in each of two monitoring wells, MW-16 and 29B. And the top unit we used as a control unit. So trying to replicate natural conditions, not putting any amendments or cultures into this unit, so we can look at what the natural site conditions would be. So inside this top PVC unit, we had three different components that were there. There was a passive diffusion bag to help us look at the contaminants of concern, a Bio-Trap so we can evaluate the microbial processes, what organisms are naturally there at the site, and then a geochemistry vial so we can look at the sulfate concentrations, we can look at volatile fatty acids, ethene, dissolved gasses that we may be interested in. And then this bottom is just a plastic PVC spacer that helps us to have these in that screened interval of our PVC.

Now, the middle PVC unit contained testing for bioaugmentation cultures. So inside these units we had sponges with a fermentable carbon that was adsorbed. And these sponges were placed on each end within the units. There was, again, a passive diffusion bag so we could look at our contaminants and a Bio-Trap for the microbial community, and, again, a vial for the geochemistry. Now, this time our Bio-Trap was pre-inoculated with a culture of halorespiring organisms. So a Dehalococcoides culture was loaded onto this so we could see what the survival was gonna be in situ.

Finally, the bottom units of this was also to test the bioaugmentation culture, but this time there was a different substrate added. And in each of the monitoring wells, there was some different amendments that were tested. So what you see here is an MW-16. The sponges were loaded with a biostimulant plus ZVI. In 29B, the sponges were loaded with ferrous gluconate and a baggie containing sulfate. And then we had the passive diffusion bag to look at the contaminants of concern, the Bio-Traps again loaded with that same Dehalococcoides culture, and those geochemistry vials.

So due to time today, I’m not gonna get into all the data and show you all the results, but the end result of the study was that the middle unit showed the highest likelihood for success. In that unit, there were drops in the VOC concentrations, an increase in the enrichment of daughter products based on CSIA analysis. We saw decreases in sulfate and increases in our volatile fatty acids. Along with end product production, seeing ethene and acetylene being produced in the unit.

And when we looked at the microbial analysis, we had a really important note that we want to take into consideration as we move to the field. What we saw was that dehalobacter was low in all of the units of the site. Now dehalobacter is an important organism for reductive dechlorination of chlorinated ethane. So it’s something that I want you to note and we’ll talk about a little bit more when we get to that pilot study.

So this is where the Aqua-ZVI comes into play. Aqua-ZVI can directly react with chlorinated compounds like we have at this site to degrade them, or it can be used to form iron sulfides in situ. So a little bit about this product. This is a pre-mixed liquid that disperses widely in the subsurface. It’s an in situ chemical reduction reagent that promotes the destruction of many pollutants like these chlorinated compounds that are of interest here. This is a micron-scale zero-valent iron that delivers enhanced reactivity via multiple pathways. Aqua-ZVI can stimulate anaerobic degradation by creating a more reducing environment, plus we get the added benefit of abiotic degradation, as well.

So based on the in situ microcosms, the first injection in the source area was performed. And the site manager saw that this treatment was effective in the majority of the monitoring wells, but he also saw that those CVOC concentrations in MW-29 and 29B rebounded. So in 2014, a second injection also was performed. Now, because a rebound could indicate the presence of NAPL in the fractured limestone in this location, careful monitoring was performed in MW-29B for diagnostic purposes.

So let’s talk about the data that was generated from this monitoring and how we can use those for next steps. What’s gonna happen next in our playbook? So if you’ve heard me talk before, you know I’m a big proponent for multiple lines of evidence. At Microbial Insights, this is something that we talk about a lot. So here’s a visual that kinda shows you the tools that we could be using at this site and how they, kind of, interact with each other.

So if we look at our contaminant concentrations, that’s, kind of, the first place I like to start. Do we see daughter products forming, which could indicate biotic degradation? If we don’t see daughter products, do we think that there might be some abiotic degradation? What’s the extent of the degradation, if we do some mole fraction analysis? What are we seeing with our contaminant concentration? Then moving to looking at the geochemistry at the site, how much sulfate is present? Are we seeing volatile fatty acids being produced? How reducing are we at this point?

And then if we use DNA with our QuantArray analysis to look at different microorganisms of interest, do we have organisms that can degrade both our chlorinated ethenes and our chlorinated ethanes? So are those halorespiring organisms present? What does their abundance look like? Do we have the appropriate functional genes that we need to get complete degradation at these sites? And then what about our terminal electronic testing processes? We’ve got sulfate present, and we saw at this site in particular we have high concentrations of sulfate. So do we see sulfate-reducing bacteria that can generate those sulfides that we’re looking for?

So by looking at this information and combining it with the information from the Microbial Insights database, we can really start to learn what the likelihood is that we’re gonna get complete reductive dechlorination at the site. We can also use tools like compound-specific isotope analysis or CSIA to help us prove that degradation is occurring under those current conditions. And then I’m gonna walk you through how we can use the contaminant concentrations over time to help us elucidate what pathway may be happening.

So one of the tools that we find to be very helpful with Aqua-ZVI is called compound-specific isotope analysis or CSIA. In nature, 99% of our carbon is a 12-carbon. And only 1% is a 13-carbon. So microorganisms will preferentially break that weaker 12-carbon bond. So in biotic degradation, we start to see an accumulation of our 13-carbon in our compounds of interest. We can also use the ratio of our 13-carbon to 12-carbon fractionation to help us understand the degradation pathways. The change in our 13-carbon value occurs in a very predictable manner. So we can use this tool to understand if our compound of interest is degrading and also help us understand what mechanism is likely causing this degradation.

So I’m gonna show you that with some of the data from this site. So we’re looking again at well 29B in that fractured limestone. And CSIA was performed on a number of target compounds to see if they were degrading and whether or not an NAPL source was replenishing those compounds in the ground water. So here’s the TCE data that was monitored in this well between December of 2014 and June of 2017.

Now, notice as we move from the left to the right on our X-axis that we see our TCE mole fraction decrease and our delta value increase. In fact, we certainly see the delta value increase by more than 2 per mil. So based on the CSIA guidance documentation provided by the EPA, this is clear evidence. We want at least a 2 per mil shift. We have far more that with what we’re seeing here in the data over time. So we can conclude that degradation is occurring.

But what else can this data tell us? Even better, our TCE mole fraction and the delta value changes are consistent. So this indicates that there likely isn’t a NAPL source nearby. If there were, we would likely see a lot of fluctuations in this data. We wouldn’t be able to draw a line so clearly. And, in fact, this plot can provide even more information about what can be happening at a site. When we plot the natural logarithm of the contaminant mole fraction against our delta 13-C value, as I’ve shown here, the slope of the trend is equal to something we call isotopic enrichment. This factor, which uses the symbol epsilon, can provide a lot of information for us at a site. So if you take your slope of the line, that’s gonna be equal to your isotopic enrichment factor or that epsilon value that you’ll see.

So this isotopic enrichment factor, it relates the isotopic enrichment factor that increase in the delta factor o the extent of degradation at our site. This enrichment factor is specific to the degradation mechanism. And it’s really useful for us in evaluating our parent compounds at the site. And we have to be careful if we try to do this with daughter products, they’re a lot more tricky. Daughter products are being formed and degrading at the same time. So because both of those things are happening, their isotopic values and concentration can fluctuate drastically, so it’s less predictable. But we can certainly use this enrichment factor to help us with our parent compounds and make some good assessment at the site.

So let me show you this put into practice here at this location. Again, we’re looking at MW-29B. Remember, I said that the enrichment factor is the slope of the line. So let’s look at the slope of the line for our TCE concentrations over time. And what we can do is compare the enrichment factor, calculate it from our site data to enrichment factors published in literature, and we can get an idea of whether it’s biotic or abiotic degradation for the dominant mechanism. So if we take that TCE data that I showed you earlier, we see that the enrichment factor is -1.5. That’s the slope of our line. And if we look at our published data for biotic degradation, it looks like we’re closest to that. So now, we know that we have a 2 per mil positive change in our TCE delta value over time. So it’s proof of degradation. And then, based on the slope of the line, that epsilon value is telling us that it is likely biotic degradation that’s happening.

So let’s do this same thing for another compound, the 1,1-DCE at the site. In the data, we only have enrichment factors for biotic degradation that are shown, but lucky for us, our slope of the line is -5.62. So it falls right in line with what we see in literature for biotic degradation. Again, if we look at our delta 13-C values over time, we see that there’s more than a 2 per mil change in our delta value. And so we can prove degradation of 1, 1-DCE. And then we can, again, say that the dominant mechanism, based on the CSIA results, is likely biotic.

Okay. So finally, let’s look at this for 1,1,1-TCA at this site, and this is an MW-29B still. And we can see that if we plot the slope and look at our literature values, that our epsilon value for isotopic enrichment is -11.6. That’s almost exactly in the middle of the range that’s published for abiotic degradation. So when we look at this, we can, again, prove degradation because we had more than a 2 per mil change in our delta 13-C value. And in this case, it looks like the dominant mechanism is likely abiotic

So this makes sense based on what we learned from our in situ microcosm study. If you remember back in the beginning, I pointed out that dehalobacter concentrations and other halorespirers that do chlorinated ethane degradation were really low at this site. So now, we have the CSIA data showing us TCA is degrading, but we also see that it’s likely degrading abiotically. And we have evidence that with this, that the ZVI injection is contributing to the contaminant degradation. And that may not have occurred otherwise, since we didn’t have the right microbiology to get biotic degradation.

So let’s look at our starting lineup of microorganisms. Which ones are present at the site? Did they have the ability to degrade the contaminants that are present? And are there numbers increasing or decreasing? Let’s see how this fits with all of the data that we have. So what I’m showing you here in this graph as looking at it over time. The dotted lines are our two injection points. We have the sulfate concentrations in this orange line that’s coming down here. The bars on our graph, the blue is Dehalococcoides, which is important for chlorinated ethene degradation. The TCEA reductase gene is a TCE gene found in Dehalococcoides. And then the purple bar is the vinyl chloride reductase gene, also found in Dehalococcoides. And this goldish-brown color is our sulfate-reducing bacteria.

So what do we see over time, if we look at this? After the second injection, we see that the sulfate-reducing bacteria, again, that brownish color, increased. This coincides with a dramatic decrease in our sulfate concentration indicating that iron sulfide is likely forming in situ, as planned. Meanwhile, we see that our Dehalococcoides increased after the second injection, and they continue to increase as sulfate drops off.

Now, we see this in a lot of sites very commonly. As sulfate reducers start to stabilize at the site, hydrogen becomes bio-available to these halorespiring organisms. You start to see them increase. And we’re seeing a good long maintenance of these organisms over that entire monitoring period. We also see dramatic increases in our TCE reductase gene and our vinyl chloride reductase gene. So this helps to tell us that we’re gonna get efficient reductive dechlorination and get completely to ethene without a lot of accumulation of daughter products.

So the next thing I’d like to show you is the chemical data. So let’s see if our chemical data agrees with what we learned from the microbiology and the CSIA. So notice how the chlorinated ethenes are producing daughter products that are very consistent with biotic degradation. We’ve even seen ethene concentrations increase at this site, which we would expect based on those vinyl chloride reductase numbers. So biotic degradation happening with our chlorinated ethenes based on our chemistry. If we look at our chlorinated ethanes, on the other hand, it’s consistent with what we would expect to see with abiotic degradation mechanisms. We see a decrease in our parent compound and very little daughter product production. So that ZVI injection worked, as would be expected.

So these consistent chemical, geochemical, microbial, and isotopic monitoring events tell us that that strategy and the injection, our game plan for this site, worked as expected. NAPL doesn’t appear to be a problem and that no change in our strategy is needed. This thorough assessment of the site and regular observation really gave way to a creative treatment that was tailored specifically for this site. And we were able to reach the target goals.

So by understanding our players, the plays that are happening, along with a good understanding of the field that we’re playing on, the site manager was able to hit the target goal for the site. And as I gave you the spoiler alert earlier, it really paid off. There was no money or time that was wasted on dead end treatments. And the entire source area was cleaned up through the pilot study. And he achieved the desired remediation goals for all of the wells of the site, as you can see here from this data.

So what do we conclude that was happening? Key take-home points? In Situ microcosms helped to really evaluate the site and answer the questions shown before moving to that full field scale implementation. Then the added microbial analysis throughout the treatment period helped show that we had sufficient sulfate reducers to generate the sulfides that we wanted to form for abiotic degradation of ethane. And we had enough Dehalococcoides to get efficient reductive dechlorination to occur.

The chemical and geochemical data, we’ve looked at it. It agreed with everything we were seeing from the other analysis, that degradation products were being formed, that the concentrations weren’t really rebounding any more over time, and the conditions were still favorable for this chosen treatment. And then the CSIA really helped us to elucidate the dominant degradation pathways and proof that the compounds were degrading. So compounds like Aqua-ZVI or Micro-ZVI can be very effective in rapidly reducing contaminants through in situ chemical reduction. And it stimulates anaerobic biological degradation by rapidly creating a reducing environment that’s favorable for reductive dechlorination. So applying these products to sites can really give us a dual benefit where we enhance those biotic and abiotic degradation.

So let’s look at another product that we have in our playbook before we close out today. I wanted to give you a quick example of some data that we’re seeing with PlumeStop. You know, PlumeStop is a really interesting product that I’m excited about and we’ve seen some really good field data that’s been coming in from this particular product. I want to share just a little bit about that with you today before we close.

So just to make sure everyone’s on the same page, I’ll start with a brief introduction about colloidal activated carbon. You know, the colloidal in colloidal activated carbon is referring to the size in that this activated carbon is very small, between one nanometer to one micrometer in size. These particles are distributed and dispersed throughout some type of medium. And the activated carbon allows for sorption of contaminants to these colloids. So in remediation practice, it’s common to use this in conjunction with an electron donor at a chlorinated site, or an electron receptor at a petroleum hydrocarbon site. And the product that I’m gonna be talking about today is PlumeStop, which is a stabilized form of these colloidal particles that’s capable of widely distributing throughout the subsurface while rapidly reducing dissolved phase contaminant concentration. And we simultaneously see biodegradation happening at these sites.

The diagram here represents your subsurface. There are two key areas that I want you to observe. There’s the higher permeability zone and the lower permeability zone. Contaminants are typically transported through this higher permeability zone. And over time, they diffuse into that lower permeability zone where the contaminant remains. It’s much easy… Well, it’s relatively easy for us to remediate contaminants in the higher permeability zone, but much more difficult to remediate those in lower permeability zones. So when the PlumeStop is applied to the site, the colloids are distributed in that high permeability scene, and this allows for rapid sorption of contaminants out of those high permeability zones with the ability for us to capture back diffusion out of those lower permeability zones.

So when we’re talking about using PlumeStop at a site, we have a couple of diagnostic objectives. We want to know if our contaminant concentrations are decreasing, and we can look at our chemical data to see that. Are we getting sorption to our colloidal particles? And then, do we have the right microorganisms present so that we can get good biodegradation so we can use QuantArray to help us understand that? And then we can start to see, is that contaminant being degraded? Most of the time, we would typically look for daughter products, and I’m gonna show you how that can be a little more difficult when things are adsorbed because those daughter products we see seem to go away very quickly. If we have products available, we can use compound-specific isotope analysis to prove if we’re getting degradation. And we can start to understand more about the back diffusion.

So I want to give you a quick case study before I close today. This is a former dry cleaners site. And the reason that this one was picked is there was a lot of historical brown water monitoring data for this site back to 2001. The baseline conditions were generally Aerobic. And since Dehalococcoides is an anaerobic organism, it wasn’t doing very well. It wasn’t detected at all at this site. And there have been no evidence for daughter product formation prior to this study. So it represents a bit of a challenge in terms of the redox conditions, but with no prior evidence for degradation, it’s a great way for us to potentially see the impact of the treatment. So what is our game plan doing, if we apply it to our field of play? Finally, this is a sandy aquifer, and there’s about 10 meter per year groundwater velocity. So this site is a great representation of where we’re gonna need to stop migration, making PlumeStop a great potential play for the site.

So here’s an example, this is the contaminant concentrations at the function of time just before and after the PlumeStop, HRC and Dehalococcoides injections. That’s represented by the dash line there. You see the PCE concentration drops immediately, from over 500 micrograms per liter to non-detect. This demonstrates effective adsorption. But now we’re interested what’s going on with the biodegradation? Are there organisms that can utilize these compounds that are adsorbed through the colloids?

So here I’m showing you an addition with the Dehalococcoides in a blue bar chart on there. And we see the Dehalococcoides abundance on the right Y-axis. After injection of PlumeStop, biostimulation with HRC, and bioaugmentation with our Dehalococcoides, we see that we have concentrations that are greater than that 10 to the 4 cells per milliliter And we know based on the Microbial Insights database and the paper that this is the critical concentration that we need for effective biodegradation.

Then we see that the concentration of Dehalococcoides was maintained for at least nine months at that greater than 10 to the 4 number even with the removal of the chlorinated hydrocarbons from the dissolved phase. Now, I’m gonna add in this orange line showing the electron donor concentration, as indicated by TOC, the total organic carbon. And we can see that that began to decrease. By 15 months, TOC had decreased to less than 10 milligrams per liter, and we see Dehalococcoides populations decreased by approximately an order of magnitude. We also saw similar trends in the vinyl chloride reductase gene found in Dehalococcoides, where it dropped from around 10 to the 3 to less than 10 to a 100 copies per milliliter. So at this point, Dehalococcoides concentrations were decreasing likely because electron donor availability was becoming more limited at the site.

The first thing I want you to notice here is I rescaled the left Y-axis so we could take a closer look at the daughter product concentration. So what happened? Electron donors completely consumed. And unfortunately, you see I don’t have the Dehalococcoides data for these last couple of monitoring events, but if we assume that the observed trend where we see the Dehalococcoides going down in concentration continued and the results make sense because there was no electron donor to support Dehalococcoides and redox conditions started to become less favorable as indicated by a decrease in ferrous iron and an increase in sulfate. So once Dehalococcoides decreased, we see a little bit of vinyl chloride that’s starting to be detected, but look how low, very low concentrations, but it does provide some additional information and very convincing evidence that reductive dechlorination of PCE was, in fact, occurring even though we really couldn’t see it during most of our monitoring period to get those normal daughter products we would find.

So the conclusion of this first case study is that PlumeStop, combined with HRC and bioaugmentation, resulted in effective adsorption and biodegradation. You know, the Dehalococcoides is an obligate halorespiring organism. And there’s no way that a population of 10 to the 4 cells per mil is gonna be maintained for 9 months, if these organisms aren’t active. Eventually, vinyl chloride was detected at very low concentration, providing evidence that there was sequential reductive dechlorination that was happening, but it’s important to remember that vinyl chloride was only detected when Dehalococcoides concentrations decreased due to that limited donor availability. So if additional electron donor or a second injection, we likely would never have seen vinyl chloride detected at all, which leads me to my next point.

If you’re doing this type of play at your site, microbial monitoring via tools like QuantArray is really important because you can’t really justify that biodegradation is happening any other way. We may not see daughter products being formed because the adsorption happens, and biodegradation can happen so effectively when the PlumeStop is applied. We may not see a lot of daughter product accumulation. So we need some way to document that we have the right microorganisms there, and that this play is working and we’re gonna reach out end target goal.

So hopefully, this presentation gave you some examples of how you can optimize your plays at a site, no matter what you’re testing out from your playbook. As I mentioned at the beginning, we can use molecular tools with any other products that REGENESIS offers, whether it’s a chlorinated site, a petroleum site, or even more emerging compounds. The goal is really to remember that we want to create actionable data at a site. And we realize this isn’t a science project. And I know there’s a lot of tools that are available out there.

If you have a chlorinated site, we can look at in situ microcosms to help understand if the treatment strategy, which one we want to select is gonna be most effective for that site. We can use tools like QuantArray to be able to look at the halogenating organisms, to see if we’ve got the right abundance there, to be able to likely get effective remediation of the site. And then we can use tools like compound-specific isotope analysis to help us prove that that degradation was, in fact, occurring.

If you have sites with petroleum hydrocarbons, typically you’ve got a mix, maybe some BTX, maybe some PAHs, or alkanes that are present at the site. And we know a lot about the degradation pathways of these organisms. And we have some really great tools that can help us assess these types of site. For instance, we can do something call stable-isotype probing where we use that heavier 13 carbon as a tracer to prove degradation. If you’re interested in this, you can go to our website to download more information or email us. We’d be happy to share that with you. We can use tools like QuantArray to help us look at these aerobic and anaerobic processes for petroleum degradation and really get a better understanding of what’s happening at these hydrocarbon sites.

And then if you have 1,4-Dioxane, we do know a couple of targets that we can do for Dioxane degradation based on research that Dr. Mike Hyman, what NC State has done, that Shaily Mahendra has done. We have a few targets and genes that can be of interest for you at those sites. We can also use stable isotope probing again to prove degradation of the compounds.

And then what if you have emerging contaminants? Maybe we don’t know the pathways yet. We don’t have a tool, a qPCR assay. We don’t know the organism or the particular pathway. It hasn’t been elucidated yet in science. What do we do? In those cases, I typically recommend that we do something called next generation sequencing. And this is where we can look at the entire microbial communities. And if we look at forced area versus non-contaminated, we can see what is causing selective pressure from those contaminants to figure out which microorganisms are present, what might be important for us to monitor for these emerging pathways.

So the key goal of this, I know you don’t want to create a science project, and that your time and money is very valuable. So we really want to use these tools to create actionable data to help us elucidate information about our site. And if you take nothing else from my presentation today, the key take-home message would be the more that we know about our site, the more money that we can save in the long run because the cost to do these tools is gonna be a fraction of what you can save from the cost benefit what you can save from the cost benefit of what you do at the site in saving your time, saving your money, getting very effective remediation strategy.

So we have several project managers that are here at Microbial Insights. We want to help you and answer any questions that you have. I’ve listed a couple of people here. You can also contact Sam or Anna. Any of us would be happy to help you and talk to you about these different tools for your sites. You know, again, knowing your site really increases the certainty of your game plan. And it really is gonna help you get overall reduction in the cost. So a lot of great products that are available, a lot of great, great things that we can do to test and prove, you know, efficacy of what we’re doing at our site. And we can get kinda the biggest bang for our buck. So hopefully, this is helpful to you. And I will be happy to take any questions that you guys have.

Dane: All right. Thank you so much, Dora. That does conclude the formal section of our presentation. Now, at this point we’d like to shift into the Q&A portion, as Dora mentioned. Before we do this, just a couple of reminders. First, you will receive a follow-up email with a brief survey. We really appreciate your feedback. So please do take a minute to let us know how we did. And also, you’ll receive a link to the webinar recording as soon as it is available. All right, so let’s circle back to these questions here. Dora, the first question is, and this person is referring to the webinar with Matt Burns. “I saw the webinar with Matt Burns of WSP a couple months ago showing case studies on these products. Both of you mentioned the cost savings from using molecular tools to optimize performance. Do you have any information about the cost benefit that you could share?”

Dora: Okay. That’s a great question. Actually, this is something that Matt and I have been talking about. And hopefully, within the foreseeable future, we’re gonna be putting together a webinar talking about cost benefit analysis from using these tools and implementing it at your site. Hopefully, that can help you sell it to the end user and explain to them the value of it. So check back with us. We’re gonna put some information together. And you should see some emails about webinars and other things that will be happening on that very topic.

Dane: Okay, great. The next question here is, “Can you explain a little about the QuantArray analysis? Do we use the same QuantArray for both chlorinated and petroleum sites?”

Dora: Sure. So QuantArray is the same thing as what you’ve probably heard for the last 15 years, 20 years, qPCR. So it’s taking qPCR and doing it in a smaller volume. So qPCR is tried and true. We know it’s the best way to quantify our microbial targets at a site and get accurate concentration of these organisms and functional genes. And that’s really the key. We can get functional genes too with this. So with the QuantArray analysis, we can look at a lot of different pathways that we can be interested in and a lot of different microorganisms all in a single analysis because we’re using such small volumes. And we get independent numbers in little through holes that don’t interact with each other. So we can really count on these results to be very reliable quality data.

And so we have different QuantArrays that are designed, one, for chlorinated sites where we look at all the known pathways for reductive dechlorination, co-metabolism, and terminal electron processes that can be important for those sites. For petroleum sites, we have both aerobic and anaerobic genes that are on there. For a variety of petroleum contaminants from BTX to alkanes to PAHs. So there are two different QuantArrays that are available, and they would be specific to that contaminant of concern.

Dane: All right. Okay. So the next question here is, “What stage of site design should we start using these molecular techniques? Only in the beginning of site design or can they be used throughout the process?”

Dora: That’s a good question. You know, really you can use them throughout the whole process. I would encourage you to be consistent in what you do with your monitoring. You can do the in situ microcosms like we showed here to help you evaluate treatment strategies. And then know which key processes and organisms are gonna be important. And then you can continue to monitor them throughout whatever your treatment strategy is. And that way, if you need to make optimizations or there needs to be changes in the amendment or a slight change at the site, you can make those very effectively by looking at those concentrations and combining it with your other data.

Dane: All right, continuing to get some more questions here. This one is, “In the slide called MNA (control) units…” And I believe this was pretty early on, maybe 10 minutes into your presentation, the question is, “In the slide called MNA (control) units, how is the geochemical vial collected? Is it a Snap Sampler?”

Dora: Okay. Yeah, that’s a good question. It’s not a Snap Sampler. It’s just a vial that has a passive membrane on top. And we’ve tested this. And we get good exchange through that membrane. So you get equilibration of the water that’s originally in there, the Nanopure water that’s sent out in the vial with the site groundwater. And we can measure a lot of things from that vial looking at dissolved gases, anything that’s happening within that unit. If there’s changes in the geochemistry over time, we also see that in the vial, as well, but it’s a passive membrane on top of that.

Dane: Okay, great. So the next question here is, “What concentrations do you need to do CSIA? And what elements can you do CSIA on?”

Dora: CSIA is most helpful… What we’ve found, especially for chlorinated compounds, there’s not really a good way because these compounds are used as electron acceptors by microorganisms. The contaminant concentration we can measure with CSIA, the values that we can get, are very low. It depends on the compounds. If you want to shoot us an email with which compound, we can give you the particular detection limit, but we’re talking ppb values there, so very low detection limits for those. So for chlorinated compounds, most any of the isomers that are out there are daughter products that are being formed. You can also use it for compounds like 1,4-Dioxane and some others. Some of the fractionation processes aren’t as well defined for those compounds, but it can be used for a pretty big variety.

Now, for petroleum hydrocarbons, the fractionation is a little less seen in some of those compounds. And so, stable-isotope probing is a better tool. And there’s a video on our website that talks about when to use CSIA and when to use stable-isotope probing. That might be helpful, if you’d like better information about that. But for petroleum hydrocarbons, I would probably recommend the stable-isotope probing.

Dane: Okay, great. The next question here is, “What is the cost of the in situ microcosm study?”

Dora: Okay. It varies depending on what test that we do inside. And we can, you know, make these different, depending on the questions we’re trying to answer, but typically each of those units from the whole package, buying the unit itself, all the analysis that goes into it, and a report telling us from the microbial perspective what we think is happening at the site. It’s around $1,000 to $1,500 per unit.

Dane: All right. Let’s see. The next question here is, “The in situ microcosm case study, in that case study presented, was the testing done before the initial injection or following the first and before the second?”

Dora: Oh, good question. It was before anything was done at the site. So this was the initial test to decide what injections were gonna be performed at the site.

Dane: Okay. Let’s see. The next question here is, “In the slide called ‘microbial analysis, with the bar chart, what is the acronym SRB? And why was it only reported in that last sampling event?”

Dora: Okay. We did sulfate-reducing bacteria is what that was for. And it was in the initial events when sulfate concentrations were higher. And what you saw in that slide is that sulfate-reducing bacteria were present at the site. And then once we saw… After that first injection, you saw sulfate concentrations, the sulfate itself, rapidly decrease, and we saw an increase in sulfate-reducing bacteria. And the reason we monitored that early on is we wanted to make sure that we were getting those sulfate chains to sulfide so we could get that iron sulfide formation.

Dane: All right. Thanks, Dora. Let’s see. The next question here is, “Application of those baffled units looked very interesting. How was long-term monitoring done using those baffled units?”

Dora: So typically we would use these for a short-term study. You would deploy them for 60 to 90 days in most cases. And we have done it for slightly longer. The only thing you’ve gotta worry about is if we do it for a longer period, we can’t add enough electron donor or acceptor to last for those long periods of time. So we might have to reload those. And so in that case, if you have to pull them out, it makes the baffles not effective, right? So we would have to, you know, only do one type of treatment being tested in a well. In that way, if we had to reload that treatment, we have done it where we’ve left them for 180 days at a sight to see what was gonna happen, but we had to reload additional electron donor at a middle point in that. So that kinda limits us in being able to test different remediation strategies. So the typical would be two to three units that are deployed in a well. Always have a control unit so we can benchmark what we’re seeing for comparison. And then the other one to two units in that well would be typically different treatment strategies. And they would be left for 60 to 90 days.

Dane: All right. The next question here is, “What is the typical scale of projects these products have been successfully used on?”

Dora: I’m not sure if I understand that. Do you have a good feel for what they’re trying to gain there, Dane?

Dane: I’m not… Scale of projects, meaning I would think the size of the project perhaps, the size of the? I’m not sure.

Dora: Yeah, maybe they’re talking about the number of samples that are typically done at a site. So for the typical site, I would say the microbial characterization, you usually pick three to five representative samples. You want to collect in the source area. You want to collect down gradient. If it’s a large site, you might want to get a couple of additional cross-gradient samples, but get, kind of, a good distribution. The rule of thumb I use is if you see big changes in the geochemistry or if you see an order of magnitude change in your contaminant concentration, collect samples there. So hopefully, that’s what you’re talking about.

If you’re meaning how often are these applied, this is becoming more routine. We see this that the regulatory agencies are really accepting these tools as a third line of evidence. They’re really valuable in accepting these different treatment strategies and feeling like we’re doing a much better job of characterizing and understanding what we’re doing at the site. So these are more readily accepted on a larger scale by sites around the world. We’re seeing other countries performing this type of analysis, as well. U.S. is really the leader in this, that we now see this really branching out at sites all over the world.

Dane: Okay. Great. Thanks so much, Dora. Let’s see here. The next question is, “I think you stated that only the parent product should be tested for fractionation. Was it CSIA? And is this due to the intermediate formation and destruction? Yet daughter products were discussed in the presentation.” So this person is just asking for clarification about what you meant by that.

Dora: Sure. So what I actually meant by that is I would recommend looking with CSIA of both the parent and daughter products, if you’re looking to prove degradation of the parent-daughter compound. But if you want to elucidate which pathway, it can be difficult to get that isotopic enrichment factor. So that’s what I was meaning there. If you’re trying to get that epsilon value where we plotted the slope of the line and we looked for that isotopic enrichment to tell us if it’s biotic or abiotic, you can do that for parent very easily, but it’s much more tricky for daughter products. So use CSIA for parents and daughter products both to indicate that degradation is occurring. But if you’re looking to elucidate the pathway of biotic versus abiotic, then only do that on the parent compounds.

Dane: All right. So okay. All right. Okay. The next question here is, “How do you interpret bio or abiotic when your slope lies within the range of both epsilons for a particular compound?”

Dora: Oh, that can be difficult. We didn’t see that in the case here. You would probably have to do additional testing. And if it actually lies… I don’t think I’ve ever seen where those biotic ranges overlap with the abiotic ranges, but if that were to happen, we’d have to look at other lines of evidence to see, do we have microorganisms there that are potentially available to do the degradation? If those are absent, then maybe, you know, look at, you know, what chemical geochemistry looks like at the site. You know, do we have magnetite or other compounds available that can do abiotic degradation? So take a comprehensive look at those multiple lines of evidence. And try to elucidate it a little better. And there can be times where you’ve got both degradation pathways that are occurring, but that other data would help you to figure that out.

Dane: Okay, great. We are close to running out of time, but maybe one more question here. And that is, “Would high concentrations of aluminum,” and they say, “up to several percent, in saturated site soils cause a problem for TCE degradation?”

Dora: For aluminum, I’m not sure. I haven’t seen anything in literature that would talk about that in particular. If you want to email me directly about that after this webinar, we can do some lit searches. And we can get back with you to let you know about that in particular, but I don’t think I’ve seen anything in literature that talks about that interfering with TCE degradation.

Dane: Okay, great. Thank you so much, Dora. That’s gonna be the end of our chat questions. If we did not get to your question, someone will make an effort to follow up with you. If you would like to learn more about molecular biological tools from Microbial Insights, please visit www.microbe.com. If you need immediate assistance with a remediation solution from REGENESIS, please visit www.regenesis.com to find your local technical representative, and they’ll 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: Thank you very much.