 Okay, thank you guys. We're just gonna wait for another minute or so just so we get Disciplines in and then we'll make a start. Thank you. Okay, you've got about 23 or so so we might kick it off Thank you all for joining us once again for another of our Hydroterra webinar series our focus for today's session is the selection process of biological tools for assessment with Contaminant degradation and this will be in conjunction with microbial insights to that So today we have Michelle Kant and our general manager that Hydroterra who we overseen this presentation and making sure things are running smoothly Myself Karl McLaren. I'm the sales manager here at Hydroterra and We're very fortunate enough to be joined by a special guest Dora Taggart who was the president microbial insights based in Knoxville, Tennessee Dora's Guidance has sort of led microbial to become the leading molecular. So environmental laboratory Without seven locations globally She's become a global ambassador for the environmental restoration industry and regularly presents at the keynote speaker environmental and corrosion conferences hundreds of sort of technical workshops worldwide and As well as co-authoring some guidance documents for the vast array of advisory groups So we're very thankful for her time today just a quick bit of Housekeeping you'll see at the top there during this presentation the Q&A at the top feel free throughout this process to type any questions you may have and at the end we'll Leave sort of five or ten minutes after the presentation just to run a bit of a Q&A session and filter any questions You may have as we go along here Our goals at Hydroterra as always is to assist our vast client base with you know showcasing the amazing technologies from our specialist monitoring suppliers that we represent here in Oz and Microbial insight is absolutely no exception to this We aim to facilitate for the education In order to empower our clients to make more informed decisions on technology integration in the future and we also use this platform to You know understand the industry needs that are currently out there. So You guys are seeing what you guys are seeing and how our Showcases might help with particular projects you've faced or are going to to face in the future So just the format I'll just run through a bit of an introduction as I'm currently doing here And then we'll hand over to Dora to do the technical presentation side of things And then once Dora is finished with that then we'll just Filter through and do a bit of a Q&A session at the end there So I'll let Dora probably talk more about microbial insights in their history as a whole But I will just talk about our relationship and what we at Hydroterra assist with here in Australia So we've represented microbial here in Oz since 2015 and the majority of the customers in Australia Have utilized their services I guess to assist their work on contaminated sites Specifically in around the ground water space the services provided by Microbial has been very valuable in the designing of groundwater mediation strategies We help microbial With logistics and and sales support So the shipping and storage of the sampling supplies of the bio flow filters and biotraps, which may be familiar with Essentially the logistics I suppose of routine Sampling clients will you know email requests for sampling supplies to be shipped which we will then facilitate and Once of the samples are collected in the field they'll be shipped directly from the client to the laboratory in Adelaide for analysis and Microbial you know periodically Stock supply of bio flow filters and biotraps at Hydroterra to have on hand to fill our requests and keep and we keep these Frigerated in storage We also facilitate, you know technical support through the assistance of microbial insights to help our client make more sort of informed decisions. So that's really our relationship with microbial and At this point, I'll hand over to Dora to present And we'll finish it off as I said at the end there with any questions and I'll just facilitate a bit of a Q&A So thanks Dora. I'll hand it over to you All right. Thank you very much Kyle. Go ahead and share my screen here Okay, can you see my slides Kyle? I can yep Perfect. All right. Hello everyone. Thank you for joining You're a little bit ahead of me. It's around 10 30 at night here in the US So I'm very grateful to be able to do this presentation for you guys and and for the attendants and Excited to be able to come back to Oz. Hopefully soon. Maybe sometime next year. I'll be able to come down for another visit Today, I'm going to be talking to you About how you can select the right tool Some of this will be refreshers for those of you that have used the biotraps before We'll talk about ways to use them for in situ understanding of contaminant degradation But I also have a few new tools that we've brought online this year that that might be of interest. I'll introduce those to you as well So for those of you that aren't familiar with microbial insights we have been Around for almost 30 years. I've been with the company for almost 20 years at this point We are One of the leading molecular labs around the world and as Kyle mentioned, we do have seven locations Hydrotera is our sales representative there in Australia So they can provide you with all the sampling supplies and information and help facilitate Collection at sites any questions you might have on that end and then we do also have a lab down in Adelaide That you know, does the preparation work for the analysis of the samples that makes shipping very easy But it's really my goal as president am I to make sure that we're taking all of these tools that are created through other industries or in academia and Making them commercially available so that you can get results that you can Actually truly rely on and make decisions that are actionable based on the data So making sure we have good QA QC and I'm really excited. We just got our ISO accreditation for QPCR and to my knowledge were the first lab that actually has it for QPCR in this industry So really excited to just finish that and and to be able to get that done I've been trying to get an ISO for almost 20 years now And they finally had a certification process that could do it for the QPCR and not for some other analytical tool So really am I what we focus on is using those molecular tools? So we're not growing cultures in the lab. We're not doing traditional chemical analysis We're doing these specially kind of boutique analysis that can be a third line of evidence to help you really understand What's happening in the environment and really provide you with that additional line of Evidence to be able to make decisions at your site And over the course of my career. I've seen a big evolution in the tools We started doing quantitative PCR 20 years ago where we could Quantify numbers of particular organisms or functional genes We now have more than 150 assays that we can do for that a lot of different organisms a lot of different pathways So we can really understand and illuminate what the biological potential is in our sites So I'm going to talk a little bit about that today on petroleum hydrocarbon site and a little bit on the chlorinated solvents We'll also talk today about stable isotope probing Which is one of my favorite tools because it gives us definitive proof of degradation of Compounds that the organisms uses a carbon and energy source. So when we're trying to degrade petroleum hydrocarbons or 1-4 dioxane Those types of compounds. This is a great tool that we can use We also have compound specific isotope analysis that I won't really get into today, but I can answer questions if you have Questions about that. It's really good to document degradation of your chlorinated compounds So if you have a PCE or TCE site and we've used this on quite a few sites there in Australia So it's a great tool that we can use to help you get more understanding and prove degradation of your compounds and Then today I'm going to show you a couple of examples of some in situ microcosm So if you have these different amendments that are available or a culture that you're interested in Microbial insights does not sell any amendment. We don't sell any cultures But what we do is we work with all of the companies that have these options And we can load these in these bio trap samplers And I'm going to show you that today with these different amendments or with those cultures and we can test to see what's going to happen In situ it's about a fifth of the cost of a traditional Bench scale microcosm study and you're actually doing it in situ So it's a great alternative to some of the bench scale studies that are available So as I mentioned I've been doing this for longer than I'd care to admit at this point And I've seen a good evolution of these tools from the chemical compounds looking at the PLFA analysis of the cell membranes Introducing QPCR in the early 2000s Stabilized to help probing coming online with a lot more use of the bio trap samplers and then the evolution of QPCR Where we can look at a lot of different organisms and compounds all at one time through a platform called Monterey and then today there's new things that are coming online that we're working on we do some we're doing some work in proteomics We're funded by the National Institute of Health here in the US looking at metabolomics and how we can use that to better understand chlorinated solvent degradation to start with and then moving into other compounds of interest So if we have a cyst ECE stall or there's certain metabolites that we can see that are predictive that we might have those stalls That happen at our sites. So some of these new tools that are coming on We're really involved heavily in the research end of that as well to make sure that we make those commercially available as quickly as possible And this year more than any other year, I think for all of us 2020 has been definitely a challenging year With COVID and the things that we've encountered It's really made me think about how important accurate data is and you know I was thinking last year about what our theme would be and crossroads was what came to mind because you know having good Evidence when we get to a decision point is really critical. And if we don't have good data to give us direction It's really hard to make accurate decisions and cost effective decisions No matter what the challenge is and that's definitely been true for this year So when you think about bio remediation, we really want to understand the chemistry at the site What are those contaminant concentrations doing? What's the trending that's happening there? What's the mass flux look like? What's the geochemistry? Are we seeing electron acceptors being consumed? Do we have enough donors to make our processes efficient and then what micro organisms are present and how Active are those microbial communities and what competing organisms? What is that biological community doing when we make an injection into our subsurface? So trying to get into that black box so we can really understand what's happening So when we look at molecular biological tools, there's a lot of questions that we can answer And as I mentioned in the beginning we can use tools like QPCR or the platform of quanta ray to look at a lot of different genes and Organisms to get a concentration of different contaminant Degraders so we can get a lot of information from that tool. Who's present? What's the potential for degradation at our site? Then we can use tools like stable isotope probing or compound specific isotope analysis to dive in a little further and Help us prove that biodegradation that compound itself is actually changing at our site And then for compounds that we don't know a lot about like PFOS is still something that those Perforated compounds we're trying to understand we're getting better at being able to analyze for the compounds themselves But we still don't have a good grasp of what microorganisms are present So what do we do at those sites? Well in those cases we can do things like next generation sequencing Metagenomic type of approaches where we can look at all the microbial community that's present and see what the selective pressure of a particular Contaminant is doing to our microorganisms. So that's a great tool that I won't get into today But if you guys have questions, I'm happy to answer that at the end And then as I mentioned in the beginning we can also use these things like our biotraps as an in situ Microcosm to help us understand if a treatment strategy is going to be effective And one of our our clients here in the us I love what he says he uses the in situ microcosm. So he fails on a small scale So something doesn't work. He tests it in the field knows that you know, it's a very inexpensive way to evaluate a Particular strategy that he's thinking see how effective it's going to be before he moves on to a pilot scale or something more cost costly So my presentation today I wanted to kind of give you a as broad a perspective as I can in a short period of time and still hopefully make it digestible We're going to talk about biotraps samplers because that's kind of a tactile Tool that you can use for a collection of your microbial community. It's unique to microbial insights We'll talk about the evolution of those and using them as in situ microcosm So we can evaluate those different electron donors and acceptors and cultures Um, I'm going to give you an example of stable isotope probing Like I mentioned one of my favorite tools to prove contaminant degradation And through this I'm going to show you some case studies and talk about our database Which is another one of my little pet projects that I'm excited to share with you guys oops So let's start with a biotrap sampler. So for those of you that aren't familiar with us I know when I was looking at the registration it looked like there were quite a few people that we already work with So thank you for listening to me and now some of you have used the biotraps there in australia But these are a passive sampling tool. They were developed by the university of tulsa and by duPont As a way to collect microorganisms and get an integrated view of what's happening in a monitoring well over a period of time So we can use this as a way to collect the microorganisms with any of the molecular tools that we offer. We can use it to do um, you know Qpcr we can use it for stable isotope probing. We can use it in the in situ microcosm So there's a lot of ways that we can use this particular tool to answer questions for us And the real key to these biotrap samplers are the little beads that are inside. They're called biosep beads They're three to four millimeters in diameter. So pretty small They're composed of 75 percent pattern activated carbon. So that gives us absorption capability So it'll absorb our compounds that are naturally occurring or that we're interested in that are in our monitoring wells So that we can take a look at those and see how they affect our microbial communities The other component to the biosep bead is something called nomex That's what a fire retardant suit is made from so it gives us the ability to sterilize these at very high temperatures And the real key to the nomex is not only the sterilization aspect But it kind of acts like chicken wire in holding that pack together So that when you put the nomex and the pack together it forms what you can see here on the right hand side of your slide That cross section these really nice little circular beads Um, but they're very porous. Um, so organisms can kind of get into these little surfaces There's a 600 Square meters of surface area per gram of these beads So tons of surface area for these little microbes to grow form biofilms and to be able to do degradation processes So we can kind of capture it inside these bio trap samplers So the way that it works is just like if you were going to go out to collect a microbial sample or a chemical sample You're going to purge that monitoring well Then you're going to suspend the bio trap from the top of your casing By a line down into the monitoring well unless you'd like to fish make sure you do suspend it from the top of that casing We've had some same some instances where that's happened. They've dropped them in the well and had to retrieve them but We leave them in for a period of 30 to 90 days depending on the questions We're trying to answer and then you'll pull them back up and send them to the laboratory for analysis And you want to deploy them at the depth that you're interested in so down in that screened interval Not inside the napple But either above or below it depending on the question that you're trying to answer The other great thing that you can do with this if you have tidal influence or you have somewhere that you're seeing a well That's getting a lot of water fluctuation We can suspend this with a float From the top of the casing and that float will give us the ability to keep it at that certain depth in your monitoring well So it's a way to kind of overcome some of those challenges So that's kind of the premise for how the standard biotrap works One of the most common questions that I get is where should we put these at a site? So just like when you're collecting your chemistry and your geochemistry you want to look where there's changes in their contaminant concentration So you definitely want to put a biotrap in the source area So we can see if those high concentrations are having a negative impact on the microbial community And then as you move down gradient by order of magnitude changes If there's receptors that are particularly of interest that are further down gradient or cross gradient We might want to put biotraps there And then sometimes a background sample can be useful and that's something that feel free to always shoot us an email Give us a call. We're happy to talk to you about those, you know The great thing with these zoom meetings is we can be face-to-face all around the world at any point So we're happy to talk to you about when a background would be useful if you're doing petroleum hydrocarbon remediation And we're doing qpcr sometimes. It's nice to have a background sample for comparison So we can say here's what's naturally occurring Where the petroleum is we see the selective pressure has caused an increase in this particular group So they're likely utilizing our contaminant of concern Things like that so we can add some context to it. So your typical site you're going to collect two to three samples across the site To get kind of representations of what we think the microbial community looks like in a particular area An evolution on that and something that we've done quite a few of in australia is something called an in situ microcosm That's what I was talking about earlier. It's where we can look at different treatment options Rather than doing a bench scale study where we have to take it out of the field and do it on the lab and try to control All those conditions we can use these in situ microcosms in the field So we get truly representative samples of what's happening in situ So typically in a monitoring well, you're going to put a couple of these into one location so that you might look at things like What's naturally happening under the regular conditions in the environment? What happens if I add a particular electron donor or acceptor and stimulate those conditions? Or if you have a culture that you're using from from one of the labs there in australia I know there's a couple of bio augmentation cultures that are available there You could test those by load we can load it onto one of the bio trap samplers And put them in to see if that's going to survive in situ So you're going to typically deploy those for 60 to 90 days pull them out and send them to the lab So These units are slotted pvc as you can see in the picture here and inside those slotted pvc You can see in the center here what those look like They're about 15 inches in length and the key is on each end of these units There are baffles that are based on the diameter of your monitoring well So those baffles are used to section off a portion of the monitoring well, so we don't get that circular Groundwater circulation that happens within a monitoring well so that you get just the flow through each unit So you can put multiple units down into one location to be able to test what's happening And one valuable thing we learned there in australia was if we're going to do these In pvc, it works really well We can get them sealed off if you have metal wells that have been installed The baffles do not work well to seal it off So we need to put those microcosms each one into a separate location with similar chemistry and geochemistry So that we could evaluate different treatment options. So there's ways we can get around it We've learned a lot over the 20 years of doing this So, you know, give us a call and we can help you. So we're going to set these up custom So these do come from the us to australia because we're going to custom make these so hydro tarot doesn't keep these in stock But they'll work with us to get whatever you need Quickly so that you can have those ready for field deployment. So you can put them in a treatment Like a little train of them to go down in the monitoring well Where you could look at maybe a control unit different treatment options and inside each unit We're going to put different components depending on the question that we want to answer So for instance, if we have a liquid electron donor that we want to look at Say an hrc or an eos or whatever it may be We're going to load that electron donor into a supplier sponge and put those on either end of the unit Then we're going to have a passive diffusion bag That will collect our Contaminants of concern so we can do a standard 82 60 analysis off of this bag To be able to see how our contaminants are affected within each unit We'll have a bio trap sampler so we can look at how our microbial community changes within each unit And then a standard vova vial that has a passive membrane on the top and we use that vova vial To be able to look at the geochemistry. So if we're say adding a sulfate for a petroleum site Then we can make sure that we are seeing increases in our sulfate concentrations Or if we want to see if we're moving From nitrate reducing conditions, we can start to see if our nitrate is getting depleted Different questions that we want to answer about the electron acceptors through that geochemistry vial So we're going to Look at multiple lines of evidence and that's kind of a key point that if you've heard me speak before You've probably heard me say it, but I'm really a big advocate for looking at all three lines of evidence We can look at our chemistry look at those trends look at the geochemistry to see what's being utilized And then look at the microbiology to see who's there and doing the processes So it really gives us a lot of information These in-situ microcosms have been used as I mentioned for almost 20 years now And we've done almost every electron donor that you can imagine Um electron acceptors again We don't sell any of these products But we work with all these vendors to be able to make them available and deliver them The best way within the units and then when we get to stable isotope probing We can bait these biotraps with different compounds that we're interested in and do heavy weighted versions of them That i'm going to talk to you about in just a moment and we can see how those compounds track Into our microorganisms into those microbial processes. And so we've done a slew of contaminants over the years Anything that the organism uses as a carbon and energy source is a great option For stable isotope so we can load and put those into in-situ microcosms as well So let me give you a quick example of this. It's one of my favorite examples because it's easy and short This is a location where they had Btex contamination you can see the site map there They picked two monitoring wells at the location. It was a kind of a standard gas station site Btex residual that was there and what they wanted to know is if we add sulfate Are we going to enhance anaerobic Btex degradation in these two locations? So inside each monitoring well, there were two in-situ microcosms that were put together One was a control unit to look at the natural Um processes that are happening in that well and then a biostimulation unit where they added a powdered sulfate amendment called EAS these were left in each monitoring well for 60 days and then were recovered for analysis So inside each unit you can see the two units there on the left hand side of your screen to show how they were linked together In the biostimulation unit, there was a passive diffusion bag to look at our chemical contaminants a biotrap To look at those anaerobic processes of interest A geochemistry vial again to make sure we're delivering that sulfate and how the natural sulfate conditions looked And then a baggie that contained that powdered EAS was put into the biostimulation unit within each monitoring well Again, those baffles are going to section that off so that we can see what happens within each of these two units So just a real quick summary of the results here This is a portion of the quanta ray analysis Looking at qpcr targets that are important for anaerobic degradation of petroleum hydrocarbons The blue bars are our two m&a units The biostim units are the two red bars that you see from mw 19 and mw 23 So if we go look at the right hand side of your graph to start We want to enhance sulfate reducing bacteria right because we're adding sulfate That's one good indicator to make sure we're delivering sulfate. So you can see with two blue bars We have some sulfate reducing bacteria that are naturally present in both locations But when we add the EAS amendment, we are increasing the concentrations of these sulfate reducing bacteria So that was a good indication. But more importantly, are we stimulating anaerobic vtex degradation? So this first gene on the left hand side here bcr is an intermediate gene for anaerobic vtex degradation A lot of pathways kind of funnel through this gene You can see that we certainly went from not seeing anything in our m&a units To pretty high concentrations in our amended unit. So we're getting around 10 of the four and 10 of the six Cells per bead that are being detected of this particular gene And then in the other two genes that we are looking at this bss is benzels succinate synthase This is an anaerobic gene for the text degradation. So the toluene ethyl benzene and xylene components You can see that only one monitoring well is stimulated here an anaerobic benzene ABC anaerobic benzene carboxylase gene for anaerobic benzene degradation is stimulated in the other well So when I first saw these microbial results, I was a little perplexed at while We weren't seeing those genes stimulated in both monitoring locations with that sulfate amendment But when I looked at the the chemical data of it It turns out that this monitoring well that we're seeing here the mw19 well Was the only text compounds that were being detected in that location So it makes sense. We're stimulating the pathway for anaerobic text degradation And benzene was the compound that was remaining in the other location So we stimulated the gene for that degradation So this is a very quick study. That's an easy way before you move to a full field scale or even to a pilot scale study To make sure that you're going to get stimulation of the right microbial populations at the site So a pretty low cost investment to get the answer to that and they did move to a full scale um amendment with the EAS and they were very successful in cleanup at this location So it was a real easy way to be able to do that initial evaluation So that kind of shows you a little bit of how you can use those in situ microcosms for petroleum hydrocarbons in particular I think it's a great tool. We can also do it for chlorinated compounds. We've done quite a few studies there in australia with both petroleum and chlorinates for this looking at different donors different electronic acceptors and seeing how they respond with those in situ microcosm So I think a very useful tool for us Another tool that I love and we've done this a few times in australia as well. It's called stable isotope probing So as I mentioned in the beginning, this is a tool that's particularly useful for any compound that the organism uses as a carbon and energy source So for petroleum hydrocarbons in particular, it's a great tool So the reason the way that this works the premise is kind of like if you've done any compound specific isotope analysis It's based on that premise. So with csia analysis. We're looking at how carbon changes In our compounds with biodegradation. So in the environment standardly our carbon 12 is around 99 percent of the carbon the stable carbon molecules that are present in For instance, any of our chlorinated compounds any of our petroleum compounds. It's about 99 12 carbon 1 percent is 13 carbon. So if we look for natural shifts, what we're doing is looking to see if these Compounds become more enriched in our heavy weighted carbon. So naturally the carbon 13 bonds are a little stronger than carbon 12 bonds So they break a little slower with bioremediation if it's dilution or dispersion you're going to see the same You know amount ratio of your carbon 12 to carbon 13 But when it's biodegradation You're going to see it become more enriched or heavier amount or more amounts of our 13 carbon. So heavier compounds So what we can do is take that natural process that's happening And we're going to flip it around and use this as a tracer So we're going to make compounds that are 99 heavy weighted carbon So when we look at the ratios we if our organisms are utilizing our heavy weighted carbon, we should see Very dramatic changes in the weight of our compounds or our microorganisms or our byproducts Based on utilizing this particular compound of interest So essentially becomes a tracer to understand the fate of our contaminant So we can do it for b-tex for oxygenates from the fuels like mtbe tba for naphthalene 1 4 dioxane Anything that the organism uses as a carbon energy source. We've done it for sulfylene for a variety of compounds So what we're going to do the way it works We're going to synthesize a heavy weighted carbon compound like a benzene for instance We're going to load it onto our biocet beads. So Remember the biocet bead is made of powdered activated carbon which absorbs these compounds really well, but they're also Bio available for microorganisms. So we're going to load them a certain concentration of this 13 carbon compound onto our bead Put it into our biotrap housing put it in our monitoring well for 30 to 60 days The organisms that are naturally in that well are going to colonize onto those beads And they're going to form biofilms Then if they're capable of utilizing that compound for instance the benzene They're going to either build new biomass with it make more microorganisms and divide and grow new microbial biomass And utilize that heavy weighted carbon so we can track that into the organism itself Or for some compounds or some parts of the process They're going to mineralize it and make waste products So we can look at carbon dioxide or methane to see if we have heavy weighted Carbon in those compounds which is proof that the organism utilized our compound of interest So it's one of the few tools that you can do one analysis one time improve that an organism used a compound So we know how much of our 13 compound we load onto the biotrap sampler We know how much we retrieve at the end more important than that Is where does that heavy weighted carbon go? Do we see it incorporated into that carbon dioxide or into the cell membranes the lipid membranes of the microbial cells? So that's definitive proof that the organism utilized this compound of interest So let me show you a quick case study on this. This is an industrial site that was done here in the united states in new jersey It was a very standard site With a leaking underground storage tank the main contaminant that they were concerned about in this particular case was para xylene So what we did was to make some biotraps that were loaded with a 13 carbon 9 13 carbon para xylene and we deployed it into four monitoring wells So what they wanted to understand is if we add an oxygen amendment into the well Are we going to see an increase over what naturally is happening at this location? So this one well m2 e2 that you see over here on the right hand side of your screen that has 10 ppm Concentration of para xylene naturally was used as our control So there was no oxygen amendment that was being affected within this particular system these other three monitoring wells had varying concentrations of para xylene 1 10 and 100 ppm And they were in the radius of influence of this oxygen system that was installed at the site So putting four biotraps loaded with the 13 carbon para xylene into the mon each of these monitoring wells Left them for 30 days And then let me show you some data from this So we know how much we loaded onto the biotrap how much we recovered at the end So you can see in our control unit. We had about a 6 loss Whereas in the biostimulation with oxygen we saw 16 to 40 loss of the compound So that's great. You can get some kind of relative rates of where things are happening Where you're seeing more metabolism of these particular compounds You know at the different wells at the site, but more importantly, where did it go? So for those of you that aren't familiar with this notation This is a delta value a delta value is our ratio of 13 carbon to 12 carbon of our compound Compared to an international standard. That's how we get this value Typically in nature remember 99 percent is 12 carbon So typically our values for carbon dioxide are going to be minus 20 to minus 30 delta value And for shifts that are natural, you're going to see very small changes It's going to become more positive by one or two delta values So very small incremental changes that happen naturally in the environment But because our biotrap and our para xylene is loaded with so much heavy weighted carbon If we have carbon dioxide that's heavy We know that had to come from our compound that was loaded onto the biotrap samplers So this m2 e2 location that had the 10 ppm para xylene In that look that monitoring well, we can see that our dissolved inorganic carbon or carbon dioxide values are Plus 600 Delta value, there's no way that that could happen in nature unless the organisms are utilizing That heavy weighted para xylene from our biotrap Same thing with those oxygen amended units. Look how much higher the incorporation values are There's no mistake that this came from that 13 carbon para xylene on the biotrap samplers So we can see a couple things one It's naturally happening where we're getting Respiration processes where it's mineralizing that para xylene the organisms that are naturally present in that monitoring well Are working under normal conditions, but at a lower Delta value than the locations where it's affected by the oxygen So it does look like oxygen is the limiting factor. Of course, we see that commonly at sites So if we can get oxygen bio available to these microorganisms get them in contact with the para xylene You're going to see degradation of that para xylene Same thing looking at our biomass a kind of a different way to look at it here So if we look at our total biomass in this first column the amount of biomass that's enriched with our 13 carbon You can see there's a little bit of enrichment that happens our delta values are plus 48 So they're making new biomass to some degree under natural conditions But look how much higher it's happening when oxygen is available. So the microorganisms are getting really stimulated building new cells Very active community that's happening then at that point So again definitive proof that the compound that we are interested in is being degraded in the environment And you know, I'd love to tell you that you can calculate a rate for your site based on this and you can get Some assumption of what rates are but this is more because we're putting it on a biotrap It's really bio available to those microorganisms and bioremediations about contact, right? It's a contact sport So we need to Look at the relative rates between locations to understand and make decisions about what we should do So if we're concerned that the geochemistry is really different in one area We could do stabilized tote probing in that area compared to another location To look at what those differences in our incorporation rates are And that way we can start to get a better understanding of what's happening at our site as a whole So you can't get a direct rate from it that you can put into a model and calculate But you can get differences across your site to make those adjustments Um before I move on from petroleum compounds I wanted to touch on this just really briefly and I'll answer questions that you guys have at the end So we've talked about with petroleum compounds. We can do quantitative pcr We can look at all these different pathways that are present We can do in situ microcosms to evaluate different donors and acceptors We can do the stable isotope probing that I just showed you to prove that a particular compound We're interested in degrading But what happens on some of our sites where you have an initial spill and you're not exactly sure who's responsible Or how long that's been there and you're doing some initial site characterization Well earlier this year microbial insights bought a lab record laboratory in the u.s. Called pace energy services Pace did some csa analysis. Um, so they had some of the same instruments that we do Plus they did this petroleum forensics where they did, you know, whole oil fingerprinting or Piano analysis to be able to look at all of the different components that are within the gasoline range To be able to add some context, you know, is it predominantly gasoline or is it diesel mix? Are there heavy-weighted, you know motor oils that are present in our sample? You can kind of help to figure out who might be responsible The age of compounds because they're blending agents or bledded compounds that have been added over the years So we can look at all of those types of things in this analysis To help put some contact text to where that sample originated from so this is kind of a new Testing it's been around for a long time in our industry But now microbial insights has the capability to be able to do that So if you're interested you can reach out to me Or to the folks the hydratera and they can can direct you to the right people to talk to in our company That can kind of give you some more information on this But I just wanted to kind of give you a little glimpse of that because i'm i'm super excited that we have this ability I think it's a nice compliment to what we were already doing for the industry Okay, so I know i've given you a lot so far, but just one more quick thing I wanted to talk quickly about chlorinated solvent sites and and we've worked on You know some of this here in australia So I wanted to just give you a quick case study to show you if you have a chlorinated sites and things you can do This is kind of a typical case study from a chlorinated solvent side It had a mixture of ethanes and ethanes They were seeing some daughter products that were forming but mostly they were seeing cysts cce So they were a little concerned What's going to happen? Am I going to see vinyl chloride accumulate? Am I going to get past the cysts even? What's going to happen at the site the conditions at the site were mildly reducing But they had some sulfate that was present. So they were really concerned So now they needed that third line of evidence that microbiology to help us really understand what's happening at that chlorinated site Oh I got mixed in there. Sorry about that So the questions they wanted to know, you know, what organisms do we have naturally present? You know, because if we don't have to helicocoides or we don't have some of the ones that can do the chlorinated ethanes If we add an electron donor, are we going to really see anything happen? Do we need to bio augment and add a culture in there? So the the first thing that we did at this site was to do some qpcr analysis looking at the quanta ray to look at different Pathways to understand the microorganisms that are present So this is the first glimpse of the quanta ray data This is on the x-axis. You can see different organisms and genes for the anaerobic portion of our quanta ray analysis Looking at dehalo coquities and some functional genes Then on the chlorinated ethane side looking at dehalobacter dehalogenomonas Some of these organisms that can do those chlorinated ethanes or higher chlorinated ethanes like the sulfuromonas So if we look at this and we get our initial baseline event here We can see we have a little bit of dehalo coquities and some dehalobacter and sulfuromonas So that really explains why we're seeing cis accumulate We just don't have enough dehalo coquities to really get that efficient reductive dechlorination So is it likely under the current conditions? Probably not unless we do something Do we need to bio augment not necessarily we have some dehalo coquities? So that can be a site decision. Do we have time to be able to stimulate and get effective? bioremediation of our compounds Or you know, do we need to to speed things up by adding a culture? Well in this case They decided they wanted to try bio stimulation first to see what happened And so how do I know that that 10 to the 1 is Not going to be enough to get efficient reductive dechlorination So one benefit with microbial insights is we've been doing this for a long time We have more molecular data than probably anyone in the world We've have for the last 20 years. We have over 50,000 samples in our database So we've gotten samples from every state in the united states all the u.s. Territories We have samples from every continent on earth and from about 45, I think different countries at this point With australia being a big portion of samples that that we've seen as well So we can take all of those samples from all over the world and look at samples where we've analyzed for dehalo coquities And you can see out of these samples where we look where vinyl chloride and ethane form We need at least 10 to the 4 cells of dehalo coquities to see efficient degradation to see vinyl chloride Form so you we've seen these same data published in some papers, but it's usually from one or two sites So this is looking at an enormous amount of sites around the world different locations And we can see that that 10 to the 4 are higher is really where we want to achieve to get good efficient chlorinated solvent degradation So if we go back to our baseline event put my little line there, that's the gold mark So let's see what happens once we add an electron donor So if we want to know is the electron donor effective are we seeing Increases in our degraders that are present and then you know, are we going to get efficient reductive? to happen at the site So the red bars here are the first quarterly event that happened after the injection And you can see our dehalo coquities concentrations have greatly increased We're almost to that 10 to the 4 mark not quite And what these numbers above tell you is when you do qpcr analysis with microbial insights you'll get a login to our database and That's something i've been super passionate about in my career was trying to develop this and we have this for qpcr and csia and i'm working on one for the stable isotope probing But to give you a way to put context to the data So i can't always tell you this is the magic number you need Dehalo coquities is kind of a unique organism that i can say that 10 to the 4 thresholds really important But for other organisms we can't so being able to put context to it Are you on the low end or the high end? Are you in the middle compared to sites around the world? So if i look here with dehalo coquities i'm In the 68th percentile so not bad for just a quarter past my injection But look how much i've stimulated these other degraders for the higher chlorinated compounds and the chlorinated ethanes We're in the top 10 percent of sites around the world for these particular organisms. So very effective post injection strategy But what we had here is the regulatory committee looked at it and said You know, you're still shy of the mark that you told me you wanted to hit So how do I know that you know the donor is not going to burn off and you're going to still continue to get effective Reductive dechlorination. So one of the great things about the quanta ray platform is not only do we look at all these anaerobic degraders and pathways that are available for this reductive dechlorination But we also look at competing microorganisms. So when we add a substance into the subsurface Like an electron donor. It's going to have to ferment right so that we can get that hydrogen produced And when that happens, we're not just stimulating the guys we want to stimulate We're stimulating anyone that can utilize that hydrogen So in this case sulfate reducers and methanogens always get stimulated and after looking at this data for 20 years I can tell you very commonly I see an initial spike in sulfate reducers and methanogens Really depends on our sulfate concentrations at the site and then you'll see three to six months Down the road once those concentrations start to stabilize Then you have your anaerobic degraders that really get stimulated So in this case, you can see we stimulated sulfate reducers We stimulated methanogens and that's good news because we're getting more reducing conditions getting into that sweet spot For anaerobic degradation pathways But it helps to explain why we're seeing a little bit of a lag with our reductive dechlorination So the Site managers were able to go back to the regulatory agency and say, you know, this is why what we think is going on Give us one more sampling sampling event to see what's going on So the second quarterly event look at this We're up to almost 10 of the six in our dehalococcoides and really positive Now we're seeing vinyl chloride that's getting generated and look at our vinyl chloride reductase gene So not only do we stimulate dehalococcoides, but we stimulated strains that have the ability to utilize Vinyl chloride very efficiently at the site so we can get really efficient degradation of vinyl chloride when it's generated So really positive results and in this case the qpcr kind of helped to prove that one the donor was having a positive effect And then helped to explain with the competing microorganisms what could be causing the lag in it So I would really encourage you if you do qpcr data to go in log into that database add some context to the data Because getting a concentration of a gene if you don't know What that concentration means it's hard to make an actionable decision But if you put some context to it, is that a high number? Is that a mid number or low number every gene's a little different Some organisms are more prevalent in the environment some or not So this helps you put some context to it so you can start to make stronger statements And um, you know, I kind of think about it like googling something if when my son was little he's 20 now Which is greatly aging me kind of grew up with the company with microbial insights And when he was little and thought I knew Stuff which he doesn't think I know anything now But when he thought I knew stuff and he would come to me I would google it so I could learn more about a subject to talk to him Same thing if you use this data, you can go into the mi database Put your information in add some context to your data So when you talk to your clients or to the public and you're doing presentations, you can really put some context What does this mean to locations around the world that are experiencing similar problems? So I wouldn't really encourage you to use the database. It's something I'm really proud of And then we continue to build and evolve and to make it more user-friendly So I hope you will give it a try Today I've kind of Given you kind of a quick introduction for petroleum and chlorinated compounds Of course, this can be used for a variety of contaminants whether it's you know immobilizing metals or Looking at abiotic degradation pathways or looking at total microbial communities like we talked about There's a lot of tools that are out there and new tools that are emerging We just finished the phase one of that metabolomics project with NIH that i'm really excited about And we're going to submit for a phase two so we can do some further field validations And I think we're coming up with some metabolomic markers that could be important predictive mechanisms We're seeing groupings where we can find the background samples compared to where things are actively Declonating compared to where there were stalls that were happening at the site So we want to kind of apply this to a broader scale and make sure but I think we've got some real promising evidence That metabolomics could be a useful tool for us in our site assessments I'm excited. Hopefully within the next year or two. I'll be able to bring you an update on that So it is constantly evolving. There's a lot of tools that are out there and we know that Not all of you are going to be microbiologists or know about all these tools So feel free to reach out to us. I love to talk to you guys Happy to talk about any tool pluses and minuses really more than selling you a tool I'm a nerdy scientist by heart And I want to be sure that whatever you use gives you valuable information that you can take action on so that it helps improve our industry We've done this not only for remediation. We do a lot of work in other markets We've been doing this for a long time on the microbial side Almost 30 years. So we do a lot of work in oil and gas in the upstream paper and pulp industries Making sure if they have corrosion that's from microorganisms in their assets. How do they protect that asset? How do you make sure the biosides effective? We do work in agriculture looking at nematode populations and at nitrogen cycling We also use the nitrogen cycling tools for wastewater tools. We've done a lot with landfills We do a lot of work with NASA looking at extreme environments. We've analyzed moon rocks And interesting we've done a lot of work in art restoration looking at How do you preserve samples of the silk road? They were seeing fungi growing in the silk road They we helped to identify the fungi that was present fungi on the salk institute We did samples from king tuts tomb. Was this fungi new fungi or was it old? Fungi that was from you know back in the days when the tomb was originally painted So we've been involved in some really interesting art restoration projects So I say all that to let you know that no matter what the question is that you have From a microbial standpoint, we want to be a resource for you. So feel free to reach out. I've extracted samples from some really disgusting things From snail poop in the rainforest to see how they were utilizing compounds in the rainforest To the moon rocks to king tuts tomb. So I've handled a lot of different samples So we definitely know how to recover biomass from a lot of different matrices So reach out to us if you have questions on even oddball projects happy to help We really want to be part of your team hydratera is a great component for you That's right there and anything that is more specialized and unique to us They'll get in touch with us or help you get in touch with us so that our team can can help you with the data And it really is my goal to have you generate actionable data. That's useful at your site I know I beat this in the ground a little bit on this presentation, but I really would encourage you to use that database I think it's a valuable tool for us And trend your data a lot of times people call me and they send me microbial data from one sampling event And my magic eight ball can't predict that well So it's really hard for me to tell you from one event what it means But if we can trend that data just like you do the chemistry and geochemistry It's a really valuable predictive mechanism for what's happening at the sites and understanding pathways and Convergence and changes that happen So there's a lot we can do when we trend the data The only thing that I can do one sample one event and really give you some definitive information is that stable isotope probing But the other stuff if you can trend it, it's really helpful Um, as I mentioned any matrix I've seen almost anything that you can imagine Typically we do a lot of groundwater samples if you reach out to hydratera They keep these filters that you see on the left hand side here in stock You just connect it to your peristaltic pumps and pump up to a liter two liters through depending on the biomass in your samples Cap that filter send it to our lab and adelaide. They're going to extract those those filters for you We can also do soil solid samples the bio traps. If you have vapor intrusion We can talk about ways that we can look at the indoor air So pretty much any matrix that you have Samplings easy Just in case you couldn't tell from my accent. We're based in Tennessee in the united states a little bit southern So hopefully some southern hospitality going on there But we do have locations as I mentioned there hydratera works You know they're in victoria with you guys We have the lab there in adelaide that does the preparation of the samples We also have labs in canada belgium italy germany In china so a lot of locations no matter where The samples are coming from we can get them to a location to make shipping easy for you guys And we do work with companies all over the world. We do have a pretty good global presence I saw a lot of your company names or companies that we commonly work with In other countries and definitely a lot here in the u.s So reach out or we can put you in contact with colleagues that we've worked on projects with if you'd like references You've heard a lot from me today and I've been to australia quite a few times I think i've been at least eight times in the past to your beautiful country and would love to come back So you'll probably see my face more often than you care to but i'm dorah And um, there's a whole team of people that travel and speak and do workshops and education and our project managers or lab directors That are very knowledgeable that can really help you with your project so you can reach out to any of us We really want to be available to you Um, so at this point I will take any questions that you guys have Excellent. Thanks dora. That was uh, that was amazing. Um Just bring up The presentation So we'll just filter through a couple of quick questions. Um, just quickly We have one from Giuseppe Greco that says, uh, what's the most common application to use as technology to monitor an Uncontaminated site like a natural forest and how the results can be compared With the carbon storage capacity Do you have anything to Yeah, so we can use it on uncontaminated sites to look at like the natural, um, microbial communities that are present We've done, you know, root analysis to look at what's being uptaken into different environments. We've done a lot of work um in fisheries and You know along coastal areas So there's a lot of different things that you can do on the microbial side to understand the community Some of that's hard on carbon storage capacity It's a little limited in like a calculation that we can do from that But we can tell you the main types of microorganisms that are present Yeah Um, just has another one, um after bushfire event the temperatures reached in the top soil are often sufficient to affect soil Microorganisms and other soil properties related to the postfire microbial recolonization Um in extreme cases the top soil can undergo complete sterilization How the technology in a monitoring well can help us to understand the scale of that damage Is that something you've worked with before in bushfire events and It is we've actually done a lot of work with that, you know, especially here right now california is having a lot of that impact with Reforestation from all the fire damage that we're experiencing here in the us So we have done that, um, you know, we think that we can sterilize the top soil with those high temperatures But what we find is that once things return to more ambient conditions That the microbial community tends to migrate to those areas anything that can survive in extreme And then as things start to break down and change and and we do Revegetation you can see dramatic increases in those microbial communities and a great example of that Is that these sites where we're doing thermal, right? We think that we're going to go in and we're going to heat it to these really high temperatures We're going to burn off the contaminants that we have present in the environment And that we might sterilize our microbial community But what we find is that doesn't actually happen you see that the microbes greatly reduce during that time But once conditions return to more normal Then you start to see that um, the the microorganisms kind of return to those areas or grow back into those areas or migrate back to those areas And you can get some good stimulation that happens after that because you've made other compounds bio available So you definitely get shifts in the community But I don't I rarely see anything that gets completely sterilized even extreme brine or extreme heat events. I've seen Um, you know microbial communities rebound fairly quickly after those events yep Alex Patterson has just asked that you spoke of historical biases in microbial data Can you explain that a bit more and how you avoid it? Sure, so I think stable isotope probing is a great example of that So if you do qpcr analysis and you find that you have all of these genes present in the environment And we look at the dna of microorganisms How do we know that that's actively happening at that point in time and it's not historical So that it wasn't that they utilize these contaminants before but it's not as active right now So there's two options. We can do RNA analysis So we're looking at RNA which is only made as the organism needs it and it degrades very quickly So that's one option or the stable isotope probing because if that happens To the compound when we add it right at that moment, you know during that deployment period of that bio trap That it's not historical. That's active degradation that was happening. So that's kind of two options to help overcome that Yep, excellent. And I just have a final question here Richard Selwyn Campbell has asked you say that bioremediation is a contact sport Is there any appreciable for bioremediation that occurs within the free phase itself? Um, yes, I think that there's some that happens there. Um, it depends on the compound and the um The organism that we're talking about how active they're going to be in that free phase Um, so a lot of it kind of happens on the peripheral boundary around our napple compounds So, um, you'll see kind of that water phase where concentrations aren't so harsh is where the biggest amount happens But we have seen, you know, even in napple Solutions and very very high concentrations. I've seen some really active degradation that surprised me Mm-hmm Excellent. Um, if we have no other questions there, um It seems to be the filtering going through but uh, you know, we have this available through recording. So if you Guys, um, have any other questions that, you know, take a bit of time to digest. Um, what Doris presented today Feel free to To contact us here at hydrogen terra in the uh, contact information that you see there And we'll be able to Feel through any questions through to dora and the team at microbial as well. Um to get you guys the answers that you need Um So I think that's probably it uh for today. So thanks so much dora for your time and I know it's uh Really late. So it might be bed time for yourself, but uh, do appreciate Um, you're taking the time today to talk to us and um, thank all our participants that have come through today So thank you very much for everything All right, thank you so much kyle michelle for putting this on and thank you everyone for attending and feel free to reach out If you have additional questions Yeah, excellent. Yep. No worries. Okay. Thank you very much everybody and uh, appreciate Appreciate all your time. All right. Thank you. Thanks dora