DAVE ADAMSON: We already spent some time looking at isotope data earlier in our lectures on CSIA. Now we're going to shift to a discussion of how to use isotope data in combination with molecular biological tools. So in our field, it's a technique that's been commonly referred to as stable isotope probing. Similar types of techniques have been around for a long time in more purely microbiological studies, but SIP is gaining traction in environmental applications, particularly in MNA. CHUCK NEWELL: So maybe one way to start is to define what is stable isotope probing, and then how do you define what it's not? So it's not the same thing as CSIA. Remember that CSIA is the measurement of naturally occurring isotopic ratios in a contaminant and then tracking them over time and distance. The panel on the right shows what we want to see when degradation occurs. Preferential utilization of the lighter isotope results in a shift that increases the abundance of the heavier isotope. DAVE ADAMSON: So that's CSIA. SIP is different, because it's no longer the naturally occurring abundance that's of interest. Instead, you're actually adding a radio labeled contaminant, one with lots of C13, for example, and seeing if it accumulates in biomarkers or byproducts over time. So if you see this accumulation, then you've got a strong line of evidence that the organisms are actually growing on the contaminant. So that's what's important. If the organisms are growing, then they're degrading the contaminant. So this flow charge on the right, it's from an excellent ITRC guidance on EMDs, environmental molecular diagnostics, that includes stable isotope probing. So it sort of outlines the stepwise process that you go through. And note that there are some different options as you get towards the end of this. You can either quantify the amount of radio labeled compound that has made its way into general biomarkers, like PFLA. CHUCK NEWELL: OK, now wait, what's PFLA? DAVE ADAMSON: PFLA is phospholipid fatty acids. So that's an important biomolecule for all organisms, and it's comprised of lots of carbon, so including carbon that would come from this containment destruction. So you could also take the other pathway and try to identify biomarkers where accumulation of radio label is taking place. So that's shown on this next slide. So besides PFLA, you could also go and look for isotopic enrichment from DNA or RNA, as well as different proteins. But you might use PCR or DGGE methods to do this. And you can get a lot of different diagnostic information about the types of organisms that are responsible for the degradation, either through standard phylogenetic analyzes or metagenomics, looking closer at some of the functional elements, as well. But an important thing to remember is that these are culture independent methods, meaning you don't has to plate out or try to grow the organisms. You're just analyzing what's there. CHUCK NEWELL: OK, great. So I know that there are a few different isotopes that can be used for these applications. This slide right here shows carbon, and nitrogen, and oxygen. And again, we're talking about stable isotopes, so only those that are not radioactive, like C14. So that gets used in the lab, but it's not really good for field applications. DAVE ADAMSON: Yeah, And C13 is probably the most common for these sorts of applications, since we're tracking organic contaminants. So remember, we're interested in measuring accumulation of heavier isotopes, so something that bugs aren't necessarily going to want to do. To overcome this, what's commonly done is basically using a really large amount of radio label in the compounds that you're adding. So at least 10%, and even sometimes approaching up to 100%. CHUCK NEWELL: OK, and I also know that you have some options in how many labeled isotopes are included. The bottom graphic shows various molecules of RDX, the contaminant, the explosive that were used in a SERDP study by Paul Hatzinger, Mark Fuller of CBNI, Bella Chu at Texas A&M, Jawal Hawari, and others. They're showing how this RDX is being degraded at Navy sites. DAVE ADAMSON: So they tested a whole suite of different ways to radio label and see what kind of responses they got out of that. So RDX and other explosives like TNT are just a few of the compounds that can be tested using stable isotope probing. So this slide shows some of the others. CHUCK NEWELL: OK, looks like we've got several hydrocarbons, like benzene and thiolene, phenol, pHHs, MTBE, Dioxane, vinyl chloride using the aerobic pathway. But what's this graph on the right? DAVE ADAMSON: That's the 1,4-Dioxane plume at the Air Force Plant 44 site, sort of a famous site in Tucson where there's been a lot of characterization of really long co-occurring Dioxane in a chlorinated solvent plume. So a study published, Dora Chang was the lead author on this, a lot of other people on that paper. They showed how stable isotope probing was used to establish Dioxane degradation at some portions of this site. CHUCK NEWELL: OK, now what about all these compounds have in common? DAVE ADAMSON: Well, the one thing that they have all in common is they were all basically carbon or energy sources for microbes. So they're degraded by these things-- the microbes want to degrade them, so they're accumulating these radio labels within their cell structure as they're degrading it. And that's basically what makes SIP important in these cases, and that's what we're trying to measure. CHUCK NEWELL: OK, so right. But I'm afraid it also means that there are a bunch of compounds that don't fill this same role that cannot be tested using SIP. I think you got some of these listed here. It includes a lot of the ones that I'm really interested in, most of the chlorinated solvents and perchlorates. So sort of disappointing in some ways. DAVE ADAMSON: Yeah, a little bit. It's basically related to what's going on in terms of metabolism here. So these compounds are primarily serving as electron acceptors, so there's not actually any assimilation of them into the biomolecules while they're being degraded. I would note that there's some other pathways that had been tested using SIP, so TCE has got a little star on that because oxidation called co-metabolism, that has been tested. It turns out there really wasn't an easy way to distinguish. And since there isn't any really direct stimulation during that initial steps of degradation in that pathway. CHUCK NEWELL: OK. But for the compounds that you can use SIP in the field, these methods are actually fairly straightforward. Application for MNA has shown on this figure from microbial insights, who markets these bio traps for SIP. DAVE ADAMSON: So these are more generally known as passive microbial sampling devices, because you're just sticking them in a monitoring well or exposing them to sort of natural groundwater flow through that well. So this graphic here that they've created they're basically baiting these beads that are inside these devices with radio label benzene. The bugs grow on those traps during that deployment period, which might be something, 30 days, maybe shorter, maybe longer. Then you pull them out and measure what's happening on those beads. CHUCK NEWELL: I love the term baited beads, to sort of understand MNA. But you can use the residual benzene to understand the rate of degradation after correcting for the amount lost just due to groundwater flow. You can also measure whether there's an accumulation of C13 in the inorganic carbon as an indirect measure of activity, like biological anaerobic oxidation. And of course, you can measure the accumulation of C13 in the biomass that's grown on those beads. DAVE ADAMSON: Yeah. So That's sort of the basic approach, and it has a lot of advantages. We list a few of them here. The C13 accumulating in the biomarkers, it's a pretty strong line evidence for attenuation. And so an example of that's shown in this little graphic again from the Air Force Plan 44 study on Dioxane, we're showing there is a big change in sort of that pre-deployment, the blue to the post-deployment carbon isotope abundance is shown for different locations from that site, where bio traps were installed. If you look at that sort of green line in the top, about 80% to 90% removal, in this case. So importantly, SIPC confirms also that this pathway is biological. In some cases, you can even use it to identify who might be responsible. CHUCK NEWELL: OK, and since the deployment is occurring inside the monitoring well and it's not in some lab reactor, it really attempts to mimic these into two conditions. It can also be used to support rate calculations. As we mentioned before, it's culture independent method. DAVE ADAMSON: And then the final strength that's listed on that side is basically complementing existing sort of monitoring programs that you have. You're using monitoring wells to gather this data. Unfortunately, that sort of also contributes one of the limitations of these methods, and we go through a few of those here on this next slide. So we've already mentioned that not all compounds, including most chlorinated solvents being degraded are really testable using SIP. There's also some concern that SIP will lead to false positives or an indication that degradation is occurring when it's really not. That's because again, we're using monitoring wells. They're easy to use, but then you sort of think about this, you're creating preferential conditions for growth. There's some concern if you talk to people about whether these data are really realistic, especially the rate information. Another reason why false positives could occur are because of cross-feeding. That's sort of shown in this little cartoon. You have different bacterial populations, so you might not actually be certain about who is actually responsible for the degradation. CHUCK NEWELL: OK, and just looking at some of the other reasons listed here-- there's obviously a cost, some extra labor analysis associated with using these methods. And there are really only a limited number of labs that really support these SIP studies. The ones that can do provide some guidance on the protocols, but there's still some uncertainty on the proper deployment times and that sort of thing. And finally, there's this issue of stakeholder familiarity. These are relatively new methods, and there are some education that might be necessary for everybody to be comfortable with the results. DAVE ADAMSON: OK. That sort of wraps up our discussion of stable isotope probing, so let's look at a few of the key points from this lecture. Stable isotope probing, or SIP, involves elements of both CSIA and MBTs. CHUCK NEWELL: These radio labeled compound is added to a lab microcosm or a passive microbial sampling device, such as the biotrap. And there's this accumulation of these radio labeled compound of the biomarkers over time, and it just serves as indication that degradations occurng. DAVE ADAMSON: And then, finally, as IP works for compounds that serve as carbon sources. It doesn't necessarily work for compounds that serve as electron acceptors.
