Research Feature: A "Smart Tracer" to Detect Suburface Microbiological Activity

Link to the Paper

Haggerty, R., A. Argerich, and E. Martí. 2008. Development of a "smart" tracer for the assessment of microbiological activity and sediment-water interaction in natural waters: The resazurin-resorufin system. Water Resources Research 40:W00D01 (August 2008).

In August, the journal Water Resources Research published a paper by geosciences professor Roy Haggerty and colleagues from Spain about their work to develop a “smart tracer” that can detect microbiological activity in stream and groundwater systems. In this short Web interview, Roy describes the concept of a “smart tracer” and his experiments with resazurin, a nontoxic compound historically used to detect spoiled milk. 

Conservative hydrologic tracers are compounds that can be injected into a stream or groundwater system and used to track flow paths and measure flow velocity.  What makes a “smart tracer” different?

The idea with a “smart tracer” is to be able to measure the same things as a conservative tracer but also have the tracer provide some more information about the processes going on in between where you inject the tracer and where you detect it again. Someday maybe we’ll have nanoparticles with computer chips that can record information as they flow through the system, but right now, the way to get this extra information is with a chemical compound that changes irreversibly into a second compound when it encounters the particular conditions you want to investigate. So you start out injecting compound A and downstream you end up with compounds A and B and the proportion of the two tells you something about the process you are studying. 

Your paper is the first to propose using resazurin as a hydrologic tracer – how did you get the idea to use the compound and what is the reaction that makes it work? 

Initially, I was looking for a tracer that could help me determine the proportion of stream flow that travels through the hyporheic zone, the near subsurface underneath and adjacent to streams. One of the characteristics that is different between surface and subsurface flow is the abundance of bacteria – there are usually far more microbes in subsurface waters than in surface waters because sediments form a substrate where bacteria can grow. So I thought that if I could find a compound that would react when bacteria were abundant, then perhaps it could be used as a tracer that could distinguish between the two environments.

When I looked for compounds that are used to detect or count bacteria, I found resazurin. It is a compound that irreversibly degrades to resorufin in the presence of living bacteria. It was first reported in the 1870’s and began to be used in the 1930’s to detect when milk had spoiled. It is still used commercially to count the number of living cells in cultures. The mechanism that triggers the reaction isn’t well understood but it probably has to do with the use of oxygen by the bacteria – anaerobic bacteria don’t trigger the reaction and the reaction will also not take place in anoxic waters without a reduction reaction.

Your paper reports on the feasibility studies you did with resazurin – what did you find?

We did a series of column and batch experiments to characterize the decay, sorption, reaction, and transport behavior of resazurin in water and sediment taken from several streams. We found that resazurin degraded to resarufin about 1000 times faster in sediment that was colonized by bacteria than in stream water alone, so it has potential to detect differences in flow paths. Rezazurin and resarufin also met most of the other key criteria needed for a conservative hydrologic tracer.  They are not toxic and can be easily measured – resazurin and resarufin are fluorescent (in different spectra) so samples can be analyzed with a spectrofluorometer.

We also identified a couple of things that make resazurin problematic as a tracer. It is an adsorbing compound, so it can bind to sediments, and resarufin, the degradation product, also degrades. Both of those characteristics make the interpretation of the data more complicated than we would prefer.

We also realized that while I was initially interested in tracking subsurface flow, the tracer is better described as an indicator of metabolically active transient storage – that is, it is an indicator of all of the areas of the system with a concentration of bacteria. That's usually the subsurface but it could also be other areas, for example the bottoms of pools or on biofilms on rocks. 

What plans do you have for future work?

Resazurin field experiment.

A resazurin field experiment in Catalunya, Spain.

We have submitted a proposal that shifts more to the field and looks at different systems to see whether we can replicate what we have seen so far. We have to see how much difference there is in resazurin reduction between systems and determine whether those observations correlate to known differences in metabolic activity in the systems. We have begun this work with some field experiments in Spain, in Austria, at the H.J. Andrews Experimental Forest, and in a stream near Salem. We have also had an SBI intern, Marc Nabelek, working to replicate the feasibility experiments in the WRR paper with sediments and water from different systems. If the proposal is funded, it would allow one of my co-authors from Barcelona, Alba Argerich, to come to OSU as a post-doc and continue to work on these questions. 

Another possibility that I haven’t explored very much yet is the potential to use resazurin as a tracer to detect hotspots of microbial activity in the subsurface. I’m interested in collaborating with others who would be interested in exploring resazurin's potential as a tracer in groundwater. 

If you have questions about the project or would like to learn more, please contact Roy Haggerty.