Does the Mass Balance of the Reactive Tracers Resazurin and Resorufin Close at the Microbial Scale?

Abstract

Resazurin (Raz) is a phenoxazine dye that can be reduced irreversibly to the daughter compound resorufin (Rru) by aerobic respiration. Previous hydrologic studies using the Raz‐Rru reactive tracer system to quantify water‐sediment interactions and metabolic activity have reported that dilution‐corrected masses of Raz and Rru recovered are smaller than the mass of Raz injected. This lack of mass balance closure has been reported as a nonideality of this tracer system and, to date, it is still unclear what drives incomplete recovery. We used controlled laboratory experiments varying the initial concentrations of Raz, the duration of the experiments, and the type of microbial communities present to quantify mass balances of Raz and Rru under conditions that removed other suspected causes of incomplete recovery in field experiments, i.e., sorption to sediments and photodecay. We used the summation of Raz and Rru concentrations over time to assess mass recovery and variability and found mass recoveries in the range of 85.6–110.4%, with a maximum standard deviation of 7.5%. In three of the four experiments, no strong temporal trend in mass recovery is present. In an experiment with Bacillus subtilis bacteria, lower recovery and evidence of a temporal trend in recovery only occurred after 13 hr past the complete transformation of Raz (i.e., beyond the duration of most field experiments). These results suggest that the lack of mass recovery in field studies is likely associated with physical or chemical mechanisms rather than biological interactions with the Raz‐Rru tracer system.

Spatiotemporal Variability in Transport and Reactive Processes Across a First‐ to Fifth‐Order Fluvial Network

“…Raz is a redox‐sensitive phenoxazine dye that can be used as a “smart” tracer to estimate microbiological activity associated with stream water‐sediment interactions (González‐Pinzón et al, 2012; Haggerty et al, 2008; Knapp et al, 2018). In the presence of mildly reducing conditions, Raz undergoes an irreversible reduction to its daughter product resorufin, Rru, and experimental work with microbial communities has shown that (1) the transformation of Raz to Rru is proportional to oxygen consumption (González‐Pinzón et al, 2012) and (2) the mass balance of Raz and Rru closes over time scales relevant for field experiments (Dallan et al, 2020). Therefore, the transformation from Raz to Rru has been used as a proxy for in situ aerobic metabolism and water‐sediment interactions in the last decade (Argerich et al, 2011; González‐Pinzón et al, 2014, 2016; González‐Pinzón et al, 2015b; Knapp & Cirpka, 2018; see review by Knapp et al, 2018).…”

“…BTCs were sampled for periods ranging from 4 to 10 hr after injection. These sampling periods were deemed sufficient to capture the Raz to Rru reaction time scale (<6 hr) (Dallan et al, 2020 ; González‐Pinzón et al, 2012). Surface water grab samples were collected from the thalweg at each of the sampling stations at intervals ranging from every 1 to 30 min throughout our tracer BTCs using 60 ml plastic syringes and were filtered through 0.45 μm pore size Whatman Nylon filters (25 mm diameter).…”

“…(2) the mass balance of Raz and Rru closes over time scales relevant for field experiments (Dallan et al, 2020). Therefore, the transformation from Raz to Rru has been used as a proxy for in situ aerobic metabolism and water-sediment interactions in the last decade (Argerich et al, 2011;González-Pinzón et al, 2014González-Pinzón et al, 2015b;Knapp & Cirpka, 2018; see review by .…”

“…These tests were done in five different stream orders along the Jemez River, New Mexico (Figure 2a), during two different flow regimes (i.e., summer baseflow and spring higher flow). The Raz‐Rru system was chosen for this study because the irreversible transformation of Raz to Rru provides an in situ estimation of hyporheic zone processing as a fraction of riverine processing through an estimation of metabolic activity (Dallan et al, 2020 ; Haggerty et al, 2008; González‐Pinzón et al, 2012, 2014, 2016; González‐Pinzón et al, 2015b; Knapp & Cirpka, 2018). While not all the parameters describing reactive transport across the Jemez River network varied consistently with stream order or discharge, we found a trend toward more reaction‐limited conditions with increasing discharge across all stream orders, as Damköhler numbers consistently decreased along the fluvial network in response to decreasing processing rate coefficients.…”

Physical and stoichiometric controls on stream respiration in a headwater stream

“…Also, we use the transformation of Raz, which occurred at the same spatiotemporal scales of the nutrient additions, to calculate how changes in stoichiometric conditions and discharge affect respiration. Briefly, the reactive tracer Raz (blue in color) is irreversibly reduced to resorufin (Rru, red) under aerobic respiration, and the relationship between Raz transformation and oxygen consumption is linear (González-Pinzón et al 2012Knapp et al 2018;Dallan et al 2020). Table 1 shows the masses injected and the discharges observed during the studies.…”