Species-Level Variability in Extracellular Production Rates of Reactive Oxygen Species by Diatoms

Biological production and decay of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide (O2-) likely have significant effects on the cycling of trace metals and carbon in marine systems. In this study, extracellular production rates of H2O2 and O2- were determined for five species of marine diatoms in the presence and absence of light. Production of both ROS was measured in parallel by suspending cells on filters and measuring the ROS downstream using chemiluminescence probes. In addition, the ability of these organisms to break down O2- and H2O2 was examined by measuring recovery of O2- and H2O2 added to the influent medium. O2- production rates ranged from undetectable to 7.3 × 10−16 mol cell−1 h−1, while H2O2 production rates ranged from undetectable to 3.4 × 10−16 mol cell−1 h−1. Results suggest that extracellular ROS production occurs through a variety of pathways even amongst organisms of the same genus. Thalassiosira spp. produced more O2- in light than dark, even when the organisms were killed, indicating that O2- is produced via a passive photochemical process on the cell surface. The ratio of H2O2 to O2- production rates was consistent with production of H2O2 solely through dismutation of O2- for T. oceanica, while T. pseudonana made much more H2O2 than O2-. T. weissflogii only produced H2O2 when stressed or killed. P. tricornutum cells did not make cell-associated ROS, but did secrete H2O2-producing substances into the growth medium. In all organisms, recovery rates for killed cultures (94–100% H2O2; 10–80% O2-) were consistently higher than those for live cultures (65–95% H2O2; 10–50% O2-). While recovery rates for killed cultures in H2O2 indicate that nearly all H2O2 was degraded by active cell processes, O2- decay appeared to occur via a combination of active and passive processes. Overall, this study shows that the rates and pathways for ROS production and decay vary greatly among diatom species, even between those that are closely related, and as a function of light conditions.

. Schematic of O 2 setup for the FIA system. O 2 setup shows the experimental medium and MCLA reagent being pumped at 3.0 mL min -1 into the FIA flow cell where a photomultplier tube detects the chemiluminescence signal. The phytoplankton cells are immobilized on the filter where they could be exposed to light or dark conditions. The filter was positioned as close to the flow cell as possible with the filter disk parallel to the floor. All tubing except that used in the peristaltic pump was black PEEK tubing.

S1.2 Calibration
A primary O 2 stock solution was made fresh for each calibration point by adding a small amount of KO 2 powder to a solution of 30 µM DTPA and 0.032 M NaOH (nominal pH 12.5) (Heller and Croot, 2010 (Bielski et al., 1985). A known volume of the primary O 2 stock solution was withdrawn immediately after the absorbance measurement (before addition of superoxide dismutase) and transferred into a solution of 10 µM DTPA and 1 mM NaOH to make the working stock. A small volume of the working stock was then spiked into the ASW (at a ratio not exceeding 100 µL: 100 mL ASW), and the chemiluminescence signal was monitored over time. A maximum of 3 min passed between the absorbance measurement of the primary O 2 stock and the addition of the working stock spike to the sample. Microsoft Excel's Solver function was used to minimize the sum of the squares of the differences between the actual data and the model to determine best fit values of R BL, R t=0 , and k loss,O2-in Equation (1) in the main text.

S.1.3 Experimental runs
The experimental run was started by pumping fresh ASW over an acid-washed 25-mm 0.45-µm cellulose acetate filter for ~4 min to get a steady baseline signal (region 1 in Figure S2), R ASW . The pump was then briefly stopped (< 3 s) while the tubing was switched to the culture.
The cells were loaded onto the filter through the pump, which took less than 2 min (region 2 in Figure S2). The pump was again briefly stopped (< 3 s) while the tubing was switched back to the ASW. The signal was then monitored for ~10 min (region 3 in Figure S2), with R cell obtained as the average of the 500 counts taken at the end of this time period. A O 2 spike was then added to the ASW from a freshly prepared working stock solution, as described for the calibration procedure above, and the signal was monitored for ~5 min (region 4 in Figure S2). Finally, superoxide dismutase (SOD) was added to the ASW at a final concentration of ~0.24 U mL -1 (~1.6 nM) (region 5 in Figure S2). R SOD was obtained as the average of 100 counts taken as soon as the signal stabilized after the SOD addition.  A""""""""""""""""""B""""""""""""C""""""D" 6:00"AM" 6:14"AM" 6:28"AM" 6:43"AM" 6:57"AM" 7:12"AM" 7:26"AM" 7:40"AM" 7:55"AM" Figure S5. Calculated H 2 O 2 production rates in C. cryptica and P. tricornutum. Error bars represent one standard deviation. None of the rates was significantly different from zero.

S4. Surface-area normalized production rates
To determine whether production rates of O 2 and H 2 O 2 were a function of cell surface area, surface-area normalized production rates were calculated as follows. Average cell surface area (SA avg ) for a given diatom was calculated by using average cell dimensions provided by the National Center for Marine Algae and assuming that diatoms were perfect cylinders (Table S1).
Total cell surface area (SA tot ) in the filter was then calculated by multiplying average cell surface area by the estimated number of cells on the filter: where d culture is the density of the culture in cells mL -1 and V culture is the volume of culture loaded on the filter. Surface-area normalized production rates ( Figures S6 and S7

S5. Surface-area normalized ROS decay
To compare the relative abilities of different organisms to break down H 2 O 2 and O 2 -, we converted our measured recovery percentages to decay coefficients. The equations for this conversion can be derived by treating the dead volume in the filter from the flow-through method as a well-mixed reactor at steady state (Hemond and Fechner-Levy, 2000).
Using the well-mixed reactor model, the change over time in the concentration of H 2 O 2 within the filter, [H 2 O 2 ], can be written as: where m H2O2,in and m H2O2,out represent the rate at which H 2 O 2 is transported into and out of the filter, respectively, in units of nM hr -1 , while the production and decay terms represent the result of biological activity within the filter. Equation S2 can be rewritten as: where [H 2 O 2 ] in is the concentration of H 2 O 2 in the analytical medium entering the filter, Q is the flow rate in L hr -1 , V is the dead volume of the filter in L, P' H2O2 is the biological production rate in nM hr -1 and k' loss,H2O2 is the first-order decay coefficient of the H 2 O 2 within the filter. At steady state ( can be rewritten as: Combining Equations S5 and S6 gives a solution for k loss,H2O2 : which can be rewritten as where Rec H2O2 is the recovery calculated by Equation 7 in the main text. The cell-surface area normalized decay coefficients for each ROS (Figures S8 and S9) were calculated by dividing k' loss for each ROS by the total cell surface area in the filter as calculated by Equation S1. For cell-density normalized decay coefficients (Table S2), we divided k' loss by the cell density on the filter instead. Figure S8. Calculated surface-area normalized O 2 decay rate coefficients for Thalassiosira spp. Error bars represent one standard deviation. Figure S9. Calculated surface-area normalized H 2 O 2 decay rate coefficients for all 5 diatoms studied. Error bars represent one standard deviation. Observed recoveries for killed controls of T. oceanica were over 100%, indicating insignificant decay. Table S2. Cell--density--normalized decay rate coefficients for diatoms. Uncertainties represent one standard deviation.