Experiment design and bacterial abundance control extracellular H2O2 concentrations during four series of mesocosm experiments
Authors: Hopwood Mark J. , Sanchez Nicolas , Polyviou Despo , Leiknes Øystein , Gallego-Urrea Julián Alberto , Achterberg Eric P. , Ardelan Murat V. , Aristegui Javier , Bach Lennart , Besiktepe Sengul , Heriot Yohann , Kalantzi Ioanna , Terbıyık Kurt Tuba , Santi Ioulia , Tsagaraki Tatiana M. , Turner David 
Published: March 2020

Abstract. The extracellular concentration of H2O2 in surface aquatic
environments is controlled by a balance between photochemical production and the microbial synthesis of catalase and peroxidase enzymes to remove
H2O2 from solution. In any kind of incubation experiment, the
formation rates and equilibrium concentrations of reactive oxygen species (ROSs) such as H2O2 may be sensitive to both the experiment design, particularly to the regulation of incident light, and the abundance of different microbial groups, as both cellular H2O2 production and catalase–peroxidase enzyme production rates differ between species. Whilst there are extensive measurements of photochemical H2O2 formation rates and the distribution of H2O2 in the marine environment, it is poorly constrained how different microbial groups affect extracellular H2O2 concentrations, how comparable extracellular H2O2
concentrations within large-scale incubation experiments are to those
observed in the surface-mixed layer, and to what extent a mismatch with
environmentally relevant concentrations of ROS in incubations could
influence biological processes differently to what would be observed in
nature. Here we show that both experiment design and bacterial abundance
consistently exert control on extracellular H2O2 concentrations across a range of incubation experiments in diverse marine environments. During four large-scale (>1000 L) mesocosm experiments (in Gran Canaria, the Mediterranean, Patagonia and Svalbard) most experimental factors appeared to exert only minor, or no, direct effect on H2O2
concentrations. For example, in three of four experiments where pH was manipulated
to 0.4–0.5 below ambient pH, no significant change was evident in
extracellular H2O2 concentrations relative to controls. An
influence was sometimes inferred from zooplankton density, but not
consistently between different incubation experiments, and no change in
H2O2 was evident in controlled experiments using different
densities of the copepod Calanus finmarchicus grazing on the diatom Skeletonema costatum (<1 % change in
[H2O2] comparing copepod densities from 1 to 10 L−1). Instead, the changes in H2O2 concentration contrasting high- and low-zooplankton incubations appeared to arise from the resulting changes in bacterial activity. The correlation between bacterial abundance and extracellular H2O2 was stronger in some incubations than others (R2 range 0.09 to 0.55), yet high bacterial densities were consistently associated with low H2O2. Nonetheless, the main control on H2O2 concentrations during incubation experiments relative to those in ambient, unenclosed waters was the regulation of incident light. In an open (lidless) mesocosm experiment in Gran Canaria, H2O2 was persistently elevated (2–6-fold) above ambient concentrations; whereas using closed high-density polyethylene mesocosms in Crete, Svalbard and Patagonia H2O2 within incubations was always reduced (median 10 %–90 %) relative to ambient waters.