It has been assumed that photo-oxidation of chromophoric dissolved organic matter is the primary source of superoxide (O2-) and hydrogen peroxide (H2O2); however, there are indications that biological sources may play a significant, if not dominant, role in the production of these reactive oxygen species (ROS). Because the presence of these ROS in the open ocean can affect the redox cycling of iron, copper, and manganese which thereby influencing their bioavailability and H2O2 has been shown to inhibit the growth of Prochlorococcus ecotypes in the mixed layer, it is important to understand the biological controls on O2- and H2O2. For this reason, scientists at the Colorado School of Mines and Harvard University will test the following hypotheses: (1) dark biological production is an important contribution of H2O2 and O2- to oligotrophic surface waters; (2) spatial and temporal variations in biological production and decomposition rates contribute to the observed variability in H2O2 concentrations; (3) biological production of H2O2 in oligotrophic waters proceeds via formation of O2-, and the yield of H2O2 from O2- is variable depending on the major sink reactions (e.g. with metals) of O2-; and (4) activities of NADH (nicotinamide adenine dinucleotide) oxidase enzymes from a phylogenetically and ecologically diverse group of bacteria, including cyanobacteria and heterotrophs, are significant sources of ROS to marine waters. To test the first three hypotheses, a series of field observations in oligotrophic waters of the subtropical North Pacific will be carried out to establish biological production rates of H2O2 and O2- and assess their relationship to photochemical production and H2O2 concentrations in the euphotic zone. In addition, laboratory studies will also be conducted to examine the stoichiometric yield of H2O2 from O2- in the presence of different O2- sinks. For hypothesis 4, the diversity of microorganisms involved in ROS production will be identified, as well as the proteins involved which will enable the interpretation of community-level transcriptomic data for regions illustrating variable contributions of biological activity to ROS production.
As regards broader impacts, the lead proponent plans to present a lecture at the Colorado Science Conference for Professional Development on the importance of biogeochemical cycles. If successfully received, she proposes to develop this material into a one day workshop and offer it via the Colorado School of Mines Teacher Enhancement Program. One postdoc and one graduate student from the Colorado School of Mines and one postdoc, one graduate student, and one undergraduate student from Harvard University would be supported and trained as part of this project.
Reactive oxygen species (ROS) are key components in the physiology of organisms, allowing for cell development, cellular communication, and wound repair. They can also be toxic. ROS degrade DNA, proteins, and cell membranes. Formation of ROS is an unfortunate consequence of breathing oxygen, so all oxygen-breathing organisms, including humans, must strictly control the concentration of ROS inside our cells in order to survive. This is accomplished by producing enzymes (proteins) that degrade ROS to benign products so they can be excreted. If cells don’t properly remove these ROS, there are consequences – cancer and premature aging in humans, cell death in macro- and micro-organisms. The ROS superoxide and hydrogen peroxide are also ubiquitous in the ocean, where reaction between sunlight and organic matter has been presumed the primary source. Phytoplankton (algae, diatoms) have also been shown to produce ROS – yet, their abundance and distribution in nature is also fundamentally constrained by sunlight. More recent discoveries have revealed that the majority of ROS production in the ocean is produced within the dark – yet the sources and reasons for this production were unknown. ROS are key controls in biogeochemistry, where for instance they influence the bioavailability of metals to support microbial life, degrade carbon to more labile sources that may then enter into the foodweb, and induce the bleaching and demise of coral reefs. It is therefore of great need to obtain a better understanding of the processes that control their formation and lifetime in marine systems. The goals of this research were to determine if ROS are common to dark marine waters and, if so, who is making ROS in the ocean and why. We focused here on the oligotrophic ocean as these nutrient-poor regions represent key areas where carbon-mediated photochemical ROS production is limited. As a comparison, we also studied deep coastal waters, where carbon is not limited but light will be attenuated. This research involved a number of research cruises in the Pacific and Atlantic Oceans, seasonal sampling in coastal waters off Cape Cod, Massachusetts, and laboratory incubations of key microbial species. In brief, we discovered that dark production of superoxide is ubiquitous in marine systems, where both bacteria and phytoplankton are key sources. Interestingly, for the common nitrogen-fixing microorganism Trichodesmium obtained from various sites in the oligotrophic Sargasso Sea, we find that superoxide production is decoupled from photosynthesis and has an inverse relationship with colony density. These results hint at a role for superoxide in cell signaling and communication in these organisms. Trichodesmium forms substantial blooms in oligotrophic ocean waters, where superoxide production has been implicated in programmed cell death and subsequent bloom demise. Thus, our findings point to a complex role for superoxide in phytoplankton bloom dynamics. We have also identified the extracellular enzymes involved in superoxide production by a common marine bacterium and two diatom species. Surprisingly, the enzymes responsible for superoxide production by these organisms differ but are all classified as antioxidants, enzymes that are expected to degrade (not produce) ROS. This research has revealed the importance of microbial processes and extracellular enzymes in the formation of superoxide in dark regions of the ocean – regions that have historically been assumed free of ROS. It has been long appreciated that these same ROS are important for controlling the biogeochemistry of the atmosphere and surface ocean – we now need to think about them in the 95% of our global habitat that is in the dark. We also need to revisit the function of enzymes that are canonically considered antioxidants – in fact, these enzymes may modulate between degrading and producing ROS. Further classifying these enzymes and the triggers impacting their activity is therefore of great importance, not only for understanding ocean biogeochemistry but also human health. This research supported the educational and scientific development of three postdoctoral scientists at the Woods Hole Oceanographic Institution and one visiting undergraduate student from Italy. Three of these scientists were women, two of which were from underrepresented groups in STEM. This research was also part of a collaborative effort with Dr. Tina Voelker and her students at the Colorado School of Mines. Outreach associated with this research included presentations to elementary school children and K-12 teachers in Colorado and undergraduate students from underrepresented groups in STEM in Massachusetts.
