Intellectual merit: The reactive oxygen species (ROS), superoxide, hydrogen peroxide, and hydroxyl, can have significant effects on the cycling of metals and organic compounds in aquatic systems. Although it is known that fungi, algae and bacteria are all capable of extracellular ROS production, it has generally been assumed that biological ROS production is negligible in freshwater systems, compared to photo-oxidation of natural organic matter. However, recent results from the Voelker lab indicate that dark biological production of H2O2 is more important than photochemical production. The overall goal of this research is to elucidate the biological role in ROS production in freshwater systems. The specific objectives are to determine which types of organisms are significant sources of reactive oxygen species to freshwater systems, which environmental variables affect ROS production rates, and which biogeochemical processes are likely to be affected by biological ROS production. The PIs will focus on three hypotheses: (i) Biological production of ROS is widespread, and specific microbial groups (which may be bacterial, algal or fungal) are responsible. (ii) Biological production rates by a specific organism can change as a result of changes in the environmental conditions such as available nutrients and light intensity. (iii) Superoxide and Fenton products are generated as a result of biological ROS production, at rates sufficient to affect the cycling of organic compounds and metals. The PIs will cultivate and isolate organisms responsible for ROS production at selected field sites, targeting algal, bacterial, and/or fungal groups. The PIs will then use their work on the cultivated organisms to design molecular techniques to examine the relationship between ROS production rates by natural assemblages of organisms.
Broader Impacts. The proposed work will provide an educational opportunity for several graduate students and undergraduates. The PIs have a strong track record in advising minorities and women, and student recruitment efforts for this proposal will continue to be focused on underrepresented groups in science and engineering. In addition, the PIs will draw on the results of this research to develop a series of lectures on the role of microorganisms in the production of reactive oxygen species and their impact on the health of microbial ecosystems and aquatic biogeochemistry. The PIs will present the lectures at the annual Professional Development Workshop for Science Educators entitled ?Windows on the Invisible World? coordinated by the Microbial Sciences Initiative at Harvard University.
Hydrogen peroxide and superoxide are two "reactive oxygen species". Their presence in natural waters is important because they may be toxic to some species, and because they can interact with toxic or nutrient metals, changing the metals’ bioavailability and environmental transport properties. Hydrogen peroxide and superoxide can also be sources of a third reactive oxygen species, hydroxyl radical, which is capable of breaking down otherwise recalcitrant organic compounds such as some organic pollutants. While it has long been known reactive oxygen species in natural waters are produced during the interaction of sunlight with dissolved natural organic matter, recent studies indicate that they can also be produced biologically. The goal of this study was to learn more about biological production of reactive oxygen species: whether it is important in many natural waters or only in a select few, which organisms may be involved, and how and why these organisms are producing reactive oxygen species. Studies performed at the Colorado School of Mines concentrated on quantifying rates of non-photochemical production of hydrogen peroxide in lakes and ponds in Massachusetts and Colorado. We found that biological production is ubiquitous in fresh surface waters, and that the rates are fast enough to make biological production significant compared to photochemical production by sunlight. The rates were most strongly related to the total amount of biological activity in the water samples, which suggests that many different organisms contribute. Filtering usually decreased production rates to near 0, but there were a few exceptions, indicating that under some circumstances, dissolved species may be capable of producing reactive oxygen species. In summary, the main impact of this work has been to show that biological production of reactive oxygen species is ubiquitous in natural waters. This means that one needs to consider that the chemical effects of reactive oxygen species production, for example metal redox cycling and oxidation of recalcitrant organic compounds, can occur in natural water systems even when photochemistry is absent. Because of the importance of superoxide in aging and many oxidative stress related diseases, as well as in the immune system and cell signaling in multicellular organisms, our finding (discussed in more detail in Colleen Hansel's report) that production of extracellular superoxide is a common phenomenon among bacterial species may turn out to be of importance in the health sciences as well. Outreach activities of this research group included two presentations (fall 2011 and fall 2012) on biogeochemical cycling at the Colorado Science Conference (organized by the Colorado Association of Science Teachers and attended by K-12 teachers from throughout the state) and a spring 2013 chemistry presentation at a local elementary school’s "Science Night." This was a collaborative project; the principal investigator of the partner project was Colleen Hansel of Harvard University and Woods Hole Oceanographic Institution. Additional project outcomes centered on activities taking place in Colleen Hansel’s laboratory are described in her report.
