ODU Researchers Recognized for Cyanate Assay Innovation
June 12, 2014
Widner (left) with Mopper and Mulholland
Scientists at Old Dominion University have devised a new way to measure minute quantities of cyanate found in aquatic environments, and this development is being hailed as a breakthrough in the study of the oceanic nitrogen cycle and the evolution of microbial life on Earth.
Cyanate metabolism appears to have evolved early in Earth's history suggesting this compound was included in the "primordial soup" from which the earliest microbial life emerged.
The research by ODU oceanographer Margaret Mulholland and chemist Kenneth Mopper, together with their Ph.D. student Brittany Widner, is the subject of a National Science Foundation (NSF) Highlight article currently featured on the NSF Science, Engineering and Education Innovation website. See http://go.usa.gov/8MwY.
The ODU scientists worked on this project in collaboration with Anton Post at the Marine Biology Laboratory in Woods Hole, Mass.
As the Highlight articles states, "Previously, it was impossible to evaluate the importance of cyanate in aquatic environments because detection methods lacked the sensitivity to identify the compound in chemically dilute environments like the ocean. Cyanate is a simple form of organic nitrogen available to phytoplankton with the appropriate enzyme to metabolize it."
The article adds that the research team measured nutrient-like concentrations of cyanate in seawater relative to the abundance of organisms expressing genes that take up and metabolize it. "These are the first steps toward understanding the role cyanate plays in global nitrogen cycling and the productivity of marine food webs," according to the article.
Nitrogen is an important nutrient in the world's oceans because, among other things, it is a major component of living organisms. The element often limits the growth of marine phytoplankton, the foundation of oceanic food webs.
For nearly two decades, Mulholland, professor of ocean, earth and atmospheric sciences, has been on the trail of aquatic nitrogen. She has studied the complex dynamics between nitrogen availability and the growth of phytoplankton, which in some cases can result in harmful algae blooms and the creation of oxygen depleted waters. She has also analyzed the relationships between climate change and the cycle of nitrogen between natural waters and the atmosphere.
Her research on nutrient cycles and algal blooms led to her election to the Scientific and Technical Advisory Committee of the Chesapeake Bay Program, which is a coalition of mid-Atlantic states and the U.S. Environmental Protection Agency. The work also has garnered her support from numerous funding agencies, including the NSF.
This latest serendipitous breakthrough in laboratory methodology promises to provide a significant boost to nitrogen-cycling research worldwide.
The journal Analytical Chemistry published a paper last year written by Widner, a Ph.D. student mentored by Mulholland and Mopper, professor of chemistry and biochemistry, that describes the method they developed to measure the low concentrations of cyanate found in marine environments.
Although the researchers' breakthrough started with a simple idea, there was a lot of ingenuity in the steps required to actually measure this simple compound in seawater, as suggested by the title of their journal article: "Chromatographic Determination of Nanomolar Cyanate Concentrations in Sea and Estuarine Waters by Precolumn Fluorescence Derivatizations." Widner, with an undergraduate degree in chemistry, tenaciously led that charge.
Potential sources of cyanate in natural waters include the decomposition of organic matter, photochemical production, release of cyanate as a byproduct of microbial cellular metabolism, industrial wastewater inputs, and herbicide or urea runoff from urban and agricultural landscapes.
Cyanate may be a critical component in the aquatic nitrogen cycle. It can provide a source of nitrogen in a nitrogen-impoverished environment that is readily useable by phytoplankton. If, indeed, cyanate does facilitate microbial growth, this could contribute to primary production.
Several factors have made cyanate (a compound of oxygen, carbon and nitrogen) almost impossible to measure in sea water. The ocean is a chemically dilute environment, the amounts of cyanate typically present in sea water are very small and the salt in seawater interferes with most chemical analyses. Consequently, most compounds are more difficult to detect in seawater than elsewhere in the environment.
The ODU researchers zeroed in on an alternative assay to measure cyanate, which was based on a fluorescence detection that the biomedical community has used to detect cyanate in blood. They took advantage of a reaction between 2-aminobenzoic acid and cyanate in the water that produces a highly fluorescent compound that can be easily separated from other compounds and quantified by high-performance liquid chromatography (HPLC). "We applied this method to measure OCN (cyanate) concentrations in estuarine and seawater samples from the Chesapeake Bay and coastal waters from the mid-Atlantic region," the researchers state in their Analytical Chemistry article. "OCN concentrations ranged from 0.9 to 41 nM (nanomolar)." A nanomolar is a thousand-millionth of a molar, which is a standard measurement of substance concentration in a liquid. Other cyanate measurement methods typically have a detection limit at the micromolar level, 1,000 times higher than the nanomolar level.
Levels of cyanate the ODU researchers found in and around the Chesapeake Bay were considerably higher than those they found in water samples from the Atlantic Ocean off the mid-Atlantic seaboard. This is not surprising because nutrient concentrations are generally lower further offshore.
Mopper, was pivotal in "working out the kinks in developing the HPLC method," Mulholland said. He is an expert in the chemical analysis of natural waters by means of HPLC and other methods, and is a veteran investigator of oceanic and global carbon cycling.
Post, the researcher at the Marine Biological Laboratory who collaborated on the project, told Chemical and Engineering News that the ODU scientists' work is "no small feat," and that it finally allows researchers to measure cyanate at levels that are relevant to the marine environment, something that he suspected all along based on reverse genomics.
In addition to her work in the laboratory, Widner has been on recent research expeditions in the North Atlantic and the Eastern Tropical North and South Pacific oceans. There she used her new method to measure cyanate concentrations in diverse oceanic environments and along vertical gradients which characterize marine environments. She also measured rates of cyanate uptake by naturally occurring microorganisms during these studies. The oceanographic expeditions were part of NSF- and NASA-funded research projects for which Mulholland is a principal investigator.
Said Mulholland, "Widner's advance in methodology allows us to begin to explore the role of cyanate in the marine environment, as well as microbial evolution."
Widner received her master's in oceanography from ODU in 2011 and is the winner of the university's Jacques S. Zaneveld Endowed Scholarship and the Dorothy Brown Smith Travel Scholarship (three times).