Award: OCE-1634630

Dissolved organic nitrogen is an important component of the marine carbon and nitrogen cycles. Creatine, a small molecule containing both carbon and nitrogen, is regarded as a component of the dissolved free amino acid pool. This molecule is generally produced by animal cells. However, recent genetic studies suggest that some phytoplankton may also have the ability to produce creatine. In our laboratory studies, we analyzed marine algae and detected creatine and other metabolites. In particular, we studied algae at the single-cell level. We further developed novel single cell mass spectrometry techniques to extend our research capabilities. We also analyzed water samples collected from different regions to detect creatine. In the past several years, we have performed studies in several different areas. (1) To understand amino acid and peptide cycling in marine environments. Like creatine, ammino acids and peptides are regarded as dissolved organic nitrogen. Many findings from these studies are tightly related to the behavior of creatine in the marine systems. (2) To understand the physiological adaptions related to creatine metabolism in marine algae. We performed genetics studies to reveal the potential of marine diatoms to participate in urea cycle, which was previously regarded as a property only for metazoans. Our discovery is novel. (3) Single cell studies of marine algae. What role creatine and/or creatine phosphate might play in algal physiology remains unknown. In an effort to gain better insights into this question, we continued to develop methodologies to conduct single cell mass spectrometry (SCMS) studies of marine algae. Importantly, we detected creatine from marine algae for the first time. In addition, we studied algae metabolisms in different environments such as nitrogen-rich vs nitrogen-lean and light vs. dark. We observed significant changes in cellular lipid compositions, with individual lipids being both up- and downregulated under conditions. (4) To study creatine metabolism across different bacterial and algal clades. We used bioinformatics tools to survey existing bacterial genome and algal transcriptome data for the existence of creatinase and creatine kinase genes. (5) Study of creatine uptake in different North American regions. We collected samples from the coastal Alaskan Arctic, an upwelling region of coastal Baja, and a brackish estuary of Chesapeake Bay. We found that potential creatine specific uptake rates were detectable in all environments. We demonstrated the aquatic microbial community’s potential to utilize dissolved metabolites, such as creatine, could be widespread. (6) Develop novel single cell mass spectrometry techniques. Mass spectrometry is a powerful analytical technique that can be used to sensitively detect and accurately identify molecules at ultra-low abundances. As a cutting-edge method, single cell mass spectrometry has a great potential to revolutionize research in different areas. In addition to applying our existing methods to different projects, we developed novel techniques. We were able to conduct chemical reactions for single cells, and then use mass spectrometry to determine the carbon-carbon double bonds in unsaturated lipids. We integrated fluorescence microscopy with mass spectrometry to study cell-to-cell interactions. Because mass spectrometry data from single cells are complex and have large sizes, advanced data analysis methods are necessary to extract essential information from the big data. We used multivariate analysis and machine learning algorithms to rapidly and efficiently analyze our single cell data. We also developed novel bioinformatics tools to quantify cell subpopulations and extended the capabilities of single cell mass spectrometry techniques. This grant also supported outreach activities, which connect research scientists with K-12 students through research experiences for teachers and lesson development. The outreach activities were carried out through the collaboration with the K20 Center for Educational and Community Renewal, a statewide research and development center located on the research campus of the University of Oklahoma. The grant integrated research with inquiry-based teaching of rural secondary science teachers through Authentic Research Experiences in oceanography and microbial ecology. Due to the pandemic influence, we redesigned our outreach programs. These activities involved several different types of interactions: on-site lectures, virtual professional development, collaboration days throughout the summer, and a final day of sharing and reflection in August. The professional development sessions included a review of the grant content, project goals, and research-based instructional practices relevant to the teachers’ skill sets and project needs. The collaboration days provided an avenue for discussion of effective instructional strategies and timely support for the teachers as they created high-quality lessons. The final day of professional development was to share and reflect on the developed lessons. The teachers’ lessons will be published to the K20 LEARN website (https://learn.k20center.ou.edu/ ). Last Modified: 01/27/2023 Submitted by: Zhibo Yang