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Abstract
Here, we present considerations and strategies for the use of nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic flux in marine microorganisms. Phytoplankton perform half the carbon fixation and oxygen generation on Earth. A substantial fraction of fixed carbon becomes part of a metabolite pool of small molecules known as dissolved organic matter (DOM), which are taken up by marine bacteria proximate to phytoplankton. Little is known about the metabolite-mediated, structured interactions occurring between phytoplankton and associated marine bacteria, in part due to challenges of studying high-salt solutions on various analytical platforms. NMR spectroscopy analysis is problematic due to the high-salt content of both natural seawater and culture media for marine microbes, which degrades the performance of the radio frequency coil, reduces the efficiency of some pulse sequences, limits signal-to-noise, and prolongs experimental time. We first describe a method to reproducibly extract low molecular weight DOM from small-volume, high-salt cultures as a promising tool for elucidating metabolic flux and genetic screening between marine microorganisms. Next, we describe the utility of the method at elucidating differential flux between several clades of marine bacteria cocultured with phytoplankton. We end with our latest work observing real-time metabolic flux using continuous in-vivo metabolism by NMR (CIVM-NMR). This method shows promise for future investigations into substrate utilization by marine bacteria and could aid in better understanding the metabolic flux crucial to sustaining life as we know it.