Identification and quantification of water-solublemetabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied tomicrobial fermentation

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultra sensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted ¹H and ¹³C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 μM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous (¹H₂O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5×longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of ¹³C-labeled versus unlabeled metabolites were determined from integral ratios of ¹H peaks that exhibit ¹³C-¹H J-couplings and independently confirmed by direct measurement of areas of corresponding ¹³C resonances. ¹³C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima.