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.
All Science Journal Classification (ASJC) codes
- Materials Science(all)
- Arthrospira (Spirulina)
- Water suppression