Identification and quantification of water-soluble metabolites by cryoprobe-assisted nuclear magnetic resonance spectroscopy applied to microbial 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 ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted (1)H and (13)C 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. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 microM 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 ((1)H(2)O) 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 x 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 (13)C-labeled versus unlabeled metabolites were determined from integral ratios of (1)H peaks that exhibit (13)C-(1)H J-couplings and independently confirmed by direct measurement of areas of corresponding (13)C resonances. (13)C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima.
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
Damian
D
Carrieri
Kelsey
K
McNeely
Ana C
AC
De Roo
Nicholas
N
Bennette
István
I
Pelczer
G Charles
GC
Dismukes
D003080:Cold Temperature; D000458:Cyanobacteria / Q000145:classification / Q000378:metabolism; D005285:Fermentation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D012995:Solubility; D014867:Water / Q000737:chemistry*
D014867:Water