Identification and quantification of water‐soluble metabolites by cryoprobe‐assisted nuclear magnetic resonance spectroscopy applied to microbial fermentation

Abstract

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.

Boosting Autofermentation Rates and Product Yields with Sodium Stress Cycling: Application to Production of Renewable Fuels by Cyanobacteria

“…6). Figure 5 represents a working model for metabolic pathways for glycolysis, oxidative pentose phosphate, and pyruvate fermentation predicted from the draft genome of A. maxima (http://genome.ornl.gov/microbial/amax/) and corroborated by the present and previous data from NMR-detected metabolites (8). The genome draft has at least 99% sequence coverage of the completed genome and is thus useful for assembling this model.…”

“…In addition to glycogen, these molecules can serve as substrates for fermentation in cyanobacteria (22). Both glucosyl-glycerol and trehalose are present in Arthrospira platensis (29) and A. maxima CS-328 (8). It was shown that a 4-fold increase in carbohydrate content can be achieved by growing A. platensis in media supplemented with additional 1 M NaCl relative to no additional NaCl (28).…”

“…5. Proposed autofermentative scheme for A. maxima based on observed excreted products (white circles), previously observed substrates (black circles) (8), and identified enzymes present in the A. maxima genome (GenBank accession numbers [gray letters]). Pathways with multiple steps (glycolysis and oxidative pentose phosphate [OPP] pathways) are given as dotted lines.…”

“…A total of 540 l of the solution was pipetted into Pyrex nuclear magnetic resonance (NMR) tubes (New Era Enterprises, Inc., Vineland, NJ), to which 60 l of D 2 O containing 30 g/ml TSP [3-(trimethylsilyl)propionic-2,2,3,3-D 4 acid sodium salt] (Sigma-Aldrich) was added, and tubes were mixed by inversion and vortexing. Water-soluble end products were then measured by 1 H nuclear magnetic resonance spectroscopy as described elsewhere (8). We should note that our method assumes that the major fractions of organic acids and alcohols are not retained intracellularly during fermentation.…”

Metabolome Remodeling during the Acidogenic-Solventogenic Transition in Clostridium acetobutylicum

“…Amino acids were derivatized with benzyl chloroformate before their quantitation by negative-mode liquid chromatography (LC)-ESI-tandem mass spectrometry (MS/ MS) (28). For the quantitation of major fermentation products (i.e., butyrate, acetate, butanol, acetone, and ethanol), we used cyroprobe-assisted 1 H-nuclear magnetic resonance ( 1 H-NMR) spectroscopy (10,23). The determination of absolute intracellular metabolite concentrations was performed using an isotope ratio-based approach described previously (7).…”