Novel monosaccharide fermentation products in Caldicellulosiruptor saccharolyticus identified using NMR spectroscopy.

BACKGROUND: Caldicellulosiruptor saccharolyticus is a thermophilic, Gram-positive, non-spore forming, strictly anaerobic bacterium of interest in potential industrial applications, including the production of biofuels such as hydrogen or ethanol from lignocellulosic biomass through fermentation. High-resolution, solution-state nuclear magnetic resonance (NMR) spectroscopy is a useful method for the identification and quantification of metabolites that result from growth on different substrates. NMR allows facile resolution of isomeric (identical mass) constituents and does not destroy the sample. RESULTS: Profiles of metabolites produced by the thermophilic cellulose-degrading bacterium Caldicellulosiruptor saccharolyticus DSM 8903 strain following growth on different monosaccharides (D-glucose, D-mannose, L-arabinose, D-arabinose, D-xylose, L-fucose, and D-fucose) as carbon sources revealed several unexpected fermentation products, suggesting novel metabolic capacities and unexplored metabolic pathways in this organism. Both 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were used to determine intracellular and extracellular metabolite profiles. One dimensional 1H NMR spectral analysis was performed by curve fitting against spectral libraries provided in the Chenomx software; 2-D homonuclear and heteronuclear NMR experiments were conducted to further reduce uncertainties due to unassigned, overlapping, or poorly-resolved peaks. In addition to expected metabolites such as acetate, lactate, glycerol, and ethanol, several novel fermentation products were identified: ethylene glycol (from growth on D-arabinose), acetoin and 2,3-butanediol (from growth on D-glucose, L-arabinose, and D-xylose), and hydroxyacetone (from growth on D-mannose, L-arabinose, and D-xylose). Production of ethylene glycol from D-arabinose was particularly notable, with around 10% of the substrate carbon converted into this uncommon fermentation product. CONCLUSIONS: The present research shows that C. saccharolyticus, already of substantial interest due to its capability for biological ethanol and hydrogen production, has further metabolic potential for production of higher molecular weight compounds, such as acetoin and 2,3-butanediol, as well as hydroxyacetone and the uncommon fermentation product ethylene glycol. In addition, application of nuclear magnetic resonance (NMR) spectroscopy facilitates identification of novel metabolites, which is instrumental for production of desirable bioproducts from biomass through microbial fermentation.

Constituents with independence from growth temperature for bacteria using pyrolysis-gas chromatography/differential mobility spectrometry with analysis of variance and principal component analysis.

Four bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus warneri, and Micrococcus luteus, were grown at temperatures of 23, 30, and 37 degrees C and were characterized by pyrolysis-gas chromatography/differential mobility spectrometry (Py-GC/DMS) providing, with replicates, 120 data sets of retention time, compensation voltage, and ion intensity, each for negative and positive polarity. Principal component analysis (PCA) for 96 of these data sets exhibited clusters by temperature of culture growth and not by genus. Analysis of variance was used to isolate the constituents with dependences on growth temperature. When these were subtracted from the data sets, Fisher ratios with PCA resulted in four clusters according to genus at all temperatures for ions in each polarity. Comparable results were obtained from unsupervised PCA with 24 of the original data sets. The ions with taxonomic features were reconstructed into 3D plots of retention time, compensation voltage, and Fisher ratio and were matched, through GC-mass spectrometry (MS), with chemical standards attributed to the thermal decomposition of proteins and lipid A. Results for negative ions provided simpler data sets than from positive ions, as anticipated from selectivity of gas phase ion-molecule reactions in air at ambient pressure.

Analysis of bacteria by pyrolysis gas chromatography-differential mobility spectrometry and isolation of chemical components with a dependence on growth temperature.

Pyrolysis gas chromatography-differential mobility spectrometry (py-GC-DMS) analysis of E. coli, P. aeruginosa, S. warneri and M. luteus, grown at temperatures of 23, 30, and 37 degrees C, provided data sets of ion intensity, retention time, and compensation voltage for principal component analysis. Misaligned chromatographic axes were treated using piecewise alignment, the impact on the degree of class separation (DCS) of clusters was minor. The DCS, however, was improved between 21 to 527% by analysis of variance with Fisher ratios to remove chemical components independent of growth temperature. The temperature dependent components comprised 84% of all peaks in the py-GC-DMS analysis of E. coli and were attributed to the pyrolytic decomposition of proteins rather than lipids, as anticipated. Components were also isolated in other bacteria at differing amounts: 41% for M. luteus, 14% for P. aeruginosa, and 4% for S. warneri, and differing patterns suggested characteristic dependence on temperature of growth for these bacteria. These components are anticipated to have masses from 100 to 200 Da by inference from differential mobility spectra.

Analysis of bacterial strains with pyrolysis-gas chromatography/differential mobility spectrometry.

Eight vegetative bacterial strains and two spores were characterized by pyrolysis-gas chromatography with differential mobility spectrometry (py-GC/DMS) yielding topographic plots of ion intensity, retention time, and compensation voltage simultaneously for ions in positive and negative polarity. Biomarkers were found in the pyrolysate at characteristic retention times and compensation voltages and were confirmed by standard addition with GC/MS analyses providing discrimination between Gram negative and Gram positive bacterial types, but no recognition of individual strains within the Gram negative bacteria. Principal component analysis was applied using two dimensional data sets of ion intensity versus retention time at five compensation voltages including the reactant ion peaks all in positive and negative ion polarity. Clustering was observed with compensation voltage (CV) chromatograms associated with ion separation in the DMS detector and little or no clustering was observed with the reactant ion peaks or CV chromatograms where ion separation is poor. Consistent clustering of Gram positive B. odysseyi and Gram negative E. coli in both positive and negative polarities with the reactant ion peak chromatograms and key CV chromatograms suggests common but unknown common chemical compositions in the pyrolysate.