Novel monosaccharide fermentation products in Caldicellulosiruptor saccharolyticus identified using NMR spectroscopy

Abstract

BackgroundCaldicellulosiruptor 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.ResultsProfiles 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.ConclusionsThe 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.

Engineering redox-balanced ethanol production in the cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii

“…The production of acetoin as a side-product of fermentation at sub-optimal growth temperatures, as observed here with C. bescii, has also been reported with other Caldicellulosiruptor species, C. sacharolyticus (T opt 70 °C: ( Isern et al, 2013 )) , and also with the extremely thermophilic archaeon Pyrococcus furiosus (T opt ~100 °C: ( Nguyen et al, 2016 )). Acetoin is generated from pyruvate by the action of acetolactate synthase and the abiotic decarboxylation of acetolactate to acetoin at high temperatures (above 60 °C).…”

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

“…However, the reports on thermophilic enzymes for acetoin biosynthesis are still limited. It has been confirmed that Caldicellulosiruptor saccharolyticus produced acetoin when grown in media supplemented with different monosaccharides, indicating that there were some candidate genes for acetoin formation in the genome 23 . While only putative ilv genes encoding ALS were identified in the genome of Caldicellulosiruptor sp.…”

“…While only putative ilv genes encoding ALS were identified in the genome of Caldicellulosiruptor sp. by bioinformatics analysis, the ALDC encoding gene alsD was not found 23 . As an alternative, an alsD gene was identified in the genome of B .…”

Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms

“…Both methods of attachment, along with the large inventory of glycolytic enzymes, give this genus its impressive ability to degrade a wide variety of lignocellulosic substrates . Caldicellulosiruptor species are capable of breaking down cellulose and hemicellulose (hexoses and pentoses), both as simple monosaccharides and complex biomasses . Unlike many cellulolytic organisms, they do not exhibit carbon catabolite repression, a process by which certain sugars are preferentially metabolized, while excluding the usage of others .…”