Hydrogen Production by Caldicellulosiruptor species: The Organism and the Metabolism

Sammanfattning: Hydrogen holds a great promise as an efficient and clean future energy carrier due to its high energy density and lack of pollutant generation. Anaerobic degradation of organic substrates by heterotrophic microorganisms enables the production of hydrogen from a wide spectrum of agricultural feedstock and waste products. Using thermophiles in this process is preferable for maximizing the substrate conversion efficiency. In this work, the hydrogen production efficiency of three extreme thermophilic bacteria of the genus Caldicellulosiruptor was evaluated. In pure cultures, the organisms could produce hydrogen at high yields and co-metabolize glucose and xylose, the major constituents of lignocellulosic biomass. In addition, we designed two co-cultures of different Caldicellulosiruptor species that produced higher hydrogen yields than the individual organisms. The maximum hydrogen yield obtained by the co-culture of C. saccharolyticus and C. kristjanssonii represents 93% of the theoretical yield of dark fermentation and is substantially higher than the yield reported for any mixed culture to date. This co-culture exhibited remarkable stability under continuous culture conditions, even when growth was limited on only one substrate. Both experimental and mathematical modelling results suggested an altruistic cooperation in the co-culture that may explain the ability of the two closely-related organisms to stably coexist rather than only competing for the same nutrients. For cost-effective hydrogen fermentation, it is vital to obtain high yields under high partial pressures of hydrogen. Conditions for achieving this property in C. owensensis and C. saccharolyticus could be identified. At proper conditions, C. owensensis and C. saccharolyticus were capable of growing and producing hydrogen under an atmosphere of 44 and 67 kPa of hydrogen, respectively. Physiological and thermodynamic aspects related to this behaviour are analysed for understanding the underlying mechanisms of hydrogen tolerance in Caldicellulosiruptor species. For achieving systems-level understanding of hydrogen synthesis patterns, the metabolic network of C. saccharolyticus was reconstructed on a genome-wide scale. This was achieved by integrating available genome sequence data, current knowledge on the organism’s physiology and newly generated experimental measurements of enzyme activities, metabolite fluxes and cellular composition. The reconstructed network consisted of 575 reactions involving 507 genes and 596 metabolites. The reconstruction process helped to identify the main nutritional requirements of C. saccharolyticus, allowing for growing the organism in a chemically defined medium for the first time. Constraint-based methods were used for analysing the network and filling its gaps, resulting in a predictive in silico model that correctly captured the behaviour of the cells during chemostat growth. This reconstruction represents a structured knowledge base for C. saccharolyticus that should benefit researchers not only in the biohydrogen field, but also in other biotechnological areas exploiting this organism.