Characterization and Development of Osmotolerant Caldicellulosiruptor Strains Targeting Enhanced Hydrogen Production from Lignocellulosic Hydrolysates

Abstract Background The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H 2 /mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high osmolarity conditions, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C. owensensis , C. kronotskyensis , C. bescii, C. acetigenus and C. kristjanssonii , were developed to tolerate higher osmolarities through an adaptive laboratory evolution (ALE) process. The developed strain C. owensensis CO80 was further studied accompanied by the development of a kinetic model based on Monod kinetics. Results Osmotolerant strains of Caldicellulosiruptor were obtained with C. owensensis adapted to grow up to 80 g/l glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. C. owensensis CO80 was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/l glucose but with reduced volumetric hydrogen productivities (Q H2 ) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of C. owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical osmolarity of the cells increased fourfold when cultivated at higher osmolarity. When co-cultured with the osmotolerant strain C. saccharolyticus G5 at a hydraulic retention time (HRT) of 20h, C. owensensis constituted only 0.09-1.58% of the population in suspension.Conclusions The adaptation of members of the Caldicellulosiruptor genus to higher osmolarity established that the ability to develop improved strains via ALE is species dependent, with C. owensensis adapted to grow on 80 g/l, whereas C. kristjanssonii could only be adapted to 30 g/l glucose. Although, C. owensensis CO80 was adapted to a higher osmolarity medium, the strain demonstrated reduced Q H2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated osmolarities, this approach does not result in improved fermentation performances at these higher osmolarities. Moreover, the observation that planktonic culture of CO80 was outcompeted by an osmotolerant strain of C. saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of strain CO80 should be improved for industrial application .