Exploring strategies to improve volumetric hydrogen productivities of Caldicellulosiruptor species

Abstract: Ongoing consumption of fossil-based fuels generates a massive amount of greenhouse gases. This may lead to global warming that is currently threatening human society and wild animal habitats. Hydrogen is an energy carrier with the highest energy content per weight compared to other all fuels and no carbon dioxide is released when combusted. Thermophilic bacteria belonging to the genus of Caldicellulosiruptor have the ability to produce hydrogen from an array of substrates such as poly-, oligo-, di-, and monosaccharides, including lignocellulosic material. Caldicellulosiruptor species have the capacity to produce hydrogen at nearly the maximum theoretical yield of 4 mol⋅mol-1 hexose. In this work, pure and co-cultures of Caldicellulosiruptor species degraded and fermented heat-treated wheat straw. The outcome indicated that the performance of C. kronotskyensis is superior and it is thus promising candidate for utilizing wheat straw through consolidated bioprocessing. Therefore, the physiology of C. kronotskyensis was further investigated using defined media containing glucose and xylose mixtures corresponding to the sugar ratio present in wheat straw hydrolysate. Interestingly, growth of C. kronotskyensis did not possess a diauxic-like growth pattern during its growth on glucose and xylose mixtures like was observed with C. saccharolyticus. This phenomenon was determined by both the volumetric productivity profile of hydrogen (QH2) and carbon dioxide (QCO2). The maximum growth rate (µmax) of C. kronotskyensis on xylose was 0.57 h-1 which is twice the µ max on glucose (0.28 h-1). C. kronotskyensis was grown on sugar mixtures i.e. xylose-cellobiose and glucose-cellobiose. The uptake of xylose and cellobiose occurred concurrently. However, for glucose and cellobiose mixtures, C. kronotskyensis consumed cellobiose faster than glucose. These results indicated that C. kronotskyensis has adapted to pentoses andoligosaccharides. Cell immobilization and co-cultures offered a promising technique for retaining cells in the system. During this work, chitosan and rubber were used as a carrier to retain biomass, thereby improving volumetric hydrogen productivity (QH2). Chitosan exhibited the property to retain C. saccharolyticus and C. owensensis but did not improve the QH2. Acrylic fibres filled in a homemade stainless-steel cage was introduced in continuous stirred tank reactors (CSTR). Notably, the highest QH2 obtained was 30 ± 0.2 mmol⋅L-1⋅h-1 at a dilution rate (D) of 0.3 h-1 with a pure culture of C. kronotskyensis with acrylic fibres and chitosan. In the co-culture of C. kronotskyensis and C. owensensis with acrylic fibres, the population dynamics indicated that C. kronotskyensis was the dominant species in the biofilm fraction, whereas C. owensensis was the dominant in the planktonic phase. Bis-(3',5')-cyclic di-guanosine-mono-phosphate (c-di-GMP) is an intracellular messenger correlated with planktonic and biofilm lifestyle. C. owensensis is a high producer of c-di-GMP, while C. kronotskyensis produced less during its fermentations. In this study, a co-culture of C. kronotskyensis and C. owensensis without carrier obtained the highest concentration of c-di-GMP at 260 ± 27.3 µM. In conclusion, this study revealed that immobilization of Caldicellulosiruptor species improved the QH2 . Secondly, it revealed the superior performance of C. kronotskyensis in relation to consolidated bioprocessing, biofilm formation and QH2. Therefore, it is recommended to carry out more research with C. kronotskyensis to pursue a breakthrough in cost-effective hydrogen production.