Appraisal of strategies to improve thermophilic hydrogen production exploiting Caldicellulosiruptor species

The transition from a fossil to a bio-based economy is of vital importance to stem the effects of ongoing climate change.This bio-based economy will necessitate the production of both biofuels and chemical compounds from biologicalsources. Hydrogen is a promising candidate as a renewable energy carrier due to its high energy density, carbonneutrality when combusted and its potential use as a reducing agent to produce biochemicals. Caldicellulosiruptor is agenus of extreme thermophilic bacteria capable of producing hydrogen close to the theoretical maximum of 4 molH2/mol hexose from an array of different mono-, oligo- and polymeric sugars permitting the utilisation of a diverse rangeof lignocellulose substrates. Although promising, the advancement of Caldicellulosiruptor towards becoming a modelorganism for hydrogen production relies on overcoming the intrinsic hurdles that exist in this genus, such asosmosensitivity, low QH2 and the high financial cost of nutrient addition.Throughout this work, techniques for improving the Caldicellulosiruptor process were studied in detail. Adaptivelaboratory evolution was employed as a technique to increase osmotolerance by incremental adaption to elevatedglucose concentrations, thereby facilitating development of osmotolerant strains belonging to five species ofCaldicellulosiruptor. Notably, the degree of adaption to higher osmolarity varies depending on the species. Theosmotolerant strain C. owensensis CO80 was demonstrated to grow at glucose concentrations up to 80 g/l;nevertheless, the highest QH2 values for this strain were observed with lower osmolarity media. C. owensensis CO80was further implemented with the osmotolerant strain, C. saccharolyticus G5, as a co-culture to produce hydrogen fromconcentrated hydrolysates, in which the wild-type strains are unable to be cultivated.Several strategies to optimise of nutrient addition to the fermentation process were studied, including designed coculturewith Coprothermobacter proteolyticus, vitamin removal and the design of a new trace element solution. Theimplementation of a novel trace element solution (EB-1) permitted a phosphorus reduction of 90% compared toprevious cultivations. However, further optimisation of the trace elements and vitamin addition are required to increaseQH2.Process integration was undertaken by further fermentation of the effluent of Caldicellulosiruptor cultivated onlignocellulose hydrolysates to yield methane and polyhydroxybutyrate by a methanogenic consortium and Ralstoniaeutropha, respectively. These studies illustrate that acetate produced by Caldicellulosiruptor can be further fermentedinto industrially relevant compounds.In addition, several key physiological attributes of C. saccharolyticus fermentation were also studied. Although, C.saccharolyticus co-ferments sugars, a diauxic-like production of hydrogen occurs when C. saccharolyticus is batchcultivated with a mixture of glucose, xylose and arabinose. This effect is amplified when wheat straw hydrolysate isused as a substrate. It was also observed that xylose is fully consumed after 25 hours while full glucose consumptionrequires over 80 hours. In addition, continuous cultivation of osmotolerant strains on the studied hydrolysates displayedfull xylose consumption, while a significant portion of glucose remained in the effluent. Co-culturing of C.saccharolyticus with the proteolytic hydrogen-producing Coprothermobacter proteolyticus marginally increased QH2compared to the mono-culture. However, a significant reduction in QH2 was observed when C. saccharolyticus wascultivated at 60°C, due to the lower growth temperature causing a significant metabolic shift from acetate to lactate.The compiled findings of this thesis opens up new avenues of research and will act as a stepping stone for furtherstrain development, media optimisation and process integration exploiting the Caldicellulosiruptor genus. (Less)