Identifying and Overcoming the Factors Limiting Growth and Substrate Utilization in <i>Caldicellulosiruptor bescii</i>

Caldicellulosiruptor bescii is an anaerobic extreme thermophile being studied for production of lignocellulosic biofuels due to its potential for plant biomass deconstruction. It can grow on a wide range of substrates and co-metabolize C5 and C6 sugars. However, incomplete biomass utilization and low cell growth, among other bottlenecks, majorly limit its bioconversion potential. The work in this dissertation aimed at identifying and overcoming the factors that hinder growth and substrate utilization in C. bescii and focused on low pH and high osmolarity as the investigated conditions that may serve as inhibitors. An RNA-seq data analysis pipeline was developed using a Bacillus thuringiensis data-set that determined essential parameters such as required number of reads and replicates for achieving results with high statistical confidence. This was further used for examining the physiological and systems level responses of C. bescii to acidic pH using integrated omics. In this study, lowering pH from 7.2 to 6.0 in mid-log and post stationary growth phases demonstrated lowered membrane potential/proton motive force (PMF) as a cause of these limitations. Dramatic increase in growth, improved substrate utilization and higher product generation was observed upon alleviating the PMF limitations post-acid addition. Patterns of elevated membrane potential and higher ATP pools further supported the hypothesis. In a follow-up study using liquid and crystalline cellulose it was demonstrated that C. bescii also benefits from the lowered pH on solid substrates indicating PMF limitation exists irrespective of the substrate and alleviation of the limitation under lower pH improves growth. Moreover, this study revealed osmolarity as the next immediate factor limiting the bioconversion potential of C. bescii once PMF limitation is alleviated. The ability of C. bescii to maintain growth at pH 5.5 (0.1 hr-1 dilution rate) in chemostat on Avicel was also displayed here which has not been previously reported, extending its growth pH range (5.5-7.3). Finally, an attempt to expand the genetic tools available for C. bescii was made, exploring RNA interference (RNAi) technology as a basis for developing a genome-wide screening tool in the future, which would aid to identify genetic elements that could confer robustness under various stress conditions.