Caldicellulosiruptor Saccharolyticus: an Ideal Hydrogen Producer?

Abstract: Caldicellulosiruptor saccharolyticus is an extremely thermophilic, strictly anaerobic, Gram-positive and cellulolytic microorganism with a natural ability to produce hydrogen (H2) at nearly theoretical maximum yield, i.e. 4 mol/ mol of glucose. Due to its CO2-free combustion and high energy density, among other desirable properties, H2is touted as a fuel of the future. Biological H2 production by thermophilic microorganisms utilizing waste biomass holds a huge potential as the most environment friendly process for commercial H2production. For this reason, it is imperative to identify and develop a microorganism with the most beneficial properties as an ideal H2producer may possess. During this work, the physiology and metabolism of C. saccharolyticus was studied in detail, which revealed that – i) it can sustain its growth in the absence of any means of removal of H2from the reactor, ii) it can utilize the sugars in wheat straw hydrolysate effectively for its growth and H2production with 67% conversion efficiency, iii) Methane can replace N2as a sparging gas for removal of H2from the culture, without affecting the growth and H2production by C. saccharolyticus, iv) by-products of its fermentative metabolism can be converted to methane by anaerobic digestion, v) it is capable of assimilating sulphate as a primary sulphur source, vi) can form biofilms when co-cultured with C. owensensis, which can be an effective means of retaining biomass in the reactor, and vii) Up-flow anaerobic (UA) reactor with granular sludge offer better alternative that a continuously stirred-tank reactor for improving its volumetric H2productivity (QH2). Moreover, to improve the QH2further, the methods of evolutionary engineering were used to develop osmotolerant mutant strains of C. saccharolyticus: i) C. saccharolyticusG10, capable of growing in a medium containing up to 100 g/L of glucose, and ii) C. saccharolyticusAG6 capable of growing in a medium containing approx. 13.2 g/L of sodium aceate and 30 g/L of glucose. Indeed, the cultivation of one of the osmotolerant strains in an chemically optimized medium improved the QH2. Furthermore, experimente were performed to understand the barriers to genetic modification of C. saccharolyticus. The studies revealed that the methylation of the foreign DNA by C5-cytosine-specific methyltransferase may help overcome the restriction modification system of C. saccharolyticus. In addition, uracil-auxorophic strains of C. saccharolyticus were developed, which can be used as a host to perform genetic modifications. In conclusion, the knowledge and newfound properties obtained in this study should be combined to create a strain of C. saccharolyticus that will fulfil nearly all the requirements to make it into an ideal H2producer.