Physiology of Caldicellulosiruptor saccharolyticus: a hydrogen cell factory

A high substrate conversion efficiency is a prerequisite for an economically feasible biohydrogen production. Caldicellulosiruptor saccharolyticus is a strictly anaerobic extreme thermophilic bacterium that is able to convert the theoretical maximum of 4 mol/mol glucose to H2. It can grow and produce H2 on a broad spectrum of sugars ranging from monomers (hexoses and pentoses) to more complex sugars such as lignocellulosics, thereby rendering it industrially interesting. Moreover, it is capable of maintaining a growth and production of H2 at elevated partial H2 pressures (PH2), which significantly reduces the cost of gas upgrading. These qualities, which make C. saccharolyticus a superior H2 cell factory, may be attributed to its adaptation to a monosaccharide-poor environment. Characteristics that it has developed to survive in this harsh environment include: i) the generation of cellulolytic and (hemi)-cellulolytic enzymes which can degrade complex polymers, ii) a possession of a high number of high affinity ATP binding cassette (ABC) transport systems to translocate a large spectrum of monomers and dimers, and iii) a lack of glucose-repression enabling co-metabolization of several types of sugars. To fuel this high affinity transport system, they are forced to oxidize glucose to acetate to generate adequate amounts of ATP. H2 is an electron sink, formed to reoxidize NADH and reduce ferredoxin in this process.

In addition, C. saccharolyticus can conserve energy by using pyrophosphate (PPi) as an additional energy carrier. It lacks cytosolic PPase activity, but is instead able to utilize the energy in the PPi bond by i) a membrane-bound proton-translocating PPase that generates a proton motive force, ii) PPi-phosphofructokinase (PPi-PFK) that uses PPi instead of ATP, and/or iii) pyruvate phosphate dikinase (PPDK) that uses PPi and PEP to generate pyruvate and ATP. Moreover, the ATP/PPi and NADH/NAD ratios increase when the growth rate decreases in the transition to the stationary phase.

To ensure a high acetate flux, the enzymes around the pyruvate node are strongly controlled in C. saccharolyticus. Lactate dehydrogenase (LDH) is constitutively expressed but strongly regulated at the enzyme level by both the ATP/PPi and the NADH/NAD ratios. These experimental data were used to derive a kinetic model over LDH rendering it possible to simulate lactate formation during batch growth in C. saccharolyticus. Such lactate formation occurs in the transition to the stationary phase as a result of the combination of an increased osmotic pressure and a raise in PH2. This is attributed to the fact that, in conditions promoting high growth rates, LDH is inactive, leading to a low lactate formation. However, LDH is activated when the ATP or NADH levels increase and the PPi levels decrease, leading to a partial metabolic redistribution to lactate. Lactate is a vital extra electron sink for maintaining a high glycolytic flux since this flux is inhibited by increased NADH levels.

However, C. saccharolyticus also possesses several shortcomings which need to be addressed. These include i) elevated dissolved H2 concentrations and osmotic pressures triggering lactate formation although C. saccharolyticus is more tolerant to PH2 than many other microorgansims, ii) sparging with CO2 which inhibits growth and H2 productivities, iii) a sensitivity to increased osmotic pressures resulting in cell lysis and decreased H2 productivities, iv) a low cell number compared to mesophilic co-cultures resulting in a low volumetric H2 productivity, and v) the addition of CO2 or acetate addition as a requirement to initiate growth on xylose and arabinose. The latter does not constitute a problem provided that a sugar mixture with both pentose and hexose sugars is used.

Despite these shortcomings, C. saccharolyticus can be considered a superior H2 cell factory. The aspects of its many positive qualities for a biohydrogen process and possible origins for these qualities are discussed in this thesis.