Biological hydrogen formation by thermophilic bacteria

A.A.M. Bielen

Research output: Thesisinternal PhD, WU



Hydrogen gas (H2) is an important chemical commodity. It is used in many industrial processes and is applicable as a fuel. However, present production processes are predominantly based on non-renewable resources. In a biological H2 (bioH2) production process, known as dark-fermentation, fermentative microorganisms are able to generate H2 from renewable resources like carbohydrate-rich plant material or industrial waste streams.

Because of their favourable biomass degrading capabilities and H2-forming features both Caldicellulosiruptor saccharolyticus and Thermotoga maritima have become model organisms in the study of thermophilic H2 production. Novel insights in substrate usability, associated fermentation pathways and the mechanism involved in H2 formation will provide steps forward in the application of these organisms for H2 production and sustainable biological H2 formation via dark fermentation in general.

Elevated H2 levels are known to inhibit H2-formation during dark fermentations. The response of C. saccharolyticus to the exposure of elevated H2 levels is investigated in different chemostat cultivation setups. Analysis of the fermentation profiles and transcriptome data associated with low and high H2 levels provides insight into this organism’s strategy to deal with elevated H2 levels. In addition, several chemostat studies were performed to elucidate the effect of increased H2 levels on the fermentation profile of C. saccharolyticus with respect to i) growth on ammonium deficient media and ii) low/high substrate loads. Furthermore, the thermodynamics of H2 formation is discussed with respect to the dissolved H2 concentration. Overall the dissolved H2 concentration was shown to be a dominant process determinant in causing the fermentation profile to shift away from maximal H2 yields. To be able to uphold desirable features for a H2 production process under high sugar load conditions, in terms of high H2 yields and productivities, the dissolved hydrogen concentrations should be kept below the fermentation switch threshold. This is only achievable via proper reactor design and certainly required for the efficient up-scaling of this bioH2 production process.

The role of inorganic pyrophosphate (PPi) in the energy metabolism of C. saccharolyticus is investigated. In agreement with the annotated genome sequence PPi-dependent phosphofructokinase, pyruvate phosphate dikinase and membrane bound pyrophosphatase activity can be detected in glucose-grown cultures. Pyrophosphate is demonstrated to inhibit pyruvate kinase activity. Furthermore, the dynamics in ATP and PPi levels throughout batch growth is discussed.

A genomic distribution profile of PPi-dependent glycolytic enzymes and their genomic co-occurrence with soluble or membrane-bound pyrophosphatases in 495 fully sequenced genomes is given. An ab initio classification of enzyme-subtypes, which elaborates on known classifications systems and incorporates characterized protein features e.g. catalytic site residues and allosteric regulatory site residues, is presented. The potential functional role of the PPi-dependent enzymes and membrane-bound pyrophosphatases is discussed. Overall the presented data indicates that the involvement of pyrophosphate in glycolysis/gluconeogenesis is a widespread phenomenon throughout the three domains of life.

Glycerol is formed as a by-product during biodiesel formation. Given the highly reduced state of carbon in glycerol this low cost substrate is of special interest for sustainable biofuel production. The use of glycerol for H2-formation by T. maritima is investigated. Growth on glycerol is demonstrated in both batch and chemostat cultivation setups.The observed H2 yields nearly reach the theoretical maximum of 3 H2 per glycerol, which is 3 times the yield generally observed for mesophilic conversions In addition, the route of glycerol fermentation and the exceptional bioenergetics associated with H­2 formation from glycerol in T. maritima are discussed.

For the future application of bioH2 production by C. saccharolyticusand T. maritima,research should focus on i) the factors limiting complex sugar degradation, ii) the specific nutritional requirements to sustain growth during high sugar loads, and iii) specific mechanisms to make the organism more robust against stresses like elevated dissolved H2 concentrations or osmotic stress. Moreover, for the incorporation of these desirable traits the development of genetic systems is required.

Original languageDutch
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • van der Oost, John, Promotor
  • de Vos, Willem, Promotor
  • Kengen, Servé, Co-promotor
Award date10 Jun 2014
Place of PublicationWageningen
Print ISBNs9789461739360
Publication statusPublished - 2014


  • biological production
  • caldicellulosiruptor
  • thermotoga maritima
  • fermentation
  • pyrophosphates

Cite this