Hydrogen is a promising renewable energy carrier that can contribute towards a low carbon economy. Fermentative hydrogen production from carbohydrate-containing feedstock, such as glucose, sucrose and starch, has been extensively studied [1
]. However, second generation hydrogen fermentation technologies are presently emerging as promising and more cost-effective solutions [1
Lignocellulosic material must be pre-treated prior to fermentation to hydrogen in order to remove lignin and hemicelluloses, reduce the cellulose crystallinity and increase the surface area of the material to enhance the release of sugars [4
]. Physico-chemical pre-treatment of lignocellulosic material, such as the application of acid, alkaline or oxidative conditions at ambient or elevated temperatures, yields a mixture of pentoses and hexoses [1
]. Efficient microbial fermentation of hexoses and pentoses is, therefore, the key step for hydrogen production from plant biomass. However, combined fermentation of mixtures of hexoses and pentoses is often prevented due to catabolic repression; in the presence of glucose, pentoses might be converted to a lesser extent thereby decreasing overall fermentation yields [5
]. Moreover, efficient hydrogen production from sugars is dependent on the different possible fermentation pathways (Figure ).
Figure 1 Major metabolic pathways for glucose and arabinose fermentation in mixed cultures (adapted from [7,8]).
Most of the extreme thermophiles from the phylum Clostridia
use the Embden-Meyerhof pathway to metabolize hexose sugars to pyruvate [9
]. Biohydrogen can be then formed via decarboxylation of pyruvate to acetyl CoA, in which reduced ferredoxin (Fdred
) is generated and acts as a direct electron donor for proton reduction to hydrogen (Figure ). Maximum hydrogen yield, both from hexoses or pentoses, is obtained with acetate as the fermentation product (equations 1 and 2). Low yields are associated with the formation of more reduced end products compared to acetate, such as butyrate, propionate and alcohols (ethanol, butanol) and lactic acid.
Environmental parameters such as pH, hydrogen partial pressure and temperature have been documented as key factors in hydrogen fermentation [10
]. The pH of the medium is known to regulate the shift to solventogenesis during the fermentation of sugars [7
]; the effect of low pH in the inhibition of methanogenic archaea is also recognized and could be potentially used as a selective pressure in mixed culture systems. Metabolic pathways of hydrogen formation are sensitive to hydrogen partial pressure (P
) and are subject to end-product inhibition [11
]. In addition, fermentation processes operating under thermophilic (45 to 60°C) and extreme thermophilic (65 to 80°C) could possibly result in higher hydrogen yields due to favorable thermodynamics and lower variety in soluble by-products [13
]. High temperatures inhibit the growth of methanogenic archaea and homoacetogenic bacteria [13
]; this is an important advantage when using mixed-cultures for hydrogen production because it prevents consumption of hydrogen by these microbial groups (as is often the case in mesophilic fermentation). Also, higher hydrolysis rates of cellulosic material have been observed in studies performed under thermophilic conditions, with the concurrent formation of higher amounts of fermentable sugars [14
]. Hydrogen production by mixed culture fermentation is more suited for industrial applications, when compared to pure culture fermentation. Some of the advantages are: (i) no need for sterile cultivation, (ii) presence of high microbial diversity, which offers increased adaptation capacity, (iii) possibility of mixed substrates co-fermentation, and (iv) higher capacity for continuous processing [15
]. However, and although there is a considerable number of studies on H2
production at extreme thermophilic conditions using pure cultures, studies using mixed-cultures are lacking [17
]. Also, the effect of pH and hydrogen partial pressure has been described in several pure cultures of thermophiles and extreme-thermophiles but the effect in mixed cultures is not yet clear [17
In the present study, the conversion of a C5-sugar (arabinose) and a C6-sugar (glucose) to hydrogen, using anaerobic mixed-cultures under extreme thermophilic conditions (70°C), was studied in continuous expanded granular sludge bed (EGSB) reactors. Microbial diversity in arabinose- and glucose-fed bioreactors was assessed using a PCR-DGGE (denaturing gradient gel electrophoresis) approach. Additional batch experiments were performed with extreme-thermophilic mixed cultures to study the effect of hydrogen partial pressure and pH on hydrogen production from arabinose and glucose.