Molecular hydrogen has a tremendous potential as a future energy carrier due to its pollution free combustion. However, current industrial hydrogen production processes contribute to the greenhouse effect. In contrast, a CO2 neutral H2 production using renewable energy sources can be obtained during dark anaerobic fermentation. Bacteria of the genus Clostridium can ferment sugars to H2 and CO2 with acetic and butyric acid as the main electron sinks. However, depending on the strain/bacterial co-culture and environmental conditions, more reduced products can be obtained e.g. ethanol, lactate, what reduces substantially the final H2 yield. Additionally, the different metabolic pathways and the regulatory circuits leading to H2 production in clostridia are not well resolved.
In this study, we focused our research on two main topics. On one hand, we investigated different bacterial co-cultures composed of Clostridium spp. in different H2-producing bioreactors. By monitoring the co-cultures of C. butyricum and C. pasteurianum with FISH (Fluorescence in situ hybridisation) and qPCR (quantitative real-time PCR), we have shown that both species stably co-existed during fermentations of different sugars in two different bioreactors. On the other hand, by using Clostridium butyricum CWBI1009 as a model species, we aimed to resolve the complex H2 metabolism in clostridia. The discovery of multiple novel [FeFe] hydrogenase genes in the sequenced genomes changed our perspective on how these microbes produce H2. Indeed, using different molecular tools, e.g. 2D-DIGE, RT-qPCR and RNA-seq, we have shown that in different environmental conditions, different hydrogenases may contribute to H2 production. Additionally, under N2 atmosphere during glucose fermentation in non-regulated pH conditions, for the first time in clostridia, nitrogenase was proposed to contribute to the overall H2 production. Surprisingly, despite the fact that clostridia seem to be perfectly equipped to produce hydrogen, they probably developed this capacity to quickly adapt to the changing conditions, namely decreasing pH value. We concluded that, in order to maintain a constant pH inside the cell, they excrete protons (presumably in form of H2) into the medium. At the same time, they get rid of the excessive reducing equivalents produced during glucose fermentation.
Altogether, the obtained results shed more light on the complex hydrogen metabolism in clostridia. Nevertheless, a challenge ahead is to characterize the key enzymes of hydrogen metabolism and, by means of metabolic bioengineering, to develop optimal microbial systems for biomass conversion to hydrogen.