Correlation of the Nifh gene Expression and a Metabolic Model for the Hydrogen Production By a Phototropic Mixed Culture

Anaerobic Digestion (AD) as a process for the treatment of organic solid wastes and the production of energy is a promising technology. During this process, hydrogen (H2) and methane (CH4) are produced as by-products in addition to volatile fatty acid (VFA). Because H2 is considered a clean fuel and it contains a high energy content, its production is preferred. In recent years, the use of the volatile fatty acids (VFA) generated during AD for H2 production by photoautotrophic bacteria has been explored to increase the overall production yield. Unfortunately, the mechanisms and regulation systems for H₂ production are not completely understood due to the complexity of the metabolic networks in photo-fermentation systems. Although several studies have been performed using single-strain cultures, they could present limitations during the scale-up of the process because the nature of the feedstock. The use of mixed cultures certainly provides a more robust system to be applied in larger scales. Here, we proposed a condensed metabolic model to describe the metabolic changes in an already characterized photo-autotrophic mixed culture during the H2 production. From previous results in our research group, the analyzed mixed culture is composed majorly by Rhodopseudomonas spp (85% of the bacterial distribution by the end of the fermentation). The information of the biochemical reactions involved in H₂ production during the photo-fermentation was gathered from KEGG and BRENDA databases, using Rhodopseudomonas spp and Rhodobacter spp global metabolic maps as the reference. Anaerobic fermentations were performed in a 5 L stirred tank photo-reactor. Acetate was used as carbon source and light intensity was set to be 1000 lux. Samples were taken regularly and analyzed by HPLC. By applying Flux Balance Analysis (FBA), the model predicts an increase in the reaction rates for the key enzymes (nitrogenase and hydrogenase) that participate in H2 production. The model predictions were validated by gene expression analysis. Previously, it was reported that H2 production in photo-bacteria happens as a way to keep the redox balance, and this mechanism is regulated by two enzymatic complex: nitrogenase and hydrogenase. Our results showed that for the mixed culture, the increase in hydrogen production is related to an increment in the expression of the nitrogenase complex from Rhodopseudomonas rather that the hydrogenase complex, as we observed an increase in the expression levels of the nifH. The model qualitatively mimics the nitrogenase flux as a response of the environmental conditions that favor nitrogenase transcription, which in turn increase the H2 production. Our results are a first approach to understand the mechanism involved in H2 production in a mixed culture. Even our results showed that Rhodopseudomonas it’s the major contributor to H2 production, still the interactions with the other population should be considered in the context of whole metabolic interactive network.