Essential Nutrients for Improving the Direct Processing of Raw Lignocellulosic Substrates Through the Dark Fermentation Process

The dark fermentation process is a base platform in which lignocellulosic substrates are biologically pretreated producing a gas mixture of H2/CO2, volatile fatty acids as intermediate chemicals, and solids enriched in cellulose. Optimal amounts of nutrients are required by mixed cultures to hydrolyze and ferment the substrate at once requiring optimal amounts of nutrients. In this study, two statistical approaches were used to screen essential nutrients for the direct conversion of lignocellulosic substrates into hydrogen (the chosen response variable) and to optimize such variable. First, a Plackett-Burman design of seven factors at two levels screened the main nutrients for the direct fermentation of the substrate. Urea, CaCl2, and KH2PO4 present a significant effect. The low levels of these nutrients in batch fermentation with 2% of total solids (90, 2, and 37 mg/L, respectively) improved 4-fold the hydrogen production in comparison with the control treatment containing no nutrients. Then, a central composite design with three factors at five levels predicted that the optimum levels of nutrients for the highest hydrogen production of 504 mL/L were 1018 mg/L of urea (a C/N ratio of 17.5), 109 mg/L of KH2PO4 (a C/P ratio of 338), and 411 mg/L of CaCl2. The usefulness of this optimized medium formulation was demonstrated using three lignocellulosic substrates where the hydrogen production increased 1.2- to 3.5-fold in comparison with the control treatments containing no nutrients.