Enhanced biohydrogen production and substrate utilization by co-culture fermentation with reduced extracellular electron shuttles

Abstract

Hydrogen is a promising energy carrier due to no greenhouse gas (GHG) emission during combustion and the highest conversion efficiency in fuel cells and highest energy content per unit mass compared to carbon-based energy carriers. However, three key challenges for large-scale biohydrogen production are to increase i) the hydrogen production rate, ii) the hydrogen molar yield, and iii) the extent of substrate utilization. A co-culture system of C. beijerinckii and G. metallireducens with extracellular electron shuttles was developed and evaluated for improved biohydrogen production. To enhance biohydrogen production, Clostridium beijerinckii was co-cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone-2, 6-disulfonate (AH2QDS). In the co-culture fermentation system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, and 39.0% for substrate utilization extent were observed compared to a pure culture of C. beijerinckii without AH2QDS. G. metallireducens grew in the co-culture system, resulting in a decrease in acetate concentration under co-culture conditions and a presumed regeneration of AH2QDS from AQDS. These co-culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production. This co-culture system was further applied to ferment complex substrates from hydrolysates of lignocellulosic biomass as well as to utilize compounds including indigo dye, juglone, lawsone, fulvic acids and humic acids as alternative extracellular electron shuttles. The observed improvements in utilization of lignocellulosic hydrolysates and particularly utilization of xylose III support the feasibility of applying this co-culture system to lignocellulosic hydrolysates, especially xylose-rich ones, in industry. In addition, the replacement of AH2QDS by alternative extracellular electron shuttles, such as humic acids, makes the co-culture with extracellular electron shuttle system more economical and flexible.