Sustainable Biorefining in Unsterilized Wastewater By Engineering of the Extreme Alkaliphile Bacillus Marmarensis

Water usage, contamination susceptibility, and inability to co-utilize pentoses and disaccharides are some major challenges to industrialization of sustainable biorefining in lieu of fossil resource consumption. We have built tools for and engineered the unexplored, extreme alkaliphile Bacillus marmarensis as a platform for new bioprocesses which meet all these challenges. With development of a transformation technique, a genetic overexpression system, and RNA knockdown strategies, we engineered B. marmarensis to produce ethanol at 40 g/l titers at 65% the theoretical maximum yield in unsterilized glucose medium. Transitioning towards industrial substrates and conditions, we next achieved ethanol titers of 12 g/l at a 50% yield from xylose and cellobiose mixtures in unsterilized seawater and algal-contaminated wastewater media. B. marmarensis presents a promising approach for the contamination-resistant biorefining of a wide range of carbohydrates in unsterilized, non-potable seawater from this initial investigation.