Engineering mutualism via nitrogen exchange in mixotrophic cocultures between Clostridium acetobutylicum and Clostridium ljungdahlii

We have previously shown that mixotrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii – using sugars and H ₂ as substrates – increase sugar-substrate carbon and electron conversion via CO ₂ and H ₂ capture and synthesize valuable products, such as isopropanol and 2,3-butanediol, that neither species can make independently. In this pairing, growth of C. ljungdahlii is constrained by C. acetobutylicum , since C. ljungdahlii relies on C. acetobutylicum to convert glucose into CO ₂ , which C. ljungdahlii can use as a carbon and electron sources. However, this dependence is unilateral; C. acetobutylicum’s growth is not constrained by C. ljungdahlii . Consequently, population ratios between the two species can vary substantially throughout the course of fermentation and in different fermentation setups, typically with the faster growing C. acetobutylicum outcompeting C. ljungdahlii . Population ratio is an important variable because it influences metabolite yields and productivity and likely also impacts the initiation and frequency of the heterologous cell fusion events we have documented between C. acetobutylicum and C. ljungdahlii . Thus, developing methods to rationally control and maintain the population ratio are important for both biotechnological applications and fundamental study of this coculture pairing. In this study we show that the different nitrogen utilization capabilities of these two organisms enable engineering of a mutualistic mixotrophic syntrophy in which C. ljungdahlii relies on C. acetobutylicum for carbon and electrons and C. acetobutylicum relies on C. ljungdahlii for nitrogen. First, we confirm that C. ljungdahlii , but not C. acetobutylicum , can convert nitrate into biologically useful ammonium, enabling the design of a culture medium in which C. acetobutylicum can only grow in the presence of C. ljungdahlii . Second, we test different ratios of nitrate to ammonium in batch cocultures and demonstrate that rapid nitrate utilization by C. ljungdahlii prevents C. acetobutylicum from becoming nitrogen-limited at any point in batch fermentation. Finally, we show that feeding different rates of nitrate to cocultures in fed-batch mode enables control of the coculture growth rate, maintenance of stable population ratios, and higher isopropanol and butanol yields in cocultures between C. acetobutylicum and C. ljungdahlii .