Bastek, P. A. | bioRxiv Science

bioRxiv2025

Enabling strong acetogenic growth on CO2 and H2: H2 solubility limits Clostridium ljungdahlii growth on CO2 and H2

Due to their ability to convert CO2, a greenhouse gas, into useful products, certain acetogenic bacterial species, such as Clostridium ljungdahlii, have been proposed as promising platform strains for renewable, carbon-negative chemical production. C. ljungdahlii, and similar acetogens, grows slowly and produce primarily acetate when grown on CO2 with H2 as the electron donor, but it grows quickly and can produce ethanol when grown on higher energy substrates, notably CO or fructose. Here, by utilizing different mixing strategies (and notably the first time use of roller bottles) to modulate the volumetric gas interfacial mass transfer coefficient (kLa), we show that, under both mixotrophic (sugar and gas utilization) and autotrophic conditions, C. ljungdahlii growth and CO2 fixation are primarily electron-limited due to the low solubility of H2 relative to CO and CO2. We demonstrate that, with sufficiently high H2 mass transfer, C. ljungdahlii can grow at similar high rates using CO2 as its sole carbon source compared to CO or fructose, a finding with significant implications for the use of acetogens in CO2-negative biomanufacturing, especially because at least 50% of CO used is oxidized and released as CO2. We also show that accumulation of fructose inhibits CO2 utilization by C. ljungdahlii under mixotrophic growth conditions, suggesting that a non-classical "catabolite repression" by fructose inhibits CO2 utilization.

bioengineering↗

bioRxiv2025

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 H2 as substrates - increase sugar-substrate carbon and electron conversion via CO2 and H2 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 CO2, which C. ljungdahlii can use as a carbon and electron sources. However, this dependence is unilateral; C. acetobutylicums 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.

bioengineering↗