Biology subjects

Otten, J. K.

Publications and source records attributed to Otten, J. K..

3 recordsLinked to original sources

bioRxiv2026

Biological upgrading of C1-C2 products of electrocatalytic CO 2 reduction to C4-C6 carboxylates

BackgroundMicrobial chain-elongation by Clostridium kluyveri using the products (acetate and ethanol) derived from the electrocatalytic CO2 reduction reaction (CO2RR) represents a unique sustainable strategy for producing C4-C6 chemicals from CO2. However, direct integration of electrocatalytic effluents with anaerobic bioprocesses is often impeded by the physiological incompatibility between electrocatalytic product streams and microbial metabolism. Specifically, CO2RR effluents commonly contain formate, which cannot be utilized by C. kluyveri for chain elongation and therefore reduces the overall carbon efficiency of CO2 conversion to C4-C6 chemicals. Moreover, both formate and the elevated phosphate concentrations typical of electrochemical reaction solutions may inhibit microbial growth. ResultsWe show that formate at concentrations of up to 50 mM did not inhibit the growth of or the chain elongation by C. kluyveri. Based on this finding, we developed a modular two-step bioprocess. In the first step, the acetogen Clostridium ljungdahlii converts formate in CO2RR product mixtures into acetate, thereby generating additional substrates for second-step C. kluyveri-driven chain elongation, thus increasing the CO2RR carbon-conversion efficiency to C- C6 chemicals. To address the issue of C. ljungdahliis inhibition by high phosphate concentrations in electrocatalytic solutions, we explored the use of C. ljungdahlii biofilms for the first, i.e. the formate-conversion, step. C. ljungdahlii biofilms exhibit tolerance to concentrated electrolytes, enabling the conversion of up to 50 mM formate in CO2RR solutions. ConclusionsThe demonstrated two-step process constitutes the basis for the development of a robust and carbon-efficient biological process for the scalable upgrading of C1-C2 CO2RR products into higher-value C4-C6 chemicals.

synthetic biology↗

bioRxiv2025

Cross-talk between engineered Clostridium acetobutylicum and Clostridium ljungdahlii in syntrophic cocultures enhances isopropanol and butanol production

There is a need for efficient and sustainable production of essential chemicals such as isopropanol and butanol - the focus of this study - from renewable sugar feedstocks. Microbial fermentations use glycolysis, and as result, a third of the sugar carbon is lost to CO2 through pyruvate decarboxylation to acetyl-CoA, the starting intermediate for the biosynthesis of most microbial metabolites. In nature, microbes exist in syntrophic consortia, allowing for mutually-beneficial interactions, the production of novel products and the realization of novel benefits - including better carbon conservation - not seen in monocultures. Here, for increased acetone production, we transformed Clostridium acetobutylicum with a plasmid (p95ace02a) expressing a synthetic acetone pathway made up of four native genes. This engineered C. acetobutylicum was cocultured with Clostridium ljungdahlii to capture the waste CO2 and H2 generated due to glucose catabolism by C. acetobutylicum, and to convert acetone into isopropanol. We examined the impact of starting cell densities, the gas atmosphere (N2, H2, or H2/CO2) and coculture species ratios (using a recently-developed RNA-FISH flow cytometric assay) on metabolite production, yields and sugar-carbon utilization. Metabolic flux analysis identified the complex patterns by which the two species alter each others metabolism in a cell-density and gas-atmosphere dependent manner. For example, C. ljungdahlii activated the dormant acetate uptake in C. acetobutylicum, while coculture density dramatically impacted species ratios, electron management, and C. ljungdahliis H2 utilization. We achieved exceptionally-high concentrations of our desired products - 246 mM isopropanol and 148 mM butanol - in 64 hours, with about 85% of the production occurring before 32 hours. We reached maximum productivities of 13.9 mM isopropanol/hour and 10.4 mM butanol/hour with 0.9 mol alcohol produced per mol of sugar consumed. Total product yields reached 84.7% on a C-mol basis, versus 65.6% that can be reached in a C. acetobutylicum monoculture.

microbiology↗

bioRxiv2025

Enabling supratheoretical isopropanol yields from carbon-negative glucose fermentations with Clostridium acetobutylicum-Clostridium ljungdahlii cocultures

Synthetic microbial cocultures, which combine the unique capabilities of multiple microbes into one process, have significant potential for sustainable production of fuels and chemicals. Most studies of defined cocultures have tested relatively low cell densities in lab-scale batch cultures, not the high cell density fed-batch or continuous processes with cell retention typically required to achieve industrially-relevant volumetric productivities. Here, we explore the impact of increased cell density on isopropanol production from the syntrophic coculture of genetically-modified Clostridium acetobutylicum [CACas9 {Delta}hbd (p95ace02_atoB), with deleted 4-C metabolism expressing an acetone-formation pathway on the plasmid] with WT Clostridium ljungdahlii using first a pseudo-perfusion approach followed by perfusion culture. CACas9 {Delta}hbd (p95ace02_atoB) produces acetone without any 4-C metabolites and C. ljungdahlii converts that acetone to isopropanol. To explore the mechanism by which these cultures enable supratheoretical isopropanol yields, we first identified NADH-driven hydrogen conversion in CACas9 {Delta}hbd (p95ace02_atoB) as the thermodynamically-limiting step for acetone and thus isopropanol production. We then demonstrated the ability of C. ljungdahlii to mitigate this issue by eliminating detectable hydrogen accumulation in the coculture. Pseudo-perfusion cocultures showed that high cell densities combined with a high population fraction of C. ljungdahlii enable dramatic increases in isopropanol yields beyond the thermodynamic limitation imposed in CACas9 {Delta}hbd (p95ace02_atoB) monocultures. Finally, we demonstrate carbon-negative fermentation of glucose to isopropanol as the sole alcohol product in a perfusion bioreactor.

bioengineering↗