Biology subjects

Willis, N. B.

Publications and source records attributed to Willis, N. B..

5 recordsLinked to original sources

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↗

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↗

bioRxiv2025

Separate, Separated and Together: the Transcriptional Program of the Clostridium acetobutylicum- Clostridium ljungdahlii syntrophy leading to interspecies cell fusion

Syntrophic cocultures (hitherto assumed to be commensalistic) of Clostridium acetobutylicum and Clostridium ljungdahlii, whereby CO2 and H2 produced by the former feeds the latter, result in interspecies cell fusion involving large scale exchange of protein, RNA and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane separated versus unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in C. ljungdahliis autotrophic Wood-Ljungdahl-Pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by C. acetobutylicum and are metabolized by C. ljungdahlii to its growth advantage. These and other findings, and notably upregulation of C. acetobutylicum ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO2 and H2, C. acetobutylicum feeds C. ljungdahlii with growth boosting amino acids, while benefiting from the H2 utilization by C. ljungdahlii. IMPORTANCEThe construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each individual monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies "touching" on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

systems biology↗