(174bd) Elucidation of Toxicity and Tolerance Mechanisms to Lignocellulosic Biomass Hydrolysate for Clostridium Acetobutylicum Using Transcriptomics and Metabolomics

Preprocessing of the largest available renewable feedstock, lignocellulosic biomass, creates toxic inhibitors which prevent production of butanol by Clostridium acetobutylicum. Identifying cellular responses to inhibitors may provide a rational approach for developing inhibitor resistant strains of C. acetobutylicum, which could make renewable production of petrochemicals economically viable. Prevalent inhibitors derived from phenyl and furan derivatives may destabilize the cell membrane, generate reactive oxygen species, and compete with metabolism associated proteins to slow growth and butanol production. Transcriptomic and metabolomic profiles give insight into differentially expressed genes that confer tolerance, which future research can utilize to develop economically viable petrochemical production on renewable feedstock.

This study investigates the impact of inhibitors on C. acetobutylicum growth and transcriptome. Specifically, growth inhibition via 4-hydroxybenzaldehyde and furfural is characterized in increasing inhibitor concentration until cell death. Cultures with inhibitor dosage corresponding to 50% inhibition of max growth rate are sampled during acetogenesis and solventogenesis growth phases for metabolite analysis. Metabolite analysis via HPLC during acetogenesis and solventogenesis growth phases identifies changes in growth and metabolism due to inhibitor stress. A population of C. acetobutylicum undergoes mutagenesis via 1-methyl-3-nitro-1-nitrosoguanidine and subsequent exposure to increasing levels of 4-hydroxybenzaldehyde as a selective pressure to isolate inhibitor resistant strains. Multiple strains that underwent selection followed by screening on agar plates with 4-hydroxybenzaldehyde showed higher growth rates than the wild type C. acetobutylicum strain. This may reveal rational engineering approaches to create a genetic variant of C. acetobutylicum that can effectively utilize lignocellulosic biomass hydrolysate.

We discuss metabolic analysis performed in conjunction with mRNA-seq showing the relationship between butanol production and differentially expressed genes. First time application of mRNA-seq to acetogenesis and solventogenesis growth phases of C. acetobutylicum in the presence of inhibitors is described and may reveal differentially expressed genes potentially responsible for inhibitor tolerance and increased butanol production.