(667c) Towards Industrial Robustness: Understanding of Wild-Type and Populus Hydrolysate- Tolerant Mutant Strains of Clostridium Thermocellum

An industrially robust microorganism capable of degrading lignocellulosic biomass in the presence of inhibitory compounds produced from the pretreatment process in a CBP scheme is needed to reduce the cost of cellulosic biofuels. Clostridium thermocellum has the ability to convert biomasses complex carbohydrates into ethanol by fermentation. However, C. thermocellum is inhibited by by-products from the biomass pretreatment process. To increase C. thermocellum’s industrial robustness, a 17.5% v/v Populus hydrolysate-tolerant mutant (PM) was produced by 117 successive transfers of C. thermocellum 27405 strain into increasing concentrations of the hydrolysate. A single colony culture of the PM was then produced and the genome has been sequenced. A study comparing the wild-type (WT) and PM strains of C. thermocellum was completed with growth on cellobiose examining sugar utilization and end product formation, and growth on Avicel examining protein concentration. The growth of the PM in 17.5% v/v Populus hydrolysate had a 20% higher optical density, a 33% faster sugar utilization rate (g glucan/L/hr), and produced 34% more ethanol (g ethanol/ g glucan) than the WT grown without hydrolysate. The PM retained these characteristics when grown without hydrolysate with a 37% higher optical density, a 40% faster sugar utilization rate and produced 20% more ethanol than the WT grown under the same conditions. The WT was severely inhibited when grown in the presence of 17.5% v/v Populus hydrolysate with a barely detectable optical density, 7% of sugar utilization rate, and produced 7.5% of the expected ethanol versus when it was grown without hydrolysate. A combination of the genomic mutations of the PM and gene expression profiles from triplicate fermentations in various concentrations of the Populus hydrolysate will be used to determine which mutations in the genome might be the cause of the increased tolerance to the hydrolysate.