Butanol production from switchgrass using novel strains of Clostridium beijerinckii

Abstract

Biobutanol is a promising fuel that can be blended with gasoline and improve its octane number. Biobutanol has been produced from corn starch and molasses via acetone-butanol-ethanol (ABE) fermentation. However, the increase in food-based feedstock prices resulted in a switch to synthesis of butanol from petroleum. Sugar substrates from cheap and sustainable feedstock such as lignocellulosic biomass can compete with chemical synthesis-derived butanol. However, pretreatment of biomass is required to allow enzymes to convert the cellulose and hemicellulose to fermentable sugars. Hydrothermolysis is a promising biomass pretreatment method, which generates lignin-degraded compounds that could inhibit ABE fermentation.The objective of this study is to evaluate butanol production using novel Clostridium beijerinckii strains with improved tolerance to lignocellulosic derived microbial inhibitory compounds (LDMICs) and to evaluate fermentation strategies to enhance butanol production. To achieve this, hydrothermolysis pretreated Alamo switchgrass was hydrolyzed by means of enzyme (Accellerase 1500) to produce enzymatic hydrolysate which contains approximately 65 g/L glucose and 2 g/L xylose. Interestingly, the concentration of furfural and hydroxymethylfurfural (HMF) in the non-detoxified hydrolysate was 60 mg/L and 4 mg/L respectively while the phenolic inhibitory compounds were present within the range of 4 -8 mg/L. Ferulic acid was not detected in the non-detoxified hydrolysate. However, 600 mL of the non-detoxified hydrolysate was detoxified using activated carbon, as a result, 80% of the furan and phenolic compounds were removed from the hydrolysate.

Consequently, ABE fermentation in 150 mL bottles using 50 mL working volume with 6% (v/v) inoculation level was performed anaerobically under N₂ / H₂ (95% / 5%) atmosphere in an anaerobic chamber. The ABE fermentation media used were P2 glucose medium, detoxified switchgrass hydrolysate medium, and non-detoxified switchgrass hydrolysate medium. Furthermore, wild type, AKR and SDR strains of C. beijerinckii were used as biocatalysts. The fermentation results show that AKR and SDR strains consumed about 30% more glucose in detoxified hydrolysate medium compared to P2 glucose medium. Consequently, about 20% more glucose was utilized by AKR and SDR strains in the non-detoxified hydrolysate medium with intermittent feeding than without intermittent feeding. Furthermore, about 20 g/L total ABE was produced by the SDR strain in the non-detoxified hydrolysate medium with intermittent feeding while the same strain produced 17 g/L total ABE in the detoxified hydrolysate medium. These results show that using inhibitor tolerant AKR and SDR strains and the intermittent feeding fermentation strategy eliminated the need for detoxification and improved ABE fermentation by about 15% when compared with the results of ABE fermentation in the detoxified switchgrass hydrolysate medium. These results showed there is a high potential of increased butanol yield from non-detoxified hydrolysate, which makes ABE fermentation more feasible.