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Background: Interannual variability in precipitation, particularly drought, can affect lignocellulosic crop biomass yields and composition, and is expected to increase biofuel yield variability. However, the effect of precipitation on downstream fermentation processes has never been directly characterized. In order to investigate the impact of interannual climate variability on biofuel production, corn stover and switchgrass were collected during 3 years with significantly different precipitation profiles, representing a major drought year (2012) and 2 years with average precipitation for the entire season (2010 and 2013). All feedstocks were AFEX (ammonia fiber expansion)-pretreated, enzymatically hydrolyzed, and the hydrolysates separately fermented using xylose-utilizing strains of Saccharomyces cerevisiae and Zymomonas mobilis. A chemical genomics approach was also used to evaluate the growth of yeast mutants in the hydrolysates. Results: While most corn stover and switchgrass hydrolysates were readily fermented, growth of S. cerevisiae was completely inhibited in hydrolysate generated from drought-stressed switchgrass. Based on chemical genomics analysis, yeast strains deficient in genes related to protein trafficking within the cell were significantly more resistant to the drought-year switchgrass hydrolysate. Detailed biomass and hydrolysate characterization revealed that switchgrass accumulated greater concentrations of soluble sugars in response to the drought and these sugars were subsequently degraded to pyrazines and imidazoles during ammonia-based pretreatment. When added ex situ to normal switchgrass hydrolysate, imidazoles and pyrazines caused anaerobic growth inhibition of S. cerevisiae. Conclusions: In response to the osmotic pressures experienced during drought stress, plants accumulate soluble sugars that are susceptible to degradation during chemical pretreatments. For ammonia-based pretreatment, these sugars degrade to imidazoles and pyrazines. These compounds contribute to S. cerevisiae growth inhibition in drought-year switchgrass hydrolysate. This work discovered that variation in environmental conditions during the growth of bioenergy crops could have significant detrimental effects on fermentation organisms during biofuel production. These findings are relevant to regions where climate change is predicted to cause an increased incidence of drought and to marginal lands with poor water-holding capacity, where fluctuations in soil moisture may trigger frequent drought stress response in lignocellulosic feedstocks. [ABSTRACT FROM AUTHOR]

Background: Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18-25 % of the total soluble sugars in the hydrolysate and 12-18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7-9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production from cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results: Oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes. Conclusion: The carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme. [ABSTRACT FROM AUTHOR]