Inhibition of microbial biofuel production in drought-stressed switchgrass hydrolysate

Abstract

BackgroundInterannual 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.ResultsWhile 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0657-0) contains supplementary material, which is available to authorized users.

Pre-senescence Harvest of Switchgrass Inhibits Xylose Utilization by Engineered Yeast

“…Imidazoles and pyrazines are two classes of inhibitory compounds that are produced during Maillard reactions of soluble sugars with ammonia during AFEX pretreatment (Chundawat et al, 2010) and can contribute toward inhibition of yeast fermentation of switchgrass hydrolysates (Ong et al, 2016). Very low levels of imidazoles and pyrazines were detected in the low AFEX severity pretreatment, most likely due to the low temperature used (80 • C), which is a major factor in the formation of these compounds (Shibamoto and Bernhard, 1976;Klinger et al, 2013).…”

“…Composition analysis of the bulk biomass was performed according to the NREL laboratory analytical procedures for biomass composition analysis (Hames et al, 2008;Sluiter et al, 2008aSluiter et al, ,b,c, 2012, with some modifications, as described previously (Ong et al, 2016).…”

“…The biological route of lignocellulosic fuel production uses a thermochemical pretreatment to open up the cell wall structure, followed by enzymatic hydrolysis to release cell wall sugars that are converted to fuel by fermentative microorganisms. Certain thermochemical pretreatments, such as ammonia fiber expansion (AFEX) are able to retain minerals and protein through the process (Lau and Dale, 2009;Lau et al, 2012;Ong et al, 2016). This means that the nutrients harvested with the biomass are not necessarily a hindrance to conversion, but can be utilized by the fermentation microorganism, eliminating the need for nutrient supplementation (Lau and Dale, 2009).…”

“…However, this benefit of nutrients from earlier harvests may be offset by a higher soluble sugar content in the biomass. Recent work has found that pretreatment reaction products of soluble sugars, which are generally at higher levels earlier in the growing season (Adler et al, 2006;Lindsey et al, 2013), contribute to fungal inhibition during fermentation (Ong et al, 2016). It is possible that instead of having a positive impact on fermentation through increased nutrients, harvesting early could negatively impact fermentation through formation of inhibitors.…”

“…Pretreated samples were subjected to high solids enzymatic hydrolysis (6% glucan loading) followed by ethanolic fermentation using an engineered xyloseutilizing yeast strain, S. cerevisiae 424A. (Oates et al, 2016;Ong et al, 2016), and the mean temperature and precipitation were 5.6 • C and 782 mm, respectively for the entire year of 2014, and were 15.3 • C and 470 mm, respectively for the growing season from 22 May to 13 November 2014. Switchgrass was planted in June 2008, and samples were manually harvested in 2014 from five replicate field plots (R1 -R5), roughly every 2 weeks from peak biomass (20 August 2014) until after the killing frost (7 November 2014).…”