Water-soluble saponins accumulate in drought-stressed switchgrass and may inhibit yeast growth during bioethanol production

Background: Developing economically viable pathways to produce renewable energy has become an important research theme in recent years. Lignocellulosic biomass is a promising feedstock that can be converted into second-generation biofuels and bioproducts. Global warming has adversely affected climate change causing many environmental changes that have impacted earth surface temperature and rainfall patterns. Recent research has shown that environmental growth conditions altered the composition of drought-stressed switchgrass and directly influenced the extent of biomass conversion to fuels by completely inhibiting yeast growth during fermentation. Our goal in this project was to find a way to overcome the microbial inhibition and characterize specific compounds that led to this inhibition. Additionally, we also determined if these microbial inhibitors were plant-generated compounds, by-products of the pretreatment process, or a combination of both. Results: Switchgrass harvested in drought (2012) and non-drought (2010) years were pretreated using Ammonia Fiber Expansion (AFEX). Untreated and AFEX processed samples were then extracted using solvents (i.e., water, ethanol, and ethyl acetate) to selectively remove potential inhibitory compounds and determine whether pretreatment affects the inhibition. High solids loading enzymatic hydrolysis was performed on all samples, followed by fermentation using engineered Saccharomyces cerevisiae. Fermentation rate, cell growth, sugar consumption, and ethanol production were used to evaluate fermentation performance. We found that water extraction of drought-year switchgrass before AFEX pretreatment reduced the inhibition of yeast fermentation. The extracts were analyzed using liquid chromatography–mass spectrometry (LC–MS) to detect compounds enriched in the extracted fractions. Saponins, a class of plant-generated triterpene or steroidal glycosides, were found to be significantly more abundant in the water extracts from drought-year (inhibitory) switchgrass. The inhibitory nature of the saponins in switchgrass hydrolysate was validated by spiking commercially available saponin standard (protodioscin) in non-inhibitory switchgrass hydrolysate harvested in normal year. Conclusions: Adding a water extraction step prior to AFEX-pretreatment of drought-stressed switchgrass effectively overcame inhibition of yeast growth during bioethanol production. Saponins appear to be generated by the plant as a response to drought as they were significantly more abundant in the drought-stressed switchgrass water extracts and may contribute toward yeast inhibition in drought-stressed switchgrass hydrolysates

STUDYING EFFECT OF DROUGHT ON SWITCHGRASS AND IDENTIFYING ASSOCIATED MICROBIAL INHIBITORS

Recent research reports newer sustainable technologies for bioenergy generation that would serve as an alternative to conventional petroleum sources. There is abundant research reported on individual subsets of the study that has linked growth parameters to biomass yield, treatment conditions to bioethanol production or pretreatment conditions to microbial inhibitors. However, there is a lack of literature that undertakes the entire field-to-fuel process conditions in a single study. In this thesis, we focus on isolating and characterizing secondary metabolites provoked by drought in switchgrass, an upcoming bioenergy crop, during the mid-western drought of 2012. To characterize these inhibitors, switchgrass was separately solvent extracted before and after the pretreatment method followed by sequential hydrolysis and fermentation using yeast. Our analysis concluded that adding a water extraction step prior to AFEX-pretreatment overcame the inhibition, and saponins, a class of plant-generated triterpene glycosides, potentially ceased yeast growth in the drought-stressed switchgrass. Using non-targeted mass spectrometry (negative ESI/MS and positive ESI LC-MS), we identified many known and unknown compounds in relatively higher amounts in the drought-stressed switchgrass compared to control. A molecular networking mathematical calculation was performed using an R-based software, MFAssignR, for the detected compounds (abundance, m/z and retention time) to get a better insight on empirical formula, oxidation state and aromatic index. Further, we studied the effect of drought-like water-stress on switchgrass, simulated using rainout shelters, harvested on five “marginal lands” located across latitudinal gradient throughout Michigan and Wisconsin. In 2018, the 60% roof occlusion failed to induce water-stress on switchgrass under the shelters compared to ambient samples at the all sites except for the Hancock site. The water-stress imposed by the rainout shelters compounded with relatively low soil moisture holding capacity and high soil temperature due to sandy nature of the soil at the Hancock site resulted in reduced fermentability of switchgrass than ambient samples. However, there was no significant reduction in the biomass yield between the paired rainout and ambient samples. Hence, in the subsequent years the shelters were modified to 100% roof occlusion resulting in reduction of the biomass yield for the rainout samples compared to ambient samples