A high solids field-to-fuel research pipeline to identify interactions between feedstocks and biofuel production

Background: Environmental factors, such as weather extremes, have the potential to cause adverse effects on plant biomass quality and quantity. Beyond adversely affecting feedstock yield and composition, which have been extensively studied, environmental factors can have detrimental effects on saccharification and fermentation processes in biofuel production. Only a few studies have evaluated the effect of these factors on biomass deconstruction into biofuel and resulting fuel yields. This field-to-fuel evaluation of various feedstocks requires rigorous coordination of pretreatment, enzymatic hydrolysis, and fermentation experiments. A large number of biomass samples, often in limited quantity, are needed to thoroughly understand the effect of environmental conditions on biofuel production. This requires greater processing and analytical throughput of industrially relevant, high solids loading hydrolysates for fermentation, and led to the need for a laboratory-scale high solids experimentation platform. Results: A field-to-fuel platform was developed to provide sufficient volumes of high solids loading enzymatic hydrolysate for fermentation. AFEX pretreatment was conducted in custom pretreatment reactors, followed by high solids enzymatic hydrolysis. To accommodate enzymatic hydrolysis of multiple samples, roller bottles were used to overcome the bottlenecks of mixing and reduced sugar yields at high solids loading, while allowing greater sample throughput than possible in bioreactors. The roller bottle method provided 42–47% greater liquefaction compared to the batch shake flask method for the same solids loading. In fermentation experiments, hydrolysates from roller bottles were fermented more rapidly, with greater xylose consumption, but lower final ethanol yields and CO2 production than hydrolysates generated with shake flasks. The entire platform was tested and was able to replicate patterns of fermentation inhibition previously observed for experiments conducted in larger-scale reactors and bioreactors, showing divergent fermentation patterns for drought and normal year switchgrass hydrolysates. Conclusion: A pipeline of small-scale AFEX pretreatment and roller bottle enzymatic hydrolysis was able to provide adequate quantities of hydrolysate for respirometer fermentation experiments and was able to overcome hydrolysis bottlenecks at high solids loading by obtaining greater liquefaction compared to batch shake flask hydrolysis. Thus, the roller bottle method can be effectively utilized to compare divergent feedstocks and diverse process conditions

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

High temperatures and low soil moisture synergistically reduce switchgrass yields from marginal field sites and inhibit fermentation

‘Marginal lands’ are low productivity sites abandoned from agriculture for reasons such as low or high soil water content, challenging topography, or nutrient deficiency. To avoid competition with crop production, cellulosic bioenergy crops have been proposed for cultivation on marginal lands, however on these sites they may be more strongly affected by environmental stresses such as low soil water content. In this study we used rainout shelters to induce low soil moisture on marginal lands and determine the effect of soil water stress on switchgrass growth and the subsequent production of bioethanol. Five marginal land sites that span a latitudinal gradient in Michigan and Wisconsin were planted to switchgrass in 2013 and during the 2018–2021 growing seasons were exposed to reduced precipitation under rainout shelters in comparison to ambient precipitation. The effect of reduced precipitation was related to the environmental conditions at each site and biofuel production metrics (switchgrass biomass yields and composition and ethanol production). During the first year (2018), the rainout shelters were designed with 60% rain exclusion, which did not affect biomass yields compared to ambient conditions at any of the field sites, but decreased switchgrass fermentability at the Wisconsin Central–Hancock site. In subsequent years, the shelters were redesigned to fully exclude rainfall, which led to reduced biomass yields and inhibited fermentation for three sites. When switchgrass was grown in soils with large reductions in moisture and increases in temperature, the potential for biofuel production was significantly reduced, exposing some of the challenges associated with producing biofuels from lignocellulosic biomass grown under drought conditions

Code from: High temperatures and low soil moisture synergistically reduce switchgrass yields from marginal field sites and inhibit fermentation

<h1>Code from: High temperatures and low soil moisture synergistically reduce switchgrass yields from marginal field sites and inhibit fermentation</h1> <p>This README file was generated on 2024-01-11 by Rebecca Ong.</p> <h2>GENERAL INFORMATION</h2> <p><strong>Title of Repository: </strong>Code from: High temperatures and low soil moisture synergistically reduce switchgrass yields from marginal field sites and inhibit fermentation</p> <p><strong>Author Information:</strong></p> <p><em>Principal Investigator/Contact Person<br></em><strong>Name: </strong>Rebecca Ong <br><strong>Institution:</strong> Michigan Technological University <br><strong>Address: </strong>Houghton, MI USA <br><strong>Email:</strong> <a href="mailto:[email protected]">[email protected]</a></p> <p><strong>Information about funding sources that supported this research:</strong></p> <ol> <li>Great Lakes Bioenergy Research Center, U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018409</li> <li>National Science Foundation Long-term Ecological Research Program (DEB 1832042) at the Kellogg Biological Station</li> <li>Michigan State University AgBioResearch</li> </ol> <h2>SHARING/ACCESS INFORMATION</h2> <p><strong>Licenses/restrictions: </strong>CC0 1.0 Universal (CC0 1.0) Public Domain</p> <p><strong>Links to publications that cite or use the code:</strong></p> <p>Chipkar S, Debrauske DJ, Kahmark K, Bohm S, Hussain MZ, Joshi L, Krieg KM, Cronk B, Burke E, Cassidy J, Aguado J, Senyk A, Robertson GP, Sato TK, Hamilton SK, Thelen KD, and Ong RG. High temperatures and low soil moisture synergistically reduce switchgrass yield and inhibit biofuel production from marginal field sites. Glob. Change Biol. Bioenergy (2023)</p> <p><strong>Recommended citation:</strong></p> <p>Chipkar S, Debrauske DJ, Kahmark K, Bohm S, Hussain MZ, Joshi L, Krieg KM, Cronk B, Burke E, Cassidy J, Aguado J, Senyk A, Robertson GP, Sato TK, Hamilton SK, Thelen KD, and Ong RG. (2023). Code from: High temperatures and low soil moisture synergistically reduce switchgrass yield and inhibit biofuel production from marginal field sites. Zenodo. <a href="https://doi.org/10.5281/zenodo.10278446" rel="nofollow">https://doi.org/10.5281/zenodo.10278446</a></p> <h2>DETAILS ON R CODE</h2> <p>R Code was used for statistical analysis and to generate figures used in the paper. All files are posted in Dryad (<a href="https://doi.org/10.5061/dryad.qnk98sfps" rel="nofollow">https://doi.org/10.5061/dryad.qnk98sfps</a>), GitHub (<a href="https://github.com/rebeccaongmtu/switchgrass-rainout-shelter-paper">https://github.com/rebeccaongmtu/switchgrass-rainout-shelter-paper</a>), and Zenodo (<a href="https://doi.org/10.5281/zenodo.10278446" rel="nofollow">https://doi.org/10.5281/zenodo.10278446</a>). All code was generated and run using RStudio Version 2023.06.1+524 (2023.06.1+524).</p> <p><strong>MLE_Weather.R</strong></p> <p>R code used to generate plots and statistics for soil properties and weather data. Requires Field_Data.csv and TempHeatMap.csv files and the readr, tidyverse, lubridate, ggh4x, reshape2, viridisLite, and viridis packages.</p> <p><strong>FermentationDataAnalysis.R</strong></p> <p>R code used to generate bar graphs and conducts ANOVAs for lignocellulosic biomass and hydrolysate composition and fermentation ethanol production and glucose consumption. Requires NIR_Composition_both_years.csv and FermentationHPLCData.csv and the readr, reshape2, ggplot2, ggpubr, and dplyr packages.</p> <p><strong>RespirometerCO2Plots.R</strong></p> <p>R code used to aggregate data from multiple CSV files into a single dataframe and then generate faceted line plots of replicates and average CO2 production from respirometer fermentation experiments. Requires the unzipped files in Respirometer_CO2.zip stored in the same folder, and the tidyverse, ggnewscale, and ggh4x packages.</p> <p><strong>Survival_Analysis.R</strong></p> <p>R code used to generate faceted survival plots annotated with p-values from a paired t-test. Requires Survival_Analysis_Data.csv and the readr, survival, ggplot2, survminer, and tidyverse packages.</p> <h2>VERSION CHANGES</h2> <p>v.1.0.1 changes</p> <ul> <li>Modified the MLE_Weather.R file to use prefiltered data (as stored in the Dryad repository)</li> <li>Modified the README file to be more descriptive</li> </ul&gt