1. Woody Invasive Species as Biomass Sources for Cellulosic Ethanol Professor J.J. VanAntwerp, Professor W. Wentzheimer, Steve Pohler, Calvin College Woody Invasive Species as Biomass Sources for Cellulosic Ethanol Professor J.J. VanAntwerp, Professor W. Wentzheimer, Steve Pohler, Calvin College Global energy demand and diminishing petroleum resources have led to increased research into alternative liquid fuels that burn cleanly and interface easily with existing infrastructure. Ethanol from biomass is one important option due to its renewable nature and an abundance of raw material for production. Plants use carbon dioxide and sunlight to produce biomass, which is based on the renewable energy from the sun. Plant material is a source of complex sugars which can be converted to simple sugars from which ethanol and other alcoholic fuels may be fermented. Some common raw materials for ethanol have been corn, corn stover and alfalfa. However, these have many disadvantages, including competition with food sources, large nutrient and energy needs, large water consumption and depletion of soil nutrients. By contrast, woody, or lignocellulosic, biomass offers many advantages. They can often grow on non-agricultural land (with soil too poor to support corn, or already depleted by corn growth) [1]. They typically do not require the fertilizer, pesticide and energy input that corn does [2]. The major disadvantage of cellulosics to corn is that while the starches in corn are easily converted to simple sugars for ethanol fermentation, cellulosics require additional processing to access the simple sugars. Still, there are a variety of commercially available and experimental techniques or pretreatments to deal with cellulosic feedstock. Pretreatments In order to maximize ethanol yield, cellulosic biomass must be subjected to pretreatment to remove the lignin and convert the cellulose to simple sugars. There are a variety of pretreatment options, including treating the biomass with dilute acid, caustics, ammonia, steam, or liquid hot water at various pressures. Dilute acid pretreatments have been the most common over the past hundred years of research and practice. Recently, enzymes have been introduced following treatments involving acids or other means, to release even more of the sugars contained in the biomass. Pretreatment advancement is a key to unlocking more sugars, increasing both ethanol yield and processing time. Our study focuses on dilute acid pretreatment at elevated pressure, followed by enzyme loading to extract sugars. This is one of the most studied treatments, providing a wide literature base for comparison. Biomass Composition Woody biomass contains three main components: cellulose, hemicelluloses and lignin. All three are natural polymers which require different processes to break apart into sugar molecules, and are then fermented by a microorganism into fuels such as ethanol. Different pretreatments are used to extract sugars from each component. Dilute acid for example is used to produce five carbon sugars (pentoses) from the hemicellulose portion of the plant, while cellulase enzymes such as Trichoderma reesei are implemented following acid addition and removal to extract six carbon sugars (hexoses) from the sample, leaving lignin behind. Leftover solids are largely lignin, which will be analyzed to determine the composition of the plant which cannot be converted to ethanol. For an industrial process, lignin would likely be burned to provide power to the plant. The goal is to determine if any of the three species are suitable for use as biomass to convert to ethanol, and where they compare to current sources economically and environmentally. 1. Huang, H.-J., et al., Effect of biomass species and plant size on cellulosic ethanol: A comparative process and economic analysis. Biomass and Bioenergy, 2009. 33: p. 234-246. 2. Hill, J., et al., Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. PNAS, 2006. 103(30): p. 11206-11210. 3. Gupta, R., Sharma, K. K., & Kuhad, R. C. (2009). Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498. Bioresource Technology, 100(3), 1214-1220. doi: 10.1016/j.biortech.2008.08.033. 10.1016/j.biortech.2008.08.033 Introduction Method Results Buckthorn cuttings were blended and the particles passed through sieves to collect a precisely sized sample of biomass. The chips or particles are subjected to dilute acid hydrolysis at elevated pressure in an autoclave. The hydrolysate, or result of hydrolysis, (red box in figure) from this process is separated from the solids and analyzed or fermented in a commercial process. The solid portion/ slurry, composed of lignin and cellulose, is then reacted with an enzyme for further hydrolysis. Following enzyme hydrolysis the solid and liquid phases are separated again and the liquid hydrolysate (blue box) is analyzed or fermented. The leftover solids are lignin, which cannot be converted to product and will be burned for energy. Sugar composition from each source is measured independently to quantify which sugars are produced and in what ratio. The pretreatment process will also be modeled using state-of-the-art engineering software to determine commercial viability and to understand the production phase better. Gas chromatography technique is used to quantify the sugars contained in each species as well as the yield from pretreatment and enzyme addition. Samples were taken from one, two and three-year old cuttings of each plant except switchgrass, which grows uniformly when mature, to determine the optimum cutting age of the plant, for composition of celluloses and lignin. Compositional sugar analysis will be done on two samples of sugars (red and blue boxes in Figure 1). With our analysis we address yield and optimal feed composition, two important considerations for commercial processes. A cellulase assay has been done to determine enzyme activity in preparation for pretreatment. When the correct activity has been determined from the standard assay curve, the acid and enzyme hydrolysis treatments will be applied to one, two and three-year old samples of buckthorn and autumn olive as well as one sample of switchgrass. Switchgrass is chosen to ensure that our study correctly determines sugar content with respect to other studies. The study will proceed during the semester with the eventual goal of determining how effective the pretreatment is in addition to the utility of each shrub as biomass source. References Conclusions Alternative fuels have come to the forefront due to global energy demand and global warming. Invasive species, requiring low input and high yield have the potential to be a renewable way to cleanly harvest energy. Cellulosic waste goes largely unused, is more appropriate for certain growth conditions and climates, and is therefore a cheap and widely available source, suitable for energy. This project also illustrates the benefit of collaboration between scientific disciplines. The input from biology Professor David Dornbos and student researcher Jennie Heidmann was and is instrumental in our approach to the problem. Purpose Autumn olive and buckthorn studies have not been evaluated to date. The purpose of this study was to determine sugar content with respect to better documented species so that a meaningful comparison between species may be done. The study has been conducted using reproduced methods [3] from studies of other common benchmark species in order to compare sugar yields as accurately as possible to aid in economic and environmental feasibility assessments. Figure 1. Process for Biomass to Ethanol Conversion for a Cellulosic Species. Mechanical Treatment Acid Hydrolysis Woodchips Biomass Pentoses from hemicellulose Cellulose and Lignin slurry Hexoses from cellulose Lignin slurry Fermentation/ GC Analysis Enzyme Hydrolysis Ethanol Fermentation/ GC Analysis Ethanol Analyzed/ Burned for Energy Power for plant For this project, we have selected cellulosic species that are compatible with Michigan growing conditions. Michigan is host to a cooler and cloudier environment than regions such as the plains where an option such as switchgrass is particularly prevalent. Our intention is to find a species that requires little human input, while maximizing output and growth rate. Though switchgrass can claim that for plains states, the shorter and cloudier growing season in Michigan is more suitable for hardier species which can continue to grow well into autumn. The three species that were chosen for study are common buckthorn, glossy buckthorn and autumn olive. These species are invasive and grow well in Michigan on exhausted agricultural land, with little or no support. While there has been much work studying the feasibility of switchgrass and similar plains states crops, we seek in this project to evaluate these Michigan crops for ethanol fermentation feasibility. These species originate in Eurasia and are non-native to North America. They are able to flourish because they produce leaves much more quickly than native species and are therefore able to outgrow them. The plants would be harvested before they are able to seed in order to prohibit their further spread. Motivation