Due to the combination of increasing demand for oil, decreasing supply, and greenhouse gas (GHG) emissions, biofuels are increasingly being investigated as carbon neutral alternatives to petroleum fuels. Current ethanol production from corn starch is nearing the feasible maximum, and only delivers approximately 1.3 units of energy in the fuel product for every unit of fossil energy consumed in the production (1). Producing fuel from starch has the additional negative effect of competing directly with the food supply and has potential for GHG emissions from large-scale land use change. Cellulose is the most abundant biopolymer on Earth, and lignocellulosic feedstocks have the potential to provide greater than 10 units of energy in the fuel for each unit of fossil energy consumed in the production (2). However, the technology for converting cellulose to sugar is still challenged with technical barriers and much work is needed to bring the cost of production down to the point where it is economically competitive.
Michigan's Upper Peninsula comprises an area approximately 14,500 square miles situated in the north central forested region of the United States. Due to ecological constraints on the growing season, agriculture is significantly limited. However the region has historically been and remains a productive forest ecosystem which supplies much timber for construction and pulp for paper, and has the potential to provide a significant amount of lignocellulosic biomass to future biorefineries. Furthermore, abandoned agricultural lands are in abundance that could be converted to energy crop production.
The goal of this research is to determine the effects of dilute acid hydrolysis pretreatment conditions on the maximum total sugar yield (pentoses plus hexoses) after pretreatment and enzymatic hydrolysis by commercially available cellulase mixtures. An understanding of the effect one process has on subsequent processes downstream will be important to future industrial operations. The data will provide yield information on a number of important forest species and lay a foundation for future enzyme improvement studies.
Methods
Dilute acid hydrolysis is a common method of pretreating lignocellulosic feedstocks prior to enzymatic hydrolysis. Dilute acid hydrolysis removes the hemicellulose fraction – composed primarily of five-carbon or pentose sugars – leaving behind cellulose and lignin which are much more recalcitrant to hydrolysis. Enzymatic hydrolysis frequently follows pretreatment to liberate hexose sugars – primarily glucose – from cellulose, leaving behind only lignin which can be used as a boiler fuel to provide heat and electricity for the process.
The primary goal of pretreatment is to expose the cellulose for more efficient enzymatic hydrolysis. However two competing secondary considerations are also important in the overall process; pentose sugars from dilute acid hydrolysis are fermentable by some bacteria species thereby increasing the total potential ethanol yield, but degradation products from dilute acid hydrolysis – primarily furfural – decrease total sugar yields and inhibit fermentation.
Three species of locally available and commercially relevant biomass feedstocks will be considered; aspen (a hardwood), balsam (softwood), and switchgrass (model energy crop). Dilute acid hydrolysis (pretreatment) conditions to be varied include temperature (150°C to 175°C), sulfuric acid concentration (0.25%, 0.50%, and 1.00%), and biomass species (aspen, balsam, and switchgrass). Pretreatment reactions will occur at constant temperature using ten 5 ml liquid volume stainless steel tubular reactors immersed in 750 ml of a Dow Corning 550 heat transfer fluid in a Parr Instruments Model 4570 reactor. Reactor tubes will be removed at different times from the hot oil bath and placed in ice to stop the reaction. Samples of the hydrolysate will be taken from the tubular reactors, filtered (0.22 µm pore size) and analyzed by High Performance Liquid Chromatography (HPLC) for sugar content. Solid residues will be washed with distilled water and enzymatic hydrolysis will be performed on the highest sugar yielding tubular reactor for each set of pretreatment conditions. Enzymatic hydrolysis conditions will be: 1% dry weight pretreated biomass (cellulose plus lignin), 60 filter paper units (FPU) SpeezymeCP (Genencor) per dry gram cellulose plus 120 cellobiohydrolase units (CBU) beta-glucosidase (Novozyme, Novozym 188) per dry gram cellulose, 72 hr. reaction time, and 50°C.
Results
Preliminary results using a similar “mini-reactor – 2.5 ml” pretreatment experimental system exhibited a distinct effect of pretreatment conditions on xylan sugar yields from pretreatment and glucan sugar yields from enzymatic hydrolysis. More severe pretreatment reduced xylan sugar yields and increased byproduct concentrations (furfural), but also increased glucose yields from enzymatic hydrolysis, especially at early in the 72 hr hydrolysis period for hardwoods (aspen, red maple, basswood) and also at all times for balsam (softwood). Results from the proposed design of experiments will add to those already obtained to identify optimum conditions for extracting maximum total sugars from the hydrolysis of the forest biomass species included in this study. Recommendations for dilute acid hydrolysis conditions to maximize total fermentable sugar yield will be made.
References
(1) USDA. 2002. The Energy Balance of Corn Ethanol: An Update, AER-814, U.S. Department of Agriculture Office of the Chief Economist and Office of Energy Policy and New Uses.
(2) U.S. DOE. 2006. Breaking the Biological Barriers to Cellulosic Ethanol: A Joint Research Agenda, DOE/SC-0095, U.S. Department of Energy Office of Science and Office of Energy Efficiency and Renewable Energy (www.doegenomestolife.org/biofuels/).