In the United States, ethanol is produced primarily from starch in corn kernels. Most of the 4 billion gallons of ethanol produced in 2005 came from 13% of the U.S. corn crop (1.43 billion bushels of corn grain). When corn is harvested, the kernels make up about half of the above-ground biomass, and corn stover (e.g., stalks, leaves, cobs, husks) makes up the other half.
Ethanol production from corn grain involves one of two different processes: Wet milling or dry milling. In wet milling, the corn is soaked in water or dilute acid to separate the grain into its component parts (e.g., starch, protein, germ, oil, kernel fibers) before converting the starch to sugars that are then fermented to ethanol. In dry milling, the kernels are ground into a fine powder and processed without fractionating the grain into its component parts. Most ethanol comes from dry milling. Key steps in the dry mill ethanol-production process include:
Milling.
Corn kernels are ground into a fine powder called "meal."
Liquefying and Heating the Cornmeal.
Liquid is added to the meal to produce a mash, and the temperature is increased to get the starch into a liquid solution and remove bacteria present in the mash.
Enzyme Hydrolysis.
Enzymes are added to break down the long carbohydrate chains making up starch into short chains of glucose (a simple 6-carbon sugar) and eventually to individual glucose molecules.
Yeast Fermentation.
The hydrolyzed mash is transferred to a fermentation tank where microbes (yeast) are added to convert glucose to ethanol and carbon dioxide (CO2). Large quantities of CO2 generated during fermentation are collected with a CO2 scrubber, compressed, and marketed to other industries (e.g., carbonating beverages, making dry ice).
Distillation.
The broth or "beer" produced in the fermentation step is a dilute (10 to 12%) ethanol solution containing solids from the mash and yeast cells. The beer is pumped through many columns in the distillation chamber to remove ethanol from the solids and water. After distillation, the ethanol is about 96% pure. The solids are pumped out of the bottom of the tank and processed into protein-rich coproducts used in livestock feed.
Dehydration.
The small amount of water in the distilled ethanol is removed using molecular sieves. A molecular sieve contains a series of small beads that absorb all remaining water. Ethanol molecules are too large to enter the sieve, so the dehydration step produces pure ethanol (200 proof). Prior to shipping the ethanol to gasoline distribution hubs for blending, a small amount of gasoline (~5%) is added to denature the ethanol making it undrinkable.
How is ethanol produced from cellulosic biomass?
Conversion of cellulosic biomass to ethanol is less productive and more expensive than the conversion of corn grain to ethanol. Cellulosic biomass, however, is a less expensive and more abundant feedstock than corn grain; more efficient processing is needed to take advantage of this plentiful and renewable resource. The structural complexity of cellulosic biomass is what makes this feedstock such a challenge to break down into simple sugars that can be converted to ethanol.
Most plant matter consists of three key polymers: Cellulose (35 to 50%), hemicellulose (20 to 35%), and lignin (10 to 25%). These polymers are assembled into a complex, interconnected matrix within plant cell walls. See Understanding Biomass: Plant Cell Walls for an illustrated description of plant cell-wall structure. Cellulose and hemicellulose are carbohydrates that can be broken down into fermentable sugars. The cellulosic and hemicellulosic portions of plant biomass are processed separately because they have different structures and sugar content. Cellulose consists of long chains of glucose molecules (simple 6-carbon sugars) arranged into a solid, three-dimensional, crystalline structure. Hemicellulose is a branched polymer composed primarily of xylose molecules (simple 5-carbon sugars) and some other sugars. Lignin, a rigid aromatic polymer, is not a carbohydrate and cannot be converted into ethanol.
Efficiently separating and breaking down the different polymers in cellulosic biomass is an important challenge that is not an issue for corn ethanol production. One multistep process for converting cellulosic biomass to ethanol is outlined below. See Applying Genomics for New Energy Resources: From Biomass to Cellulosic Ethanol for an illustrated description of key steps in the conversion process.
Mechanical Preprocessing.
Dirt and debris are removed from incoming biomass (e.g., bales of corn stover, wheat straw, or grasses), which is shred into small particles.
Pretreatment.
Heat, pressure, or acid treatments are applied to release cellulose, hemicellulose, and lignin and to make cellulose more accessible to enzymatic breakdown (hydrolysis). Hemicellulose is hydrolyzed into a soluble mix of 5- and 6-carbon sugars. A small portion of cellulose may be converted to glucose. If acid treatments are used, toxic by-products are neutralized by the addition of lime. Since cellulose biomass can come from many different sources (e.g., grasses, wheat straw, corn stover, paper products, hardwood, softwood), a single pretreatment process suitable for all forms of biomass does not exist.
Solid-Liquid Separation.
The liquefied syrup of hemicellulose sugars is separated from the solid fibers containing crystalline cellulose and lignin.
Fermentation of Hemicellulosic Sugars.
Through a series of biochemical reactions, bacteria convert xylose and other hemicellulose sugars to ethanol.
Enzyme Production.
Some of the biomass solids are used to produce cellulase enzymes that break down crystalline cellulose. The enzymes are harvested from cultured microbes. Purchasing enzymes from a commercial supplier would eliminate this step.
Cellulose Hydrolysis.
The fiber residues containing cellulose and lignin are transferred to a fermentation tank where cellulase enzymes are applied. A cocktail of different cellulases work together to attack crystalline cellulose, pull cellulose chains away from the crystal, and ultimately break each cellulose chain into individual glucose molecules.
Fermentation of Cellulosic Sugars (Glucose).
Yeast or other microorganisms consume glucose and generate ethanol and carbon dioxide as products of the glucose fermentation pathway.
Distillation.
Dilute ethanol broth produced during the fermentation of hemicellulosic and cellulosic sugars is distilled to remove water and concentrate the ethanol. Solid residues containing lignin and microbial cells can be burned to produce heat or used to generate electricity consumed by the ethanol-production process. Alternately, the solids could be converted to coproducts (e.g., animal feed, nutrients for crops).
Dehydration.
The last remaining water is removed from the distilled ethanol.
What are key biological barriers to cellulosic ethanol production?
Compared to cornstarch ethanol production, several factors make cellulosic ethanol production more costly and less efficient. One important barrier is lower sugar yields due to the heterogeneous and recalcitrant nature of cellulosic biomass. More effort is needed to pretreat and solubilize hemicellulose and cellulose because they are locked into a rigid cell-wall structure with lignin. Harsher thermochemical pretreatments generate chemical by-products that inhibit enzyme hydrolysis and decrease the productivity of fermentative microbes. The crystallinity of cellulose also makes it more difficult for aqueous solutions of enzymes to convert cellulose to glucose.
Another barrier is the mix of sugars generated from hemicellulose hydrolysis. Microorganisms that can ferment both 5- and 6-carbon sugars exist, but they have lower production rates and exhibit less tolerance for the end-product ethanol. Broth produced from a mix of 5- and 6-carbon sugars is about 6% ethanol instead of 10 to 14% ethanol produced from cornstarch glucose fermentation.
Overcoming these and other barriers will require a more complete understanding of several biological factors that impact the conversion process:
- Understanding what aspects of plant cell-wall structure and composition make some plant materials easier to break down than others.
- Investigating regulatory mechanisms that control cell-wall synthesis so that new bioenergy crops optimized for efficient biomass breakdown can be developed. For example, minimizing lignin content would improve enzyme access to cellulose during the hydrolysis step, thus increasing sugar yields.
- Surveying natural microbial communities to discover and analyze a more diverse range of enzymes that can break down cellulose, hemicellulose, and lignin. Perhaps novel enzymes capable of breaking down lignin and hemicellulose could be used to reduce the severity and improve the effectiveness of pretreatment.
- Creating new enzyme mixtures and analyzing their collective activities to determine the best combinations needed for rapid and complete breakdown of different components of biomass.
- Identifying the many genes that determine the most-desirable traits for fermentative microbes and understanding how these genes are regulated. Some of these traits include tolerance of higher ethanol concentrations, improved uptake and conversion of all sugars generated from biomass hydrolysis, elimination of unnecessary metabolic pathways, and achieving optimal fermentation productivity at higher temperatures to prevent contamination. Identifying these genes and understanding how they are controlled will be critical to developing the ideal fermentative microbe that possesses all these traits.
- Integrating all hydrolysis and fermentation steps into a single microbe or stable mixed culture to streamline the entire process and reduce costs.
How can Genomics:GTL improve production of cellulosic ethanol and other biofuels?
Genomics:GTL (GTL) will provide systems-level biological investigations needed to rapidly develop new crops designed for bioenergy applications and to determine genetic makeup and functional capabilities of such microbial communities involved in biomass decomposition and sugar fermentation. Some advanced systems biology capabilities that GTL can provide include:
- Using advanced sequencing capabilities at DOE's Joint Genome Institute to sequence and analyze the genomes of crop plants, fermentative microbes, and microbial communities involved in biomass decomposition and soil productivity.
- High-throughput analysis of genes and proteins expressed by plants and microbes used to produce cellulosic ethanol and characterization of the conditions and regulatory systems that control expression.
- Comprehensive analysis of metabolites in plants and microbes to model cellular metabolism and define metabolic pathways relevant to biomass breakdown or ethanol fermentation.
- State-of-the-art imaging technologies covering a wide range of spatial scales to track enzymes in cells and elucidate molecular structure of plant cell walls.
Advanced computational tools that will integrate large quantities of diverse biological data and develop predictive computer-based models of plant and microbial systems.
