1. Fermentation By C Kohn Agricultural Sciences Waterford, WI Most information is based on materials from the DOEs Great Lakes Bioenergy Research Center, Madison, WI – www.glbrc.org

2. Fermentation in Society The process of fermentation is critical for… Fuel Ethanol Production Bread making (yeast causes the break to rise) Alcoholic Beverages Cheese-making (bacteria produce lactic acid which with rennet curdles the milk) and yogurt Pickles and Sauerkraut Additionally, fermentation is necessary for ruminants such as cattle to break down cellulosic vegetation. Wisconsin has a fermentation culture. Many things associated with WI rely on fermentation as a part of the process of their creation.

3. What is Fermentation? Fermentation : a form of cellular respiration done in an environment without oxygen (anaerobic) Respiration – the complete breakdown of a source of energy such as a sugar into CO2 and H2O in an aerobic environment. Yeast and bacteria are frequently used as fermenters they consume sugars for energy and release byproducts such as ethanol and carbon dioxide. Fermentation is the process by which we create ethanol from renewable plant materials. This is very different from transesterification, the process by which we create biodiesel. Transesterification is a chemical process; fermentation is a biological process

4. Fermentation Components Fermentation consists of… Substrates – the substance being acted upon (usually a sugar) Products – the substance created (ethanol, lactic acid, etc.) Ethanol is a waste product for the organism because it cannot acquire any additional energy from this molecule. Fermentation requires an organism that can break down substrates in the absence of oxygen. Yeast (saccharomyces) is most often the organism of choice. However, yeast are not the only organisms that can do this. Many species of bacteria can also ferment. Even your own cells can obtain energy through fermentation (your muscles produce lactic acid when they dont receive enough oxygen for the demand of the physical activity)

5. Substrate Products

6. Basic Fermentation So how does yeast turn sugar into ethanol? Yeast has enzymes capable of converting a simple sugar, called glucose, into ethanol as they extract energy from the molecule. Simple sugars are easy to ferment More complex carbs are more difficult. Creating ethanol from sugar cane, as they do in Brazil, is fairly straightforward because cane juice contains these simple sugars that yeast can digest.

7. Ethanol from Starch & Cellulose Ethanol production becomes more difficult when starting with corn grain or other more complex plant materials The long sugar molecules in starches (like corn and potatoes) require additional steps to complete fermentation The fermenting organism must have enzymes that chop the long chains of starch into smaller glucose units. This is known as saccharification saccharification

8. Corn Ethanol Currently, the main biofuel used in the United States is ethanol distilled from kernels of corn. There are two methods used to convert the corn kernel into a usable fuel: wet milling dry milling. The two different processes yield different by- products Steeping, the first step in the wet milling process, is what differentiates this process from dry-milling processes

9. Wet Milling In wet milling, the kernel is steeped before being separated yielding a final product of ethanol, corn oil, corn gluten, and corn starch which may be modified to corn syrup. Generally large, corporate owned ethanol plants use wet milling. Ethanol plants owned by farmer cooperatives generally utilize the dry milling process.

10. Dry Milling In dry milling, the kernel is first ground into a fine flour before adding water to produce a mash. After fermentation, the mash is separated into ethanol and stillage. The stillage is centrifuged and dried producing dried distillers grains (DDG), a high-protein, high- energy livestock feed. Dry milling is cheaper and produces DDG, whilethe process of wet milling is more flexible. Source: Cornell Univ.Cornell Univ.

11. Cellulose as a Feedstock Cellulose is another possible feedstock for fermentation into ethanol. Because cellulose makes up nearly half of all plant biomass, cellulosic ethanol is considered the largest potential source of biofuel in the near future. Like starch, cellulose is made of long chains of sugar A cellulose molecule is simply a polymer of glucose Polymer – a long chain of repeating molecules.

12. Cellulose – a Pain in the Grass Unlike starch, cellulose is far harder to ferment for two key reasons: 1. There are fewer organisms with enzymes that are capable of breaking down cellulose Without the right enzyme you cannot degrade a molecule biologically (cellulase, hemicellulase, etc). The kind of enzymes that break down starch do not work on cellulose because of their differing bonds (right). 2. Cellulose molecules are packed into a tight crystalline form and then wrapped up in lignin and hemicellulose. These two molecules sheath and protect cellulose from degradation (next page)

13. Cellulose

14. Todays Work – Untangle & Unpack To access the cellulose in order to ferment it, we first have to unwrap it from the other molecules and unpack the crystals into their individual glucose molecules. This process is called pretreatment and is currently the most expensive part of the conversion process. Researchers are looking for more efficient, less expensive ways to easily separate the cellulose from the lignin and hemicellulose. (unwrap) Researchers are also working to find enzymes or organisms that more efficiently chop the cellulose into smaller pieces of glucose for fermentation. (unpack)

15. Ruminant Fermentation In many ways, what we want to accomplish in regards to fermentation of cellulosic ethanol is very similar to what the rumen (the first stomach) of a cow already does. A cows rumen is filled with microbes that produce cellulase, the enzyme that breaks down cellulose. A cows rumen is the perfect environment for these microbes to thrive A cows rumen is a good model for what we want to be able to do with cellulosic feedstocks for ethanol The work of the rumen produces far more energy than it consumes

16. Rumen Advantages The top benefit of being a herbivore is the ability to extract energy from cellulose in plant cell walls. Cellulose makes up about half of the dry weight of stems, roots, and leaves. A cow can meet almost all of its dietary needs from the vegetation that surrounds it in a pasture. Few animals are better equipped to obtain large amounts of energy from cellulose. We would like to do the same, but for fuel.

17. Bovine Fermentation Think about what a cow must do to break down cellulose 1. Obtain the food (graze) 2. Grind the food to increase the surface area (chew) 3. Chemically break down the food (with enzymes in the saliva such as amylase) 1. This would known in chemistry as hydrolysis 2. Hydrolysis: a reaction with water that breaks larger molecules into smaller ones (e.g. example breaking cellulose into glucose). 4. Ferment the food in their rumen with microbes 5. Absorb and digest the carbohydrates in their stomachs and intestines. The steps in making cellulosic ethanol are very similar.