Biofuels are fuels that are derived from biological matter, such as plants. Biofuels serve as an alternative to fossil fuels, as they can be sourced from crops in many parts of the world. Additionally, they are cleaner burning, thereby reducing greenhouse gas emissions.

One of the most widely used biofuels is ethanol derived from plant biomass, typically sugar cane and corn. In the US, the majority of ethanol biofuel is produced from corn.

The use of corn crops as a feedstock is controversial, as corn is energy intensive to grow, uses a large quantity of fertilizers, and its use as a feedstock removes a large quantity of corn from the food supply, especially feed for livestock. As a result, the use of other plant materials, or lignocellulosic materials, such as grass, leaves, paper, and non-edible parts of crops is increasing.

This video will cover the basics of deriving ethanol from lignocellulosic material, and demonstrate the production of ethanol from lignocellulosic feedstocks in the laboratory.

Lignocellulosic biomass refers to plant matter with woody cell walls. This type of plant matter is one of the most abundant raw materials available, as it is frequently a waste product of agriculture and manufacturing.

The cell walls are composed of the highly crosslinked polymer, lignin, and two complex carbohydrates, hemicellulose, and cellulose. Cellulose is the primary source of fermentable sugars, such as glucose, but it must be first separated from the lignin and hemicellulose components.

The first step in processing of the lignocellulosic material is to finely grind the dry plant matter into a powder. The ground feedstock then undergoes pretreatment to break down the lignin and hemicellulose barrier in the cell wall, and enable access to the cellulose.

Next, the cellulose is treated with hydrolytic enzymes, such as cellulase and hemicellulase. The enzymatic hydrolysis breaks down cellulose into glucose. Finally, the glucose is fermented with yeast to produce ethanol.

The following experiment demonstrates this step-wise method of producing ethanol from cellulosic biomass through the removal of lignin and hemicellulose, followed by the enzymatic treatment of cellulose, and the fermentation of glucose to produce ethanol.

In this experiment, ethanol will be produced from corn stover, the leaves and stalks from corn plants. Using a ball mill grinder, grind the feedstock into a fine powder, and ensure that no large pieces remain.

Weigh 1 g of feedstock, place it into a 50-mL centrifuge tube, and label it. Label a second tube as the control sample, and do not add any feedstock. To pretreat the samples, set up a 500-mL beaker with approximately 400 mL of water, and bring it to a gentle boil.

Add 25 mL of distilled water to the two prepared centrifuge tubes and cap them loosely. Swirl the tubes to mix. Place the tubes in the boiling water, and ensure that the water from the bath does not leak into the tubes. Allow them to boil for 30 min, then remove and let them cool to room temperature.

Once the tubes have cooled, add 1 mL of cellulase enzyme to both tubes. Place the tubes in an incubator for 24 h. After 24 h, remove the tubes and allow them to cool to room temperature. Ethanol is produced from the digested cellulosic material through fermentation by yeast. To begin this process, add 1 g of active yeast to each of the centrifuge tubes, and swirl to mix.

Place an airlock on the centrifuge tubes. The airlock allows the carbon dioxide that is generated during the fermentation to escape so pressure does not build up in the tube. Place the centrifuge tubes in a rack, and place in an incubator at 37 °C. Once fermentation is complete, use an ethanol sensor to measure the ethanol concentration in the control and sample tubes.

To make biofuels a competitive energy source, certain questions about the structure and performance of the feedstocks must be answered.

It is important to understand the distribution of lignin in various plants, so its removal can be performed efficiently. In this example, the lignin distribution in plant cell walls was analyzed by slicing thin layers from a plant stem. The thin slices were then imaged using confocal microscopy with 532 nm laser light to create three-dimensional images of the plant stem.

Lignin content was determined using Raman spectroscopy. By combining the confocal images and the Raman spectra, a three-dimensional map of lignin distribution was generated.

In order to maximize the amount of bioethanol derived from plant feedstocks, the types of feedstocks must be compared. In this example, ethanol was produced from cardboard, and compared to corn stover.The cardboard was prepared as shown previously, where the ground cardboard was subjected to pretreatment, followed by enzymatic digestion in order to separate lignin and hemicellulose from the material and break down the cellulose to glucose. The extracted glucose was then fermented with yeast to produce ethanol. Cardboard proved to be a superior feedstock to corn stover, as it produced more than double the concentration of ethanol in solution.

In the United States, the vast majority of bioethanol is produced from corn. While the production of ethanol from corn is energy intensive, it is less complex than the production of ethanol from cellulosic biomass.

In order to transition away from corn feedstocks, the yield from cellulosic biomass must be better than that of corn. In this example, cornmeal and corn stover were compared using the same procedure as shown previously.

Cornmeal produced a higher concentration of ethanol than corn stover, showing that corn is a slightly better feedstock than the corn stalks themselves. However, corn stalks and other cellulosic feedstocks, are more plentiful and inexpensive and may provide a viable alternative.

You've just watched JoVE's Introduction to Biofuels. You should now understand the production of ethanol from plant feedstocks, and the challenges associated with the process. Thanks for watching!