Biofuels: Producing Ethanol from Cellulosic Material

Environmental Science
 

Overview

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

In this experiment, cellulosic material (such as corn stalks, leaves, grasses, etc.) will be used as a feedstock for the production of ethanol. The cellulosic material is first pretreated (ground and heated), digested with enzymes, and then fermented with yeast. Ethanol production is monitored using an ethanol probe. The experiment can be extended to optimize ethanol production by varying the feedstock used, pretreatment conditions, enzyme variation, yeast variation, etc. An alternative method of monitoring the reaction is to measure the carbon dioxide produced (using a gas sensor) instead of the ethanol. As a low-tech alternative, glucose meters (found in any drug store) can be used to monitor the glucose during the process, if an ethanol probe or carbon dioxide gas sensor is not available.

With an increased emphasis on ‘inquiry-based learning”, scientific probes are becoming more popular. Handheld devices like the Vernier Lab Quest used in conjunction with a variety of probes (such as those for conductivity, dissolved oxygen, voltage, and more) allow for less focus on collecting data and/or making graphs and more on analyzing the data and making predictions. Another advantage is that these are small and lightweight and can be taken into the field for measurements.

Cite this Video

JoVE Science Education Database. Environmental Science. Biofuels: Producing Ethanol from Cellulosic Material. JoVE, Cambridge, MA, (2017).

Principles

The United States is looking to wean itself off of fossil fuels, especially petroleum used in gasoline. Global climate change, dependence on foreign countries, and increased political instability around the world are only a few reasons why. One possible way to decrease dependence on petroleum as a transportation fuel is by using more ethanol. Currently, regular gasoline contains approximately 10% ethanol as an additive. Special flex-fuel vehicles can use E85 gasoline, which is 85% ethanol.

In Brazil, ethanol is made by using sugarcane as the feedstock. The main product of sugarcane is sucrose, which is a disaccharide of glucose and fructose. Most species of yeast have the enzyme sucrase and are able to cleave the glucose – fructose bond. The sucrose (C12H22O11) is fermented by yeast to produce ethanol and carbon dioxide. The overall chemical reaction is shown in Equation 1.2,3

Equation 1

However, in the US, ethanol is made from corn. Corn stores carbohydrates as a polysaccharide called starch. This requires more effort than making ethanol from sugarcane because yeast cannot cleave the bonds in starch. To make ethanol from corn, first corn kernels need to be ground up, then the ground corn treated with enzymes to convert the starch in the corn to glucose. After this step, the process continues as in the sugarcane method above by using yeast to ferment the glucose into ethanol and carbon dioxide. The chemical reaction is shown in Equation 2.2

Equation 2

The production of ethanol from corn is problematic, however. For one thing, it takes some corn out of the food supply, especially feed for livestock, thus driving up prices. It is also energy and fertilizer intensive to produce corn, decreasing its desirability as a transportation fuel alternative to petroleum. Therefore, scientists are increasingly turning to cellulosic material to make ethanol. These materials include wood, grasses, and non-edible parts of plants. These are more desirable as they do not impact food supply. However, in order to release the glucose from the cellulosic material, much more effort is needed, as the glucose from cellulosic material is bound up in cellulose, which is then wrapped with hemicelluloses and lignin. First the cellulose needs to be extracted from the hemicelluloses and lignin bindings. This is done through a pretreatment of grinding and acid hydrolysis. Then, the cellulose is treated with enzymes to break it up into its component glucose. Finally, the glucose can be fermented with yeast to produce ethanol and water. The process can be summarized as shown in Equation 3.

Equation 3

This is currently too energy intensive to make it a practical method for large-scale ethanol production. However, research is underway to make the process better.2,3

Procedure

1. Sample Preparation

  1. Select cellulosic material to be used as feedstock. This can be corn stalks, grasses, leaves, pet bedding, or paper. 
  2. Using a ball mill grinder (or coffee grinder if ball mill grinder is not available), grind feedstock into a fine powder with no large pieces remaining.
  3. Measure 1.0 g of feedstock and place in a 50-mL centrifuge tube. Label the tube with the feedstock chosen.
  4. Label a second 50 mL centrifuge tube as “Control”. Do not put any feedstock in this tube.

2. Pretreatment

  1. Set up a 500-mL beaker with approximately 400 mL of water on a hotplate and heat to a gentle boil.
  2. Add 25 mL of distilled water to the 2 centrifuge tubes. Swirl to mix. Put the cap on the centrifuge tubes loosely.
  3. Put the centrifuge tubes in the beaker full of gently boiling water. Be sure that water from the water bath does not leak into the tubes. Let boil for 30 min.
  4. Let tubes cool to room temperature.

3. Enzymatic Digestion

  1. Add 1 mL of cellulase enzyme to both tubes.
  2. Put tubes in an incubator at 50 °C for 24 h.
  3. Allow the tubes to cool to room temperature.

4. Fermentation

  1. Add 1.0 g of active yeast (regular grocery store yeast is ok) to each of the centrifuge tubes. Swirl to mix.
  2. Put an airlock on top of the centrifuge tube. The airlock allows CO2 to escape, keeping the pressure low in the centrifuge tube.
  3. Place the centrifuge tubes in a rack and put in an incubator at 37 °C for 24 h.
  4. Using an ethanol sensor, measure the ethanol concentration in the control tube (Figure 1). Ethanol sensors can be purchased through Vernier or PASCO for approximately $100 each.
    Figure 1
    Figure 1. Ethanol probe measuring the ethanol concentration in the control tube.
  5. Using an ethanol sensor, measure the ethanol concentration in the sample tube (Figure 2).
    Figure 2
    Figure 2. Handheld tablet displaying % of ethanol.

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!

Results

The % ethanol in the solution will be displayed on the handheld tablet screen using the software related to the brand of the ethanol sensor used (Figure 2).

Representative results of the percent ethanol produced by various feedstocks can be seen in Table 1.

Feedstock Ethanol produced
Sawdust 0.70%
Corn Stalks 0.60%
Cardboard 1.67%
Switchgrass 0.37%
Control 0.11%

Table 1. Representative results of the percent ethanol produced by various feedstocks.

Applications and Summary

The Energy Independence and Security Act of 2007 set into law a renewable fuel standard. It created a phase-in for renewable fuel volumes starting at 9 billion gallons in 2008 and ending at 36 billion gallons in 2022. Of that 36 billion, it was expected that 16 billion of that would come from cellulosic materials. For 2014, the original proposal was for 18.15 billion gallons of renewable fuel, 1.75 billion of that coming from cellulosic material. Unfortunately, based on the volume of cellulosic ethanol that is feasible to be produced currently, this number has had to be reduced to 17 million gallons according to a recent EPA proposal.1 Improving the process of creating ethanol from cellulosic material is currently a very hot area of research. In this experiment, students will be emulating the scientific practices that scientists in the top research labs are following. 

A variety of biomass feedstock materials can be used to produce cellulosic ethanol for transportation. The U.S. Department of Energy’s Bioenergy Technologies Office is focused on developing cellulosic feedstock from non-food based plant material and the related technologies that will allow an economically feasible conversion of this biomass to transportation fuel. They are investigating biomass sources ranging from agricultural residue, herbaceous energy crops, and forest materials to waste materials and algae. In this laboratory activity, students can vary the feedstock they use and compare the amount of ethanol that results. Possibilities include corn stover, grasses, leaves, cardboard, newspaper, paper, flowers, etc.

One roadblock to large scale production of cellulosic ethanol is the costly nature (both in terms of money and energy) of the pretreatment process. Much research is being done on reducing these costs and making the breakdown of the cellulosic material easier. Enzyme companies are spending a lot of time and money developing new enzymes to increase the yield of ethanol. In this laboratory activity, students can vary the enzymes they use and compare the amount of ethanol that results. A variety of Cellulase enzymes can be purchased from chemical companies that sell to schools, such as cellulase from Aspergillus niger or cellulase from Trichoderma virde. Or proprietary enzymes can be purchased from specialty enzyme companies, however these are costly. Other enzymes can be used, like amylase, to compare their ability of producing ethanol from cellulosic material to that of the cellulases.

Another emerging area of research is improving the ability of yeast to convert cellulosic biomass into ethanol. Lignocellulose has evolved in plants to provide stability. This is due to the crosslinking between cellulose and hemicellulose and the ester linkages in lignin. It is necessary to separate the cellulose from the lignin and then treat the cellulose to break it down into a monosaccharide. In addition, the hemicellulose has a high percentage of pentoses like xylose, which is more difficult to ferment than a hexose like glucose.4 

References

  1. The Energy Independence and Security Act of 2007. United States Congress, Washington DC. January 4, (2007).
  2. Balat, M., Balat, H., Oz, C. Progress in bioethanol processing. Progress in Energy and Combustion Science. 34 (2008).
  3. Ragauskas, A.J., Williams, C.K., Davison, B.H., Britovsek, G., Cairney, J., Eckert, C.A., Frederick Jr, W.J., Hallett, J.P., Leak, D.J., Liotta, C.L., Mielenz, J.R., Murphy, R., Templer, R., Tschaplinski, T. The Path Forward for Biofuels and Biomaterials. Science. 311 484 (2006).
  4. Demirbas, A. Bioethanol from Cellulosic Materials: A Renewable Motor Fuel from Biomass. Energy Source. 27 327-337 (2005).

1. Sample Preparation

  1. Select cellulosic material to be used as feedstock. This can be corn stalks, grasses, leaves, pet bedding, or paper. 
  2. Using a ball mill grinder (or coffee grinder if ball mill grinder is not available), grind feedstock into a fine powder with no large pieces remaining.
  3. Measure 1.0 g of feedstock and place in a 50-mL centrifuge tube. Label the tube with the feedstock chosen.
  4. Label a second 50 mL centrifuge tube as “Control”. Do not put any feedstock in this tube.

2. Pretreatment

  1. Set up a 500-mL beaker with approximately 400 mL of water on a hotplate and heat to a gentle boil.
  2. Add 25 mL of distilled water to the 2 centrifuge tubes. Swirl to mix. Put the cap on the centrifuge tubes loosely.
  3. Put the centrifuge tubes in the beaker full of gently boiling water. Be sure that water from the water bath does not leak into the tubes. Let boil for 30 min.
  4. Let tubes cool to room temperature.

3. Enzymatic Digestion

  1. Add 1 mL of cellulase enzyme to both tubes.
  2. Put tubes in an incubator at 50 °C for 24 h.
  3. Allow the tubes to cool to room temperature.

4. Fermentation

  1. Add 1.0 g of active yeast (regular grocery store yeast is ok) to each of the centrifuge tubes. Swirl to mix.
  2. Put an airlock on top of the centrifuge tube. The airlock allows CO2 to escape, keeping the pressure low in the centrifuge tube.
  3. Place the centrifuge tubes in a rack and put in an incubator at 37 °C for 24 h.
  4. Using an ethanol sensor, measure the ethanol concentration in the control tube (Figure 1). Ethanol sensors can be purchased through Vernier or PASCO for approximately $100 each.
    Figure 1
    Figure 1. Ethanol probe measuring the ethanol concentration in the control tube.
  5. Using an ethanol sensor, measure the ethanol concentration in the sample tube (Figure 2).
    Figure 2
    Figure 2. Handheld tablet displaying % of ethanol.

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!

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