Science Buddies: "Ask an Expert"

My science fair group is taking cellulose from plants, converting it to yield glucose, then fermenting the glucose to produce ethanol. We also thought of using the ethanol to power something small like a lawnmower (just to show that the ethanol really is useful).We thought of putting the plant in a blender and just using the "mush" to convert to glucose but I'm not sure this is the best idea because the concentration of cellulose might be minimal. In converting the cellulose to glucose, I thought of hydrolysis as an option, but I'm not exactly sure how to do it. In fermenting the glucose to produce ethanol, we thought of putting the glucose in a closed container and leaving it in an oven for about 2-3 days. So here are my questions:

1) Is blending the plant for cellulose ok or should we try something else?
2) How do we convert the cellulose to glucose?
3) Is our method of fermenting ok or we should we try something else?
4) Will we have enough concentration of ethanol to power a lawnmower?

Thank You

Hi,

This is a challenging, but very interesting project. Your idea to blend the plants to grind the cellulose into very small particles to increase the surface area is a good idea. However, the cellulose will still exist as a polysaccharide of many subunits of glucose that cannot be fermented to ethanol directly. This site shows the structure of cellulose:

http://en.wikipedia.org/wiki/Cellulose

Cellulose, the polymer, must be broken down by dilute acid or by enzymatic techniques before it can be fermented to ethanol. The following Wikipedia article is an excellent review of the topic and includes references that you should check out.

http://en.wikipedia.org/wiki/Cellulosic_ethanol

You will need to decide how to hydrolyze the cellulose first. Are you going to use acid or enzymes? Once you have glucose, how are you going to convert the glucose into ethanol? You should do more background reading to decide exactly what to do before setting up your first experiment.

Also, before you start, make sure you can enter this project into competition. Have you checked with your teacher to make sure the project complies with all of the local rules and regulations?

Donna Hardy

Since I don't think we can obtain any enzymes, acid hydrolysis seems to be the way to go for converting cellulose to glucose. However, what acid should we use? Do we just pour the acid on the blended plant and hope that the result is glucose? If not, how do we successfully free the cellulose from the lignin in order to perform hydrolysis? In the cellulosic ethanol wikipedia article that you sent me, it talked about inhibitors such as furfural that could be harmful to the experiment? Also, can this experiment be done at home?

Hi,

Here are some additional comments on your previous questions and answers to your latest questions:

1. Blending the cellulose is a good way to prepare the sample because if breaks the sample down to very small pieces of cellulose.

2. If you don’t have cellulase, then you will need to use acid hydrolysis. Here is a method that uses 5% sulfuric acid.

http://www.eng.umd.edu/~nsw/ench485/lab4.htm

Sulfuric acid is sold an auto supply stores as battery acid, which is usually about 30% sulfuric acid, so you would dilute this acid with one volume acid plus 5 volumes of water. Since this is a science project, I recommend using distilled or deionized water if you can get it. You should verify the concentration of acid that you purchase. Don’t put the acid in the blender and use containers that have secured caps. You should working under the supervision of an adult who has experience in diluting acid.

Normally, samples are heated for hydrolysis, but if you are not working in a laboratory, I would recommend hydrolyzing the sample overnight at ambient temperature.

When working with the acid, you should wear safety glasses, gloves, and old clothes (acid can burn a hole through clothing), and follow all safety precautions. Work slowly and carefully and make sure no pets or small children are around.

Once the sample is hydrolyzed, you will need to neutralize the acid with base, such as sodium hydroxide. For this you can use a drain cleaner that does not contain any additives. I am assuming you have had chemistry, so you should be able to calculate how much sodium hydroxide you will need to neutralize your sample. Let me know if you have any questions about this step, or if you cannot obtain the sodium hydroxide... It would be helpful to have pH paper or a pH meter to verify the sample is neutralized. You will need to use the same safety precautions when working with NaOH.

At this point you will have glucose in a high concentration of sodium sulfate. If possible, you should measure the glucose concentration using the method suggested in the link above. Also, the sodium sulfate will inhibit the fermentation, so you will need to deionize the sample. This is usually done with a deionization or mixed bed ion exchange resin. Can you this item through your school? Let me know if this is a problem.

The furfural in the plant sample can inhibit fermentation. Here is a reference that studies the mechanism behind the inhibition.

http://www.chemeng.lth.se/exjobb/012.pdf

It is possible to separate glucose from furfural using chromatography techniques, but you would need access to specialized laboratory equipment. Furfural is an aldehyde so is volatile. Perhaps it would be possible to gently heat and evaporate the hydrolyzed glucose sample for a day or two to allow as much furfural as possible to evaporate before starting the fermentation step. Here is a paper that reviews methods for preparing cellulose that do not contain fermentation inhibitors. Please read this and see if you can get any ideas for your project:

http://www.ijabe.org/index.php/ijabe/ar ... ile/168/83

Once you ferment the sample, the maximum concentration of ethanol will be about 5-10% if you are lucky. You will need to distill the sample to concentrate the ethanol you have a way to do this? Do you have a lawnmower that runs on ethanol? What volume of ethanol will you need to run the lawnmower for a few minutes? You can back calculate and determine what quantity of cellulose you need to start with.

I recommend that you write out a detailed protocol for the procedure before you start. This will help you plan the experiment.


Donna Hardy

Once we obtain the ethanol from fermentation, is there a way to measure the percentage of ethanol obtained? Also, I don't think we will be able to obtain the mixed bed ion exchange resin.

Hi,

It sounds like you are making progress. Yes, there are a number of ways to measure alcohol. Most analytical methods require specialized equipment, usually a gas chromatography system or a high pressure liquid chromatography system. However, it is possible to use a hydrometer to measure ethanol by measuring the density of the sample during fermentation. The density of the liquid will decrease as the sugar is converted to ethanol.

http://en.wikipedia.org/wiki/Hydrometer

I recommend that you do some additional internet searches and find out what type of hydrometer would be suitable for your sample. You may know someone who has a hydrometer you can borrow. Let me know if you need more help on this.

You will not be able to obtain a significant amount of alcohol from your cellulose sample if it contains high salt, so you need to try to either find a source of enzymes or the mixed bed resin. Do you have an ethanol fermentation plant in your area that you could contact and ask for help with materials? Can you search the internet and find companies that sell cellulases or mixed bed resin and call their technical support groups and ask for a donation of materials? Is there a water treatment company in your community that you can contact for assistance with deionizing the sample? Is there an analytical laboratory in your community that has a gas chromatograph or HPLC system that can be used to analyze your samples? When you contact a company, you can explain that you are working on a science fair project and you are calling to ask for either a donation of supplies or a suggestion for solving the problem. Some companies will not be interested in helping at all, but I’m sure there will be someone who will be willing to help you. When you call, be prepared with the name and address of your school so that any donations can be mailed to your teacher’s attention. Hopefully my questions here will give you some ideas on what to do


Assuming you will be able to solve the technical problems with the fermentation, you will need to distill the sample to obtain ethanol that is pure enough to use for a fuel. Have you made any plans for this part of the project, or do you need suggestions?


Donna Hardy

We had an extension on our project deadline, so we have a bit more time to complete the project. Is there a way to isolate the glucose from the excess products once acid hydrolysis is performed? Also, how do we distill the ethanol to obtain a purer sample?

Hi,

It’s good that you have some additional time to complete your project. You don’t need to worry about removing anything except the sulfuric acid from your sample; the other components should not interfere with ethanol fermentation as much as high salt. The best way to remove an acid like this from your sample is to use a weakly basic anion exchange resin with a tertiary amino functional group. I’m sure you are not familiar with this type of product, but it is used in industrial and laboratory applications and would be available from a laboratory supply company or perhaps from a water purification company. Perhaps you could ask your teacher for help in finding this type of product. Here are some possible resins you could use from Sigma-Aldrich, a scientific supply company:

http://www.sigmaaldrich.com/catalog/Loo ... N25=0&F=PR


If you can get this resin, let me know and I’ll explain how to use it. If you can’t, then let me know and I’ll make another suggestion for your project. Please let me know how much time you have left. If you can't remove the acid or salt from your sample, then there won't be enough ethanol produced for distillation.


Donna Hardy