Science Buddies: "Ask an Expert"

I am going to be doing a long-term lab about cellulose degradation into glucose through enzymatic hydrolysis. The general context for this lab is the production of bio-ethanol. I will only be investigating conversion of cellulose into glucose as this is the only part of bio-ethanol production that I am really interested in for this lab and going all the way to ethanol just introduces more places for error. One of the only real holes, and it is a major one, that I can foresee in the lab will be the ability to accurately measure glucose. I will be controlling many different variables such as the sources of the biomass to the enzymes and the different environments that are necessary for each different enzyme, so i will be taking many different test samples. I will need to fairly quickly and accurately measure each test sample. I know that there are many procedures for measuring glucose levels in a solution, but many are very time consuming (titrations for each sample etc.) I know that there are many different glucose meters out on the market for people with diabetes that are fairly cheap and I was thinking that these might be the best option. There appears to be something like what I am looking for already on this website:

https://www.sciencebuddies.org/science- ... ml#summary

It looks basically like it discusses what I intend to do at least in regard to measuring glucose. One of the questions that I had after reading the project's explanation is the reasoning of why it is recommended to "NOT use the kind of test strip designed to test glucose in blood." I was under the impression that the urine samples were less accurate than the blood samples as most of the blood samples are digital while the urine samples are mostly still done by matching the test strip to a color chart. If someone could please help explain this to me and/or give me a better alternative I would greatly appreciate it.

Thank you in advance!

Hi dshaw,

Welcome to Science Buddies. This is a really great project idea and you have an excellent question. Measuring glucose will be very important for monitoring the fermentation of the samples. I don't have an easy solution for you, but here are some ideas that I hope will be helpful.

In the Science Buddies project idea, dilutions of known concentrations of glucose are made and the glucose test strips are used to detect the glucose. You establish a calibration curve and you could determine the sensitivity and accuracy of the method.

I'm not sure why the glucose monitoring used for blood samples are not recommended for your type of sample. I know that they are calibrated for use with whole blood samples and using a different sample matrix would affect the test results. I don't know if they can be recalibrated for a different type of sample. I will inquire further about this question.

How will you be hydrolyzing the cellulose samples? Here is a detailed protocol from a college level experiment that uses a colorimetric method for measuring glucose. Do you have access to a spectrophotometer for this type of analysis? Here's the reference for the glucose method. You would need to purchase the chemicals for this method.

Measure the glucose concentrations of the samples with the dinitrosalicylate colorimetric method. (Reference: Gail Lorenz Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Analytical Chemistry, 31, 427, 1959.)

Do you have access to a hydrometer? This is an instrument that measure the specific gravity of samples, and there are hydrometers available for different applications. In the fermentation of your sample, you would see a decrease in the specific gravity of your sample as the glucose is converted to ethanol:

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

In ethanol-producing companies, glucose analysis is typically done by HPLC (high pressure liquid chromatography) analysis. Do you have access to a laboratory that does this type of analysis, or do you think you could find a lab? Perhaps you could find someone with a suitable column and HPLC system to help you with the analysis. Here is a description of a commercial product for this application.

http://www.waters.com/webassets/cms/lib ... 1896en.pdf

How are you going to measure the ethanol?

Please post again if you have more questions.

Donna Hardy

I will be hydrolyzing the cellulose samples with some cellulase enzyme, but I have yet to decide which exact ones. I looked over doing the DNS colorimetric method, and it seems to be better than Bennets quantitative reagent and some of the other tests for reducing sugars. I do have access to the high school's spectrophotometer, although I was unsure of how long this method would take to measure each sample.

I am generally familiar with them, but have never actually used them in a class. How many different wavelengths are usually measured? If one must go wavelength by wavelength through the entire spectrum, I have a feeling that the process would take hours for every sample. Are there certain wavelength's that are indicators so that one only has to check a certain number of them?

I also have access to a hydrometer, although I will not be going all the way to the fermentation of ethanol. I will be focusing solely on the degradation of cellulose into glucose, so I won't have to worry about measuring anything other than glucose.

Thanks for your help

Hi,

Your question about why glucose meters cannot be used for your application is a good one and here is some additional information on this question:

Here is a paper that describes how different types of glucose meters work.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2141585/

The glucose meter is calibrated for a specific lot of test strips and the glucose in the sample is converted in the presence of oxygen enzymatically to gluconic acid and hydrogen peroxide and the results are measured colorimetically or by a change in conductivity. The meters measure glucose in a range of 0 to 450 mg/dL.

I don’t know why you could not use these strips for your samples, except I’m sure you would have to recalculate the results because the programming on the meters is designed for a blood matrix. It seems like you should be able to test a series of known concentrations of glucose to establish a calibration curve, and then test your samples and calculate the results. . However, part of the technology on these meters is proprietary and there may be something about the design of the system that would make it difficult to adapt to cellulose samples.

You would need to make sure that nothing in your sample interfered with the chemical reactions, so please let me know about the chemical composition and the enzymes that you are planning to use? What concentration of ions will be in the sample? Blood contains about 20 mM phosphate buffer plus 150 mM sodium chloride.

If you have a specific glucose meter available to use, I think it would be worthwhile to contact the manufacturer and ask about using it for your samples, or just try a sample of cellulose spiked with a known concentration of glucose and see what happens. Since there are several different types of glucose meters, it is possible that one or more would be suitable for your samples. Being able to use the glucose meter would make your experiment easy to do, although you would need lots of test strips, which would add to the cost of doing the project.

Here is a paper that includes a detailed protocol on how to do a glucose assay with a spectrophotometer:

You would need magnesium chloride, ATP, NADP, glucose 6-phosphate dehydrogenase, Tris buffer, hexokinase, pipettes, disposable cuvettes, and a spectrophotometer that measures at 340 nm. Please note that the authors report the range of their assay, the correlation coefficient, and the coefficient of variation. You would want to do something similar for your project.

http://www.hindawi.com/journals/biomed/2009/430426/

Sigma offers a kit with all of the reagents based on the method described above. Do not order these reagents until you confirm that your sample will not interfere with the analysis:

http://www.sigmaaldrich.com/etc/mediali ... k20bul.pdf

Here is the detailed protocol for the dinitrosolicylic colorimetric method, which is recommended for cellulose samples. You would need dinitrosalicylic acid, phenol, sodium sulfite, sodium hydroxide, potassium sodium tartrate, pipettes, disposable cuvettes, a hot water bath and a spectrophotometer that will read at 575 nm.

http://www.eng.umd.edu/~nsw/ench485/lab4a.htm

Here is an abstract from an older paper that compared different methods of glucose analysis. The authors found that that reducing methods overestimated glucose concentrations and that the enzymatic methods gave the most accurate results.

http://www.clinchem.org/content/28/12/2405

You can go to Google Scholar and do a search for glucose assays and related inquiries and you will find many more references that will provide you with good background information for your project.

One of the spectrophotometer methods would take an hour or two and you would have to set up a standard curve every time. However, you would get faster after the first time. If you were doing experiments over a period of time, you might want to freeze samples and test the glucose concentration for lots of samples at one time.

If you have access to the hydrometer, you might consider testing your samples to see if there is any difference in density when the cellulose is converted to glucose.

If you do try the glucose meter, let me know what happens.

Donna Hardy