Science Buddies: "Ask an Expert"

Hi i'm Deo,
I have a research study that i really need help in. The aim of my study is to investigate the hydrogen production rates ofEscherichia coli isolated from different sources: confectionery wastes, fecal wastes and sewage wastewater. In my methods, i plan on measuring the hydrogen gas volume produced by using th Hydrogen Low Partial Pressure Assay described in "Metabolic engineering to enhance bacterial hydrogen production"http://www.aiche.org/uploadedFiles/SBE/ ... terest.pdf by Maeda and others(2008). The problem is, i do not undestand how the whole set-up works and how it is prepared. That is why i have some questions concerning the set-up, Hydrogen Low Partial Pressure Assay
(1) how will the set-up measure hydrogen gas volume? how exactly, like what processes are involved in the isolation of hydrogen gas from the biogas produced by the bacteria?
(2) how will i prepare the set-up?
(3) why is the graduated cylinder inverted?
(4) How will i inoculate the containers with e coli?
(5) How will i quantify the hydrogen production of e.coli?
(6) what will the hydrogen gas look like in the inverted graduated cylinder?
(7) how will i prepare the complex-glucose medium?
(8) how will i prepare 1M of NaOh in the container? is it a solution? will i mix NaOH with water?

There are also some problems in my methods. I can't seem to get a decent source or article or any manual that could explain to me how to isolate e.coli.. I have another set of questions that also need some enlightenment.
(1) how will i isolate Escherichia coli from different sources: confectionery wastes, fecal wastes and sewage wastewater in LB agar plates.
(2) do you know any confirmation tests that will help assure me that the microbe that i've isolated is e. coli?
(3) Can i ask a link to a site or pdf file that may have a manual or article or instruction guide on isolating e.coli in LB agar plates?

Please i really need the answers.. if you know an answer to any of the questions , even how little as it may seem, please do tell me. even a bit of information or advice could help me in my time of need.. i ask for your help

Hi Deo,

This is a really great topic for a science project, and I will try to answer your questions so you can get your project on track. Thank-you so much for including your reference as it did help me understand what you are asking about. Here are answers to your questions:

(1) how will the set-up measure hydrogen gas volume? how exactly, like what processes are involved in the isolation of hydrogen gas from the biogas produced by the bacteria?

1. The set-up shown on page 5 of the reference shows a graduated cylinder that is initially filled with water and inverted in a beaker of water. Tubing from the culture vessel on the stir plate ends in the graduated cylinder, so any gas produced from the culture will be released in the cylinder and the water will gradually be replaced by hydrogen gas, and you can measure the volume of gas produced.

(2) how will i prepare the set-up?

2. You will need to obtain containers that are as close to the ones in the picture as possible, but you could adapt the set-up to other types of containers. The culture vials will need to have a cork with glass or plastic tubing so that any gas produced cannot escape. Ask your teacher for help if you need help in locating these containers.

(3) why is the graduated cylinder inverted?

3. Look at the picture on page 5 and follow the tubing from the culture vial to the glass cylinder. The only possible path for hydrogen gas to escape is through the tubing, which ends in the glass cylinder. The gas will rise in the water and displace it, and since the gas will be in a graduated cylinder, you can measure the volume. This set-up allows capture of all of the hydrogen gas.

(4) How will i inoculate the containers with e coli?

Go to the science fair project guide of the science buddies website and read all of the information about growing and inoculating microorganisms; and also ready the important safety information about working with microorganisms. You will be transferring a small amount of culture from a plate or culture broth into the medium used to grow the hydrogen producing E. coli.

(5) How will i quantify the hydrogen production of e.coli?

This will be the volume of gas in the graduated cylinder.

(6) what will the hydrogen gas look like in the inverted graduated cylinder?

Hydrogen gas is clear; it looks like air. You can verify that the gas is probably hydrogen by inverting the cylinder and quickly lighting it with a lighter or match with a long handle while wearing safety glasses and non-flammable clothing in an outdoor location. Hydrogen gas is flammable and you see a flash of flames as the hydrogen burns.

(7) how will i prepare the complex-glucose medium?

I may have missed it, but I don’t see an exact description of the medium used to culture the E. coli in the reference. You could probably use a general all purpose nutrient broth with 1% glucose added from a supplier that sells microbiological media. Ask your teacher for help if you don’t know where to obtain this.

Or, since this is a recent publication, you can probably contact the authors of the paper directly and ask. The reference says that you should correspond with the primary author, Thomas Wood. His e-mail address is [email protected] , phone number 1-979-862-1588. You will find that authors of papers are generally very helpful and the knowledgeable source of information about the paper.

You should also ask Dr. Wood if he will send your teacher a sample of one of the recombinant strains of E.coli that was used in the paper. Normally, E coli don’t produce much hydrogen gas, so you would need to use one of the especially genetically-engineered strains to obtain similar results.

(8) how will i prepare 1M of NaOh in the container? is it a solution? will i mix NaOH with water?

1 M NaOH is a solution containing 1 mole of sodium hydroxide dissolved in a liter of water. It is usually made by dissolved sodium hydroxide pellets in distilled water, but it can be purchased as a solution from a scientific supply company. To make a solution, you would add up the molecular weight of sodium, oxygen, and hydrogen to obtain the grams per mole. Then dissolve this number of grams in one liter of water, or one-tenth the number of grams per mole in 100 ml of water. I am assuming you have had some chemistry, so how many grams of NaOH would you use?

The purpose of the NaOH in the experiment is to remove carbon dioxide from the hydrogen gas produced by the E. coli. If you didn’t include this step, then the gas collected would be a combination of carbon dioxide and hydrogen gas. You will need to use fresh NaOH for each experiment.

Do you need any other information to help you get started on this challenging project?

Donna Hardy

Hi Deo,

I just posted my reply and noticed that I missed your last 3 questions. Here are some more answers to your questions about growing and identifying E. coli.

Here is a website that describes how to isolate and identify E. coli. http://www.foodsafety.gov/~ebam/bam-4.html
As you read through this, I'm sure you will realize that this part of your project would be a complete project on its own. It would be much better if you could obtain a genetically engineered strain of E. coli that produces hydrogen gas. Garden variety E. coli found in sewage doesn't normally produce significant amounts of hydrogen gas. The identification of E. coli is partially based on its ability to ferment a sugar called lactose, and it will produce carbon dioxide from this process, which is a non-flammable gas.

Do you have access to a microbiology lab in a local college or company? It would help a lot if you could find a local mentor who could show you how to do the basic microbiological techniques, and it would be safer to do this project in a regular laboratory.

Donna Hardy

Miss Donna Hardy,

I really appreciate your answer... it really helped me understand better the set-up that i plan to have..
I would like to thank you for taking some of your time to enlighten with such stuff... in the future, i may have some more questions and i hope that you would be there to be at my aid.. Thank you and more power to you!!!

-Deo

Hi Deo,

You are more than welcome! I would be more than happy to answer any other questions you might have.

I do have one additional suggestion. This is the type of project that can be done in stages. If you are not a senior in high school, you could do the project this year and learn the techniques for growing bacteria and optimize your set-up for collecting hydrogen gas. I'm sure this would be a very good project. Then, you could do the project again, developing the experiments based on your initial results, for next year's science fair. I have seen projects evolve like this in my local science fairs, and the projects that are done the second time are generally excellent.

Good luck!

Donna Hardy