Science Buddies: "Ask an Expert"

Hi-

I mentioned earlier that I need help with my science fair experiment. It is about the oil spills. I plan to use the A. borkumensis bacterium to see what effect do the conditions in the water have on the degradation of oil in an oil spill. My independent variable is the conditions in the water. I will have one sample of water that has high concentrations of nutrients (nitrogen and phosphorus), one with low concentrations of nutrients, and one with the right amount of concentration. I know that the bacterium is found naturally in seawater conditions and can live in salinities ranging from 1.0-12.5% and in temperatures ranging from 4-35°C. My question is: how do I obtain samples of water for each concentration of nutrients while maintaining a constant pH level and temperature? Also, how do I measure how much oil the bacterium has degraded?

Thanks!

Hi,

Since Abork grows in salt water you will need to either get some water from the ocean or make imitation sea water from chemicals. I’m not sure which I would use. Hopefully some of the other experts can give their opinions here.

The bacteria normally live in seawater so using seawater would seem to be the best choice. The problem is that you don’t know the chemical composition of your seawater. There are some average compositions that you could use and from what I have read, there is not much phosphate or nitrate present normally:

http://www.seafriends.org.nz/oceano/sea ... omposition
http://en.wikipedia.org/wiki/Seawater
http://www.britannica.com/EBchecked/top ... 1/seawater

The question I would be asking now is how much nitrate and phosphate are optimal for Abork to grow. Like most bacteria, they need a source of nitrogen, phosphorus, carbon and some minerals like magnesium, calcium, potassium, sodium, etc. Abork eats oil so that is its carbon source, but nitrate and phosphate may be the limiting factors in seawater. That’s why scientists are studying bioremediation with these nutrients: http://www.int-res.com/abstracts/meps/v362/p25-36/

In the paper cited above, on page 4 of the Materials and Methods section, the authors list a synthetic salts medium for the marine bacteria that contains the following: 18.6 mM K2HPO4•3H2O; 7.2 mM NaH2PO4•H2O; 37 mM NH4Cl
There are several other ingredients, but unless you plan to make your own seawater you won’t need those.

You can use the above concentrations of phosphate and ammonium as your optimal concentration and choose some low and high values for the other tests. In growing and testing Abork, you would keep one ingredient constant at the optimal amount while varying the other. In order to keep the pH the same, you will have to measure it using a pH meter and make adjustments using acid or base. Ask your teacher if they have a pH meter you can use. If not you will have to find a lab to work in that will let you use their equipment. Almost every lab has a pH meter.

To determine how well the Abork eat oil I would simply measure their growth rate. Since the oil is their carbon source their growth will depend on how well they can use it. There are ways to measure the metabolic breakdown of oil by the bacteria but they require some pretty sophisticated chemistry and instruments. I would just do counts of the bacteria after a period of time in culture with the oil. This is pretty easy to do and we can give you the details of the method when you get to that point.

I don’t want to overwhelm you with too much info, so I will stop here. I’m sure you will have more questions and we’ll be here to help when you do.

Good luck,

Sybee

Hi-

Exactly what is the usual concentration of nitrogen and phosphorus in seawater? I didn't see it on the links you recommended. And what are the details for measuring the bacteria's growth rate?

If I bought, Petco real ocean water that is already filtered, sanitized, and pH balanced, how would I add a higher concentration of nitrogen and phosphorus? how would I lower the concentration of nitrogen and phosphate? And am I lowering the nitrate level or the ammonia level?

*Note: The salinity level is 1.024 and the pH level is exactly 8.1
Is that okay?

Hi,

The concentration of nitrogen (N) and phosphorus (P) in sea water is normally so low that it is not even included in the list of common ions: http://www.marinebio.net/marinescience/ ... sition.htm

The Petco RealOcean product is natural Pacific Ocean water that has been filtered and pH balanced to 8.1. The amounts of nitrogen and phosphorus in this water are probably so small that you can ignore them. All you have to do is add nitrogen in the form of ammonium chloride or phosphorus in the form of sodium phosphate to make your growing medium for Alcanivorax borkumensis (Abork).

Do you have pH paper or a hand-held pH meter? It would be useful to check the pH of the sea water before and after adding the N or P to be sure it hasn’t changed more than a few tenths of a unit.

Measuring bacterial growth—the increase in numbers of bacteria with time—is usually done by physically counting them under a microscope. There is another way to count bacteria and that is to make serial dilutions of the bacterial culture, plate them on agar in a petri dish and count the number of colonies forming on the surface of the agar. Counting them directly is simpler but it does require a special counting chamber slide called a hemocytometer and of course a microscope.

There is one other way to measure growth, at least of some bacteria, and that is by the turbidity or cloudiness of the culture as compared to the clear medium with no cells. The turbidity is usually measured by an electronic instrument but in the old days it was done by eye. This is not a quantitative measurement like counting cells but you can say that one bacterial culture has grown more than another if it is more turbid. What I don’t know is whether Abork grows fast enough to produce noticeable turbidity.

Let us know what counting method you are able to use and we will help you with the details.

Good luck!

Sybee

How do I expose A. borkumensis after growing it in a agar plate?
How much seawater should I use? How much oil should I use?
Should I mix the water and oil in a petri dish and then expose the bacterium?

*Note: I plan to use Saudi Arabian crude oil online.

Please tell me where I can purchase samples of crude oil for my project? Also, can you give me the details of measuring the bacterium growth with the first method you listed (serial dilutions)?

To answer all of your questions—

Abork grows normally in seawater so you need to transfer some of your agar bacterial culture into seawater. You should use a sterile wire or disposable inoculating loop (http://www.carolina.com/catalog/search- ... SearchForm) to remove a good bit of the bacteria from the plate and put it into about 5 mL of seawater in a test tube. Shake the liquid carefully but thoroughly to suspend all the bacteria.

Do you have a lab that you can work in to do this? It would be much easier if you had all the apparatus to do bacterial work and someone to show you how. Here’s a video showing how to inoculate bacteria from an agar plate to a liquid culture. The technician is using a metal wire loop and flaming it with a Bunsen burner to sterilize it. You can use the disposable plastic loops without flaming because they are already sterile. http://www.youtube.com/watch?v=93I4cmk3Z1o

After you have the bacteria suspended in the tube you will use that to inoculate each of your cultures. Let’s say you want to do 8 cultures. You would put 8.5 ml of seawater into each of 8 sterile culture tubes (http://www.carolina.com/biotechnology-l ... ture+tubes) and then add 0.5 ml of the bacterial suspension (shake gently again just before pipetting). You should have about 1 ml of bacterial suspension left over. Save it.

Now you can add the NH4Cl or Na2HPO4 to the cultures that get it. I gave you the final concentrations in a previous post. Weigh out enough of the chemical to make a 20-fold concentrated stock solution in 5 ml of seawater. Here again, weighing accurately and making solutions of specific molarity is best done in a lab with someone there to show you how to do it. It isn’t difficult but it takes a lot of explaining that is best done in person. Add 0.5 ml of the specific solution to the tubes that get it. For the control, just add 1.0 ml of seawater. For those tubes that only got 0.5 ml of N or P you need to add 0.5 ml of seawater to make the total volume equal to 10 ml. Do two tubes for each test for a total of eight.

Now you eight 10 ml cultures of Abork two of which have N, two with P, two with both and two with neither. How much oil do you add? This is where you are going to have to do some reading on your own because I have no idea. Remember that you only have 10 ml cultures so you probably only want to add maybe 0.1 ml of oil or less. I have no idea where you can buy crude oil. Search online and see what you come up with. Don’t hesitate to make some phone calls to companies or labs to ask for a sample. Many companies will help a young science student with a project.

I would need to be there to teach you how to do serial dilutions and since I can’t do that I will suggest that you watch videos of the procedure until you understand how it works. Then if you have a specific question, I can help you with that. Here’s a video from BioRad Labs on making serial dilutions and doing plate counts but there are many others on youtube: http://www.youtube.com/watch?v=pmRUBYlPMBM

Good luck!

Sybee

Thank-you so much!!!

Hi there,

The previous experts already gave you some excellent advice. I agree that the simplest way to determine how well the bacteria eat the oil (without using sophisticated chemistry and equipment) is to measure their growth rate. However, I would suggest to also make another set of the same samples of seawater + nutrients but without adding oil to them, and measure the bacterial growth in those. You want to determine whether the bacteria grows without oil present in the solutions. If the bacteria don't, then that suggests that the growth you see in the +oil samples will likely be due to them eating the oil. However, if the bacteria are able to grow in the solutions without oil, you want to compare and see whether the presence of oil allows them to grow more. If yes, that suggests that the "extra" bacterial growth is due to them using the oil. However, if growth in the solutions plus or minus oil are the same, then it's harder to say whether the bacteria are actually eating the oil at all to grow. It's important to do these controls to make sure that you can actually use growth as a proxy for bacteria eating oil. Experts, correct me if I'm wrong.

Let us know if you have any more questions!

Connie

Thanks!