Dissolving Nitrogen in A.borkumensis Living Medium

[deleted-385552]

I am a Junior conducting a research project over how Nitrogen affects A. borkumensis' ability to degrade hydrocarbons. One of my many questions about this is how would I dissolve organic Nitrogen in A.borkumensis' living medium, which will be seawater with a salinity of around 10%?

Thank you for your help on the matter, and I may have additional questions regarding my research project as well.

[deleted-332001]

Hi, LanceJaDumbly! Welcome to ScienceBuddies! What source of nitrogen are you planning to use? Nitrogen by itself is a gas at room temperature, or a liquid when it's extremely cold. I'm guessing that you will need to use a different source of nitrogen for your project.

Here's an article that discusses optimal growth conditions for this bacteria: http://www.sciencedirect.com/science/ar ... 051500100X . I've also attached the pdf.

1-s2.0-S092041051500100X-main.pdf

(634.96 KiB) Downloaded 227 times

According to the article, supplementing the growth media with 2 g/L KNO3 as a nitrogen source increased bacterial growth.

Good luck on your project!
Jen

[deleted-385552]

Thank you for helping me out. I'd like to use solid nitrogen, however that I'm afraid that it would lower my water temperature too much if I dissolve it with the bacteria in the water. So, I'd have to add solid nitrogen before I add the bacteria, and I'd need to know if the nitrogen would dissolve in the water, or if it would just evaporate.

Thanks!

[deleted-332001]

Unfortunately, it would be a lot more difficult to obtain or handle solid nitrogen (since it is colder than even most lab-grade freezers) than it would be to use a chemical that contains nitrogen, like the chemical I mentioned KNO3 (potassium nitrate). KNO3 should be very suitable for your needs because it's also used as a major nitrogen source for fertilizers. As with any lab chemical, you should wear gloves when handling it.

[deleted-385552]

Alright, thanks!

How should I go about getting that dissolved in the water? Is there any particular method or would I just drop it into the water?

[donnahardy2]

Hi LanceJaDumbly,

I see that you have already received some excellent advice from Jen. Here is an answer to your latest question:
If you want to grow bacteria that can use elemental nitrogen, they will be able to use nitrogen from the air; you don’t have to add any liquid nitrogen.

I don’t believe that A. borkumensis can fix nitrogen on its own. All of the recipes for growing this organism include either nitrate or ammonium. Here is a recipe that uses ammonium chloride.

http://aem.asm.org/content/79/14/4282.full

A. borkumensis SK2 (NCIMB 11132) was cultivated at 30°C in a modified ONR7 medium which contains (per liter) NaCl (22.79 g), Na2SO4 (3.98 g), KCl (0.72 g), NaBr (83 mg), NaHCO3 (31 mg), H3BO3 (27 mg), TAPSO {3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid} (1.3 g), MgCl2 · 6H2O (11.18 g), CaCl2 · 2H2O (1.46 g), SrCl2 · 6H2O (24 mg), FeCl2 · 4H2O (40 mg), NH4Cl (5.4 g), Na2HPO4 · 7H2O (1.78 g), and Na2HPO4 (0.94 g).
If you want to compare the effect of nitrogen on the growth of this organism, I recommend that you use either an ammonium or nitrate salt.

This sounds like an excellent project. Please let us know if you have any other questions.

Donna Hardy

[pswiatko]

Hi LanceJaDumbly,

The advice you received from jschrandt and donnahardy2 is excellent.

Using solid nitrogen will be pretty much near impossible for you. If you want to use nitrogen gas, you can just apply it directly (tube) into your solution, but really the best way to approach this is to use some of the protocols the other experts already mentioned.


Peter S.

[donnahardy2]

Hi LanceJaDumbly,

Peter S has also provided some excellent advice, and I agree that it would be impractical to use solid nitrogen for an experiment.

I have had a chance to do an additional search on Alcanivorax_borkumensis and there are no reports that this microbe can fix nitrogen from the atmosphere. The atmosphere is composed of 78% nitrogen (N2) and there are only a few microbes that can use elemental nitrogen as a nitrogen source.

Adding ammonium ions (NH4+) or nitrate ions (NO3-) will stimulate growth of this microbe, so I think that you will want to use one of these nitrogen sources for your experiment. Changing the concentration of nitrogen gas will not likely have any effect on growth.

Here are some links that will provide helpful background information for your project. First is the Wikipedia article on the nitrogen cycle; this appears to be a well-written, accurate article.

https://en.wikipedia.org/wiki/Nitrogen_cycle

Next is some general information on A. borkumensis. You need to become an expert on this organism for your project.

https://microbewiki.kenyon.edu/index.ph ... orkumensis

And, finally here is an interesting research article that reports the effect of pressure on A. borkumensis. This microbe does not grow well in deep ocean waters. The information on metabolism is good background information for your project.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981847/

Donna

[deleted-385552]

Thanks you for your help, I'll be sure to do that.

[deleted-385552]

Now, what would be the best way of determining the progress made by the bacteria? I need to be able to measure the amount of oil remaining at the end of the experiment and compare it to the amount of oil I added initially. What could I use to completely separate the oil and water (I understand the oil and water would have already separated, but I need to be very precise) to record the mass of the oil remaining.

Thanks!

[donnahardy2]

Hi LanceJaDumbly,

It’s great that you are thinking about a way to measure your results.

Do you have access to a centrifuge? Oil has a lower density compared to water, so if you centrifuge a sample at about 5000 x g for 5 minutes, the oil will float on the surface. You can measure the volume of oil remaining compared to a control with no bacteria present.

If this is not possible, then do check out the references we found and see how the authors measured oil consumption.

Donna

[deleted-385552]

Alright, I was thinking of using a centrifuge, and I should be able to obtain one, thanks.

[deleted-385552]

Another issue I will encounter is UV radiation. A. borkumensis resides primarily on the surface of the ocean, and they are exposed to quite a bit of UV radiation. I would think that that could have some effect on its progress in degrading oil. This isn't exactly an outdoors experiment, as I will have to use agitators, so what could I do?

[donnahardy2]

Hi,

Another possible method for measuring oil is extraction in non-polar organic solvents like hexane. The problem with this method is that the solvents used are very toxic, so hopefully you will be able to use a centrifuge method. However, let me know if you need an alternative method.

You should plan to run a control sample of oil with no bacteria added. This will help you confirm that the microbes are consuming the oil. The control will allow you to measure he effect of UV radiation and all other environmental factors on the oil. The control sample should be exposed to the identical conditions as the test samples, however. should contain no microorganisms.

Donna

[deleted-385552]

Alright, that sounds good. It sounds like the centrifuge method should work for me so that's taken care of. I am going to have to agitate the water, and I do not want to spend a large sum of money on a scientific agitator. I would be able to put together a magnetic agitator relatively easily, but would having a metal rod or a vortex in the water create another problematic variable? I'm not sure if I'll be able to obtain a DIY "shaker" type agitator. Is there another alternetive to creating agitation in this case?

[donnahardy2]

Hi,

You have some good ideas for agitating the water. You might also consider using an aquarium pump aerator. That would provide lots of oxygen without disturbing the sample too much. Again, the agitation of the water is one of your controlled variables, so your control and samples should all be treated the same way.

Donna

[deleted-385552]

I am planning on conducting an experiment on A. Borkumensis' ability to degrade crude oil. Will I be able to make this happen by using an artificial seawater solution like Nature's Ocean Nutri-Sea Water, or would I have to do something else? If someone could help me determine whether or not using an artificial seawater solution would produce realistic conditions, it would be greatly appreciated.

Thank you.

[donnahardy2]

Hi,

It sounds like you are working on an excellent project. Alcanivorax borkumensis and grows in water with salt concentrations from 1 to 12.5%, so should grow well in instant ocean, or diluted instant ocean. This organism uses long carbon chain molecules as a carbon source, but you will need to add nitrogen and phosphorus in addition to the oil.

https://en.wikipedia.org/wiki/Alcanivorax

Please let us know if you need any additional information.

Donna Hardy

[deleted-385552]

I am planning on ordering a freeze-dried culture of A. borkumensis to use in an experiment that will test its ability to degrade crude oil. I need to know what to do with the bacteria before I can introduce it into a living medium of seawater. I have been looking at the ATCC product information page for a while now, and I am having a hard time understanding it. Nobody at my school has had experience with this process and it would be very helpful if someone could explain this to me. Thank you!

[donnahardy2]

Hi,

You will need to open the culture tube and transfer the contents to a suitable growth medium. For information on basic microbiology techniques, please refer to the microbiology section in the reference materials from this website. You will find information on growing and storing microorganisms.

https://www.sciencebuddies.org/science- ... nces.shtml

Please refer back to your original topic and look at the article that discusses optimum growth for this microbe. You will want to maintain the bacterium using optimum growth conditions.

viewtopic.php?t=16544

I will ask the moderator to merge this topic with the original. Please post additional questions on that topic so you the experts following your topic will be notified immediately.

Donna