Bioremediation of carbamazepine

AFTER you've done your research and concluded your experiments, it is time to prepare for the science fair. Ask specific questions about preparing for a science fair, including how to set up your display board, how to prepare a presentation, etc. (Please post questions about selecting a project or conducting your experiment by posting in the appropriate "area of science" forum.)

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Bioremediation of carbamazepine

Postby nicolezhao » Tue Jul 29, 2014 12:58 pm

Hello everybody,

I am Nicole and I am going into grade 11. My project is on finding potential bacteria in my local watershed (I am from Toronto, Ontario) that can bioremediate carbamazepine (CBZ). CBZ is an anti-epileptic drug, and it is the most commonly found pharmaceutical pollutant in the great lakes basin. It is persistent in the environment, can trigger serious allergic reactions to some patients, and difficult to remove in some waste water treatment plants; however, there is no thorough risk assessment for this pollutant and minimal attention is focused on eliminating it.

CBZ is NOT water soluble, so I could not follow the common method of creating a minimal media with CBZ as the only carbon source. Instead, I got water samples from a local river, and grew the microorganisms on nutrient agar first, and then sprayed CBZ dissolved in an organic solvent (acetone) to onto the plate. the sprayed CBZ formed a cloudy layer over the plate, and after a few days, zones of clearing would appear around certain colonies. This means that those colonies are able to degrade CBZ by using it as the only carbon source, or are able to transform CBZ into some other substance. I isolated these colonies, and performed PCR (by amplifying the 16s rRNA gene) to identify them. I isolated around 6 colonies, and they were all identified as Pseudomonads (Pseudomonas fluorescence or fragi), except for one isolated colony that was purple in color and did not give a sequence.

This is what I have complete so far, and it is what I submitted to my 2014 regional science fair. I did not qualify for the Canada Wide Science Fair (CWSF), but I did receive a bronze medal for my project.

Here are some definite plans & timelines for my project for now:

-I am hoping to continue this project again this year, and hopefully qualify for CWSF 2015. If things go well, I may even try applying for Team Canada ISEF!

-I will not be able to do any experiments until school starts (end of August) because I do my experiments in my high school lab. The more advanced procedures such as PCR are performed in a university lab.

-I want to redo the whole experiment, starting from getting water samples and doing the spray plate isolation procedures. This is because I think the what I did was very poorly carried out and not very controlled. I have also gained experience from the first time, and would like to make some changes to improve the experiment.

-In addition, I want to grow some microorganisms and just spray acetone on them without CBZ dissolved in it. This is to act as a control and see how much acetone affects the microorganisms (e.g. does it kill everything?)

Here are some potential next steps of my project. I have some ideas, but I do not really know how to go about them, and I am really in need of some guidance! :?

-what I have done is a very preliminary isolation/identification of potential degraders. What can I do to further confirm the degradation?
-any suggestions for my project, suggested reading, etc.? Are there anything wrong with what I have done, or can anything be done in a different manner?

Any help would be greatly appreciated, please let me know if you require additional information on my project!!

Thank you,
Nicole
nicolezhao
 
Posts: 2
Joined: Tue Jul 29, 2014 11:35 am
Occupation: student: 11th grade
Project Question: Science fair questions, bioremediation, biology, environment, microbiology
Project Due Date: April 2015
Project Status: I am conducting my research

Re: Bioremediation of carbamazepine

Postby SandbaggerOne » Sat Aug 02, 2014 12:22 pm

Hi Nicole,

I agree that the acetone control is important, and yes it is likely killing a large fraction of your microorganisms, which is probably decreasing the sensitivity of your screen (you only find organisms that can survive the acetone AND degrade CBZ). What about using a less toxic organic solvent, like DMSO? Also, it is worth considering that for actual remediation the bacteria would have to metabolize CBZ from water, not an organic solvent. References I have read show CBZ contamniation in the range og 0.4 ppb pre-treatment and 1.5 ppb post-water treatment: http://www.health.state.mn.us/divs/eh/r ... zeinfo.pdf

Do you have the ability to measure CBZ at such low concentrations (HPLC, Mass-spectrometry, etc)? If so, after the initial high dose screen to find your bugs of interest, could you set up a system that more closely models what real CBZ remediation would look like?

Cheers,
Colin
SandbaggerOne
Expert
 
Posts: 89
Joined: Wed Aug 28, 2013 7:55 pm
Occupation: Immunologist
Project Question: Volunteer expert.
Project Due Date: n/a
Project Status: Not applicable

Re: Bioremediation of carbamazepine

Postby nicolezhao » Wed Aug 13, 2014 6:37 pm

Hello Colin,

Thank you very much for the reply! I cannot use DMSO, and many other less toxic solvents because they are not very volatile. I need a volatile organic solvent to dissolve CBZ so that after spraying, the organic solvent would evaporate and the insoluble CBZ would be left behind, forming a cloudy layer over the agar plates.

I am not sure how to measure CBZ---I think it might be difficult to find a lab that is able to measure CBZ, since it is not especially popular.

I am going back to school in less than two weeks, and then I will be able to re-do the first stage of my experiment. That is what I will do first and I'll see how it goes. I will post updates, and hopefully then I can have some help to decide what to do next!

Thank you again!

Nicole
nicolezhao
 
Posts: 2
Joined: Tue Jul 29, 2014 11:35 am
Occupation: student: 11th grade
Project Question: Science fair questions, bioremediation, biology, environment, microbiology
Project Due Date: April 2015
Project Status: I am conducting my research

Re: Bioremediation of carbamazepine

Postby SandbaggerOne » Wed Aug 13, 2014 7:03 pm

Hi Nicole,

I didn't appreciate the importance of the solvent volatility, but I understand now. It looks like you have a plan and know what you are doing. Please keep me posted and let me know if I can help in any way.

Cheers,
Colin
SandbaggerOne
Expert
 
Posts: 89
Joined: Wed Aug 28, 2013 7:55 pm
Occupation: Immunologist
Project Question: Volunteer expert.
Project Due Date: n/a
Project Status: Not applicable


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