Measuring bacterial degradation

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chebean
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Joined: Mon Oct 12, 2015 10:12 am
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Measuring bacterial degradation

Post by chebean »

I am testing the effects of different glyphosate based pesticides on the bacterial degradation of glyphosate. Glyphosate breaks down into AMPA and carbon dioxide, so I am planning on using a vernier carbon dioxide probe to measure the amount of carbon dioxide.

The vernier probe only has a resolution of 1ppm. Will this resolution be small enough to detect bacterial degradation?
SciB
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Re: Measuring bacterial degradation

Post by SciB »

Hi and welcome to Scibuddies.

Did you mean that you are using a glyphosate-based herbicide as a source of glyphosate to feed to your bacteria? Which bacteria will you be using and how will you culture them? The amount of glyphosate that is degraded is going to depend on how many bacteria are present when you add it. You may have to test a range of bacterial titers to find one that gives you a readable amount of CO2. How does your probe work? Does it measure dissolved CO2 or just in the gas phase?

I can't answer your question without some information about the organism that you are using and the growth conditions. Post again with some details of your experimental method and hopefully we can figure out if your probe has enough sensitivity. I think the only way you will really know is by trying it.

Sybee
chebean
Posts: 2
Joined: Mon Oct 12, 2015 10:12 am
Occupation: Student

Re: Measuring bacterial degradation

Post by chebean »

Thanks for you response Sybee! I will try to outline what I have set for my experiment so far.

I am using the bacteria Pseudomoas putida, which is able to degrade glyphosate according to multiple research papers.
I am using glyphosate based herbicides as a source of glyphosate for my bacteria. I will use Roundup super, Gly star plus, and compare and save. All of these glyphosate based herbicides have 41% glyphosate as their only listed ingredients and their glyphosate is in the form of isopropylamine salt. I will also use analytical grade glyphosate as a control group. I figure that I will also need a control group of only the bacteria.
I will run an approximately 30 day test where data is collected on each sample at regular intervals.
Where I am stuck is figuring out culturing the bacteria and collecting data.
Culturing: The bacteria I am ordering comes in a tube. I originally thought that since I will be adding aqueous soltions of pesticides to the bacteria, I would culture it in nutrient broth. I am flexible on this point though.

Data collection: In reading previous experiments, one of the most common methods for detecting the amount of glyphosate in a given substance was liquid chromatography (I do not have access to the equiptment need for this). The methods I may be able to use are detecting carbon dioxide through a probe (the one I have access to only measures CO2 in the gas state) or through KOH traps (I don't quite understand how these work other than that they make a precipitate). I have also considered using a photo spectrometer.
The reasoning behind measuring the CO2 is when the bacteria metabolize the glyphosate they break it down into CO2 and AMPA. The amount of CO2 would be an indirect reference to the amount glyphosate left in the culture.
I also looked into doing a precipitation reaction with AMPA and measuring that with a photo spectrometer, but that reaction had a very low percent yield.
SciB
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Posts: 2066
Joined: Fri Feb 01, 2013 7:00 am
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Re: Measuring bacterial degradation

Post by SciB »

Wow! Good job developing a difficult project. I hope you post the results because you got me curious about how good P putida is at degrading Gly.

I have never cultured Pseudomonas but most bacteria will grown in nutrient broth. However, since in the real world the Gly would be in soil I wonder if there would be a way to inoculate the bacteria into soil and apply the Gly to that. You could also do liquid cultures for comparison, but I think having a tray of dirt inside a sealed container with a CO2 monitor hooked up to it registering gas coming off would be way cooler.

Do some more reading on P putida to see how other researchers have set up experiments with it. I took a quick look and found this paper in which P putida was inoculated into soil contaminated with naphthalene for bioremediation: http://femsec.oxfordjournals.org/content/54/1/21

I think you could do the same for Gly-contaminated soil.

Is there a way to calculate how much CO2 that a certain number of P putida cells make over time? You really need to know that so you have some idea if your CO2 meter is sensitive enough to measure the amount of CO2 released by the bacteria. Measuring gases is tricky because you have to capture them in a closed container somehow. As you said, KOH can be used to trap the gas and maybe that would be accurate enough and easier, but I would think the culture set-up would still have to be isolated from the atmosphere, otherwise the CO2 in the air would be continuously reacting with the KOH.

I can see you are going to have lots of questions on this project! It is very interesting but technically challenging.

Good luck!

Sybee
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