Hi CMS,
Collecting soil sample: I understand your concern about insects and sticks. You certainly wouldn’t want to put a live worm or bug into the MFC. Perhaps you could dig down a couple of inches and get a sample below the bug zone. You don’t know how hard the dirt will be, so this might be difficult. If you can find a sample with some organic material, there will be more anaerobic bacteria present. I guess you should leave the details of this step open for the moment and describe what you do at the time you collect your sample.
Since this is a public garden, do you think you should check with someone and get permission to collect your samples?
The protocol is good to turn in for approval, but I will continue with comments so you can think about the details of what you will do. It’s very important to have a clear idea of what you will do, so the procedure can be clarified and revised before you actually start.
Back to comments on original method proposal. b. You have a lot of work to do in this project and I think it will be important to focus on your main objective of making the MFC, so the bacterial plating step on the original soil sample could probably be omitted. Doing plate counts is a lot of work, and you may not get a lot of useful data from doing it.
Most of the papers we have read report bacterial analysis of the MFC after it has been operating for a while and the authors analyze the population of bacteria that are actively growing, not the original population in the soil sample.
We have read a few statements in the papers that indicate that the number of bacteria in the MFC is proportional to the amount of current generated. So, if you had two MFC’s running under identical conditions, except one contained lead-contaminated soil and the other contained no lead, you could use the voltage and current measurement to show the effect of the lead on the growth of the bacteria. This is what Feng et. al did with copper in their paper; when copper was added to the anode chamber, the generation of current decreased the this was interpreted as inhibition of growth. Voltage and current would give you a measurable result that you could use to show the effect of lead on bacterial growth.
Perhaps to test the lead tolerance of the microbes, you could set up a MFC with no lead or with the lead-contaminated soil, and then add some lead in increments every day to measure the effect of increasing lead on the current and voltage output.
c. This step is not clear. In the Motos copper paper, the authors set up the MFC with a standard aerobic chamber and monitored it until current was flowing, then they switched the cathode chamber from using oxygen as a final electron acceptor to using copper under anaerobic conditions. I think this is a good idea because if you do not see any evidence of lead being reduced, you need to know if the problem is with the set up of the cathode chamber or with the growth of the bacteria in the anode chamber.
Perhaps to measure the conversion of Pb+2 to Pb (0), you could start with a known concentration of lead (Pb+2)acetate in the cathode chamber and then measure the decrease in Pb+2 (as it is reduced to Pb)0) using the lead inspector kit It’s going to be hard to see lead plated onto a carbon electrode, but you should be able to measure the lead left in solution.
Notes: This is good to mention that you are going to feed the microbes during the experiment and use phosphate buffer to maintain the pH. Your final protocol should include the quantity of acetic acid and phosphate buffer added, and you should include pH measurements with your results.
I don’t think you mentioned temperature monitoring. You should measure the temperature of the MFC’s as you go along. If you see some variation in the output of the MFC’s from one experiment to the next, you might be able explain the results if you have recorded a difference in the temperature.
II) a. Your experiment is about lead, not about the amount of acetate added to the chamber. In a science experiment, you can only vary one parameter at a time. Many authors have used several concentrations of acetate to optimize this parameter, so you should be able to find an optimum concentration in the literature that you will add to your MFC’s. Your control should be an MFC without any lead. Unfortunately, you will have to use a soil sample from a non-contaminated source to do this, so it won’t be a perfect control.
The reduction of Pb+2 to Pb(0) measurements. Again, I think that starting with a known concentration of lead acetate in the cathode chamber, and then measuring the decrease in the lead in solution in the cathode chamber will be the best way to do this. If you weigh the cathode electrode before you start, and then dry it and weigh it after the experiment, you might be able to measure the addition of lead to the carbon paper. However, this will be challenging to do, and you may not be able to measure results if your balance is not sensitive enough. The microscopic observation is a qualitative test, but you should definitely include this as it is key for your experiment.
II) b. The culturing and sRNA sequencing analysis would certainly be a good addition to the experiments after a month. By this time, the MFC’s will contain the populations of bacteria that are exoelectrogenic.
Further research: Quick chemistry lesson. Pb (lead) is an element and cannot be degraded and its intrinsic elemental properties cannot be changed. Otherwise, you could work on a project to remove 3 protons and turn it into gold (Au). Pb has 82 protons and 82 neutrons, and can have either 80 or 82 electrons. Pb+2, with 80 electrons, can be reduced to Pb(0) (82 electrons) and Pb(0) can be oxidized to Pb+2 . (There is also a Pb+4 with 78 electrons, but it is rare).
The acetate that you will be adding as a carbon (energy) source will be metabolized, oxidized, or degraded in the MFC to hydrogen and carbon dioxide to produce energy for the bacteria to grow. This process is called anaerobic respiration and the electrons produced by the oxidization process are the basis of the MFC operation. Here is a paper that describes the mechanism of acetate metabolism.
aem.asm.org/content/72/7/5138.full.pdf
https://en.wikipedia.org/wiki/Anaerobic_respiration
Further research: If you can demonstrate the reduction of Pb +2 to Pb0, perhaps you could devise a way to scale up the application to treat lead contaminated sites. The biochemical pathway research would also be interesting, but a plan to solve the world’s lead contamination problems would be very significant.
This is a complicated project with three distinct experiments (bacterial lead tolerance, using MFC to reduce Pb+2, and 16S RNA analysis of lead tolerant bacteria.) I recommend that you sketch a display board and plan what you are going to present, including the graphs. This exercise will help you focus on what you need to do and help eliminate distractions.
When you prepare your display board in the future, it will be helpful to have photos or drawing of each step of the experiments. to help the reader understand quickly what you did.
I would also be helpful, to rewrite the experiments as separate experiments so you can follow each one step-by-step. Try to imagine what you will be doing at each step. This will help you anticipate possible problems in advance and ensure that you have all materials needed for the experiment.
I hope you meet all of your deadlines. Good luck!
Donna
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. It seems like there is a lot of useful information in it though. I will try to find more papers on those subjects later. By the way I asked my science teacher if had Lead Acetate in the lab and we do! My general school proposal is due on Monday, but we only have to write about the variables and our expected results.
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. Plus the Carolina containers come in packs of 16 so I can conduct many trials with many samples. They also come in small volumes so I can fit many mfcs in an incubator.
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