Yes I chose the Fraser River because I saw a few studies that found lead contamination in the river. I am planning to get the samples near Oak St.
This study shows the concentrations they used: http://file.scirp.org/pdf/ABB20120300017_83240845.pdf.
What does mM mean? They reported that there were only colonies that grew on dishes containing 1mM and 2mM. By the way should I add the metals to the chambers gradually at low concentrations so as to not disturb the bacteria too much? However if I do that, it would be hard to find the rate of reduction...
For the standard curve, I meant the lead concentrations for the test kit . Sorry I should have made it more clear. I watched a few videos on how to do the bacterial dilutions already.
Very interesting articles. Here are some comments that are relevant to your project.
1. Chatterjee et. al. This bioremediation paper characterized two strains of lead-resistant bacteria isolated from sewage. The bacteria were grown in nutrient agar with 2 mM lead acetate at 37 degrees Centigrade. No bacteria grew in greater than 2 mM lead. There is a detailed protocol for DNA isolation and PCR analysis for 16s rDNA. Both strains were Gram-positive spore forming bacteria that produced a polysaccharide and the authors measured the optimum temperature, pH, salt concentration. Atomic Absorption Spectroscopy was used to quantitate lead absorption. The authors observed lead disappearing from the culture medium. (The authors did not try anaerobic conditions).
2. For the Roane paper, I can only see the abstract, so no experimental details are available. The author characterized two lead-resistant bacteria, a Gram-negative Pseudomonas and a Gram-positive Bacillus species. The bacteria grew ins 0.3 and 0.1 mM lead. The author used electron microscopy to analyze the mechanism of lead resistance. The Psudomonas produced a large amount of the extracellular polysaccharide that is associated with lead-resistance, but the Bacillus species did not.
Quick chemistry lesson: 1 mM is 1 millimole per liter. Lead acetate is available in an anhydrous or hydrated form. The molecular weight of the anhydrous form is 325 grams per mole or 325 milligrams per millimole. . Please check the bottle of lead acetate you have available and check the formula on the label to see if it is the hydrated form. If so, the formula will include 3 H2O (water) molecules.
One possibility would be to make 100 mM lead acetate and then add 1/100th of the total volume of the total volume of the MFC to dilute to make a 1 mM, or dilute the 100 mM 1:10 in water to make 10 mM and them make a 1:100 dilution to make a 0.1 mM solution. What is the volume of the anode chamber of your MFC?
To make 100 mL (0.1 liter ) of 100 mM lead acetate:
0.1L x 100 mM/L x 325 mg/mM = 3250 mg lead acetate, or 3.25 grams.
For the lead test kit standard curve, you should probably do a 5 point standard curve the first time to make sure it is linear. If you find the absorbance values very consistent, you could reduce it to 2-3 dilutions in the future.
The bioremediation experiment does not actually require an MFC. You could grow bacteria in containers and measure the quantity of free lead in solution to determine if the bacteria are sequestering it. This would allow you to grow multiple samples; perhaps try 0, 0.1, 0.5, 1 and 2 mM lead acetate at the same time. Perhaps you could do one sample in the MFC to measure the effect if adding the lead on current and voltage.
Good news! My project proposal was approved! I can begin my project right after I gather some materials.
Thank you for the quick chemistry lesson! I will look at the lead I have access to right before I start my experiment (and do the calculations). The volume of my chamber is going to either be a litre or 450 mL (depending on what's available at Walmart). I have decided to not get the containers from carolina because it would take too long for them to ship.
I completely forgot that the bioremediation portion of my study does not need a mfc. However I will still need to do a comparision of the power outputs from both mfcs so as to know if Pb2+ is a better electron acceptor than oxygen. I will probably do them seperately.
I am still not sure if I should add lead to the anode mfc gradually. This way it will not affect the bacteria too much. However it would be hard to find the rate of degradation. Also it would not seem very controlled because I am not gradually adding lead to the cathode for the second mfc. What would you suggest?
Congratulations on getting your project proposal approved. This is excellent news.
Since your primary objective now is a bioremediation project and an investigation into lead=resistant bacteria, my first idea is that you can do multiple samples with different concentrations of lead. it can be used to monitor the growth rate of the bacteria. Set it up first and get it running with no lead added, then add a low concentration first and measure the power output. You can get multiple containers from Walmart, and just set one pair up as an MFC.
I will go back and review your project proposal again. Can you confirm that your original version was approved?
I was thinking that I can first compare the lead reduction rates from both mfcs and further investigate the mfc with the highest reduction rate. In that case I would run several experiments using only containers (if it is not the cathode reduction mfc).
Looking back on the sciencebuddies project idea: https://www.sciencebuddies.org/science- ... #procedure, it said that not sifting the soil would leave rocks that would aerate the soil. You did mention that most of the active bacteria in the mfc would be facultative anaerobes. Do you think that sifting the soil will affect them too much?
Also, should I work with test tubes or larger containers if I'm doing the bacteria bioremediation of lead?
Your plan should work well; using the MFC to monitor bacterial growth will make your project unique, and using additional containers without electrodes will give you more data. So definitely proceed with your plan to use both types of containers. The bacteria will be able to grow equally well with or without a cathode chamber.
The procedure for preparing the soil in the Science Buddies Project description is certainly detailed and would be an excellent way to make a consistent twig-free, pebble-free soil sample, but would definitely expose all of the bacteria to oxygen. This would not harm facultative anaerobes, but it would kill the obligate anaerobes. All of the papers we have read on MFC bacteria have described the growth of facultative anaerobes, but I don’t recall any that specifically investigated the role of obligate anaerobes . And the papers we have read have not included the details on sample collection. If everyone sifted their samples, they would have only facultative anaerobes remaining.
Obligate anaerobes do not use oxygen as a terminal electron acceptor, so would have to use some other cation, such as lead. I recommend minimal handling of the soil samples in order to allow the obligate anaerobes to survive. You might be able to discover something new if you include facultative as well as obligate anaerobes in your samples. Whatever you do, be sure to describe the details, and make every sample as identical as possible.
Where are you going to collect your mud samples? The Fraser River looks like it’s a big river. Is there a shallow location with a soft muddy bottom where you could safely collect your samples? Please do not collect river samples by yourself, as this is a potentially hazardous activity.
I just entered my project into experiment, which is an online funding website where many people (not nescessarily scientists) fund a project together. Currently I am waiting for approval. Here is the website: https://experiment.com/projects/cqxypipoiyaswicztosm.
I am not quite sure what the fraser river near where i live looks like. I will probably go after I collect all the materials needed for the mfc, since I don't want the bacteria to die off when im making it. I also need to start building the incubator, which will take a lot of time.
I just did a bit of research into obligerate anaerobes. Apparently there aren't obligate anaerobes in soil? I don't want to risk digging mud under the fraser river because it's humongous and has strong currents. If there really aren't obligate anaerobes in the soil, should I risk sifting it?
I'm impressed that you checked out the references. Good work!
Yes, you would probably need to collect a benthic mud sample to get obligate anaerobes. Since your safety is the most important consideration here, collect samples that are safe and easy to get. The soil/mud sample is one of your controlled parameters, so should be the same for all of your samples. Do describe the location and technique you use to collect the sample and take a photo or two if possible. However, if you have a choice between some loose, aerated soil and some mud that has been sitting undisturbed for a while, choose the mud.
Thanks for the link on the experimental protocol. I will look at the link next.
I just read your grant proposal and it's great! What is the time frame for approval?
If you do get approval for the grant, do invest in a catalyst for the cathode chamber, as this will increase the power output for the mfc.
And, yes, do wait until you have your mfc set up and tested for leaks before you collect the samples. After you collect the samples, add them to the mfc and other containers as quickly as you can get home.
Are you going to wait 45 days before you start? You have a lot of work to do, and it would be good if you could go ahead and set up the MFC and other containers and test for lead. and start enriching for lead-resistant microbes. You could save the 23s RNA testing, and perhaps the plate counts until later.
The funds are just used to pay off my "debt" to my parents, so I will begin the project before the 45 days I'm collecting some materials tomorrow, and I'll update you on what I got.
For Experiment Crowfunding, it requires at least one endorsement. I was wondering if it was possible that you can give an endorsement to my project? I don't really know who to ask .
If it is not possible for you to write one, should I ask my science teacher?
I'm going to my local pet store to look for aquarium pumps today. I'm hoping to start my experiment in roughly one week, and I'll keep you updated on any questions or discoveries.
. Sorry I should have made it more clear. I watched a few videos on how to do the bacterial dilutions already.
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