Science Buddies: "Ask an Expert"

Hi Donna,

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...

Another study also had similar results: https://www.ncbi.nlm.nih.gov/pubmed/10227879.

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.

Thank you!
CMS

Hi CMS,

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.

Donna

Hi Donna!

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?

Thank you,
CMS

Hi CMS,

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?

Donna

Hi Donna,

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?

Yes the original proposal was approved.

Thank you!
CMS

Hi CMS,

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.

Donna

Hi Donna,

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.

https://experiment.com/

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.

Thank you!
CMS

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?

Thank you!
CMS

Hi CMS,

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.

Donna

Hi CMS,

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.

Good luck!

Donna

Hi Donna,

I will keep that in mind when I collect my soil samples.

I set a time range of 45 days to raise funds. Approval takes about 24 hours? I just got it reviewed and I would need to make some minor changes.

Thank you!
CMS

H CMS,

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.

I want to make sure you have time to get results.

Donna

Hi Donna,

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.

Thank you!
CMS

Hi CMS,

That's great news. I'm glad you don't have to wait too long before you start. Do let me know what materials you can obtain.

Donna

Hi Donna,

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 .

Here is their article on asking for endorsements:

https://experiment.com/guide/extra#requ ... dorsements

And this explains how to write an endorsements: https://experiment.com/guide/extra#writing_endorsements

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.

Thank you so much!
CMS

Hi CMS,

I would be happy to write an endorsement for your project, and I will do it on Sunday. Thank-you for asking.

Since Science Buddies is an on-line forum and I don't know you personally, it would not hurt to ask your science teacher for a personal reference, if you think this would help. But I can certainly write an endorsement to support both you and your project.

Donna

Hi Donna,

That's great! Thank you so much!

I was thinking that I wanted to begin testing the bacterial bioremediation portion of my experiment first, since it only requires some containers, the lead test kit, and an incubator. Just wondering: if I incubated the soil samples at 25 degrees in an anaerobic environment before adding it to the anode chamber, will it speed up the formation of a biofilm?

Thank you!
CMS

I'm also planning to buy this for my incubator: http://www.ebay.ca/itm/182071212734?_tr ... EBIDX%3AIT

It's a temperature controller that I can put in an insulating box.

At first, I wanted to build a whole incubator: http://www.instructables.com/id/Digital ... /?ALLSTEPS
But then I had no idea how/where to start...

Thank you!
CMS

I just thought of a problem with bacterial culturing. When I'm transferring bacteria to the dishes, wouldn't the obligate anaerobes be killed? I don't know how long they can survive without oxygen...

Thanks!
CMS

Hi CMS,

Good thinking. You are correct; the culture conditions you will be using are suitable for facultative anaerobes and microaerophilic bacteria, but would not a;;ow obligate anaerobes to grow. Culturing obligate anaerobes requires using an anaerobic chamber for handling the the microbes, and is definitely outside of the scope of your current project.

The 23S RNA analysis would detect obligate anaerobes. You are interested in anaerobes for this project because they do not use oxygen, so might be able to use Pb+2 as an electron acceptor.

Donna

Hi CMS,

I have the endorsement written, but I can't find a place to post it on your project proposal. Did I miss something, or should I post it here for you to copy?

Your plans for an incubator should work. If you have a thermostat and some insulation, and perhaps add a small light bulb, you should be able to maintain a temperature that would support the growth of bacteria.

Donna

Hi Donna,

I checked the website. Apparently I would need to send an "invite" to endorse the project through email. Should I send an email to sciencebuddies?

For insulation, I will use cardboard because I don't have styrofoam.

For lead remediation rates, how long should I check lead concentrations? Should I check every 24 hours?

Thank you!
CMS

Hi CMS,

Yes, send the invitation to Science Buddies and reference this topic. Hopefully, I'll be able to forward the endorsement so it can be posted properly.

Cardboard is a good insulator, so I'm sure that will be fine.

I don't remember the incubation times from all of the references we checked. We should go back and check them again to see if the incubation times were included in the methods sections. It seems to me that 24 hours is a little too short; maybe 2 or 3 times a week would be good.

Donna

Hi Donna!

I will send the email as soon as my proposal gets approved! The past few days I have just been gathering some more background infomation on my topic .

I'm confused about the "electrolyte" used in the paper below. Do I have to dissolve my lead in the acid electrolytes? I'm not too sure that I want to use acid though... Also, what does the hydrolysis mean in this case?

http://www2.bren.ucsb.edu/~dturney/port ... ing/08.pdf

The paper said that lead precipitation rates increase with high ph, especially a ph of 1. Should I consider adding some acid to alter the pH of the solution?

Thank you!
CMS

Hi CMS,

Thanks for the article; this is a good one.

In this paper the electrolyte refers to the acid or base added to the solution used for electroplating Pb out of solution. Electrolytes are atoms or molecules that have a charge when in solution. For example, the perchloric acid is a strong acid with a formula of HCL04. In solution, the molecule dissociates into H+ and CLO4-. The acid or bases described in the article are the electrolytes used for electroplating.

Yes, electrolytes are added to the plating solution containing the lead. The tables in the paper list different combinations of electrolytes that have been used for lead plating. The electrolytes used for lead increase the solubility of the lead, and since electricity is used for electroplating, they increase the conductivity of the solution.

When I suggested trying the electroplate Pb in your MFC, I did not realize that strong acids or bases would be required. Unfortunately, all of the electrolytes listed in the paper are too hazardous for you to use at home. You would need access to a lab or an electroplating facility to use any of these chemicals, so I recommend delaying this part of the experiment until you can devise a safe way to do the experiment. You will still have everything else.

I don’t know if lead can be plated from acetic acid, but this would be much safer to work with.
You should not add acid or base to the anode chamber, as this would inhibit microbial growth. What were you planning to put in the anode chamber besides the mud sample?

Lead ions (Pb+2_ precipitates with many anions, including phosphate, chloride, and sulfate. Lead acetate is fairly soluble.

The hydrolysis described in the paper, for example under the amidosulfonate paragraph, refer to the degradation of the electrolyte and this interferes with the electroplating process. . In hydrolysis, a molecule is split in half by added a water molecule. Amidosulfonic acid is hydrolyzed to ammonia and sulfate.

Donna

Hi Donna,

Is it absolutely necessary to use acids in the cathode? My project is trying to be sustainable, so I don't really want to be using these acids...

http://www.explainthatstuff.com/electroplating.html: This paper only mentionned that acids are used to dissolve certain metals.

http://pubs.acs.org/doi/abs/10.1021/j15 ... e=jpchax.2: This paper said that acetic acid can be used. I want to add a small amount of the acid to my cathode.

One more thing: All the papers I have read so far stated that stirring the cathode would speed up the process? Should I try doing this?

Thank you!
CMS

Hi CMS,

These are great references. The first one gives a general description and explanation for electroplating. The second article (did you notice it was published in 1906?) contains the information you needed. It confirms that lead can be electroplated from a solution of lead acetate and acetic acid. So this is good news for your project, I think you could proceed with this idea for your project.

All of the electroplating articles use chemicals that ensure the metal ions are soluble and that are highly conductive to electricity. .

Donna

Hi Donna,

I'm so happy that I can use acetic acid now! I'm pretty sure that if I used the acids mentionned in the paper, I would burn a hole in my hand.

Currently, I'm having trouble finding how much acetic acid to add to the cathode. I can't seem to find any papers on it! Should I just drip the acid into the chamber until it reaches a pH of around 4? I know I can't get above 1 (the optimum pH) because I don't have access to strong acids except for HCL, and vinegar has a pH of around 2.4.

Just wondering, for the lead tolerance tests in bacteria, should I use a) varying concentrations of Pb2+ in agar dishes, b) mfcs set up with varying concentrations of lead, or c) several containers with varying concentrations in which I will then conduct OD readings or colony counting? I think we initially agreed on mfcs with varying concentrations, but I realized that setting up that many mfcs would be a lot of work and supply-consuming. Varying concentrations of lead in petri dishes will be tedious without an analytical balance or micropipette. Unless if I use large concentrations as mentionned in this paper: https://www.ncbi.nlm.nih.gov/pubmed/24416938, I would use this method. The paper was actually quite interesting as I didn't know that bacteria could be that tolerant to lead.

I personally like method b) and c) more. b) would lead to quick and accurate results, while c) would give accurate results, although requiring much longer wait-time.

As for the funding, I am still waiting for Experiment to approve my proposal. I got some feedback the last time I submitted the proposal, so I made a lot of changes. Here's the link again: https://experiment.com/projects/cqxypipoiyaswicztosm

Thank you so much for taking the time to write an endoresement! I will send the email to sciencebuddies as soon as my project gets approved.

Thank you!
CMS

Hi Donna,

Winter break is approaching, so I will have a lot of time to work on my project. I have been thinking, what if I transfer some of the lead acetate from the cathode after plating to the anode, and let the bacteria "absorb" the lead? This would elimate all of the lead used in the project. I realized that this would be much like an innovation project than an experiment.

Is this a good idea?

Thank you!
CMS

Hi CMS,

Sorry for the delay in responding.

Yes, it’s good news that you can use acetic acid. I have not been able to find any information on the concentration that would be suitable either. Vinegar is about 5% acetic acid. Why don’t you plan on using that and ask your teacher is there if any glacial acetic acid available, just in case it’s needed? This is pure acetic acid, about 17 moles/liter.

If you use Pb in your agar, you will be testing for the lead tolerance of the bacteria that are growing in the MFC. Since I think you can only set up one MFC, I think you should start it with no Pb at first in the MFC, and then if growth and current production is good, add a low concentration (0.5) and measure the effect of adding lead on current production. If current production continues in the MFC, then you could increase it to 1 ppm. If you can set up two MFC's, then do the experiment in duplicate to help verify your results. Most science projects are done with just one MFC.

To start your MFC, use aerobic conditions as a positive control until you know the MFC is working, then switch to lead acetate, acetic acid, and anaerobic conditions all at once in the cathode chamber. It would be good if you could run a trial experiment just on the electroplating on the side, but you may have to try the experiment with your MFC.

So, I agree with you’re a approach. Adding Pb to the agar will measure the lead resistance of the bacteria you have collected from a site that is known to be contaminated with lead. You will actually have a complete project with this experiment. If you add lead to the anode chamber, the mfc will not work if the bacteria are inhibited. So definitely, dothe experiment one small step at a time.

Thanks for the link to your new experimental protocol.

Donna