Soil-Based Microbial Fuel Cells

[Crazy_Mad_Scientist]

Hi Donna,

I will go with the carbon cloth electrodes. However it may take some time for it to ship (around two weeks).

Sorry I couldn't access the paper . 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.

I am having trouble with finding ways to sequence bacteria... I don't really understand the "16S Rna sequencing" that this paper used: http://www.nature.com/articles/srep30455. Of course if I'm going to identify any mutations in bacteria species, I would have to have a genetic sequence to compare on Blast. Unfortunately I have not learned much genetics in school yet, and do not know the methods behind sequencing and their costs. I will put more research into genetic sequencing tomorrow.

Thanks!
CMS

[Crazy_Mad_Scientist]

Here is part two of my methods:

2. Substrates of 10 mL of a 0.05% dilution of glucose/vinegar plus 10 µl of Pb+2 will be added to the mfc every 3-5 days. If needed, ph will be adjusted to around 7: https://en.wikipedia.org/wiki/Microbial_fuel_cell.

After 2-4 weeks, bacteria from the mfc will be removed and streaked onto agar dishes in serial dilutions. (Agar dish methods: Agar will be mixed with water and will be autoclaved) Limited exposure to oxygen will be preferred. A series of agar dishes with varying levels of lead will be created (10 µl and up). Dishes will be incubated at 25 degrees celcius in an anaerobic chamber. Colonies will be counted via a computer imaging code that I will make. The top three largest colonies will be transferred to a liquid nutrient broth with a small amount of Pb+2. (The nutrient broth will be boiled to remove oxygen.) After 24 hours of incubation in an anaerobic chamber, lead concentrations will be measured using the kit. A decrease in lead will hopefully be observed. Additionally turbidity of the broths will be measured before and after the 24 hours.

4 groups will be established: R1, R2, and R3 consisting of a bacteria strain each, and R4 consisting of all 4 bacteria. Observation of lead degradation efficiency will be observed.

An attempt to find the mechanism behind lead remediation will be performed if there is extra time:

One experiment will be conudcted: To find if the lead is reduced to a less toxic form or if lead is absorbed into the bacteria:
1. Daphina Magna will be exposed to a positive control (only lead), Dapnia Magna + a pre-made solution of bacteria and lead incubated for 24 hours, Daphnia Magna + distilled water (negative control). If the solution with Daphnia + the bacteria and lead solution affects less Daphnia than the positive control, it can be concluded that the lead was reduced to a less toxic form. Observation of insoluble Pb will also be observed.

[donnahardy2]

Hi CMS,

You are making great progress.

I will see what I can do to answer the sequencing question and upload a copy of the review article by tomorrow morning.

The bottle of lead acetate is great news.

Donna

[donnahardy2]

Hi CMS,

The problem with the reference that you found is that the authors did not describe how to d0 16s rRNA sequence analysis; they probably assumed that all of their readers would know how to do this.

Here is a review article that gives more background information on this technique. This technique is used to identify microbes in a mixed culture, so should be ideal for your MFC.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC523561/

And here is a commercial website that offers products for 26S sequencing for clinical applications; the website includes details on how to extract the DNA, prepare the library, do the sequencing and interpret results.

http://www.illumina.com/areas-of-intere ... ncing.html

The link for reference for the review article that describes how a microbial fuel cell works was missing some letters. Sorry. Here is the link again, but let me know if you still can’t access it.

http://www.sciencedirect.com/science/ar ... 6815000484

Donna

[Crazy_Mad_Scientist]

Hi Donna,

Over the past few days I'll been wondering if I should run multiple mfc trials. I might end up with different bacteria for each trial since there is a lot of bacteria in soil. Should I just run one trial with the mfcs but do many trials for the tests I'm going to do?

I will finish reading the sequencing papers by tonight.

Thank you!
CMS

[donnahardy2]

Hi CMS,

It's always best to do at least two trials if you have time. However, MGC experiments take a long time; it takes several days for the bacteria to start growing and form a biofilm on the electrode, which starts the flow of electrons in an MFC and then a few more days for maximum power generation. So, you can plan to do two trails, but you may run out of time and only be able to do one. Just wait and see how it goes. The same applies to the sequencing part of the experiment.

It's great if you have similar results with two trails; if you don't then you would need to provide a reasonable explanation for the science fair judges. Since you are doing so much background reading, you should be able to compare your general results with other researchers.

Donna

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[Crazy_Mad_Scientist]

Hi Donna,

If I have a lot of extra time I will do two to three trials for the mfcs. Thanks for the clear-up!

I will attach a link to all the materials I will need for the mfc:

Containers: http://www.carolina.com/stc-secondary-r ... =container

Or

http://www.walmart.ca/en/ip/ziploc-twis ... 0016942453

Compression Fitting: https://www.lowes.ca/flexible-pvc-fitti ... 93129.html

Carbon Cloth: http://www.ebay.ca/itm/3K-200gsm-Real-P ... 1#shpCntId

I did a little reading on heavy metal contaminated soils in Vancouver, and I found that most native soil in community gardens contain hign concentrations of lead. I will find a local community garden, or my schools garden to collect some soil after building the mfc.

I read through the websites you attached last night! Next-generation sequencing seems very useful since I don't really need a whole culture. This pdf explains the whole procedure: http://www.illumina.com/content/dam/ill ... 4223-b.pdf

I'm really happy to know that 16S RRNA sequencing costs about $30 - 40 dollars and not $2000.00 like I previously thought .
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832449/.
However that paper stated that conventional identification could cost as low as $2 per sample. I will look into bacteria identification and find which methods I should use. Since I am dealing with anaerobic bacteria, it will be difficult to conduct biochemical tests without oxygen.


Thank you Donna!
CMS

[donnahardy2]

Hi CMS,

\You are making great progress.

I like the ziplock containers, but it will probably be easier to seal the containers from Carolina, since they have a screw capped top. MFCs have to be completely sealed to keep them anaerobic.

Here is a report from a group of students from Buce Logan’s lab on inexpensive materials for MGC’s. They used 1 liter soda pop bottles and plastic milk containers. The problem with these containers is that it would be difficult to get a sample for testing during the experiment.

http://www.engr.psu.edu/ce/enve/logan/b ... al-mfc.pdf

Before you buy the carbon cloth, check to make sure it has a high surface area. I can’t tell from the description on e-bay. The description of the carbon cloth used in this Bruce Logan paper is:

The anode was either ammonia-treated or plain carbon cloth (non-wet proofed, type A, E-TEK)

https://pdfs.semanticscholar.org/a2a3/e ... 7e0317.pdf

The platinum carbon cloth used by the students for the cathode electrode would enhance performance of your MFC. However, the students said that they had to buy a yard of the cloth, and you don’t need nearly that much. See if you can buy a small quantity of platinum carbon cloth as a reasonable price.

The paper on the 16S RNA sequencing is great. I can’t tell if the authors were including labor and materials, or just material cost. They probably did not the cost of the lab set up. Do you have access to a lab where sequencing is done, or will you need to get all of the materials?

Donna

[Crazy_Mad_Scientist]

Hi Donna,

Yeah that was what I was thinking . 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.

I read that paper a while ago and I found it very useful! However I don't think I would use soda bottles for that reason, and also because I don't want to drink that much soda to get the bottles.

About the carbon cloth, I will contact the dealer to find the surface area of the cloth. What do you mean by surface area exactly? Is it the surface area of the carbon particles? I tried finding platinum cloth but the cheapest one was about $9.00 per 4 cm2. That would be out of my budget since I would need at least 4 pieces that big.

Yes I think the paper did not cover the lab set up and the labour costs. I don't think I want to buy all the equipment since it would kind of be a one - time use thing... (I don't know if I need them for next year's project). Plus they're really expensive. I will try to find access to a lab at UBC.

Thanks!
CMS

[donnahardy2]

Hi CMS,

Good thinking. The Carolina containers should work very well. I’m happy that you don’t want to drink that much soda to get soda bottles.

When you call the carbon cloth dealer, ask them if it is similar to non-wet proofed, type A, E-TEK.

The platinum carbon cloth is definitely too expensive.

For the sequencing, you definitely need access to a microbiology lab with sterile pipettes and other special equipment. When you get to that point, you can decide how many samples to do. Maybe just one or two samples from the MFC’s that perform the best would be sufficient.

Donna

[donnahardy2]

Hi CMS,

Here is a paper that used manganese dioxide as an alternative catalyst to platinum in a microbial fuel cell. The procedure sounds easy enough; just mix the MnO2 with graphite and PVDF and coat the carbon cloth with a brush.

https://www.researchgate.net/profile/Zh ... b7aa0d.pdf

The catalyst increases the rate of oxygen reduction in the cathode chamber of most MFC’s. Since you will be reducing Pb+2, we don’t know if a catalyst will be essential at this point. If you don’t see any reaction with your MFC, then you may need to add a catalyst. So file this paper away for possible future reference.

Here is another reference to file away for possible future reference. MFC cells are usually powered by mixed bacterial cultures, and these authors wanted to know if was possible for one organism to degrade cellulose and generate electricity. They started with a mixed culture from a paper recycling plant, and diluted the culture to extinction 19 times until they obtained a pure culture that worked well. Since you are interested in the identity of the bacteria, you could use this technique to develop of pure culture, maybe next year. You can’t add anything to your experiments for this year.

http://pubmedcentralcanada.ca/pmcc/arti ... 600-08.pdf

Donna

[Crazy_Mad_Scientist]

Hi Donna,

Thank you so much for the paper! I'm not quite sure if my school has any polyvinylidene fluorine or meganese dioxide. This will definately be a consideration if my school has these materials!

Yeah I was thinking about running a few microbial fuel cells with the most efficient lead-reducing isolated bacteria so that I can look at the voltages they generate. However I realized that it may be way too much work .

How would I be able to determine the mechanisms behind the bacteria reduction of lead? I know that they could use them as final electron acceptors, undergo cell accumulation, enzymatic detoxification, acid production, and a few biotransformation reactions: http://pubs.sciepub.com/plant/1/3/4/.

Of course if this step is too complicated or time-consuming, I may leave it for next year's project. I have a few ideas to detect the reduction of Pb+2 to insoluble lead, bioaccumulation, and acid production by the bacteria, but I have no idea how to detect enzymatic detoxification or biotransformation reactions. Here is my summary of ideas:

1. Detection of Pb+2 to insoluble lead: use a spectrophotometer to run through a liquid culture of bacteria grown with Pb+2 before and after the addition of lead. Potential concerns: bacteria may grow and cause turbidity, thus results are not valid. The idea is that insoluble lead will sink to the bottom of the culture, thus absorbance will be reduced.

2. Bioaccumulation: Measure lead content before and after addition of lead to liquid bacteria culture. Tests for insoluble and soluble lead will be used. (I kind of hope that the bacteria I isolate can undergo this, as the heavy metals can be extracted for recycling from the cells) The idea is that the bacteria will absorb all the lead (or most).

3. Acid production: Measure pH and find acid responsible for the pH. Then measure lead concentrations.

Please note that I have done no research on the validity of those methods. I will do more research on this later.

Thank you! Your advice so far is really helpful!
CMS

[donnahardy2]

Hi CMS,

The reference you found is a general all purpose review of bioremediation, but does not have the details that we need. The two citations that supposedly have the details about lead reduction (43 and 44) do not come up in a Google search, not even the abstracts, so there may have been an error in the manuscript. Or, since the authors are from a non-English speaking country, perhaps the original papers were not written in English. You could try sending an e-mail message to the author of the review paper asking for assistance since the two references might be very useful for your project.

You can keep looking, but a literature reference describing the mechanism of lead reduction by microorganisms does not seem to exist. In general, anaerobic bacteria use metal ions as an electron acceptor in energy metabolism, similar to the way that humans use oxygen. You will need to be able to discuss anaerobic respiration for the science fair judges, but you won’t have the specific details for lead. You probably need to save this part of the project for the future.

The test kit that you are ordering will measure Pb+2. If you skip the first step in the protocol, then the concentration of soluble Pb+2 will decrease over time in your sample. Pb(0) is a solid and should not be detected by the test kit.. Please do check with the lead test kit manufacturer to verify this assumption.

And, since you will presumably be plating Pb onto the cathode electrode, the sold lead should appear on the electrode. If you measure the dry weight of the electrode before you set up the MFC, and then dry it and weigh it after the experiment, the increase in weight should reflect the accumulation of Pb (0). You would need access to an analytical balance for this step.

A microscope with camera might also be useful to show the formation of solid lead.

Measuring the pH of both the cathode and anode chambers is an excellent idea. Do you have a multimeter to measure voltage and current? And a thermometer to measure temperature?

Donna

[Crazy_Mad_Scientist]

Hi Donna,

I just sent the email asking the manufacture of the lead test kit about the lead it measures! I also asked if anything will interfere with the results and a few other questions.

I took a look at the detection limits of the kit, and I realized that my measurements would be really qualitative. Should I first make a few dilutions of lead and use the kit, and then run the finished samples through a spectrophotometer so that I can estimate the lead contents quantitatively? Alternatively I can use a multimeter to measure conductivity and not use the kit at all.

Here is the chart:


I am thinking of measuring the tolerance to lead of the bacteria samples when I first collect them so I know what concentrations of heavy metals I should put in the mfcs. I will do a serial dilution on lead-enriched agar dishes and put them in an anaerobic chamber.

What do you mean by plating Pb onto the electrode? This sounds pretty interesting. I can then observe the electrodes under the microscope.

My dad tried teaching me electronics over summer break, so I have a really fancy multimeter. My friend has a ph meter that I can borrow, and my school has a bit over 300 thermometers that I can borrow .

Thanks Donna!
CMS

[Crazy_Mad_Scientist]

Here is another Lead test kit that seems more accurate: http://www.ebay.ca/itm/281537710793?_tr ... EBIDX%3AIT. However it is more expensive, and will be more difficult to use a spectrophotometer to measure the lead content.