Finding a Bacteria for my Microbial Fuel Cell

[brindhaaa]

My plan is to add the compost into the anode, which has the mud, and mix it around. This is so that the anaerobic bacteria contained in the mud can use the nitrate from the compost and, from my understanding, generate more amino acids to grow. The more amino acids there are, the more proteins will be created, and the more proteins are present, the more bacteria are present. An increase in bacteria also means more anaerobic respiration and electrons given off. Therefore, more power will be generated.

This is simply my understanding, but I wanted to confirm before I began my testing.

Thank you!
Brindha

[koneill18]

Hi Brindha,

It's really great that you're taking the time to understand the rationale behind your project before you get started! I think you've pretty much got it. Nitrogen is one of the basic building blocks that make up microbial cells. It's a crucial component of the amino acids, proteins, and nucleic acids that are necessary for normal cell growth and function. Supplying the bacteria with nitrogen will increase the growth of the bacterial population, which will increase overall energy output. Additionally, nitrate is an electron acceptor in the electron transport chain that's used by bacteria during anaerobic respiration. The cells use anaerobic respiration to produce ATP, which the cell uses as a source of energy. So by exposing the bacteria to nitrate, you're enhancing their ability to carry out anaerobic respiration.

I hope this answers your questions. It sounds like you're well prepared to start your experiment!

[brindhaaa]

Thank you!

Yes, I wanted to fully understand the background behind my experiment before beginning, and thanks to your and Dr. Payne's help, I am!

One last question before I begin...

If I have the bacteria out of the water and in the containers for more than 24 hours wouldn't they die? In this case, would the fuel cell still run for 30 days?

Thank you!
Brindha

[koneill18]

Hi Brindha,

I think the reason they tell you to use the mud within 24 hours is because once you remove the mud from the stream, it gets exposed to oxygen. Obligate anaerobic bacteria live in the absence of oxygen, so exposing them to the air can be toxic for them. The anode chamber of the fuel cell is an oxygen-free environment, so it should keep the bacteria alive for an extended period of time.

I hope this helps!

[brindhaaa]

Hi,

Today I assembled my fuel cell, but unfortunately, I did not get any readings. I only assembled one fuel cell to start with in case there were any problems. Do you know what could have gone wrong?

Thank you,
Brindha

[brindhaaa]

Also, are the results expected to increase throughout the 30 days I test the fuel cell? I am a bit confused about what to look out for.

Thanks,
Brindha

[brindhaaa]

Hi,

Today I assembled my fuel cell, but unfortunately, I did not get any readings. I only assembled one fuel cell to start with in case there were any problems. Do you know what could have gone wrong?

Thank you,
Brindha

Regarding this, with the multimeter, I also tested the circuit connectivity and it was 100%, so that means there is no problem with the circuit, I suppose. So my only guess is that the bacteria may have died, but I am not sure.

Thanks again!
Brindha

[koneill18]

Hi Brindha,

Have you checked to make sure there are no air bubbles trapped in the mud? That air could prevent the bacteria from growing properly. If there are any small rocks or twigs trapped in the mud, you should remove those since they can cause air bubbles too. Also, check to make sure that the room you have the fuel cell in isn't too cold. The ideal growth temperature for the bacteria is 66–77° F. Once you set up the fuel cell somewhere, you should avoid moving it since that can disrupt bacterial growth.

[brindhaaa]

Yes, I will be sure to try all of those. The period that I used the mud in was very close to 24 hours, so I was worried that the bacteria may have died. If those tips do not work, I think I will just go back to the park and get some more mud.

Regarding the type of mud, I just collected a sample of mud from the bottom of a lake, but I am not sure if that is benthic mud. Would it still work?

Thank you so much,
Brindha

[koneill18]

Hi Brindha,

I think that should work. The term benthic means that the mud is taken from the benthic zone, which is the bottom sediment surface of a body of water. So as long as you're getting the mud from the floor of the lake, it should work fine!

[brindhaaa]

Ok, great!

Yesterday I visited a marsh/wetland and dug up some rich mud. It smelled a little but I think that is good because it means the mud has lots of bacteria. The marsh did not really have much water I could take so I bought 2 gallons of distilled water to put in my cathode chambers. The SciBuddies procedures say to miz 6 tbs of salt into 1 gallon of stream water for the cathode chamber, but since I am using distilled water should I still mix in salt?

Thank You,
Brindha

[brindhaaa]

Hello,

I built my constant MFC today and am in the process of testing it. I have connected the multimeter for a while because the reading keeps increasing! My constant MFC is currently generating 205 millivolts and still rising!!

I am planning to test all 3 of my MFCs for 3 weeks, but am curious what the readings should look like going forward. Are they supposed to increase? stay the same? decrease? I want to be ready to know what to look out for.

Thank you so much for all the help!

Brindha

[koneill18]

Hi Brindha,

I'm so happy to hear that things are going well! It's hard to predict what your readings are going to be over the course of the 30 days since your experimental system is unique. Something you can do is do an online search for "microbial fuel cell compost" and look at the research articles that come up. You can look at those researchers' data to get an idea of what your data might look like. Here's one paper that might be helpful to you. Let me know if you're not able to access it and I can attach the PDF for you.

https://www.sciencedirect.com/science/a ... 3X14005200

If you scroll down to figure 3, there's a graph showing how the voltage in the microbial fuel cells changes over the course of 120 days. Buckets 3 and 4 were the ones that had compost in them, so the readings from those buckets might be more similar to what you're going to see.

Let us know if you need more help finding information!

[brindhaaa]

Hi again!

I have now been testing my fuel cell for a little over 2 weeks, and I have 6 more days left to go! So far, my 5% nitrate addition MFC is producing the most energy out of all fuel cells. In the last reading I took, it was up to 514.3 mV with the pump! I think that's good . However, a few things happened to my others MFCs. My constant fuel cell's electrode broke. The copper wire detatched from the carbon cloth, but ever since that hapened, it has been producing way more energy! Overall, I think my project is going great!

I do, although, have a few questions I was hoping you could help me with.

1. My original though was that the more carbon cloth there is for the electrode, the more area would be available to collect electrons coming from the anode. However, since the electrode in my constant MFC broke, and the copper wire is not touching the carbon cloth, how is electricity still being produced?

2. I have read that microbial fuel cells can be used as a method of treating wastewater. Other than producing energy from the bacteria living in the wastewater/mud, how would an MFC remove contaminants from the matter?

3. I am currently working on my Data and Results sections for my research paper, and my teacher has made it very explicit to have lots of statistics in my Data section and not just graphs and tables. With my project, since I simply measured voltage readings, do you have any recomendations as to how I would present statistics anywhere else except in my graphs and tables?

Thank you so much for all the help!

Brindha

[koneill18]

Hi Brindha,

I’m glad to hear your project is going well! I attempted to answer your questions to the best of my ability:

1. I don’t really know enough about physics to answer this question properly. Dr. Payne or another expert on the Physics forum might be able to help explain why you’re still getting readings even though the copper wire isn’t connected to the electrode. However, if you haven’t already, you should definitely try to reattach the copper wire to the carbon cloth! When you’re performing an experiment, you want to make sure that all of your experimental groups are exactly the same except for the independent variable you’re trying to test. If the copper wire is still attached to the carbon cloth in your other two fuel cells, then it should be attached in the control cell as well. If you see a difference between your control fuel cell and your fuel cells that have nitrate, you won’t be able to tell if the difference is due to the nitrate or to the fact that the electrodes are made differently.
2. Waste water is full of organic matter that needs to be removed during waste water treatment. This organic matter is a food source for bacteria, and they’re able to clean up the waste water by digesting the organic matter and leaving clean water behind. An added bonus is that the bacteria release protons and electrons from the organic matter during the digestion process. This generates electricity that can be used as a power source at water treatment plants. Water treatment normally requires a lot of power, so having bacteria that can produce energy while also eating up organic contaminants from the water would save a lot of money and resources.
3. I’m including a link to the Science Buddies guide to data analysis. This document, and the documents linked within it, will help you learn about different types of statistical analyses that you can perform on your data.
https://www.sciencebuddies.org/science- ... e-projects

At the end of your experiment, you will have the voltage readings you collected, and the power output for each fuel cell, which you calculate using the voltage and the resistance. There are a bunch of options for statistical analyses that you can perform on your data that range from simple to more complex. A simple form of analysis would be calculating the mean power output for each of your fuel cells and comparing those values in graphs. You can also calculate the standard deviation for each of your fuel cells and show that on the graphs as well. The Science Buddies guide I linked talks about all of these analyses and explains how to do them. If you want to take things up a notch, you can try performing a T test on your data to test for differences between each of your nitrate treated fuel cells and your control fuel cell. Here’s a good article that explains the basics of the T test.
https://sciencing.com/how-to-interpret- ... 24290.html

Statistics can be a source of confusion even for experienced scientists, so don’t hesitate to let us know if you need more assistance!

I hope this helps answer your questions!

[brindhaaa]

Thank you for that!

I will most likely go with calculating and graphing the mean, standard deviation, and performing the T-test. In addition, I have 3 different graphs and tables with the readings with and without the air pump and when the readings were taken (morning, afternoon, noon). However, regarding calculating the power output, I did not get any readings with the resistor, so I do not think I can calculate the overall power output since the formula includes the amount of resistance applied while testing.

I have a quick question regarding denitrification if you are able to help with that:

I read the articles you provided me about the topic of denitrification. From reading, my understanding is that denitrification is a type of anaerobic respiration that occurs when soil bacteria use nitrate for their respiration in the place of oxygen. Since the anode is anaerobic, only a limited amount of oxygen is available, therefore, nitrate is used as a terminal electron acceptor instead of oxygen. The articles mentioned that nitrate will be reduced into various forms of nitrate. I am a bit confused about what this has to do with anaerobic respiration overall. After all the reduction, the final output is dinitrogen, but how does N2 contribute to anaerobic respiration? I am completely lost

Thank you for helping me!

Brindha

[koneill18]

Hi Brindha,

That sounds like a good plan to me!

Sure, I’ll try to explain the process a little bit better. Anaerobic cellular respiration is a process that bacteria use to break down fuel molecules like glucose into energy molecules. The energy molecules that they produce are called adenosine triphosphate (ATP). To produce the ATP, the bacteria start by breaking down the fuel molecules to release electrons. Those electrons then get passed through an electron transport chain. As the electrons get passed down to each member of the chain, they create an electrochemical gradient that drives the synthesis of ATP. In aerobic respiration, oxygen acts as the final electron acceptor, but in the absence of oxygen, inorganic compounds like nitrate or sulfate take over the job. In the process of denitrification, nitrate is the final electron acceptor. The step-wise reduction of nitrate to nitrite, then nitric oxide, then nitrous oxide, and finally dinitrogen is what generates the electrochemical gradient that drives the whole respiration process. The bacteria aren’t specifically trying to make nitrogen, they’re just using the reaction itself to drive energy production. Does that make sense?

[brindhaaa]

Ohhh, I think I have a much better understanding now. So the passing of electrons to each member of the chain is, basically, the process of making ATP, while in denitrification, the last terminal electron acceptor is nitrate instead of oxygen due to the anoxic condition, correct?

Thank you so much for that wonderful explanation. I don't think any article or YouTube video could help me understand like that

Also, when the air pump is used in the cathode, would it be possible for you to explain why the readings are supposed to increase? From the articles I read, the air pump is supposed to aerate the solution in the cathode with oxygen, thus generating electricity. However, I have no clue how this is supposed to work! I am trying to better understand this concept mainly because my readings only increased a little bit, if not at all, when the air pump was active, compared to the readings taken without the air pump, so I am a bit confused why this happened.

Thank you!

Brindha

[koneill18]

Hi Brindha,

I’m sorry, I totally missed the detail that this fuel cell uses an air pump! I think I misunderstood your experimental setup. Let me re-do my explanation of how the fuel cell is working.

In this fuel cell, the air pump is pumping oxygen into the cathode, and the oxygen is acting as the final electron acceptor. So, the bacteria in your anode are eating the carbon and the nitrogen from the compost, or whatever nutrients were in the mud in your control fuel cell, and releasing electrons and protons. The electrons move out of the anode through the external circuit and into the cathode where the oxygen accepts them. The protons travel across the salt bridge and combine with the electrons and the oxygen to make water. These oxidation and reduction reactions are driving electricity production. Since you’re adding oxygen to the system, the bacteria wouldn’t have to use an alternative compound like nitrate like they normally would in the absence of oxygen.

Some researchers have tried adding nitrates to the cathode instead of oxygen because nitrates are also good electron acceptors. But in your fuel cell, you didn’t put any nitrate in the cathode did you? If I understand correctly, you only have nitrogen in the anode. The bacteria are eating the organic matter in the compost in the anode and extracting electrons from it. Those electrons are then traveling to the cathode where they’re being accepted by the oxygen.

The compost is made up of organic matter that is essentially acting as an electron donor since electrons are being released from it as the bacteria digest it. Both the carbon and the nitrogen in the compost are important because they’re nutrients that the bacteria need to grow and carry out their metabolic activities. The more nutrients you add to the anode, the more bacterial growth you’ll have and the more electricity they’ll produce. That’s how adding the nitrogen helps increase the power output in the fuel cell.

Just to confirm, can you tell me what exactly was in the compost that you put in your anode chamber? I just want to make sure I’m giving you the right explanation of what’s going on!

[brindhaaa]

Thank you again, for that amazing explanation!

In the anode, I added coffee grounds as my source of nitrate. I chose this because they have a very fine texture, which is great for the process of respiration. In addition, it has a C:N ratio of 20:1, and the optimal C:N ratio for anaerobic bio-digestion is between 20:1 and 30:1. Also, they have a 2% nitrogen content by volume.

In the 5% MFC, I added 5% of coffee grounds, by the weight of the anode. In the 10% MFC, I added 10% of coffee grounds, by again, the weight of the anode.

I have one more question, but this is regarding the statistical aspect of my project. I have been watching a few tutorials on how to find the p-value of a T-Test with an excel spreadsheet. However, there are options for 1 and 2-tailed T-Tests. I researched a bit on these, but I still do not understand the difference.

P.S: When determining the p-value in a 1-tailed T-Test, I got 0.000020584. With a 2-tailed T-Test, my p-value was 0.000041169.
After reading the article you linked a few posts ago about different statistical tests, I think this is a sufficiently significant value.

Thank you,
Brindha

[koneill18]

Hi Brindha,

Perfect, so everything I said in my last post should be accurate for your experimental setup. I can tell a lot of research and preparation went into this project. I know it’s going to turn out great!

I’m glad you’re learning how to perform a T test in Excel! That’s much easier than calculating it by hand.

So, the purpose of the T test is to determine if the mean of your data from the experimental group is significantly different from the mean of your data from the control group. If your p value is less than 0.05, that means the difference between your groups is significant. If the difference is significant, that means it’s statistically unlikely to be due to chance alone. If your P value is 0.000041169, that’s definitely significant!

When you’re looking at the relationship between data from a control group and an experimental group, you have the option to look for a relationship in only one direction or in both directions. In your experiment, your fuel cell that has the coffee grounds can either produce significantly more voltage than your control fuel cell or significantly less voltage than your control fuel cell.

Let’s say you wanted to do a T test to see if your fuel cell with the coffee grounds produces significantly more voltage than the control fuel cell. If you did a one tailed T test, all of your statistical power would be devoted to calculating if the experimental fuel cell produces significantly more voltage than the control. If it actually produced significantly less voltage than the control, you would miss that if you were only doing a one tailed test.

If you choose to do a two tailed T test, then it will test for significant differences in both directions. The two tailed T test is almost always the better option. The only exception would be if missing a relationship in one direction doesn’t influence the outcome of the experiment at all. But in your case, the two tailed T test is more appropriate since missing a relationship in one direction could lead to misinterpretation of your data.

If you have any more questions about your data or your statistical analysis, don’t hesitate to ask them here!

I hope that helped!

[brindhaaa]

Ok! In that case, as you said, I think a 2 tailed T-test would be more appropriate, especially since one of the MFC's did not produce more energy than the control.

I have been researching ways to graph the p-value, and I came across 2 specific graphs I am considering. One is a T-distribution graph, and the other is a normal distribution graph. Do you have a recommendation as to which one I should use?

Thank you!

Brindha

[koneill18]

Hi Brindha,

In scientific papers, researchers don’t typically graph the p value. They just graph their data and write the p value on the graph. In your experimental procedure, you can include a step that explains what statistical test you used to calculate your p value. You could say something along the lines of, “Comparisons between the experimental and control fuel cells were measured by a T-test (two tailed distribution and equal variances between the two groups) using Excel software. Feel free to use your own wording of course! You just want to give a detailed description of what you did so that another researcher would be able to repeat it if they wanted to.

To display the p value on the graph, scientists usually use asterisks to represent the value. This is the key that most scientists typically use:

* means that p<0.05
** means that p<0.01
*** means that p<0.001
**** means that p<0.0001

Since your p value was less than 0.0001, you would use four asterisks. To show the p value on the graph, you draw a line above your data and put the asterisks on top of the line. I attached an example picture to this post with some random made-up numbers so you can see what I’m talking about. Let me know if you have trouble seeing it. The vertical lines coming out of the bars on the graph represent the standard deviation. You could also just write out the whole p value above the graph if you’d prefer. If you decide to use the asterisks, just make sure to include a key below the graph so everyone knows what it means.

Do you have one graph comparing the 5% MFC to the control and another graph comparing the 10% MFC to the control? Keep in mind that you can only use the T-test to compare each experimental MFC to the control. If you wanted to also compare the two experimental MFCs to each other, you would have to use an ANOVA test. That’s a much more complicated test that you probably wouldn’t learn about until you got to college. If you really want to compare the two experimental groups to each other, I can try to help you through it, but if you’re okay with just comparing each experimental MFC to the control, then the T-test is fine.

Let me know if you need more clarification!

[koneill18]

I just saw that someone in a different message thread shared a video explaining how to do an ANOVA in Excel. It seems pretty good, so I'll share it here in case you decide you want to learn more about it.

https://www.youtube.com/watch?v=tPGPV_XPw-o&t=6s

[brindhaaa]

Average Readings Per Day (mV) PDF.pdf

(55.67 KiB) Downloaded 283 times

Average Readings Per Day (mV) PDF.pdf

(55.67 KiB) Downloaded 283 times

Hi,

I watched the video for the ANOVA test, but I got completely lost! I, although I very much do appreciate the help with it, do not think it is the right level for me .

However, I did take a look at the sample graph you prepared (which btw was extremely helpful) with the p-value indicated in asterisks. I followed this style and make a graph just like it with three bars graphed according to the overall average voltage output, along with error bars and asterisks across the top.

After analyzing my readings on a line graph (I have attached the graph below), I noticed that the constant MFC had the most fluctuation in readings. I think this is because, compared to the 5% coffee ground addition MFC, it did not have as stable a supply of nutrients and electron acceptor. I wanted to confirm this before I put it in my presentation and papers.

Also, I read in a few other research papers that the anode chamber of an MFC serves as the terminal electron acceptor. However, I thought oxygen was the terminal electron acceptor. If what is read in the research articles is correct, does that mean I technically replaced the nitrate as the terminal electron acceptor instead of the anode?



Thank you,
Brindha

[koneill18]

Hi Brindha,

I don’t blame you. Understanding the ANOVA can be difficult if you haven’t taken a statistics class! Even the t-test is complex for 9th grade, so it’s great that you’re learning about it already! When you did your t-test in Excel, did you do a paired or unpaired t-test?

I think it’s perfectly valid to propose that the fluctuations in voltage in the control cell could be due to differences in the nutrient supply. You can also think about whether or not there were any changes to the experimental conditions that could have influenced the readings from that fuel cell. For example, I know you said there was a point where the copper wire detached from the carbon cloth. Maybe that contributed to the voltage fluctuations?

Could you send me links to some of the papers where it says the anode acts as the terminal electron acceptor? I don’t have any experience with microbial fuel cells, so there could be some experimental setups that I’m not familiar with. But from my understanding, the anode has the electron donor and the electron acceptor is in the cathode. The donor releases electrons in the anode and then those electrons travel through the external circuit and into the cathode where the terminal electron acceptor picks them up. I don’t think the terminal electron acceptor should be at the anode because then it’ll pick up all the electrons and they won’t make it into the circuit. I could be missing something though since I’m not a physics person. If you send me the articles, I can check them out!

[brindhaaa]

Hi,

In excel, I did an unpaired t-test because the data did not directly depend or relate to each other.

Regarding the electrode breaking, I replaced it immediately so that the experimental groups were built the same way. But maybe that time away from a proper electrode could have caused fluctuations within the readings, so I will definitely look into that.

Here is one specific link that I really looked into regarding the anode being an electron acceptor. I think if you scroll down and read the first paragraph of the intro, you can see where it talks about it.

https://www.researchgate.net/publicatio ... _Materials

I am not sure if you remember, but with the 220 Ohm resistor connected to the external circuit, no readings were produced for any of the fuel cells, at least that's what I thought. The last few days, only for the 5% MFC, I got readings of 1.7-1.9 mV (I am pretty sure this can't power anything!), but I think that it is because the resistor was too powerful, that's why I did not get readings at the beginning of my testing.

Also, if you had any more recommendations for any statistical tests, I am more than ready to try them, since I am aiming to have lots of data to display to the judges. I must have all my papers, presentations, and data in by Tuesday, Feb 6, so I am currently editing everything now.

Thank you again for all the help!

Brindha

[koneill18]

Hi Brindha,

Great, I think the unpaired t-test was the right call.

This article is confusing because there are some parts that say oxygen is an electron acceptor at the cathode and there are other parts where they say the electron acceptor is in the anode. They might be talking about different types of MFCs than the one you built. I would just go with what it says in the Background section of the Science Buddies project guide since that’s the exact MFC that you built. The Background section has a graphic showing how electrons and protons are released in the anode and then travel into the cathode where they’re accepted by the oxygen to form water. Since you followed the procedure from this project guide, their background information should be the most applicable to your project.

In terms of statistical tests, I think I told you all the ones I know of that would work well with your data. I’ll let you know if anything else comes to mind, but I think you’ve already analyzed it pretty thoroughly!

[brindhaaa]

Yes, I think I will rather follow the ScienceBuddies background info instead.

I am currently working on my discussion section and reasoning as to why the 10% coffee ground addition MFC began the decrease in voltage readings rapidly. I have read in articles that one significant symptom of an excess of nitrate (unbalance in the C:N ratio) is when the compost/matter becomes stinky and slimy. This is exactly what happened to the sludge in my 10% coffee ground MFC.

However, I want to get to know the science behind this, but I cannot really find much online of why this specifically happens. If you are able to give me another one of your wonderful explanations, that would be so helpful.

Thank you so much for all your help with this project. I am so close to finishing, and so happy my experiment succeeded!

Brindha

[koneill18]

Hi Brindha,

It’s actually not a super complicated explanation! Compost needs to have a high enough amount of carbon to give the bacteria enough energy to incorporate the nitrogen into their cells. If this doesn’t happen, the nitrogen starts to build up in the form of ammonia (NH3). The ammonia is what makes the compost start to rot and give off a bad smell.

It’s been my pleasure helping you out in any way I can! You’ve put a lot of hard work into this project and I’m so happy it turned out well!