Help With Microbial Fuel Cell

[brindhaaa]

Hello there!

My name is Brindha and I am a sophomore high school student. I am enrolled in my school's science research program and am thinking of an idea for my project. I won at my city science fair last year, but this year I really want to do a complex project and try to make it to the County or Regional fair

Last year I worked with microbial fuel cells. I tested the effect of nitrogen on the efficiency of MFC's by adding different concentrations of coffee grounds to the MFC's. Working with MFC's really interested me in that field, and I have decided that this year I would also like the work with MFCs. I looked at the ScienceBuddies Microbial Fuel Cell page and read through it. I believe I have a good understanding of how MFCs work. The problem is, I don't know what I want to do! If someone could please help me think of what to do, and possibly guide me throughout the way, that would be really amazing!

Btw, my pre-approval is due Nov 5, so I need to have a basic idea of all my procedures laid out before then.

Thank you so much!
Brindha

Moderator removed personal identifiers, per Science Buddies policies.

[17eugenekim]

Hi Brindha,

Welcome back to Science Buddies! It's great to hear that you've developed an interest in expanding on your science fair project. Let's see what we can do before your deadline.

The first thing I'm going to link is our Project Guide overview: https://www.sciencebuddies.org/science- ... ience-fair. This might be old news for you, but I always like to start by making sure students understand the basics of building and conducting an experiment for a project. Be sure to at least look through all the pages in the first tab, and then browse through some of the relevant sections of the Advanced Project Guide tab, as they may also be useful to you.

Which Microbial Fuel Cells pages have you seen from Science Buddies so far? It looks like there's a few, which might help kickstart your ideas (see top 4): https://www.sciencebuddies.org/science- ... fuel+cells

Ultimately, however, the project idea should come from some question you are interested in answering. What is it about MFCs that you want to answer? Based on what you understand about how MFCs work, how do you think they can be "improved" or "optimized"? For instance, last year, you said you tested the importance of nitrogen content in MFC efficacy. Are there other variables that you want to try and tweak? Perhaps something about the type of medium the microbes grow on, or something about aerobic vs anaerobic bacteria? Just throwing out ideas. Get creative, and remember we're thinking about the scientific question itself -- we'll worry about details of experimental procedure later (but it's of course helpful if you think about both at the same time, too!).

Best of luck -- keep us posted in this thread!

[brindhaaa]

Hey there!

Thank you so much for getting back to me, it means so much!

I looked at those links you threaded and I have read through both of them. Thank you for that!

Also, getting back to your "how do you think they can be improved" or optimized" question, I think this year I am going to add different concentrations of a specific enzyme to catalyze the anaerobic respiration level/speed. I researched a bit about which enzymes are involved in the overall process, and the two most important ones I found were pyruvate decarboxylase and lactate dehydrogenase. Do you have any feedback on this idea to add either of these enzymes to my experiment? I would love to hear anything you have!

Thank you so much for your help
Brindha

[17eugenekim]

Hi Brindha,

Glad to be of some help.

It sounds like you're heading in the right direction with these questions! Enzymes are certainly a variable you could test, and the ones you've listed (pyruvate decarboxylase / lactate dehydrogenase) are the critical ones that are used in anaerobic metabolism.

Now, it's probably not feasible to measure out concentrations of enzyme and dump it into the MFC build. You'd have to get your hands on some kind of extract or purified mix of the enzyme you want, and that may be a logistical obstacle if you're not doing research in a lab. It's probably pretty pricey that way, too.

However, while purified pyruvate decarboxylase might be hard to get, live yeast is much easier -- home bakers & brewers use it so you can get it from a lot of retailers. Live yeast (Saccharomyces cerevisiae) uses pyruvate decarboxylase to ferment glucose into ethanol. If you go this route, you should read up more on yeast and pyruvate decarboxylase, and fermentation as well. I found this source on yeast as an additive in MFCs: https://www.intechopen.com/chapters/56779

Where do you plan on getting the microbes for your MFC? I think the types of microbes involved in the design can also affect the relationship between enzyme activity and metabolic rate. Just a side thought.

Lastly, I want to highlight a secondary variable that may be of interest to you. Enzymes are protein machines that function best at an "optimal temperature" -- which in a majority of cases is about 37 C. If they get too cold, they can be significantly less effective or just stop working entirely. If they get too hot, they can undergo denaturation, breaking down and also not working. I don't know what the exact temperature parameters are for these enzymes, but if you can get access to an incubator or have another way of maintaining various stable temperatures, I think this might be a worthwhile experiment to test out. Up to you.

Hope that helps!

-Eugene

[brindhaaa]

Hey,

I think you are right, as those two enzymes would be very hard to gain access to outside of a lab. Also, I took a look at the link about adding yeast to an MFC. I love that idea! However, I was researching a bit more about that and it looks like there is already a lot of experiments and studies supporting the effect of yeast on an MFC. Are you aware of any other enzymes involved in anaerobic respiration that are reasonably easy to access? I have been researching a lot recently but unfortunately, I haven't come across anything I can use : (

Last year, I obtained a benthic mud sample from a lake in my city. I got great results with it, and I plan to get my microbe sample from there this year as well.

Also, regarding the idea of testing temperature as a variable, that's a good idea, however, I am trying to find something a little more complex to test. But that is a very important factor to keep in mind. I think I might borrow an incubator from a teacher's lab or purchase one to ensure that the MFCs are in the same condition.

Thank you so much,
Brindha

[17eugenekim]

Hi Brindha,

I don't want you to get too discouraged by an abundance of existing research on similar topics as an idea that you want to do, because that does tend to happen at the level of a lot of high school science fairs. That said, I understand your concern. This is actually partly the reason why I brought up temperature as a secondary variable. I wonder if you could integrate yeast AND temperature to conduct a more complex series of experiments.

If that's still too simple for your tastes, we can look for alternatives. I'm not entirely sure how to add lactate dehydrogenase in a controlled way. However, I do think we can increase production rate of lactate, the reactant in the lactate dehydrogenase reaction. Lactobacillus bacteria, particularly Lactobacillus acidophilus, is a commonly found probiotic that conducts lactic acid fermentation. You can find probiotic capsules with live bacteria in some grocery stores in the health & wellness section, which should be nice for experiments. I wonder if you could even use yogurt, as long as it says it has "live cultures" -- which would be cheaper & a bit more intriguing, but also I'm not sure how much I trust yogurt companies' claims.

Maybe reading up on fermentation will give you more ideas. At the moment, though, in terms of conducting enzyme-based experiments using commercially available tools, I think those are your best bets -- probiotics and yeast. Of course I'll let you know if I think of anything else.

Hope that helps.

-Eugene

[brindhaaa]

Hi there!

So I looked into Lactobacillus bacteria, but I am a little bit confused. I read articles about it but from my understanding, it's a bacteria that undergos fermentation. So is it not something that I would add to the anode chamber of my MFC to enhance performance? Do you think you could elaborate just a bit on this?

Thank you so much!
Brindha

[17eugenekim]

Hi Brindha,

Yes, it is bacteria that produces lactate via fermentation. I am thinking it could be worthwhile to put in different amounts of said bacteria into your MFC and see if it produces a measurable effect by changing the available levels of lactate. The idea is that you are adding some quantity of a bacteria that you know can perform fermentation, rather than having a whole cell of unknown bacteria. I'm honestly not sure how it will turn out! Maybe it'll be effective, or maybe it'll do the opposite by messing up the microbial ecology.

Also keep in mind two things: 1) fermentation requires a source of sugar/carbs, so make sure the bacteria don't run out of that, and 2) most fermentation likes to happen anaerobically, so keep air out of the design. I think both of these are elements you would have thought about in your first MFC design, but just bringing them up again just in case.

[brindhaaa]

Hey!

That makes so much more sense! But I am just unclear about one more aspect of fermentation: How will an increase in lactate eventually lead to an increase in electricity? Does an increase in lactate promote fermentation levels?

Also, I was looking at where I can find Lactobacillus Acidophilus bacteria. It seems like the only place it can commonly be found in is yogurt, particular places in the human body, or pills. Are you aware of any other sources where I can get this bacteria?

Thank you so much!
Brindha

[17eugenekim]

Hi Brindha,

Sure. In an earlier post in this thread, you mentioned one of your potential ideas was to increase the electrical output by increasing the available lactate dehydrogenase, an enzyme that performs fermentation. In response to this, I suggested that, while acquiring the dehydrogenase itself may be difficult, adding Lactobacillus may 1) contribute to more fermentation in general, and 2) specifically increase the available lactate, which is the reactant that lactate dehydrogenase uses. That's how we got to this point in the design. Like I said, I'm really not sure what the end result will be.

Is there a particular reason you want to look elsewhere? One of the other reasons I suggested adding Lactobacillus was because it is relatively easy to procure. Probiotic pills and yogurt indeed often contain live bacteria that you may be able to use, which I think should be fine. It won't be fantastic in terms of determining exact quantitative amounts that are being added to the cells, but it's better than nothing. (If you are interested, you could probably even pivot to a different variable to test: do various probiotic brands actually contain the bacteria that they claim they do? You could test this with slight modifications to your MFC setup...etc.)

[brindhaaa]

Hello!

That makes things much clearer! But also, how exactly does the bacteria contribute to more fermentation. You said that it increases lactate, which is a reactant in LDH, but could you please explain some of the intricate processes (like how it converts pyruvate to lactate faster and stuff) so that I can maybe get a better understanding?

Also, thank you so much for your suggestions. I agree, it would be better if I could find an exact quantity of how much bacteria I am adding. But I think I am going to be adding yogurt with live Lactobacillus cultures in them, and measuring the concentration of yogurt to the mud by weight.

Thank you so much!
Brindha

[17eugenekim]

Sure. When I said Lactobacillus can contribute to more fermentation, I should have been more clear. Lactobacillus bacteria itself can ferment sugars into lactate (in an anoxic environment). This fermentation pathway is not the same as the LDH pathway, and by itself it probably won't have much of an effect on your fuel cell's electrical output. (Or maybe it will, indirectly, by altering the substrate environment that all the muddy bacteria are living in and impacting metabolism of other bacteria. But for simplicity's sake right now, I am disregarding this indirect effect.) However, it will generate excess lactate, which changes the reactant pool for LDH and affects LDH activity overall.

Here I have to admit I have made an error. For LDH, it's the pyruvate-to-lactate reaction that frees up electrons, and the lactate-to-pyruvate reaction consumes electrons. I mixed this up and thought it was the reverse. LDH does both reactions (favoring pyruvate-to-lactate in anoxic conditions), but high levels of lactate will inhibit the pyruvate-to-lactate reaction, which would actually reduce overall electrical output for the MFC. In other words, I should think adding Lactobacillus will decrease MFC efficiency. I'm so sorry for not catching that sooner.

That leaves us with a bit of a dilemma, though I think this would still be a worthwhile experiment if you want to go forward with it. It would provide evidence that LDH is what drives the electrical output, and not some other enzymatic pathway that you hadn't taken into account because the mud contains a diverse pool of bacteria. From a scientific inquiry standpoint, this is still an interesting direction to take. However, an alternative would be to investigate how optimizing pH could improve MFC productivity. This leaves a lot of room for playing around with as well.

I apologize for the mix-up, and please let me know how you'd like to proceed. There's still a lot of directions we can take this project and I want to know what you think.

[brindhaaa]

Hello!

I understand the science behind everything now, so thank you so much for the explanation! However, I do see the problem with this. You said that high levels of lactate will inhibit the pyruvate to lactate reaction. But I built 3 MFCs: one as the constant, one with 5% of yogurt, and one with 10% yogurt. I haven't started testing yet, but since the amount of yogurt is very little, especially in the 5% MFC, would the reaction still be inhibited?

The only problem is that I have bought all the materials according to this experiment and am not really sure what to do now.

I'm also intrigued by your suggestion to optimize the pH in each microbial fuel cell. How exactly would I do that? I am also open to any other suggestions, as I will be researching a bit on my own as well!

Thank you again,
Brindha

[17eugenekim]

I'm so sorry for being late to catch it!

When you say 5% and 10%, what percent is this referring to? 5% by volume of the whole MFC? Regardless, I think there may still be measurable effects. Even aside from the presence of lactobacillus, yogurt in general contains lactic acid. But we'll see! I think it'll still be worthwhile.

As for pH, lactic acid is, well, acidic. I would wonder if adding a base to neutralize said acid would affect the MFC. This is a super-simplified way of thinking about the role of acidity in bacterial metabolism, but I'd still find it interesting enough, personally. In this experiment, you would add some sort of base to the MFC to raise the pH and make the substrate more alkaline. Just a thought for now, though let me know if you want to take that further.

[brindhaaa]

Don't worry about it, no problem at all!

Sorry for the confusion, I mean 5% of the weight of the mud of the MFC. So I weighed the amount of mud in the anode chamber of the MFC, calculated 5% of that amount, added that much yogurt if that makes sense.

And unfortunately, I have already mixed the mud with the yogurt and I won't be able to resample mud because the location that I went to is really far away, so I do not think I will have time. That being said, I'm not sure if I can proceed with the pH experiment, as much as I would love to. However, I think it would be worthwhile to just go with this experiment. But the good part is that now if my results don't go as expected, I will have scientific evidence to back it up

Also, I have another question. I built my control MFC and started testing it yesterday, but I have noticed that when I add the air pump into the cathode readings actually decrease. I'm not entirely sure why this keeps happening. Do you think you can help me figure out this problem?

Thank you so much!
Brindha

[17eugenekim]

This is the air pump that keeps the environment anaerobic? I'm not too sure. If you're comparing electrical output between anaerobic and aerobic states (with/without pump), then I find it reasonable to find aerobic output higher than anaerobic output, because aerobic metabolism is more effective. Does that match what you're seeing? Also, is this different from what you observed in your previous experiments with MFCs?

As long as you're getting a nonzero reading with the MFC fully assembled, I think it makes sense from a theory standpoint. Wherever that nonzero reading settles, it becomes your control.

I'll think on it. In the meantime, if you have additional context, do let me know. Best of luck!

[brindhaaa]

Sorry for being unclear, but I meant the air pump that I inserted into the cathode, which is aerobic . For my data section, another thing I want to analyze is also how much the air pump affects each MFC. I have readings every day that I have been taking for the past few weeks 3 times a day, and with and without an air pump. I expected readings to be much higher with the inclusion of an air pump in the cathode, but readings only increase by about 5-10 mV, and I am not sure why.

I am about 1-2 weeks from finishing my testing, and I am starting to begin to enter data into graphs and tables. I was thinking of running a T-test on my data to analyze its significance and credibility. Do you have any other suggestions as to what statistical tests I can do?

Thank you so much!

[17eugenekim]

I'm not too sure, either. Maybe there are obligate anaerobes contributing to the overall metabolism that are inhibited by air? Really not sure, sorry. An interesting question to look into, though, and at least mention in your results/conclusions!

How do you envision presenting your data? I don't know your exact methods for data collection, but still I can think of a few ways you could organize it. You could do line plots of each MFC with electrical readouts over time, showing confidence intervals around each timepoint. Also, because you have more than two groups, you may want to look into doing a one-way ANOVA. It's conceptually similar to a t-test but can do more than just pairs of datasets.

[brindhaaa]

Hey!

I think it may be due to the fact that the air pump wasn't left in the water for too long before obtaining readings. But I will definitely make sure to implement this in my research paper!

Also, my results are looking fantastic! I have two more days left of testing (I tested for 3 weeks in total) and so far the 5% yogurt addition MFC has been peaking in results and surpassing both other MFCs. However, the 10% yogurt addition MFC began to increase in readings but at around day 12, rapidly began to decrease (I think this is due to the excess amount of lactate, which inhibited the enzymatic processes).

I have also finished graphing my results (besides for the two days that are left) and I noticed that the constant and 10% yogurt addition MFCs have been experiencing lots of fluctuation in readings. In other words, readings are rising then falling in a pattern. However, the 5% yogurt addition has been experiencing very little fluctuation. Do you think it would be accurate to say that in the 5% Yogurt MFC, anaerobic bacteria received a steady quantity of Lactobacillus and other probiotics, which led to a stabilization in anaerobic respiration levels?

One other thing I noticed is that in all three MFCs, my digital multimeter detected no readings on days 1 and 2, but suddenly detected a minimum of 100 mV starting from day 3. Do you know the reasoning behind this? I was researching a bit, and could it be due to the fact that the protons took some time to diffuse across the PEM for the circuit to close?

Finally (I apologize for the super long message ), I think I am going to go with the ANOVA test as you suggested. I discovered that there is such thing as a one-way and two-way ANOVA test...do you know which one would be best for my experiment? And also since I am doing an ANOVA test, I think I will scratch the t-test, since it is basically the same thing, just with fewer groups.

Thank you so much for all the help!
Brindha

[17eugenekim]

Your data & initial analysis of it seems very interesting, and definitely headed in a good direction! It shows that you're very capable of thinking critically through the results of your experiment, so kudos! And glad the data collection hasn't been too frustrating.

I'm not sure what you mean by the 5% readings stability comment. To say it received a "steady quantity" confuses me, because the quantity of bacteria you added was always constant - you added 5% at the beginning. (Same goes for other groups, with different numbers.) Perhaps you meant something about the quantity of their metabolic products? Like there's an equilibrium of some kind regarding the outputs of Lactobacillus and the inputs of anaerobic respiration?

Remember that your current hypothesis centers around the activity of the LDH enzyme, which needs to turn pyruvate into lactate to free up electrons. Pyruvate is the product of simple glycolysis, which almost every cell can do as long as they have sugar. With that in mind, I'm actually wondering if the effect that you see on the 5% MFC is due to the added "fuel" - i.e. the sugar content of the yogurt - displacing the inhibitory effects of added lactate-making bacteria. I have no idea if that's correct, but what do you think? And what do you think you can say about the 10% MFC if we follow this line of reasoning?

I don't think this accounts for the fluctuation in your readings, by the way, which are also an interesting facet of your data. You said they have a cyclical pattern? How clear is it? I think we can keep throwing out ideas for why this is happening while we have the time. Also, from a presentation standpoint, you may want to call it something else, because "fluctuation" tends to imply less of a pattern in my experience.

As for ANOVA, one-way and two-way are what you'd expect from their names. Two-way ANOVA is what you do when you want to analyze how one variable is affected by two others (i.e. 2 independent variables, 1 dependent variable). It also assesses whether the effects of those two variables are independent from each other. I don't think you have 3 variables in your data, unless you want to do something with time. One-way is probably what you want for your hypothesis, but if there's more hypotheses you want to test, please go ahead and we can talk about the analysis required for that.