URGENT:Help on environmental/microbiology project!

[heychristinee]

Hi, my name is Christine and I am currently working on a science project called "The study of different bacteria on the degradation of components of commercial disposable diapers". I chose this project in order to challenge myself for the Science Fair this year after seeing Daniel Burd's science project about the finding bacteria that can degrade plastic at a fast rate. Here's his research paper on the experiment: http://wwsef.uwaterloo.ca/archives/2008 ... Report.pdf. Anyways, after seeing his project, I wondered if I would be able to degrade the diapers at a much faster rate since landfills are filled with them. Using some soil from my backyard (I was unable to come in contact with any soil from landfills sadly), I tried to decompose the different components of the diaper, such as plastic, cotton, etc. After a month, I found that the cotton decomposed significantly while the others didn't. I decided to focus on decomposing the cotton for my experiment, thereafter. Some online sources told me that by creating conditions of the environment that the cotton lives in (hot, dry soil), they say that I could get rid of the other bacteria that won't be able to consume the cotton, which is exactly what I did. I dried my soil in the temperature that cotton can survive and grow in. However, when I plated the bacteria in the resulting soil, many, many colonies of different bacteria grew, making it impossible for me to isolate the bacteria that would consume the cotton fastest. In Daniel Burd's experiment (fyi. he happened to use a shaker, while i didn't), he found 4 DISTINCT colonies when he plated them. So here are my questions:

1. How can I limit the amount of bacteria growth to only those that will consume cotton?
2. Are there any ways to find unknown bacteria through experimentation?
3. What is a shaker used for and could not using a shaker have affected my experimentation?
4. I am also trying to better the conditions of the bacteria so that it will consume cotton at a faster rate once I find them. Are there specific nutrients that I should use to better their conditions, like how Daniel Burd did as well?
5. Another factor that was different between Daniel Burd and I was time. Should I have prolonged my experiment longer?

I may have more questions that I can't think of at the moment.
Thank you so much for reading and I will be waiting for your response!

[donnahardy2]

Hi,

This is an excellent topic for a science fair project, but a very complex as well. Thanks for posting the previous project. It sounds like you are trying to develop an original project based on previous research and this is a good approach. However, please let me know if you are trying to write up results of the experiment you have completed.

Here are some answers to your questions and some suggestions for continuing your project.

1. Daniel Burd used polyethylene as the only carbon source for selecting and growing his isolates and very carefully documented that polyethylene would support the growth of pure cultures of each bacterium. . If you are going to concentrate on the cotton, then you would need to demonstrate that the bacteria you isolate will grow with cotton as the only carbon source. Cotton is composed of the polysaccharide cellulose, which contains glucose, so the bacteria you grow would need to be able to break down the chemical bond between the glucose units.
http://en.wikipedia.org/wiki/Cellulose

The specialized enzymes required for this are found in some obligate anaerobic bacteria found in the digestive tract of ruminants like cows.

Here is a Science Buddies project that provides background information for collecting and growing this type of bacterium:

https://www.sciencebuddies.org/mentorin ... p027.shtml

2. Yes, the best way to find unknown bacteria that will do what you want them to do (like hydrolyze cellulose) is to collect samples where the specific material has been left in contact with soil or water. That’s why the sample of landfill soil was a good choice for the diaper project; landfills are full of diapers so there has been a natural selection for organisms that can utilize the various components of diapers. Once the sample is collected, then the bacteria are incubated in the specific food source to select for specific organisms.

3. The shaker was used to increase the concentration of oxygen. The bacteria that degraded polyethylene were aerobic organisms (catalase positive) so would grow faster with more oxygen. You did not aerate your samples, so conditions probably were microaerophilic or anaerobic. With the conditions of your experiment, you would select for bacteria that could grow in low oxygen conditions; Daniel Burd used a shaker so selected for aerobic bacteria. Since cellulose is hydrolyzed by anaerobic organisms, the conditions you used were actually better for the specific sample. I think that it would be worthwhile to try aerobic and static (no air) conditions and compare results. If you don’t have a shaker, you could simply mix the sample by hand 3-4 times days to increase the concentration of oxygen in the sample.

Since the carbon source you were using was cellulose, once it was hydrolyzed to glucose, there were probably many other species of bacteria present that could utilize the glucose. Daniel Burd was careful to use inorganic salts to provide all other elements needed for bacterial growth. I think that you should do some additional background reading on this topic and look for ideas from the literature. The inorganic salts plus the miniscule amount of yeast extract that Daniel used would be a good place to start; simply make this mixture of salts and add the cellulose from the diapers: 0.1% (NH4)2SO4, 0.1% NaNO3, 0.1% K2HPO4, 0.1% KCl, 0.02%, MgSO4, and 0.01 % yeast extract was prepared in 1L of tap H20.

4. The time you used was probably adequate. Multiple transfers of the culture into new growth medium would improve the selectivity of the conditions.

Now, here are some questions for you:

1. Are you trying to finish up your project, or will you continue with it over the summer?
2. What type of laboratory facilities do you have access to?

3. Do you really want to do a project on cellulose degradation? You could do this, but I think you would want to add something to make it more original, like use the diapers to make biofuel, for example.

4. This website lists the components of a disposable diaper:

http://www.babynaturale.com/whindidi.html

Daniel Burd’s project was original because he worked on the non-degradable portion of the diaper. The cellulose portion of the diaper is considered biodegradable, so a science project on a faster way to degrade cellulose is not as intrinsically interesting and not as challenging. You could switch to a different component of the diaper for the next phase of your project. How about the polypropylene or the polyacrylate?

http://www.babynaturale.com/whindidi.html

5. What background reading have you done on your topic? If you are going to continue this project for next year, I recommend that you stop and do more reading before continuing. For example, if you continue to work on the cellulose, you need to know more about cellulose-degrading bacteria.

I hope this helps. Let me know if you have additional questions.


Donna Hardy

[donnahardy2]

Hi,

If you are interested in doing more background reading, here are some articles that will provide good information for your topic. You could apply the information and techniques to the disposable diapers:

Here is an article on microbial decomposition of cellulose at sea. Perhaps if you live near an ocean, you could study the decomposition of disposable diapers by ocean microorganisms:
http://downloads.sage-hindawi.com/journ ... 941810.pdf

Here’s an article reporting the decomposition of cellulose by fungi;

http://webs.uvigo.es/jdguez/old/documen ... osting.pdf

Here’s an article that reports the decomposition of cellulose by the gut flora of millipedes. These microorganisms were aerobic, so would grow faster with oxygen.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC242008/

Here is a review article on decomposition of hydrocarbons; the information here might be useful for the study of decomposition of polyethylene and polypropylene:

http://dodreports.com/pdf/ada327756.pdf

Here is a review article on microbial decomposition of various polymers, including polyacrylamide, which is one of the polymers in disposable diapers:

http://nopr.niscair.res.in/bitstream/12 ... 86-193.pdf

Let me know if you have any questions about the content of any of these articles.


Donna Hardy

[heychristinee]

Thank you SO MUCH for your quick & very specific response! I actually have been researching quite a lot on bio-degradation of landfill materials, and since my biggest problem lied in the isolation of bacteria, I have been researching on that as well. Honestly, I did research on cotton as well but had no clue that it was composed of cellulose. When I was on wikipedia, all I saw was that it was made of fibers and such so your stating that cotton is composed of cellulose will DEFINITELY help me improve my experiment.

1 & 4. The truth is, I was supposed to have this project done by the time of the science fair, which was in the end of March. However, since I only came up with this project in late-January, I was unable to finish it in time. I tried to continue this til now but many problems came up when too many bacterial colonies grew on the agar platings, so now I'm looking for help from experts like you! Thanks again for your help. Anyways, the school year is coming to a close soon, so I'm guessing that I will be restarting this project during the summer. I was thinking of decomposing the components of the diaper during the duration of summer break so that I can see if the components that didn't decompose as much will significantly decompose over the 3 months. Decomposing the polypropylene or the polyacrylate portions would be a great addition to the experiment!
2. I have access to an incubator and some of the nutrients listed to improve the conditions for the bacteria in my research classroom. There are also some basic laboratory equipments, such as a very sensitive weight measurer, beakers, flasks, etc.
3. I'm very interested in challenging myself since I haven't done so in my previous year of research. I actually am very interested in cellulose bio-degradation and attempting to make biofuel from it will certainly be VERY interesting. Thank you so much for your suggestion!
5. As I said before, I did much research on bio-degradation and isolating of bacteria, etc. I've been doing research on things from here to there so I guess I haven't gone too in depth with anything else. so, I will definitely need to spend a significant amount of time researching on this experiment more during the summer.

Additionally, Thank you so much for the website suggestions!

Do you think that I will need to work with a mentor over the summer or during the next school year for my experiment?

[heychristinee]

Actually, I have a few more questions.

1. At one point in my research, I found that manure has an effect on degradation. Is this true and if so, would it be a good idea to add that to my experiment?

2. Also, I found that trash in landfills are closed with a clay cap and surrounded by a liner. Even with those, is it possible to harbor aerobic bacteria in there?

3. On wikipedia, it stated "Researchers are yet to find a practical application" for Daniel Burd's findings. Why is this? I would like my experiment to have a practical application in landfills, if possible.
http://en.wikipedia.org/wiki/Polyethylene

Thank you so much for your time

[donnahardy2]

Hi,

It sounds like you found a closed landfill that had been sealed off and I don’t think you should try to get a sample from a sealed landfill. What you need is a landfill in progress, or perhaps just a compost pile from someone’s garden. Cellulose is a very abundant natural polymer found in all plants so any place that dead plants have been sitting in contact with moist soil would provide a good source of natural cellulose-degrading bacteria. For example a bale of straw that had been left on the ground over the winter would be a good source of bacteria Cow manure is a good source of cellulose-degrading bacteria since cows eat grass, however, could also be a source of E. coli, so I’m not sure I would recommend using it as a source of microorganisms. Since cellulose is common, there are many microorganisms that can hydrolyze this polymer. Polyethylene is much less common and it not a natural polymer, so bacteria that can hydrolyze it are much rarer. If you are planning to continue this project over and work on it for next year, you can bury a couple of unused diapers in some garden soil, maybe add some manure and let it compost for a few weeks. Sometimes people use polyethylene barriers in their gardens to block the growth of weeds, so if you want to enrich for polyethylene-degrading bacteria, try to find soil that has been in contact with polyethylene for a source of soil for your experiment. This will allow natural selection of disposable diaper-decomposing microorganisms.

I recommend that you focus on one component of the diapers for your project. Studying the decomposition of all of the components would be a vast project and beyond the scope of a single science fair project. If you can work on this project over the summer and do some preliminary experiments, you will be in a good position to complete an outstanding project by next March. It would be good to have a local mentor to provide advice, but since you are familiar with the basic techniques you could also do the work at home if you follow safety precautions. You should talk to your teacher before school ends and find out what resources you have available and take advantage of all opportunities.

Daniel Burd’s project was the preliminary experiment to show that bacteria could degrade polyethylene. It is very exciting research with new results and you could continue his research and demonstrate a practical application. Research on cellulose-degrading microorganisms has been published for many years. You could do either project, but you would need to review all of the previous research on each topic and then select an experiment that would answer a unique question. I think you are on the right track, but you need to do more background reading. Since you are not working on a deadline, you have the advantage of time to use to select a specific topic that no one has done before.

Donna Hardy

[heychristinee]

Hi!

I am SO sorry about the late response. I just got into summer and was on vacation and was focusing on summer school.

Anyways, thanks so much for your help once again. Your advice has been a HUGE help to me. I have to start writing my proposal for this experiment so that I can get permission to begin. I am looking for the right soil but because our town/city does not have local landfills, it's been difficult to find some. The soil from my own backyard is excessively dry so I don't feel that my backyard soil would be of much help. Would commercial brand soil contain the same bacteria that would be present in the conventional soil (from backyards)?

Additionally, I spoke to my teacher about the polypropylene degradation idea suggested by you and he thought it would be brilliant. From my research, I saw that there has been studies on the polymer's degradation by heat and such. Would degradation by soil and bacteria be original enough or do you know of studies that already found a type of bacteria that can consume it?

If I were to experiment on cellulose-consuming bacteria, would I have to change the methods I was originally trying to use to experiment (using diapers & soil, etc.) ?

[donnahardy2]

Hi,

Welcome back! It’s good that you have time to focus on your science project again. I hope the following comments will help you finalize your experimental plan. Degradation of polypropylene takes time, so you do need to get started.

Less research has been done on polypropylene degradation, so I think this would be would be an excellent topic. If you are going to try to isolate polypropylene-degrading bacteria you are right that your dry backyard would not be a good source of polypropylene-degrading bacteria. However, is there any polypropylene (playground equipment, plastic garbage can) that has been sitting in contact with the soil? You need soil that has been in contact with polypropylene and moist enough for a few months so that there will have been a natural selection of polypropylene-degrading microorganisms. If you want to try and isolate these organisms, please look in your local environment for some polypropylene (a trash bag, weed block in a garden, etc. that has been sitting in contact with soil for a while) and collect a sample of the soil that has been touching the polypropylene. There would be no reason that commercial garden soil would contain high levels of polypropylene-degrading bacteria.

Since your project is degradation of commercial disposable diapers, you do not have to limit yourself to bacteria. You could compare thermal, UV, and microbial degradation using soil with bacteria

According to this Wikipedia article, which does include some scientific references, it takes 25 years to degrade a disposable diaper. So if you could plan to do an experiment that would reduce the time required it would definitely be a unique project.

http://en.wikipedia.org/wiki/Biodegradation

Here is an article that includes an experimental set up for measuring degradation of polypropylene. You can use the outline of this article for inspiration for your project.

http://pcc5050.pcc.usp.br/Artigos/Degra ... ycling.pdf

Maybe you could solve the problem by developing an alternative to the use of polypropylene in disposable diapers using biodegradable nonwoven materials:

http://www.technica.net/NF/NF3/biodegradable.htm

This article used a number of techniques to verify degradation on polypropylene by bacteria. How will you measure your results?

http://www.ncbi.nlm.nih.gov/pmc/article ... 0-0191.pdf

You have done lots of background reading, so since you have time now, go back and reread everything and think about what you think you would do to solve the disposable diaper problem.

Please post back and let us know if you need more information.


Donna Hardy