Introduction: How to Isolate Plastic Degrading Bacteria From Soil!
With all of the publicity about how plastics are piling up in landfills and the ocean there has been an assumption that the plastics are incapable of breaking down. During my undergrad as a Microbiology major at Cal Poly SLO I was interested in environmental microbiology. During this time I was given a link to an article about a Canadian teenager, Daniel Burd, who found an aerobic (grows in presence of oxygen) bacteria that can degrade plastic. Well this got me thinking, maybe the whole idea that plastics could be degraded actually made sense, I mean there are some really complex molecules in nature that can be broken down, why not the simple design of plastics. So I started planning an experiment, looking for anaerobic (grows without oxygen) bacteria that degrade plastic using a Winogradsky Column which consists of soil at the bottom of the column and liquid at the surface of the column, limiting the amount of air that can diffuse into the soil. I did this with the hope that if I could find plastic degrading bacteria I could make a microbial fuel cell which would essentially breakdown plastic and produce electricity at the same time. Yes, a little ambitious but worth a try. If you are interested in how I went through developing the procedure and results please feel free to read the following paragraphs. If you just want to get straight to business skip to the the next page!
With a vague idea of what Daniel Burd had done with his aerobic experiment I decided to design a similar experiment. In just a small sample of soil there are literally millions of bacteria, most are unable to be cultured in a lab. These bacteria and their relationships are so complicated that we may never be capable of understanding the exact ecology of a population of microbes in a spoonful of soil. With this general idea in mind it made sense to me that if put into an extreme environment, the microbes would adapt in order to survive. The nice thing with microbes is their ability to adapt quickly, turning on and off a variety of metabolic pathways to best utilize the resources available to them. That is exactly why microbes can be found almost everywhere on the planet. So my idea was to put soil microbes in an environment full of all the tasty elements they use to grow. I used Bushnell Hass Broth which is composed of Magnesium Sulfate, Calcium Chloride, Monopotassium Phosphate, Dipotassium Phosphate, Ammonium Nitrate, and Ferric Chloride. Or if you can see it better in terms of elements (Mg, S, O, Ca, Cl, K, N, H, Fe). You will notice that there is one KEY element missing from this solution, Carbon. Well lucky for us plastic is made up of Carbon (and Hydrogen) in long chains. Since Carbon is absolutely essential for microbes to grow (and all known life really) I was hoping that with all the other necessary ingredients available to them the microbes would use the Carbon found on the plastic to grow and luckily I was correct. So I will show you in just a few really simple steps how you can find plastic degrading bacteria from soil (I used landfill soil but I believe any soil would work).
Currently I am working on becoming a teacher and it is my personal belief that publishing this research for profit would really defeat the purpose.This would take away access to most people (who would have to fork over some serious cash just to look at it), especially when this could make a real, noticeable difference for a real environmental problem we face today. So I invite you to perform this experiment on your own, see what kind of results you get and collaborate our results. Please leave feedback for me in the comments, because this is my first instructable and I would like to make it the best it could possibly be. Thanks :)
Step 1: Materials
First you need your materials:
- At least 1 empty 2 liter Soda Bottle (cut the top off, see pictures below. I used 4)
- Bushnell Hass Broth (http://www.sigmaaldrich.com/catalog/product/FLUKA/B5051?lang=en®ion=US)
- Soil (try to dig down a few feet for a better chance of finding anaerobic bacteria)
- An accurate scale (preferably accurate to the thousand ex: 2.008g)
- Cut up strips of plastic bags
- Petri dishes and agar (I used TSA, Oatmeal agar, and Czapek agar just to have a nice variety)
- Sealable test tubes
- Sterile gloves
- Bunsen Burner
- Inoculating Loop
Step 2: Making the Winogradsky Column
Making a Winogradsky Column for this experiment is very simple.
1. Place the soil you collected into the empty plastic bottles.
2. Add the Bushnell Hass Broth into the plastic bottle so that it the liquid extends several inches above the top of the soil.
3. Take your plastic you will be testing and weigh it ( the more accurate the scale the better)
4. Place the plastic into the soil (it may be easier if you use a utensil, like a knife or fork)
5. Seal the top part of the bottle back on
6. And wait....(choose whatever amount of time you would like, I did 4 months). Be sure to make observations and take a lot of pictures of your column. You will soon start seeing all kinds of interesting things growing in the column. I also observed a lot of gas formation in the soil from the anaerobic bacteria growth.
Step 3: Results
*If you would like to just see if the plastic lost weight and are not interest in finding the microbes responsible, simple remove the plastic, rinse the soil off, let them dry, then weigh. If not than continue below*
1. Prepare several sterile test tubes with Bushnell Hass broth using an autoclave. (dont forget to label them!)
2. After you have waited a given length of time you have to open up your Winogradsky columns, by removing the top that you taped on after you made them.
3. Using sterile gloves remove the plastic from the Winogradsky columns and place them in the appropriately labelled test tube.
4. Seal the test tubes and shake them around to help remove any microbes that were growing on their surface.
5. Using aseptic technique, take flame sterilized tweezers and remove the plastic and place it on a drying material, like papertowels or filter paper. The plastic is ok to be contaminated at this point with the filter paper or paper towels but be sure to seal the test tube back up immediately! Now you hopefully have only your plastic eating microbes in your test tube with no contaminations.
6. Using aseptic technique with an inoculating loop, open the test tube and dip the inoculating loop into the liquid. Then remove the inoculating loop and steak onto a labeled agar plate (whichever kind you decided to use), using standard streaking techniques. Plates can be stored at room temperature or in a incubator.
7. While wearing gloves run some water over the plastic to help clean off any dirt that may still be present, then place back onto paper towels or filter paper. Allow the plastic to sit for up to a few days so that it is completely dry! It is super important to get the plastic to be completely dry.
8. Weigh-in time. Using the same scale used before for the plastic, weigh them again. Jump up and down if they lost weight. Scratch your head and look perplexed if they gained weight.
9. Observe the plates for growth, Anything growing could represent a true plastic degrader.
10. (Optional from this point on) If you would like to find out exactly which microbes on the plate were responsible for the weight loss you can attempt to isolate them on their own plates by streaking out a single colony.
11. Continue until you have isolated colonies of the microbes. Prepare Bushnell Hass broth tubes. (do not sterilize yet)
12. Cut up new plastic bags to use, weigh them and then place them in the appropriate test tube. Be sure to label one test tube control.
13. Sterilize all of the test tubes, with the plastic in them. Top off the test tubes so that the broth completely fills the tube.
14. Inoculate each test tube with a different isolated microbe from your isolation plates. (be sure to hang onto the isolation plates)
15. Wait the same length of time you choose to use at the first part of this experiment.
16. Shake the test tubes and then, using aseptic technique, remove the plastic, like in step 5. Briefly rinse the plastic with water and dry over several days.
17. Weigh the dry plastic pieces to hopefully observe weight loss. Be sure to factor in the control plastic (i.e. if the control plastic lost 0.05 g of weight, then an experimental piece of plastic that seems to have lost 1.85 g actually only lost 1.80 g)
18. Any isolates that degraded the plastic are now easy to isolate (from the isolation plate, or the test tube) and can be identified using various biochemical tests or testing kits.
Step 4: My Experiment / Cool Calculations
I did the first part of the experiment over summer between my spring and fall quarter. It was approximately 4 months long and in that time I was able to get plastic weight loss in all four of the columns I tested. Of course the results varied (0.38%, 0.39%, 0.08%, and 2.05 %). I got a significant amount of isolates, but decided to test 19 randomly selected isolates. After four months of waiting I weighed these strips and found that 6/19 isolates I tested showed significant plastic degradation ( 7.59%, 8.10%, 8.15%, 7.51%, 8.60%, 7.28%). Unfortunately I got these results during my final undergraduate days so I was unable to continue the experiment and identify the isolates. However, with the data I obtained I made some very cool calculations, which I will show below.
Plastic is a simple molecule (see image below), it is essentially a huge molecule with a 2 Carbon: 4 Hydrogen ratio.
The weight ratio is 3.98x10^-23g Carbon : 6.68x10^-24g Hydrogen
The ratio in % is 85.6% Carbon : 14.4% Hydrogen
The approximate number of carbon atoms per E.coli cell = 7x10^9 cells
Weight of Carbon in a single cell= 1.40x10^-13g
Using this information the weight of the Carbon atoms lost from the plastic can be calculated. Divide that number by the number of days the experiment took place to get the weight loss per day. And although this is a rough estimate, the weight of the Carbon loss can be translated into the number of microbial cells that grew based on that weight, and then the growth of these cells per day can be calculated as well. :)
Step 5: The Next Step
Since I now longer have access to the materials at my college, I was unable to make (or attempt to make) a microbial fuel cell out of just plastic bags. Assuming the bacteria/fungi isolated were anaerobic or facultative (can live in the presence or without the presence of oxygen) a microbial fuel cell can theoretically be developed (there are plenty of how to videos online). If any of you consider taking that step please please please contact me. I am very interested in the possible results! Even if you dont go for the microbial fuel cell I would love to collaborate with anyone who actually carries out the experiment. You can contact through the comments or through my instructable account. Look forward to talking with you! :)