Introduction: How to Isolate Plastic Degrading Bacteria From Soil!

Picture of 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

Picture of 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&region=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)
- Autoclave
- Sealable test tubes
- Sterile gloves
- Bunsen Burner
- Tweezers 
- Inoculating Loop


Step 2: Making the Winogradsky Column

Picture of 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

Picture of 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

Picture of 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! :)


-Ryan

Comments

MallikaB1 (author)2016-06-16

Hi Ryan,

I'm an undergraduate microbiology student. I've to do a 6 month project as part of my coursework. I'm really interested in working on plastic degraders. I would like to know in detail about the isolates you got, incubation time, problems faced etc.

SHOE0007 (author)2016-06-01

Of topic but I am testing soil from mushrooms and they have a unique bacteria.

Sucrose: - Fructose: + Glucose: + Lactose: + Maltose: + Stevia powder: + Glycerol: + Other Alcohol sugars: +

No protien was added and this was done with air exposure. A gram stain indicated that they were gram positive and a endospore stain indicated that they were endospore stain negative.

SHOE0007 (author)2016-05-12

Here I have bought mushroom compost and I will (soon) be extracting the bacteria from these packaged soil.

SHOE0007 (author)2016-02-19

I am thinking of testing Triethanolamine salicylate at different concentrations: 10%, 15% and 20%. Then I will with my condenser kit I will make the following oils:

Pepper Oil

Garlic Oil

Red hot pepper Oil

Other compounds

Tetrachloroethylene, Pine Oil, Tree tea oil.

These will all be tested with fresh samples of lactobacillus retuni.

Daniel.

SaheliR (author)2016-01-14

Hi Ryan. I am a college student too and I need to design some experiments for my course. I was thinking about designing an anaerobic soil bacteria genome that can multiply fast. I would like it to be viable outside the lab, so that once released in soil, it can convert the age-old plastic present there on its own. Can you help me by pointing out any disadvantages of these naturally-occuring plastic feeders? Based on them, I will try improving the genome designing.
Thanks, in advance.

SHOE0007 (author)2016-01-13

Here is a link to what silver does to bacteria.

http://www.saltlakemetals.com/Silver_Antibacterial...

Thank You

Daniel.

SHOE0007 (author)2016-01-13

Here is another experiment with silver 380 mg and sodium fluorescence under UV light.

Silver metal with 2.5 g milk protien powder, 5.3 g glucose, 100 ml of

tap water and 2 g silver at 87.5% water with 40 mg sodium fluroescence

powder solution with 95% ethanol.

Keeping in dark until I expose it to the UV light. UV light from lamp keep 1 meter away. Set UV for 3 hours a day!!

Daniel

SHOE0007 (author)2016-01-09

Perie water. with 480 ppm HCO3. This will make 155 mg

silver carbonate!!

Solubility in water: 0.032 g per L. /10 = 3 mg carbonate at

25 degrees C. Rest insoluble silver carbonate.

Soluble in silver chloride = 250 ug in a 1L. At 25 degrees C.

Extremely insoulbe in water.

Not very effective

25 ml or 0.425 g silver by silver chemcial plating!!

100 g * 0.017% = 1.7 g silver nitrate.

Ag+2(aq) + Cu(s) >> Ag (s) + Cu+2(aq).

AgNO3 >> Ag+ + NO3

Cu(s) + 2AgNO3(aq) → Cu(NO3)2(aq) + 2Ag(s)

1.7 g/169.87 g * 107.8682 g = 1.07 g silver with trace amounts of copper nitrate.

http://evises.evi.com.hk/chem/defaul3/chapter3_1.h...

Copper nitrate

Copper nitrate salt.

so add 1.7 g silver and 20.06 g copper nitrate/2 = 10.03 g

1.07 g silver metal. This is if you use more than 1.7 g of copper metal. Like 2 grams copper. Thus a 1.7 g silver mixed with copper nitrate (10.03 g) as a disinfectant property for 100 ml. Using 5 ml

10.03 g *5/100 g = 0.5 g copper nitrate.

1.07 g * 5 g/100 g = 0.0535 g silver.

SHOE0007 (author)2016-01-09

Perie water. with 480 ppm HCO3. This will make 155 mg

silver carbonate!!

Solubility in water: 0.032 g per L. /10 = 3 mg carbonate at

25 degrees C. Rest insoluble silver carbonate.

Soluble in silver chloride = 250 ug in a 1L. At 25 degrees C.

Extremely insoulbe in water.

Not very effective

25 ml or 0.425 g silver by silver chemcial plating!!

100 g * 0.017% = 1.7 g silver nitrate.

Ag+2(aq) + Cu(s) >> Ag (s) + Cu+2(aq).

AgNO3 >> Ag+ + NO3

Cu(s) + 2AgNO3(aq) → Cu(NO3)2(aq) + 2Ag(s)

1.7 g/169.87 g * 107.8682 g = 1.07 g silver with trace amounts of copper nitrate.

http://evises.evi.com.hk/chem/defaul3/chapter3_1.h...

Copper nitrate

Copper nitrate salt.

so add 1.7 g silver and 20.06 g copper nitrate/2 = 10.03 g

1.07 g silver metal. This is if you use more than 1.7 g of copper metal. Like 2 grams copper. Thus a 1.7 g silver mixed with copper nitrate (10.03 g) as a disinfectant property for 100 ml. Using 5 ml

10.03 g *5/100 g = 0.5 g copper nitrate.

1.07 g * 5 g/100 g = 0.0535 g silver.

SHOE0007 (author)2016-01-09

I have found more evidence that silver salts even if they are not soluble can actually increase the killing properties of disinfectants or they say. To test this I will add 10 ml or 10 g of 0.1 molar solution of silver nitrate to tap water which produces slightly less soluble compounds. Then add 0.5 ml to 5 ml of a 5 g cobalt ammonia chloride solution.

SHOE0007 (author)2016-01-09

I have found more evidence that silver salts even if they are not soluble can actually increase the killing properties of disinfectants or they say. To test this I will add 10 ml or 10 g of 0.1 molar solution of silver nitrate to tap water which produces slightly less soluble compounds. Then add 0.5 ml to 5 ml of a 5 g cobalt ammonia chloride solution.

SHOE0007 (author)2016-01-08

I bought 0.1 molar solution of silver nitrate. It will be mixed with equal lye to produce Silver oxide. Reacting with ammonia will generate silver ammonia hydroxide complex. With salts though it will form silver chloride which is not soluble in water. Will there be an effect of ammonia hydroxide and silver generating silver molecules that may inhibit the growth of bacteria?

SHOE0007 (author)2015-12-06

Here is a picture of a broth of glucose 5 g, 5 g of Knox beef extract and 100 ml of tap water.

SHOE0007 (author)2015-12-06

Graph of data R2 is 0.586 out of 1 which isn't very good and time, and temperature reading with very cold temperatures of water while diluting may have a result. Also I will use more precise tools this is just a rough trial.

SHOE0007 (author)2015-12-06

More data on my project.

Hours 72, Amplified ph 2.6

Hours 97, Amplified ph 3.3

SHOE0007 (author)2015-12-04

I have tested for tolerance of ammonia cobalt chloride solution weak only 20 mg. The broth is made from tap water, Ox powder half a tab, and 5 g of glucose.

Here are the solutions ph of three data points

Time in hours pH of solution amp by 2000.

0 4.30

24 4.10

48 3.00

I need to record data for 72 hrs and 96 hours. 5 data points are needed for good statistics of these values. These will continue. This will be redone with improvements around the probe and tested again to see if I get similar results since each time you do this there is a small error due to pressure and temperature change but it is below the range of error of the ph meter. I will consider it part of the data though.

SHOE0007 (author)2015-12-01

Did I mention that you can properly test ph solutions by dilution factor. I am thinking of diluting a sample by 2000 times. I have only tested weak solutions and not growth of BACTERIA.

willf3 (author)2014-12-28

this is helpful for my research and experiment. thank you

terrefirma (author)willf32015-09-12

Is anyone doing this type of project on a larger scale? I'm pretty sure that if it does work someone will find a way to capitalize on it. I'm a little concerned about the end result whereby a lot of anaerobic bacteria is unleashed. Any thoughts?

sbkenn (author)terrefirma2015-10-03

Anthrax exists naturally in most soil too, just don't come in contact with concentrates

rch121 (author)terrefirma2015-09-18

Well I hope that this kind of project can be done on a larger scale, but that has yet to be seen. Based on how I set up the experiment all of the anaerobic bacteria is the experiment are naturally found in soil. All you do is provide them with an environment to grow up to larger numbers, but since these are naturally occurring there shouldn't be any issues if they were "unleashed" as you described it.

Bstard (author)2015-10-03

Great Experimental work :-)

Good idea to use landfill soil, successful micro-organisms would already be tolerant for toxins / harsh environments.

However has anyone stopped to consider that we ourselves consist of about 18% Carbon, the second most plentiful element in the body. And as stated the adaptive ability of micro-organisms (and the rate at which generations progress) such Carbon hunting Bacteria may adapt to using us as a source, especially as we would provide a better source of all round nutrients and a warm environment. - and to those who say 'then why hasn't that already happened' - the answer is that the bacteria have not been required to find a new sources (of Carbon) due to the abundance already available in nature.

SHOE0007 (author)2015-09-24

I tried this with a ph paper but it was too difficult to read the intervals. I had to buy a brand new ph meter. Then dilute the sample of bacteria by 1:1000. Then for precautions clean the ph probe with 0.1% bleach or 0.05% sodium dichloroisocyanurate solution. Ph probe due the build up of protein requires 1g pepsin in 100 ml of 0.1% Hydrochloric acid.

SHOE0007 (author)2015-09-19

They are both kept at 10 degrees C to prevent abnormal growth and to slow down growth. At room temperature they will be tested again. Pehaps cold temperature and chemicals may have an effect???

SHOE0007 (author)2015-09-19

Hi I have been testing my samples and the one with cobalt salts have a constant ph of 6.5 for 2 days while the Chromium salts Cr3+ are less effective and the ph goes from 6.5 to 6.0 in two days. These are still a bit premature to make an conclusion but it sounds good to me. (This is for resistance or death of bacillus reuteni bacteria).

SHOE0007 (author)2015-09-19

I am thinking of getting a 100 ul pipette so 0.1 ml /100 ml of sample (for diluting solution of metal salt with bacteria could be measured with a ph meter.

Error 0.1 ml/100 ml * 100% = 0.1%

If you don't care that much about accuracy and just want ball park range then 0.5 ml in 100 ml will do.

Error = 0.5%!

SHOE0007 (author)2015-09-18

Another option is to make Potassium or Sodium aluminate by reacting Aluminum with either sodium or potassium hydroxide. Then you can see if the aluminum complex and lye are effective to kill bacteria or will the bacteria build up tolerance.

SHOE0007 (author)2015-09-18

I am think of adding 1 ml of sugar solution diluting it by 200 ml of water to dilute the ph reading.

Then use the amplified ph H+ concentration and amplify it by 200. This may work too.

Example ph 8

H+ = 10^-8. * 200 = 2*10^-6.

ph of 2.00*10^-6 = 5.69!

Daniel Out..

SHOE0007 (author)2015-09-18

I think that you could test the sugar solutions if it is possible to filter bacteria and sugars from samples. This would make it more accurate for use of a pH meter.

Sticky solutions can destroy a ph meter (Even good ones) quickly that is why I use percison ph paper as an alternative.

SHOE0007 (author)2015-09-17

I took bacteria (I found lactobacillus Reuteri) and I am growing them in a medium beef ox extract with 5 ml of the following chemicals: Copper ammonia sulfate, Cobalt ammonia chloride, Chromium ammonia chloride, and Zinc ammonia hydroxide.

Warning:

Both Cobalt and Chromium compounds may be carcinogenic with prolonged exposure ,so handle them with the proper PPE. Copper and zinc compounds are corrosive and may cause burns with skin and eyes. All the chemicals I use here are corrosive.

1 M ammonia hydroxide is added to cobalt chloride, copper sulfate, zinc sulfate, and chromium chloride.

rch121 (author)SHOE00072015-09-18

Hey thanks for the comment. It seems like this experiment you are describing is more about metal tolerance of your Lactobacillus. Is there a link from what you have done to what I did? Thanks!

majesticpegasus. (author)2015-09-07

hello! i'm so glad to have found this page! i wanted make a microbial fuel cell using plastic degrading bacteria but i can't find any anaerobic plastic degrading bacteria, that is, until i found this page! thank you very much for sharing :D

rch121 (author)majesticpegasus.2015-09-18

Glad you came across the page! Where are you doing this research?

willf3 (author)2014-12-28

this is helpful for my research and experiment. thank you

rch121 (author)willf32015-09-18

Thank you! What is your research?

SHOE0007 (author)2015-09-17

I forgot to mention that accurate ph paper 0-13 intervals of 0.5 ph will be used to determine the concentration of ammonia salt and too see if an acid from the sugar Glucose is produced. The level of salt may slow down the process.

willf3 (author)2014-12-28

this is helpful for my research and experiment. thank you

willf3 (author)2014-12-28

this is helpful for my research and experiment. thank you

willf3 (author)2014-12-28

this is helpful for my research and experiment. thank you

willf3 (author)2014-12-28

this is helpful for my research and experiment. thank you

amelia.m.mayer (author)2014-12-25

Greetings! This is an excellent article and ties well with my project in Uganda.

I just have two quick questions, the first might answer the second.
Firstly, I am not a scientist of any sorts, so if these questions seem like the answers are obvious, please excuse my ignorance. What I AM good at is asking questions until I overstand. I'm just a simple grassroots organizer, searching for possibilities to the scourge of plastic on our environment.

Question One: When the plastics have been broken down, does the remnant lose its toxicity? As in, will this byproduct be safe for the environment? How should one go about putting it to use? Can we dump it close to areas we are farming in and not have to worry about any toxic compounds entering into our food source?

Question Two: We are looking at TONS of plastic in Uganda. How easy would it be to upscale this system? Would a simple ratio increase work or is it more complex than that?

I can be emailed with details at dreadymayer@hotmail.com

You can find our project on facebook at "zuukuka!", which is the umbrella organization, and the actual project is called "Projekt Cavella". (Cavella is the local name for plastic bags here.)

Much appreciation for the free spreading of knowledge. If everyone had such a mentality, the world would be much different.

Bless,
DreadyBear

gravityisweak (author)2014-12-08

Great work, this is really cool! Do you have any updates of your own or from people who have contacted you about their own research?

rch121 (author)gravityisweak2014-12-19

Thank you so much! Unfortunately I never got a chance to continue this research in any capacity but quite a few people have talked to me about trying it themselves. However, most haven't gotten back to me after their initial contact. Hopefully that changes at some point but I am just glad so many people have read about this experiment, over 26k now...so crazy!

pmahatta.bokakhat (author)2014-11-20

hello sir. I am a student of msc microbiolgy. And i am very interested in this plastic degradable microbes. If i get chance i will do this expwriment. Thanks for your update. If i have getting prblem i will contact you. And salute for your nice job ...

rch121 (author)pmahatta.bokakhat2014-12-19

Hey. thanks a lot! It's great to hear that you will be giving this experiment a try. I wish you the best of luck and let me know if there is any way I can help!

produ (author)2014-03-25

Did this in college as well; got some cool SEM images of it, too!

rch121 (author)produ2014-12-19

Hey thanks for the comment. Sorry for the ridiculous response time, but any chance you still have those images? I would be interested to see what they look like!

lanerenner (author)produ2014-10-10

hello

lanerenner (author)2014-10-10

ok...

lanerenner (author)2014-10-10

block bacteria!

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