Evolution of Plastic Degrading Bacteria

Project Title

Evolution of Plastic Degrading Bacteria

Project Area of Specialization Biomedical EngineeringProject Summary

Plastic pollution is one of the biggest environmental problems of the present time. Millions of tons of disposable one-use plastic has accumulated in our lands and seas due to its slow degradation rate. It is carcinogenic and it takes around 500 to 5000 years to degrade.

In 2016, a group of researchers led by Kohei Oda of Kyoto Institute of Technology and Kenji Miyamoto of Keio University discover Ideonella Sakaiensis (a plastic eating bacterium) originally isolated from a sample of PET-contaminated sediment near a plastic recycling facility in Sakai, Japan.

Being inspired by the above mentioned researchers, our objective is to evolve such a bacterium which has the ability to degrade PET plastic. The bacteria which are considered to evolve as the plastic degrading bacteria are those which do not have endospore formation process. The specification is due to the reason that the bacteria with endospore formation encapsulate their cell organelles into a capsule when there is lack of nutrition and do not consume unfavorable nutrition. Such bacteria are usually gram-positive.

We are supposed to gradually decrease the nutritional supply of the sample bacteria population and provide them culture medium such as agar added with plastic nano-particles. As bacteria has the ability to survive in all sorts of conditions, we hypothesize that in a struggle to survive, some of the sample bacteria population will learn to consume the PET plastic contaminated culture medium and then on PET plastic itself and consequently release such enzymes which can help to degrade plastic. The enzymes PETase and MHETase will be released by the bacteria to help in the breakdown of PET plastic nano-particles as expected. These enzymes will help to break the long chains of PET plastic particles into shorter chains or monomers.

We plan to iteratively culture and reculture the select population of bacteria that starts producing such enzymes, in a medium with greater proportions of PET plastic nano-particles, untill we have a population of evvoled bacteria that can thrive on PET plastic only. 

This will help to increase the rates of decaying activity by increased enzyme release by the bacteria. Bacteria with no endospore formation volunteers in this process because it has the ability to survive in all sorts of conditions and low nutritional environments. To obtain it’s nutrition in the conditions of decreased growth medium the gram-negative (or bacteria with no endospore formation) try their level best for survival. The cultured sample will be provided with PET plastic nano-particles in increasing proportions. With time, the bacteria will start to degrade PET plastic nanoparticles for obtain their nutrition. This is how bacteria can help to degrade PET plastic. This can be implemented on a large scale as a solution to the increasing plastic waste threat.

Project Objectives

The following are the objectives to be achieved.

1. Cultivate (culture) and Evolve plastic degrading bacteria in a laboratory environment.
2. Work out techniques of large scale but controlled culture of the bacteria that can help degrade PET plastic and can function in atmospheric conditions akin to Pakistan.
3. Identify environmental and health hazard that may possibly be caused by such bacteria.

Project Implementation Method

Creating a laboratory environment that provides favorable conditions for the bacteria to thrive on plastic

This can be made possible by altering the nutritional intake of the bacteria by gradually increasing the amount of nanoparticles of PET plastic into the bacterial sustenance. We will observe that do the bacteria alters its chemical properties to help decompose plastic.

We will use gram-negative bacteria for this purpose as these bacteria do not have endospore formation. Such bacteria have the ability to alter their nutritional intake according to the provided growth media and try their best to strive. It is therefore expected that slowly and gradually the gram-negative bacteria will start consuming PET nanoparticles as it has the ability to adapt to all sorts of nutritional environment. Regularly providing PET nanoparticles will facilitate the bacteria to produce such chemicals/enzymes which will assist in the breakdown of plastic nanoparticles. The enzymes being produced may include MHETase and PETase (as expected) which will empower with time to work along with long chained particles, and if yes, we will find the decomposition rate which it provides. We will also find if there is a production of any toxic material during the process.

Steps to be followed:

1. Get a petri dish and an incubator.
2. Make up  the agar mixture.
3. Fill up the petri dish with agar nutrition solution.
4. Let the solution cool and harden for around an hour.
5. One can use a cotton swab to collect bacteria, and place it on the petri dish.
6. Cover the lid for safety purposes. You can even visualize the growth.
7. Place the petri dishes in the oven or incubator (temperature about 37 degree C and pH around 6.6 to 7.5).
8. Give them enough time to grow (around 2-7 days).
9. Inoculating loop can be used to pick the growing bacteria up for further analysis.
10. In the next culture, use the bacteria from the grown colony.
11. Do the procedure for 3 different types of Gram-Negative bacteria.
12. Keep adding the amount of PET plastic nanoparticles in increasing proportions in every next culture.
13. The amounts of PET nano-particles added, and later the leftover quantity in each batch will be noted. The Electron microscope will be used to examine the changes In the provided and the leftover nutritional samples to examine the bacterial activity.
14. The results will be compared after each trial batch.   
15. Repeat the process till the bacteria thrive completely on plastic.

Benefits of the Project

Some of the benefits that can be achieved from the implementation of this project are:

• It offers clean solution to the problem of plastic waste accumulation.
• There are prospects of implementing it on large scale, which can be used for public health sector.
• It is the most environment-friendly process of plastic disposal.
• It is highly economical solution to the deadliest threat of PET plastic accumulation.

Plastic pollution is one of the gravest environmental problems facing the Earth. Millions of tons of disposable one-use plastic have accumulated on the land, in the landfills, and in our oceans due to its extremely slow degradation rate. It takes around 500 to 5000 years to degrade. Our project will help to degrade the PET plastic in comparatively less time than the natural time required. 

It can be very helpful to control marine pollution and help to save sea life as PET plastic accommodation is highly toxic for marine life as it is adding up into our food chain, as these marine species are later being utilized by humans. The consumption of such infected edible marine products is causing the production of chemicals like Bisphenol A and Dioxin which are really dangerous and even carcinogenic for human health. Proper disposal technique of PET plastic can help save the ecosystem. 

Technical Details of Final Deliverable

At the end of our project, we expect to have samples of gram-negative bacteria which have evolved themselves as plastic degrading bacteria.

We will then find out the time required for such bacteria to hydrolyze the long chains of PET plastic polymers into smaller chains or monomers. The amount of  sample being hydrolyzed will also be noted. And hence, we will find out the time estimate of degradation of a given amount of PET bottles in usual use with a given number of bacteria.

All this is aimed at developing a practically viable and environment friendly large scale facility of PET disposal.

Final Deliverable of the Project Hardware SystemCore Industry HealthOther Industries Others Core Technology Clean TechOther Technologies OthersSustainable Development Goals Good Health and Well-Being for People, Clean Water and Sanitation, Sustainable Cities and Communities, Life Below Water, Life on LandRequired Resources

Item Name	Type	No. of Units	Per Unit Cost (in Rs)	Total (in Rs)
			Total in (Rs)	80000
Electronic Microscope	Equipment	1	49000	49000
Laboratory glassware	Equipment	34	170	5780
Culture media	Equipment	2	7610	15220
Disposibles	Miscellaneous	2	500	1000
Transport	Miscellaneous	20	300	6000
Print	Miscellaneous	3	1000	3000