A Novel, Fast, Low-Cost Approach to Achieve Near 100% LDPE Degradation: Phanerochaete chrysosporium Landfill Bioremediation

Summary

Every year, 200 million metric tons of single use plastics are produced. Low-Density Polyethylene (LDPE) is a commonly used plastic and takes over 500 years to break down.

Because current methods such as incineration are unsafe and ineffective, and less than 5.7 percent of LDPE is recycled, my goal is to develop a method to degrade the remaining 95.3 percent in landfills. 

This project's objective is to achieve maximum LDPE degradation without producing toxins in a rapid timeframe for viable landfill implementation.

LDPE was inoculated with Phanerochaete chrysosporium (PC), a white-rot-fungus. Four independent variables were tested: the presence of PC, baking, etching, and the presence of landfill leachate (carbon and nitrogen-rich wastewater). I hypothesized that the combination of these four variables would yield the highest degradation rate, and this was supported by LDPE surface area reduction, CO2 production, and microscopic image data. The experimental group in which LDPE was baked, etched, inoculated with PC, and exposed to synthetic leachate exhibited 99.5 to 99.9 percent degradation rate over 6 days for 50 samples. The results were statistically significant (using one-way-ANOVA) with pvalue < 0.001.

Furthermore, ESI-MS and Carolina Water Quality testing of post-PC-treated leachate showed conversion of ammonia to nitrates and remediation of salts over 12 days.

Further experimentation confirmed the industrial viability of this solution. Its implementation would reduce landfill LDPE waste and leachate treatment expenses.

Future experiments include expansion of this procedure to High-density-polyethylene and Polyethylene-terephthalate and maintaining the viability of pretreatment methods given the landfill waste compaction process.

Question / Proposal

Primary Question: Is it possible to achieve near 100% LDPE degradation without producing toxic byproducts in a rapid timeframe?

LDPE is difficult to break down because of its strong CH2 backbone and highly resilient branching structure. It takes over 500 years to degrade in a landfill and currently, no safe, reliable, and inexpensive methods are available to break it down more rapidly. I found that common white-rot fungus Phanerochaete chrysosporium (PC) degrades lignin, a tough aromatic hydrocarbon with aliphatic side-chains similar in structure to LDPE. Additionally, it has an aerobic metabolism that converts CH2 to COin lignin.

Synthetic leachate (SL) is a major waste problem created when rainwater percolates through a landfill. It is a liquid that contains high amounts of ammonia and carbon, both of which are difficult to remove in wastewater treatment. However, studies indicate that nutrients in SL promote PC growth.

Research shows that baking LDPE rearranges its chains and increases its population of crystallites, making it more brittle. Additionally, chemically etching LDPE roughens its film and increases its surface area, making it more available for fungal anchorage dependency.

Hypothesis: If LDPE is baked, etched, inoculated with Phanerochaete chrysosporium (PC), and exposed to synthetic leachate, then it will degrade more rapidly than it would in the absence of these variables. I predict that leachate will stimulate the growth of PC. PC will break down LDPE which has increased in surface area due to etching and weakened due to baking. This will result in increased LDPE degradation.

 

 

Research

Past research on LDPE biodegradation has included microbes of genus Pseudomonas, Aspergillus, and Lysinibacillus[1,2] which take between 6-24 weeks to degrade 10-30% LDPE. These studies did not use pretreatments to make LDPE more susceptible to degradation. Additionally, the studies’ methods are applicable in a lab setting but not viable in a landfill.

I chose Phanerochaete chrysosporium (PC), a white-rot fungus that has been used in aromatic hydrocarbon degradation[6,7,8]. Although LDPE is aliphatic[9], I wondered if PC could biodegrade its CH2 backbone like it degrades lignin’s CH2 chains in nature.

LDPE Pre-treatment: 

Baking: LDPE consists of long -CH2- repeating chains[9]. Research shows that when LDPE is heated to 110°C - 120°C, there is an increase in crystallites and a rearrangement of its chains[10].

Etching: Research has found that etching creates irregularities on LDPE, greatly increasing its surface area[11].

Baking was chosen as an independent variable because it decreases LDPE tensile strength. I hypothesized that this would make it more susceptible to enzymes[12]. Etching was chosen as independent variable because the increase in surface area gives rise to more sites for fungal anchorage. 

PC's Metabolism: 

PC’s ability to degrade pollutants is related to the production of two enzymes: lignin peroxidase (LiP) and manganese peroxidase (MnP). Under aerobic conditions, these enzymes have high redox potential and produce CO2 and H2O. They catalyze the oxidation of various organic substrates in the presence of H2O2 as an electron acceptor[19].

This enzymatic system of PC is activated during secondary metabolism of fungal growth and is regulated by the availability of nutrients, oxygen, trace metals and pH[16]. (Secondary metabolites are enymatic products that are not fundamental to an organism's primary survival ex. antibiotics).

Background on Landfills:

The image below illustrates the typical landfill. Waste is compacted into “bales”, covered in soil,  and placed in cells.

Presence of Leachate: Landfill Leachate is a wastewater that forms when water percolates through a landfill and mixes with the waste. Treating it can cost landfills up to $600,000 a year. The use of leachate in this project has three significant benefits: its presence simulates the landfill environment, its high Carbon/Nitrogen ratio in leachate greatly enhances fungal growth for LDPE degradation, and PC itself reduces the levels of ammonia nitrogen, soluble chemical oxygen demand (COD) and color in leachate.

There are four phases of microbial activity of which Phase 1 is aerobic[26]. Phase 1 typically lasts around one year, during which aerobic organisms degrade hydrocarbons and release CO2. This stage provides the ideal environment for this project’s implementation.

Landfill temperatures range between 75 and 115 °C in deeper and older cells[25]. These temperatures have sparked interest in channelling landfill heat to above-ground spaces. Baking is a key pretreatment method in this project. It would be feasible to transfer heat from cells 100 feet below to new LDPE bales using pumps.

Overall, this study has impactful implications for the environment and health. It offers an on-site landfill solution that remediates leachate and degrades LDPE at about the same rate as weekly trash pickup.

Method / Testing and Redesign

Experiment 1: Identify group that achieves near 100% LDPE degradation

All experiments were performed at a temperature of 20 degrees Celsius. Potato-dextrose agar was used in each sample.

Constants:

  • Amount of Phaneochaete chrysosporium (PC) used to inoculate each sample
  • LDPE pieces cut from the same bag
  • Composition of Synthetic Leachate (SL)

Control: Untreated LDPE placed in agar

Independent Variables:

  • Inoculation with pure culture of PC
  • Presence of SL
  • Baking
  • Etching

Dependent Variables:

  • LDPE degradation (by surface area change, miscroscopic images, CO2 production)
  • Leachate remediation (by change in chemical composition)

Sample Size: 10 per group

The four independent variables gave rise to 8 experimental groups with 10 samples each when used in different combinations (see Slide 1 below Experimental Method).

Experimental Method:

After preparation, all samples were placed in a petri dish. The observation period was every 6 days for a 12-day period.

Data collection:

The following data was collected in this experiment, after 6 days:

  • Surface Area (using ImageJ)
  • Microscopic Images (taken daily for 6 days)
  • Carbon dioxide Production Measurements (using Pneumatic trough)

The following data was collected in this experiment, after 12 days:

  • Electrospray Ionization Mass spectrometry
  • Ammonia, Nitrate, and Nitrite Concentration Changes over 12 days (using indicator solutions)

Experiment 2: Industrial Viability in a Landfill

The goal of this experiment was to test the industrial viability of this project while mimicking a landfill model. A soil mixture (5:1 ratio subsoil to sawdust) was poured into 500 mL capacity beaker such that it was one-inch deep. 10 mL of synthetic leachate was poured into the soil mixture. LDPE pieces were placed on top of the first layer of soil and 5 mL of the 3 percent citric acid etchant was poured on top. The pieces were subsequently covered by the second layer, as illustrated below:

Control: LDPE alone

Independent Variables: 

  • PC
  • Baking
  • Etching

*Note: Leachate was not an independent variable in this experiment. As it is present in landfills, it was incorporated into the soil mixture.

Dependent Variable:

  • LDPE Degradation (by change in surface area)

Below is the experimental setup of Group 3:

Surface area measurements of all ten plastics in each beaker were taken before adding the second soil covering using an iPhone camera and Image J. The exact location of the LDPE pieces was marked on the side of the beaker by a strip of tape. The three samples were observed for six days and surface area measurements were taken again. The percentage change was calculated.

Safety Measures Followed:

Gloves, a long-sleeved shirt, full-length pants, and protective eyewear were worn while handling PC and synthetic leachate. Oven gloves were worn while handling oven trays. PC cultures were treated with dilute bleach-water solution before disposal. Leachate was placed in Presentation High School's hazardous waste disposal bin. Following experimentation, all work areas and counters were cleaned.

All experimentation was done at the chemistry lab of Presentation High School supervised by Dr. Tracy Hughes. ESI-MS data was collected at Stanford University under the supervision of PhD candidate Ms. Katherine Walker.

 

Results

Experiment 1: Identify group that achieves near 100% LDPE degradation

Surface Area Change: The percentage change in surface area between day 1 and day 6 was calculated. The group in which LDPE was inoculated with PC, exposed to Synthetic Leachate, baked, and etched, henceforth referred to as Group 8, had the highest percentage of degradation at an average of 99.5% reduction over 10 samples. Group 7 (LDPE + PC + Etching) experienced a 61% decrease in surface area. Group 6 (LDPE + PC + Baking) reduced in surface area by 57%. The remaining groups fell under 50% reduction.

One-way ANOVA was done using StatPlus for all 80 samples and showed a p value < 0.001. This statistical significance confirmed that Surface Area measurement using Image J is a good measure of degradation with a high correlation to LDPE degradation. It also established the validity of the results.

Change in LDPE Surface Area Over 6 Days

CO2 production: The rise in the water level of the pneumatic troughs was measured after 6 days. (The magnitude of the rise corresponds to amount of CO2 produced.) Group 8 had the greatest increase in water level at 8 mL. Group 7 rose 4 mL and Groups 4-6 rose 3 mL. The change in water level in Groups 1-3 was 2 mL. Ideal Gas Law calculations confirmed that in Group 8, 5 mL could be attributed to the degradation of CH2 in LDPE and 2 mL could be attributed to the CaCO3 in leachate. The remaining 1 mL was correlated to fungal respiration and/or error in reading.

Microscopic Images: Microscopic images were taken daily using 4x magnification:

Electrospray Ionization Mass Spectrometry: Comparison between Day 0 and Day 12 ESI-MS graphs showed that several peaks have reduced to trace levels or zero. Carbonates, Ammonia, and Chlorides decreased to insignificant levels. Manganese became more soluble. LDPE degradation could be observed in the form of trace levels of organics. Overall, the ESI-MS data clearly shows that PC remediated the leachate and LDPE was degraded.

*Note: While some of the peaks appear to have increased in height for “LDPE + SL + PC Day 12 ESI-MS Data”, this is because ESI-MS provides relative measurements. For example, because all other peaks have decreased in level, Peaks 198.0964 and 203.0518 appear larger.

Ammonia, Nitrate, and Nitrite Levels: Table 4 shows a high presence of Ammonia (over 8ppm) and absence of Nitrates in leachate on Day 0.  After 12 days, PC converted 8 ppm ammonia into 5ppm of benign nitrates. Nitrite levels remained 0 ppm throughout.

Experiment 2: Industrial Viability in a Landfill

Surface Area Change: Surface Area measurements of Group 3 showed a 99.52% reduction in surface area. Group 2 decreased by 14.25% and Group 1, which lacked any pretreatment methods, showed no change.

Conclusion

Both experiments 1 (Group 8) and 2 (Group 3) support the hypothesis that when LDPE is baked, etched, exposed to synthetic leachate, and inoculated with PC, it experiences the highest degradation rate of all combinations. 

In Experiment 1, this group (Group 8) experienced a decrease in surface area of 99.5-99.9%. Degradation was confirmed by an 8 mL rise in water level of the pneumatic trough. This occurred due to degradation of CH2 in LDPE and CaCO3 in leachate into CO2. Trace levels of organics were observed in leachate on Day 12 ESI-MS graphs. Leachate was significantly remediated, experiencing a 69% decrease in ammonia, reduction in salts, and increase in manganese solubility.

In Experiment 2, this group (Group 3) experienced a 99.52 % reduction in surface area under landfill conditions, demonstrating the industrial viability of the solution.

This project was successful in several areas:

1. It achieves near 100% degradation of LDPE in 6 days, which is 600 times faster than current degradation in landfills.

2. It preliminarily remediates leachate by decreasing ammonia, carbonate, and salt concentrations, thereby reducing the environmental and health impact of leachate pollution.

3. It offers a low cost solution that can degrade the annual influx of 21 million metric tons of LDPE and eliminate the activated sludge leachate treatment step, saving large landfills $330 dollars every day.

4. No toxic compounds are produced by this approach.

5. The results are statistically significant with a p value < 0.001.

The data collection methods of this project were confined by financial and feasibility limitations and could be improved if high caliber instruments are available. ESI-MS analyzes all chemical compounds at once. In the pharmaceutical industry, it is very common to study the side products of drugs using large scale separation techniques. Such techniques could be used to further identify specific compounds. However, with the available resources, this project was conducted in the most accurate possible way.

Further experimentation currently being conducted includes expansion of this project to High-Density Polyethylene and Polyethylene terepthalate, both of which belong to the same plastic family as LDPE (polyethylene). Since LDPE in landfills is compacted into bales, my next research question will test whether the landfill waste compaction process (bales) affects the effectiveness of this solution.

This year, 500 billion single use plastic bags will be produced, each with a working life of approximately 15 minutes.

Let's not let them stay for long.

 

 

About me

My name is Shloka Janapaty and I am a junior at Presentation High School in San Jose, California.

My first science fair project in environmental science was inspired by the California drought and investigated the effect of changing watering frequency on moisture retention in soiI. I have done science projects since 6th grade  - testing the conductivity of metals and measuring the pH of soda - but never has science felt more rewarding than when I explore a real-world problem.

My greatest inspiration is marine biologist and environmentalist Rachel Carson, whose book Silent Spring ultimately led to the banning of DDT. To me, she embodies an action-oriented individual who used science to create positive change.

I hope to study environmental microbiology at a research institution and pursue a PhD. I aspire to be a scientist researching ways to remediate waste and reduce pollution.

Winning Google Science Fair would be an incredible honor. I would be humbled by the recognition of my work by such experienced and inspiring scientists and would use the award to further develop my project for implementation in a landfill setting. I hope that if I were to win, more attention would be brought to the important issue of plastic pollution.

In my free time, I love hiking with my family, listening to Latin pop, and working in the art room after school.

Health & Safety

All experimentation was done at Presentation High School. Safety precautions were followed while conducting experiments and collecting data. The following are the health protocols:

  • Gloves, long-sleeved shirts, full pants, close-toed shoes, and protective eyewear were worn during experimentation.
  • Tables and counters were wiped down immediately preceding and following lab work.
  • Oven gloves were worn while placing trays into the oven and taking them out.
  • Protective gloves were worn while handling 3 percent citric acid (not hazardous).
  • All components of leachate were handled by my supervisor, Dr. Tracy Hughes.
  • Sterile techniqe was used when handling Phanerochaete chrysosporium (a Biosafety Level 1 fungus).
  • The Orbitrap (ESI-MS instrument) uses high voltage to ionize compouds. Because it is difficult to operate and is the property of Stanford University, measurements and setup were done by my mentor, Ms. Katherine Walker.
  • Protective gloves was worn when handling indicator solutions (not hazardous).
  • Leachate solutions were disposed of in the school's hazardous waste disposal bin.
  • Fungal culture were pretreated with bleach-water solution before disposal.
  • All nondisposable equipment (forceps, spatulas, etc.) were sterilized after usage.

Dr. Hughes was present during all experiments and data collection.

Adult Mentor Contact Information:

Dr. Tracy Hughes- thughes@presentationhs.org

Ms. Katherine Walker- kwalker7@stanford.edu

 

Bibliography, references, and acknowledgements

Acknowledgments:

I would like to sincerely thank my school, Presentation High School for support, funding, and lab space. I am also especially thankful for my teacher and mentor, Dr. Tracy Hughes, for her technical advice and supervision during experiments.

I am also extremely grateful to my mentor, PhD candidate Katherine Walker (Stanford University), for her explanations of molecular changes in LDPE structure, properties of sawdust as a bioavailable medium, and the Ideal Gas Law. I am especially thankful to her for giving me access to Stanford University's lab and helping me take ESI-MS data (special equipment).

I would also like to thank PhD candidate Nicolette Meyer (Stanford University) for her explanations of anchorage dependency and Dr. John Rowe (CSU Sacramento) for sharing his expert insights in leachate wastewater treatment.  

I sincerely thank Mr. Edward Kaiser of Penn State University for providing a pure culture of Phanerochaete chrysosporium.

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