Earth ScienceEnvironment

Biodegradation of Polyethylene Fragments and Microbeads using Bacillus subtilis and Pseudomonas putida

Abstract

More than 5 trillion plastic pieces – a major contributor being microplastics – are afloat in the world’s oceans[1]. Microplastics are less than five millimeters in length, are problematic to remove using filters, and cause other ecological damages. Over time, multiple morphologies and polymers of microplastics have increased in quantity in San Francisco Bay Water; the highest contributors being polyethylene microbeads and fragments[2][3]. Previous studies have demonstrated that different species of bacteria and fungi can reduce the level of plastics. This investigation examined two species of non-pathogenic bacteria, Bacillus subtilis and Pseudomonas putida, and their impact on the degradation of polyethylene microbeads and fragments. 108 culture tubes were each filled with twenty milligrams of plastic that were submerged in two milliliters of nutrient broth with 100 microliters of bacteria. The cultured tubes were observed over the course of six weeks. The mass of the plastics was measured by wet-sieving, disinfecting, and drying plastics to weigh on analytical balances. The results show significant plastic removal by both bacteria; however, P. putida was able to degrade an average of 13.0 milligrams of microbeads and an average of 2.3 milligrams of polyethylene fragments. B. subtilis removed an average of 8.0 milligrams of microbeads and an average of 1.7 milligrams of polyethylene fragments. This investigation has shown that the species of bacteria are able to reduce the level of microplastics.

Introduction

Locally, microplastics pose a huge threat as over the course of the years, microplastics have increased in quantity in San Francisco Bay Water[4]. Microplastics are plastic that are less than five millimeters in length. Some microplastics can be found as exfoliants in health and beauty products such as some cleansers and toothpastes[5]. Although President Obama signed the Microbeads-Free Waters Act of 2015, banning plastic microbeads in rinse-off exfoliating products, microplastics still show up in many places. They are not only able to pass through Bay Area wastewater treatment plants, even when the most advanced technologies are used, but can also absorb and give off chemicals and harmful pollutants[6][7]. Microplastics may also carry disease-causing organisms and act as a vector for diseases in the environment, resulting in “a range of potentially harmful effects on the species that drink the water”[8]. Polyethylene, one of the many polymers of microplastics, is made of long stable chains of carbon and hydrogen, is the most widely used plastic in the world; it is used in food wrap, shopping bags, detergent bottles, synthetic fibers, and more. Existing solutions have been identified to reduce microplastics including the production of bio-components (living components that affect other organisms or shape the ecosystem) and the use of multiple species of bacteria and fungi[9] as sources of degradation. However, few studies have compared two different types of bacteria in their degradation capabilities, or targeted a specific polymer and morphology. Before biodegradation occurs, physical degradation happens due to photodegradation and other weathering processes. The following are the steps in biological degradation[10]:

  1. Bio-deterioration: action of microbial communities responsible for physical and chemical deterioration
  2. Bio-fragmentation: enzymes secreted by bacteria, cleave polymeric plastics into simpler substances
  3. Assimilation: simpler substances integrated as a Carbon source into microbial metabolism
  4. Mineralization: degradation of polymer resulting in oxidized metabolites

There are also specific metabolic pathways involved in polyethylene (PE) degradation. Bacteria secrete oxygenases (enzymes capable of adding oxygen to long carbon chains) which bind to the PE substrate (lock and key model) to catalyze the oxidation process. The oxidation breaks up the PE and forms simpler carboxylic molecules. An ATP binding cassette (ABC) incorporates the carboxylic molecules into the bacteria. Next, further broken down molecules enter the energy cycle where most of the carbon in the PE produces chemical energy. This energy produces the ATP necessary to create a new biomass. The remaining carbon in the PE is mineralized into CO2 and releases H2O[11]. With this research, it was hypothesized that a change in the species of bacteria used would alter the rate of degradation of microplastics. This investigation examined two species of non-pathogenic bacteria, Bacillus subtilis[12][13][14] and Pseudomonas putida[15][16][17], and their impact on the degradation of polyethylene microbeads and fragments.

Methods

To begin the experiment, record the weight and size of the different plastic pieces. Separate the plastic pieces into groups according to morphology (fragment, foam, spheres, film, fibers, and nurdles). In this experiment, the selected morphologies were spheres and fragments. Prepare cultured tubes, adding the same amount of bacteria, broth, and plastics. A ratio of 50 ul:1 ml:10 mg was used. Place both bacteria into their respective petri cultured tubes. For the control tubes, add the same amount of broth and plastics. Label the tubes according to trial number, morphology, and bacteria. Each week, record visual observations of bacterial growth and plastic degradation. At the end of each week, record observations of the plastic: record the analytical weight of the dry plastics from each cultured tube by (1) pouring all of the substance in the cultured tube into the sieve and spraying it with alcohol, (2) placing the plastics into a watch glass and into the drying oven, (3) using analytical balances and weigh boats to weigh the plastic and recording the weight. After, dispose of that week’s cultured tubes. Each week repeat the measuring steps according to the week number on the cultured tube. At the end of six weeks, compare the initial results to the final results.

Data Analysis

Overall, P. putida was able to degrade more of the plastics (both fragments and beads) quicker than B. subtilis.

Bead Data Analysis: P. putida began to degrade the beads at week 1 measurements; whereas B. subtilis only began to degrade the plastics at week 2 measurements. Both bacteria continued to degrade the plastic over the course of the project. At the end of the project, B. subtilis degraded 51.50% and P. putida degraded 73.50% of the plastics. B. Subtilis degraded the plastics at a rate of -0.0003 g/week and P. putida degraded the plastics at a rate of -0.0004 g/week. With a constant rate of degradation, B. subtilis is expected to completely degrade the microbeads around 3 weeks later than P. putida.

Fragment Data Analysis: B. subtilis and P. putida both started to degrade the plastics at week 2 measurements. At Week 3, P. putida degraded more plastics and B. subtilis stayed the same. At the end of the project, B. subtilis degraded 11.50% and P. putida degraded 13.50% of the plastics. B. subtilis degraded the plastics at a rate of -6×10-5 and P. putida degraded the plastics at a rate of -8×10-5g/week. With a constant rate of degradation, B. subtilis is expected to completely degrade the fragments around 12 weeks later than P. putida. The results on Day 28 show a switch which could be due to an error in measurement with the analytical balance.

Comparison of Data to Existing Results

Table 1. Comparative Studies over 30 days
B. subtilis P. putida
Self Vimala[18] Notes on Vimala Self Kathiresan[19] Notes on Kathiresan
11.67% (fragment) 3.53% UV treated and biosurfactants

 

Film – choosing 41u HDPE vs fragments

33.35% (beads)

 

11.65% (fragment)

20.54% Unknown environment and morphology

Conclusion

The hypothesis was supported because the change in bacteria did change the extent of PE degradation. P. putida was able to degrade 2% more fragments and 22% more beads than B. subtilis. If microplastics are reduced on a larger scale, this could lead to safer ecosystems, potable water supply, safer food supply, and marine organisms would not be harmed by plastics. This investigation has shown that the species of bacteria B. subtilis and P. putida are able to reduce the levels of microplastics through biological degradation, and that this process has practical applications in reducing the global level of microplastics in our oceans.

References

(1) Eriksen, Mark, Laurent C. M. Lebreton, Henry S. Carson, Martin Thiel, Charles J. Moore, Jose C. Borerro, Francois Galgani, Peter G. Ryan, and Julia Reisser. 2014. “Plastic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea.” PLOS ONE: Public Library of Science. https://doi.org/10.1371/journal.pone.0111913.

(2) Eriksen, Mark, Laurent C. M. Lebreton, Henry S. Carson, Martin Thiel, Charles J. Moore, Jose C. Borerro, Francois Galgani, Peter G. Ryan, and Julia Reisser. 2014. “Plastic Pollution in the World’s Oceans: More than 5 Trillion Plastic Pieces Weighing over 250,000 Tons Afloat at Sea.” PLOS ONE: Public Library of Science. https://doi.org/10.1371/journal.pone.0111913.

(3) Sutton, Rebecca. 2016. Microplastic Contamination in San Francisco Bay – Fact Sheet. 2015, Revised 2016. SFEI Contribution No. 770.

(4) Sutton, Rebecca. 2016. Microplastic Contamination in San Francisco Bay – Fact Sheet. 2015, Revised 2016. SFEI Contribution No. 770.

(5) US Department of Commerce, National Oceanic and Atmospheric Administration. 2016.

“What Are Microplastics?” NOAA’s National Ocean Service. https://oceanservice.noaa.gov/facts/microplastics.html.

(6) San Francisco Estuary Institute. 2019. “The Pulse of the Bay.” Regional Monitoring Program for Water Quality in San Francisco Bay.

https://www.sfei.org/sites/default/files/biblio_files/The%20Pulse%202019%2010_8_19%20print%20version%202.0%20medres.pdf

(7) Sutton, Rebecca, Diana Lin, Meg Sedlak, Carolynn Box, Alicia Gilbreath, Rusty Holleman, Liz Miller, et al. 1970. “Understanding Microplastic Levels, Pathways, and Transport in the San Francisco Bay Region.” San Francisco Estuary Institute. http://www.sfei.org/documents/understanding-microplastics.

(8) UN Environment Programme. 2018. “Plastic planet: How tiny plastic particles are polluting our Soil.” UN Environment Programme. https://www.unenvironment.org/news-and-stories/story/plastic-planet-how-tiny-plastic-particles-are-polluting-our-soil.

(9) Sutton, Rebecca. 2016. Microplastic Contamination in San Francisco Bay – Fact Sheet. 2015, Revised 2016. SFEI Contribution No. 770.

(10) Jacquin J, Cheng J, Odobel C, Pandin C, Conan P, Pujo-Pay M, Barbe V, Meistertzheim AL and Ghiglione J-F. 2019. “Microbial Ecotoxicology of Marine Plastic Debris: A Review on Colonization and Biodegradation by the “Plastisphere.” Front. Microbiol. doi:10.3389/fmicb.2019.00865

(11) Jacquin J, Cheng J, Odobel C, Pandin C, Conan P, Pujo-Pay M, Barbe V, Meistertzheim AL and Ghiglione J-F. 2019. “Microbial Ecotoxicology of Marine Plastic Debris: A Review on Colonization and Biodegradation by the “Plastisphere.” Front. Microbiol. doi:10.3389/fmicb.2019.00865

(12) Claire Dussud and Jean-Francois Ghiglione. 2014. “R63 La Dégradation Des Plastiques En Mer, Par C. Dussud Et J-F. Ghiglione.” Société Française d’Ecologie. http://www.sfecologie.org/regards/2014/12/26/r63-plastiques-en-mer-dussud-et-ghiglion/.

(13) Augustyn, Adam, William Hosch, and Gloria Lotha. 2019. “Polyethylene.” Encyclopædia Britannica. http://www.britannica.com/science/polyethylene#ref283104.

(14) EPA.gov. 1997. “Attachment I – Final Risk Assessment of Bacillus Subtilis.” Environmental Protection Agency. https://www.epa.gov/sites/production/files/2015-09/documents/fra009.pdf.

(15) Vimala, P.P., and Lea Mathew. 2016. “Biodegradation of Polyethylene Using Bacillus Subtilis.” Procedia Technology.

http://www.sciencedirect.com/science/article/pii/S2212017316301153.

(16) Al-Jaillawi, Majid, and Rasha Ameen. 2015. “(PDF) Polyethylene Degradation by Pseudomonas Putida S3A.” ResearchGate. http://www.researchgate.net/publication/283459113_Polyethylene_degradation_by_Pseudomonas_putida_S3A.

(17) EPA. 2019. “TSCA Environmental Release Application (TERA) for Pseudomonas Putida (P. Putida).” Environmental Protection Agency.

https://www.epa.gov/regulation-biotechnology-under-tsca-and-fifra/tsca-environmental-release-application-tera-1

(18) Vimala, P.P., and Lea Mathew. 2016. “Biodegradation of Polyethylene Using Bacillus Subtilis.” Procedia Technology.

http://www.sciencedirect.com/science/article/pii/S2212017316301153.

(19) Kathiresan, K. 2003. “Polythene and Plastic-Degrading Microbes in an Indian Mangrove Soil.” U.S. National Library of Medicine.

http://www.ncbi.nlm.nih.gov/pubmed/15162769.

About the author

 Nithika Karthikeyan is a current junior at Notre Dame High School in San Jose. Over the past couple years, she has deepened her interest into marine biology and finding solutions for marine plastic pollution. She hopes to pursue a career in Marine Biology and Environmental Science and Policy, by attending law school.

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