October 2013 Volume 3, Issue 1

Effect of Phanerochaete chrysosporium, Pseudomonas putida, and Sphingomonas macrogoltabidus on the Degradation of HDPE Plastic with Chemical, UV, and Thermal Pre-treatments

Cara Broshkevitch1*, Anne Richards1*#, and Jacqueline Curley2
Student1, Teacher2:  The Loudoun County Academy of Science, 21326 Augusta Drive, Sterling, VA 20164 Virginia
*These authors contributed equally
#Corresponding author:  anneelizabeth.722@gmail.com

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Cited by: Mukherjee, S., & Chatterjee, S. (2014). A comparative study of commercially available plastic carry bag biodegradation by microorganisms isolated from hydrocarbon effluent enriched soil. Int. J. Curr. Microbiol. App. Sci, 3(5), 318-325.

Abstract

High-density polyethylene plastic (HDPE) is used for many everyday items such as grocery bags: the top consumer item in the world.  When disposed in landfills, this plastic takes over 100 years to degrade, filling up the landfill space, slowing the degradation of other substances, and blocking groundwater collection 1.  Phanerochaete chrysosporium fungus and the bacteria Pseudomonas putida and Sphingomonas macrogoltabidus have been shown to biodegrade low-density polyethylene plastic in a natural environment, and in 2012, research by our group established that these microbes could also use high-density polyethylene plastic as their sole carbon source4.  Biodegradation rates were also found to be higher when exposed to multiple microorganisms at once rather than individual microorganisms.  Ratios of P. chrysosporium fungus, P. putida bacteria, and S. macrogoltabidus bacteria have been manipulated to maximize the biodegradation of plastic pretreated with UV and thermal radiation.  It was predicted that the optimum combination of microorganisms would include P. putida bacteria since past research identified it as the major factor of biodegradation4.  In addition to the pretreatment of UV and thermal radiation, a second pretreatment using manganese stearate and a shorter thermal radiation was investigated to identify a pretreatment requiring less electricity.  Three ratios (½ P. putida, ½ P. chrysosporium; ½ P.  putida, ½ S. macrogoltabidus; ¼ P. putida, ½ S. macrogoltabidus, ¼ P. chrysosporium) from the three month long trials consistently degraded more than the other four ratios and the control.  An ANOVA test run on the 3-month data indicated that the degradation by the three ratios was statistically significant when compared to the control.  Also, manganese stearate pretreated plastic had a significantly higher degradation than UV pretreated plastic, as shown by Kruskal Wallis tests for five of the seven ratios.  Using the most efficient ratios and manganese stearate pretreatment process, the degradation time of HDPE plastic could be cut from over 100 years to 1 year.

Introduction

High-density polyethylene (HDPE) (#2) plastic has applications in products such as food packaging, plastic bags, plastic bottles, and pipes.  As the most commonly produced plastic, around 140 million tons of HDPE are utilized each year1.  HDPE’s durability and high melting point make it useful, but also very hard to break down.  It can take HDPE plastic over one hundred years to degrade in a landfill1.  Although some HDPE plastic is processed, recycled HDPE represents only 5% out of 1 trillion plastic bags produced annually in the United States1. These discarded plastics are filling up landfills and, as the population of our country grows simultaneously, the United States is running out of places to put this plastic waste1.

Two bacterial strains, Pseudomonas and Sphingomonas, have been discovered to digest HDPE; their metabolisms reducing it into heat, H2O, CO2, and biomass2.  This may provide a solution to the overflowing of landfills with plastic waste.  Past research isolated Pseudomonas putida from sludge in industrial waste and determined that its growth utilized o-chloronitrobenzene (o-CNB), an organic raw material, as its only carbon, nitrogen, and energy source3.  Most importantly, the highest degradation of o-CNB (85%) by P. putida was found to be at 32°C and at a pH of 8.0, the living conditions found in nature3.  Since HDPE plastic is composed of carbon like o-CNB, these findings can be translated to experiments using a high-density polyethylene polymer3.  In 2012, Broshkevitch and Richards found that P. putida and S. macrogoltabidus bacteria grow both individually and synergistically using HDPE plastic as a sole carbon source. However, the addition of vegetable starch as another carbon source has been shown to stimulate their metabolic activities further and, therefore, the rate of degradation of the HDPE plastic5.

HDPE plastic is made up of polymers with C-C single bonds (-CH2-CH2-)n.  When a plastic degrades, these polymer bonds break.  Past research has investigated the use of bacteria and fungi to facilitate this degradation process by breaking up the plastic polymers for energy.  This plastic degradation is a two-step process: first, plastic reacts with oxygen from the air, and second, biodegradation of products from this oxidation occurs2.  In one study, samples of polythene and other plastics were gathered from pollution sites such as petroleum refilling centers, industrial and construction sites, and locations in proximity to automotive businesses. Pseudomonas bacteria were found degrading these discarded plastic samples, and the strain was found to be easily cultured for continued research purposes6.  Pseudomonas’s ability to degrade polyethylene and other polymer types in a natural environment suggests that it would be able to degrade high-density polyethylene plastic in a laboratory6.

Bisphenol A is the main ingredient in polycarbonate plastic.  Although itself incapable of degrading bisphenol A, Pseudomonas was found to accelerate the degradation of bisphenol A by the Sphingomonas bacterial strain, suggesting symbiosis between the two strains7.  This research suggests that Sphingomonas may also be capable of directly degrading plastic.  When Sphingomonas was given a nutrient supplement, degradation of the bisphenol A was accelerated from 40 days to only 20 hours, further supporting this theory7.  Researchers are currently investigating the symbiotic relationships between Pseudomonas and Sphingomonas bacteria involved in this plastic degradation7.  The bacteria are so similar, however, that there are numerous instances of species of Pseudomonas being moved to the genus Sphingomonas, suggesting growing compatibility between the two strains, or misidentification of Sphingomonas strains as Pseudomonas in past investigations8.  In fact, Broshkevitch and Richards found that HDPE plastic had a greater degradation rate when the P. putida and S. macrogoltabidus bacteria were grown synergistically on HDPE than when grown individually4.

Research has also determined that Phanerochaete chrysosporium, commercially known as white-rot fungus, is able to degrade UV and thermal treated bisphenol A polycarbonate. Previous research has shown approximately a 5.4% weight loss in the polycarbonate and suggested that UV and thermal pretreatment enhanced the degradation of the plastic9.  P. chrysosporium has also been found to increase natural degradation of polyethylene films in soil.  This finding suggests that the fungus is capable of digesting a variety of plastic types2. Different strains of fungi continue to be researched for their plastic degradation properties. Other researchers have found that lignin-biodegrading fungi contribute to the degradation of polyethylene1.  Since P. chrysosporium also digests lignin, this research provides further support that it will metabolize HDPE plastic1.  Broshkevitch and Richards found that P. chrysosporium fungus could grow both individually and synergistically with bacteria using the HDPE plastic as its sole carbon source4.

Currently, research is being conducted on factors maximizing bacterial degradation of plastic. Abiotic degradation involves physical and chemical processes which change the intramolecular structure of the plastic’s polymers.  Thus, abiotic degradation will cause photo- or thermo-oxidation to the plastic’s polymers when they are exposed to UV rays or heat, respectively.  This process has been supported by past research, showing increased degradation in low-density polyethylene (LDPE) after an increased exposure to UV radiation1.  Since HDPE is more durable than LDPE, such pretreatment with UV light and heat is likely to accelerate degradation1. Polymers are susceptible to radiation throughout the UV, visible, and infrared light spectra10. More specifically, HDPE has been shown to have rapid degeneration under a UV-A lamp of 340 nm at 50°C (a UVA-340 lamp emits light from 300-400 nm)2.  Significant degradation was shown to occur after UV exposure times greater than 96 hours11; 12.  Overall, pretreatment using UV and thermal radiation has been shown to increase biodegradation of plastic because the pretreatment increases the surface area available for microbial colonization and decreases the molecular weight through photo- or thermo-oxidation13.  This decrease in molecular weight makes the plastic more accessible for microbial metabolization by reducing the polymeric chain size of the plastic14.  

In addition to UV and thermal pretreatment, pro-oxidant additives have been shown to increase biodegradation of polyethylene.  In one investigation, the samples were soaked in a pro-oxidant solution including manganese (II) stearate.  The pro-oxidants acted as a photo inducer, speeding up the degradation process by initiating a free radical chain reaction which began to break down the plastic15.  Low molecular mass oxidation products were also produced, modifying the hydrophilic surface of the plastic15.  The decreased molecular weight makes the carbon-chain backbones available to the microorganisms, creating a better environment for their growth16.  Manganese stearate pretreatment resulted in a greater degradation of the plastic when compared to pretreatment using other pro-oxidants in the pretreatment15.  Past researchers have exposed LDPE plastic to the manganese stearate for two days15, used a 1% pro-oxidant solution for the pretreatment16, and found that manganese stearate increases sensitivity of plastic to thermal degradation2.

Current research investigates the process of cultivating bacteria on the plastic’s surface1.  A biofilm on the plastic’s surface is the most efficient method determined thus far1.  It’s also been found that microbial species secrete an adhesive which fills in pores on materials to help the species stick17.  Due to the slow process of biodegradation, the biofilm is required to be active over a long period of time.  An investigation with B. sphericus and B. cereus, two bacteria less commonly used for investigation of the biodegradation of plastic, found a three and a half percent decrease in mass of HDPE in one year14.  This degradation was increased to nine percent with UV pretreated HDPE, supporting the idea that pretreatment increases biodegradation of plastic14.  Since B. sphericus and B. cereus have not been isolated at natural degradation sites, these low degradations of HDPE may also have been due to low degradation abilities of the bacteria themselves.  

Physical signs of plastic degradation include roughening of the surface, formation of cracks, and formation of biofilms.  As microorganisms start to grow on plastic, they first grow in the crevices in the plastic, facilitating visual identification of degradation5.  The presence of carbon dioxide, a product of biodegradation, is also an indicator of HDPE degradation.  Therefore, when bacteria are grown on plastic in the minimal media, CO2 tests are often used to verify this degradation10.  These tests indicate that the bacteria are digesting and being sustained by the plastic.  In 2012, researchers Richards and Broshkevitch completed carbon dioxide tests that showed statistically significant differences between the carbon dioxide levels measured in a flask containing the microorganism, a flask containing only M63 minimal media, and by a probe left to measure CO2 room levels4.  This showed that the microorganisms could grow individually on the plastic, using the polymer as the microbes’ sole carbon source.  

In the study presented here, we show our findings on accelerated degradation by a combination of P. putida and S. macrogoltabidus bacteria, and P. chrysosporium fungus. Pretreatment process was varied, using two different procedures as follows:  (1) UV radiation at 360 nm for 144 hours and thermal radiation at 115°C for 48 hours, (2) exposure to manganese stearate for 48 hours and thermal radiation at 115°C for 1 hour.  The second pretreatment procedure was designed to use less electricity to decrease any negative environmental impact. The plastic was exposed to combinations of P. putida and S. macrogoltabidus bacteria, and P. chrysosporium fungi for 1 month and 3 month periods.  The ratio of the three species was varied to determine the best combination resulting in the greatest percent decrease in HDPE mass.  Each of these ratios was tested for viability by plating the organisms after application to the plastic, and checking for growth of each species. Degradation rates were also compared to determine which pretreatment process causes the greatest amount of degradation of the plastic.  

The purpose of the investigation was to determine the optimum procedure for large-scale biodegradation of plastic providing a more environmentally friendly alternative to landfills. There has been little research with data on the relationship between the manganese stearate pretreatment and the degradation of the plastic.  Moreover, the trials with UV and thermal pretreatment lasted 3 months versus a previously shorter duration time for similar pretreatment levels.

It was hypothesized that if the ratios of P. chrysosporium fungus, and the bacteria P. putida and S. macrogoltabidus, were varied, then a maximum decrease in HDPE mass would be obtained. It was also hypothesized that if the pretreatment process was changed from UV and thermal to manganese stearate and thermal, then microbial biodegradation would increase and be more environmentally friendly, because manganese stearate acts as a thermo-inducer and reduces the pretreatment’s electricity requirement.

Materials and Methods

Pseudomonas putida (ATCC Catalog Number 4359), Sphingomonas macrogoltabidus (ATCC Catalog Number 51380), and Phanerochaete chrysosporium (ATCC Catalog Number 34541), all with a biosafety level of 118, were rehydrated following the ATCC protocols.  The microorganisms were then incubated: P. chrysosporium fungus at 24°C, P. putida bacteria at 26°C, and S. macrogoltabidus bacteria at 30°C. The two bacteria were subcultured throughout the investigation by adding an inoculating loop full of culture to a test tube with 12 mL of the nutrient broth.  The fungus was subcultured by cutting of a small piece of fungal growth with a scalpel and placing it in the new test tube to grow.  Every 2 weeks throughout the experiment, new cultures were established and maintained for future use.  Subcultures of P. putida bacteria and P. chrysosporium fungus were kept at room temperature, and subcultures of S. macrogoltabidus bacteria were kept in the incubator.  After the new cultures had been established, the original cultures were moved to the refrigerator to slow growth.  

The optical density of each bacteria culture was measured at 540 nm, and diluted or concentrated until an optical density of 0.44 AU for P. putida and 0.46 AU for S. macrogoltabidus was obtained12.  A scalpel and sterilized pick were used to remove the film of P. chrysosporium fungus under the laminar flow hood.  This film was placed on a weigh boat, and brought to the appropriate mass (See Table 1).

 

Microorganism Ratios and Abbreviations

Ratio of Microorganisms

Abbreviation

Control (no M63 or microorganisms, just HDPE plastic)

C

1/2 P. chrysosporium (0.25 grams), 1/2 P. putida (2.5 mL)

HPP-HPC

1/2 P. chrysosporium (0.25 grams),1/2 S. macrogoltabidus (2.5 mL)

HS-HPC

1/2 P. putida (2.5 mL), 1/2 S. macrogoltabidus (2.5 mL)

HPP-HS

1/3 P. chrysosporium (0.17 grams), 1/3 P. putida (1.66 mL), 1/3 S. macrogoltabidus (1.66 mL)

Third

1/2 P. chrysosporium (0.25 grams), 1/4 P. putida (1.25 mL), 1/4 S. macrogoltabidus (1.25 mL)

HPC

1/4 P. chrysosporium (0.125 grams), 1/2 P. putida (2.5 mL),1/4 S. macrogoltabidus (1.25 mL)

HPP

1/4 P. chrysosporium (0.125 mL), 1/4 P. putida (1.25 mL), 1/2 S. macrogoltabidus (2.5 mL)

HS

Table 1. Volume of bacteria and mass of fungi added to flasks for each combination of microorganisms.

 

54 samples of HDPE plastic were cut from a grocery bag in 0.25-gram increments, using an analytical balance.  Strips with a 12 cm width were cut and then the length adjusted to maintain a relatively equal shape for all samples.  Each of the 54 HDPE samples was exposed to UV radiation with a wavelength of 365 nm2 for 144 hours12 using a UVA lamp set up 20 cm above the plastic.  The samples were then exposed to thermal radiation at 115°C for 48 hours13 using an oven, and massed after the completed pretreatment process.

Each of the 54 HDPE samples of plastic was sterilized.  45 mL of pre-prepared M6321 were then poured into 52 of the 54 Erlenmeyer flasks.  The appropriate volume of P. putida bacteria at an OD of 0.44 AU was pipetted into each of the flasks 1-49.  The appropriate volume of S. macrogoltabidus bacteria at an OD of 0.46 AU was added to each of the flasks 1-49.  Tweezers were used to drop the appropriate grams of fungi into flasks 1-49 (See Table 1) (throughout the investigation, the fungi was assumed to keep a relatively constant number of cells as long as the newest subcultures were used to avoid spores).  No microorganisms were added to the M63 minimal media in flasks 50-52.  No microorganisms or M63 minimal media were added to flasks 53-54.  Placement of the microorganisms was as follows: P. putida, S. macrogoltabidus, P. chrysosporium, flasks 1-49; M63 minimal media control, flasks 50-52; and NO M63 minimal media control, flasks 53-54.  There were 7 flasks per combination of the three microorganisms (See Table 1 for combinations).

The HDPE plastic samples were exposed to the microorganisms for 3 months.  After these three months, the HDPE plastic samples were removed and soaked in bleach for 1 hour, soaked in distilled water for 30 minutes, and then rinsed gently with a pipette and tap water.  A cell scraper was used to scrape off as much bacteria as possible still adhering to the plastic samples.  The plastic was treated as if it was bacteria (since although dead, not all of the bacteria were probably scraped off the HDPE) when it was saved for further observations.

The dry mass of each sample was then measured using the analytical balance.  The percent change in dry mass of the plastic was calculated, comparing mass after pretreatment to mass after exposure to the microorganisms.  Each percent change in dry mass was averaged from the seven trials for each combination.  

The above experimental steps were repeated with the same combinations of microorganisms but with an exposure length of 1 month for the trials. The process was then repeated for a second time, using an alternate pretreatment and a one month exposure time with the original combinations.  In a glass container closed with Parafilm, each of the 54 HDPE samples was soaked in a 1% solution16 of manganese stearate19 for 48 hours at room temperature (25°C).  The manganese stearate15 was dissolved in hexane by grinding the tablets and then stirring the powder into the hexane on a stir plate.  After 48 hours, the plastic samples were rinsed with distilled water to remove the hexane20.  Each of the 54 HDPE samples was then exposed to thermal radiation at 115°C for 1 hour using an oven.

The percent change in mass of the plastic in different situations was compared.  First, data from the plastic exposed to different ratios of the microorganisms after UV and thermal pretreatment were compared together and tested for presence of the initial microorganisms to determine viability of the symbiotic relationship.  Then, data from the plastic exposed to different ratios of the microorganisms after manganese stearate and thermal radiation pretreatment was compared with the data from the original pretreatment.

An ANOVA test was used to evaluate which combination of microorganisms resulted in the greatest amount of degradation of the HDPE plastic.  This test also compared the percent change in mass of combinations of microorganisms with the control to determine if their degradation of the HDPE was statistically significant.  Growth tests were used to test for presence of the initial microorganisms to determine viability of the symbiotic relationship.  When cultures from each experimental flask were plated after the one or three month degradation period, bacterial and fungal growth was seen for each trial, indicating that the microorganisms could coexist.  Kruskal-Wallis tests were then used to determine which plastic pretreatment resulted in the greatest amount of HDPE degradation.

Results

As seen in Figure 1, three ratios exposed for three months to HDPE with UV and thermal pretreatment  separated themselves from the rest of the seven ratios and the control (with no exposure to M63 or microorganisms).  These ratios were:

HPP-HPC; ½ P. putida and ½ P. chrysosporium

HPP-HS; ½ P. putida and ½ S. macrogoltabidus

HS; ¼ P. putida, ½ S. macrogoltabidus, and ¼ P. chrysosporium

 

P. chrysosporium

Figure 1. Three ratios (½ P. putida and ½ P. chrysosporium (HPP-HPC); ½ P. putida and ½ S. macrogoltabidus (HPP-HS); ¼ P. putida, ½ S. macrogoltabidus, and ¼ P. chrysosporium (HS)) exposed for three months to HDPE with UV and thermal pretreatment separated themselves from the rest of the seven ratios and the control (with no exposure to M63 or microorganisms).

 

As seen in Figure 2, the ranges of standard deviation around the means for these combinations of microorganisms were completely negative and did not overlap zero.  Therefore, there was a significant difference between the percent change in mass of the plastic exposed to these three ratios and that of the control plastic exposed to neither M63 minimal media nor microorganisms.  Thus, biodegradation by these ratios of microorganisms differed significantly, suggesting their suitability for accelerated plastic degradation.  As seen in Figure 2, these results were supported by the P-value of 0.015 after a one-way ANOVA test.  Because 0.015 < 0.050, the null hypothesis (HO) that there would be no statistically significant difference could be rejected, and the test hypothesis (HT) that there would be a statistically significant difference could be accepted.  Because each of the three combinations which separated themselves contained P. putida, this suggests that P. putida is the primary contributor to biodegradation of HDPE plastic.

 

Phanerochaete chrysosporium

Figure 2. Confidence Intervals for Means and Deviations of Percent Changes in Mass of HDPE for different Ratios of the Bacteria P. putida and S. macrogoltabidus and the Fungus P. chrysosporium after 3 Months of Exposure.  The ranges of standard deviation around the means for these three combinations of microorganisms were completely negative and did not overlap zero, indicating a significant difference between microbial treated plastic and control plastic.  A P-value of 0.015 was obtained when a one-way ANOVA t-test was performed, indicating that the percent change was statistically significant.

 

As shown by the non-parametric Kruskal-Wallis statistical tests for small sample sizes in Table 2, the rate of degradation of plastic pretreated with manganese stearate and thermal radiation had a statistically significant difference from that pretreated with UV and thermal radiation after exposure to microorganisms for one month.  For five of the seven trials, the P-value comparing the percent change in mass from biodegradation after each of the two pretreatment methods was 0.002.  The trials were:

HS-HPC;½ S. macrogoltabidus and ½ P. chrysosporium

HPC; ¼ P. putida, ¼ S. macrogoltabidus, and ½ P. chrysosporium

HPP; ½ P. putida, ¼ S. macrogoltabidus, and ¼ P. chrysosporium

HS; ¼ P. putida, ½ S. macrogoltabidus, and ¼ P. chrysosporium

THIRD; ⅓ P. putida, S. macrogoltabidus, and ⅓ P. chrysosporium

 

P-values and Means for Comparison of the Percent Changes in Mass of HDPE with either UV and Thermal or Manganese Stearate and Thermal Pretreatmentafter 1 Month of Exposure 

Manganese Stearate

Table 2. The rate of degradation of plastic pretreated with manganese stearate and thermal radiation was greater than that of plastic pretreated with UV and thermal radiation after 1 month.  Because 0.002 < 0.050, there was a statistically significant difference between the percent changes in mass of HDPE after different pretreatment procedures.

 

Because 0.002 < 0.050, the null hypothesis (HO) that there would be no statistically significant difference could be rejected, and the test hypothesis (HT) that there would be a statistically significant difference could be accepted.  For the ½ P. chrysosporium, 1/2 P. putida and ½ P. putida, 1/2 S. macrogoltabidus combinations, the P-values were 0.085 and 0.180 respectively. Although these P-values were not < 0.050 and, therefore, the null hypothesis could not be rejected and a statistically significant difference found, the mean percent change in mass for the manganese stearate and thermal pretreatment was visibly lower than that for the UV and thermal pretreatment in both cases (-3.174% vs. -1.323% and -3.581% vs. -1.166%).  These means suggest the same trend even though the two P-values do not show a statistically significant difference.  Moreover, the visibly lower mean percent changes in mass for the manganese stearate and thermal pretreatment compared to those for the UV and thermal pretreatment demonstrates that manganese stearate is a better option than UV radiation.  Because the manganese stearate pretreatment was accompanied by only 1 hour of thermal radiation, while the UV pretreatment was accompanied by 48 hours of thermal radiation, even an equal effect on biodegradation would prove manganese stearate more effective because it required less thermal radiation compared to UV radiation for the same effect on biodegradation.

Discussion

It was hypothesized that if the ratios of P. chrysosporium fungus, and the bacteria P. putida and S. macrogoltabidus, were varied, then a maximum decrease in HDPE mass would be obtained.  It was also hypothesized that if the pretreatment process was changed from UV and thermal to manganese stearate and thermal, then microbial biodegradation would increase and be more environmentally friendly.  Results supported both of these hypotheses, suggesting that the large-scale biodegradation of plastic waste pretreated with manganese stearate and thermal radiation could provide a more environmentally friendly alternative to landfills.

A one-way ANOVA test with a P-value of 0.015 revealed a significant difference between the percent change in mass of the plastic exposed to HPP-HPC, HPP-HS, and HS ratios and that of the control plastic exposed to neither M63 minimal media nor microorganisms.  Thus, these ratios of microorganisms would be optimal for large scale HDPE degradation.  Each of the three combinations which separated themselves contained P. putida, suggesting that P. putida is the primary contributor to biodegradation of HDPE plastic.  However, the HS combination which was only ¼ P. putida and the HPP combination which was ½ P. putida did not indicate a significant difference.  These results suggest that trials longer than three months would be necessary in order to confirm the significance of P. putida.

A non-parametric Kruskal-Wallis test produced P-values of 0.002 for five of the seven trials.  This revealed that there was a statistically significant difference between the rate of degradation of plastic pretreated with manganese stearate and thermal radiation from that pretreated with UV and thermal radiation after exposure to microorganisms for one month.  These trials- HS-HPC, HPC, HPP, HS, and THIRD- demonstrate that manganese stearate is a better option than UV radiation. Two of the ratios, HPP-HPC and HPP-HS, showed a visibly lower mean percent change in mass for the manganese stearate and thermal pretreatment, but no statistically significant difference.  However, these were two of the combinations which showed a significant difference for the three month trials with UV and thermal pretreatment.  These results suggest that the degradation rates of the microorganisms may vary by pretreatment, and that future research should investigate degradation rates after manganese stearate and thermal pretreatment over a longer time period.

The manganese stearate and thermal pretreatment was more environmentally friendly when its electricity usage/cost was measured against that of the UV and thermal pretreatment.  For the UV and thermal pretreatment, UV costs approximately $0.06 per trial and thermal costs approximately $3.84 per trial for a total of about $3.90 per trial.  For the manganese stearate and thermal pretreatment, manganese stearate costs $0.00 per trial and thermal costs approximately $0.08 per trial for a total of about $0.08 per trial.  Thus the manganese stearate and thermal pretreatment would decrease the fossil fuels burned to create electricity- an important attribute because high electricity usage could counteract the environmental benefits gained from the biodegradation of plastics.  Although relatively expensive itself, manganese stearate can be reused.

Although the percent changes in mass resulting from biodegradation were ranging between -1% and -8% in the raw data, trials were run only over 1 month and 3 month intervals.  Thus, over the longer time periods, a greater effect would be seen as biodegradation continued.  Even at a degradation rate of 1% every three months using the less efficient UV and thermal pretreatment would reduce the degradation time of a plastic sample from over 100 years to 25 years.  At a degradation rate of 8% every one month using the manganese stearate and thermal pretreatment, the degradation time of a plastic sample could be reduced to slightly over 1 year.

Future work could examine the degradation rates of plastic with UV and thermal pretreatment exposed to microorganisms for time periods longer than three months in order to investigate the role of P. putida.  Also the degradation rates of plastic with manganese stearate and thermal pretreatment could be examined when exposed to microorganisms for time periods longer than one month.  Investigation is necessary to determine the maximum time manganese stearate can be reused before losing its effectiveness as a pretreatment.  The effect of combined microbial degradation and varied pretreatment on water bottles instead of plastic bags could also be investigated.  This would cut down on experimental costs because the bottle would be both the container and plastic for the trial, eliminating the requirement of glass Erlenmeyer flasks.

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