Disposal of end-of-life low density polyethylene (LDPE) in land ll structures poses an ecological threat through leaching, fragmentation, and additive migration. The present study examines the mycodegradation of morphologically modi ed low- density polyethylene (LDPE) lms by Phanerochaete chrysosporium , a lignolytic basidiomycete. Three physicochemical treatments were employed: thermo-oxidation, chemical etching, and synthetic leachate. Surface area imaging over 6 days revealed bioerosion, qualifying P. chrysosporium as a suitable agent for LDPE degradation. Analysis of crystallinity indices showed that thermo-oxidation and chemical etch- ing induced conformational changes to the polymer surface, increasing surface area reduction by 19% and 22% respectively. Synthetic leachate (SL) was associated with a 36% increase in surface area reduction. In combination, the three treatments achieved a 99% increase in surface area reduction. These trends were corroborated by gaseous evolution in parallel, attributable to the metabolization of fungal iso- lates. Electrospray Ionization Fourier Transform (FTMS + pESI) pro les observed signs of leachate remediation and organic byproducts. A molecular mechanism for degradation was subsequently proposed based on SL composition. KEYWORDS mineralization, secondary metabolism, cross-linking, ligninolysis, bioremediation 1. Introduction Low density polyethylene (LDPE) is a versatile plastic of immense industrial utility. However, of the 64 million metric tons manufactured every year, only 5.7 percent is recycled [1,2]. Other methods of disposing end-of-life LDPE are often unsafe or counterproductive [3]. As a result, most LDPE accumulates in land lls, where its carbon-to-carbon backbone and branched structure render it recalcitrant and pose an ecological threat through leaching, fragmentation, and additive migration [4,5]. In 1978, Hiroyuki et al. proposed two solutions to this polymeric design challenge: engineering biodegradable materials and enhancing microbial degradation [6]. While the former holds long-term potential, this study focuses on biodegradation as a means of addressing LDPE entering existing solid waste structures. Previous literature has established that fungi exhibit higher enzyme activity than bacteria [7]. Microbial species in genera Aspergillus ,Ideonella , and Trichoderma are svj2113@columbia.edu SV JANAPATY particularly e ective at eroding the surface of tertiary sites on polyethylene [8{11]. Studies have also reported that small product degradation is easier than large prod- uct degradation, in addition to considering morphological changes to polymer sur- faces [7]. However, literature on the impact of physicochemical surface pretreatment on biodegradation is scarce. The objective of this investigation is to enhance LDPE biodegradation by inducing morphological changes to its surface. 1.1. Phanerochaete chrysosporium Phanerochaete chrysosporium is a white-rot basidiomycete that degrades aromatic hy- drocarbons, predominantly lignin, under aerobic conditions [12{14]. Lignin's aliphatic side chains share structural similarities with LDPE, leading to the novel hypothesis that P. chrysosporium 's two key enzymes, lignin peroxidase (LiP) and manganese per- oxidase (MnP), may possess the ability to mineralize LDPE [15,16]. These enzymes generate H 2O2through an oxidation reaction to degrade organic pollutants, viable in pH range 2.5 <pH<6 and over a wide range of temperatures, 25 to 60C [16,18]. It is capable of growth in both shallow stationary cultures and agitated ask cultures [17]. P. chrysosporium metabolization of various contaminants is shown in Table 1. Dur- ing secondary metabolism, bio lm formation occurs based on nutrient, oxygen, and trace metal availability [18{22]. Table 1. P. chrysosporium Metabolization Substrate Mineralization Organics CO 2 Nitrogen Compounds NO 32- Hydrogen Compounds H 2O Heavy Metals Ion Adsorption This study examines the degradation of morphologically modi ed LDPE using P. chrysosporium . The selection of physicochemical pre-treatments is described in the section that follows. 1.2. Morphological Treatments LDPE consists of long aliphatic chains with repeating CH 2| units [4], resulting a closely-packed, branched network. Under normal conditions, this structure interferes with crystallization. However, Drummond et al. found a rise in melting crystallites in the 110C to 120C temperature regime [23,24]. Mijovic also reported that chemical etching impacts polymer mechanical proper- ties and surface area roughness, facilitating subsequent fungal anchorage [25]. Land ll leachate is wastewater formed by the percolation of rainwater through a land ll. A high C:N ratio and substrate adsorbability suggest activation of the sec- ondary enzymatic mechanism necessary for LiP and MnP secretion [26,27]. The author also examined 11 other pretreatment methods in preliminary experi- ments, omitted for brevity. 2 SV JANAPATY 2. Methods The aim of this study was to identify physicochemical treatment combinations that maximize LDPE mycodegradation: P. chrysosporium inoculation, synthetic leachate, etching, and thermo-oxidation. Table 2 displays the con guration. Table 2. Treatment Combinations Composition Group LDPE PC Thermo-oxidation Etching Synthetic Leachate Control15 1 5 5 2 5 5 5 3 5 5 5 4 5 5 5 5 5 5 5 5 6 5 5 5 5 7 5 5 5 5 8 5 5 5 5 5 1No morphological or mycoremedial treatment. 2.1. Experimental LDPE was obtained from commercially-available lms of uniform thickness ( 15m) and cut into strips of surface area 115 mm2and mass 3 mg. The lms were then sterilized with a bleach-water solution. 2.1.1. Thermo-oxidation LDPE was heated in a convection oven at 120C for 4 minutes. The surface area reduction for each lm as a result of this procedure was recorded, averaging 7%. 2.1.2. Chemical Etching Anhydrous citric acid was used to prepare a 30% citric acid solution, in which LDPE lms were immersed for 10 minutes. After removal from solution, the solvent was allowed to evaporate o the polymer surface. In preliminary experiments, LDPE rinsed with distilled water proved less susceptible to biodegradation, so this procedure was eliminated. 2.1.3. Synthetic Leachate Synthetic leachate (SL) composition was adapted from Azhar et al. to simulate land ll wastewater conditions [28] at pH 5.6 and comparable COD:TN:TP ratios. SL was boiled and set with small amounts of potato-dextrose agar (PDA) for 24 hours. The C/N ratio of the SL-PDA medium was approximately 25:1 with a starting pH of 5.7. 2.1.4. P. chrysosporium Inoculation P. chrysosporium was incubated on a PDA slant at 27CC for 24 hours. After sporula- tion, SL-PDA plates were subsequently inoculated with 2 107fungal spores per gram. LDPE lms were placed in their corresponding media (PDA or SL-PDA) for an ob- servation period of 6 days every 12 days. After the experimental procedure was termi- 3 SV JANAPATY nated, LDPE was retrieved using lter paper, sterilized with a bleach-water solution, and analyzed for degradation. 2.2. Surface Area and Crystallinity Surface area and crystallinity changes were quanti ed over 6 days using the Java image-processing program, ImageJ . Surface area reduction indicates polymer bioero- sion, and crystallinity is associated with spherulitic nucleation and roughness in LDPE morphology [29]. Statistical analyses were performed by StatPlus . 2.3. CO 2Evolution It is well-known that polyethylene degradation yields gaseous byproducts, predomi- nantly CO 2, due to the increase in low molecular weight material and reduction in carbonyl groups. CO 2evolution was measured by water displacement in a pneumatic trough apparatus. 2.4. Electrospray Ionization Fourier Transform (FTMS + pESI) Changes in leachate composition in PDA, SL-PDA, and Group 8 samples over 12 days were investigated using an Orbitrap and fourier transform (FTMS + pESI) techniques at 5L/min and 60,000 resolution. ESI was performed at 30 V capillary voltage and 60 V tube voltage, low mass conditions. Compounds were identi ed by the ChemBioDraw software ( 0.0001 m/z). NH 4+, NO32-, and NO 2-data were corroborated by ancillary indicator tests. 3. Results and Discussion All results concern the identi cation and analysis of morphological pre-treatments for enhanced LDPE biodegradation and were statistically signi cant ( p>0.05). 3.1. Crystallinity Index Figure 1 shows trends in relative percentage crystallinity induced by thermo-oxidation (Group 2) and thermo-oxidation with etching (Group 4). For both surface modi cations, the initial spike occurred on Day 1 after pretreat- ment. Thermo-oxidation causes a cross-linking reaction where tie chains undergo chain scission and cilia chains recrystallize [30]. The increase in cross-linking is likely respon- sible for the sharp increase in percentage crystallinity. Elongation of the amorphous region was visually observed, in agreement with the ndings of Veitmann et al. [31]. Chemical etching performed prior to thermo-oxidation further enhanced crystal- lization, a result shared by Miyagawa et al. who corroborated that chemical etching crystallizes amorphous regions [30]. After initial treatment, the population of crystallites declined due to surface bioero- sion as a function of exposure time. Percentage crystallinity remained fairly unchanged byP. chrysosporium and SL, and when there were no treatments. 4 SV JANAPATY Figure 1. Trends in relative percentage crystallinity induced by thermo-oxidation (Group 2) and thermo- oxidation with etching (Group 4). 3.2. Surface Area Reduction and Gaseous Evolution Figure 2 tracks the change in average surface area reduction of 3 mg LDPE lms after 6 days, with a baseline comparison against Group 1. Prolonged exposure to P. chrysosporium (Group 1) yielded a surface area reduction of 5%. Figure 2. Surface area reduction and corresponding gas production (Groups 1-8). An increased surface area loss of 14% was observed in samples pretreated by thermo- oxidation (Group 3) at 120C. The procedure causes cross-linking, which breaks carbon-hydrogen bonds and gives rise to carbon-carbon single bonds. These tertiary sites have lower bonding energies and are therefore vulnerable points in LDPE mor- phology to extracellular enzymes [31]. Etching (Group 3) increased surface area reduction by 17% over a 6 day observation period. This result accompanies previous ndings that on a molecular level, chemical- etching cleaves bonds in amorphous polymeric regions. The presence of citric acid may also be responsible for enhanced microbial activity by increasing surface bonding sites for hyphal extension and adding various polar groups [31]. 5 SV JANAPATY An increase of 36% in surface area reduction was noted in leachate samples (Group 5). While the mechanism for this remains unclear, it is well-known that ligninolytic activity catalyzed by P. chrysosporium generally occurs in nitrogen-starved environ- ments [32]. The SL-PDA growth media had a high C:N ratio, was sulfur-limited and phosphorus abundant, and contained Mg2+and Ca2+, trace metals known to cat- alyze secondary metabolic responses in P. chrysosporium . Carbohydrate levels were also low. SL did contain abundant NH4+, but Je ries et al. previously reported that it does not signi cantly suppress ligninolytic activity [28,33]. This leads to the fol- lowing proposed mechanism: the synthetic composition of leachate met ligninolytic conditions, facilitating high levels of LDPE mineralization. In conjunction, the three morphological treatments (Group 8) increased surface area reduction by a maximum of 99%. This data was corroborated by an analogous trend in gas evolution associated with fungal respiration and degradation products, quanti ed by water displacement in a pneumatic trough. Displacement in a sample with no fungal isolates was 0.7 mL. Group 8 showed an 8 mL change in water level, and Ideal Gas Law calculations based on LDPE and SL composition approximately con rmed the surface area reduction results [34,35]. 3.3. Electrospray Ionization Fourier Transform Figures 3, 4, and 5 show the FTMS + pESI pro les of SL, SL exposed to P. chrysospo- rium for 12 days, and SL with LDPE lm exposed to P. chrysosporium for 12 days respectively. Signi cant decreases were observed in the CO 32-peak at 121.0278 m/z and NH 4+ peak at 180.0859 m/z after 12 days of exposure. Heavy metal solubility appeared to rise. For example, Mn2+at 350.9300 m/z can be observed in three unique peaks on Day 12. A number of organics were also observed in samples containing degraded LDPE products. ESI results suggested some signs of SL remediation, later con rmed by indicator tests for NH 4+, NO 32-, and NO2-. This aligns with conclusions drawn by previous research on P. chrysosporium metabolism and biodegradative potential for leachate [36{38]. 6 SV JANAPATY Figure 3. FTMS + pESI pro le of Synthetic Leachate on Day 0. Figure 4. FTMS + pESI pro le of Synthetic Leachate after 12 days of exposure to P. chrysosporium . Figure 5. FTMS + pESI pro le of Synthetic Leachate and degraded LDPE product after 12 days of exposure toP. chrysosporium . 7 SV JANAPATY 4. Conclusions This study expands upon the limited body of research amalgamating physicochemical surface treatments and biodegradative approaches for e ective mineralization. Surface area imaging revealed bioerosion, qualifying Phanerochaete chrysosporium as a suit- able candidate for LDPE degradation. Trends in the crystallinity index over 6 days show that thermo-oxidation and chemical etching induced conformational changes to polymer surface morphology, and that crystallinity values decreased as a function of mycoremedial exposure. Thermo-oxidation was associated with a 19% increase in surface area reduction, etching showed a 22% increase, and synthetic leachate me- dia showed a 36% increase. Together, they achieved a 99% increase in surface area reduction. These results were accompanied by a rise in gaseous evolution associated with CO 2and the respiration of fungal isolates. Therefore, the following molecular mechanism was proposed. Cross-linking and amorphous crystallization increased sur- face vulnerability to ligninolytic enzymatic activity, and P. chrysosporium secondary metabolites were produced in response to appropriate chemical conditions in leachate. FTMS + pESI pro les of leachate after 12 days of exposure to P. chrysosporium suggested signs of leachate mycoremediation: reductions in CO 32-and NH 4+levels, improved heavy metal solubility, and the presence trace organics were observed. This approach holds promise for attenuating challenges in LDPE disposal, especially in land ll structures. Acknowledgements The author thanks Drs. K. Walker and T. Hughes for technical guidance, along with the Department of Chemistry at Stanford University for ESI instrumentation. 8