Biodegradation of Physicochemically Treated Polycarbonate by Fungi

Publication Date (Web): December 7, 2009. Copyright © 2009 American Chemical Society. * To whom correspondence should be addressed. Tel.: +91 44 2257...
0 downloads 0 Views 2MB Size

Biomacromolecules 2010, 11, 20–28

Biodegradation of Physicochemically Treated Polycarbonate by Fungi Trishul Artham and Mukesh Doble* Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India Received July 17, 2009; Revised Manuscript Received October 29, 2009

Two fungal strains isolated from soil and a commercial white-rot fungus, Phanerochaete chrysosporium NCIM 1170 (SF2), were tested for biodegradation of untreated, UV-, and thermal-treated bisphenol A polycarbonate (PC). The isolated strains based on 18S rDNA analysis were characterized as Engyodontium album MTP091 (SF1) and Pencillium spp. MTP093 (SF3). About 5.4% weight loss and 40% reduction in Mn were observed for UV-treated polycarbonate in one year with SF2 strain. An increase in surface energy and oxygen content and a reduction in methyl index indicated oxidation of PC during this period. PC exposed to the SF1 strain showed a 15 °C decrease in glass transition temperature, indicating an increase in the number of chain ends and, hence, an increase in the free volume of polymer. No bisphenol A, the monomer of PC, was detected during the study. NMR and FTIR spectra showed the formation of methyl groups due to pretreatments. EDAX analysis exhibited surface oxidation of the PC. The current study advocates that biodegradation of PC can be enhanced by pretreatments.

1. Introduction Plastics have become an indispensible part of human life due to their versatility. PC (bisphenol A polycarbonate) is one of the widely used engineering thermoplastics and has found wide applications in several fields, including electronics, automobile, optics, and so on. About 2.7 million tons of PC is produced annually.1 The steep rise in its production and its durability has led to increasing problems in its disposal and challenges in waste management. Chemical recycling of polycarbonate waste has gained great importance during recent years.2,3 This is not an environmentally benign process, so it is essential to address the waste disposal problem through biological means because biodegradation offers an efficient benign and “green” solution to tackle the waste management. Fungi are widely used in bioremediation due to their robust nature and for their great source of diverse enzymes.4 One of the widely reported fungi, Phanerochaete chrysosporium, commonly known as white-rot fungus, is able to degrade broad range of persistent pollutants and xenobiotics under nutrient limited conditions because of its robust enzyme machinery. While not many reports are available on fungal mediated degradation of polycarbonate, a Geotrichum-like fungus isolated from a biodeteriorated compact disk made of polycarbonate was able to degrade its components.5 The biodegradation of bisphenol A, a monomer of PC, by fungi has also been reported.6,7 To enhance the degradation rate of polycarbonate, several pretreatment strategies have been suggested.8 Photo and thermal treatments might increase the rate of biodegradation. These treatments generate free radicals, which are able to oxidize the polymer molecule resulting in the breakage of the chains. Enhancing the biodegradation of polyethylene9-11 and polypropylene12 by pretreatments have been reported. Recently, an article has been published on the photo and thermal degradation of polycarbonate in which the mechanisms of the process have been discussed.13 In vitro biodegradation of polycarbonate has * To whom correspondence should be addressed. Tel.: +91 44 2257 4107. Fax: +91 44 22574102. E-mail: [email protected].

been recently reported with marine bacterial consortia.14 Changes in its physicochemical properties due to exposure to marine environment have also been observed.15,16 Here the biodegradation of UV and thermally pretreated PC using two mesophilic fungi isolated from soil along with a strain from a culture collection are reported.

2. Materials and Methods 2.1. Materials. Poly(bisphenol A-carbonate), a Lexan grade resin, with a weight average molecular weight of 57800, was a gift from GE Plastics, India. A 3% solution of the polycarbonate resin in chloroform (HPLC grade, Merck Limited, Mumbai, India) was prepared and it was poured on a glass plate to form a film of 0.125 mm thickness. The solvent was allowed to evaporate and it was then dried overnight in a vacuum oven at 50 °C. The resulting films were cut into 60 × 10 mm strips for the in vitro studies. 2.2. Physicochemical Treatments. Two different pretreatment strategies were tested. In the first, PC films were thermally treated at 100 °C in an atmosphere of air for 30 days to induce oxidation, and in the other, they were subjected to UV light (UV-C, >300 nm wavelength) for 10 days. These pretreated samples along with the untreated polycarbonate films were used as the sole carbon source for the fungi. 2.3. Enrichment of Polycarbonate Degrading Fungi. Soil samples were collected from a plastic dumpsite (near Pallikarani, Chennai, India). A total of 1 g of the soil sample was suspended in 10 mL of sterile Milli-Q water and vortexed for 15 min. Nearly 100 µL of suspension was used as inoculum. Erlenmeyer flasks containing 100 mL of mineral salt medium, 20 mg of untreated polycarbonate, 0.01% (w/v) glucose, and 1 mL of inoculum were used for maintaining the first preculture. The later subcultures did not contain glucose but only the polymer as the sole carbon source. After three successive subcultures, in which microorganisms were grown in presence of PC and without glucose, pure cultures were isolated on potato dextrose agar plates (Himedia Limited, Mumbai, India) containing 50 mg of Chloramphenicol to avoid bacterial contamination. Two strains, isolated during this process, along with a commercial strain, Phanerochaete chrysosporium NCIM 1170 (procured from NCIM, Pune, India), were evaluated for their efficacies to biodegrade polycarbonate under submerged conditions.

10.1021/bm9008099 CCC: $40.75  2010 American Chemical Society Published on Web 12/07/2009

Biodegradation of Treated Polycarbonate by Fungi 2.4. Identification of PC Degrading Fungi. Fungal strains were identified by the morphological features of their colony and conidia using microscopic examination. Samples of 10 mm2 of the polymer film with mycelium were cut and fixed with 3% glutaraldehyde followed by successive dehydration with varying concentrations (20, 50, 70, and 90%) of ethanol in water. The SEM micrographs (FEI Quanta 200) of the overnight dried films were obtained at a current of 30 mA for 50 s and were observed at 15 KV and at a magnification of ×3000. DNA isolation, extraction, and sequencing of the conserved 18s rRNA region ITS1-5.8S rDNA and ITS2-28S rDNA for the two fungal strains (SF1 and SF3) isolated during the course of study were performed by ACME ProGen Biotech Ltd., India. The aligned sequences were compared with the sequences of standard nucleotide databases (EMBL, Genbank) using FASTA3 sequence homology searches. 2.5. Submerged Culture with Polycarbonate as Sole Carbon Source. A mineral salt medium containing (per liter): 1 g KH2PO4, 0.2 g NaH2PO4, 0.5 g MgSO4 · 7H2O, 0.1 g CaCl2, and 0.169 g (1 mM) MnSO4 · H2O, 1 g yeast extract, and 1 mL of vitamin solution was used in all the experiments.17 About 20 mg of untreated or pretreated polycarbonate films in triplicate were suspended in aliquots of 100 mL of this medium in 500 mL Erlenmeyer flasks. Fresh mycelium of the previously grown fungus on potato dextrose agar plates was scraped from the plate and suspended in 10 mL of sterile water and vortexed. Around 1 mL of this suspension was inoculated into the flasks. They were then incubated at 30 °C and at 200 rpm on an orbital shaker (Scigenics Pvt. Ltd., India) and were monitored for twelve months. The pH of the medium was adjusted to 7 using 0.1 N HCl or 0.1 M KOH. Sampling was done every 4 months, in which the polycarbonate films were taken out under aseptic conditions washed in sterile water and air-dried before further analysis. Noninoculated flasks acted as abiotic control, while flasks with the pure culture but without the polymer films served as the biotic control. All the controls were treated the same way as biotic samples. All the experiments were done in duplicate and the mean and standard deviation are reported here. 2.5.1. Total Biomass. About 1 mL of the culture was transferred into a 1.5 mL micro centrifuge tube and pelleted down at 12000 rpm at 4 °C for 25 min. The cell-free supernatant was used for enzyme assays. The pellet was dried overnight at 50 °C and the dry weight of the resulting biomass was calculated. Sampling was done in triplicates. 2.5.2. Total Protein and Reducing Sugars in the Culture Supernatant. After removing the films from the media, the total protein concentration in the supernatant was determined according to the method reported by Bradford.18 Bovine serum albumin (Himedia Limited, Mumbai, India) solutions were used as standards and the absorbance was measured with a spectrophotometer (JASCO V550, Japan) at 595 nm. The total carbohydrates were analyzed according to the method suggested by Dubois et al.19 Glucose was used as the standard (Himedia Limited, Mumbai, India) and the absorbance was measured at 495 nm. 2.5.3. Correlation Analysis. Correlation between the biofouling parameters such as biomass, total protein, and total carbohydrates were determined using Microsoft Excel 2003 software. 2.5.4. Enzyme ActiVity Measurement. Two different enzyme assays were performed during the course of the study. The hydrolytic activity of lipases in the supernatant was evaluated using p-nitrophenyl palmitate (pNPP) assay.20 A reaction mixture containing 1 vol of 16.5 mM solution of pNPP in acetonitrile was mixed just before use with 9 vol of 50 mM Tris-HCl buffer (pH 8) containing 0.4% (w/v) Triton Xl00 and 0.1% (w/v) arabic gum. After the addition of 0.1 mL of supernatant, the reaction mixture was incubated for 15 min at 37 °C. The variation in the absorbance at 410 nm against a blank without the enzyme was measured. One enzyme unit is the amount of protein liberating 1 µmol of p-nitrophenol per minute at these conditions. Laccase activity was determined by monitoring the conversion of 14 µmol of ABTS [2,2′-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] to ABTS cation radical in glycine-HCl (50 mM) buffer at a pH

Biomacromolecules, Vol. 11, No. 1, 2010


of 3, monitored at 436 nm (ε ) 29300 M-1 cm-1).21 Laccase activity is expressed in nanokatals mL-1 of culture supernatant. 2.5.5. Electrophoresis of Extracellular Proteins. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed at room temperature with 10% resolving polyacrylamide gel and Tris-glycine buffer (pH 8.5) at 100 V for 90 min, as reported by Laemmli et al.22 After electrophoresis, the gel was fixed and stained using silver nitrate to visualize the protein bands. The molecular mass of the extracellular proteins were evaluated using molecular mass standards [β-galactosidase (116.0 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), lactate dehydrogenase (35 kDa), restriction endonuclease Bsp981 (25 kDa), β-lactoglobulin (18.4), and lysozyme (14.4 kDa); Fermentas, U.S.A.]. 2.6. Polymer Characterization. The changes in the physical, chemical, and surface properties of the treated polymer were determined by following methods. 2.6.1. Weight Loss. Weight loss of a polymer is a direct measure of its degradation. Polymer films were removed under sterile conditions from the culture medium at regular intervals, washed, dried, and weighed using a sensitive balance with an accuracy of (0.01 mg (Sartorius CP64D, Germany). Average % weight loss of the films with respect to their initial weights was calculated. 2.6.2. Surface Energy Calculation. Surface energy is a measure of interactions between various interfacial forces acting on the surface, while the hydrophobicity correlates with the water contact angle (θ) to the surface. Both the approaches attempt to infer the structure-property relationship between surface energy/wetting and some biological response. In general, the higher the surface energy of a surface, the lower is its hydrophobicity. Surface energy was measured using the sessile drop method with an Easy Drop Contact Angle Measuring System (Kruss, Germany). The polymer film was supported on a glass slide and a drop of the probing liquid was placed on it using a syringe. Double distilled deionized water, diiodomethane (Sigma, India), and formamide were used as test liquids. The image of the drop and the angle it made with the polymer surface were measured, and the surface energy was calculated according to Owens method.23 The total surface energy of a solid, γs, can be expressed as the sum of contributions from dispersion γsd and polar γsp force components of the polymer. These can be determined from the contact angle (θ) of polar and nonpolar liquids with known dispersion and polar parts by the equation d p γlv(1 + cos θ) ) 2√γds γlv + 2√γps γlv

where γlv, γlvd, and γlvp are the surface tension, dispersive components, and polar components of the surface energy, respectively, of the probing liquid. 2.6.3. Surface Changes. Surface morphology and composition of the polymer were investigated with a scanning electron microscope (SEM-EDAX; FEI Quanta 200). A 10 × 10 mm piece was cut from the polymer sample and placed on the sample holder and was scanned within an area of 100 µm2 at a magnification of 500×. Chemical analysis (microanalysis) of the polymer surface was performed by measuring the wavelength and intensity distribution of X-ray signals generated by a focused electron beam on the specimen with the energy dispersive spectrometer (EDS). The sample preparation for this analysis was the same used for SEM analyses. The voltage was set at 20 kV, and the magnification was set at 200×. 2.6.4. Thermal AnalysissDifferential Scanning Calorimetry. The glass transition temperature (Tg) and the change in melting point (Tm) of the polymer were estimated with a NETZCH Phoenix DSC-7 differential scanning calorimeter, calibrated with indium standard. About 5-7 mg of the sample was used in the study. The scanning was performed over a temperature range of 50-300 °C at a heating rate of 10 °C min-1 under a nitrogen atmosphere. 2.6.5. Chemical ChangessFTIR Analysis. A Jasco N4200 (Japan) Fourier transform infrared spectrophotometer (FTIR) was used for


Biomacromolecules, Vol. 11, No. 1, 2010

Artham and Doble

detecting the formation of new functional groups or changes in the amount of existing functional groups. The spectra were recorded at a resolution of 4 cm-1 in the frequency range of 4000-400 cm-1, calibrated with polystyrene standards. The analysis was performed using HATR (horizontal attenuated total reflectance) mode by accumulating 32 scans. The readings were taken in triplicates. The methyl index16 for polycarbonate was calculated by taking the ratio of absorbances at 2915 and 1508 cm-1. The former peak corresponded to the stretching frequency of the methylene carbon and the later to the bending frequency of the aromatic sp2 carbon. Carbonate carbonyl index (CC index) was calculated by taking a ratio of the absorbance of the carbonate bond at 1778 cm-1 and aromatic CH stretching at 1508 cm-1. 2.6.6. Gel Permeation Chromatography. A GPC (model Shimadzu 20A, Japan) was used to measure the molecular weight and the distribution of the polycarbonate films dissolved in HPLC grade tetrahydrofuran (Merck Pvt. Ltd., India) to make up a concentration of 1 mg mL-1. A Styragel guard column (4.6 × 30 mm) and two PLgel columns (PL mixed B and PL 10E4 with effective molecular weight ranges of 200-2000000 and 400-400000 g mol-1, respectively, 300 × 7.6 mm, Polymer Laboratories, U.K.) were used for the analysis. The samples were run in the GPC with the following settings: injection volume of 20.0 µL, flow rate of 1 mL min-1, and a temperature of 35 °C. The GPC was calibrated using polystyrene standards having a narrow molecular weight distribution (Easical, Polymer Laboratories, U.K.). All molecular measurements were performed relative to these standards. The refractive index (RI) detector (model RID10A) was used for the measurements. The UV variable wavelength detector at 254 nm (model SPD 20A, Japan) was used since the aromatic groups in polycarbonate absorb strongly near this wavelength. 2.6.7. 1H NMR Studies on Degraded Polycarbonate. 1H NMR spectra of the polymer were measured at room temperature with a Bruker Avance III spectrometer (Massachusetts, U.S.A.) operating at 500 MHz, using CDCl3 as the solvent and tetramethylsilane as an internal standard. 2.7. Statistical Analyses. Two-way ANOVA was performed using MINITAB (v 14.0) on all the data to determine the statistical significance of the effect of pretreatments on the observed differences in the results. The significance has been calculated within 95% confidence level and designated as significant (*) if p < 0.05 and highly significant (**) if p < 0.01.

3. Results and Discussion 3.1. Fungal Identification. The isolates, SF1 and SF3, were identified using partial rDNA sequences and also comparing the microscopic morphological (Figure 1) features with the published data. The ITS sequence of SF1 had 94% similarity with its nearest homologue Engyodontium album (Gen bank acc. No. AB106650). The later has been recently reported to degrade polyesters.24 The ITS sequence of the fungus SF3 had 99.66% of homology with the published strain, Pencillium adametzii NRRL 8972 (Gen bank acc. No. AF034459). Pencillium spp. is a well-known fungus in bioremediation and has also been reported to degrade polyesters.25 For the isolate SF3, the microscopic examination showed that the conidiophores originate from the foot cell located on the supporting hyphae and terminate in a vesicle at the apex. The morphology on the plate was of radial growth and the conidiophore was slightly greenish in color. The SF1 strain formed a silver matte on the potato dextrose plate and was nonsporulating bearing elongate to subcylindrical conidigeneous cells. The other commercial strain tested here was Phanerochaete chrysosporium NCIM 1170 (SF2). This strain is widely reported in various biodegradation studies.26 3.2. Total Biomass. Fungal biomass is a direct measure of the growth of the strains in the medium. Figure 2 shows the variation in biomass during 4, 8, and 12 months, respectively,

Figure 1. SEM pictures of the fungal strains used in the study: (A) Engyodontium album MTP091 [SF1], (B) Phanerochaete chrysosporium NCIM 1170 [SF2], (C) Pencillium spp. MTP093 [SF3].

for the three fungal species with untreated, UV-, and thermaltreated PC. The fungal biomass, for all the three strains, was higher in the medium where physicochemically treated polycarbonate was used as the sole carbon source when compared to untreated polycarbonate. At the end of one year, a higher biomass was observed for the strains SF1 and SF2 incubated with pretreated polycarbonate, while for SF3 higher biomass was observed with untreated PC. There is a statistically significant difference between the way each organism responds to the pretreatment (interaction effect, p < 0.0001). This significance was also observed between the various treatment

Biodegradation of Treated Polycarbonate by Fungi

Figure 2. Total biomass of three fungal strains as a function of time with physicochemically treated and untreated polycabonate (dotted bar, 4 months; gray bar, 8 months; striped bar, 12 months; UT ) untreated; UV ) UV-treated; TT ) thermal-treated).

Figure 3. Total carbohydrates excreted by the three fungal strains as a function of time with physicochemically treated and untreated polycabonate (dotted bar, 4 months; gray bar, 8 months; striped bar, 12 months; UT ) untreated; UV ) UV-treated; TT ) thermal-treated).

strategies (p < 0.0001). Moreover, biomass production by these strains was significantly influenced by the pretreatments (p < 0.0001). The detailed statistical analysis of pretreatment effect on various biological parameters is presented in Supporting Information (Table S1). The hypothesis is that physicochemical treatments of the polymer leads to its oxidation and subsequent breakdown assisting in the easy assimilation by the fungus and, hence, can be effectively used as a pretreatment strategy before subjecting the polymer to biodegradation.12 Albertsson et al.27 observed a synergistic effect between photo-oxidation and biodegradation in the case of polyethylene exposed to soil and water for various study periods. The oxidized polymer helps in adhesion of microorganisms (due to probable changes in the hydrophobicity of the polymer surface), which is a prerequisite for biodegradation.8 Hence, a higher biomass was observed in the case of SF1 and SF2 on the pretreated samples. 3.3. Total Carbohydrates and Total Protein in the Culture Supernatant. Figure 3 represents the amount of total carbohydrates, in terms of reducing sugars (such as glucose in the current study), produced by the three different fungal strains during 12 months of exposure to the samples. In general, its content increased throughout the study for all three strains. Highest carbohydrates were observed with SF1 fungus after 12 months. Carbohydrate content was also highest for thermally treated samples when compared to UV-treated samples incubated with SF1 and SF2 strains. After 12 months of incubation, the changes in the production of carbohydrates were highly

Biomacromolecules, Vol. 11, No. 1, 2010


Figure 4. Extracellular proteins in the supernatant of three fungal strains as a function of time with physicochemically treated and untreated polycabonate (dotted bar, 4 months; gray bar, 8 months; striped bar, 12 months; UT ) untreated; UV ) UV-treated; TT ) thermal-treated).

significant among three strains (p < 0.0001), while the pretreatment had a significant effect on carbohydrate production for each strain (p < 0.005). The response of each organism toward pretreatment was also significant (p < 0.05). Because carbohydrates in the medium constitute the main energy source for their growth and metabolism during the nonavailability of readily assimilable carbon source,28 a relatively higher amount of carbohydrates were observed on untreated samples. Apart from being used as an energy source, they also adhere to the polymeric surface during the formation of the biofilm, which is essential for bringing about degradation. Carbohydrates are also involved in negating the effect of surface charge, on colonization, produced during the physicochemical treatments of polymer.29 Total protein in the supernatant of the cell-free culture was different for each treatment and each strain. There was an increase in protein content as a function of time in the case of SF1 (Figure 4), while it was random with the other two strains. SF2 exhibited highest protein content after the eighth month with UV-treated sample. In the case of SF3, highest protein amount was observed for the thermally treated sample after 12 months. There is a highly significant difference (p < 0.0001) among the three difference strains in the production of extracellular proteins into the culture medium and the way each organism responds to pretreatment (interaction effect, p < 0.0001). The biodegradation of polymers normally refers to an attack by microorganisms and the process is heterogeneous. Microorganisms are unable to transport the polymeric material directly into the cells due to the lack of its solubility in water and its size. The microorganisms excrete extracellular enzymes which aid in the degradation of polymers outside the cells.30 These enzymes constitute the total protein in the supernatant, and it can be hypothesized that due to lack of readily available carbon source, the fungal strains excrete higher amount of enzymes when compared to the control flasks with no polymer. Similar observations were reported earlier.31 Hence, higher biomass, higher carbohydrates, and higher protein are generally observed when microorganisms grow on physicochemically treated polycarbonate. 3.4. Correlation Studies. A positive correlation (correlation coefficient ) 0.71) was observed between carbohydrates and proteins and an inverse relation between the proteins and the biomass in the culture supernatant for all three fungal strains together (correlation coefficient ) -0.45) at the end of 12 months. There was an increase in the mycelial growth, while


Biomacromolecules, Vol. 11, No. 1, 2010

the total protein content decreased during the same period. There was no statistically significant effect of pretreatments on protein concentration (p < 0.5), probably due to degradation of these proteins during the long incubation periods. These fungi are known to produce proteases that eventually degrade the proteins in the culture supernatant. Similar observations have been reported for the Pencillium spp.32 and white-rot fungi.33 A negative correlation (correlation coefficient ) -0.57) was observed between the biomass and the carbohydrates at the end of 12 months. After 12 months there was high negative correlation (correlation coefficient ) -0.99) between biomass and carbohydrates for untreated samples, suggesting that higher polysaccharides were released with a less biomass growth. For the UV- and thermal-treated samples, a positive correlation between biomass and carbohydrates was observed (correlation coefficient ) 0.81 and 0.39, respectively), suggesting higher carbohydrates and higher biomass after 12 months. These correlations help us to understand whether pretreatments have any influence on the metabolic behavior (in this case proteins and carbohydrates) of fungi during degradation of polycarbonate. 3.5. Enzyme Assays. The polycarbonate consists of hydrolyzable carbonate bond. Lipases belong to hydrolase class of enzymes known for cleaving esters and carbonates. Following this rationale, the lipase activity in the cell-free culture supernatant was measured by p-nitrophenyl palmitate assay. The activities measured in all the cases were quite low. Highest activity (nearly 2 U mL-1) was observed for the strain SF1 with the untreated polycarbonate. SF3 exhibited nearly 1.8 U mL-1 activity with thermally treated polycarbonate after 12 months. This fungus is reported to degrade polyesters and exhibit lipase activity.25 No correlation was observed between the physicochemical treatments to the polycarbonate and enzyme activity for all the three strains. Laccase activity was highest (0.91 nanokatals mL-1) for SF2 after 12 months with the UV-treated sample. One of the strains used in the current study, SF2, is a white-rot fungus. It is a known source for laccases (phenol oxidases) and this strain is reported to degrade a number of recalcitrant xenobiotics such as polycyclic aromatic hydrocarbons, synthetic dyes,34 synthetic polymers, and lignin.35 Because the PC used in the current study is an aromatic polycarbonate (bisphenol A polycarbonate), laccase activity was also observed. The SDS-PAGE profile of the extracellular proteins in culture supernatant is shown in Figure S1 (see Supporting Information). It is interesting to observe that the expression of total proteins for the same fungus varied with the type of pretreatment given to the polycarbonate. An extracellular protein band with a molecular weight of nearly 35 kDa was observed in all three fungi and was in higher amounts for untreated and thermally treated samples when compared to other treatments. Moreover, laccase activity was also higher in these respective systems, suggesting that this protein could possibly be a putative laccase. 3.6. Polymer Analysis. 3.6.1. Weight Loss. Weight loss of pretreated and untreated polycarbonate over the study period is shown in Figure 5. Nearly 5.5% of weight loss was observed for the UV-treated samples exposed to SF1 or SF2 strains. Thermal treatment had resulted in only about 2-3% weight loss in 1 year. These results suggest that UV treatment has the highest effect on the biodegradation of PC, which was statistically significant when compared to the control sample (p < 0.05). 3.6.2. Surface Morphology Studies Using SEM/EDAX. The fungus colonized the physicochemically treated samples within weeks of inoculation. Electron microscopic examination (Figure

Artham and Doble

Figure 5. Weight loss (%) of the polycarbonate exposed to three fungal strains as a function of time with physicochemically treated and untreated polycabonate (dotted bar, 4 months; gray bar, 8 months; striped bar, 12 months; UT ) untreated; UV ) UV-treated; TT ) thermal-treated).

6) showed that the hyphae of SF1 had adhered to PC (Figure 6b), while SF2 penetrated the polymer matrix (Figure 6c) in the untreated samples after 12 months. The material shows clear crack initiation points, indicating that the polymer has become brittle in nature. Also, the microbial propagation has been initiated from these cracks. Such colonization and adhesion by microorganisms are a fundamental prerequisite for biodegradation of the polymer. Cavities were also observed on the polycarbonate surface. Penetration and cavities were higher for untreated samples while cracks were observed on physicochemically treated samples. Physiochemical treatment induces oxidation and, hence, the polymer becomes brittle, which eventually leads to cracks due to the action of fungi. In addition, capsular structures were also observed on the thermally treated samples. Microorganisms that colonize the polymer surface can probably adhere by means of extracellular polymeric substances (Figure 6d), which are mainly constituted by polysaccharides. This forms a sheath that is bonded to the polymer and plays an important role in transporting the depolymerising enzymes to its surface.36 Physicochemically treated samples exhibited higher oxygen weight content when compared to untreated samples (Table 1). SF1 and SF2 showed maximum oxidation with UV-treated sample, while SF3 showed maximum oxidation with the thermally treated sample. Ohtake et al.37 observed that the superficial growth of hyphae on the polymer surface was a function of the oxidation levels of treated sample. 3.6.3. Surface Energy Changes. The surface energy of a polymer is an important parameter in biodegradation studies.16 An increase in surface energy was observed for the UV- and thermally treated control samples, which were not exposed to fungus (POSUV and POSTT in Table 2), indicating a possible modification of the polymer surface. Physicochemical treatments increase surface energy probably by creating new hydrophilic functional groups. An increase in the surface energy from 34.63 to nearly 45.13 mN/m (Table 2) was observed for the thermally treated sample with the SF1 strain after 12 months with respect to the control. A positive correlation (0.67) was observed between surface energy and % elemental oxygen content as measured by SEM-EDAX analyzer. Surface energy, which arises due to a combination of polar and dispersive components, has higher contribution from the polar component in the case of untreated samples exposed to the SF1 and SF3 strains. This suggests a change in its surface chemical composition due to the formation of hydrophilic functional groups. Samples exposed

Biodegradation of Treated Polycarbonate by Fungi

Biomacromolecules, Vol. 11, No. 1, 2010


Figure 6. SEM pictures of the degraded polycarbonate after twelve months in submerged culture conditions. (A) Control sample, (B) thermally treated, SF1, (C) penetration of hyphae of SF2 into the grooves of untreated sample, and (D) formation of cracks due to SF2 on thermally treated sample. Table 1. Elemental Analysis of Polycarbonate Samples Removed after 12 Months of Exposure to Various Fungal Strains Using SEM-EDXa element (wt %)












83.84 16.12

73.02 20.55

68.66 26.06

70.79 25.8

72.70 18.43

72.79 23.99

77.84 22.16

80.16 19.84

79.54 19.05

75.24 22.43

a Control indicates polymer unexposed to fungus. UT ) untreated sample; UV ) photo-aged samples; TT ) thermally treated samples; SF1 ) Engyodontium album MTP091; SF2 ) Phanerochaete chrysosporium NCIM 1160; SF3 ) Pencillium spp. MTP093.

to SF2 strain after UV and thermal treatment showed a higher contribution from polar component. Similar observations of higher contribution from polar component toward increase in surface energy after pretreatments were reported earlier.38 3.6.4. Thermal BehaVior of Treated Polycarbonate. The effect of biodegradation on the bulk properties such as crystallinity, glass transition temperature and melting temperature, of the polymer are monitored by differential scanning calorimeter and are presented in Table 2. UV-treated and untreated samples exposed to SF1 exhibited largest reduction in Tg. Similar reduction in Tg due to degradation was observed by many researchers.17,36,39 In contrary to the above observations, an increase in glass transition temperature was observed for the thermally treated when compared to untreated polycarbonate. According to the Flory’s theory, the Tg of a polymer depends on the electronic interactions between the macromolecules and the free volume surrounding them. Webin et al.40 observed that the oxidative degradation of PC might lead to the formation of

a stiff layer localized at the surface leading to increase in its rigidity and subsequently in its glass transition temperature. Crystallinity of the degraded polymer increases further when thermally treated samples are exposed to SF2 for 1 year (11%) when compared to UV-treated or untreated samples with other fungal strains. Alizadeh et al.41 reported secondary crystallization due to thermal treatment, which led to multimelting behavior in polycarbonate. In the present study, such multiple melting behavior was observed in the case of thermally treated polycarbonate exposed to SF2 for the duration of 12 months. Each fungal strain exhibited a different behavior toward the physicochemically treated samples. The strain SF1 increased the crystalline content of untreated and thermally treated polymer while it reduced the crystalline content of UV-treated sample. It is well documented11,42 that microorganisms attack the amorphous phase of the polymer cleaving it randomly and introducing shorter chains leading to its degradation. These chains can fall back into lamellae and


Biomacromolecules, Vol. 11, No. 1, 2010

Artham and Doble

Table 2. Physicochemical Analysis of Degraded Polycarbonate after 12 Months in Submerged Conditionsa GPC -1

DSC -1

surface energy

sample code

Mn (g mol )

Mw (g mol )


Tg (°C)

crystallinity %

Tm (°C)

total (mN/m)




37087 26780 27104 28827 27942 22240b 26989b 23912b 29317 27571b 26124b 25811b

57693 54163 53859 55237 53976 49606 50273 49297 56400 54788 50782 53892

1.56 2.02 1.99 1.92 1.93 2.23 1.86 2.06 1.92 1.99 1.94 2.09

143.1 127.4 142.6 133.6 142.7 128.8 143.4 143.0 144.4 151.6 142.5 151.7

3.44 4.60 2.13 4.30 3.23 2.32 2.60 2.98 7.77 9.07 11.02 4.00

226.6 220.3 217.1 219.3 218.9 216.7 214.4 216.3 221.9 216.8 221.5 208.3

34.63 40.96 35.61 39.47 38.83 41.11 45.07 41.35 38.59 45.13 38.72 43.57

33.14 25.78 27.22 25.32 34.79 39.37 33.83 38.92 36.2 41.22 27.16 39.64

1.49 15.18 8.39 14.16 4.04 1.74 11.23 2.43 2.39 3.91 11.56 3.93

a POS-UT ) unexposed and untreated polycarbonate sample (abiotic control); POS-UV ) unexposed and UV-treated polycarbonate sample; POS-TT ) unexposed and thermal-treated polycarbonate sample; SF1 ) Engyodontium album MTP091; SF2 ) Phanerochaete chrysosporium NCIM 1160; SF3 ) Pencillium spp. MTP093; UT ) untreated sample; UV ) photo-aged samples; TT ) thermally treated samples. b p < 0.05, at 95% confidence intervals.

Figure 7. Methyl indices of the physicochemical-treated and fungaltreated polycarbonates after 12 months.

contribute to the increase in the relative crystalline content of the polymer. These newly formed crystals are generally small in size and they are the next targets of the microbial attack. This could be one of the reasons for an increase observed during the initial phases of degradation followed by reduction in the crystallinity of the polymer. Because each microorganism possesses a different secondary metabolism, the kinetics of the formation of crystalline content varies for each species. 3.6.5. Chemical Changes in Polycarbonate. The change in functional groups of polycarbonate was monitored by ATRFTIR, and the methyl index (MI) and carbonate carbonyl index16 (CCI) were calculated and presented in Figure 7 and Figure S2 (refer to Supporting Information), respectively. With the increasing incubation time, the MI varied without any trend. There was an initial decrease and later an increase in the methyl index. The overlay of the FTIR spectrum (Figure 8) of the physicochemically treated with untreated polycarbonate samples clearly showed a shift in the absorbance of the methyl group. Relative decrease in the intensity of the sp3 carbon-hydrogen peaks (in the range of 2900 cm-1), while an increase in intensity of peaks in the range of 2700-2800 cm-1 are observed after 12 months of incubation with different fungal strains. A shift in methyl intensities implies that they might have been oxidized.43 Because this functional group has the lowest bond dissociation energy in the polycarbonate, in all possibility this could be the functional group where the fungus can attack. The carbonate carbonyl indices show a gradual increase during the current study. It is reported44 that polycarbonate undergoes hydrolysis to yield bisphenol A in the presence of hydrolytic enzymes such as lipases, thereby showing a reduction in the carbonate index, but on the contrary, an increase in CCI (Figure S2, refer to Supporting Information) was observed for most of

Figure 8. Overlay of methyl region for (A) untreated, (B) UV-treated, and (C) thermally treated polycarbonate [(a) polycarbonate control and (b) PC exposed to SF1, (c) SF2, and (d) SF3].

the samples. It is reported that during physicochemical treatments many carbonyl groups, such as ketones, aldehydes, and acids, are produced.45 These functional groups fall in the absorbance region of the carbonate bond (a region from 1690 to 1800 cm-1) and are generally masked by the strong absorbance of carbonate bond. Hence, we observe negligible changes in the carbonate index.

Biodegradation of Treated Polycarbonate by Fungi

Biomacromolecules, Vol. 11, No. 1, 2010


Figure 9. Proposed mechanism of degradation of polycarbonate by fungus based on NMR studies. McNeils and Ricons scheme for pretreatment of polycarbonate followed by oxidation by fungus.

3.6.6. Molecular Weight Distribution of Polycarbonate. A change in the molecular weight distribution is a key parameter in evaluating the degradation of the polymer. The untreated polycarbonate was used as the reference sample for evaluating the changes produced by the physicochemical and biological treatments (Table 2). Nearly 40 and 27% reduction in the number average molecular weight (Mn) was observed for the UV-treated and untreated samples, respectively, when exposed to the strain SF1 for 1 year. Thermally treated polycarbonate exposed to SF3 exhibited a 30% reduction in Mn during the same period. The differences in the decrease in molecular weight observed between physicochemically treated when compared to the untreated polymer are statistically significant (p < 0.05, at 95% confidence intervals). The pretreatments by themselves were able to initiate the degradation with 24 and 20% reduction in Mn were observed for UV- and thermally treated polycarbonate control samples (POSUV and POSTT in Table 2). An increase in the polydispersity index is observed, which could be due to the extracellular enzymes acting upon the polymer by an exotype, which is end-chain scission, leading to the fragmentation of higher molecules. Formation of oligomers was observed in the GPC chromatograms of degraded polycarbonate (Figure S3, see Supporting Information), which were not characterized. 3.6.7. NMR Studies of Degraded Polycarbonate. The expanded 1H spectra for the untreated and physicochemically treated polycarbonate samples after 12 months are presented in the Supporting Information (Figure S4). The proton NMR spectra of the control polycarbonate sample displayed an aromatic A2B2 pattern at 7.16 and 7.24 ppm and a singlet peak corresponding to the gem-dimethyl at 1.69 ppm, relative to tetramethylsilane (CH3)4Si. A new singlet peak was observed in the UV-treated samples at δ 1.56 ppm, which corresponded to a methyl proton. A triplet (δ 1.26 ppm) and a quartet (3.73 ppm) confirm the observations made by Neill and Rincon,46 namely, that the isopropylidene group is cleaved, followed by migration to the benzene ring. Figure 9 shows the possible mechanism of degradation of PC due to the synergy between pretreatments and exposure to fungi. The methyl groups could have been oxidized by the fungal enzymes, leading to the degradation of polycarbonate. While the FTIR data shows the presence of new methyl groups, elemental analysis indicates the oxidation of polycarbonate.

4. Conclusions Two fungal strains able to degrade Bisphenol A polycarbonate (PC) were isolated and characterized using 18S rDNA analysis.

They were identified as Engyodontium album MTP091 (SF1) and Pencillium spp. MTP093 (SF3). These strains, along with Phanerochaete chrysosporium NCIM 1170 (SF2), were used to study the biodegradation of intact PC and the physicochemically (UV and thermal) treated polymer. A higher biomass, total protein, and total carbohydrate content in pretreated samples implies easy colonization when compared to untreated samples. About 5.4% loss in the physical weight of UV-treated polycarbonate was observed for SF2 strain. Nearly 40% reduction in Mn was observed, with the same at the end of 12 months, while an increase in surface energy, oxygen content, and reduction in methyl index indicated the oxidation of polycarbonate. Thermal analysis on polycarbonate exposed to the SF1 strain showed a loss of 15 °C in glass transition temperature, which is interpreted as a decrease in molar mass and an increase in the number of chain ends, thus, leading to an increase in the free volume. No bisphenol A, monomer of PC, was detected during the study. The differences in the amount of degradation due to different pretreatments clearly indicate that physicochemical treatments enhance biodegradation. Enhancement in degradation cannot be generalized based on pretreatment alone, as it is strain specific. Moreover, only a limited knowledge about the detailed mechanism of biodegradation of PC is available to date. Studies with pure enzymes under different environmental conditions have to be carried out to elucidate the degradation mechanism of this polymer. Acknowledgment. Authors thank The Sophisticated Analytical Instrumentation Facility, IIT Madras, for DSC and NMR analysis and the Dept. of Metallurgy, IIT Madras, for SEMEDAX analysis. Supporting Information Available. Information about the protein profile of the extracellular supernatant after 12 months shown using SDS-PAGE; carbonate carbonyl index from FTIR studies; GPC chromatograms showing the oligomer formation; the 1H NMR spectra of the degraded polycarbonate by various fungal strains; and a statistical analysis of the effect of pretreatments on various biological parameters. This material is available free of charge via the Internet at

References and Notes (1) Shinsuke, F.; Tojo, M.; Hachiya, H.; Aminaka, M.; Hasegawa, K. Polym. J. 2007, 39, 91–114. (2) Pin˜ero, R.; Garcı´a, J.; Cocero, M. J. Green Chem. 2005, 7, 380–387. (3) Lin, C. H.; Lin, H. Y.; Liao, W. Z.; Dai, S. A. Green Chem. 2007, 9, 38–43. (4) Lowe, D. A. In Handbook of Applied Mycology, Fungal Biotechnology; Arora, D. K., Elander, R. P., Mukerji, K. G., Eds.; Marcel Dekker: New York, 1992; pp 681-706.


Biomacromolecules, Vol. 11, No. 1, 2010

(5) Romero, E.; Speranza, M.; Garcı´a-Guinea, J.; Martı´nez, A. T.; Martı´nez, M. J. FEMS Microbiol. Lett 2007, 275 (1), 122–129. (6) Lee, S. M.; Koo, B. W.; Choi, J. W.; Choi, D. H.; An, B. S.; Jeung, E. B.; Choi, I. G. Biol. Pharm. Bull. 2005, 28 (2), 201–207. (7) Kang, J. H.; Katayama, Y.; Kondo, F. Toxicology 2006, 217, 81–90. (8) Artham, T.; Doble, M. Macromol. Biosci. 2008, 8 (1), 14–24. (9) Hasan, F.; Shah, A. A.; Hameed, A.; Ahmed, S. J. Appl. Polym. Sci. 2007, 105, 1466–1470. (10) Hanaa, A.; El-Shafei.; Nadia, H.; Nasser, A. E.; Kansoh, A. L.; Ali, A. M. Polym. Degrad. Stab. 1998, 62, 361–365. (11) Manzur, A.; Gonza´lez, M. L.; Torres, E. F. J. Appl. Polym. Sci. 2004, 92, 265–271. (12) Arkatkar, A.; Arutchelvi, J.; Bhaduri, S.; Uppara, P. V.; Doble, M. Int. Biodeterior. Biodegrad. 2009, 63, 106–111. (13) Rivaton, A.; Mailhot, B.; Soulestin, J.; Varghese, H.; Gardette, J. L. Polym. Degrad. Stabil. 2002, 75, 17–33. (14) Artham, T.; Doble, M. J. Polym. EnViron. 2009, DOI: 10.1007/s10924009-0135-x. (15) Artham, T.; Sudhakar, M.; Doble, M.; Umadevi, V. R.; Viduthalai, R. R.; Kumar, K. S.; Murthy, P. S.; Venkatesan, R. Open Macromol. J. 2008, 2, 43–53. (16) Artham, T.; Sudhakar, M.; Venkatesan, R.; Nair, C. M.; Murty, K. V. G. K.; Doble, M. Int. Biodeterior. Biodegrad. 2009, 63, 884– 890. (17) Friedrich, J.; Zalar, P.; Mohorcˇicˇ, M.; Klun, U.; Krzˇan, A. Chemosphere 2007, 67, 2089–2095. (18) Bradford, M. M. Anal. Biochem. 1976, 72, 248–254. (19) Dubois, M.; Gilles, K.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Anal. Chem. 1956, 28, 350–356. (20) Pencreac’h, G.; Baratti, J. C. Enzyme Microb. Technol. 1996, 18, 417– 422. (21) Srinivasan, C.; D’Souza, T. M.; Boominathan, K.; Reddy, C. A. Appl. EnViron. Microbiol. 1995, 61, 4274–4277. (22) Laemmli, U. K. Nature 1970, 227, 680–685. (23) Owens, D. K.; Wendt, R. C. J. Appl. Polym. Sci. 1969, 13, 1741– 1747. (24) Tokiwa, Y.; Calabia, B. P. J. Polym. EnViron. 2007, 15 (4), 259–267.

Artham and Doble (25) Kim, D. Y.; Rhee, Y. H. Appl. Microbiol. Biotechnol. 2003, 61 (4), 300–308. (26) Gusse, A. C.; Miller, P. D.; Volk, T. J. EnViron. Sci. Technol. 2006, 40, 4196–4199. (27) Albertsson, A. C.; Andersson, S. O.; Karlsson, S. Polym. Degrad. Stab. 1987, 18, 73. (28) Gottlieb, D.; Etten, J. L. V. J. Bacteriol. 1964, 88 (1), 114–121. (29) Katsikogianni, M.; Missirlis, Y. F. Eur. Cells Mater. 2004, 8, 37–57. (30) Mueller, R. J. Process Biochem. 2006, 41, 2124–2128. (31) Bhatt, R.; Panchal, B.; Patel, K.; Sinha, V. K.; Trivedi, U. J. Appl. Polym. Sci. 2008, 2, 975–982. (32) Oda, Y.; Yonetsu, A.; Urakami, T.; Tonomura, K. J. Polym. EnViron. 2000, 8, 29–32. (33) Bonnarme, P.; Asther, M. J. Biotechnol. 1993, 30, 271–282. (34) Novotny´, Cˇ.; Svobodova´, K.; Erbanova´, P.; Cajthaml, T.; Kasinath, A.; Lang, E.; Sˇasˇek, V. Soil Biol. Biochem. 2004, 36 (10), 1545– 1551. (35) Sutherland, G. R.; Haselbach, J.; Aust, S. D. EnViron. Sci. Pollut. Res. 1997, 4, 16–20. (36) Sepu´lveda, T. V.; Castan˜eda, G. S.; Rojas, M. G.; Manzur, A.; Torres, E. F. J. Appl. Polym. Sci. 2002, 83, 305–314. (37) Ohtake, Y.; Kobayashi, T.; Asabe, H.; Murakami, N. J. Appl. Polym. Sci. 1995, 56, 1789. (38) Vernhet, A.; Fontaine, M.N. B. Colloids Surf., B 1995, 3, 255–262. (39) Weiland, M.; David, C. Polym. Degrad. Stab. 1994, 45, 371. (40) Weibin, G.; Shimin, H.; Minjiao, Y.; Long, J.; Dan, Y. Polym. Degrad. Stab. 2009, 94, 13–17. (41) Alizadeh, A.; Sohn, S.; Quinn, J.; Marand, H. Macromolecules 2001, 34 (12), 4066–4078. (42) Sudhakar, M.; Doble, M.; Murthy, P. S.; Venkatesan, R. Int. Biodeterior. Biodegrad. 2008, 61, 203–213. (43) Jang, B. N.; Wilkie, C. A. Polym. Degrad. Stab. 2004, 86, 419–430. (44) Sivalingam, G.; Madras, G. J. Appl. Polym. Sci. 2004, 91, 2391–2396. (45) Rivaton, A. Polym. Degrad. Stab. 1995, 49 (1), 163–179. (46) McNeill, I. C.; Rincon, A. Polym. Degrad. Stab. 1993, 39, 13.