Materials
Poly [4,4'-methylenebis (phenyl isocyanate)-aH-1,4-butanediol/di (propylene glycol)/ polycarbonate] was purchased from Sigma-Aldrich, GmbH, Germany. Impranil® DLN w50 was a gift from Bayer Company. Modified M9 medium was used for Impranil® and PU degradation assays which contains (g/l): Na2HPO4 (13.0), KH2PO4 (3.0), NH4Cl (1.0), NaCl (0.5), agar (15) and 1% Impranil® DLN w50 (sterilized separately by 0.45 μm filter and was added after autoclaving) [42] or PU films as the sole source of carbon. Urea agar was used for urease activity assay which contains (g/l): peptone (1), NaCl (5), KH2PO4 (2), phenol red (0.012), glucose (1) and agar (15), Urea (20) in 1 litter of distilled water (Urea was sterilized separately by 0.45 μm filter and was added after autoclaving) [43]. Tributyrin Agar (TBA) was used for the esterase activity assay. For TBA preparation, 20 gr tributyrin agar base (containing yeast extract and peptone) was added to 1 litter of distilled water [44]. Internal transcribed spacers (ITS) amplification was performed using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primer sets [45].
Sample collection
To isolate polyurethane degrading yeasts, 21 samples were collected from 5 to 10 cm depth of Saravan forest soil (Gilan province, Iran), and wastewater of edible oil from Ladan Oil Company (Tehran province, Iran). Soil and wastewater samples were collected in sterilized zip plastic bags and sterilized glass bottles respectively. Samples were transferred quickly to the laboratory at 4°C and immediately used for enrichment culture.
Preparation of polyurethane films
Polyurethane films were prepared by dissolving 0.25 gr Poly [4,4'-methylenebis (phenyl isocyanate)-aH-1,4-butanediol/di (propylene glycol)/ polycarbonate], in 25 ml Tetrahydrofuran (THF) solvent. The prepared solution was poured into glass Petri dishes and allowed to evaporate under a fume hood. After 24 h, polyurethane films were cut into quadratic pieces of 2×2 cm2 [5].
Isolation of polyurethane degrading yeasts
Environmental yeast strains were isolated via streak plate technique on Rose Bengal Agar. This technique was performed to enrich and isolate yeast strains with the potential of producing polyurethane degrading enzymes. Further purification was performed on GPY (Glucose-Peptone-Yeast Extract) culture medium until complete purification of all isolates. Purified isolates were stocked in 20% glycerol containing TSB media and stored at -20°C.
Preliminary screening
Protease, urease, esterase, and impralinase activity assays were investigated for screening and selection of isolated strains. To detect the protease activity, isolated strains were cultured on skim milk agar [46] and Esterase activity was evaluated on TBA medium. Inoculated media were incubated at 30°C for 48 h. colonies with clear halo zones were considered protease and esterase positive. Slant urea agar was used for urease assay. Prepared culture media was inoculated and incubated at 30°C for 48 h and color changes were monitored (Roberts et al. 1978). Impranilase assay was performed using a modified M9 medium with impranil® as the sole source of carbon to screen polyurethane degrading yeast strains. Culture plates were incubated at 30°C and monitored frequently for halo zone formation. For quantitative analysis of Impranil® degradation activity, 50 ml of M9 liquid culture media containing 1% Impranil® were inoculated with 5% inoculum and incubated at 30°C for 14 days at 120 rpm. In 48 h intervals, 2 ml of the media was recovered from culture for colony counting on GPY agar and Impranil® concentration, measured by spectrophotometer (Shimadzu-UV160, Japan) at 600 nm. The standard curve of Impranil concentration was constructed with different concentrations of Impranil®. It is worth mentioning that all experiments were repeated three times and quantitative results were confirmed by t-test analysis (P<0.05). Statistical analysis was performed by SPSS for windows (version 16).
Molecular identification of selected yeast strains
Strain identification was performed through PCR amplification of the ITS region of the rRNA gene. DNA was extracted from biomass pellets according to Zhang et al. protocol [47]. Amplification was performed by ITS1 and ITS2 primer sets, in thermocycler with the following program: initial denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final elongation step at 72°C for 10 minutes. The amplified PCR products were sequenced by Macrogen (South Korea). Sequences were blasted with National Center for Biotechnology Information (NCBI) sequences and the closest identity results were reported.
Polyurethane degradation assays
To investigate polyurethane degradation, the prepared polyurethane films were added into Erlenmeyer flasks, containing 20 ml of M9 culture medium and each flask was inoculated with 5% of inoculum. Inoculated flasks were incubated at 30°C in a shaker incubator at 120 rpm for 30 days. After 30 days, possible changes in polyurethane film structure were investigated by the following tests: Sturm test, Fourier transform infrared (FTIR) analysis, scanning electron microscopy (SEM), and Proton nuclear magnetic resonance (H-NMR) spectroscopy. Inoculum-free medium containing PU films was used as a negative control.
Carbon dioxide (CO2) production assay (Sturm test)
Sturm test was performed to measure CO2 production as an indicator of growth and PU degradation. This test was carried out in silicon-sealed caped vessels with oxygen inlet and CO2 outlet. Three pieces (2×2 cm2) of polyurethane film were added into vessels, containing 50 ml M9 culture medium. Oxygen gas was blown into the medium through a silicon hose and a 0.45 μm filter to remove the carbon dioxide completely. Then, the outlet and inlet valves were closed and vessels were incubated at 30°C in a shaker incubator at 120 rpm for 30 days. To measure the CO2 production, barium chloride buffer (0.1M) was added to KOH buffer (1M), instill. Then, the CO2 outlet was connected to a buffer chamber and white precipitates of barium carbonate were dried by dry heat at 80°C. The amount of CO2 was measured by the difference in the weight of samples dried precipitates and control. This is an indirect way to measure accumulated CO2 in the sample, according to this equation: BaCO3 → CO2 + BaO. The test was repeated three times and Inoculum free M9 medium vessels (containing polyurethane film) were used as control.
Fourier Transform Infra-Red (FTIR) analysis
FTIR analysis was performed for chemical bonds and functional group changes elucidation in polyurethane films. For this purpose, after 30 days of incubation, polyurethane films (2×2 cm2) were recovered from culture media and washed with distilled water. Treated films were placed in a sample plate of FTIR (Bruker Vector22-Germany) and spectra were recorded within 400-4000 cm-1 wavelengths. The intensity was determined based on absorption (%) and polyurethane films incubated in inoculum free M9 medium were used as control.
Scanning electron microscopy (SEM)
Scanning electron microscopy was performed to evaluate structural changes on the surface of polyurethane films. Polyurethane films were recovered from culture media and fixed with 2% v/v glutaraldehyde for 2 hours. After fixation, polyurethane films were dehydrated by 25%, 50%, 75%, 90%, and 100% ethanol (2 hours for each concentration). Dried polyurethane films were coated with a thin layer of gold and observed by scanning electron microscope (EVO18; Zeiss) [48]. The polyurethane films incubated in an inoculum-free M9 medium were used as control.
Proton nuclear magnetic resonance (H-NMR) spectroscopy
Polyurethane polymer has got many hydrogen atoms in its structure, so H-NMR 500 MHz was used as one of the chemical changes assessments in this experiment. For H-NMR spectroscopy, 30 days treated PU films, were recovered from culture media, washed with distilled water, and dissolved in 0.6 ml of deuterated tetrahydrofuran solvent. After complete dissolution of polyurethane films in the solvent, the sample was placed in Bruker (Germany) instrument and investigated by H-NMR spectroscope. The polyurethane films, incubated in inoculum-free M9 medium were used as control.