2.1. Materials
The AA and AM monomers were purchased from Thai Mitsui Specialty Chemicals (Thailand). Potassium hydroxide (KOH), ammonium persulfate (APS) and hydrogen peroxide (commercial grade) were purchased from Ajax (Australia). N, N’-methylene-bisacrylamide (MBA, Fluka, analytical grade) and N, N, N′, N′-tetramethylethylenediamine (TEMED, Sigma-Aldrich, ≥99.5% purity) were used as a crosslinking agent and co-initiator, respectively. Acetone and nitrogen gas (N2) gas (99.99% purity) were purchased from Zen point and Praxair (Thailand), respectively. All chemicals were used as received without further purification. Crude peroxidase was produced from Trametes versicolor CBR-04 according to Bankeeree et al. [12].
2.2. Synthesis of acrylate superabsorbent polymers
Two types of superabsorbent polymers, poly(potassium acrylate) (P(KA)) and poly[acrylamide-co-(potassium acrylate)] (P(Am-co-KA)), were synthesized via a free radical chain polymerization in aqueous solution of monomers with MBA cross-linker while APS and TEMED were used as an initiator and a co-initiator, respectively. All the reactions were performed under N2 atmosphere. A KA solution was prepared by neutralization of 27% (v/v) AA in deionized water (110 mL) with 40 mL of 30% (w/v) KOH. The suspension was stirred at room temperature (25±3°C) for 90 min and then the polymerization of KA was conducted by adding APS (2.7 mg), MBA (17.2 mg) and TEMED (0.18 mL). The reaction was heated up to 65°C with vigorous stirring for 90 min to form a viscous gel.
For the copolymer synthesis of P(Am-co-KA), the monomer solution was prepared by dissolving AM (3 g) in KA solution at the ratio of 5:95 and stirring at room temperature (28±3°C) for 90 min. APS (14.3 mg), MBA (16.1 mg) and TEMED (0.18 mL) were then added and stirred at 65°C for 10 min to form a viscous gel. Each viscous gel was soaked in acetone to remove the absorbed water and unreacted monomers. The gels were oven dried at 65°C until a constant weight was reached, and then ground to reduce the particle sizes and collected the size portions by sieving through a set of ASTM E11-certified steel test sieve of 63, 108 and 212 mm (Retsch, Germany).
2.3. Polymer structure and property determination
The functional groups of P(KA) and P(Am-co-KA) were examined by Fourier transform infrared spectroscopy (FTIR; Perkin Elmer, Spectrum One, USA) with KBr disc for a direct compression over a wavenumber ranging from 400 to 4,000 cm−1, with 32 scans. For water absorbency analysis, the dry P(KA) and P(Am-co-KA) weighing 0.1 g were each immersed in deionized water (500 mL) at room temperature for 24 h to reach an equilibrium swelling. The residual water was removed by filtration through an 80-mesh stainless steel screen. The retained polymer was left on the screen for at least 1 h to completely drain off the unabsorbed water. The water absorption was determined using Eq. (1) as follows:
Water absorbency (g/g) = (Ws – Wd)/Wd (1),
where Wd is the weight of the dried SAP and Ws is the weight of the swollen sample.
2.4. Biological degradation
The biodegradations of P(KA) and P(Am-co-KA) were carried out using a soil burial method (Sharma et al., 2014). Five samples (150 g each) of each swollen gel were separately put in a 14 ´ 14 cm linen bag and buried in the natural garden soil at a depth of ten cm with a three-cm spacing between bags. The soil moisture was maintained by adding 200 mL of tap water every 24 h. To determine the extent of biodegradability, the samples were taken out from the soil after ten weeks, rinsed thoroughly with distilled water to remove the contaminated soil particles and dried in hot air oven at 60°C till a constant weight was obtained. The percentage of weight loss was evaluated based on the following Eq. (2):
Weight loss (%) = [(Wi – Wf) / Wi] x 100 (2),
where Wi and Wf are dry weights of the polymer before and after the treatment, respectively. The structural changes were determined by comparing the FTIR spectra of each sample before and after the biological degradation. Further the changes in the morphological appearance were evaluated by scanning electron microscopy (SEM) and the presence of element was identified by energy dispersive X-ray spectrometry (EDXS) (JSM-6610; JEOL, Tokyo, Japan).
2.5. Effects of peroxidase, hydrogen peroxide and temperature on SAP degradation
The equilibrium swellings of each P(KA) and P(Am-co-KA) were performed by soaking 0.1 g of each SAP in deionized water (100 mL) for 24 h, filtrated through an 80-mesh screen and the products were used as initial polymers for the following studies. These swollen SAPs were incubated in a culture of T. polyzona (100 mL of minimal medium) under a static condition at room temperature (28±3°C) for 20 days. Then, the treated SAPs were filtrated and dried. The degradation efficiency was investigated by the weight loss and water absorbency as described above. To evaluate the enzymatic degradation, the crude peroxidase at different concentrations (0, 5, 10, 15 and 20 U/g) in the same working volume of 50 mL was incubated with each SAP at room temperature for 16 h. The effect of hydrogen peroxide pretreatment was investigated by adding 50 mL of hydrogen peroxide with the different concentrations at 0%, 4%, 8% and 12% (v/w of the dry polymer) to each SAP and incubated at room temperature for 2 h. Weight loss and water absorbency were analyzed from each treatment and the best effective concentration of hydrogen peroxide was used as a basis in the next optimization step. The impact of temperatures was investigated by incubation of the SAP with hydrogen peroxide at the selected concentration for 2 h at different temperatures from 40 to 60°C. The degradation was evaluated according to weight loss and water absorbency reduction as mentioned above. All experiments were performed in triplicate and the results shown are means ± one standard deviation.
2.6. Optimization of SAP degradation
The optimal conditions for SAP degradation were separately determined for the swollen P(KA) and P(Am-co-KA) by the Response Surface Methodology (RSM). The Box-Behnken experimental design with three levels of the three variable factors including three replicates at the center point is shown in Table 1 (Box and Behnken, 1960). The percentages of SAP degradation were taken as the response in each reaction. Statistical analysis of the data was performed by Design-Expert (version 8.0.7.1, Stat-Ease, Inc., Minneapolis, USA) to evaluate the analysis of variance (ANOVA) and to determine the significance of each term in the equations. The fitted polynomial equation was then expressed in the form of three-dimensional plots to illustrate the main and interactive effects of the independent variables on the dependent variables. To verify the accuracy of the predicted model, the experiment was repeated in triplicate using the predicted optimal condition. The structures of the treated SAPs at the optimal condition were identified by 1H-NMR using a Bruker spectrometry (400 MHz).
2.7. Toxicity assessment of the products from SAP degradation
The phytotoxicities of the untreated and treated SAPs were evaluated via the seed germination test using mung bean (Vigna radiata L.), and sweet corn (Zea mays L.). The certified seeds were purchased from Chia Tai Co., Ltd. (Thailand) and imbibed in distilled water overnight before use. The experiments were carried out at room temperature by placing 25 seeds in each petri dish (15 ´ 100 mm.) containing 10 mL of either distilled water, the untreated or treated SAPs (5 g/L) and incubated for three days in the dark. All experiments were performed in five replicates and the results obtained are means ± one standard deviation. The percentages of germination were calculated using the following Eq. (3):
Germination (%) = (seed germinated/total seed) ´ 100 (3),
All the experiments were performed in triplicate and the results so obtained are means ± one standard deviation.