Cellulose filter paper

In batch experiments using Milli-Q water and secondary effluent from a wastewater treatment plant, the effectiveness of LMPPs for removing emerging contaminants from an aqueous solution was examined. Before processing the wastewater samples, they were filtered using Whatman cellulose filter paper (47 mm circle) and the pollutants were spiked in the samples. In the tests, 100 mg of LMPPs was added to 40 mL of a pollutant mixture (50 mg/L, from each) and mixed at 150 rpm at room temperature. Samples of 2 mL were collected every half an hour, and then a 24-h sample was collected. The purpose of this step was to determine when the system reach equilibrium. Consequently, the system attained equilibrium at 120 min after evaluating the whole measurements. Based on the initial and final aqueous phase concentrations, the removal efficiency was calculated. The LMPPs were cleaned with Milli-Q water and dried, and the cycle continues (every 120 min). The removal of pollutants by MPPs (without laccase) due to adsorption was also examined to understand the contribution of physical removal and degradation. This was accomplished by adding 100 mg of MPPs to 40 mL of a pollutant mixture (50 mg/L) and mixing at 150 rpm at room temperature. Samples of 2 mL were withdrawn every 30 min for two hours.

Cellulose filter paper (541, Whatman) with average pore size ~22 μm, 125 mm diameter, and ~154 μm thickness, acted as a neat cellulose substrate and was purchased from Sigma-Aldrich, India. The AgNW suspension (10 mg/ml) was purchased from Zhejiang Kechuang Advanced Materials Co., Ltd (Zhejiang, China), with an average diameter of 30 nm and an average length of 15 µm. Hydrophobic inorganic ceramic agent (GN-704, silicon dioxide as the main component, isopropyl alcohol as the solvent) was supplied by Guangzhou Yina Co. Ltd (Guangzhou, China).

Semi-solid agar was prepared using a mixture of 1% peptone, 0.5% NaCl and 0.25% Bacto-agar [21 (link)]. A total of 100 mg/L of BSA, SA (Sigma-Aldrich, USA), and HA were dropped on a cellulose filter paper (2.5 µm pore size, Whatman, Maidstone, UK) and allowed to diffuse from the membrane into the semi-solid media for 16 h at RT. P. aeruginosa PAO1 cells were incubated in an Luria–Bertani (LB) medium (BD, USA), for 16 h at 30 °C. A total of 10 µL of cultured bacteria (10⁵ cells/mL) were inoculated at the center of the plate. The tip with the bacteria solution was inserted into the agar and ejected bacteria solution when the tip was pulled up through the media [21 (link)]. After inoculation, the agars were incubated at 30 °C, for 24 h. The diameter of the distance moved from the edge of the membranes was measured and averaged (n = 7).

The well-studied F. graminearum wild-type strain PH-1 (NCBI: txid229533) was used. It was routinely grown on defined Fusarium medium (DFM) agar medium [20 (link)], with NO₃ as the sole nitrogen source, consisting of glucose (12.5 g/L), NaNO₃ (20 mM), MgSO₄ 7H₂O (2.1 mM), KH₂PO₄ (11.2 mM), KCl (7 mM), and trace minerals (Na₂B₄O₇ 40 μg/L, CuSO₄ 5H₂O 400 μg/L, FeSO₄ 7H₂O 1200 μg/L, MnSO₄ 1H₂O 700 μg/L, NaMoO₂ 2H₂O 800 μg/L, ZnSO₄ 7H₂O 10 μg/L), and the pH set to 6.5 after autoclaving. For agar media, 2% agar was added. PH-1 and the FgHis1mCherry reporter strain were used as a non-bacterial background strain, and the Fg-FEB fungal strains containing FEB were grown, subcultured, and kept on DFM solid agar medium supplemented with gentamicin (0.512 mg/L). The fungal cultures were incubated at 24 °C with 12 h of light and dark cycles. For long-term storage, fungal cultures grown on small cut pieces of Whatman cellulose filter paper were dried and stored in sterilised envelopes with silica gel at 4 °C. To clarify, Table S1 contains descriptions and explanations for naming gene constructs and fungal strains containing bacteria with softened versions since this had to be introduced to avoid frequently repeated extended expressions.

Cellulose filter papers (diameter 125 mm, Whatman, Hangzhou, China) were cut into (2 × 5 mm) pieces and dried overnight at 105 °C before use. Tetrabutylammonium acetate (Sigma-Aldrich, Santa Clara, CA, USA, 97%), Dimethyl sulfoxide (BDH, 99.7%), Succinic anhydride (Alfa Aesar, Shanghai, China, 99%), and Isopropyl Alcohol (BDH, Radmanovic, PA, USA, 99.5%) were used without further purification. All other chemicals were purchased from commercial sources and were of reagent grade.