Depletion of Cr(VI) from aqueous solution by heat dried biomass of a newly isolated fungus Arthrinium malaysianum: A mechanistic approach

For the first time, the heat dried biomass of a newly isolated fungus Arthrinium malaysianum was studied for the toxic Cr(VI) adsorption, involving more than one mechanism like physisorption, chemisorption, oxidation-reduction and chelation. The process was best explained by the pseudo-second order kinetic model and Redlich-Peterson isotherm with maximum predicted biosorption capacity (Q m) of 100.69 mg g−1. Film-diffusion was the rate-controlling step and the adsorption was spontaneous, endothermic and entropy-driven. The mode of interactions between Cr(VI) ions and fungal biomass were investigated by several methods [Fourier Transform-Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD) and Energy-Dispersive X-ray spectroscopy (EDX)]. X-ray Photoelectron Spectroscopy (XPS) studies confirmed significant reduction of Cr(VI) into non-toxic Cr(III) species. Further, a modified methodology of Atomic Force Microscopy was successfully attempted to visualize the mycelial ultra-structure change after chromium adsorption. The influence of pH, biomass dose and contact time on Cr(VI) depletion were evaluated by Response Surface Model (RSM). FESEM-EDX analysis also exhibited arsenic (As) and lead (Pb) peaks on fungus surface upon treating with synthetic solutions of NaAsO2 and Pb(NO3)2 respectively. Additionally, the biomass could also remove chromium from industrial effluents, suggesting the fungal biomass as a promising adsorbent for toxic metals removal.


Methods
Isolation and molecular identification of the fungus. We isolated a novel fungus from the growing mat of another mushroom Termitomyces clypeatus MTCC-5091. The isolated fungus was routinely cultured in YPG media. After appropriate growth in the culture flasks, the fungal pellets were harvested through centrifugation (6000 rpm for 30 min) and washed with deionized water until pH of the water reached to 6.8-7.0 approximately.
For molecular identification, the fungal genomic DNA was isolated using the GeneJET genomic DNA purification kit according to the manufacturer protocol and the 18s rRNITS region was amplified by gradient PCR using customized primers [ITS1-'TCCGTAGGTGAACCTGCGG' and ITS4-'TCCTCCGCTTATTGATATG']. PCR (per 50 µl reaction mixture) conditions has been shown in tabulated form (Table S6a,b).Post PCR, the product was purified using QIAquick PCR purification kit following manufacturer protocol. The sequenced regions were submitted to the GenBank under the accession number KY007521.1. For quality checking purposes, approximately 125 ng of genomic DNA and 200 ng of purified PCR samples were separated by standard 1% agarose gel along with 1 kb DNA ladder (GeneRuler TM 1 kb DNA ladder, thermo fisher scientific). After electrophoresis, the gel was visualized with ethidium bromide staining and photographed accordingly.
Equilibrium adsorption isotherm. The adsorption study of chromium on heat dried fungal biomass was carried out by batch equilibrium experiments. In brief, a known mass (~0.2 g) of adsorbent was suspended in 25 mL Cr (VI) solutions of different concentration (50-1200 mg L -1 ) in a stoppered 250 mL flask and kept under isothermal condition (30°C) for 8 h at pH 3.0 in a shaking incubator (EYELA, Tokyo Rikakikai, Japan). We set up an individual flask for obtaining each of the data and therefore no correction was needed due to pipeting out of sampling volume.
The concentration of hexavalent chromium in the solution was determined spectrophotometrically at 540 nm (Thermo Scientific-Multiskan Go) after an equilibrium time.
Adsorption kinetic study. Kinetic data were obtained at designated time points (0-360 min) at initial chromium concentration of 100 mg L -1 with biomass dose of 8 g L -1 . At different time intervals, the solution was separated from the adsorbent material and the concentration of Cr (VI) in the solution was analyzed. The amount of dye adsorbed, Q t (mg g -1 ) at time t on the adsorbent, was calculated by equation (2). The linear equations for all the tested kinetic models and the methods of calculating the model parameters are presented in Table S7.
Batch biosorption experiments using Response Surface Methodology. Another batch experiments, designed through RSM, were conducted as described earlier. [1][2][3][4] In brief, experiments were conducted in 250 mL Erlenmeyer flasks containing 25 mL K 2 Cr 2 O 7 (100 mg L -1 ) solution and appropriate amount of dried AMB by shaking (150 rpm) at 30°C. All the experimental data was performed and analyzed using Design Expert software (Version 10.0, stat-Ease, Inc., Minneapolis, United States). Software was used for fitting the equations that is developed along with regression and graphical analysis for evaluating the statistical significance.
The fittest and the accuracy of the model were evaluated by F-value and the regression coefficient (R 2 ). The optimal condition for hexavalent chromium (Cr +6 ) uptake by the fungal strain was obtained by solving the regression equations and the 3D responses for the variable parameters using Design-Expert software version 10.0. The mathematical model obtained using RSM was validated by conducting experiments on given optimal conditions. The differences in Cr(VI) concentration before and after biosorption were used to find out the efficiency (R) of metal removal [percentage of hexavalent chromium adsorbed] by the biomass as equation (1).The amount of adsorbed Cr (VI) per gram biomass was obtained using equation (2).
For each study, the control flask (without biomass) was also maintained. At the end of each experiment solutions were separated from the biomass by filtration through filter paper (Whatman no. 1), for analysis of Cr(VI) ions left in solution after biosorption. Care was taken to wash all glassware used for experimental purpose with extran (phosphate free) 50% (v v -1 ), followed by 60% (v v -1 ) nitric acid and subsequent rinsing with double-deionized water to remove any possible interference by other metals.         Nitro compounds and disulfide groups