Aspergillus tamarii mediated green synthesis of magnetic chitosan beads for sustainable remediation of wastewater contaminants

The release of different hazardous substances into the water bodies during the industrial and textile processing stages is a serious problem in recent decades. This study focuses on the potentiality of Fe3O4-NPs-based polymer in sustainable bioremediation of toxic substances from contaminated water. The biosynthesis of Fe3O4-NPs by A. tamarii was performed for the first time. The effect of different independent variables on the Fe3O4-NPs production were optimized using Plackett–Burman design and central composite design (CCD) of Response Surface Methodology. The optimum Fe3O4-NPs production was determined using incubation period (24 h), temperature (30 °C), pH (12), stirring speed (100 rpm) and stirring time (1 h). The incorporation of Fe3O4-NPs into chitosan beads was successfully performed using sol–gel method. The modified nanocomposite exhibited remarkable removal capability with improved stability and regeneration, compared to control beads. The optimal decolorization was 94.7% at 1.5 g/l after 90 min of treatment process. The reusability of biosorbent beads displayed 75.35% decolorization after the 7th cycle. The results showed a highly significant reduction of physico-chemical parameters (pH, TDS, TSS, COD, EC, and PO4) of contaminated wastewater. The sorption trials marked Fe3O4-NPs-based biopolymer as efficient and sustainable biosorbent for the elimination of hazardous toxic pollutants of wastewater in a high-speed rate.


Results and discussion
Genetic confirmation of the fungal strain identity. The purified fungal strain EG-MO7 was morphologically and microscopically identified as Aspergillus sp. (Fig. 1A,B), based on the identification key 23 . The morphological identity was further ascertained based on its sequence of ITS fragments. The size of PCR amplicons was found to be around 571 bp (Fig. 1C, uncropped gel image of PCR product in Fig. S1). In the NCBI database, the non-redundant BLAST search revealed that the fungal strain EG-MO7 was closely related to Aspergillus tamari and its ITS sequence was deposited in the database of NCBI with accession number: OL824549.1. The phylogenetic tree of such ITS sequence was constructed via the Neighbor-joining method using a confidence level of 1000 bootstrap (Fig. 1D). The A. tamarii OL824549.1 displayed a similarity percentage of 99% with A. tamarii retrieved database deposited isolates MH2793821.1, KP784375.1, MH859187, and MN128231.1 with E-value of 0.0 and query coverage of 100%.

Optimization of process factors for Fe 3 O 4 -NPs production. The effects of five independent variables
on the process of magnetic nanoparticles production were screened using the two factorial Plackett-Burman design, which is important for reducing the number of repetitive trials during process optimization 24 . The values of the maximum and minimum level of the input parameters and the multiple-regression analysis of the process are shown in Table 1. The regression equation from PBD, after omitting the non-significant factors (P > 0.05), was as the following The optimum magnetic nanoparticle production by harnessing the biomolecules of A. tamarii was recorded at the trial number 2, with the contribution of incubation period (24 h, −1), temperature (30 °C, + 1), pH (12, + 1), stirring speed (100 rpm, −1) and stirring time (1 h, −1). A minimum production of the myco-synthesized magnetic nanoparticle was detected in the 1st run. The highest effect (87.04%) on the production process was recorded for the input factor (pH, X 3 ) as shown in Fig. 2A, followed by the incubation period (X 1 ) and the stirring  www.nature.com/scientificreports/ time (X 5 ). A negative sign indicates antagonistic effect, whereas a positive one represents a synergistic effect. The main effects of the investigated variables on Fe 3 O 4 -NPs production using PBD was graphically illustrated as a Pareto chart in Fig. 2B. The main influential variables affecting the nanoparticle production process was optimized by the application of response surface methodology (RSM) using CCD analyses. The three remarkable factors with positive effect on the dependent variable (response) were applied in CCD as central values. The significance of the RSM model was assessed by ANOVA and the results are briefly listed in Table 2. The results shown that the significant model parameters (P < 0.05) are X 1 , X 3 , X 5 , and X 3 *X 5 . The interactive effects among two independent factors, when the third factor is at optimal level, were presented by 2D-contour plots ( Fig. 3A-C). The 2D plots demonstrated that the production of nanoparticles was significantly increased in the middle value for each factor; however, the production process was significantly reduced by the variation in the factors levels, beyond the optimum level.
The biosynthesis of magnetic nanoparticles is of great significant in the biomedical and biotechnological applications. Different physic-chemical properties are remarkably affected the biosynthesis process. The optimization of the influential factors is significantly desired. PBD and RSM are considered as one of the important statistical analysis which provides adequate information on the significance of real variables in the Fe 3 O 4 -NPs 25,26 . Such techniques are applied for the determination of the major variables affecting the process and the interactive connections among the output and input parameters 25 . The significant influences of temperature, stirring time and pH have been reported by other researchers 26,27 .  www.nature.com/scientificreports/ The results displayed a maximum peak height of 3.68 (Table S1), which was incredibly analogous to the predicted value by using CCD analysis and remarkably close to the best trial (Run no. 3). The experimental and predicted values were found to be satisfactorily correlated, indicating that the relationships between the investigated variables and the Fe 3 O 4 -NP s biosynthesized by A. tamari can adequately described by the empirical model derived from CCD. Therefore, such model could be employed for the prediction of the biosynthesis of Fe 3 O 4 -NP s . Several researchers were approved the exo metabolites of fungai, bacteria, actinomycetes and plants as stabilizing and capping agents for the green synthesis of various nanoparticles 17,28,29 . Characterization UV-vis spectra, FT-IR and TEM analyses. The capability of the fungal mycelial exo-metabolites for the formation of biosynthetic Fe 3 O 4 -NP s was initially observed by the color transformation of reaction mixture to deep black ( Fig. 4A(a-c)). The formation of magnetic nanoparticles was then ascertained by UV-visible spectra. A characteristic absorption peak at 325 nm, which is corresponding to magnetite nanomaterial was observed (Fig. 4A) in agreement with [30][31][32] . The color change is probably attributed to the presence of various biomolecules in fungal extract, which delivers the reduction of different metal ions and the surface plasmon resonance of  www.nature.com/scientificreports/ Two peaks at 1383 and 1027 cm -1 are corresponding to C-N and C-O stretching bands, respectively. A peaks at 1641 and 1148 cm -1 were assigned, respectively, to C=O stretching vibration of amide group and C-O-C of glycosidic bridge. In the MchiBs spectrum, a very low intensity peak at 1083 was determined which is related to Fe-OH stretching vibration. The peak at 527 cm -1 related to the stretching band of Fe-O group. Reactive red 198 selected for studying the decolorization capability using the MchiBs (Fig. 4c). The appearance of some new peaks at a range of 400-700 cm -1 may attributed to the successful adsorption of dye onto the chitosan-Fe 3 O 4 -NPs beads. The peaks become generally broad with slight shift and the intensity decreased after the sorption of dye onto MchiBs. Overall, the FT-IR spectra ascertained the incorporation of Fe 3 O 4 nanoparticles into chitosan beads and the successful biosorption of dye by MchiBs. Similar results have reported by other researchers 1,4,34,35 .
The average size of the nanomaterial is one of the major parameters, evidently related to its activities 35,36 . Herein, the size and shape of Fe 3 O 4 -NPs were determined using TEM. The TEM image showed that the average size of the Fe 3 O 4 -NPs were from 5 to 22 nm with homogenous distribution and spherical morphology ( Fig. 4C(a, b)) 8 observed that the biosynthesized Fe 3 O 4 -NPs had uniform spherical shapes with homogenous distribution. The potentiality of the metabolites produced by A. niger for the green synthesis of Fe 2 O 3 -NPs was confirmed by 37 . SEM and EDX analyses. The digital images of control chitosan beads (CchiBs) and MchiBs have illustrated in (Fig. S2A-D). After the incorporation of magnetic nanoparticles, it's observed that the color of CchiBs changed from white to whitish-black or grayish-white. The textural and surface morphology of both CchiBs (Fig. 5A,B) and MchiBs (Fig. 5C) were examined by SEM. The SEM images showed spherical irregular surface and porous structure. The surface area was increased by incorporating Fe 3 O 4 -NPs. The SEM pictures of the CchiBs and MchiBs before adsorption showed quite smooth surface with small grains and high porosity; however, the surface of MchiBs loaded with reactive red 198 as a model dye, shows the formation of aggregated dye particles, which were interconnected to each other (Fig. 5D). Several researchers stated that the behavior and biosorption efficiency is mainly dependent on the surface area as well as porosity of the nano-sorbent particles 10,19,[38][39][40] (Fig. 7A,B). The results reveal the decolorization percentage increased on continuing elevation in the MchiBs quantity from 0.25 to 1.5 g/l. The optimal removal percentage (94.7%) was determined at 90 min using 1.5 g/l of biosorbent (Fig. 7A). Whereas, the minimum decolorization percentage after 90 min incubation found to be 56.7% using 0.25 g/l of nanosorbent. The bioremediation process showed no remarkable difference when the process prolong for 150 min. The probable reason is the presence of more available sites for the uptake of dyes at the beginning of the batch experiment using biosorbent beads. The developed modified beads have a surface rich in the protonated -OH and -NH 2 groups due to chitosan network. The sulfonic groups of dyes are vulnerable and show a low penetration ability in chitosan, which increase the accessibility of such groups to adsorption sites 1,13,36,45,46 .
The successful reusability of biosorbent for multiple cycles is very important parameter for the sustainability and potentiality of biosorbent in the bioremediation processes. Therein, the decolorization potentiality of MchiBs for seven repeated treatment cycles was assessed using textile wastewater at the optimal sorbent concentration and the optimal incubation time. The decolorization efficiency using MchiBs displayed 97.18, 91.14, 85.91, 80.98, 77.46 and 75.35% during successive decolorization trials for textile wastewater (Fig. 7C). After the 7th cycle, 75.35% decolorization was determined 5 reported the enhancement of dye removal efficiency by using magnetic chitosan beads. One of the most important features of the magnetic chitosan beads is the ease retrieve from treatment solution and use for the consequent contaminants removal 35,47,48 .
The treatment process of textile wastewater using MchiBs was also evaluated through determination of pH, TDS, TSS, COD, EC, and PO 4 as compared to untreated samples (control) under the same conditions (Table 3a). The treatment process conducted using 1.5 g/l biosorbent dose for 90 min. The results showed a highly significant reduction of TDS and COD from 1471.64 to 492.67 mg/l and from 1062.1 to 75.67 mg/l, respectively. In addition, the MchiBs could respectively reduce TSS EC, and PO 4 , related to the control textile wastewater samples. The removal percentage of textile wastewater contaminants using MgO-NPs was 72.2, and 92.1% for TDS, and COD. In addition, Fe 2 O 3 -NPs displayed 47.6, and 82.8%, respectively; however, the other tested parameters were considerably reduced, compared to untreated samples 2,41 . The polyaluminum chloride exhibited a reduction percentage of 84% for TDS 49    The real industrial and textile wastewater have high organic contents with non-degradable nature, which are lost during dyeing process in the effluent 37,45 . This may attributed to the high value of various physicochemical features (pH, TDS, TSS, COD, EC, and PO 4 ) of untreated effluents 2,50 . The removal of contaminants (heavy metals) using the Fe 3 O 4 -NPs incorporated into chitosan and regeneration of beads was reported by 1,4,5 . The physicochemical characters of textile wastewater remarkable reduced by treating with the nanoparticles, particularly COD, as determined by other researchers 37,51 . Similar results have reported for the significant reduction of industrial and textile wastewater parameters when treated with various biosorbent systems 4,36,37 .

Materials and methods
The overall process for the synthesis of control chitosan beads (CchiBs), magnetic chitosan beads (MchiBs), and the proposed adsorption mechanism for a dye pollutant model clearly shown in Fig. 8. Fungal strain. The fungal strain EG-MO7, which was previously isolated from a soil sample (Benha, Egypt), was employed in the current study for the synthesis of magnetic nanoparticle. In brief, the isolation was performed by inoculating 1.0 ml of 10 -5 soil dilution onto Czapek's-Dox agar (CDA) medium. The plate was incubated at 30 °C for 5 days. The developed purified strain was preserved on CDA medium slant for further study 52 . Morphological and molecular identification of fungal strain. The fungal isolate was recognized according to its morphological and macroscopically features using standard key of the genera Aspergillus sp. 53,54 . Such primary identification of fungal strain was affirmed by sequencing its internal transcribed spacer (ITS-rDNA) segment 28,55 . The fungal genomic DNA was extracted according to 56 . In the PCR, the gDNA was employed as template for the primers of ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') using 2 × PCR master mixture (AlphaDNA Co, Canada). The PCR was performed in a Solgent EF-Taq, PCR Machine name: 9700(ABI), MJ research thermal cycler (USA). PCR amplification was conducted for 3 min at 95 °C, followed by 35 cycles for 30 s at 95 °C, 50 °C for 30 s and 72 °C for 90 s and next a final extension for 5 min at 72 °C. The PCR amplicon was investigated throughout 1% agarose gel electrophoresis, and then was sequenced by the same primer sets. The nucleotide sequence obtained from the ITS sequence was related to the ITS sequences in the GenBank database. Multiple sequence alignment were performed using ClustalW muscle algorithm of MEGA-X 11 and a phylogenetic tree was conducted by applying the neighborjoining method with 1000 bootstrap analysis.  www.nature.com/scientificreports/ dark conditions for 24 h. The preparation was heated at 60 °C, stirred for 1 h at 100 rpm and the pH was retained at pH 12. The resulted intense black color was monitored using UV/visible spectrophotometer. Subsequently, the preparation was kept at lab temperature for 1 h and then settled by centrifugation at 5000 × g for 20 min, and washed several times by distilled H 2 O. The as-formed Fe 3 O 4 -NPs were oven-dried at 60 °C for 12 h.

Optimization of Fe 3 O 4 -NPs production using two-factorial Plackett-Burman design.
In order to investigate the influences of different significant variables in Fe 3 O 4 -NPs production, five selected main variables namely incubation period (X 1 ), temperature (X 2 ), pH (X 3 ), stirring speed (X 4 ) and stirring time (X 5 ), were analyzed using Plackett-Burman design (PBD) as illustrated in Table S3. The investigated variables were selected based on literature reports on the Fe 3 O 4 -NPs production 26,27 . Each independent variable was checked at 2-levels namely: high level (+1) and low level (−1), based on the fact that the biosynthesis of nanoparticles is remarkably regulated by the physicochemical parameters 10,26,57 . PBD depends on a 1st order model: Y = β 0 + ∑β i www.nature.com/scientificreports/ X i where, Y was the height of absorbance peaks (response), β 0 and β i were the constant coefficients and X i was an independent variable. The experiments were conducted in duplicate and the process optimization was investigated by spectrophotometrically measuring the height of absorbance peaks at the previously detected surface plasmon resonance 2 . Analysis of variance (ANOVA) was employed for testing the significance of each variable in the model. The investigated variables which exhibiting the major positive effects were chosen according to the results of Pareto chart, followed by further optimization throughout the Central composite design (CCD) of Response Surface Methodology. Further, three selected independent variables were optimized using CCD with α value of ± 1.681. The correlation between the response data and the variables were analyzed using a second order polynomial equation. 2D response plots were plotted, to investigate the major effects as well as the interactive ones between the dependent variable (response) and the independent ones.

Synthesis of magnetic-chitosan gel beads (MchiBs).
The production of magnetic-chitosan gel beads was performed according to 1,35 via the modified sol-gel method (Fig. S3). In brief, a desirable amounts of chitosan powder was dissolved using magnetic stirring for 15 min in 50 ml of CH 3  was added to wastewater samples. The systems were incubated in dark at ambient temperature with continuous agitation to attain proper oxygenation. The decolorization efficiency was determined at various time intervals (30,60,90, 120 and 150 min) by withdrawing 1 ml of each treatment, centrifuged for 15 min at 5,000×g, and the residual concentration of the dye was monitored at λ max = 530 nm using UV-visible. The decolorization percentage (D.P., %) was calculated using the following equation 28,58 : www.nature.com/scientificreports/ where C i and C f are the initial and final dye concentration. The reusability of the investigated MchiBs was assessed for textile wastewater treatment through seven consecutive cycles under the optimal conditions according to 28,52,59 with some modification. Afterward the completion of the 1st biosorption cycle as declared above, the MchiBs were separated using external magnet, next rinsed many times with distilled water and then used for the next cycle. The relative decolorization percentages were calculated, related to the first degradation cycle 28,52 .
The physicochemical parameters; total dissolved solids (TDS), pH, total soluble salts (TSS), chemical oxygen demand (COD), conductivity (EC), phosphate and sulfur were determined before and after the treatment with MchiBs according to 60 at the optimal time of decolorization and biosorbent concentration. Deposition of the fungal strain. The fungal strain A. tamari, was deposited into the GenBank under accession number OL824549.1.

Data statistical analysis.
All experiments were carried out in triplicates and data was articulated as mean ± standard deviation. The process of nanoparticle production was optimized by screening the selected independent variables using the Placket-Burman design. The analysis was conducted using MINITAB 18.0 statistical software package, USA. In order to explore significant differences at a confidence level of 95% (P < 0.05), the paired t test was carried out. Statistical Package for Social Sciences (SPSS) version 25 (IBM, Armonl, Ny, USA) was used for conducting the data processing statistical analysis.

Conclusions
The successful biosynthesis of Fe 3 O 4 -NPs by harnessing the exo-metabolites present in the fungal filtrate of A. tamarii EG-MO7 was performed. Based on PBD and CCD, the optimum magnetic nanoparticle production was determined with the contribution of incubation period (24 h), temperature (30 °C), pH (12), stirring speed (100 rpm) and stirring time (1 h); however, the highest effect was recorded for the pH. The Fe 3 O 4 -NPs impregnation into chitosan beads, which was successfully, performed using sol-gel method. The modified beads exhibited a remarkable decolorization rate with considerable regeneration property and a highly significant reduction of various physico-chemical parameters (pH, TDS, TSS, COD, and PO 4 ) in short treatment period (90 min). The exact mechanism of the fungal exometabolites incorporate in the nanoparticle synthesis should be performed in further studies. The performance of Fe 3 O 4 -NPs-based polymer in the bioremediation process was found to be simple, satisfactory, and perform potential advantages in the bioremediation of wastewater contaminants. However, the detailed adsorption mechanism, kinetics and isotherms of the modified beads for the dye removal should be performed in future studies. Large-scale production of the above-mentioned chitosan composite, especially in the developing countries, required further research for reducing the biosynthetic cost. The formation of secondary pollutants due to the usage of chitosan composite in bioremediation processes is a major drawback during the recycling and regeneration of chitosan nanocomposite.

Data availability
All data generated or analyzed during this study are included in this article and its additional file.