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Innovative method for encapsulating highly pigmented biomass from Aspergillus nidulans mutant for copper ions removal and recovery

['Ailton Guilherme Rissoni Toledo', 'Department Of Biochemistry', 'Organic Chemistry', 'Institute Of Chemistry', 'São Paulo State University-Unesp', 'Araraquara', 'Sp', 'Jazmina Carolina Reyes Andrade', 'Mauricio Cesar Palmieri', 'Itatijuca Biotech']

Date: 2021-12

Biosorption has been considered a promising technology for the treatment of industrial effluents containing heavy metals. However, the development of a cost-effective technique for biomass immobilization is essential for successful application of biosorption in industrial processes. In this study, a new method of reversible encapsulation of the highly pigmented biomass from Aspergillus nidulans mutant using semipermeable cellulose membrane was developed and the efficiency of the encapsulated biosorbent in the removal and recovery of copper ions was evaluated. Data analysis showed that the pseudo-second-order model better described copper adsorption by encapsulated biosorbent and a good correlation (r 2 > 0.96) to the Langmuir isotherm was obtained. The maximum biosorption capacities for the encapsulated biosorbents were higher (333.5 and 116.1 mg g -1 for EB10 and EB30, respectively) than that for free biomass (92.0 mg g -1 ). SEM-EDXS and FT-IR analysis revealed that several functional groups on fungal biomass were involved in copper adsorption through ion-exchange mechanism. Sorption/desorption experiments showed that the metal recovery efficiency by encapsulated biosorbent remained constant at approximately 70% during five biosorption/desorption cycles. Therefore, this study demonstrated that the new encapsulation method of the fungal biomass using a semipermeable cellulose membrane is efficient for heavy metal ion removal and recovery from aqueous solutions in multiple adsorption-desorption cycles. In addition, this reversible encapsulation method has great potential for application in the treatment of heavy metal contaminated industrial effluents due to its low cost, the possibility of recovering adsorbed ions and the reuse of biosorbent in consecutive biosorption/desorption cycles with high efficiency of metal removal and recovery.

Competing interests: The company did not fund this research or participate in the study design. Also, they did not participate in the data collection and analysis or publication decision or manuscript preparation, did not fund this research or provided any research materials, did not provided financial support in the form of authors’ salaries either and there is no competing interest in this work. Dr. Mauricio Cesar Palmieri, who was one of the inventors of the new biomass encapsulation method proposed, only participated in this study in his spare time. Therefore, based on these statements, it was not altered our adherence to PLOS ONE policies on sharing data and materials.

Funding: -AGRT -R$1500.00 per month, from 03/01/2016 to 02/01/2018 -Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - https://www.gov.br/capes/pt-br -The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. -JCRA -R$2200.00 per month, from 16/08/2014 to 16/08/2018 -Asociación Universitaria Iberoamericana de Postgrado (AUIP) - https://auip.org/es/ --The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Rissoni Toledo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this context, the development of a cost-effective immobilization technique for metal removal/recovery is essential to improve the competitiveness of industrial processes, decreasing the process cost and dependence on a continuous supply of biosorbent [ 2 , 11 , 40 , 47 ]. In the present study, a new method of reversible encapsulation was developed using cheap, nontoxic and readily available semipermeable membrane capsules, in which the fungal biomass can freely float inside the capsule filled with deionized water, without blocking its binding sites and also allowing the passage of copper ions from the solution into the capsule containing the biosorvent immersed in the aqueous phase. Batch biosorption experiments were conducted to evaluate copper ion removal by an encapsulated biosorbent. The biosorption mechanism was investigated using kinetic and isothermal models, as well as scanning electron microscopy with an energy dispersive X-ray analytical system (SEM-EDXS) and FT-IR spectroscopy. The potential of our encapsulated biosorbent for a practical application in wastewater treatment was evaluated in relation to the efficiency of metal recovery in several biosorption/desorption cycles.

For the application of fungi biomass in large-scale processes, immobilization of biosorbent is a necessary step to increase the efficiency of metal adsorption on the surface of biomass, the removal and recovery of metals, as well as to regenerate and reuse the biosorbent in subsequent cycles. The free microbial cells are generally small particles that have low density, poor mechanical strength and little rigidity, which may cause solid–liquid separation problems, possible biomass swelling, inability to regenerate/reuse, clogging of filter parts and a reduction in high pressure required to generate suitable flow rates in a packed or fluidized column mode [ 5 , 35 , 36 ]. To address these issues, microbial biomass immobilization systems, including entrapment, adsorption, cross-linking, covalent bonding to the carrier and encapsulation, have been studied [ 5 , 37 – 46 ]. In most studies, biomass is immobilized in polymeric matrices, such as sodium alginate, polysulfone, polyacrylamide and polyurethane, with an appropriate mechanical strength porosity and size [ 40 ]. Nevertheless, this type of immobilization has some disadvantages, including mass transfer limitations and high cost of these matrices, which may not allow its application in large scale processes. Furthermore, these matrices may reduce the removal capacity, obstructing or damaging the metal-binding sites due to irreversible binding between the biosorbent and the immobilizing matrix [ 40 , 47 ].

Studies in our laboratory showed that the highly pigmented biomass produced by the MEL1 mutant from Aspergillus nidulans has a higher biosorption capacity for neodymium than the unpigmented biomass [ 28 ]. We characterize this pigment as 3,4-dihydroxyphenylalanine (DOPA)-melanin according to its physicochemical properties and tests with melanin biosynthesis inhibitors [ 29 ]. In the literature, other studies have also suggested that biomass from fungi pigmented can be considered a promising biosorbent due to the fact that melanin acts as a metal chelator, significantly enhancing the biomass-metal interaction and consequently its biosorption capacity [ 18 , 30 – 34 ].

As the biosorption consists of the adsorption of metals into the cellular surface of the biomass, the metal binding capacity depends mainly on the components present on the cell surface and the spatial structure of the cell wall [ 5 , 6 , 22 , 23 ]. The fungal cell walls are complex macromolecular structures predominantly consisting of chitin, glycans, mannans, which have various functional groups (amine, imidazole, phosphate, sulfate, sulfhydryl and hydroxyl) that are potential metal-binding sites. Furthermore, some fungal species produce a dark-brown pigment closely associated with chitin, known as melanin that contains many groups including carboxyl, phenolic and alcoholic hydroxyl, carbonyl and methoxyl, which have a vital role in metal adsorption, significantly increasing the efficiency of the biosorption process [ 18 , 24 – 27 ].

Among the various types of biosorbents, fungal biomass has been considered as a cost-effective adsorbent for treating metal-contaminated wastewaters because it can be easily obtained in large quantities from industrial processes or organisms of rapid growth using simple and inexpensive cultivation techniques [ 2 , 7 , 13 – 16 ]. Several studies have reported fungal biomass as a promising biosorbent for heavy metal removal from industrial wastewater [ 9 , 11 , 13 , 17 – 21 ].

Compared to conventional methods (precipitation, flocculation, ion exchange and membrane filtration), biosorption has been considered a promising alternative to treat large amounts of industrial effluents containing heavy metals in low concentrations [ 3 , 5 , 6 ]. The main advantages for biosorption applications in industrial processes are the low cost of biosorbents, their high efficiency for metal removal (especially in low-concentration solutions), regeneration/reuse of biosorbents, potential metal recovery, and the non-generation of secondary residues [ 7 – 12 ].

Various anthropogenic and industrial activities generate bulk quantities of waste containing considerable concentrations of heavy metals, which have detrimental effects on terrestrial and aquatic environments for all living beings [ 1 , 2 ]. Copper ion is one of the most common heavy metals in effluents from different industries and it can become toxic to cells when its concentrations surpass certain optimal levels, causing adverse human health effects [ 3 , 4 ]. After introducing more restrictive laws for wastewater disposal contaminated with metals, economic, effective and eco-friendly technology needed to be developed to remove toxic metals from wastewater before disposing of it safely.

The results were presented as the mean ± standard deviation (three independent experiments, n = 3). Root-mean-square deviation (RMSD) and linear regression analysis were used as a measure of the goodness-of-fit of the mathematical models. Small RMSE values and values of R 2 close to 1.0 indicate better curve fitting. All kinetic and isotherm parameters of the models were evaluated by linear regression analysis of the experimental data using the Microsoft Excel 2016, version 2102, software ( S1 Appendix ).

To evaluate the biosorption/desorption cycles, the capsules containing the metal-free biosorbent were exposed to copper solution (initial concentration of 750 mg L -1 ). After the time required to reach biosorption equilibrium, the capsules were removed, washed with distilled water and treated with HCl solution (0.05 mol L -1 ) for metal desorption. This sorption-desorption cycle was repeated five times to determine the reusability potential of the biosorbent. From the second biosorption cycle, the pH of the copper solution containing regenerated encapsulated biosorbent was corrected again at 5 using the 0.2 mol L -1 NaOH solution and the system was allowed to reach equilibrium once more. The cycles were performed using the same batch of capsules. The desorption capacity and recovery efficiency of the metal ions were calculated according to the following Eqs 7 and 8 , respectively: (7) (8) where q des (mg g -1 ) is the desorption capacity expressed in milligrams of metal ions desorbed per gram of biosorbent, C f (mg L -1 ) is the final copper ion concentration in solution, V (L) is the volume of the solution, m (g) is the mass of the biosorbent, m des (mg) is the mass of desorbed metal ions, m bios (mg) is the mass of biosorbed metal ions and d (%) is the recovery efficiency expressed as a percentage of recovered metal. The m bios value was obtained from the biosorption capacity calculation.

After the biosorption assay, the encapsulated biosorbent (EB30) was collected from the copper solution (initial concentration of 750 mg L -1 and pH 5) and the capsule containing the metal-loaded biomass was washed with distilled water and treated with HCl solutions at different concentrations (0.05, 0.1 and 0.2 mol L -1 ). This mixture was allowed to stand at room temperature under constant agitation (150 rpm) for 30 to 360 min to determine the time required to reach chemical equilibrium. The copper ion concentration was determined by Atomic Absorption Spectroscopy.

The functional groups present in the fungal biomass were investigated by Fourier-transform infrared spectroscopy (FT-IR). FT-IR spectra of metal-free and copper-loaded biosorbents were obtained using a Nicolet iS5 FTIR Spectrometer (Thermo Scientific). The washed and dried biomasses were mixed with KBr, pressed in a pastillator (6 tons) under vacuum for 1 min and analysed with a resolution of 2 cm -1 in the range of 4000–400 cm -1 .

The biosorbent morphology before and after copper ion sorption was analysed by scanning electron microscopy (SEM, JEOL, JSM-7500F, Japan) using secondary electrons and elementary analysis of these samples was performed by dispersive X-ray spectroscopy (EDXS). Before these analyses, biosorbent samples were washed with distilled water dried at 55°C for 24 h and then coated with carbon using a vacuum system.

The linearized mathematical expressions of the Langmuir and Freundlich isotherms are represented in Eqs 5 and 6 , respectively, as shown below: (5) (6) where C eq (mg L -1 ) is the metal concentration at equilibrium, q eq (mg g -1 ) is the biosorption capacity, amount of metal adsorbed by the biosorbents at equilibrium, q max (mg g -1 ) is the maximum biosorption capacity, K L (L mg -1 ) is the Langmuir constant related to the adsorption energy, K F (mg 1-1/n L 1/n g -1 ) is the Freundlich constant related to the adsorption capacity and 1/n is the Freundlich constant related to the heterogeneity of the surface.

The adsorption properties of encapsulated biosorbents at an equilibrium condition were studied by the Langmuir and Freundlich isothermal models. The Langmuir model assumes a homogeneous monolayer adsorption surface, in which the adsorption energy remains constant and the maximum adsorption capacity occurs when only a saturated layer of solute is present on the adsorbent surface [ 51 ]. The Freundlich model is widely used to describe heterogeneous multilayer adsorption surfaces with different interaction energies leading to a logarithmic decrease in affinity during surface coverage [ 52 ].

From the experimental data, the pseudo-first-order and pseudo-second-order kinetic models were applied using their respective linear mathematical expressions [ 2 , 49 , 50 ]. (3) (4) where k 1 (min -1 ) and k 2 (g mg -1 min -1 ) are the kinetic constants of pseudo first and pseudo second order of adsorption, respectively, q eq and q t (mg g -1 ) represent the amounts of solute adsorbed at equilibrium and time t (min), respectively.

The biosorption capacity and the removal efficiency of metal ions were calculated according to Eq 1 and 2 : (1) (2) where q (mg g -1 ) is the biosorption capacity, m (g) is the mass of biosorbent, V (L) is the volume of the copper solution, C 0 (mg L -1 ) is the initial copper concentration in the solution and C f (mg L -1 ) is the copper concentration in the solution at the time of sampling.

Isothermal studies using free and encapsulated biosorbents (EB10 or EB30) were conducted with a copper solution at different initial concentrations until the time required for the system to reach equilibrium, as determined by biosorption kinetics. During this period, the pH of this solution remained relatively constant at 5.0 ± 0.1 by adding small amounts of NaOH (aq) . After the equilibrium time, the remaining copper concentration was measured by the method below.

Biosorption kinetic experiments were performed in capped plastic bottles containing one capsule (EB10 or EB30) and 300 mL of aqueous copper solution at an initial concentration of about 100 mg L -1 with pH adjusted to 5.0 ± 0.1. The bottles were incubated on a rotary shaker under constant agitation of 150 rpm at room temperature (25 ± 2°C). Afterwards, a bottle was removed at each different time of incubation and the copper ion concentration in the solution was determined by Atomic Absorption Spectroscopy (Agilent Technologies 200 Series AA). Control biosorption assays were performed using a capsule containing only water (without the biosorbent) to evaluate a possible adsorption of metals by the cellulose membrane.

The capsules were prepared using two biosorbent concentrations. The capsule referred to as “EB10” contained 33.3 mg of biosorbent and one denominated as “EB30” contained 100 mg of biosorbent, both containing 3.33 mL of deionized water inside the capsule, whose final concentrations were 10 mg mL -1 and 30 mg mL -1 , respectively.

The fungal biomass was enclosed in a cellulose semipermeable membrane measuring 21 mm wide and 33 mm in length containing deionized water and the system was sealed with a nylon line, forming the encapsulated biosorbent ( Fig 1 ).

The biomass obtained after the growth of the MEL1 mutant of the fungus Aspergillus nidulans, characterized as an overproducer of the DOPA-melanin pigment [ 29 ], was used as a biosorvent in this study. This microorganism belongs to the culture collection of the Filamentous Fungi Laboratory at the Department of Biochemistry and Organic Chemistry, the Institute of Chemistry, São Paulo State University-UNESP in Araraquara, Brazil. Cultivation of the MEL 1 mutant was conducted as described by Sponchiado et al., 2018 [ 48 ]. After the growth period, the biomass was harvested by filtration, washed with deionized water, dried at 55°C until constant weight, crushed and sieved to obtain an adsorbent with a uniform particle size. The fraction with a diameter less than 0.42 mm was selected to be used in the sorption experiments.

Results and discussion

For the application of biosorption in the removal of heavy metals from industrial wastewater, it is important to use immobilized biosorbent to facilitate the collection of the metal-loaded biosorbent for metal ion removal and recovery by desorption, as well as to regenerate and reuse the metal-free biosorbent for the next biosorption-desorption cycle.

In this work, the capacity of the MEL1 mutant pigmented biomass from Aspergillus nidulans encapsulated in a semipermeable cellulose membrane (as shown in Fig 1) for copper ion removal in aqueous solution was evaluated.

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