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Efficient mycosorption of anionic azo dyes by Mucor circinelloides: Surface functional groups and removal mechanism study
Journal of Environmental Chemical Engineering 6 (2018) 4114–4123
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Efficient mycosorption of anionic azo dyes by Mucor circinelloides: Surface functional groups and removal mechanism study Ehsan Azin, Hamid Moghimi
T
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Department of Microbial Biotechnology, School of Biology, College of Science, University of Tehran, Tehran, 1417864411, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Azo dye Biosorption Chitin Chitosan Functional groups Mycosorption
In this study, fungal isolates were purified from contaminated soil and dyestuff wastewater samples. Among 58 fungal isolates, Mucor circinelloides with 94% Congo red removal capacity at the concentration of 150 mg/L was selected as the superior isolate. Comparison of wet and dried wet biomass showed that dye adsorption of wet biomass was 39% more than that of dried wet biomass. The maximum Langmuir adsorption capacity was calculated 169.49 mg dye/g wet biomass of M. circinelloides. Pretreatment of biomass with sodium hydroxide enhanced dye removal for 19%. Acetone 50% and 0.1 M acetic acid had the highest and the less effect on desorption of dye molecules from fungal cells, respectively. Light and scanning microscopy imaging, Fourier transform infrared spectroscopy, pretreatment and zeta potential analysis indicated that dye removal was performed through biosorption process and hydroxyl and amine groups in the cell wall polymers have the main role in dye biosorption. M. circinelloides could produce crude chitin as 57% of its dry biomass. Chitin and chitosan in zygomyceta cell wall such as Mucor genus are probably main adsorption contributing surface polymers.
1. Introduction Dyes are organic and soluble compounds that may be synthetic or natural [1]. There are more than 10,000 types of dyes with a worldwide annual production of 70,000 tones. The textile industry alone consumes 20% of the dyes, from which 10–15% is discharged into wastewater [2–4]. The release of textile wastewater into the environment not only disturbs it visually, but also significantly reduces water quality and the rate of photosynthesis in aquatic environments; it also alters water pH, increases COD levels, and gives a drastic colouration to the water [5,6]. Azo dyes are the biggest and the most diverse group of synthetic dyes, which are characterized by the presence of nitrogen double bond (eN]Ne) [7]. They are recalcitrant xenobiotic compounds which usually cannot be remediated from wastewater by common refinement methods [8]. Moreover, most of azo dyes and the intermediate compounds formed during their degradation are highly toxic and carcinogenic to living organisms [7]. These characteristics highlight the need for developing a proper approach for the removal of azo dyes. The methods employed for treating coloured wastewater are physical, chemical, and biological. Due to some drawbacks in the physical and chemical methods, such as production of large amount of sludge, low efficiency, ineffectiveness in different dyes removal, limited applicability, production of toxic substances, and high cost of performance, they may not be suitable for wastewater treatment [5].
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Corresponding author. E-mail address: [email protected] (H. Moghimi).
https://doi.org/10.1016/j.jece.2018.06.002 Received 23 January 2018; Received in revised form 26 May 2018; Accepted 1 June 2018 2213-3437/ © 2018 Elsevier Ltd. All rights reserved.
Studies have shown that various microorganisms like bacteria, mold, yeast, and algae are capable of biosorption, bioaccumulation, and biodegradation of azo dyes [9]. During biodegradation, microorganisms break down azo dyes using their degradative enzymes including laccase, lignin peroxidase, and manganese peroxidase. So far several studies have been carried out on azo dyes biodegradation ability of various fungi including Phanerochaete chrysosporium, Trametes versicolor, Coriolus versicolor, Irpex lacteus, and Trametes hirsute [7]. Despite the high efficiency of biodegradation based removal methods, this enzymatic remediation suffers from some serious problems such as its metabolism dependent nature, low degradability of many dye molecules and formation of highly toxic products [7]. In contrast to biodegradation, biosorption is defined as a process of metabolically independent adsorptive uptake which can be performed using dead or living biomass [10]. Generally, adsorption process is divided into two categories of physisorption and chemisorption. In the chemisorption, the adsorbent and the adsorbate are joined by strong forces and generally, these connections are irreversible [11]. However, in physisorption, there are weak reversible forces between adsorbent and adsorbate. The adsorption process is more suitable than other methods because of its flexibility, design simplicity, ease of operation, lack of toxic compound production, and lack of sensitivity to toxic compounds. So far, many studies have been done on various kinds of adsorbents for dye adsorption [12].
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2.3. Molecular and morphological identification of the selected isolate
Among various biosorbents, use of fungal biomass in wastewater treatment was considered as an attractive, cheap, eco-friendly, and more publicly acceptable approach [12]. The large fungal cell surface makes them efficient biosorbent agents, and the presence of various polysaccharide polymers in the cell walls makes fungi competent and applicable adsorbents for different types of environmental contaminants, such as heavy metals and synthetic dyes. An adsorption study of Alizarin Blue Black B and alkali lignin by Haematonectria haematococca BwIII43 showed the most adsorption capacity of 247.47 and 161 mg/g, respectively [13]. Also, Penicillium ochrochloron AMDB12 demonstrated adsorption potency yield of 55% and 66% for Reactive Blue 13 and Reactive Blue 72, respectively [17]. I another study Penicillium janthinellum could adsorb 344.83 mg/g of Congo red on its pellets [3]. There are a large number of articles in the area of biosorption of dyes, but most of them have introduced various biosorbent and evaluating their removal capacity and they have not focused on removal mechanism specifically. Therefore, studying the adsorption mechanism and properties by adsorbent and pollutants are very important for design the wastewater treatment plants. In order to explore and improve the adsorption characteristics of an isolated fungal strain, the corresponding functional groups were identified and evaluated through various methods in this paper.
Morphological features were investigated using slide cultures of the fungi. For molecular identification, 200 mg of fungal biomass was ground to a fine powder in liquid nitrogen, and DNA was isolated and purified using standard protocols. PCR reaction was conducted using primer ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and primer ITS4 ( 5′-TCCTCCGCTTATTGATATGC-3′) [15] and the PCR product was sequenced (Macrogen, South Korea). The sequence was compared with those of other validated species using the BLAST program. 2.4. Assessment of growth and biosorption rate in different dye concentrations The media containing different concentrations of Congo red (150, 600, 1000, 2000 and 3000 mg/L) were prepared to study the growth and mycosorption rate. The inoculated flasks were shaken at 170 rpm and 28 °C for 48 h. After 48 h, the absorbance of cell-free culture supernatant was measured at 500 nm wavelength. The remaining biomass was dried in the oven and weighted as dry cell weight (DCW). The experiment was repeated three times. 2.5. Biosorbent preparation and comparison of wet and dry biomass biosorption capacity
2. Materials and methods To prepare mycelial pellets, a fragment of 1 cm2 was inoculated from PDA to PDB (potato dextrose broth), and the flasks were shaken at 28 °C and 170 rpm. After five days, mycelial pellets were harvested and washed with sterile dH2O and then preserved at 4 °C. In order to compare the biosorption capacity of wet and dried wet biomass, 0.2 g wet biomass and 0.2 g dried wet biomass (weighted before drying) were added to 20 ml Congo red solution (1000 mg/L), individually. The flasks were shaken at 170 rpm for 3 h, following which the absorbance of cell-free supernatant was measured at 500 nm.
2.1. Soil samples collection and fungal isolation Widespread use of azo dyes in various industries results in the release of huge amount of dye-containing wastewater into the environment. At present, utilization of microorganisms for dye removal is under investigation as an alternative to conventional remediation methods, which suffer from high costs and low efficiency [14]. Contaminated soil and wastewater samples were collected from regions surrounding the textile manufacturing units in different provinces of Iran, and kept at 4 °C during transportation to the lab. The collected soil samples were ground to a fine powder with mortar and pestle. Fungi isolation was performed using the enrichment method in a culture medium containing (g/L of DW): yeast extract, 0.5; glucose, 20; Na2HPO4, 0.05; KH2PO4, 1; FeSO4·7H2O, 0.01; ZnSO4·7H2O, 0.001; MnSO4·4H2O, 0.001; CaCl2, 0.01; CuSO4·5H2O, 0.002 and MgSO4·H2O, 0.5. The pH was adjusted to 7 ± 0.2 before sterilization. In order to prevent bacterial growth, 100 mg/L tetracycline was added to the culture medium. Congo red (benzidinediazo-bis-1-naphtylamine-4-sulfonic acid, C.I. 22120, diazo dye) was used as a representative of azo dyes for mycosorption evaluation. Afterwards, 0.1 g soil or 1 ml wastewater samples were added to 100 ml Erlenmeyer flasks with 20 ml medium and 150 mg/L Congo red. Flasks were shaken at 170 rpm and 28 °C for seven days. Then, 100 μl of bleached fermentation broth was cultured using the spread plate method on PDA. Plates were incubated at 28 °C for 14 days. The fungal isolates were purified on PDA and stored at 4 °C.
2.6. Analysis of adsorption involving functional groups In order to investigate the mycelial pellet surface, light and scanning electron microscopy imaging and Fourier transform infrared spectroscopy (FTIR) analysis were performed before and after dye biosorption. An optical and scanning electron microscope (ZEISS) images were taken before and after the adsorption by fungal mycelial pellets. For FTIR analysis, KBr discs of dye-loaded and unloaded mycelial pellets were prepared and the FTIR spectra of the mycelial pellets was obtained using Perkinelmer spectrometer in the range of 400 to 4000 cm−1. To determine the biomass surface charge, Zeta potential was measured by Malvern Zetasizer instrument before and after dye biosorption, at a pH value of 8. 2.7. Evaluation of temperature effect on dye removal To study the effect of increasing temperature on dye removal efficiency by the fungal wet cells, removal experiments were conducted at 25 °C, 50 °C and 75 °C in 2000 mg/L. The experiment was conducted in triplicate. The enthalpy parameter was calculated by thermodynamic study in different temperature [16].
2.2. Screening and superior isolate selection Superior isolate was selected based on the percentage of dye removal from the medium. For this purpose, fungal isolates were cultured in the medium containing 150 mg/L Congo red. Flasks were incubated at 28 °C in the shaker incubator (170 rpm). After seven days, the cells were harvested by centrifugation at 4000 rpm for 10 min, and the absorbance of supernatant was measured at 500 nm wavelength. To calculate dye removal percentage, Eq. (1) was used:
(Ai − At ) D= × 100 Ai
2.8. Evaluation of pre-treatment effects on dye biosorption by M. circinelloides To evaluate the effects of different physical and chemical pretreatments on dye biosorption, several pre-treatments were performed on the fungal mycelial pellets, including autoclaving and incubation with 10% (v/v) acetic anhydride, 50% (v/v) methanol, 10% (v/v) formaldehyde [17], and 0.1 M sodium hydroxide solution [18]. Finally, the biosorption capacity of pre-treated biomass was compared with that
(1)
where D is dye removal percentage, Ai is initial absorbance, and At is absorbance after a specific time. 4115
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3. Results and discussion
of untreated biomass in a solution of 3000 mg/L dye.
3.1. Isolation and superior isolate selection 2.9. Desorption study In order to find efficient fungal strains in dye removal, we isolated samples from textile-industry effluents and contaminated soils. A total of 64 fungal isolates were purified from which, 58 isolates belonged to contaminated soil samples, and six isolates belonged to textile-industry wastewater samples. The isolates were screened on the basis of percentage of dye removal and 13 isolates were found to be able to remove more than 50% of Congo red in 150 mg/L dye solution after the incubation period of seven days. The isolate M32 was selected as the superior strain with 94% dye removal capacity in the previously explained condition.
To study the desorption process, solutions of 1000 mg/mL Congo red with 0.2 g fungal biomass were added to acetone 50% v/v, methanol 50% v/v, 0.1 M, 0.01 M and 0.001 M NaOH and 0.1 M, 0.01 M and 0.001 M acetic acid solutions and incubated at 28 °C for 2 h then, the biomass of each flask was collected and the absorbance of supernatants were measured at 500 nm.
2.10. Evaluation of chitin production
3.2. Molecular and morphological identification of the selected isolate
To evaluate the amount of chitin produced by M. circinelloides, the strain was inoculated in PDB medium and the flasks were incubated in the shaker incubator (170 rpm) at 28 °C. After five days, mycelial pellets were harvested by centrifugation and washed with sterile dH2O. In order to assist the drying process, mycelial pellets were placed at 50 °C overnight. Then 1 M NaOH ratio of 1: 30 (w/v) was added to the powdered biomass and flasks were incubated at 40 °C for 2 h. Chitin extraction was performed through the protocol suggested by Alvarez [19].
Morphological characteristics such as sporangium and columella structures revealed that the selected isolate belongs to the zygomycota group. Molecular identification revealed that M32 has 100% similarity with a strain of Mucor circinelloides in the ITS gene. The isolate and the related partial ITS sequence were deposited in GenBank under the accession numbers, KR263057. The selected isolate has the closest relationship to M. circinelloides f. circinelloides.
2.11. Evaluation of different dye biosorption by M. circinelloides
3.3. Cell toxicity and biosorption rate of M. circinelloides at different dye concentrations Congo red toxicity and its ability for dye biosorption were evaluated. Growth and biosorption rate studies indicated that M. circinelloides has high capacity for Congo red adsorption. According to our findings, 95% of the dye was removed at 1000 mg/L concentrations within 48 h (Fig. 1A). Fungal growth at different dye concentrations demonstrated the ability of M. circinelloides to grow and remove 95% of Congo red even in high dye concentrations (up to 1000 mg/L). Increasing dye concentration to 3000 mg/L resulted in 31% dye biosorption. Considering that 3000 mg/L is a high concentration of dye in actual wastewater and dye concentration is lower than this amount, M. circinelloides can be introduced as an efficient biosorbent for dye removal in industrial samples. Congo red has a benzidine group in its structure, which is known as a carcinogenic agent and causes genetic mutation in organisms. Čeněk Novotny et al. Half-maximal effective concentration (EC50) of 1623 and 4.8 mg/L for Congo red were reported in a bacterial luminescence flash test (Vibrio fischeri) and algal growth inhibition test (Selenastrum capricornutum), respectively [20]. M. circinelloides was able to grow in high concentrations of Congo red, and increasing dye concentration did not affect fungal growth and biomass production significantly (Fig. 1B). The notable point is that the same study on Aspergillus flavus reported that increasing Congo red concentration from 0 to 1000 mg/L reduces the production rate of fungal biomass from 6.5 to 1.44 g/L [12].
Seven anionic azo dyes with different structures and absorption wavelengths such as Direct blue 71 (594 nm), Acid blue 161 (600 nm), Reactive black b (600 nm), Reactive blue 222 (600 nm), Reactive yellow 145 (454 nm), Reactive red ME43 (547 nm) and Congo red (500 nm) were studied in order to evaluate the biosorption capacity of M. circinelloides. For this purpose, single, double, and triazo types of dyes and also reactive and non-reactive dyes were examined. Mycelial pellets of 0.2 g were added to 20 ml dye solution (300 mg/L). Flasks were shaken at 170 rpm for 3 h and the supernatant absorption was measure.
2.12. Adsorption equilibrium To determine adsorption isotherm, 0.2 g mycelial pellet were added to 20 ml dye solution with concentrations of 150, 300, 600, 800 and 1000 mg/L. The flasks were shacked at 170 rpm for 1 h. The supernatant absorbance was measured at 500 nm. The biosorption capacity of the biomass qe (mg/g) was calculated using the Eq. (2):
qe =
(C0 − Ct ) ×V m
(2)
where qe is biosorption capacity, C0 and Ce (mg/L) are dye concentration in solution at initial and t time and V (L) is solution volume and m (g) is weight of used wet biomass. Isotherm studies were performed using two models of Langmuir and Freundlich. The equations of these models are as follow (Eq. (30)):
1 1 1 = + qe qm kl Ce qm Langmuir equation
log qe = log kf +
3.4. Comparison of wet and dry biomass biosorption capacity Since previous studies have shown that the adsorption capacity for dry biomass and wet mycelial pellets are different [21], we analysed the ability of wet and dry cell biomass for dye biosorption, separately. The biosorption capacity of 0.02 g dried wet biomass (equal to 0.2 g wet biomass) at 1000 mg/L Congo red was 56.17% while it was measured 95.2% for wet biomass, indicating a 24% increase in Congo red removal by same amount of wet biomass as compared to dried wet biomass in the same dye concentration.
1 log Ce n
Freundlich equation (3)
where qe is biosorption capacity, qm is maximum adsorption, kf and kl are Freundlich and Langmuir isotherm constant and Ce (mg/L) is dye concentration at equilibrium time.
3.5. Surface functional group analysis before and after dye biosorption Light and scanning electron microscopy analysis showed cell 4116
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Fig. 1. A) Biosorption rate of M. circinelloides at different dye concentrations B) Dry cell weight of M. circinelloides at different dye concentrations.
Fig. 2. Light microscopy (×400) and scanning electron microscopy (×5000) images of M. circinelloides mycelia B, D) before and A, C) after dye biosorption.
considerable changes in biosorption peaks, including eOH and/or eNH stretching and NeH bending of primary amines. Therefore, it can be concluded that these functional groups may play a significant role in the adsorption of dye on the fungal mycelial pellet surface. These results indicate that eOH and/or eNH stretching and NeH bending of primary amines from peaks of 1645 and 3415 cm−1 shifts to 1636 and 3430 cm−1, respectively. In addition, the intensity of these peaks decreased after biosorption process. These data confirm the presence of Yoshida interaction (interaction between hydroxyls and amines hydrogens of cell surface and aromatic rings of dye molecules) and
biosorption of Congo red by M. circinelloides mycelia (Fig. 2). Optical and scanning electron microscope images of mycelia taken before and after dye adsorption confirmed the attachment of dye molecules to fungal cells. Similar results were found on P. janthinellumin Congo red removal [3]. FTIR spectra are shown for colourless and coloured biomass between 400 to 4000 cm−1 wavelength in Fig. 3. These spectra indicate the structures of surface functional groups in detail. The band positions in the FTIR spectra of the mycelial pellets before and after dye biosorption are shown in Fig. 3. The results of FTIR analysis indicate 4117
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Fig. 3. FTIR spectra of M. circinelloides mycelial pellets before and after Congo red adsorption. A: eCN (Aliphatic amine), B: eCH bending/CeC stretching, C: NeH bending of primary amines, D: eCH stretching (Alkanes), E: eOH and/or eNH stretching.
zeta potential value, the adsorbent was still in stable condition, showing that this fungal strain could be appropriate for azo dye adsorption.
dipole–dipole hydrogen bonding (interaction between hydrogen of the hydroxyls and amines on cell surface and aromatic rings of dye molecules) [22]. FTIR peak shift of more than 4 cm−1 is considered significant [17]. The presence of high levels of chitin and chitosan in M. circinelloides wall structure has been previously reported. Both of these are carbohydrate polymers containing large numbers of hydroxyl and amine groups. The results of FTIR analysis showed that amines, amides, sulfonic acids, and carboxyls groups are probably responsible groups for Congo red and Acid blue 172 biosorption by Penicillium YW 01 [23]. The zeta potential, which is related to the surface charge of fungal biomass, is one of the main factors in pollutant adsorption. The existence of carbohydrate polymers, phosphates, and amino groups on the microorganism’s cell surface gives them an external charge. Enhancement of the solution pH increases the available sites for negative charge on the fungal cell surface. The zeta potential result showed that the surface external charges of fungal biomass are −34.8 and −30.1 mV before and after adsorption, respectively. The dye adsorption decreases the surface external charge, due to the presence of sulfonic acid groups in the structure of Congo red. According to the results, it can be concluded that the mechanism involved in dye removal is biosorption. Zeta potential value indicates the stability of the colloidal system. If the zeta potential value of particles is high in the system (more positive than +30 mV or more negative than −30 mV), they do not acquire the tendency to flocculate. In the case of low zeta potential value, the particles tend to reach an unstable state and flocculate. Since the results confirmed a decrease in zeta potential value after adsorption, it can be deduced that the tendency of the adsorbent for flocculation has increased during the adsorption process. However, considering the
3.6. Evaluation of temperature effect on dye removal By increasing the temperature of removal experiment from 25 °C to 75 °C, the removal average percentage of 1000 mg/L dye increased, as the results were 73.8%, 89.9% and 94.6% at 25 °C, 50 °C and 75 °C, respectively. The improvement in removal capacity by increasing the temperature shows that the reaction is of endothermic type and the adsorption process is heat dependent and consequently is chemisorption. The results indicated that enthalpy amount is 31634 kJ/mol. 3.7. Effect of pre-treatment on the biosorption capacity of M. circinelloides Generally, various pre-treatment methods were performed to remove impurities from the biomass surface, break down the cell membranes, and change the surface-binding sites in order to increase adsorption efficiency [20]. The effect of different pre-treatments on mycelial pellets of M. circinelloides is shown in Fig. 4. The results show that pre-treatment with sodium hydroxide lead to 19% increase in biosorption capacity, and have the maximum positive effect on the adsorption process. In addition to sodium hydroxide, other pre-treatments such as incubation with acetone, dimethyl sulfoxide, calcium nitrate, and autoclaving also increased dye biosorption capacity (Fig. 4). Treatment of mycelial pellets with sodium hydroxide, autoclave, Ca (NO3)2, acetone, and DMSO enhanced dye biosorption capacity for 4118
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hydroxyl groups in its structure, the high adsorption capacity of M. circinelloides mycelial can be attributed to the presence of these compounds. Given that the extraction and purification of these compounds produced by fungi and insects or chemicals synthesis are costly and time-consuming, direct use of microorganisms with high abilities to produce these compounds can be a cost-effective approach for pollutant-removal from wastewater. Fig. 5 shows the proposed functional groups involved in Congo red adsorption on the surface of M. circinelloides cell wall. There are two types of hydrogen bonding depicted in Fig. 5. The first type is formed between amine groups hydrogen and the biomass surface with nitrogen and oxygen atoms in Congo red. The second type of hydrogen bonding, which is called Yoshida bonding, is formed between the hydroxyl groups on the surface of biomass with aromatic ring of dye molecules [22]. Different studies have shown that the maximum Congo red adsorption capacities are 14.16, 6.7, 22.73, and 14.63 mg/g for A. niger [32], coir pith carbon [33], wheat bran, and rice bran [34], respectively. In the present study, the maximum Langmuir adsorption capacity was calculated 169.49 mg dye/g wet biomass of M. circinelloides. The result shows that this isolate has higher adsorption capacity than the most biological and chemical sorbents (Table 2).
19%, 13%, 15%, 8%, and 7%, respectively in comparison to untreated samples. Biosorption improvement followed by autoclaving can be attributed to further exposure of available functional groups on the fungal mycelial pellets [24]. Given that the dead biomass is able to adsorb dye at a high percentage, the mechanism of adsorption by M. circinelloides may be independent of fungal metabolism. In addition, the alkaline pretreatment with sodium hydroxide increases the exposure of available surface functional groups and their hydroxylation. On the other hand, sodium hydroxide causes the deacetylation of chitin into chitosan and probably creates free amino functional groups on the surface of mycelial pellets. This can be a reason behind the enhancement of dye adsorption [25]. Acetic anhydride, acetic acid, and formaldehyde treatment decreased dye biosorption for 35%, 43%, and 30%, respectively. Hydrogen ions binding to biomass functional groups can be considered as a reason for the reduction in adsorption of acetic acid pre-treated fungal biomass. This process changes the biomass surface electronegativity and thereby reduces the absorption capacity for some pollutants [26]. The decreased adsorption capacity after acetic anhydride pre-treatment is probably due to acetylation of hydroxyl and amino groups of chitin and chitosan. Formaldehyde causes interaction among adjacent hydroxyl groups in glucose [18]. Moreover, methylation of first and second amines in biomass surface cause a decline in azo dye adsorption [17]. The results indicate that methanol does not have a significant effect on capacity adsorption. As per the pre-treatment results, it can be concluded that the OH and NH2 groups play a vital role in the biosorption of Congo red by M. circinelloides, which goes with the FTIR results. FTIR analysis determines involved functional groups in the adsorption process, which provides essential data for choosing an efficient pre-treatment approach in order to improve dye adsorption. Table 1 presents some studies on fungal biomass pre-treatment for the biosorption of various contaminants. Since studies on fungal biomass pre-treatment for dye biosorption are limited, the effects of various pre-treatment approaches on the removal of different contaminants, including heavy metals, are summarized in Table 1. Fungal cell walls have many polymers, including chitin and chitosan, which can be used as suitable adsorbent agents for pollutants. As the results of FTIR and biomass pre-treatment show, hydroxyl and amine groups play an important role in dye adsorption (Figs. 3 and 4). Chitosan is produced by deacetylation of chitin, which is found in zygomycota cell wall. During the exponential growth phase, most chitosan molecules are free, but in the stationary phase, the molecules are anchored to other polymers in the cell wall, which makes them difficult to be extracted from the fungal biomass [31]. Therefore, it can be concluded that using fungal strain directly as an adsorbent is more practical than using pure polymers. Since chitosan has amino and
3.8. Desorption study Comparison of Congo red desorption percentage from wet biomass of M. circinelloides as the bioadsorbent was performed using different solutions. The results showed that acetone 50% and acetic acid (0.1 M) had the highest and the less effect on desorption of dye molecules from bioadsorbent, respectively (Fig. 6). Since using methanol and acetone resulted in higher desorption efficiency in comparison to acetic acid and NaOH, it can be concluded that hydrogen bonding or n-π interactions played more important role than electrostatic interactions in dye biosorption process [22]. 3.9. Evaluation of chitin production The chitin produced by M. circinelloides was quantified as 57% (w/ w) of the total dried biomass. Many studies have shown that chitosan and chitin, which are the major components of fungi cell wall, can be applied as efficient adsorbents for the elimination of contaminants such as heavy metals and dyes in industrial effluent. Chitosan forms more than 50% of the cell wall of zygomycetes members [39]. It has been shown that M. circinelloides was able to produce high amounts of chitin (64 mg/g) and chitosan (500 mg/g) [31]. A study on chitosan production capacity in strains of Aspergillus niger, Rhizopus oryzae, Zygosaccharomyces rouxii and Candida albicans revealed that the volume of produced chitosan using soybean residue (0.4–4.3 g/kg) was more than
Table 1 Some studies on the fungal biomass pre-treatment to various contaminants removal. microorganism
Pretreatment type
Effect
Reference
M. rouxii
Autoclave and alkali treatment
[27]
Penicillium digitatum
Heat, DMSO, alkali and formaldehyde treatment
Neurospora crassa M. rouxii Aspergillus oryze and Rhizopus oryzae A. niger
Acetic acid, DMSO and alkali treatment alkali treatment Acidic treatment
Autoclave: decrease Alkali: increase Formaldehyde: decrease Heat, DMSO, alkali: increase Increasing biosorption capacity Increasing biosorption capacity Increasing biosorption capacity
M. circinelloides
Autoclave, DMSO, alkali, acetic acid and formaldehyde treatment
Autoclave, DMSO, alkali, acetic acid, acetone, methanol, acetic anhydride, calcium nitrate and formaldehyde treatment
4119
Autoclave: decrease DMSO, alkali, formaldehyde and acetic acid: increase Acetic anhydride, acetic acid, and formaldehyde: decrease Autoclave, calcium nitrate, DMSO, alkali, acetone: increase
[24] [21] [28] [29] [30]
This study
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Fig. 4. A) Effects of different pre-treatments on dye biosorption by M. circinelloides in 3000 mg/L dye solution B) Alkaline pre-treatment with sodium hydroxide, which causes acetylation of chitin into chitosan.
Fig. 5. Proposed functional groups involved in Congo red adsorption on the surface of M. circinelloides cell wall.
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microorganisms, there are strains that have the potential to adsorption of reactive dyes. Aspergillus versicolor biomass was shown to be able to adsorb reactive black 5. The maximum reactive black 5 adsorption capacity was calculated 227.27 mg/g [44]. In addition, Rhizopus arrhizus NCIM 997 has maximum biosorption capacity of 133.63 mg/g for Reactive Orange 13 in optimum condition [45].
Table 2 The comparison of the dye adsorption capacities of different chemical and biological adsorbent. Type of adsorbent
Adsorption capacity (qm)
References
Bentonite Kaolin Zeolite Coir pith carbon Waste red mud CS/TX-100 beads CS/SDS beads Ball-milled sugarcane bagasse Aspergillus niger Penicillium YW 01 M. circinelloides
35.84 5.44 3.77 6.7 4.05 378.79 318.47 38.2 14.16 411.53 169.49
[35] [35] [35] [33] [36] [37] [37] [38] [32] [24] This study
3.11. Adsorption equilibrium Determination of regression coefficients showed that the biosorption process followed both Langmuir and Freundlich adsorption isotherms (Fig. 8). However, the results showed that the adsorption isotherm agreed well with Freundlich isotherm. As mentioned in Fig. 8, qm was 169.49 mg/g. 3.12. Mechanism of dye removal
that of mungbean residue (0.5–1.6 g/kg). The strain of R. oryzae with chitosan yield of 4.3 g/kg was concluded as the most efficient fungi in chitosan production [40]. Therefore, the high ability of M. circinelloides to produce these polymers can be one possible reason behind the high capacity of this strain for dye adsorption. M. circinelloides is able to produce ethanol [41], extracellular proteases [42] and gamma-linolenic acid [43].
Analysis the FTIR spectra of pre and post removal indicated that the hydroxyl and amine groups are two determining agent in biosorption process. Since chitin and chitosan, the major components of fungal cell walls contain many hydroxyl and amine groups in their structure, Congo red removal by M. circinelloides can be related to adsorption by these polymers. Also, dye adsorption was improved significantly by NaOH pretreatment while, acetic acid pretreatment results in adsorption capacity reduction. In acidic pH, more protons are available so protonated groups of amines are increased resulting in a decrease of the number of accessible dye binding sites [46]. Shifting the pretreatment agent from acid acetic to NaOH, caused an enhancement in the adsorption capacity. Considering that OH− ions released from NaOH results in deprotonation of hydroxyl groups on the fungal biomass. To evaluate the Congo red bioremediation mechanism of M. circinelloides, the removal efficiency of live and autoclaved biomass was compared and the results showed that autoclaved biomass could remove a high percentage of dye. Since biosorption is metabolism-dependent in contrast to bioaccumulation which depends on metabolism, the results indicate the adsorption nature of dye remediation by the fungi. Zeta potential value represents the electrical potential difference between the inner and outer sides of the surface. Regarding the observed changes of zeta potential before and after dye removal by the fungi, it is clear that an adsorption on an adsorbent was happened. Studying the effect of NaCl different concentrations in Congo red solution showed about 30% increase in dye removal percentage (from 65% to 95%) at
3.10. Biosorption of different dyes by M. circinelloides Evaluating the potential of M. circinelloides biomass for different dye adsorption showed that this strain is highly capable of biosorption of Congo red, Direct blue 71, and Acid blue 161 with 40.9, 21.9, and 20.6 mmol/g removal percentage in 300 mg/L dye solution, respectively (Fig. 7). Our results indicate that M. circinelloides does not have a significant capacity for biosorption of reactive dyes. The lowest adsorption capacity observed among the tested dyes was that of 2.8 mmol/g for Reactive red 43. This value may be related to reactive groups in reactive dye structures. Considering these variable results, we can conclude that the anionic colour didn’t show significant effect on the biosorption. Furthermore, it is clear that the azo bond had no effect on dye adsorption. However, the results show that adsorption of reactive dyes is less than other dyes, and the reason for this can be related to the presence of reactive groups in these dyes. However, the different fungal strains have a different ability to adsorption of dyes and among these
Fig. 6. Comparison of dye desorption from M. circinelloides using different solutions. 4121
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Fig. 7. Biosorption percentage of different dyes by M. circinelloides mycelia.
Fig. 8. Parameters of Langmuir and Freundlich isotherms in Congo red biosorption by M. circinelloides.
4. Conclusion
5% NaCl (W/V). By increasing salt concentration from 5%, no significant effect in dye removal was observed. The present of salt in dye solution increases the fungal cell wall thickness and therefore, increases dye biosorption capacity [3]. Na+ and Cl−, created from dissolution of NaCl, had not any negative effect on dye adsorption efficiency. So, it can be deduced that adsorption mechanism is not of electrostatic type and hydrogen bonding or n-π interactions may play significant role in adsorption process. Dye adsorption could be achieved by various mechanisms such as the formation of ionic interaction, hydrogen, and covalent binding or van der Waals forces. Due to the fact that under acidic conditions eNH2 groups are converted to eNH3+ if the interaction was ionic, then dye adsorbent should be increased. This is while acidic conditions reduced the percentage of dye adsorption. So, it can be concluded that removal mechanism is not ionic interaction and probably the other interactions such as hydrogen bonding or n-π interactions are involved in dye adsorption.
In this study, the contribution of functional groups on the surface of M. circinelloides in azo dye removal from dye solution was studied. On the basis of SEM images, zeta potential, FTIR, pre-treatment and evaluating dye adsorption by dead and living biomass results, it was concluded that the adsorption was the main responsible process in Congo red removal in the aqueous dye solution. In addition, hydroxyl and amine groups probably play an important role in dye adsorption. In addition, the adsorption of reactive dyes was compared with other groups of azo dyes is poorly performed on M. circinelloides biomass. Our findings proposed that characterization and identification of important binding groups involved in dye adsorption can be applied to different treatments on adsorbents, for improving adsorption capacity. In addition, deep insight about important binding groups leads to the selection of appropriate adsorbent that contain these groups in their structure. This approach can be used to determine methods of functional group 4122
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analysis to develop fast and low-cost ways of choosing a suitable adsorbent.
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This project was financially supported by University of Tehran. References
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Contents lists available at ScienceDirect
Journal of Environmental Chemical Engineering journal homepage: www.elsevier.com/locate/jece
Efficient mycosorption of anionic azo dyes by Mucor circinelloides: Surface functional groups and removal mechanism study Ehsan Azin, Hamid Moghimi
T
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Department of Microbial Biotechnology, School of Biology, College of Science, University of Tehran, Tehran, 1417864411, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: Azo dye Biosorption Chitin Chitosan Functional groups Mycosorption
In this study, fungal isolates were purified from contaminated soil and dyestuff wastewater samples. Among 58 fungal isolates, Mucor circinelloides with 94% Congo red removal capacity at the concentration of 150 mg/L was selected as the superior isolate. Comparison of wet and dried wet biomass showed that dye adsorption of wet biomass was 39% more than that of dried wet biomass. The maximum Langmuir adsorption capacity was calculated 169.49 mg dye/g wet biomass of M. circinelloides. Pretreatment of biomass with sodium hydroxide enhanced dye removal for 19%. Acetone 50% and 0.1 M acetic acid had the highest and the less effect on desorption of dye molecules from fungal cells, respectively. Light and scanning microscopy imaging, Fourier transform infrared spectroscopy, pretreatment and zeta potential analysis indicated that dye removal was performed through biosorption process and hydroxyl and amine groups in the cell wall polymers have the main role in dye biosorption. M. circinelloides could produce crude chitin as 57% of its dry biomass. Chitin and chitosan in zygomyceta cell wall such as Mucor genus are probably main adsorption contributing surface polymers.
1. Introduction Dyes are organic and soluble compounds that may be synthetic or natural [1]. There are more than 10,000 types of dyes with a worldwide annual production of 70,000 tones. The textile industry alone consumes 20% of the dyes, from which 10–15% is discharged into wastewater [2–4]. The release of textile wastewater into the environment not only disturbs it visually, but also significantly reduces water quality and the rate of photosynthesis in aquatic environments; it also alters water pH, increases COD levels, and gives a drastic colouration to the water [5,6]. Azo dyes are the biggest and the most diverse group of synthetic dyes, which are characterized by the presence of nitrogen double bond (eN]Ne) [7]. They are recalcitrant xenobiotic compounds which usually cannot be remediated from wastewater by common refinement methods [8]. Moreover, most of azo dyes and the intermediate compounds formed during their degradation are highly toxic and carcinogenic to living organisms [7]. These characteristics highlight the need for developing a proper approach for the removal of azo dyes. The methods employed for treating coloured wastewater are physical, chemical, and biological. Due to some drawbacks in the physical and chemical methods, such as production of large amount of sludge, low efficiency, ineffectiveness in different dyes removal, limited applicability, production of toxic substances, and high cost of performance, they may not be suitable for wastewater treatment [5].
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Corresponding author. E-mail address: [email protected] (H. Moghimi).
https://doi.org/10.1016/j.jece.2018.06.002 Received 23 January 2018; Received in revised form 26 May 2018; Accepted 1 June 2018 2213-3437/ © 2018 Elsevier Ltd. All rights reserved.
Studies have shown that various microorganisms like bacteria, mold, yeast, and algae are capable of biosorption, bioaccumulation, and biodegradation of azo dyes [9]. During biodegradation, microorganisms break down azo dyes using their degradative enzymes including laccase, lignin peroxidase, and manganese peroxidase. So far several studies have been carried out on azo dyes biodegradation ability of various fungi including Phanerochaete chrysosporium, Trametes versicolor, Coriolus versicolor, Irpex lacteus, and Trametes hirsute [7]. Despite the high efficiency of biodegradation based removal methods, this enzymatic remediation suffers from some serious problems such as its metabolism dependent nature, low degradability of many dye molecules and formation of highly toxic products [7]. In contrast to biodegradation, biosorption is defined as a process of metabolically independent adsorptive uptake which can be performed using dead or living biomass [10]. Generally, adsorption process is divided into two categories of physisorption and chemisorption. In the chemisorption, the adsorbent and the adsorbate are joined by strong forces and generally, these connections are irreversible [11]. However, in physisorption, there are weak reversible forces between adsorbent and adsorbate. The adsorption process is more suitable than other methods because of its flexibility, design simplicity, ease of operation, lack of toxic compound production, and lack of sensitivity to toxic compounds. So far, many studies have been done on various kinds of adsorbents for dye adsorption [12].
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2.3. Molecular and morphological identification of the selected isolate
Among various biosorbents, use of fungal biomass in wastewater treatment was considered as an attractive, cheap, eco-friendly, and more publicly acceptable approach [12]. The large fungal cell surface makes them efficient biosorbent agents, and the presence of various polysaccharide polymers in the cell walls makes fungi competent and applicable adsorbents for different types of environmental contaminants, such as heavy metals and synthetic dyes. An adsorption study of Alizarin Blue Black B and alkali lignin by Haematonectria haematococca BwIII43 showed the most adsorption capacity of 247.47 and 161 mg/g, respectively [13]. Also, Penicillium ochrochloron AMDB12 demonstrated adsorption potency yield of 55% and 66% for Reactive Blue 13 and Reactive Blue 72, respectively [17]. I another study Penicillium janthinellum could adsorb 344.83 mg/g of Congo red on its pellets [3]. There are a large number of articles in the area of biosorption of dyes, but most of them have introduced various biosorbent and evaluating their removal capacity and they have not focused on removal mechanism specifically. Therefore, studying the adsorption mechanism and properties by adsorbent and pollutants are very important for design the wastewater treatment plants. In order to explore and improve the adsorption characteristics of an isolated fungal strain, the corresponding functional groups were identified and evaluated through various methods in this paper.
Morphological features were investigated using slide cultures of the fungi. For molecular identification, 200 mg of fungal biomass was ground to a fine powder in liquid nitrogen, and DNA was isolated and purified using standard protocols. PCR reaction was conducted using primer ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and primer ITS4 ( 5′-TCCTCCGCTTATTGATATGC-3′) [15] and the PCR product was sequenced (Macrogen, South Korea). The sequence was compared with those of other validated species using the BLAST program. 2.4. Assessment of growth and biosorption rate in different dye concentrations The media containing different concentrations of Congo red (150, 600, 1000, 2000 and 3000 mg/L) were prepared to study the growth and mycosorption rate. The inoculated flasks were shaken at 170 rpm and 28 °C for 48 h. After 48 h, the absorbance of cell-free culture supernatant was measured at 500 nm wavelength. The remaining biomass was dried in the oven and weighted as dry cell weight (DCW). The experiment was repeated three times. 2.5. Biosorbent preparation and comparison of wet and dry biomass biosorption capacity
2. Materials and methods To prepare mycelial pellets, a fragment of 1 cm2 was inoculated from PDA to PDB (potato dextrose broth), and the flasks were shaken at 28 °C and 170 rpm. After five days, mycelial pellets were harvested and washed with sterile dH2O and then preserved at 4 °C. In order to compare the biosorption capacity of wet and dried wet biomass, 0.2 g wet biomass and 0.2 g dried wet biomass (weighted before drying) were added to 20 ml Congo red solution (1000 mg/L), individually. The flasks were shaken at 170 rpm for 3 h, following which the absorbance of cell-free supernatant was measured at 500 nm.
2.1. Soil samples collection and fungal isolation Widespread use of azo dyes in various industries results in the release of huge amount of dye-containing wastewater into the environment. At present, utilization of microorganisms for dye removal is under investigation as an alternative to conventional remediation methods, which suffer from high costs and low efficiency [14]. Contaminated soil and wastewater samples were collected from regions surrounding the textile manufacturing units in different provinces of Iran, and kept at 4 °C during transportation to the lab. The collected soil samples were ground to a fine powder with mortar and pestle. Fungi isolation was performed using the enrichment method in a culture medium containing (g/L of DW): yeast extract, 0.5; glucose, 20; Na2HPO4, 0.05; KH2PO4, 1; FeSO4·7H2O, 0.01; ZnSO4·7H2O, 0.001; MnSO4·4H2O, 0.001; CaCl2, 0.01; CuSO4·5H2O, 0.002 and MgSO4·H2O, 0.5. The pH was adjusted to 7 ± 0.2 before sterilization. In order to prevent bacterial growth, 100 mg/L tetracycline was added to the culture medium. Congo red (benzidinediazo-bis-1-naphtylamine-4-sulfonic acid, C.I. 22120, diazo dye) was used as a representative of azo dyes for mycosorption evaluation. Afterwards, 0.1 g soil or 1 ml wastewater samples were added to 100 ml Erlenmeyer flasks with 20 ml medium and 150 mg/L Congo red. Flasks were shaken at 170 rpm and 28 °C for seven days. Then, 100 μl of bleached fermentation broth was cultured using the spread plate method on PDA. Plates were incubated at 28 °C for 14 days. The fungal isolates were purified on PDA and stored at 4 °C.
2.6. Analysis of adsorption involving functional groups In order to investigate the mycelial pellet surface, light and scanning electron microscopy imaging and Fourier transform infrared spectroscopy (FTIR) analysis were performed before and after dye biosorption. An optical and scanning electron microscope (ZEISS) images were taken before and after the adsorption by fungal mycelial pellets. For FTIR analysis, KBr discs of dye-loaded and unloaded mycelial pellets were prepared and the FTIR spectra of the mycelial pellets was obtained using Perkinelmer spectrometer in the range of 400 to 4000 cm−1. To determine the biomass surface charge, Zeta potential was measured by Malvern Zetasizer instrument before and after dye biosorption, at a pH value of 8. 2.7. Evaluation of temperature effect on dye removal To study the effect of increasing temperature on dye removal efficiency by the fungal wet cells, removal experiments were conducted at 25 °C, 50 °C and 75 °C in 2000 mg/L. The experiment was conducted in triplicate. The enthalpy parameter was calculated by thermodynamic study in different temperature [16].
2.2. Screening and superior isolate selection Superior isolate was selected based on the percentage of dye removal from the medium. For this purpose, fungal isolates were cultured in the medium containing 150 mg/L Congo red. Flasks were incubated at 28 °C in the shaker incubator (170 rpm). After seven days, the cells were harvested by centrifugation at 4000 rpm for 10 min, and the absorbance of supernatant was measured at 500 nm wavelength. To calculate dye removal percentage, Eq. (1) was used:
(Ai − At ) D= × 100 Ai
2.8. Evaluation of pre-treatment effects on dye biosorption by M. circinelloides To evaluate the effects of different physical and chemical pretreatments on dye biosorption, several pre-treatments were performed on the fungal mycelial pellets, including autoclaving and incubation with 10% (v/v) acetic anhydride, 50% (v/v) methanol, 10% (v/v) formaldehyde [17], and 0.1 M sodium hydroxide solution [18]. Finally, the biosorption capacity of pre-treated biomass was compared with that
(1)
where D is dye removal percentage, Ai is initial absorbance, and At is absorbance after a specific time. 4115
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3. Results and discussion
of untreated biomass in a solution of 3000 mg/L dye.
3.1. Isolation and superior isolate selection 2.9. Desorption study In order to find efficient fungal strains in dye removal, we isolated samples from textile-industry effluents and contaminated soils. A total of 64 fungal isolates were purified from which, 58 isolates belonged to contaminated soil samples, and six isolates belonged to textile-industry wastewater samples. The isolates were screened on the basis of percentage of dye removal and 13 isolates were found to be able to remove more than 50% of Congo red in 150 mg/L dye solution after the incubation period of seven days. The isolate M32 was selected as the superior strain with 94% dye removal capacity in the previously explained condition.
To study the desorption process, solutions of 1000 mg/mL Congo red with 0.2 g fungal biomass were added to acetone 50% v/v, methanol 50% v/v, 0.1 M, 0.01 M and 0.001 M NaOH and 0.1 M, 0.01 M and 0.001 M acetic acid solutions and incubated at 28 °C for 2 h then, the biomass of each flask was collected and the absorbance of supernatants were measured at 500 nm.
2.10. Evaluation of chitin production
3.2. Molecular and morphological identification of the selected isolate
To evaluate the amount of chitin produced by M. circinelloides, the strain was inoculated in PDB medium and the flasks were incubated in the shaker incubator (170 rpm) at 28 °C. After five days, mycelial pellets were harvested by centrifugation and washed with sterile dH2O. In order to assist the drying process, mycelial pellets were placed at 50 °C overnight. Then 1 M NaOH ratio of 1: 30 (w/v) was added to the powdered biomass and flasks were incubated at 40 °C for 2 h. Chitin extraction was performed through the protocol suggested by Alvarez [19].
Morphological characteristics such as sporangium and columella structures revealed that the selected isolate belongs to the zygomycota group. Molecular identification revealed that M32 has 100% similarity with a strain of Mucor circinelloides in the ITS gene. The isolate and the related partial ITS sequence were deposited in GenBank under the accession numbers, KR263057. The selected isolate has the closest relationship to M. circinelloides f. circinelloides.
2.11. Evaluation of different dye biosorption by M. circinelloides
3.3. Cell toxicity and biosorption rate of M. circinelloides at different dye concentrations Congo red toxicity and its ability for dye biosorption were evaluated. Growth and biosorption rate studies indicated that M. circinelloides has high capacity for Congo red adsorption. According to our findings, 95% of the dye was removed at 1000 mg/L concentrations within 48 h (Fig. 1A). Fungal growth at different dye concentrations demonstrated the ability of M. circinelloides to grow and remove 95% of Congo red even in high dye concentrations (up to 1000 mg/L). Increasing dye concentration to 3000 mg/L resulted in 31% dye biosorption. Considering that 3000 mg/L is a high concentration of dye in actual wastewater and dye concentration is lower than this amount, M. circinelloides can be introduced as an efficient biosorbent for dye removal in industrial samples. Congo red has a benzidine group in its structure, which is known as a carcinogenic agent and causes genetic mutation in organisms. Čeněk Novotny et al. Half-maximal effective concentration (EC50) of 1623 and 4.8 mg/L for Congo red were reported in a bacterial luminescence flash test (Vibrio fischeri) and algal growth inhibition test (Selenastrum capricornutum), respectively [20]. M. circinelloides was able to grow in high concentrations of Congo red, and increasing dye concentration did not affect fungal growth and biomass production significantly (Fig. 1B). The notable point is that the same study on Aspergillus flavus reported that increasing Congo red concentration from 0 to 1000 mg/L reduces the production rate of fungal biomass from 6.5 to 1.44 g/L [12].
Seven anionic azo dyes with different structures and absorption wavelengths such as Direct blue 71 (594 nm), Acid blue 161 (600 nm), Reactive black b (600 nm), Reactive blue 222 (600 nm), Reactive yellow 145 (454 nm), Reactive red ME43 (547 nm) and Congo red (500 nm) were studied in order to evaluate the biosorption capacity of M. circinelloides. For this purpose, single, double, and triazo types of dyes and also reactive and non-reactive dyes were examined. Mycelial pellets of 0.2 g were added to 20 ml dye solution (300 mg/L). Flasks were shaken at 170 rpm for 3 h and the supernatant absorption was measure.
2.12. Adsorption equilibrium To determine adsorption isotherm, 0.2 g mycelial pellet were added to 20 ml dye solution with concentrations of 150, 300, 600, 800 and 1000 mg/L. The flasks were shacked at 170 rpm for 1 h. The supernatant absorbance was measured at 500 nm. The biosorption capacity of the biomass qe (mg/g) was calculated using the Eq. (2):
qe =
(C0 − Ct ) ×V m
(2)
where qe is biosorption capacity, C0 and Ce (mg/L) are dye concentration in solution at initial and t time and V (L) is solution volume and m (g) is weight of used wet biomass. Isotherm studies were performed using two models of Langmuir and Freundlich. The equations of these models are as follow (Eq. (30)):
1 1 1 = + qe qm kl Ce qm Langmuir equation
log qe = log kf +
3.4. Comparison of wet and dry biomass biosorption capacity Since previous studies have shown that the adsorption capacity for dry biomass and wet mycelial pellets are different [21], we analysed the ability of wet and dry cell biomass for dye biosorption, separately. The biosorption capacity of 0.02 g dried wet biomass (equal to 0.2 g wet biomass) at 1000 mg/L Congo red was 56.17% while it was measured 95.2% for wet biomass, indicating a 24% increase in Congo red removal by same amount of wet biomass as compared to dried wet biomass in the same dye concentration.
1 log Ce n
Freundlich equation (3)
where qe is biosorption capacity, qm is maximum adsorption, kf and kl are Freundlich and Langmuir isotherm constant and Ce (mg/L) is dye concentration at equilibrium time.
3.5. Surface functional group analysis before and after dye biosorption Light and scanning electron microscopy analysis showed cell 4116
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Fig. 1. A) Biosorption rate of M. circinelloides at different dye concentrations B) Dry cell weight of M. circinelloides at different dye concentrations.
Fig. 2. Light microscopy (×400) and scanning electron microscopy (×5000) images of M. circinelloides mycelia B, D) before and A, C) after dye biosorption.
considerable changes in biosorption peaks, including eOH and/or eNH stretching and NeH bending of primary amines. Therefore, it can be concluded that these functional groups may play a significant role in the adsorption of dye on the fungal mycelial pellet surface. These results indicate that eOH and/or eNH stretching and NeH bending of primary amines from peaks of 1645 and 3415 cm−1 shifts to 1636 and 3430 cm−1, respectively. In addition, the intensity of these peaks decreased after biosorption process. These data confirm the presence of Yoshida interaction (interaction between hydroxyls and amines hydrogens of cell surface and aromatic rings of dye molecules) and
biosorption of Congo red by M. circinelloides mycelia (Fig. 2). Optical and scanning electron microscope images of mycelia taken before and after dye adsorption confirmed the attachment of dye molecules to fungal cells. Similar results were found on P. janthinellumin Congo red removal [3]. FTIR spectra are shown for colourless and coloured biomass between 400 to 4000 cm−1 wavelength in Fig. 3. These spectra indicate the structures of surface functional groups in detail. The band positions in the FTIR spectra of the mycelial pellets before and after dye biosorption are shown in Fig. 3. The results of FTIR analysis indicate 4117
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Fig. 3. FTIR spectra of M. circinelloides mycelial pellets before and after Congo red adsorption. A: eCN (Aliphatic amine), B: eCH bending/CeC stretching, C: NeH bending of primary amines, D: eCH stretching (Alkanes), E: eOH and/or eNH stretching.
zeta potential value, the adsorbent was still in stable condition, showing that this fungal strain could be appropriate for azo dye adsorption.
dipole–dipole hydrogen bonding (interaction between hydrogen of the hydroxyls and amines on cell surface and aromatic rings of dye molecules) [22]. FTIR peak shift of more than 4 cm−1 is considered significant [17]. The presence of high levels of chitin and chitosan in M. circinelloides wall structure has been previously reported. Both of these are carbohydrate polymers containing large numbers of hydroxyl and amine groups. The results of FTIR analysis showed that amines, amides, sulfonic acids, and carboxyls groups are probably responsible groups for Congo red and Acid blue 172 biosorption by Penicillium YW 01 [23]. The zeta potential, which is related to the surface charge of fungal biomass, is one of the main factors in pollutant adsorption. The existence of carbohydrate polymers, phosphates, and amino groups on the microorganism’s cell surface gives them an external charge. Enhancement of the solution pH increases the available sites for negative charge on the fungal cell surface. The zeta potential result showed that the surface external charges of fungal biomass are −34.8 and −30.1 mV before and after adsorption, respectively. The dye adsorption decreases the surface external charge, due to the presence of sulfonic acid groups in the structure of Congo red. According to the results, it can be concluded that the mechanism involved in dye removal is biosorption. Zeta potential value indicates the stability of the colloidal system. If the zeta potential value of particles is high in the system (more positive than +30 mV or more negative than −30 mV), they do not acquire the tendency to flocculate. In the case of low zeta potential value, the particles tend to reach an unstable state and flocculate. Since the results confirmed a decrease in zeta potential value after adsorption, it can be deduced that the tendency of the adsorbent for flocculation has increased during the adsorption process. However, considering the
3.6. Evaluation of temperature effect on dye removal By increasing the temperature of removal experiment from 25 °C to 75 °C, the removal average percentage of 1000 mg/L dye increased, as the results were 73.8%, 89.9% and 94.6% at 25 °C, 50 °C and 75 °C, respectively. The improvement in removal capacity by increasing the temperature shows that the reaction is of endothermic type and the adsorption process is heat dependent and consequently is chemisorption. The results indicated that enthalpy amount is 31634 kJ/mol. 3.7. Effect of pre-treatment on the biosorption capacity of M. circinelloides Generally, various pre-treatment methods were performed to remove impurities from the biomass surface, break down the cell membranes, and change the surface-binding sites in order to increase adsorption efficiency [20]. The effect of different pre-treatments on mycelial pellets of M. circinelloides is shown in Fig. 4. The results show that pre-treatment with sodium hydroxide lead to 19% increase in biosorption capacity, and have the maximum positive effect on the adsorption process. In addition to sodium hydroxide, other pre-treatments such as incubation with acetone, dimethyl sulfoxide, calcium nitrate, and autoclaving also increased dye biosorption capacity (Fig. 4). Treatment of mycelial pellets with sodium hydroxide, autoclave, Ca (NO3)2, acetone, and DMSO enhanced dye biosorption capacity for 4118
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hydroxyl groups in its structure, the high adsorption capacity of M. circinelloides mycelial can be attributed to the presence of these compounds. Given that the extraction and purification of these compounds produced by fungi and insects or chemicals synthesis are costly and time-consuming, direct use of microorganisms with high abilities to produce these compounds can be a cost-effective approach for pollutant-removal from wastewater. Fig. 5 shows the proposed functional groups involved in Congo red adsorption on the surface of M. circinelloides cell wall. There are two types of hydrogen bonding depicted in Fig. 5. The first type is formed between amine groups hydrogen and the biomass surface with nitrogen and oxygen atoms in Congo red. The second type of hydrogen bonding, which is called Yoshida bonding, is formed between the hydroxyl groups on the surface of biomass with aromatic ring of dye molecules [22]. Different studies have shown that the maximum Congo red adsorption capacities are 14.16, 6.7, 22.73, and 14.63 mg/g for A. niger [32], coir pith carbon [33], wheat bran, and rice bran [34], respectively. In the present study, the maximum Langmuir adsorption capacity was calculated 169.49 mg dye/g wet biomass of M. circinelloides. The result shows that this isolate has higher adsorption capacity than the most biological and chemical sorbents (Table 2).
19%, 13%, 15%, 8%, and 7%, respectively in comparison to untreated samples. Biosorption improvement followed by autoclaving can be attributed to further exposure of available functional groups on the fungal mycelial pellets [24]. Given that the dead biomass is able to adsorb dye at a high percentage, the mechanism of adsorption by M. circinelloides may be independent of fungal metabolism. In addition, the alkaline pretreatment with sodium hydroxide increases the exposure of available surface functional groups and their hydroxylation. On the other hand, sodium hydroxide causes the deacetylation of chitin into chitosan and probably creates free amino functional groups on the surface of mycelial pellets. This can be a reason behind the enhancement of dye adsorption [25]. Acetic anhydride, acetic acid, and formaldehyde treatment decreased dye biosorption for 35%, 43%, and 30%, respectively. Hydrogen ions binding to biomass functional groups can be considered as a reason for the reduction in adsorption of acetic acid pre-treated fungal biomass. This process changes the biomass surface electronegativity and thereby reduces the absorption capacity for some pollutants [26]. The decreased adsorption capacity after acetic anhydride pre-treatment is probably due to acetylation of hydroxyl and amino groups of chitin and chitosan. Formaldehyde causes interaction among adjacent hydroxyl groups in glucose [18]. Moreover, methylation of first and second amines in biomass surface cause a decline in azo dye adsorption [17]. The results indicate that methanol does not have a significant effect on capacity adsorption. As per the pre-treatment results, it can be concluded that the OH and NH2 groups play a vital role in the biosorption of Congo red by M. circinelloides, which goes with the FTIR results. FTIR analysis determines involved functional groups in the adsorption process, which provides essential data for choosing an efficient pre-treatment approach in order to improve dye adsorption. Table 1 presents some studies on fungal biomass pre-treatment for the biosorption of various contaminants. Since studies on fungal biomass pre-treatment for dye biosorption are limited, the effects of various pre-treatment approaches on the removal of different contaminants, including heavy metals, are summarized in Table 1. Fungal cell walls have many polymers, including chitin and chitosan, which can be used as suitable adsorbent agents for pollutants. As the results of FTIR and biomass pre-treatment show, hydroxyl and amine groups play an important role in dye adsorption (Figs. 3 and 4). Chitosan is produced by deacetylation of chitin, which is found in zygomycota cell wall. During the exponential growth phase, most chitosan molecules are free, but in the stationary phase, the molecules are anchored to other polymers in the cell wall, which makes them difficult to be extracted from the fungal biomass [31]. Therefore, it can be concluded that using fungal strain directly as an adsorbent is more practical than using pure polymers. Since chitosan has amino and
3.8. Desorption study Comparison of Congo red desorption percentage from wet biomass of M. circinelloides as the bioadsorbent was performed using different solutions. The results showed that acetone 50% and acetic acid (0.1 M) had the highest and the less effect on desorption of dye molecules from bioadsorbent, respectively (Fig. 6). Since using methanol and acetone resulted in higher desorption efficiency in comparison to acetic acid and NaOH, it can be concluded that hydrogen bonding or n-π interactions played more important role than electrostatic interactions in dye biosorption process [22]. 3.9. Evaluation of chitin production The chitin produced by M. circinelloides was quantified as 57% (w/ w) of the total dried biomass. Many studies have shown that chitosan and chitin, which are the major components of fungi cell wall, can be applied as efficient adsorbents for the elimination of contaminants such as heavy metals and dyes in industrial effluent. Chitosan forms more than 50% of the cell wall of zygomycetes members [39]. It has been shown that M. circinelloides was able to produce high amounts of chitin (64 mg/g) and chitosan (500 mg/g) [31]. A study on chitosan production capacity in strains of Aspergillus niger, Rhizopus oryzae, Zygosaccharomyces rouxii and Candida albicans revealed that the volume of produced chitosan using soybean residue (0.4–4.3 g/kg) was more than
Table 1 Some studies on the fungal biomass pre-treatment to various contaminants removal. microorganism
Pretreatment type
Effect
Reference
M. rouxii
Autoclave and alkali treatment
[27]
Penicillium digitatum
Heat, DMSO, alkali and formaldehyde treatment
Neurospora crassa M. rouxii Aspergillus oryze and Rhizopus oryzae A. niger
Acetic acid, DMSO and alkali treatment alkali treatment Acidic treatment
Autoclave: decrease Alkali: increase Formaldehyde: decrease Heat, DMSO, alkali: increase Increasing biosorption capacity Increasing biosorption capacity Increasing biosorption capacity
M. circinelloides
Autoclave, DMSO, alkali, acetic acid and formaldehyde treatment
Autoclave, DMSO, alkali, acetic acid, acetone, methanol, acetic anhydride, calcium nitrate and formaldehyde treatment
4119
Autoclave: decrease DMSO, alkali, formaldehyde and acetic acid: increase Acetic anhydride, acetic acid, and formaldehyde: decrease Autoclave, calcium nitrate, DMSO, alkali, acetone: increase
[24] [21] [28] [29] [30]
This study
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Fig. 4. A) Effects of different pre-treatments on dye biosorption by M. circinelloides in 3000 mg/L dye solution B) Alkaline pre-treatment with sodium hydroxide, which causes acetylation of chitin into chitosan.
Fig. 5. Proposed functional groups involved in Congo red adsorption on the surface of M. circinelloides cell wall.
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microorganisms, there are strains that have the potential to adsorption of reactive dyes. Aspergillus versicolor biomass was shown to be able to adsorb reactive black 5. The maximum reactive black 5 adsorption capacity was calculated 227.27 mg/g [44]. In addition, Rhizopus arrhizus NCIM 997 has maximum biosorption capacity of 133.63 mg/g for Reactive Orange 13 in optimum condition [45].
Table 2 The comparison of the dye adsorption capacities of different chemical and biological adsorbent. Type of adsorbent
Adsorption capacity (qm)
References
Bentonite Kaolin Zeolite Coir pith carbon Waste red mud CS/TX-100 beads CS/SDS beads Ball-milled sugarcane bagasse Aspergillus niger Penicillium YW 01 M. circinelloides
35.84 5.44 3.77 6.7 4.05 378.79 318.47 38.2 14.16 411.53 169.49
[35] [35] [35] [33] [36] [37] [37] [38] [32] [24] This study
3.11. Adsorption equilibrium Determination of regression coefficients showed that the biosorption process followed both Langmuir and Freundlich adsorption isotherms (Fig. 8). However, the results showed that the adsorption isotherm agreed well with Freundlich isotherm. As mentioned in Fig. 8, qm was 169.49 mg/g. 3.12. Mechanism of dye removal
that of mungbean residue (0.5–1.6 g/kg). The strain of R. oryzae with chitosan yield of 4.3 g/kg was concluded as the most efficient fungi in chitosan production [40]. Therefore, the high ability of M. circinelloides to produce these polymers can be one possible reason behind the high capacity of this strain for dye adsorption. M. circinelloides is able to produce ethanol [41], extracellular proteases [42] and gamma-linolenic acid [43].
Analysis the FTIR spectra of pre and post removal indicated that the hydroxyl and amine groups are two determining agent in biosorption process. Since chitin and chitosan, the major components of fungal cell walls contain many hydroxyl and amine groups in their structure, Congo red removal by M. circinelloides can be related to adsorption by these polymers. Also, dye adsorption was improved significantly by NaOH pretreatment while, acetic acid pretreatment results in adsorption capacity reduction. In acidic pH, more protons are available so protonated groups of amines are increased resulting in a decrease of the number of accessible dye binding sites [46]. Shifting the pretreatment agent from acid acetic to NaOH, caused an enhancement in the adsorption capacity. Considering that OH− ions released from NaOH results in deprotonation of hydroxyl groups on the fungal biomass. To evaluate the Congo red bioremediation mechanism of M. circinelloides, the removal efficiency of live and autoclaved biomass was compared and the results showed that autoclaved biomass could remove a high percentage of dye. Since biosorption is metabolism-dependent in contrast to bioaccumulation which depends on metabolism, the results indicate the adsorption nature of dye remediation by the fungi. Zeta potential value represents the electrical potential difference between the inner and outer sides of the surface. Regarding the observed changes of zeta potential before and after dye removal by the fungi, it is clear that an adsorption on an adsorbent was happened. Studying the effect of NaCl different concentrations in Congo red solution showed about 30% increase in dye removal percentage (from 65% to 95%) at
3.10. Biosorption of different dyes by M. circinelloides Evaluating the potential of M. circinelloides biomass for different dye adsorption showed that this strain is highly capable of biosorption of Congo red, Direct blue 71, and Acid blue 161 with 40.9, 21.9, and 20.6 mmol/g removal percentage in 300 mg/L dye solution, respectively (Fig. 7). Our results indicate that M. circinelloides does not have a significant capacity for biosorption of reactive dyes. The lowest adsorption capacity observed among the tested dyes was that of 2.8 mmol/g for Reactive red 43. This value may be related to reactive groups in reactive dye structures. Considering these variable results, we can conclude that the anionic colour didn’t show significant effect on the biosorption. Furthermore, it is clear that the azo bond had no effect on dye adsorption. However, the results show that adsorption of reactive dyes is less than other dyes, and the reason for this can be related to the presence of reactive groups in these dyes. However, the different fungal strains have a different ability to adsorption of dyes and among these
Fig. 6. Comparison of dye desorption from M. circinelloides using different solutions. 4121
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Fig. 7. Biosorption percentage of different dyes by M. circinelloides mycelia.
Fig. 8. Parameters of Langmuir and Freundlich isotherms in Congo red biosorption by M. circinelloides.
4. Conclusion
5% NaCl (W/V). By increasing salt concentration from 5%, no significant effect in dye removal was observed. The present of salt in dye solution increases the fungal cell wall thickness and therefore, increases dye biosorption capacity [3]. Na+ and Cl−, created from dissolution of NaCl, had not any negative effect on dye adsorption efficiency. So, it can be deduced that adsorption mechanism is not of electrostatic type and hydrogen bonding or n-π interactions may play significant role in adsorption process. Dye adsorption could be achieved by various mechanisms such as the formation of ionic interaction, hydrogen, and covalent binding or van der Waals forces. Due to the fact that under acidic conditions eNH2 groups are converted to eNH3+ if the interaction was ionic, then dye adsorbent should be increased. This is while acidic conditions reduced the percentage of dye adsorption. So, it can be concluded that removal mechanism is not ionic interaction and probably the other interactions such as hydrogen bonding or n-π interactions are involved in dye adsorption.
In this study, the contribution of functional groups on the surface of M. circinelloides in azo dye removal from dye solution was studied. On the basis of SEM images, zeta potential, FTIR, pre-treatment and evaluating dye adsorption by dead and living biomass results, it was concluded that the adsorption was the main responsible process in Congo red removal in the aqueous dye solution. In addition, hydroxyl and amine groups probably play an important role in dye adsorption. In addition, the adsorption of reactive dyes was compared with other groups of azo dyes is poorly performed on M. circinelloides biomass. Our findings proposed that characterization and identification of important binding groups involved in dye adsorption can be applied to different treatments on adsorbents, for improving adsorption capacity. In addition, deep insight about important binding groups leads to the selection of appropriate adsorbent that contain these groups in their structure. This approach can be used to determine methods of functional group 4122
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analysis to develop fast and low-cost ways of choosing a suitable adsorbent.
[22]
Acknowledgment [23]
This project was financially supported by University of Tehran. References
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