Soil emendation with nano-fungal chitosan for heavy metals biosorption

Soil emendation with nano-fungal chitosan for heavy metals biosorption

BIOMAC-10147; No of Pages 4 International Journal of Biological Macromolecules xxx (2018) xxx–xxx Contents lists available at ScienceDirect Internat...

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BIOMAC-10147; No of Pages 4 International Journal of Biological Macromolecules xxx (2018) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Soil emendation with nano-fungal chitosan for heavy metals biosorption Sultan F. Alsharari a, Ahmed A. Tayel b,⁎, Shaaban H. Moussa a,c a b c

College of Science and Humanitarian Studies, Shaqra University, Qwaieah 11971, Saudi Arabia Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, Egypt Genetic Engineering and Biotechnology Research Institute, University of Sadat City, Egypt

a r t i c l e

i n f o

Article history: Received 17 May 2018 Received in revised form 7 July 2018 Accepted 16 July 2018 Available online xxxx Keywords: Bioremediation Nano-chitosan Soil matrix

a b s t r a c t The bioremediation of water and soil, from heavy metal (HM) contamination, is a continuing worldwide demand. Chitosan, as a promising bioactive polymer, was produced from grown fungal (Cunninghamella elegans) mycelia and had a molecular weight of 112 kDa and a deacetylation degree of 87%. Sodium tripolyphosphate was applied for the synthesis of chitosan nanoparticles (NCt) from fungal chitosan (Cts); the particle size of produced NCt was in range of 5–45 nm. The produced biopolymers were used for HM absorption, Pb2+ and Cu2+ at concentration range of 100–300 ppm, from aqueous solution and soil matrix. Both Cts and NCt had high adsorption capacity toward the examined HM, with higher affinity as adsorbents to Pb2+ than to adsorb Cu2+ from water or after amendment of soil matrix. The produced NCt particles were highly effective than bulk Cts for the remediation and biosorption of contaminant metals, Pb2+ and Cu2+. Both Cts and NCt could be effectually applied as amendments in HM-contaminated soils for their bioremediation. © 2018 Published by Elsevier B.V.

1. Introduction The water and soil contaminations, with heavy metals (HM), originating from diverse sources worldwide and considered from the extremely hazardous environmental problems [1, 2]; the remediation approaches for HM-contaminated soils may be categorized into three classes: chemical, physical and biological. The main rationales of bioremediations (biosorption) include their high efficacy, cost effectiveness, potential metals recovery, chemical and biological mud minimization and biosorbent restoration [3–6]. From the continuing efforts toward remediation of HM-contaminated soils, traditional techniques for excavation and landfilling were proved to be expensive; whereas soil modifiers (amendments) was from the best cost-effective techniques for soil stabilization that cause least environmental disruption [7]. Frequent industrial and agricultural wastes and byproducts could be employed for in situ HM stabilization/remediation in contaminated soils; from the most recommended is chitosan (Cts) [8, 9]. Chitosan (deacetylated chitin with ≥70% deacetylation degree) could be originated from several sources such as shellfish wastes and fungal biomass, and magnificently applied in numerous fields, including HM biosorption [10, 11]. Cts is biodegradable and non-toxic for human, animal and plant; thus it is regarded as a safe amendment agent for safety and health perspectives [12].

⁎ Corresponding author at: Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University, El-Geish St., 33516 Kafrelsheikh City, Egypt. E-mail addresses: [email protected] [email protected] (A.A. Tayel).

The Cts potentialities, as powerful antimicrobial, biodegradable, biochelator, eco-friendly and biocompatible polymer, gave it great advantage for HM ions' sorption through its reactive chemical structure that has many amino and hydroxyl groups [13–16]. The Cts was suggested to remediate HM-contaminated soil through active extraction/ removal of HM ions/complexes or via HM ions immobilization [2]. The solid form of Cts, and its crosslinked derivatives, were applied as soil amendments for immobilizing contaminant and to adsorb HM ions in moist soil, thereafter they could reduce the HM availability and toxicity for living organisms [10, 17]. Cts was also economically used for remediating wastewater from electroplating processes [18], and for recovering treasurable metals from wastewater of mining [19]. Nanotechnology has been progressively explored as an ideal alternative for traditional approaches to treat and remediate contaminated water with diverse pollutants [20]. Because of the very minute size (b100 nm), nanomaterials have additional chemical, physical and biological characteristics, compared to corresponding larger counterparts, with micro- and macro-particle size [21, 22]. Nanomaterials have greater surface/volume ratios and, accordingly, higher intensity from surface reaction sites for each mass unit [20]. Compared to their customary used micro- and macro-sized particles, in water clarification practices, adsorbents with nano-size particles possess greater performance because of their smaller size, higher specific surface area and quantum size impact, which may give them increased capacities for HM adsorption [23]. The chitin and Cts were illustrated to be easily processed into various forms, with additional activities, e.g. membranes, gels, beads, scaffolds, microparticles, nanofibers and nanoparticles [24].

https://doi.org/10.1016/j.ijbiomac.2018.07.103 0141-8130/© 2018 Published by Elsevier B.V.

Please cite this article as: S.F. Alsharari, et al., Soil emendation with nano-fungal chitosan for heavy metals biosorption, (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.07.103

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Therefore, the current investigation aimed to produce fungal Cts from Cunninghamella elegans biomass, to produce nanoparticles from fungal Cts and to apply the produced biopolymers for heavy metals, Pb2+ and Cu2+, adsorption from water and from amended contaminated soil. 2. Materials and methods 2.1. Fungal production and chitosan extraction The applied fungus, Cunninghamella elegans (RCMB-012002, attained from Azhar University, Regional Fungal Centre, Egypt), was propagated at 25 °C, under shaking at 80 ×g for 96 h, in Czapek Dox broth. Grown fungal mycelia, after incubation, were collected through filtration with filter paper (Whatman No. 2), then cleansed with double distilled water (DW), dried with hot air (45 °C) and weighted. The Cts extraction, from fungal mycelia, was executed using the following steps [25]: (1) deproteinization by treatment with NaOH (1 M) at 90 ± 2 °C for 120 min; (2) centrifugation (4200 ×g for 12 min), washing the precipitate with DW and recentrifugation; (3) demineralization with acetic acid (10%) for 6 h at 65 ± 3 °C and collection of acid soluble fraction; (4) deacetylation with NaOH (4 M) at 100 ± 3 °C for 100 min; (5) repeat step 2 and wash with DW then ethanol (95%) then acetone; (6) drying at 50 ± 2 °C and grinding. Cts molecular weight was determined using chromatographic gel permeation as specified by Tayel et al. [11]; whereas the degree of acetyl groups abstraction (deacetylation degree) was calculated based on Cts spectra using spectroscopic Fourier-transform infra-red (FTIR-FTS 45, Biorad, Germany) [26]. 2.2. Nano-chitosan synthesis The produced Cts solution (0.5% w/v) was prepared using diluted acetic acid (1.5%), then subjected to sonication (Braun-Labsonic, Germany) at 45 W for 22 min. A stock aqueous solution from sodium tripolyphosphate (Na-TPP), with concentration of 0.5% w/v, was made in DW. While stirring, Na-TPP was slowly and finely dropped into Cts solution until an equal volume then the solution was re-sonicated, as mentioned above. The suspension was subjected to centrifugation (at 8500 ×g for 20 min) then the sedimented pellet from nano-chitosan/ Na-TPP (NCt) was suspended in DW, re-sonicated and re-centrifuged. Both Cts and NCt were characterized using FTIR analysis at wavenumbers of 500–4000 cm−1 [26]. The synthesized NCt was further characterized using an electron microscope (transmission), using model JEM-2100, JEOL, Japan.

samples were then filtered and the soil extracts were collected and kept for analysis. The HM contents, after treatments of aqueous solution and soil matrix, were determined using a spectrometer (atomic absorption) (AAS, AAnalyst 400, Perkin-Elmer, Massachusetts, USA) according to manufacturer instructions [27]. The adsorption capacities (qe) of Cts and NCt were calculated via application of this equation: qe ¼

ðC0−C1ÞV m

where (C0) is the initial HM concentration and (C1) is the equilibrium HM concentration (mg/L), (m) is the used adsorbent amount (g) and (V) is the solution volume (L). 2.4. Statistical analysis The trials were triplicated and the calculation of their means and SD (standard deviations) was performed using Excel spreadsheet program (V 5.0, Microsoft Co., Redmond, Wash DC). The significance of mean differences was computed by t-test, using MedCalc software, (V 9.3.9.0, Mariakerke, Belgium), with CI (confidence intervals) of 95% (P b 0.05). 3. Results The produced fungal Cts and nano-chitosan (NCt) were characterized, through FTIR spectral analyses; the spectra of produced biopolymers are shown in Fig. 1. The extracted Cts molecular weight was estimated to be 112 kDa, whereas its deacetylation degree was calculated and recorded as 87%. From the FTIR spectra of Cts (Fig. 1C) and NCt (Fig. 1N), it could be noticed that both spectra were closely related and had the main characteristic bands for standard chitosan, e.g. the carbonyl (C_O) group at 1740 cm−1, the C\\H rock stretching at 1420 cm−1, and the peaks around 3455 cm−1 that indicate N\\H and\\OH stretching vibrations. The peaks at 1540 cm−1 and 1322 cm−1 designated the amide II (N\\H) bending vibration and the absorption of amide III, respectively. The sharp peaks around 1628 cm−1 are assigned to amide I. The emergence of a peak at 1267 cm−1, in NCt spectra, indicates P_O stretching that verifies the crosslinking between tripolyphosphate (negatively charged) and chitosan (positively charged). The structural morphology of produced NCt was elucidated through TEM imaging (Fig. 2). The NCt particles were almost appeared in

2.3. Absorption of HM by fungal Cts and NCt The produced fungal Cts and NCt were initially evaluated for HM absorption, i.e. lead (Pb2+) and copper (Cu2+), from aqueous solution and in soil matrix. For the aqueous solution, metal suspensions were prepared in DW from lead nitrate and copper sulfate; gradual concentrations from each metal ion (100, 200 and 300 ppm) were welldissolved by vortexing then press filtered. Cts and NCt were dissolved in 0.5% acetic acid aqueous solution then added to HM solution to have final concentrations of 0.25 and 0.5% (w/v) from sorbents. Treated HM solutions were kept at 25 °C, under shaking (120 ×g), for 450 min. For the soil treatment, about 500 g of soil matrix (~30% sand) was achieved from certified organic farm, repeatedly washed/drained with 10 volumes from deionized water, then dried in oven until dryness. HM stock solutions were added to soil until reaching the specified concentrations, homogenized well and air dried. Solutions of dissolved Cts or NCt were added to soils, with the previously mentioned concentrations, with excess addition of deionized water (up to 10 folds), then samples were agitated in an end-over-end shaker (Revolver, Labnet Int., Edison, NJ) for 120 min at room temperature. The treated soil

Fig. 1. FTIR spectra of produced fungal chitosan (C) and nano-chitosan (N).

Please cite this article as: S.F. Alsharari, et al., Soil emendation with nano-fungal chitosan for heavy metals biosorption, (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.07.103

S.F. Alsharari et al. / International Journal of Biological Macromolecules xxx (2018) xxx–xxx

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(group B) and 300 ppm (group C), the application of Cts and NCt, as adsorbent and soil amendments, resulted in significant reduction of free metals' concentration in soil extract (Table 2). NCt amendments were significantly much effectual than Cts for remediate both experimentally contaminated soils. The application of NCt, at concentrations of 0.25 and 0.5%, led to the reduction in Pb2+ concentrations by 91.7 and 98.6% for group A, 74.0 and 87.2% for group B, and 71.3 and 94.8% for group C, respectively. The corresponding reduction percentages after amendment with bulk Cts were 58.2 and 74.3% for group A, 50.6 and 65.7% for group B, and 45.6 and 60.8% for group C, respectively. Regarding Cu2+ contaminated soils, the highest reduction percentages, i.e. 97.3%, 90.4% and 88.8%, was achieved by the amendment with NCt at concentration of 0.5% into soil groups A, C and B, respectively. The subsequent effective amendments, for Cu2+ remediation, were the concentration of 0.25% from NCt, 0.5% from Cts then 0.25% from Cts. 4. Discussion Fig. 2. TEM micrographs of produced nano-chitosan.

spherical shape and smooth morphological structure. The estimated diameters of synthesized NCt were in the range of 5–45 nm as determined from the TEM scale bar (Fig. 2). The impact of HMs, i.e. Pb2+ and Cu2+, absorption by Cts and NCt, in aqueous solution, is represented in Table 1. The adsorbed amounts and adsorption capacity (qe) increased with the increase of metal concentration, from 100 to 300 ppm, for both the determined metals, using either Cts or NCt. For all used concentrations from HMs, the reduction in concentrations was significantly much higher with the application of 0.5% from Cts or NCt, than the usage of 0.25% concentration from them, as biosorbents in contaminated water (Table 1). Both Cts and NCt showed higher affinity, as adsorbents, to Pb2+ than to adsorb Cu2+. Although the adsorbed amounts from metals significantly increased with the raised concentrations from Cts and NCt, the adsorption capacity decreased. Both the adsorbed amounts and qe were remarkably higher with the application of NCt as adsorbent, compared to bulk Cts application. For the remediation of experimentally contaminated soil with Pb2+ and Cu2+ ions, at concentrations of 100 ppm (group A), 200 ppm

The bioremediation of HM-contaminated water and soil is a valued demand worldwide, especially with the application of eco-friendly agents. Chitosan was employed in this study because of its consideration as one from the best biological HM chelators [28]. Cts molecules were proved as adequately flexible for forming helical arrangements surrounding HM ions; it can also form several linkage bonds with them [10]; low molecular weight Cts that produced via oxidation or acid hydrolysis could be safer as amending agents for bioremediation of HM-contaminated soil. At low pH values, the Cts as sorbent could become physically unstable, thus Cts-based sorbents were recommended to be stabilized via crosslinkage with other chemical agent [8, 9, 29]. The amendment with of positively charged, acid-dissolved Cts could modify the physicochemical attributes of soil, and immobilize HM therein [30]. FTIR spectroscopy was recurrently used to determine molecules bioactivities by identifying the existence of certain chemical bonds/functional groups in their structures; this is because every characterized chemical bond mostly has a distinctive band indicating its energy absorption [31]. The FTIR spectra, for the produced fungal Cts, is comparable to the spectra of many previously produced [9, 11, 25], and this indicated that current Cts has equivalent bioactivities to be applied in

Table 1 The concentration reduction (cR, ppm) and adsorption capacity (qe) of Pb2+ and Cu2+ after treatment with fungal chitosan and nano-fungal chitosan in aqueous solution. Biosorbent materials

Biosorbent concentration %

Control (initial conc.) Chitosan 0.25 0.50 Nano chitosan 0.25 0.50

Pb2+ concentration (ppm) 100

Cu2+ concentration (ppm)

200

300

100

200

300

cR

qe

cR

qe

cR

qe

cR

qe

cR

qe

cR

qe

95.31 63.15 ± 3.2a 77.29 ± 5.1b 90.05 ± 4.8c 92.58 ± 5.6c

NA 25.26 15.46 36.82 19.32

192.52 105.38 ± 6.1a 137.73 ± 4.5b 148.63 ± 4.8c 169.26 ± 6.4d

NA 42.15 27.75 58.65 33.85

287.13 149.05 ± 5.4a 187.61 ± 6.1b 218.78 ± 7.3c 278.24 ± 7.1d

NA 59.62 37.52 87.51 55.65

94.31 60.53 ± 4.1a 72.66 ± 4.1b 81.34 ± 3.4c 91.32 ± 4.7d

NA 24.21 14.53 32.54 18.26

192.62 110.35 ± 5.7a 141.08 ± 3.5b 158.68 ± 6.1c 174.51 ± 7.3d

NA 44.14 28.22 63.47 34.90

289.58 142.55 ± 6.6a 188.39 ± 6.2b 222.80 ± 7.7c 264.88 ± 6.1d

NA 57.23 37.68 89.12 52.98

Values for cR are means of triplicates ± standard deviation. The different superscript letters in one column indicate significant difference at confidence interval (CI) of 95%.

Table 2 The concentration reduction (cR, ppm) and adsorption capacity (qe) of Pb2+ and Cu2+ after amendment with fungal chitosan and nano-fungal chitosan in contaminated soil. Biosorbent materials

Biosorbent concentration %

Control (initial conc.) Chitosan 0.25 0.50 Nano chitosan 0.25 0.50

Pb2+ concentration (ppm) (A) 100

Cu2+ concentration (ppm)

(B) 200

(C) 300

(A) 100

(B) 200

(C) 300

cR

qe

cR

qe

cR

qe

cR

qe

cR

qe

cR

qe

93.42 54.32 ± 3.2a 69.45 ± 3.8b 85.69 ± 4.1c 92.35 ± 3.9c

NA 21.73 13.89 34.28 18.47

184.64 93.45 ± 3.8a 121.36 ± 4.6b 136.58 ± 5.1c 161.08 ± 4.9d

NA 37.38 24.27 54.63 32.22

284.17 129.44 ± 4.2a 172.68 ± 4.4b 202.55 ± 5.6c 269.26 ± 5.9d

NA 51.78 34.54 81.02 53.85

91.26 53.57 ± 3.8a 67.84 ± 4.1b 77.61 ± 3.2c 88.79 ± 3.5d

NA 21.43 13.57 31.04 17.76

189.71 101.33 ± 3.6a 134.21 ± 4.1b 150.94 ± 3.9c 168.42 ± 4.3d

NA 40.53 26.84 60.38 33.68

285.25 137.63 ± 4.4a 182.22 ± 4.7b 213.54 ± 5.2c 257.78 ± 4.9d

NA 55.05 36.44 85.42 51.56

Values for cR are means of triplicates ± standard deviation. The different superscript letters in one column indicate significant difference at confidence interval (CI) of 95%.

Please cite this article as: S.F. Alsharari, et al., Soil emendation with nano-fungal chitosan for heavy metals biosorption, (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.07.103

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numerous fields, including HM removal and water remediation. The richness of\\NH2 groups in Cts was reported to permit it to effectually remediate toxic HM oxyanions from wastewater; HM oxyanions could be adsorbed to\\NH3+ ions, on Cts structure, through mechanisms of ion exchange [32]. The synthesis of NCt was succeeded using Na-TPP, as evidenced from the particles TEM micrographs. The appearance of sharp peak in the FTIR spectrum of NCt at 1628 cm−1 indicated that the used chitosan was not completely deacetylated, as evidenced from amide I presence [33]. The application of Na-TPP for synthesis and crosslinkage of NCt gave more activity to the synthesized nanoparticles. The Cts physical optimization was successfully conducted through its linkage with materials carrying negative charges, e.g. Na-TPP [23]; the resulted NCt-TPP complex exhibited high qe for Pb2+ sorption, due to the complex low crystallinity in addition to the presence of TPP ions. The NCt particles are assumed to have more sites for HM ions sorption, in addition to the hydroxyl and amino groups, via the resulted phosphoric groups from their crosslinkage with TPP [23]. The HM immobilizing amendments were advocated for decreasing metal ions leaching and bioavailability, in contaminant water/soil, via various sorption manners: formation of stabilized complexes; adsorption into the adsorbent surfaces; ion exchange; and surface precipitation [34]. The qe of chitosan and nano-chitosan varied toward examined heavy metal, Pb2+ and Cu2+, and this indicated the presence of affinity from these polymers as absorbent to each metal. The enhanced extraction of HM in subsurface soil, Cu2+ and Cd2+ ions, was observed from hydrochloride solution of Cts [35]; it was confirmed the raised Cts affinity for Cu2+ adsorption. The modification of Cts was reported to increase phytoremediation and extraction of contaminated HM ions in soil [17]; the introduction of thiol group, to \\NH2 groups in Cts, could improve the corn uptake of Pb2+ in contaminated soil. The sole Cts application, for contaminated soil remediation, may have less effectiveness due to Cts low affinity toward some HN ions; this matter could be managed through Cts blending with different functional groups from other materials to form composites that enhance their remediation activities [10, 17]. In current study, the Cts -TPP composite, formed through NCt synthesis, had greater HM adsorption capacity, compared to bulk Cts. Although Cts crosslinking may be expected to reduce\\NH3+ groups' content in its structure, the contrary was proved for dyes sorption capacity [36], which was augmented from crosslinked Cts, compared to native non-linked polymer. From agricultural view point, the availability of metal ions to be absorbed by cultivated plant is a critical factor for remediate contaminated soils. Glucosamine (monomeric Cts) was evidenced to develop complexes with metals, which increase their bioavailability for plant absorption [37]. This synergistic effect was attributed to the mobilization of HM ions by complexation with Cts [38]. Water-soluble HM-Cts complexes enabled greater plant absorption, possibly from increased partitioning of metal ions to the shoot tissues of the plants [17]. The amendment of soil with Cts or NCt, in this study, used low concentrations from the biosorbent materials (0.25 and 0.5%); this was for maintaining the soil structure as possible and for helping cultivated plants to effectually uptake their needs from HM-sorbent complexes. Kamari and Pulford [39] had reported that Cts application rates affect plants sorption of metals via the formation of water-soluble HM/Cts complexes; the low Cts amendment rate (up to 1%) enhanced metal sorption from plants, whereas at higher application rates (up to 10%), the metal sorption decreased from Lolium perenne.

As illustrated in the results of study, the NCt-TPP was highly effective for the biosorption and remediation of HM from contaminated water and soil; this could be principally attributed to the very small size of NCt particles and the expected increase in their functional groups content; which enable them to highly react with HM and to form many active bonds with them [20, 23, 33]. 5. Conclusion The chemical synthesis of nano-fungal chitosan could be successfully conducted using Na-TPP. The produced NCt particles were highly effective than bulk Cts for the remediation and biosorption of contaminant metals, Pb2+ and Cu2+, from water and soil. Both Cts and NCt could be effectually applied as amendments in HM-contaminated soils for their bioremediation. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39]

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Please cite this article as: S.F. Alsharari, et al., Soil emendation with nano-fungal chitosan for heavy metals biosorption, (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.07.103