Materials Science and Engineering C 52 (2015) 31–36
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
The fungicidal properties of the carbon materials obtained from chitin and chitosan promoted by copper salts Anna Ilnicka ⁎, Mariusz Walczyk, Jerzy P. Lukaszewicz Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarina St., 87-100 Torun, Poland
a r t i c l e
i n f o
Article history: Received 13 August 2014 Received in revised form 19 January 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Fungicidal properties Carbon materials Chitin Chitosan Copper Copper(I) oxide
a b s t r a c t Renewable raw materials chitin and chitosan (N-deacetylated derivative of chitin) were subjected to action of different copper modifiers that were carbonized in the atmosphere of the N2 inert gas. As a result of the novel manufacturing procedure, a series of carbon materials was obtained with developed surface area and containing copper derivatives of differentiated form, size, and dispersion. The copper modifier and manufacturing procedure (concentration, carbonization temperature) influence the physical–chemical and fungicide properties of the carbons. The received carbons were chemically characterized using several methods like low-temperature adsorption of nitrogen, X-ray diffraction analysis, scanning electron microscopy, cyclic voltammetry, elemental analysis, and bioassay. Besides chemical testing, some biological tests were performed and let to select carbons with the highest fungicidal activity. Such carbons were characteristic of the specific form of copper derivatives occurring in them, i.e., nanocrystallites of Cu0 and/or Cu2O of high dispersion on the surface of carbon. The carbons may find an application as effective contact fungistatic agents in cosmetology, medicine, food industry, etc. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, much of the research focuses on synthesizing new carbon materials which could help in reclaiming of the production waste. Chitin, i.e., poly-(1-4)-β-N-acetyl-D-glucosamine, is present in the shells of crustaceans, insects, spiders, and bat guano [1–4]. The original studies involving the use of chitosan as a precursor for the preparation of activated carbons appeared relatively recently and were carried out at the Faculty of Chemistry, Nicolaus Copernicus University in Torun [5–9]. The studies have confirmed that the proposed chemical activation leads to an effective improvement of the structural parameters compared to carbons obtained by carbonization of non-activated chitosan: surface area SBET is in the range of 440–2000 m2 g−1, high nitrogen content up to 6.5% by weight, steerable pore structure and volume. Literature reviews deliver a description of some on the biocidal activity of silver, silver derivatives, and silver containing materials [10–12]. High price and therefore a limited accessibility of silver justify the search for other metals which could exhibit similar activity [13,14]. Recently, several research attempts have been made towards the application of metallic clusters containing metals at 0 oxidation degree as well as at intermediate oxidation states like copper(I) derivatives. Moreover, some papers [15,16] have reported that the biocidal activity of such metallic materials depends strongly on their degree of dispersion. For instance, microporous carbon fibers decorated with highly dispersed metal clusters are a good biocidal material [17]. ⁎ Corresponding author. E-mail address: [email protected] (A. Ilnicka).
http://dx.doi.org/10.1016/j.msec.2015.03.037 0928-4931/© 2015 Elsevier B.V. All rights reserved.
It is known from ancient times that some metals like copper, silver, and gold exhibit bactericidal and fungicidal [13,18–23]. According to these studies, metallic copper may have a broad spectrum of antimicrobial properties if applied in contact mode [24], like the inhibition of growth of some health hazard pathogens: bacteria (Escherichia coli, Staphylococcus, Legionella pneumophila), viruses (influenza type A), and fungi. Copper(I) oxide is a derivative having fungicidal contact properties. According to the assumptions of the HSAB theory, soft acids and bases will easily react with DNA. Consequently, copper ions have a particular affinity to the DNA and can bind and disrupt its helical structure by cross-linkage within and between the DNA strands [25–27]. Thus, the insertion of copper derivatives (Cu0- and Cu+1-based) into carbon matrix seems to be crucial regarding the mentioned conditions. The amine groups which are present in chitosan chains are definitely essential for the adsorption of copper ions due to chelating properties. Fig. 1 demonstrates the formation of the chelate chitosan–Cu(II). Chitosan itself can be applied for the capture of heavy metals in the process of water and waste water purification, recovery of precious metals from solutions, and the removal of radioactive ions. The chelation effect has been proven especially in the case of transition metal ions [4,28]. In our studies we are going to employ copper(II) complexation by amino groups to enable their retention in the carbon matrix after the carbonization of chitin and chitosan containing copper compounds. To our knowledge so far, the application of chitin and chitosan has not been investigated and there are no reports published on this topic. The chief aim of our work is to elaborate the method of manufacturing new carbon–copper materials from chitin and
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2.5. Morphology The carbons were examined by scanning electron microscopy (SEM, 1430 VP, LEO Electron Microscopy Ltd.) with an energy dispersive X-ray spectrometer (EDX, Quantax 200; detector: XFlash 4010, Bruker AXS). 2.6. X-ray diffraction X-ray diffraction (XRD) spectra were measured by means of a CuKα source in the range of 2Θ from 10° to 70° (X-Pert PRO Systems, Philips). 2.7. Electrochemical studies Fig. 1. The formation of the chelate chitosan–Cu(II) [28].
chitosan and to study the fungicidal/fungistatic effect of the carbon materials.
2. Materials and methods 2.1. Materials Chitosan (CH) was purchased from Sigma Aldrich (chitosan physical form N 75% deacetylation, medium molecular weight CAS Number: 9012-76-4). Chitin (CA) was purchased from Sigma Aldrich (chitin from shrimp shells CAS Number: 1398-61-4) as purified natural product in the form of loose flakes. Cu(NO3)2·3H2O of analytical purity was applied for preparation of water solution for further modification of the carbons. Rhizopus nigricans, a mold typical for food products, was selected for biological tests.
2.2. Method of preparation of carbons Activated carbons were modified with copper in the following way. Chitin or chitosan was treated with the 0.1 or 0.05 M aqueous solution of Cu(NO3)2·3H2O (ACu0.1 or ACu0.05), then for selected samples a chemical activator ZnCl2 (Zn) was added, according to the method previously developed by the authors [9]. The mixture was heated under the flow of nitrogen at the temperature of 700 °C for 1 h. In the previous studies [5–9] it was found that the carbonization temperature of 700 °C was optimal for the preservation of nitrogen atoms on the carbon surface and development of structural parameter (surface area and total pore volume). Therefore, carbonization temperature was not considered as a parameter influencing the investigated phenomena. Carbons obtained with the activator were additionally washed in a hot ultrasonic bath. The washing was performed using successive portions of distilled water until the neutral pH was reached. Next, the rinsed carbons were dried. Furthermore, the unmodified reference carbons were also prepared after the annealing of raw chitin or chitosan. Further in text these materials are called CA and CH, respectively.
2.3. Surface area and pore structure The structural parameters of the carbon materials were characterized by low-temperature nitrogen adsorption. The relevant isotherms of all the samples were measured at 77 K on a Gemini VI volumetric adsorption analyzer (Micromeritics, USA). Before each adsorption measurement, the sample was outgassed under vacuum at 200 °C.
2.4. Elemental analysis The activated carbons were analyzed (Vario MACRO CHN, Elementar Analysensysteme GmbH) for their total carbon and nitrogen contents.
Cyclic voltammetry (CV) curves were recorded using a computercontrolled Autolab (Eco Chemie) modular electrochemical system equipped with a PGSTAT128N potentiostat, controlled by NOVA software. The measurements were carried out using a three-electrode electrochemical cell presented in one of our earlier papers [29–34]. The working, counter and reference electrodes were, respectively, a powdered carbon electrode (PACE), a Pt wire and a Ag/AgCl (3 mol L−1 KCl) electrode. After vacuum desorption (10−2 Pa), the carbon sample (mass = 50 mg) was placed in an electrode container and drenched with a deaerated solution to obtain a 2–3 mm sedimentation layer. The potentiometric responses of the carbon electrodes were measured in oxygen-free electrolyte solutions (1 M KCl; usually after 24 h). The relevant sweep rates and amplitudes are given each time in the figure captions (20 to 200 mV s− 1). All measurements were carried out in the thermostated system at room temperature (293 K). 2.8. Microbiological test The fungicidal properties of synthesized carbon materials were tested. First, thermal (2 h, 160 °C) and chemical (ethanol 70%, Meliseptol) sterilization of laboratory equipment was performed. Microfungi (molds) were grown on a solid carbohydrate microbiological substrate (pepton K/agar–agar/glucose) during incubation at 20–22 °C. Some grains of unmodified carbon materials (CA, CH) and carbon–copper materials under testing were placed in the middle of the microbiological substrate to ensure contact with growing molds. Inoculation was performed by spraying spores of mold over the test sample. 2.9. Porosity characterization and surface area Table 1 shows the results gathered by means of low-temperature nitrogen adsorption of carbons obtained by carbonization of raw precursors (chitin and chitosan), modified with copper and activated by zinc chloride. Specific surface area was determined by the Brunauer– Emmett–Teller (BET) method (SBET). Carbons obtained from the unmodified chitosan (CH) are non-porous, as evidenced by the low value of SBET equal to 3 m2 g−1. It is different in the case of chitin, where the
Table 1 Specific surface area (SBET) and elemental content for carbon materials obtained from chitin (CA) and chitosan (CH). Sample
SBET (m2 g−1)
CA CA–ACu0.05 CA–ACu0.1 CA–ACu0.05–Zn1.00 CA–ACu0.1–Zn1.00 CH CH–ACu0.05 CH–ACu0.1 CH–ACu0.05–Zn1.00 CH–ACu0.1–Zn1.00
360 455 303 1184 1287 3 123 102 905 1159
Elemental content (wt.%) N
C
H
5.7 5.1 5.0 5.6 5.6 6.9 7.8 7.9 5.8 5.0
87.9 74.3 64.6 66.2 70.2 83.1 70.7 66.7 67.4 67.8
1.3 1.5 1.4 1.4 1.3 1.1 1.5 1.3 1.4 1.2
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unmodified materials reach a surface area of 360 m2 g−1. The shape of adsorption/desorption isotherms (IV-type according to IUPAC classification) suggests that the active carbons under investigation are strictly mesoporous with a little contribution of micropores.
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should be underlined that the high content of nitrogen may be useful for further chemical modification of the surface of the tested activated carbons, such as secondary introduction (by ion exchange) of copper and/or other metals, since nitrogen atoms are supposed to act as chelation centers for metal ions.
3. Results Activation of chitin and chitosan with zinc chloride resulted in activated carbons whose surface and pore structure are very well developed. The results of our tests show that this method of activation offers the possibility of designing the pore structure, and therefore surface properties, and the content of individual elements in the synthesized material. In the case of chitin, the same activation parameters allowed us to reach a spectacular SBET value of 1287 m2 g−1 for the sample of CA–ACu0.1–Zn1.00. In the case of both precursors, the activation with zinc chloride causes the production of microporous materials (the shape of adsorption/desorption isotherms is of type I according to IUPAC classification). For the chitin (CA) samples that have been treated only with Cu(NO3)2·3H2O, there were no significant changes of the surface area, contrary to the case in which the chemical activator was applied. However, for the chitosan (CH) samples that have been treated only with Cu(NO3)2·3H2O, a significant change of the surface area (from 3 to about 100 m2 g−1) was observed. 3.1. Analysis of the elemental composition The data on elemental composition show that the materials obtained from unmodified chitosan (CH) exhibit a nitrogen content equal to 6.9 wt.%, while the sample obtained from pristine chitin (CA) showed a slightly lower amount. In the chitosan-originated samples treated only with Cu(NO3)2·3H2O (ACu) an increase of the nitrogen content to 7.9–8.8 wt.% is observed. The addition of a chemical activator, i.e., zinc chloride, decreased the value by 1–2%. However, it is still at a significant level of nitrogen content. It should be noted that a combustion analysis does not allow the determination of copper content. It
3.2. Topography of materials The application of scanning electron microscopy (SEM) allows the determination of the size and degree of dispersion of the copper clusters occurring in the carbon matrix. The type of the employed modifier was found to change the chemical composition of the surface, yet its effect was observed for the dispersion of crystallites. On the surface of carbon materials obtained without using a copper modifier (CA, CH), only carbon matrix is observed. Figs. 2 and 3 show carbon surfaces obtained from chitin/chitosan with copper modifier, where the inorganic crystallites appear uniformly distributed throughout the entire volume of the carbon material and on its surface (SEM image). All the investigated carbon–copper materials are twocomponent materials: the darker background can be attributed to the presence of a conductive carbon matrix, while the lighter spots are identified as being a less conductive inorganic matrix (a derivative of copper). Fig. 2 presents the images of the materials obtained with Cu(NO3)2·3H2O only. In the case of chitosan-derived samples (Fig. 2A and B), the copper crystallites, in various forms, have a diameter in the range of 0.1–0.9 μm. In the materials derived from chitin (Fig. 2C and D), the crystallites have diameters in the range of 0.1–0.3 μm. Fig. 3 shows the morphology of materials obtained by the means of Cu(NO3)2·3H2O and the chemical activator ZnCl2. The SEM/EDX mapping clearly shows that apparent crystallites on the carbon surface contain the copper atoms and an insignificant amount of zinc atoms. For chitosan displayed at Fig. 3A and B, the crystallites of the copper moieties have diameters in the range of 0.7–5.3 μm. The crystallites have a diameter ranging between 0.4–1.8 μm for the materials derived from chitin (Fig. 3C and D). Keeping in mind the preparation method,
Fig. 2. SEM images of carbon material surface for samples: (A) CH–ACu0.05; (B) CH–ACu0.1; (C) CA–ACu0.05; (D) CA–ACu0.1.
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Fig. 3. SEM images of carbon material surface for samples: (A) CH–ACu0.05–Zn1.00; (B) CH–ACu0.1–Zn1.00; (C) CA–ACu0.05–Zn1.00; (D) CA–ACu0.1–Zn1.00.
which introduced copper to the bulk of the precursor, it can be concluded that the ions of the metal-based crystallites are as uniformly distributed over the entire volume of the sample as on its surface. 3.3. Determination of electrochemical properties Voltammetric studies of carbon materials confirm this supposition because the results indicate the presence on the surface of some samples (e.g., CH–ACu0.1) of the electrochemically active Cu2O(s), H3O+/ Cu0(s) with formal potential of approximately 0.45 V (A1/C1 vs. SHE), and Cu2 +/Cu+ forming a stable complex compound, e.g., cyanide, with formal potential of approximately 1.16 V (A2/C2 vs. SHE) — Fig. 4. 3.4. Characterization of fungicidal properties The growth inhibition effect of R. nigricans was investigated throughout the bioassay (10–14 days until the exhaustion of nutrients).
In Fig. 5 the fungicidal activity is shown as a function of time against R. nigricans of chitin samples: CA–ACu0.1 and CA–ACu0.05 (chitin impregnated by 0.1 M or 0.05 M Cu(NO3)2 before carbonization), CA (carbonized chitin without modification), and reference sample. In the case of CA–ACu0.1 and CA–ACu0.05 samples, inhibited mycelial growth is observed. Analogical fungicidal action is observed for samples derived from chitosan, but the diameter of colony growth inhibition zone was smaller by about 2–3 mm compared to the inhibition zone of samples obtained from chitin materials. 3.5. Structural analysis of copper During the annealing in an inert gas atmosphere, the introduced copper compound underwent a transformation into the corresponding copper(I) oxide and copper at 0 oxidation state (metallic), as confirmed by the XRD X-ray test results. Fig. 6 presents an example of XRD spectrum of one of the most active materials that is CA–ACu0.05–7 sample. The visible peaks are attributed to the species identified by electrochemical investigation by metallic copper Cu0 and Cu2O. The broad peak at 24θ is attributed to graphite which is a material present in the carbons as a minor residue (002 plane). It is typical for carbon materials obtained at medium carbonization temperature (700 °C). The remaining peaks belong to a sample holder made of aluminum. 4. Discussion
Fig. 4. Cyclic voltammogram of powdered carbon electrode material CH–ACu0.1 recorded in 0.1 M KCl.
The results presented in the current study make it possible to confirm that an innovative methodology was developed for the production of chitin/chitosan-originated carbons modified with copper and characterized by well-developed structural parameters as well as intensively enriched in nitrogen. The above-presented results clearly show that the novel Cu-containing activated carbons exhibit high fungicidal properties when brought into a contact with the colony of R. nigricans. An important factor was the change in the concentration of copper nitrate(V). Reducing the concentration resulted in a slight deterioration of antifungal properties (see Fig. 5). Copper(I) oxide decomposes very slowly and is suitable for fungicidal contact applications. It has been
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Fig. 5. Fungicidal activity of different samples as a function of time against R. nigricans: CA–ACu0.1 sample (first row); CA–ACu0.05 sample (second row); CA sample (third row); reference sample blank (fourth row).
Fig. 6. XRD pattern of CA–ACu0.05–7 sample.
found that the small particle size (b2 μm) of the compound particles enable the control of pathogens. In this case the positive charge on the surface of Cu2O particles interacts intensively with the negatively charged pathogen surface [14,15,24,35]. As previously demonstrated, the surface of chitin-derived carbons is containing smaller crystallites (the smallest diameter among all tested samples) of copper and copper oxide of high concentration. Moreover, their BET surface area is greater than that of activated carbons derived from pristine chitin. These factors should expand the surface of contact between molds and copper derivatives on the surface. We assume that this may explain the effective fungicidal activity of carbons derived from chitin. The mechanism of action of the biocidal copper compounds is mainly based on the release of copper ions, which damage the cell wall by changing the pH and conductivity of the local environment. In addition, copper has complexing properties with respect to various functional groups of proteins and enzymes, leading to disturbing their structure and subsequent inactivation. Copper(I) belongs to a group of soft acids (according to the HSAB theory). Certain amino acids contain thiol (e.g., methionine, cysteine) and disulfide bonds (e.g., L-cystine), which
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are classified as soft bases. Furthermore, both the carbon material surface (especially basic) and copper compounds may generate active oxygen species (Fenton reaction), also toxic to microorganisms (induces significant cellular oxidative stress). It is supposed that the combination of the toxic properties of reactive oxygen species and copper ions leads to the destruction of proteins present in fungal cell wall, and consequently to its breakdown [26]. The research is still ongoing and the fungicidal action in respect of other strains of microscopic fungi are being considered. 5. Conclusions The proposed application of the two natural biopolymers (chitin and chitosan) is an original solution that permits the use of the proposed precursors for achieving a high dispersion of nano-clusters of copper/ copper(I) oxide in the matrix of carbon. The samples received by means of Cu(NO3)2·3H2O show the effective contact antifungal action. The tested carbon materials prevented the increase of the population of micro-organisms by contact (creating an inhibition zone around carbon samples). Microscopic examination confirmed the presence of highly and evenly dispersed crystallites of metallic copper and copper(I) oxide on the surface of the materials under investigation. However, materials derived from chitin have a better antifungal action because the crystallite size is crucial. Acknowledgments This work was partly supported by the Scholarship Program of the Marshal of the Kuyavian-Pomeranian Province “Step into the future scholarships for PhD students fifth edition” (Grant No. UM_ES.533.3.011.2014). The authors acknowledge Mr Henryk Różycki for his valuable suggestions. References [1] M. Kaya, I. Sargin, K.O. Tozak, T. Baran, S. Erdogan, G. Sezen, Chitin extraction and characterization from Daphnia magna resting eggs, Int. J. Biol. Macromol. 61 (2013) 459–464. [2] M. Kaya, O. Seyyar, T. Baran, S. Erdo an, M. Kar, A physicochemical characterization of fully acetylated chitin structure isolated from two spider species: with new surface morphology, Int. J. Biol. Macromol. 65 (2014) 553–558. [3] M. Kaya, O. Seyyar, T. Baran, T. Turkes, Bat guano as new and attractive chitin and chitosan source, Front. Zool. 11 (2014) 59. [4] M. Mucha, Wydawnictwo Naukowo-Techniczne, 2010. [5] A. Kucińska, J.P. Łukaszewicz, in: P.396955 (Ed.), Polska 2011. [6] A. Kucińska, J.P. Łukaszewicz, Nano-CaCO3 jako matryca do przygotowania z chitozanu bogatych w azot mezoporowatych materiałów węglowych, Inż. Ochrona Środowiska 16 (2013) 191–202. [7] A. Kucinska, A. Cyganiuk, J.P. Lukaszewicz, A microporous and high surface area active carbon obtained by the heat-treatment of chitosan, Carbon 50 (2012) 3098–3101. [8] A. Olejniczak, M. Lezanska, J. Wloch, A. Kucinska, J.P. Lukaszewicz, Novel nitrogencontaining mesoporous carbons prepared from chitosan, J. Mater. Chem. A 1 (2013) 8961–8967. [9] A. Kucinska, R. Golembiewski, J.P. Lukaszewicz, Synthesis of N-rich activated carbons from chitosan by chemical activation, Sci. Adv. Mater. 6 (2014) 290–297. [10] V. Melinte, T. Buruiana, D. Moraru, E.C. Buruiana, Silver-polymer composite materials with antibacterial properties, Dig. J. Nanomater. Biostruct. 6 (2011) 213–223.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
The fungicidal properties of the carbon materials obtained from chitin and chitosan promoted by copper salts Anna Ilnicka ⁎, Mariusz Walczyk, Jerzy P. Lukaszewicz Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarina St., 87-100 Torun, Poland
a r t i c l e
i n f o
Article history: Received 13 August 2014 Received in revised form 19 January 2015 Accepted 22 March 2015 Available online 24 March 2015 Keywords: Fungicidal properties Carbon materials Chitin Chitosan Copper Copper(I) oxide
a b s t r a c t Renewable raw materials chitin and chitosan (N-deacetylated derivative of chitin) were subjected to action of different copper modifiers that were carbonized in the atmosphere of the N2 inert gas. As a result of the novel manufacturing procedure, a series of carbon materials was obtained with developed surface area and containing copper derivatives of differentiated form, size, and dispersion. The copper modifier and manufacturing procedure (concentration, carbonization temperature) influence the physical–chemical and fungicide properties of the carbons. The received carbons were chemically characterized using several methods like low-temperature adsorption of nitrogen, X-ray diffraction analysis, scanning electron microscopy, cyclic voltammetry, elemental analysis, and bioassay. Besides chemical testing, some biological tests were performed and let to select carbons with the highest fungicidal activity. Such carbons were characteristic of the specific form of copper derivatives occurring in them, i.e., nanocrystallites of Cu0 and/or Cu2O of high dispersion on the surface of carbon. The carbons may find an application as effective contact fungistatic agents in cosmetology, medicine, food industry, etc. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Nowadays, much of the research focuses on synthesizing new carbon materials which could help in reclaiming of the production waste. Chitin, i.e., poly-(1-4)-β-N-acetyl-D-glucosamine, is present in the shells of crustaceans, insects, spiders, and bat guano [1–4]. The original studies involving the use of chitosan as a precursor for the preparation of activated carbons appeared relatively recently and were carried out at the Faculty of Chemistry, Nicolaus Copernicus University in Torun [5–9]. The studies have confirmed that the proposed chemical activation leads to an effective improvement of the structural parameters compared to carbons obtained by carbonization of non-activated chitosan: surface area SBET is in the range of 440–2000 m2 g−1, high nitrogen content up to 6.5% by weight, steerable pore structure and volume. Literature reviews deliver a description of some on the biocidal activity of silver, silver derivatives, and silver containing materials [10–12]. High price and therefore a limited accessibility of silver justify the search for other metals which could exhibit similar activity [13,14]. Recently, several research attempts have been made towards the application of metallic clusters containing metals at 0 oxidation degree as well as at intermediate oxidation states like copper(I) derivatives. Moreover, some papers [15,16] have reported that the biocidal activity of such metallic materials depends strongly on their degree of dispersion. For instance, microporous carbon fibers decorated with highly dispersed metal clusters are a good biocidal material [17]. ⁎ Corresponding author. E-mail address: [email protected] (A. Ilnicka).
http://dx.doi.org/10.1016/j.msec.2015.03.037 0928-4931/© 2015 Elsevier B.V. All rights reserved.
It is known from ancient times that some metals like copper, silver, and gold exhibit bactericidal and fungicidal [13,18–23]. According to these studies, metallic copper may have a broad spectrum of antimicrobial properties if applied in contact mode [24], like the inhibition of growth of some health hazard pathogens: bacteria (Escherichia coli, Staphylococcus, Legionella pneumophila), viruses (influenza type A), and fungi. Copper(I) oxide is a derivative having fungicidal contact properties. According to the assumptions of the HSAB theory, soft acids and bases will easily react with DNA. Consequently, copper ions have a particular affinity to the DNA and can bind and disrupt its helical structure by cross-linkage within and between the DNA strands [25–27]. Thus, the insertion of copper derivatives (Cu0- and Cu+1-based) into carbon matrix seems to be crucial regarding the mentioned conditions. The amine groups which are present in chitosan chains are definitely essential for the adsorption of copper ions due to chelating properties. Fig. 1 demonstrates the formation of the chelate chitosan–Cu(II). Chitosan itself can be applied for the capture of heavy metals in the process of water and waste water purification, recovery of precious metals from solutions, and the removal of radioactive ions. The chelation effect has been proven especially in the case of transition metal ions [4,28]. In our studies we are going to employ copper(II) complexation by amino groups to enable their retention in the carbon matrix after the carbonization of chitin and chitosan containing copper compounds. To our knowledge so far, the application of chitin and chitosan has not been investigated and there are no reports published on this topic. The chief aim of our work is to elaborate the method of manufacturing new carbon–copper materials from chitin and
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2.5. Morphology The carbons were examined by scanning electron microscopy (SEM, 1430 VP, LEO Electron Microscopy Ltd.) with an energy dispersive X-ray spectrometer (EDX, Quantax 200; detector: XFlash 4010, Bruker AXS). 2.6. X-ray diffraction X-ray diffraction (XRD) spectra were measured by means of a CuKα source in the range of 2Θ from 10° to 70° (X-Pert PRO Systems, Philips). 2.7. Electrochemical studies Fig. 1. The formation of the chelate chitosan–Cu(II) [28].
chitosan and to study the fungicidal/fungistatic effect of the carbon materials.
2. Materials and methods 2.1. Materials Chitosan (CH) was purchased from Sigma Aldrich (chitosan physical form N 75% deacetylation, medium molecular weight CAS Number: 9012-76-4). Chitin (CA) was purchased from Sigma Aldrich (chitin from shrimp shells CAS Number: 1398-61-4) as purified natural product in the form of loose flakes. Cu(NO3)2·3H2O of analytical purity was applied for preparation of water solution for further modification of the carbons. Rhizopus nigricans, a mold typical for food products, was selected for biological tests.
2.2. Method of preparation of carbons Activated carbons were modified with copper in the following way. Chitin or chitosan was treated with the 0.1 or 0.05 M aqueous solution of Cu(NO3)2·3H2O (ACu0.1 or ACu0.05), then for selected samples a chemical activator ZnCl2 (Zn) was added, according to the method previously developed by the authors [9]. The mixture was heated under the flow of nitrogen at the temperature of 700 °C for 1 h. In the previous studies [5–9] it was found that the carbonization temperature of 700 °C was optimal for the preservation of nitrogen atoms on the carbon surface and development of structural parameter (surface area and total pore volume). Therefore, carbonization temperature was not considered as a parameter influencing the investigated phenomena. Carbons obtained with the activator were additionally washed in a hot ultrasonic bath. The washing was performed using successive portions of distilled water until the neutral pH was reached. Next, the rinsed carbons were dried. Furthermore, the unmodified reference carbons were also prepared after the annealing of raw chitin or chitosan. Further in text these materials are called CA and CH, respectively.
2.3. Surface area and pore structure The structural parameters of the carbon materials were characterized by low-temperature nitrogen adsorption. The relevant isotherms of all the samples were measured at 77 K on a Gemini VI volumetric adsorption analyzer (Micromeritics, USA). Before each adsorption measurement, the sample was outgassed under vacuum at 200 °C.
2.4. Elemental analysis The activated carbons were analyzed (Vario MACRO CHN, Elementar Analysensysteme GmbH) for their total carbon and nitrogen contents.
Cyclic voltammetry (CV) curves were recorded using a computercontrolled Autolab (Eco Chemie) modular electrochemical system equipped with a PGSTAT128N potentiostat, controlled by NOVA software. The measurements were carried out using a three-electrode electrochemical cell presented in one of our earlier papers [29–34]. The working, counter and reference electrodes were, respectively, a powdered carbon electrode (PACE), a Pt wire and a Ag/AgCl (3 mol L−1 KCl) electrode. After vacuum desorption (10−2 Pa), the carbon sample (mass = 50 mg) was placed in an electrode container and drenched with a deaerated solution to obtain a 2–3 mm sedimentation layer. The potentiometric responses of the carbon electrodes were measured in oxygen-free electrolyte solutions (1 M KCl; usually after 24 h). The relevant sweep rates and amplitudes are given each time in the figure captions (20 to 200 mV s− 1). All measurements were carried out in the thermostated system at room temperature (293 K). 2.8. Microbiological test The fungicidal properties of synthesized carbon materials were tested. First, thermal (2 h, 160 °C) and chemical (ethanol 70%, Meliseptol) sterilization of laboratory equipment was performed. Microfungi (molds) were grown on a solid carbohydrate microbiological substrate (pepton K/agar–agar/glucose) during incubation at 20–22 °C. Some grains of unmodified carbon materials (CA, CH) and carbon–copper materials under testing were placed in the middle of the microbiological substrate to ensure contact with growing molds. Inoculation was performed by spraying spores of mold over the test sample. 2.9. Porosity characterization and surface area Table 1 shows the results gathered by means of low-temperature nitrogen adsorption of carbons obtained by carbonization of raw precursors (chitin and chitosan), modified with copper and activated by zinc chloride. Specific surface area was determined by the Brunauer– Emmett–Teller (BET) method (SBET). Carbons obtained from the unmodified chitosan (CH) are non-porous, as evidenced by the low value of SBET equal to 3 m2 g−1. It is different in the case of chitin, where the
Table 1 Specific surface area (SBET) and elemental content for carbon materials obtained from chitin (CA) and chitosan (CH). Sample
SBET (m2 g−1)
CA CA–ACu0.05 CA–ACu0.1 CA–ACu0.05–Zn1.00 CA–ACu0.1–Zn1.00 CH CH–ACu0.05 CH–ACu0.1 CH–ACu0.05–Zn1.00 CH–ACu0.1–Zn1.00
360 455 303 1184 1287 3 123 102 905 1159
Elemental content (wt.%) N
C
H
5.7 5.1 5.0 5.6 5.6 6.9 7.8 7.9 5.8 5.0
87.9 74.3 64.6 66.2 70.2 83.1 70.7 66.7 67.4 67.8
1.3 1.5 1.4 1.4 1.3 1.1 1.5 1.3 1.4 1.2
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unmodified materials reach a surface area of 360 m2 g−1. The shape of adsorption/desorption isotherms (IV-type according to IUPAC classification) suggests that the active carbons under investigation are strictly mesoporous with a little contribution of micropores.
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should be underlined that the high content of nitrogen may be useful for further chemical modification of the surface of the tested activated carbons, such as secondary introduction (by ion exchange) of copper and/or other metals, since nitrogen atoms are supposed to act as chelation centers for metal ions.
3. Results Activation of chitin and chitosan with zinc chloride resulted in activated carbons whose surface and pore structure are very well developed. The results of our tests show that this method of activation offers the possibility of designing the pore structure, and therefore surface properties, and the content of individual elements in the synthesized material. In the case of chitin, the same activation parameters allowed us to reach a spectacular SBET value of 1287 m2 g−1 for the sample of CA–ACu0.1–Zn1.00. In the case of both precursors, the activation with zinc chloride causes the production of microporous materials (the shape of adsorption/desorption isotherms is of type I according to IUPAC classification). For the chitin (CA) samples that have been treated only with Cu(NO3)2·3H2O, there were no significant changes of the surface area, contrary to the case in which the chemical activator was applied. However, for the chitosan (CH) samples that have been treated only with Cu(NO3)2·3H2O, a significant change of the surface area (from 3 to about 100 m2 g−1) was observed. 3.1. Analysis of the elemental composition The data on elemental composition show that the materials obtained from unmodified chitosan (CH) exhibit a nitrogen content equal to 6.9 wt.%, while the sample obtained from pristine chitin (CA) showed a slightly lower amount. In the chitosan-originated samples treated only with Cu(NO3)2·3H2O (ACu) an increase of the nitrogen content to 7.9–8.8 wt.% is observed. The addition of a chemical activator, i.e., zinc chloride, decreased the value by 1–2%. However, it is still at a significant level of nitrogen content. It should be noted that a combustion analysis does not allow the determination of copper content. It
3.2. Topography of materials The application of scanning electron microscopy (SEM) allows the determination of the size and degree of dispersion of the copper clusters occurring in the carbon matrix. The type of the employed modifier was found to change the chemical composition of the surface, yet its effect was observed for the dispersion of crystallites. On the surface of carbon materials obtained without using a copper modifier (CA, CH), only carbon matrix is observed. Figs. 2 and 3 show carbon surfaces obtained from chitin/chitosan with copper modifier, where the inorganic crystallites appear uniformly distributed throughout the entire volume of the carbon material and on its surface (SEM image). All the investigated carbon–copper materials are twocomponent materials: the darker background can be attributed to the presence of a conductive carbon matrix, while the lighter spots are identified as being a less conductive inorganic matrix (a derivative of copper). Fig. 2 presents the images of the materials obtained with Cu(NO3)2·3H2O only. In the case of chitosan-derived samples (Fig. 2A and B), the copper crystallites, in various forms, have a diameter in the range of 0.1–0.9 μm. In the materials derived from chitin (Fig. 2C and D), the crystallites have diameters in the range of 0.1–0.3 μm. Fig. 3 shows the morphology of materials obtained by the means of Cu(NO3)2·3H2O and the chemical activator ZnCl2. The SEM/EDX mapping clearly shows that apparent crystallites on the carbon surface contain the copper atoms and an insignificant amount of zinc atoms. For chitosan displayed at Fig. 3A and B, the crystallites of the copper moieties have diameters in the range of 0.7–5.3 μm. The crystallites have a diameter ranging between 0.4–1.8 μm for the materials derived from chitin (Fig. 3C and D). Keeping in mind the preparation method,
Fig. 2. SEM images of carbon material surface for samples: (A) CH–ACu0.05; (B) CH–ACu0.1; (C) CA–ACu0.05; (D) CA–ACu0.1.
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Fig. 3. SEM images of carbon material surface for samples: (A) CH–ACu0.05–Zn1.00; (B) CH–ACu0.1–Zn1.00; (C) CA–ACu0.05–Zn1.00; (D) CA–ACu0.1–Zn1.00.
which introduced copper to the bulk of the precursor, it can be concluded that the ions of the metal-based crystallites are as uniformly distributed over the entire volume of the sample as on its surface. 3.3. Determination of electrochemical properties Voltammetric studies of carbon materials confirm this supposition because the results indicate the presence on the surface of some samples (e.g., CH–ACu0.1) of the electrochemically active Cu2O(s), H3O+/ Cu0(s) with formal potential of approximately 0.45 V (A1/C1 vs. SHE), and Cu2 +/Cu+ forming a stable complex compound, e.g., cyanide, with formal potential of approximately 1.16 V (A2/C2 vs. SHE) — Fig. 4. 3.4. Characterization of fungicidal properties The growth inhibition effect of R. nigricans was investigated throughout the bioassay (10–14 days until the exhaustion of nutrients).
In Fig. 5 the fungicidal activity is shown as a function of time against R. nigricans of chitin samples: CA–ACu0.1 and CA–ACu0.05 (chitin impregnated by 0.1 M or 0.05 M Cu(NO3)2 before carbonization), CA (carbonized chitin without modification), and reference sample. In the case of CA–ACu0.1 and CA–ACu0.05 samples, inhibited mycelial growth is observed. Analogical fungicidal action is observed for samples derived from chitosan, but the diameter of colony growth inhibition zone was smaller by about 2–3 mm compared to the inhibition zone of samples obtained from chitin materials. 3.5. Structural analysis of copper During the annealing in an inert gas atmosphere, the introduced copper compound underwent a transformation into the corresponding copper(I) oxide and copper at 0 oxidation state (metallic), as confirmed by the XRD X-ray test results. Fig. 6 presents an example of XRD spectrum of one of the most active materials that is CA–ACu0.05–7 sample. The visible peaks are attributed to the species identified by electrochemical investigation by metallic copper Cu0 and Cu2O. The broad peak at 24θ is attributed to graphite which is a material present in the carbons as a minor residue (002 plane). It is typical for carbon materials obtained at medium carbonization temperature (700 °C). The remaining peaks belong to a sample holder made of aluminum. 4. Discussion
Fig. 4. Cyclic voltammogram of powdered carbon electrode material CH–ACu0.1 recorded in 0.1 M KCl.
The results presented in the current study make it possible to confirm that an innovative methodology was developed for the production of chitin/chitosan-originated carbons modified with copper and characterized by well-developed structural parameters as well as intensively enriched in nitrogen. The above-presented results clearly show that the novel Cu-containing activated carbons exhibit high fungicidal properties when brought into a contact with the colony of R. nigricans. An important factor was the change in the concentration of copper nitrate(V). Reducing the concentration resulted in a slight deterioration of antifungal properties (see Fig. 5). Copper(I) oxide decomposes very slowly and is suitable for fungicidal contact applications. It has been
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Fig. 5. Fungicidal activity of different samples as a function of time against R. nigricans: CA–ACu0.1 sample (first row); CA–ACu0.05 sample (second row); CA sample (third row); reference sample blank (fourth row).
Fig. 6. XRD pattern of CA–ACu0.05–7 sample.
found that the small particle size (b2 μm) of the compound particles enable the control of pathogens. In this case the positive charge on the surface of Cu2O particles interacts intensively with the negatively charged pathogen surface [14,15,24,35]. As previously demonstrated, the surface of chitin-derived carbons is containing smaller crystallites (the smallest diameter among all tested samples) of copper and copper oxide of high concentration. Moreover, their BET surface area is greater than that of activated carbons derived from pristine chitin. These factors should expand the surface of contact between molds and copper derivatives on the surface. We assume that this may explain the effective fungicidal activity of carbons derived from chitin. The mechanism of action of the biocidal copper compounds is mainly based on the release of copper ions, which damage the cell wall by changing the pH and conductivity of the local environment. In addition, copper has complexing properties with respect to various functional groups of proteins and enzymes, leading to disturbing their structure and subsequent inactivation. Copper(I) belongs to a group of soft acids (according to the HSAB theory). Certain amino acids contain thiol (e.g., methionine, cysteine) and disulfide bonds (e.g., L-cystine), which
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are classified as soft bases. Furthermore, both the carbon material surface (especially basic) and copper compounds may generate active oxygen species (Fenton reaction), also toxic to microorganisms (induces significant cellular oxidative stress). It is supposed that the combination of the toxic properties of reactive oxygen species and copper ions leads to the destruction of proteins present in fungal cell wall, and consequently to its breakdown [26]. The research is still ongoing and the fungicidal action in respect of other strains of microscopic fungi are being considered. 5. Conclusions The proposed application of the two natural biopolymers (chitin and chitosan) is an original solution that permits the use of the proposed precursors for achieving a high dispersion of nano-clusters of copper/ copper(I) oxide in the matrix of carbon. The samples received by means of Cu(NO3)2·3H2O show the effective contact antifungal action. The tested carbon materials prevented the increase of the population of micro-organisms by contact (creating an inhibition zone around carbon samples). Microscopic examination confirmed the presence of highly and evenly dispersed crystallites of metallic copper and copper(I) oxide on the surface of the materials under investigation. However, materials derived from chitin have a better antifungal action because the crystallite size is crucial. Acknowledgments This work was partly supported by the Scholarship Program of the Marshal of the Kuyavian-Pomeranian Province “Step into the future scholarships for PhD students fifth edition” (Grant No. UM_ES.533.3.011.2014). The authors acknowledge Mr Henryk Różycki for his valuable suggestions. References [1] M. Kaya, I. Sargin, K.O. Tozak, T. Baran, S. Erdogan, G. Sezen, Chitin extraction and characterization from Daphnia magna resting eggs, Int. J. Biol. Macromol. 61 (2013) 459–464. [2] M. Kaya, O. Seyyar, T. Baran, S. Erdo an, M. Kar, A physicochemical characterization of fully acetylated chitin structure isolated from two spider species: with new surface morphology, Int. J. Biol. Macromol. 65 (2014) 553–558. [3] M. Kaya, O. Seyyar, T. Baran, T. Turkes, Bat guano as new and attractive chitin and chitosan source, Front. Zool. 11 (2014) 59. [4] M. Mucha, Wydawnictwo Naukowo-Techniczne, 2010. [5] A. Kucińska, J.P. Łukaszewicz, in: P.396955 (Ed.), Polska 2011. [6] A. Kucińska, J.P. Łukaszewicz, Nano-CaCO3 jako matryca do przygotowania z chitozanu bogatych w azot mezoporowatych materiałów węglowych, Inż. Ochrona Środowiska 16 (2013) 191–202. [7] A. Kucinska, A. Cyganiuk, J.P. Lukaszewicz, A microporous and high surface area active carbon obtained by the heat-treatment of chitosan, Carbon 50 (2012) 3098–3101. [8] A. Olejniczak, M. Lezanska, J. Wloch, A. Kucinska, J.P. Lukaszewicz, Novel nitrogencontaining mesoporous carbons prepared from chitosan, J. Mater. Chem. A 1 (2013) 8961–8967. [9] A. Kucinska, R. Golembiewski, J.P. Lukaszewicz, Synthesis of N-rich activated carbons from chitosan by chemical activation, Sci. Adv. Mater. 6 (2014) 290–297. [10] V. Melinte, T. Buruiana, D. Moraru, E.C. Buruiana, Silver-polymer composite materials with antibacterial properties, Dig. J. Nanomater. Biostruct. 6 (2011) 213–223.
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