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Hybrid chitosan-silver nanoparticles enzymaticallyembedded on cork filter material for water disinfection Lina Vanesa Garcia Peña, Petya Petkova, Rosanna Margalef-Marti, Marc Vives, Lorena Aguilar, Angel Gallegos, Antonio Francesko, Ilana Perelshtein, Aharon Gedanken, Ernest Mendoza, Juan C. Casas-Zapata, Jordi Morató, and Tzanko Tzanov Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b04721 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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Industrial & Engineering Chemistry Research

Hybrid chitosan-silver nanoparticles enzymatically-embedded on cork filter material for water disinfection Lina Vanesa Garcia Peña,‡,a1 Petya Petkova,‡,b Rosanna Margalef-Marti,a Marc Vives,a Lorena Aguilar,a Angel Gallegos,a Antonio Francesko,b Ilana Perelshtein,c Aharon Gedanken,c Ernest Mendoza,d

Juan Carlos Casas-Zapata,e Jordi Moratóa and Tzanko

Tzanov*b a

AQUASOST Group - UNESCO Chair on Sustainability, Universitat Politècnica de Catalunya, 08022 Barcelona, Spain. b

Grup de Biotecnologia Molecular i Industrial, Departament d’Enginyeria Química, Universitat Politècnica de Catalunya, 08222 Terrassa, Spain

c

Department of Chemistry, Kanbar Laboratory for Nanomaterials, Institute Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel

of

d

Grup de Nanomaterials Aplicats. Centre de Recerca en Nanoenginyeria, Universitat Politècnica de Catalunya, 08028 Barcelona, Spain e

Grupo de Investigación Ciencia e Ingeniería en Sistemas Ambientales (GCISA), Facultad de Ingeniería Civil, Departamento de Ing. Ambiental, Universidad del Cauca, calle 5 Nº 4-70 Popayán Cauca, Colombia ‡These authors contributed equally to the work *Corresponding author: [email protected]

1

Permanent address: Grupo de Investigación Ciencia e Ingeniería en Sistemas Ambientales (GCISA), Facultad de Ingeniería Civil, Departamento de Ing. Ambiental, Universidad del Cauca, calle 5 Nº 4-70 Popayán Cauca, Colombia

1 ACS Paragon Plus Environment


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Abstract Microbial contamination remains a major challenge in supply of drinking water in developing regions, despite the continuous advances being made in water purification processes. The spread and transmission of pathogens due to consuming unsafe water culminate in waterborne diseases and increased number of deaths worldwide. Recently, the application of nanotechnology for water purification and in particular, the use of antibacterial nanoparticles (NPs) to control microbial contaminations, received considerable interest. In this study, antibacterial chitosan-silver NPs (CS/AgNPs) were enzymatically grafted on cork matrices to design a water purification point-of-use device. The antibacterial efficiency of the constructed filtering system was further evaluated against severely contaminated with Escherichia coli water (~107 CFU/mL). The system was tested in two operating filtration modes with varied water residence time. The antibacterial nanocomposite decreased the water bacterial contamination by 4 and 5 log CFU/mL when performing a series of continuous short disinfection cycles of 15 min residence time (Experiment I). Nevertheless, a complete bacteria removal was achieved only after increasing the water residence time in the filters up to 8 hours (Experiment II). Durability of the system was demonstrated via performing five disinfection cycles after which the hybrid CS/AgNPs still remained on the cork surface. Importantly, the antibacterial nanocomposite prevented the bacteria attachment and proliferation during all cycles of the disinfection process. Keywords: Silver nanoparticles, Chitosan, Cork, Enzymatic grafting, Water purification

1. Introduction


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Microbiologically unsafe water is among the major morbidity and mortality causes in developing countries. The World Health Organization (WHO) reports that by 2025 half of the world population will be living in water-stressed areas with access only to inadequate drinking water.1 One such example is microbiologically contaminated water associated with transmission of so-called bacteria waterborne diseases such as diarrhoea, cholera, dysentery and typhoid fever. Annually 2.2 million diarrheal disease deaths are being linked with the consumption of contaminated water.2 The concerns about these diseases have even worsened during the last years due to the effects of global warming, extreme rainfall and flooding, which contribute to the inordinate microbial proliferation in surface and groundwater.3,4 Strategies to efficiently recover wastewater acceptable for domestic or recreational purposes are therefore considered of global importance. Boiling is widely used to disinfect household water and reduces most waterborne pathogens.2 However, this method is only efficient for small volumes. Whenever the purification of larger volumes is required, the disinfection is based on chemical treatments, such as with chlorine, chloramines, iodine or ozone.5,6 The most popular, due to its efficiency and low price, chlorination, unfortunately leads also to the formation of by-products as trihalomethanes,7,8 related with developing certain cancers or adverse reproductive outcomes.9,10 Research should be therefore directed towards new and sustainable techniques to disinfect large volumes of water without endangering environment and human health. Nanomaterials, such as silver nanoparticles (AgNPs), are antimicrobials that in low concentrations do not induce harmful side effects.11–14 The antibacterial effect of AgNPs largely depends on their size and morphology, whereas the mechanism of their antibacterial activity still remains a subject of debate. The following three mechanisms



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are proposed in the literature: i) the generation of reactive oxygen species that damage the bacterial cell membrane,15,16, ii) the oxidative dissolution of Ag0 to Ag+ that irreversibly binds to several bacterial molecules crucial for its functioning,17 and/or iii) the perturbation of the bacterial cell wall, ultimately resulting in cell death.18,19 It was also demonstrated that contact killing is the predominant bactericidal route for the AgNPs when immobilized on solid support. In addition, AgNPs immobilized on surfaces display greater efficacy than colloidal AgNPs.20 In line with this and since AgNPs are more efficient bactericidals than many commonly used disinfectants,21 devices containing these nanomaterials are suggested for removal of microorganisms in water treatments22 and purification of drinking water in point-of-use paper-based devices.23 For disinfection purposes, AgNPs immobilization preferably on low-cost materials is necessary, whereas the permanent NPs binding would ensure the disinfection efficiency during long-term exploitation.24 AgNPs impregnated on different materials are applied for water disinfection and biofouling control.25–27 An example of a lowcost material is the residual cork from the wine stopper industry, considered as a source for potentially added-value products, especially for filtering purposes.28 Annually, thousands of tons of cork residues are being generated only in Spain.29 Our previous study provided the proof of concept for the generation of an antimicrobial filter material for water remediation based on residual cork enzymatically embedded with AgNPs capped with chitosan (CS).30 The rationale of this composite design consisted in the oxidative laccase-catalyzed coupling between the natural cork polyphenols and amino functionalities from CS. Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize phenolic compounds into reactive o-


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quinones, which are prone to further undergo non-enzymatic Michael addition and/or Schiff-base coupling reactions with nucleophiles such as amino groups.31,32 The objective of the current study is to validate the developed CS/AgNPs-cork filter material in a point-of-use pilot for continuous water decontamination demonstrating its potential for large-scale water disinfection. To this aim custom made filter cartridges packed with antibacterial CS/AgNPs-cork granules were designed. To evaluate their antibacterial performance, water inoculated with Escherichia coli (E. coli) as an indicator for fecal contamination in water sources, is pumped through the point-of-use filtering system and further investigated for the presence of bacteria.

2. Experimental

2.1. Materials, reagents and bacteria Granulated cork with mean particle size of ∼0.5 cm was provided by the Catalan Cork Institute (Spain). Silver nitrate (AgNO3), sodium borohydride (NaBH4), hydrochloric acid, sodium hydroxide and ethanol from analytical grade were purchased from Sigma-Aldrich (Spain). Laccase (Denilite® II S, 0.125 mg of protein per mg of solid) was provided by Novozymes (Denmark). Laccase activity (0.14 U/mg protein) was determined by oxidation of 5 mM 2,2′-azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS) substrate in 0.1 M succinic acid/succinate buffer (pH 5.0), followed by the absorbance increase at 420 nm and 50 °C, where one unit is defined as the amount of enzyme necessary to oxidize 1 µmol of ABTS per min (ε420 = 36000 M−1 cm−1). The biopolymer used in the study was medical grade CS (∼ 15 kDa, 87 % degree of deacetylation) from Kitozyme (Belgium). Gram-negative E. coli (ATCC 4157, Spanish Type Culture Collection (CECT)), Chromocult agar (Merck KGaA, Germany) 5 ACS Paragon Plus Environment


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or Coliform agar (Sigma-Aldrich, Spain) and tryptic soy broth (TSB, Merck KGaA, Germany) were used for the bacterial cultures studies.

2.2.

Preparation of CS/AgNPs and their immobilization on cork

The synthesis and the enzymatically assisted grafting of CS/AgNPs on cork were carried out as previously described.30 Briefly, for the synthesis of CS/AgNPs, 0.8% (w/v) CS solution was prepared in 1 % CH3COOH and then the pH was adjusted to 5. The synthesis of the hybrid NPs was carried out by chemical reduction of Ag+ to elemental Ag using NaBH4 (17.5 mg/mL in dH2O), starting from aqueous solutions of AgNO3 (5 mg/mL) under vigorous stirring. In the presence of CS the AgNPs are simultaneously capped with the biopolymer. The cork granules were subjected to several cleaning steps before functionalization. The functionalization reaction was carried out in 1:1 ratio of CS/AgNPs suspension to 0.1 M succinic acid/succinate buffer (pH 5) in presence of laccase (final concentration of 0.1 U/mL for each g of cork granules) for 24 h at 50 °C and 30 rpm in a laboratory dying machine Ahiba (Datacolor). For packaging of one filter cartridge 11 g of functionalized cork granules were required. Prior to filling the cartridges, the functionalized cork was washed thoroughly with distilled water to remove the loosely fixed particles and NaBH4 and finally dried at 50 °C for 12 h.

2.3. Characterization of the CS/AgNPs-cork composite The amount of the AgNPs immobilized on the cork surface was determined after extraction with 0.5 M nitric acid. The concentration of solubilized Ag+ ions after extraction from the cork was probed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) using ULTIMA JY2501 (France). The surface morphology of the composites was studied with high resolution environmental scanning electron 6 ACS Paragon Plus Environment

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microscope (HRSEM) model Quanta 200 FEG (FEI, USA). Scanning electron microscopy (SEM) was performed to examine the presence of bacteria on the nonmodified cork (control) and on the CS/AgNPs-cork composite. The micrographs were obtained using a Zeiss Neon FIB microscope (Carl Zeiss, Germany) operating in SEM mode. Additionally, the presence of AgNPs on the cork surface detected by energy dispersive X-ray spectroscopy (EDS). To this end the image was focussed at 20 kV at 10 º angle. The spectra were acquired by Inca software during 60 seconds with 1 K number of channels.

2.4.

Filtering cartridge construction

Each filtering cartridge (Figure S1, Supporting Information) used for the performance of the experiments consisted of a PVC tube (diameter: 3.2 cm, length: 20 cm) filled with cork limited with a polyethylene grid (8.08 cm2) at the upper end, and a geotextile piece (8.08 cm2) and a polyethylene grid (8.08 cm2) at the bottom end. The filtering cartridge was further enclosed by two plugs (diameter: 3.2 cm) joined to a section of polyethylene pipe (diameter: 0.6 cm). The upper pipe (length: 45 cm) was connected to the peristaltic pump (Etatron, ET-PBV4336574), which in turn was connected (pipe length: 35 cm) to the influent water container that was constantly agitated at 250 rpm, whereas the bottom pipe (length: 30 cm) ended to the effluent container. Each filter cartridge had a volume capacity of 160 mL of which 110 mL corresponded to the displacement volume of the cork (active sorbent layer volume) and the remaining 50 mL were filled with the influent water. The packaging step was carried out at the beginning of each assay, and at the end the cork was collected and stored at room temperature for further analysis.



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The filtering cartridges filled with CS/AgNPs-cork composite were incorporated in the filtering system (Figure S2, Supporting Information). To test the disinfection efficacy, the influent flow was split equally to the operating cartridges filled with non-modified cork (control) or with the hybrid CS/AgNPs-cork composite. The cartridges were arranged in a system of three filters (Figure 1) and tested in two experiments - Experiment I and Experiment II. Experiment I was set-up as a demo for the reusability of the filters, while Experiment II was used as a demo of the fastness/efficiency of the disinfection. In both experiments the materials were tested in a single assay, in order to avoid inter-assay variability and ensure reliability of the study. The influent flow rate was kept constant (3.33 mL/min), while the water residence times varied through experiments.

2.5.

Antibacterial performance of the cork material after multiple disinfection

cycles of the same residence time (Experiment I) The disinfection performance of the constructed filters was evaluated with water inoculated with E. coli. To this end, 30 mL of E. coli suspension in TSB corresponding to 109 colony forming units per milliliter (CFU/mL) was inoculated in 3 L sterile deionized water to reach bacterial concentration of ~107 CFU/mL and was then introduced to the cartridges. To determine the disinfection efficiency in terms of antibacterial performance of the hybrid nanocomposite after multiple disinfection cycles, a continuous influent flow (3.33 mL/min) contaminated with E. coli (~107 CFU/mL), corresponding to 0.25 h hydraulic residence time (which is the time needed for the first drop of the water to pass from the inlet to the outlet sides of the constructed filter), was pumped through the cartridges. A total of 5 filtering cycles were performed and 50 mL aliquots from each cycle were taken to determine the viable bacteria count. Finally, the bacterial suspensions (influent and effluent water of


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each filtering cycle) were serially diluted, plated on a Chromocult agar, and then incubated at 37 °C for 24 h to determine the number of survived bacteria. Antimicrobial activity is reported as log10 of CFU/mL surviving bacteria before and after the contact of the inoculated with E. coli water with the CS/AgNPs-cork granules. Cartridges filled with unmodified cork were used as controls. All experiments were carried out in triplicate (system of three filtering cartridges).

2.6.

Determination of the disinfection efficiency in multiple cycles with

increasing residence times (Experiment II) To assess the performance of the system, the contaminated with E. coli (~107 CFU/mL) influent water (50 mL) was pumped (3.33 mL/min) and remained during a certain residence time inside the cartridges packed with the CS/AgNPs-cork composite or with unmodified cork. The flow was then stopped (the outlet for the effluent was blocked in the beginning of the experiment) and remained inside the filter for different residence times. The system effectiveness was determined as the minimum water residence time that ensures complete water disinfection. Each filter was subjected to a series of consecutive disinfection cycles with increasing residence time: 0.5, 2, 4, 8 and 16 hours. The viable bacteria count was determined as described above. To study the viability of the bacteria accumulated on the cork surface, unmodified and functionalized with CS/AgNPs-cork pieces were incubated with E. coli contaminated water for 16 h. Thereafter, the cork samples were washed with sterile distilled water. To grow the alive bacteria, five of the washed cork granules were placed on Coliform agar and incubated at 37 °C for 24 h, while another five granules were used for SEM measurements in order to detect the presence of bacteria on the cork surface. Before the SEM analysis the bacteria accumulated on the cork were



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subjected to fixation procedure preserving their initial structure. Briefly, the samples were placed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min, then washed twice with sterile PBS, and placed in 25, 50, 75 and 96 % ethanol for 10 min. The fixation was conducted at room temperature.

3. Results and discussion Depending on capacity of the water filtering system, there are three types of constructs smaller than large-scale installations, namely, point-of-use, point-of-entry and small-scale systems.33 Point-of-use systems are applicable to treat water volumes consumed daily for drinking, e.g. 2 L per person. Point-of-entry system refers to the treatment of all the water supplied to a household and its treatment capacity is in order of 100-150 L/day. The capacity of the small-scale systems cannot be unequivocally defined, but usually varies between 1000 and 10000 L/day. Our point-of-use system (Figure 1) relies on especially designed filtering cartridges (Figure S1, Supporting Information) which simplicity allows to be up-scaled in higher water capacity systems for disinfection of larger water volumes. Moreover, the hybrid CS/AgNPs-cork material is facile to handle and can be used as disinfection element in already existing water purification systems.


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Figure 1. Experimental set-up consisting of system of three filtering cartridges packed with CS/AgNPs-cork composite or unmodified cork.

3.1. Characterization of the CS/AgNPs-cork composite The antibacterial efficiency of the filtering material and its durability are directly related to the stability of the embedded NPs during use. For example, cellulose acetate hollow fiber membrane loaded with AgNPs for water treatment were active against E. coli and Staphylococcus aureus after being immersed in water bath for 180 days, despite that 60 % of the initially loaded silver remained. However, during filtration the silver content in the hollow fiber was depleted and the material lost its antibacterial activity against both bacteria strains, meaning that silver content must be periodically replenished after permeation.34 We have previously reported that the leaching of AgNPs (coated on paper as a substrate) could be 11 ACS Paragon Plus Environment


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minimized when more uniform coating is obtain.35 The latter was attributed to the better adherence of layers of metallic silver on the primary NP layer due to the continued homogeneous coating. The amount of Ag+ in the effluent water and on the cork surface was determined by ICP-AES after the first and the last cycle of both Experiment I and II (Table 1). The effluent water was probed with and without the addition of nitric acid. The addition of nitric acid is aimed at dissolving the AgNPs, if leached from the surface. The ICPAES values were not influenced by the addition of the acid, meaning that the leached substances are solely Ag+ and not AgNPs. Regarding the amount of Ag remaining on the cork after Experiments I and II, the ICP-AES data showed that the stability of the CS/AgNPs-cork composite during the disinfection cycles was between 73 % and 76 % for the Experiment I and II, respectively (Table 1), which shows a reasonably stability of the coating, considering the long duration of the disinfection cycles performed. The comparable Ag amounts remained on the cork surface studied in two experiments with significantly different overall residence times means that some portion of the Ag was simply physically adsorbed on the cork and not removed by the washings. The Ag ions in the effluent water after the last cycle of Experiment II (16 h residence time) were also probed by ICP-AES. The results showed that the release Ag ions in the water was within the acceptance limit of 0.001 ppm,36 namely 0.00072 ppm.

Table 1. Amount of silver on the cork - initial amount and remaining after the last disinfection cycle - performing Experiment I and II. Experiment I Residence

Number

Ag (initial)

Ag


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time (h)

of cycle

*

(remained) ** 0.084

0.25

5

0.11 (76.4 %)

Experiment II 0.081 0.5h-16h

5

0.11 (73.6 %)

* Ag % wt per 100 g of cork (initially deposited) ** Ag % wt per 100 g of cork (remained after performing the cycles) HRSEM analysis of the samples from Experiment I and II was carried out to confirm the presence of hybrid NPs on the cork surface before filtration and after the last disinfection cycle. Both single hybrid NPs and larger particle agglomerates could be detected (Figure 2a, b, c, and d). The dense layer of spherical in shape CS/AgNPs observed before the disinfection cycles (Figure 2a and b) illustrates an efficient enzymatically-promoted deposition of the hybrid nanomaterial on the cork surface. The size of the individual NPs within the agglomerates ranged from 20 to 80 nm. Moreover, the EDS analysis of the nanocomposites detected the presence of Ag on the surface, thereby confirming their successful deposition (Figure S3, Supporting Information). In order to confirm the stability of the obtained antimicrobial hybrids during the filter exploitation, the surface morphology of the samples was analysed after the last disinfection cycle of each experiment set-up. The HRSEM images of the cork granules used in Experiment I (Figure 2c and d) showed that their surface remained covered with hybrid CS/AgNPs after the completion of 5 filtering cycles. This observation



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supports the enzymatic grafting of CS from the AgNPs shells to the phenolic moieties in the cork matrix, resulting in a stable hybrid material suitable for continuous water disinfection systems.30 The surface of the samples used is Experiment II also remained densely covered with NPs even after performing a series of disinfection cycles with increasing up to 16 h residence time (Figure 2e and f).

Figure 2. HRSEM micrographs of CS/AgNPs-cork composites before (a, b) and after 5 disinfection cycles (c, d; 0.25 h hydraulic retention time in each cycle, Experiment I); and after performing a series of consecutive disinfection cycles (e, f) with increasing residence time (0.5, 2, 4, 8 and 16 hours) and without changing the filter


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(Experiment II). The images on the left were taken with 10000×, whereas the rightside images were taken with 50000× magnification. Interestingly, the presence of large structures, not observed on the cork samples from Experiment I, was also evidenced. Considering the long residence time (16 h) of the contaminated with bacteria influent water in Experiment II, it is reasonable to speculate that these structures are actually E. coli accumulated on the cork surface. To confirm this, we performed an additional experiment, placing unmodified and functionalized cork granules in contact with E. coli suspension for 16 h. Afterwards their surface morphology was studied by SEM (Figure 3). The SEM analysis confirmed the presence of E. coli on the cork surface (Figure 3). However, the morphology of the bacteria found on the untreated cork and on the nanocomposite was somewhat different. Proliferating bacteria with well-formed flagella were observed on the surface of the untreated cork (Figure 3a, b), whereas the E. coli found on the CS/AgNPs-cork granules were not flagellated (Figure 3c, d). Apparently, the hybrid nanocomposite affected the bacteria structure and could prevent their attachment and proliferation on the surface. More bacteria were attached on the surface of the unmodified cork than on the surface of the CS/AgNPs-cork composite. The loss of flagella in E. coli after water disinfection with TiO2-based photocatalyst was previously reported.37 The presence or absence of flagella is important to bacterial survival and growth, since these appendages are not only driving cell locomotion, but also allow the bacteria to attach to surfaces or to epithelial cells.38 It is well established that flagellar-mediated motility and the ability to produce a number of pili are essential also for biofilm formation39 and contribute to the virulence of pathogenic bacteria.40 Moreover, in a recent study AgNPs decorated microspheres were demonstrated to prevent the formation of biofilms due to improved antifouling 15 ACS Paragon Plus Environment


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properties of the developed antibacterial composite.41 Thus, the designed herein nanocopmposite could prevent the formation of biofilms and as a consequence, to limit the bacteria spread and transmission. As a next step, the viability of the bacteria accumulated on the cork surface was tested by placing the cork pieces on Coliform agar plates specific for E. coli. The results showed that the flagellated bacteria observed on Figure 3a and b on the surface of the unmodified cork samples were viable, while bacteria growth was not observed for the CS/AgNPs–cork samples on which surface bacteria were without flagella adhered (Figure 4d). The bacteria found after the 7th disinfection cycle in Experiment II (Figure 2e, f) were not viable, demonstrating the disinfection potential of the designed nanocomposite material.

a

b

c

d

Figure 3. SEM images of E. coli accumulated on the surface of unmodified cork (a, b) and on the CS/AgNPs-cork composite (c, d) after 16 h of contact with E. coli


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suspension. The images on the left were taken with 10000×, whereas the right-side images were taken with 100000× magnification.

Figure 4. Images of cork granules after incubation in sterile distilled water (a) and water inoculated with E. coli (b); and CS/AgNPs-cork composite after incubation in sterile distilled water (c) and water inoculated with E. coli (d) for 16 h at room temperature. The images b and d are related to Figure 3a, b and Figure 3c, d, respectively.

3.2.

Antibacterial performance of the CS/AgNPs–cork hybrids after multiple

disinfection cycles (Experiment I) The verification of the microbial water quality could be based on the analysis of fecal indicator microorganisms. The presence of E. coli provides conclusive evidence of recent fecal pollution and should not be present in drinking water.2 The bacteria concentrations reported in such studies vary from 109 CFU/mL42–44 down to 103 CFU/mL.45 The disinfection efficiency of the cartridges filled with the CS/AgNPscork composite was herein evaluated against ~107 CFU/mL E. coli, corresponding to a highly contaminated fecal water. 17 ACS Paragon Plus Environment


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To find out the disinfection sustainability of the system during exploitation when applying multiple short cycles with the same duration, the antibacterial efficacy of the hybrid filter material was studied during 5 continuous filtering cycles (Experiment I). No reduction in the bacterial count was observed for the cartridges filled with unmodified cork (Figure 5). In contrast, the cartridges filled with the CS/AgNPs-cork composite decreased the bacterial contamination of water by 4 and 5 log CFU/mL without significant variations between the cycles. Indeed, the performance slightly decreased after 4 filtering cycles using the same cartridge. As suggested by the ICPAES findings (Table 1), the dissolution of the physically adsorbed AgNPs from the surface of cork may have affected the antibacterial performance of the material.

Figure 5. Bacterial load of the effluent water circulated through non-modified cork (control, black bars) and through the hybrid CS/AgNPs-cork composite (grey bars) during 5 continuous filtering cycles of 0.25 h residence time each. The initial E. coli count for the contaminated water was ~107 CFU/mL. Different studies on AgNPs-coated disinfection materials reported a significant decrease in bacteria viability (E. coli and Enterococcus faecalis) between 51% and 78% when AgNPs were incorporated on bentonite,46 while the viability dropped by


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99% when incorporated in carbon foams.47 In our study, to facilitate the overview on the efficiency, the reduction in bacteria viability for Experiment I was also calculated in percentage (Table 2). The bacteria inhibition in all cycles was above 99%. Table 2. Reduction in bacterial viability (Experiment I) Number of disinfection cycle 1

2

3

4

5

Reduction in bacteria viability (%) 99,99

99,99

3.3.

99,99

99,99

99,99

Determination of the disinfection efficiency at increasing residence

times (Experiment II) Since the objective of zero E. coli per 100 mL of water is the goal for all water supplies, even in cases of emergencies,2 and this requirement was not met by the Experiment I, we further determined the minimum residence time needed to achieve such objective. A series of experiments with increasing residence time up to 16 h were performed (Experiment II) and the bacterial count in the contaminated with E. coli water was evaluated. The cartridges filled with CS/AgNPs-modified cork decreased the initial E. coli count (~ 107 CFU/mL) by 4 and 5 log, during the first 3 cycles with 0.5, 2 and 4 h residence time (Figure 6), which corresponds to more than 99% reduction in bacteria viability (Table 3). In contrast, a complete bacterial removal was achieved after 8 h residence time with the modified cork (Figure 6 and Table 3). The antibacterial efficiency of the CS/AgNPs-modified cork is thus a function of the water residence time, reaching a total bacterial removal in 8 h. Operating for appropriate



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water residence time, the developed in this study filtering system was suitable for water disinfection.

Figure 6. Bacterial load of the effluent water circulated through the unmodified cork (control, black bars) and through the hybrid Ag/CS NPs-cork (grey bars) after 1 to 7 filtering cycles with different residence time (0.5, 2, 4, 8 and 16 h). The initial E. coli count for the contaminated water was ~107 CFU/mL.

Table 3. Reduction in bacterial viability (Experiment II) Residence time (hours) 0.5

2

4

8

16

Reduction in bacteria viability (%) 99,99

99,99

99,96

100

100

4. Conclusions Supplying microbiologically safe water in the developing countries is of paramount importance. Building on our previous investigation, we up-scaled an efficient point-ofuse water filtering system implementing cartridges filled with cork granules


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enzymatically functionalized with hybrid CS/AgNPs composite. The antibacterial potential of the novel water purification device was demonstrated under two different continuous operating filtration regimes. The durability of the antibacterial effect was first proven by maintaining the disinfection performance in a series of short disinfection cycles treating E. coli contaminated water. A complete bacterial removal was achieved within 8 h under the filtration operation mode consisting of cycles with gradually increased water residence time. The cork granules remained homogeneously covered with the hybrid NPs even after repeated disinfection cycles with long residence time. The antibacterial surface nano-modified cork filtration medium affected the structure of E. coli preventing its attachment and proliferation, and thus demonstrating the disinfection efficacy of the designed natural-based system.

Supporting Information Details of the filter cartridge design and EDS spectrum of the nanocomposite are described in Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements This work was financially supported by two European projects: REAGRITECH (LIFE11 ENV/ES/000579) and ECORKWASTE (LIFE14 ENV/ES/000460). The authors thank to the Institut Català del Suro (ICSURO) for providing the cork material used in the study. Lina Vanesa Garcia Peña acknowledge the Grupo de Investigación en Ciencias e Ingeniería en Sistemas Ambientales (Universidad del Cauca) for financing her internship. Petya Petkova thanks the Spanish Ministerio de Educación, Cultura y Deporte (MECD) for the PhD grant FPU12/06258.



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TOC:

Inoculated water

Alive bacteria

Dead bacteria

Filtered water Outlet tank


Hybrid ChitosanâSilver Nanoparticles ... - ACS Publications

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