Effective Bacterial Inactivation and Removal of Copper by Porous

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Effective bacterial inactivation and removal of copper by porous ceramics with high surface area Tanja Yvonne Klein, Julia Wehling, Laura Treccani, and Kurosch Rezwan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es3045828 • Publication Date (Web): 30 Dec 2012 Downloaded from http://pubs.acs.org on January 1, 2013

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Effective bacterial inactivation and removal of copper by porous ceramics with high surface area Tanja Yvonne Klein, Julia Wehling, Laura Treccani and Kurosch Rezwan*, Advanced Ceramics, University of Bremen, Germany. *Corresponding author – mail: [email protected], Tel. +49 (0)421 218 – 64930, Fax. +49 (0)421 218 – 64932

Abstract In this study, we present porous ceramics combining the antibacterial effect of copper with an integrated copper removal adsorbent. After preparing and characterizing the antibacterial copper-doped microbeads and monoliths (CuBs and CuMs), their antibacterial efficiency is probed against different non-pathogenic and pathogenic bacteria (Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa). An antibacterial efficiency of 100% is reached within 15 min to 3 h for all tested strains under static conditions. Dynamic tests with B. subtilis and E. coli showed high antibacterial efficiency up to 99.93% even at continuous flux. To avoid any adverse effects on the environment, continuous removal of released copper-ions is accomplished with porous, high surface area monolithic adsorbents (MAds). MAds are prepared similarly to the CuMs but without adding copper during the manufacturing process. MAds reduce the amount of copper released from the CuMs ≥ 99% during the first 15 min, ≥ 90 % up to 2 h, and after 22 h of continuous filtration up to 56% of the released copper is removed.

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Introduction Pathogenic bacteria pose a permanent sanitary risk, but the versatility and adaptability of nature makes it challenging to find the ideal antibacterial agent against all types of harmful bacteria. Human warfare against pathogenic bacteria knows numerous weapons like antibiotics, microfiltration, antibacterial molecules/enzymes or metals‒each with its own pros and cons.1-3 Amongst the antibacterial metals the use of copper (Cu) has a long tradition and even ancient civilizations benefited from its excellent antimicrobial effects.4 Nowadays, bacterial inactivation with Cu covers a wide spectrum including Cu-surfaces, Cunanoparticles or release of Cu-ions. Grass et al.5, for example, gave a comprehensive overview on the effect of Cu-surfaces against various microorganisms. Copper oxide nanoparticles to be used against waterborne bacteria were prepared from Pandey et al.6 Abou Neel et al.7 prepared antibacterial Cu-releasing, phosphate-based glass fibers for wound healing applications. Copper is known to inactivate even antibiotics resistant bacteria species like certain strains of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) or Escherichia coli (E. coli) which are, for example, main triggers for nosocomial infections.4, viruses and fungi.9,

10

8

Besides bacteria, Cu is also effective against different types of

Copper toxicity is dose dependent, in low amounts it acts as an

indispensable trace element important for growth and reproduction of many organisms. An adult human needs about 1.0 to 1.5 mg Cu each day.11 Copper is also known to have a positive effect on the angiogenesis process.12 and was used by Erol et al.13 to induce glassbased scaffolds for bone tissue engineering. Although Cu is an important trace element and an extremely powerful antimicrobial agent, one has to consider possible adverse effects on the environment that may occur by the use of Cu-ions for antibacterial purposes.14-16 Hence, for water filtration excessive Cu-ions need to be removed after killing undesirable germs. There are numerous often elaborated approaches for Cu-ion removal from water. Cu-ion adsorption can be accomplished, for example, by zeolites functionalized with metallothioneins17 or metal ion chelation polymers 18. Ganesh et al.19 also removed Cu-ions and nanocopper via activated sludge biomass. 2 ACS Paragon Plus Environment


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Previously20, we reported on the manufacturing of porous, high surface area ceramic adsorbents for purification technologies. Adsorbents, single microbeads or highly porous monoliths (MAds) were prepared by ionotropic gelation using alginate a natural, nontoxic, high-molecular weight polymer as template material. Ionotropic gelation uses Me2+ (Me2+ = Ca2+, Cu2+, etc.) as cross-linking agents. MAds were obtained by 3-dimensional alignment of wet microbeads without any additional binders. In the present study, we adapt this simple but extremely versatile technique to produce antibacterial Cu-doped ceramic microbeads (CuBs) and monoliths (CuMs). To avoid any possibly negative effects on the environment, we furthermore show that excessive Cu can easily be removed from an aqueous solution using MAds.

Experimental section Materials Porous microbeads and monoliths were fabricated using α-alumina (d50 150±8 nm, Taimei Taimicron TM-DAR, lot. 8182, Krahn Chemie, Hamburg, Germany) and silica sol (d50 7±1 nm, BINDZIL®30/220, NH3-stabilized, Akzo Nobel, Leverkusen, Germany). The d50 values have been determined by dynamic light scattering (UPA 150; Microtrac Inc., Largo, FL). Sodium alginate (lot. 8S005427, AppliChem, Darmstadt, Germany) and tri-sodium citrate dihydrate (lot. 9Z006552, AppliChem) were used as gelling agent and cross-linking moderator, respectively. Finally, the cross-linking solution was prepared from copper chloride dihydrate (lot. 1S001111, AppliChem) or calcium chloride dihydrate (lot. 0001394393, Fluka, Buchs, Switzerland) and ethanol (Ph. Eur., lot. 10E170502, VWR, Hannover, Germany) in double deionised water, conductivity 0.1 mS/cm (ddH2O, Synergy system, Millipore corp., Schwalbach, Germany). Copper-ion release was tested in different media: ddH2O, ddH2O with adjusted pH (pH 7.4)‒ adjusted with ammonia (lot. SZBA1400, Sigma Aldrich, Steinheim, Germany), and


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tris(hydroxymethyl)aminomethane, pH 7.4 (Tris-HCl, lot. MKBD9221V, Sigma Aldrich). All chemicals were used as received.

Preparation of porous ceramics Porous Cu-doped ceramics were prepared via ionotropic gelation of sodium alginate, as described elsewhere.20 In brief, a suspension containing Na-alginate, silica sol, citrate and alumina was added dropwise to the cross-linking solution, containing 0.1 mol/L copper chloride in water mixed with ethanol (80/20–v/v). After cross-linking, the resulting microbeads were rinsed with water to remove excessive Cu. Finally, single CuBs were obtained by freeze-drying the synthesized wet beads at -20°C (P8K-E-80-4 -80°C, Piatkowski, Munich, Germany). CuBs were poured into molds and dried at room temperature to prepare CuMs. For the Cu removal experiments MAds were prepared in the same manner as the CuMs but instead of copper chloride, the cross-linking solution contained calcium chloride dihydrate. CuBs, CuMs and MAds were subsequently sintered at Tsinter= 900°C or 1000°C, heating rate 120 K/h, dwell time 2 h (C42, Nabertherm, Lilienthal, Germany).

Characterization X-Ray powder diffraction (XRD, X'Pert Pro, PANalytical GmbH, Kassel, Germany), energy dispersive X-Ray spectroscopy (EDS, Gemini Supra 40, Zeiss, Oberkochen, Germany) and ICP-AES analysis (Optima 3300, Perkin Elmer, MA, USA) provide information on CuBs composition. The specific surface area (SBET) of sintered and non-sintered samples was determined by nitrogen adsorption (Belsorp-Mini, Bel Japan, Osaka, Japan) using the BETmethod.21 Densities were determined by helium-pycnometrie (AccuPyc 1330, Micomeritics, Aachen, Germany). The zeta-potential (ζ-pot) of sintered CuBs/CuMs and MAds was obtained by streaming potential measurements (SurPASS, Anton Paar GmbH, Ostfildern, Germany), using 1 mmol KCl as electrolyte. The ζ-pot was measured for single microbeads instead of CuMs and MAds due to the instrumental setup. However, preliminary tests (data not shown) indicate that microbeads and monoliths behave likewise, for example, with regard 4 ACS Paragon Plus Environment


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to SBET and surface morphology. Therefore, microbeads ζ-pot results shall be transferable to MAds and CuMs. 3-D images were obtained by computer tomography (phoenix nanotom m, GE Measurement & Control, Wunstorf, Germany). Additionally, the surface morphology of microbeads and monoliths was monitored via scanning electron microscopy (SEM, CamScan and Gemini Supra 40).

Copper release Static copper release was determined after continuous shaking of 1 g sintered CuBs in 10 mL of the test medium at room temperature. Tested media were double deionized water, pH 6.1, conductivity 0.1 mS/m; ddH2O with an adjusted pH of 7.4, conductivity 1.9 mS/m; and Tris-HCl buffer (which was also used as buffer for the bacteria tests), pH 7.4, conductivity 75.7 mS/m. For the dynamic copper release tests one CuM was fixed between two vitreous hose connectors and sealed with a head shrink tubing, respectively (BP105, BIT Bierther, Swisttal-Heimerzheim, Germany). Then 10 mM Tris-HCl buffer was pumped through the monolith using a peristaltic pump (BVB Standard, Ismatec, Germany), flux 25.0±1 L/m²*h. Copper concentrations of supernatant and eluate were photometrically determined (Xion 500, HACH LANGE, Düsseldorf, Germany) via cuvette tests (LCK 329, HACH LANGE, detection range 0.1–8.0 mg/L). The test utilizes bathocuproin disulfonic acid disodium salt to form an orange complex with Cu-ions.

Copper removal Removal tests were carried out to test the general suitability of MAds to adsorb Cu-ions (Cu) that were previously released from a CuM in a dual filter system. Copper removal was tested in two ways i) with a constant Cu-feed concentration, and ii) under release conditions with declining Cu concentrations. For the constant feed tests first Cu-ions were released from a CuM by pumping 10 mM Tris-HCl buffer through the monolith using a peristaltic pump, flux 25.0±1 L/m²*h. Tris-HCl mimicked presence of other ions in an aqueous solution which might 5 ACS Paragon Plus Environment

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influence Cu-ion adsorption. The collected eluate was diluted to a final concentration of 1.3 mg/L, in accordance with the maximum United States Environmental Protection Agency (USEPA)22 guideline value. The eluate was subsequently filtered by a high surface area MAd, flux 25.0±1 L/m²*h. Additionally, one CuM was directly combined with two MAds to test Cu removal under release conditions. Dynamic Cu-ion removal by MAds was also tested in presence of B. subtilis (flux 12.5±1 L/m²*h). Copper concentrations of eluate and filtrate were photometrically measured corresponding to the copper release tests.

Antibacterial performance tests Prior to the bacteria tests, sintered CuBs and CuMs Tsinter= 900°C and MAds Tsinter= 1000°C were heat sterilized at 180°C for 3 h (L3/11/S27, Nabertherm, Lilienthal, Germany). Static tests were carried out with 4 different bacteria strains. Non-pathogenic B. subtilis (DSM 1088, Gram-positive) and E. coli, K 12 (DSM 1077, Gram-negative) and human-pathogenic S. aureus (DSM 1104, Gram-positive) and Pseudomonas aeruginosa (P. aeruginosa, DSM 1117, Gram-negative). Additionally, dynamic tests were carried out with B. subtilis and E. coli. Bacteria from a pre-culture were grown overnight in Luria Bertani medium at 37°C under gentle shaking. Subsequently, the bacteria were harvested by centrifugation at 3000 x g for 10 min and washed two times with 10 mM Tris-HCl buffer. Then the cell pellets were resuspended in fresh buffer and diluted to an optical density OD600 of 0.1 corresponding to a cell concentration of approx. 108/mL-according to McFarland standards. Afterwards, 10 mL of each bacteria suspension were added to 1 or 2 g of CuBs for the static tests. Samples were incubated at room temperature under continuous shaking. The number of colony forming units (cfu) in the supernatant was determined via Aerobic-Count-Petrifilm TM-tests (Type 06400, 3M, Neuss, Germany) samples for plating were withdrawn after incubation times of 15 min, 1 h and 3 h, respectively. The PetrifilmsTM were incubated for one day at 37°C prior to scoring the number of cfu’s.



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For the dynamic tests a suspension containing either B. subtilis or E. coli was pumped through the CuMs (flux 12.5±1 L/m²*h) in analogy to the dynamic copper release tests with sampling at t = 0 min, 15 min, 30 min, 1 h and 3 h. To verify whether the decline in bacteria concentration is an effect of the present Cu, additional tests with MAds were performed. In order to test the reusability of copper-doped ceramics 10 mL of B. subtilis suspension were exposed to 1 g of sintered CuBs for 15 min. After each cycle the beads were washed 2 times with buffer followed by heat sterilization. The test was repeated for 3 cycles.

Results and discussion Characterization of CuBs XRD patterns (fig. 1a) of sintered CuBs show mainly α-alumina (corundum) reflexes. The increased background observed for small angles up to approx. 35°2θ results from the presence of X-ray amorphous silica. Silica crystallization (cristobalite formation) within CuBs starts at a sintering temperature of 1000°C. Neither for Tsinter= 900°C nor for 1000°C any Cucontaining phases are found, indicating that Cu forms no new crystalline phases with alumina or silica. Figure 1b depicts microbeads with and without copper. Microbeads without copper are plain white. Incorporation of Cu changes the color to turquois for non-sintered microbeads. During sintering the CuBs become greyish-brown, which will be due to the reduction of Cu2+ to Cu+ or Cu.23 Furthermore, the presence of Cu appears to accelerate sintering. Porosity and SBET of CuBs sintered at Tsinter = 900°C are comparable to the results of microbeads without Cu obtained at Tsinter= 1000°C (table 1). According to EDS-analysis, the surface of CuBs cross-linked in 0.1 mol/L Cu-solution contains about 1.9 mass-% of Cu (fig. 1c and table S.1 supplemental data). EDS-spectra of the inner core taken from a fracture surface indicate the presence of a Cu gradient with declining Cu concentrations towards the CuB center. This Cu gradient results from the ionotropic gelation process where cross-linking proceeds from the outside. Determination of the exact Cu allocation via EDS for CuBs crosslinked in 0.1 mol/L Cu-solution was not possible, as the Cu concentration in the inner core turned out to be below the EDS detection limit, which is approx. 2.0 mass-%. Finally, we 7 ACS Paragon Plus Environment

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used ICP-AES analysis (table 1) to reveal CuBs total Cu content. According to ICP-AES sintered and non-sintered CuBs contain 9.2-9.7 mg/g of Cu. Besides Cu-concentration, the electrostatic interactions between a charged filter surface and a charged pollutant play an important role in filtration and adsorption processes.24 Therefore, we characterized the electric potentials close to the surface via ζ-pot-measurements. The ζpot curves for microbeads with and without Cu-doping are rather similar (fig. 2). For both the isoelectric point (IEP)25 is located at about pH 2.1 resulting in a negatively charged surface at the bacteria testing conditions at pH 7.4. The microbeads consist of α-alumina combined with silica nanoparticles. For silica the IEP ranges from pH 1.5-3.0 whereas for α-alumina IEPs between pH 8.0 and 9.2 are reported.26 Hence, the present silica will predominantly govern MAds surface properties.

Copper release and removal Static tests: Figure 3 depicts the static Cu release of CuBs sintered at Tsinter= 900 and 1000°C, monitored for 10 d. The results of the release tests indicate Cu release of CuBs being a function of sintering temperature, pH and ionic-strength, and thereby adjustable. CuBs sintered at 900°C release a maximum of 0.01 mg/g after 10 d in double deionized water at pH 6.1. When pH and conductivity increase to 7.4 and 1.9 mS/m, respectively, the release increases to 0.03 mg/g, while in Tris-HCl at a conductivity of 75.7 mS/m and at the same pH 0.18 mg/g are released. For CuBs sintered at 1000°C (fig. 3b) the overall Cu release is less than for CuBs sintered at 900°C. In Tris-HCl, for instance, Cu release is only 0.12 mg/g after 10 d. During our tests, the ionic-strength was found to play an important role in Cu release. The distinguished effect of ionic strength on Cu-ion release was also observed by FernándezCalviño et al.27 who investigated Cu-ion release from soil at different pH and ionic strengths. However, in all cases the amount of Cu released from the CuBs during the tests is at least an order of magnitude below the World Health Organization (WHO)28 and US-EPA22 guideline values for Cu-concentrations in drinking water, which are 2.0 mg/L and 1.3 mg/L, 8 ACS Paragon Plus Environment


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respectively. No significant changes in CuBs surface morphology were observed after exposure to the different media (inset in fig. 3a and fig. S.1 supplemental data), indicating that CuBs are stable in the tested media for at least 10 d.

Dynamic tests: Figure 4 depicts examples of monoliths used for the dynamic Cu release/removal and bacteria inactivation tests. Single microbeads are about 300-700 µm in size. 3-dimensional assembly results in a highly open porous filter structure with pores inbetween the beads with average diameters ≥ 100 µm. Most bacteria are distinctly smaller than 10 µm.29 Due to the much larger pore channels risk of pore clogging during a filtering process will therefore be moderate. Figure 5a gives a schematic overview on the experimental setup used for the dynamic copper release and removal tests. Continuous Cu release was monitored for CuMs sintered at Tsinter= 900°C and 1000°C in Tris-HCl (fig. 5b). Under dynamic conditions CuMs sintered at Tsinter= 900°C release slightly higher amounts of Cu than CuMs sintered at Tsinter= 1000°C which is in accordance to the statically tests. Cu-release has its maximum during the first h for both sintering temperatures. Cu-release reaches a “quasi-plateau” after 1 h where it is almost constant up to at least 8 h. About 8.8±0.7% of CuMs total Cu-content are released after 22 h of continuous operation.

Although static and dynamic Cu-release tests show that the amount of Cu-ions released from CuBs/CuMs is rather low, one still has to consider possible accumulation effects in case larger amounts of CuBs/CuMs are used. To avoid any possibly detrimental effects, free Cuions should therefore be removed as a precaution after inactivating the bacteria. Figure 5c and d depict the results of the continuous removal tests where MAds were combined with CuMs. MAds reduced the amount of Cu in the eluate by a multiple, at t=0 min > 99% are removed and even after 22 h the removal rate of the released Cu is still 56%.


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Presence of B. subtilis slightly reduces Cu-adsorption on MAds (fig. 5d), at t=0 min only about 96% of the released Cu were removed. Bacteria can interact with any surface (cp. antibacterial efficiency section). Probably a small volume of B. subtilis either permanently or temporarily adsorbs to the surface of MAds. In this way, bacteria would “shield” parts of the surface, reducing Cu adsorption. However, the released Cu is still removed to a great extent and adjustment of the filtration conditions, for example, an increase in the filtration bed length should improve Cu removal in the presence of bacteria.

During the constant feed test, MAds drastically reduced the initial Cu concentration of 1.3 mg/L. Removal was 100% in the beginning, and after 5.7 h of continuous filtration still up to 68.8% was removed (fig. S.2 supplemental information). The amount of Cu adsorbed to the MAds corresponds to an adsorption of ~0.1 mg/g. Assuming adsorption of a single, uniformly distributed Cu-ion layer and a random close packing (64%)30 for the Cu-ions, the theoretic maximum adsorption capacity of 1 g MAds would be approximately 245 mg/g for Cu2+ and 376 mg/g for Cu+. Further investigations are needed to evaluate the influence of pH, dwell time or filter surface area as well as presence of different ions like Mg+, Ca2+, Pb2+, etc., biomolecules (proteins, drug-residues, etc.) or bacteria on MAds adsorption behavior. However, our adsorption tests strongly suggest that MAds are well suitable for Cu-ion removal in a dual filter system. In addition, preliminary tests (table S.3 and fig. S.3, supplemental information) indicate that Cu-soaked MAds are supposedly reusable to serve as antibacterial filter themselves.

Antibacterial efficiency Static tests: Figure 6 shows the decrease of active bacteria in contact with 1 and 2 g of CuBs for 3 different incubation times. An efficient antibacterial agent for drinking water disinfection needs to provide a reduction of living bacteria of log10 2 after 10 min contact time and log10 4 after 25 min,31 corresponding to an antibacterial efficiency of 99% and 99.99%, respectively. 10 ACS Paragon Plus Environment


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One g of CuBs reaches an antibacterial efficiency of 100% for B. subtilis (fig. 6a, table S.2 supplemental information) within 15 min, compared to Cu-free microbeads where only 3% of B. subtilis died at the same conditions during a preliminary test. S. aureus behaves rather similar to B. subtilis (fig. 6c). The antibacterial efficiency was 100% for all samples except for 1 g of CuBs, where after 15 min few bacteria survived in the supernatant. One g of CuBs in contact with E. coli and P. aeruginosa needed incubation times of 3 h to reach an antibacterial efficiency of 100%, while for 2 g only after 15 min living bacteria were observed in the supernatant (fig. 6b and d). Nevertheless, 1 g CuBs still reaches an antibacterial efficiency of 99.82% after 15 min for E. coli and 99.47% for P. aeruginosa after 1h.

At our test conditions, Gram-positive B. subtilis and S. aureus died faster in contact with the CuBs than Gram-negative E. coli and P. aeruginosa, suggesting that Gram-positive bacteria might be more sensitive towards Cu. Yet, the precise impact of Cu on bacteria metabolism is still unknown. Currently, there are several modes of action under debate. However, there seems to be no toxic mode of action that is true for all bacteria.32-35 In addition, there are huge variations even within one bacteria species. Many E. coli strains, for example, die in contact with Cu5, whereby the minimum inhibition concentration (MIC) needed is variable. Ruparelia et al.36 report MICs between 0.04 and 0.18 mg/mL for different E. coli strains. And there are also some strains found to be resistant against Cu.37 Basically, Cu can act on bacteria in two different ways: i) by contact of a bacterium to a Cu containing surface or ii) by uptake of Cu-ions released from the surface.38,

39

During the

incubation time, the test tubes were therefore shaken continuously, enabling release of Cuions as well as permanent contact of beads and bacteria. CuBs contain an average of 9.4 mg Cu per g (table 1) with the highest Cu concentrations found in the outer regions of the CuB and the CuB surface. Parts of the present Cu are released, but according to the static release tests, about 99 % remain within the beads after the maximum incubation time used for the bacteria tests. Therefore, we presume that for CuBs both contact mechanisms (bacteria inactivation by released Cu-ions and at the same time inactivation of the bacteria 11 ACS Paragon Plus Environment

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caused by direct contact with the Cu-rich CuB surface) or rather a combination of both is possible. In either way, the synthesized CuBs have proven to be highly efficient against all tested bacteria strains. The CuBs will also most likely be effective towards other noisome bacteria, that are reported to be sensitive against Cu like Salmonella, Listeria5 or several multidrug resistant Burkholderia species40. In contrast to other antibacterial materials, where organic molecules are utilized like immobilized proteins41 or enzymes42, CuBs can easily be recycled by heat sterilization. During the reusability tests CuBs antibacterial efficiency against B. subtilis was 100%, 100% and 99.999% after 15 min for the first, second and third cycle, respectively.

Dynamic tests: The results of the static tests affirm CuBs high antibacterial efficiency. However, a constant filtration process will decrease bacteria’s contact time by a multiple. Therefore, we performed additional filtration tests to show CuMs suitability for use in a continuous filter system. CuMs reduced the amount of living B. subtilis up to 99.93% during 3 h of continuous filtration (fig. 7a and table S.3 supplemental information). For E. coli the antibacterial efficiency decreased from 99.29% at t=0 min to 92.57% at t=3 h (fig. 7b). These results have the same tendency as the static tests, where tested Gram-positive bacteria were slightly more sensitive towards CuBs than the Gram-negative ones. Reference tests with MAds also yield a slight reduction of living bacteria. MAds antibacterial effect against B. subtilis, for example, is 15.98% at t=0 min, the value decreases to 4.89% after t=3 h. Yet, MAds antibacterial efficiency is 6-20-times (for B. subtilis) and 5-33-times (for E. coli) smaller than CuMs efficiency. Bacteria interactions with materials are very complex and can be influenced by various factors

such

as

surface

charge,

hydrophobic/hydrophilic

interactions

or

surface

heterogeneities.43, 44 Therefore, bacteria reduction by CuBs/CuMs or MAds could possibly be attributed to adsorption to the ceramic surface. The chemical surface properties of 12 ACS Paragon Plus Environment


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CuBs/CuMs and MAds are governed by the present silica (cp. characterization section). Silica as a rather hydrophilic surface is regarded to be less susceptible to bacterial adhesion than hydrophobic ones like polymers.44 All samples are negatively charged at our test conditions (fig. 2). Bacteria are generally negatively charged at neutral pH values, too.29, 45 For E. coli and B. subtilis, for example, IEP´s between pH 1.5 and 2.1 are reported.45, 46 Yet, one cannot exclude presence of surface inhomogeneities like the local accumulation of positively charged Cu (in CuBs/CuMs) or Ca-ions (in MAds). Such spots could enable bacterial adhesion to a certain extend44 and thus be responsible for the observed antibacterial effect of Cu-free MAds. However, MAds and CuBs/CuMs are virtually identical with regard to surface charge, morphology and SBET, but CuMs effect on bacteria viability is distinctly larger than the effect of MAds. Therefore, the reduction of living bacteria will predominantly be due to bacteria inactivation by the incorporated Cu.

Acknowledgements The authors thank the European Research Council for financial support of this study (project BiocerEng number 205509), and the DFG research training group PoreNet is thankfully acknowledged. Furthermore, we thank Prof. Dr. Dieter Reinscheid, from the Bonn-RhineSieg University of Applied Sciences, for the opportunity to carry out the S2 bacteria tests. We also thank Dr. Johannes Birkenstock, Dr. Maria Jose Ruiz Chancho and Petra Witte from the University of Bremen for carrying out the XRD, ICP-OES and the EDS measurements and Sabine Schulte for proofreading the manuscript. The CT-image was obtained from GE Measurement & Control, Wundstorf, Germany.

Supporting Information available Supplemental figures S.1 - S.3 and table T.1 – T.3, as mentioned in the text. This information is available free of charge via the Internet at http://pubs.acs.org/.


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References 1.

Kroll, S.; Treccani, L.; Rezwan, K.; Grathwohl, G., Development and characterisation of functionalised ceramic microtubes for bacteria filtration. Journal of Membrane Science 2010, 365, (1-2), 447-455.

2.

Martinez, J. L., Environmental pollution by antibiotics and by antibiotic resistance determinants. Environmental Pollution 2009, 157, (11), 2893-2902.

3.

Malachová, K.; Praus, P.; Rybková, Z.; Kozák, O., Antibacterial and antifungal activities of silver, copper and zinc montmorillonites. Applied Clay Science 2011, 53, (4), 642-645.

4.

Gould, S.; Fielder, M.; Kelly, A.; Morgan, M.; Kenny, J.; Naughton, D., The antimicrobial properties of copper surfaces against a range of important nosocomial pathogens. Annals of Microbiology 2009, 59, (1), 151-156.

5.

Grass, G.; Rensing, C.; Solioz, M., Metallic Copper as an Antimicrobial Surface. Applied and Environmental Microbiology 2011, 77, (5), 1541-1547.

6.

Pandey, P.; Merwyn, S.; Agarwal, G. S.; Tripathi, B. K.; Pant, S. C., Electrochemical synthesis of multi-armed CuO nanoparticles and their remarkable bactericidal potential against waterborne bacteria. Journal of Nanoparticle Research 2012, 14, (1).

7.

Abou Neel, E. A.; Ahmed, I.; Pratten, J.; Nazhat, S. N.; Knowles, J. C., Characterisation of antibacterial copper releasing degradable phosphate glass fibres. Biomaterials 2005, 26, (15), 2247-2254.

8.

Weaver, L.; Noyce, J. O.; Michels, H. T.; Keevil, C. W., Potential action of copper surfaces

on

meticillin-resistant

Staphylococcus

aureus.

Journal

of

Applied

Microbiology 2010, 109, (6), 2200-2205. 9.

Li, J.; Dennehy, J. J., Differential Bacteriophage Mortality on Exposure to Copper. Applied and Environmental Microbiology 2011, 77, (19), 6878-6883.

10.

Kaali, P.; Perez-Madrigal, M. M.; Stromberg, E.; Aune, R. E.; Czel, G.; Karlsson, S., The influence of Ag(+), Zn(2+) and Cu(2+) exchanged zeolite on antimicrobial and



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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

long term in vitro stability of medical grade polyether polyurethane. Express Polymer Letters 2011, 5, (12), 1028-1040. 11.

de Romaña, D. L.; Olivares, M.; Uauy, R.; Araya, M., Risks and benefits of copper in light of new insights of copper homeostasis. Journal of Trace Elements in Medicine and Biology 2011, 25, (1), 3-13.

12.

Barralet, J.; Gbureck, U.; Habibovic, P.; Vorndran, E.; Gerard, C.; Doillon, C. J., Angiogenesis in calcium phosphate scaffolds by inorganic copper ion release. Tissue Engineering Part A 2009, 15, (7), 1601-1609.

13.

Erol, M. M.; Mouriňo, V.; Newby, P.; Chatzistavrou, X.; Roether, J. A.; Hupa, L.; Boccaccini, A. R., Copper-releasing, boron-containing bioactive glass-based scaffolds coated with alginate for bone tissue engineering. Acta Biomaterialia 2012, 8, (2), 792801.

14.

Ytreberg, E.; Karlsson, J.; Eklund, B., Comparison of toxicity and release rates of Cu and Zn from anti-fouling paints leached in natural and artificial brackish seawater. Science of The Total Environment 2010, 408, (12), 2459-2466.

15.

Ore, S.; Mertens, J.; Brandt, K. K.; Smolders, E., Copper Toxicity to Bioluminescent Nitrosomonas europaea in Soil Is Explained by the Free Metal Ion Activity in Pore Water. Environmental Science & Technology 2010, 44, (23), 9201-9206.

16.

Berg, J.; Thorsen, M. K.; Holm, P. E.; Jensen, J.; Nybroe, O.; Brandt, K. K., Cu Exposure under Field Conditions Coselects for Antibiotic Resistance as Determined by a Novel Cultivation-Independent Bacterial Community Tolerance Assay. Environmental Science & Technology 2010, 44, (22), 8724-8728.

17.

Mureseanu, M.; Cioatera, N.; Trandafir, I.; Georgescu, I.; Fajula, F.; Galarneau, A., Selective Cu2+ adsorption and recovery from contaminated water using mesoporous hybrid silica bio-adsorbents. Microporous Mesoporous Mat. 2011, 146, (1-3), 141150.


Page 17 of 28

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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


18.

Atta, A. M.; Ismail, H. S.; Elsaaed, A. M., Application of anionic acrylamide-based hydrogels in the removal of heavy metals from waste water. Journal of Applied Polymer Science 2012, 123, (4), 2500-2510.

19.

Ganesh, R.; Smeraldi, J.; Hosseini, T.; Khatib, L.; Olson, B. H.; Rosso, D., Evaluation of Nanocopper Removal and Toxicity in Municipal Wastewaters. Environmental Science & Technology 2010, 44, (20), 7808-7813.

20.

Klein, T. Y.; Treccani, L.; Rezwan, K., Ceramic Microbeads as Adsorbents for Purification Technologies with High Specific Surface Area, Adjustable Pore Size, and Morphology Obtained by Ionotropic Gelation. J. Am. Ceram. Soc. 2012, 95, (3), 907914.

21.

Brunauer, S.; Emmett, P. H.; Teller, E., Adorption of Gases in Multimoleculer Layers. J. Am. Ceram. Soc. 1938, 60, 309-319.

22.

United States Environmental Protection Agency (EPA), Basic Information about Copper

in

Drinking

Water,

http://water.epa.gov/drink/contaminants/

basicinformation/copper.cfm - download 20.02.2012. 23.

Hollemann, A. F.; Wiberg, E.; Wiberg, N., Inorganic Chemistry - German edition. Walter de Gruyter: New York, 1995; Vol. 34 (101).

24.

Michen, B.; Meder, F.; Rust, A.; Fritsch, J.; Aneziris, C.; Graule, T., Virus Removal in Ceramic Depth Filters Based on Diatomaceous Earth. Environmental Science & Technology 2011, 46, (2), 1170-1177.

25.

Salmang, H.; Scholze, H., Ceramic (Keramik - German edition). Springer Verlag: Heidelberg, 2007; Vol. 7.

26.

Kosmulski, M., Chemical properties of material surfaces. Marcel Dekker: New York, 2001; Vol. 102.

27.

Fernández-Calviño,

D.;

Bermúdez-Couso,

A.;

Garrido-Rodríguez,

B.;

Peña

Rodríguez, S.; Arias-Estévez, M., Copper release kinetics from a long-term contaminated acid soil using a stirred flow chamber: Effect of ionic strength and pH. J. Colloid Interface Sci. 2012, 367, (1), 422-428. 16 ACS Paragon Plus Environment


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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28.

Page 18 of 28

World Health Organization (WHO), Guidelines for Drinking-water Quality - Copper in Drinking water, 2004, http://www.who.int/water_sanitation_health/dwq/chemicals/copper/en/ - download 20.02.2012.

29.

Stock, I., Bakterien, Viren, Wirkstoffe (Bacteria, Viruses, Active Agents). Govi: Eschborn, 2009.

30.

Song, C.; Wang, P.; Makse, H. A., A phase diagram for jammed matter. Nature 2008, 453, (7195), 629-632.

31.

German

Federal

Environment

Agency

(Umweltbundesamt).

Quantitative

determination of the efficacy of substances for disinfection in drinking water treatment http://www.umweltbundesamt.de/wasser/themen/trinkwasser/trinkwasseraufbereitung. htm - download 25.10.2012. 32.

Magnani, D.; Solioz, M., How bacteria handle Copper. In Molecular microbiology of heavy metals, Nies, D. H.; Silver, S., Eds. Springer, Heidelberg: 2007; pp 259–285.

33.

Solioz, M.; Abicht, H.; Mermod, M.; Mancini, S., Response of Gram-positive bacteria to copper stress. Journal of Biological Inorganic Chemistry 2010, 15, (1), 3-14.

34.

Macomber, L.; Imlay, J. A., The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proceedings of the National Academy of Sciences of the United States of America 2009, 106, (20), 8344-8349.

35.

Berg, J.; Brandt, K. K.; Al-Soud, W. A.; Holm, P. E.; Hansen, L. H.; Sørensen, S. J.; Nybroe, O., Selection for Cu-Tolerant Bacterial Communities with Altered Composition, but Unaltered Richness, via Long-Term Cu Exposure. Applied and Environmental Microbiology 2012, 78, (20), 7438-7446.

36.

Ruparelia, J. P.; Chatterjee, A. K.; Duttagupta, S. P.; Mukherji, S., Strain specificity in antimicrobial activity of silver and copper nanoparticles. Acta Biomaterialia 2008, 4, (3), 707-716.

37.

Jain, R. K., Copper-resistant microorganisms and their role in the environment. World Journal of Microbiology and Biotechnology 1990, 6, (4), 356-365. 17 ACS Paragon Plus Environment

Page 19 of 28

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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38.

Wu, X. H.; Ye, L.; Liu, K.; Wang, W.; Wei, J.; Chen, F. P.; Liu, C. S., Antibacterial properties of mesoporous copper-doped silica xerogels. Biomedical Materials 2009, 4, (4).

39.

Molteni, C.; Abicht, H. K.; Solioz, M., Killing of Bacteria by Copper Surfaces Involves Dissolved Copper. Applied and Environmental Microbiology 2010, 76, (12), 40994101.

40.

Ibrahim, M.; Wang, F.; Lou, M.-m.; Xie, G.-l.; Li, B.; Bo, Z.; Zhang, G.-q.; Liu, H.; Wareth, A., Copper as an antibacterial agent for human pathogenic multidrug resistant Burkholderia cepacia complex bacteria. Journal of Bioscience and Bioengineering 2011, 112, (6), 570-576.

41.

Jerri, H. A.; Adolfsen, K. J.; McCullough, L. R.; Velegol, D.; Velegol, S. B., Antimicrobial Sand via Adsorption of Cationic Moringa oleifera Protein. Langmuir 2012, 28, (4), 2262–2268.

42.

Kristensen, J. B.; Olsen, S. M.; Laursen, B. S.; Kragh, K. M.; Poulsen, C. H.; Besenbacher, F.; Meyer, R. L., Enzymatic generation of hydrogen peroxide shows promising antifouling effect. Biofouling 2010, 26, (2), 141-153.

43.

An, Y. H.; Friedman, R. J., Concise review of mechanisms of bacterial adhesion to biomaterial surfaces. J. Biomed. Mater. Res. 1998, 43, (3), 338-348.

44.

Ma, H.; Winslow, C. J.; Logan, B. E., Spectral force analysis using atomic force microscopy reveals the importance of surface heterogeneity in bacterial and colloid adhesion to engineered surfaces. Colloids and Surfaces B: Biointerfaces 2008, 62, (2), 232-237.

45.

Hong, Y.; Brown, D. G., Cell surface acid–base properties of Escherichia coli and Bacillus brevis and variation as a function of growth phase, nitrogen source and C:N ratio. Colloids and Surfaces B: Biointerfaces 2006, 50, (2), 112-119.

46.

Leone, L.; Ferri, D.; Manfredi, C.; Persson, P.; Shchukarev, A.; Sjöberg, S.; Loring, J., Modeling

the

Acid−Base

Properties

of

Bacterial

Surfaces:‒

A

Combined



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 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Spectroscopic and Potentiometric Study of the Gram-Positive Bacterium Bacillus subtilis. Environmental Science & Technology 2007, 41, (18), 6465-6471.


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Table 1: Properties of microbeads with and without copper doping. 75x24mm (300 x 300 DPI)



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Fig. 1: a) XRD-analysis of Cu-doped microbeads (CuBs), sintered at Tsinter= 900 and 1000°C; b) photograph of CuBs, non-sintered, and sintered at Tsinter= 900°C; c) EDS-spectrum of CuBs, sintered at Tsinter= 900 °C. 110x48mm (300 x 300 DPI)


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Fig. 2: Zeta-potential, obtained by streaming potential measurements, of sintered ceramics, with and without Cu-doping, sintered at Tsinter= 900°C and 1000°C, the dashed lines are just to guide the eye. 163x114mm (300 x 300 DPI)



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Fig. 3: Static copper-ion release of Cu-doped microbeads, sintered at a) Tsinter= 900°C, and b) Tsinter= 1000°C, over time in different media, measured at static conditions; the dashed lines are just to guide the eye. 101x37mm (300 x 300 DPI)


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Fig. 4: a) Photograph of antibacterial copper-doped monolith (CuM); b) SEM detail of monolith; c) photograph of high surface area adsorbent (MAd); d) tomographic cross-section monolith; e) SEM crosssection of single microbead. 96x36mm (300 x 300 DPI)



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Fig. 5: a) Schematic overview on dynamic test-setup, the tests were done in Tris-HCl, pH 7.4 with a constant flux of 25.0±1 L/m²; b) Copper-ion release, for copper doped monoliths (CuMs) sintered at Tsinter= 900°C and 1000°C; c) Copper-ion removal under release conditions for CuMs sintered at Tsinter= 900°C and MAds sintered at Tsinter= 1000°C; d) Copper-ion removal under release conditions in the presence of a bacteria solution. 191x137mm (300 x 300 DPI)


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Fig. 6: Effect of increasing amounts of Cu-doped microbeads, sintered at Tsinter = 900°C, on the viability of a) B. subtilis; b) E. coli; c) S. aureus and d) P. aeruginosa. The control groups contained no microbeads. 158x108mm (300 x 300 DPI)



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Fig. 7: Dynamic tests: effect of Cu-doped monoliths (CuMs), sintered at Tsinter = 900°C and Cu-free monoliths (MAds), sintered at Tsinter = 1000°C on the viability of a) B. subtilis; b) E. coli. 102x39mm (300 x 300 DPI)


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Effective Bacterial Inactivation and Removal of Copper by Porous

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