Article

pubs.acs.org/est

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
*
S Supporting Information

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 nonpathogenic 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.

■ 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 mole-
cules/enzymes, or metals − each with its own pros and
cons.1−3 Among 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, Cu-nanoparticles, 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.4,8 Besides bacteria, Cu
is also effective against different types of viruses and fungi.9,10
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 process12 and was
used by Erol et al.13 to induce glass-based 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.
Previously,20 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 3D
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 microbe-
ads (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.
Received: May 25, 2012
Revised: December 20, 2012
Accepted: December 29, 2012
Published: December 30, 2012

© 2012 American Chemical Society 1065 dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072
Environmental Science & Technology
■
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, BINDZIL30/220, 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 trisodium 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 deionized
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
tris(hydroxymethyl)aminomethane, pH 7.4 (Tris-HCl, lot.
MKBD9221 V, 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). Wet
beads 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 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, Oberko-
chen, Germany), and ICP-AES analysis (Optima 3300,
PerkinElmer, MA, USA) provide information on CuBs
composition. The specific surface area (SBET) of sintered and
nonsintered samples was determined by nitrogen adsorption
(Belsorp-Mini, Bel Japan, Osaka, Japan) using the BET-
method.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 to SBET and surface morphology. Therefore, microbeads
ζ-pot results shall be transferable to MAds and CuMs. 3D
images were obtained by computer tomography (phoenix
nanotom m, GE Measurement & Control, Wunstorf,
1066
Article
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 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 (US-EPA)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. Nonpathogenic 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 preculture were grown overnight in Luria−
Bertani medium at 37 °C under gentle shaking. Subsequently,
the bacteria were harvested by centrifugation at 3000g 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 approximately 108/mL according to McFar-
land standards. Afterward, 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-tests (Type 06400,
dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072
Environmental Science & Technology
Figure 1. (a) XRD-analysis of Cu-doped microbeads (CuBs), sintered at Tsinter = 900 and 1000 °C; (b) photograph of CuBs, nonsintered, and
sintered at Tsinter = 900 °C; (c) EDS-spectrum of CuBs, sintered at Tsinter = 900 °C.
Table 1. Properties of Microbeads with and without Copper Doping
type Tsinter density (g/cm3)a total Cu content (mg/g)b
CuB nonsintered 2.92 9.4 ± 0.5
CuB 900 °C 3.14 9.7 ± 0.8
CuB 1000 °C 3.17 9.2 ± 0.2
B 1000 °C 3.11 without Cu
a
He-pycnometry. bICP-AES. cN2 adsorption.
3M, Neuss, Germany) samples for plating were withdrawn after
incubation times of 15 min, 1 and 3 h, respectively. The
Petrifilms were incubated for one day at 37 °C prior to scoring
the number of cfu’s.
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 and 3 h. To verify
whether the decline in bacteria concentration is an effect of the
present Cu, additional tests with MAds were performed.
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 (part a of Figure
1) of sintered CuBs show mainly α-alumina (corundum)
reflexes. The increased background observed for small angles
up to approximately 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 Cu-containing
phases are found indicating that Cu forms no new crystalline
phases with alumina or silica. Part b of Figure 1 depicts
microbeads with and without copper. Microbeads without
copper are plain white. Incorporation of Cu changes the color
to turquois for nonsintered 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 (part c
1067
Article
SBET (m2/g)c BET pore volume (cm3/g)c total porosity (%)a,c
63.9 0.2059 40.8
37.5 0.1831 37.7
11.1 0.0744 15.0
37.8 0.2124 38.3
of Figure 1 and Table S.1 of the Supporting Information). EDS-
spectra of the inner core taken from a fracture surface indicate
the presence of a Cu gradient with declining Cu concentrations
toward 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 cross-linked 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 approximately 2.0
mass-%. Finally, we used ICP-AES analysis (Table 1) to reveal
CuBs total Cu content. According to ICP-AES sintered and
nonsintered 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 ζ-pot curves for microbe-
ads with and without Cu-doping are rather similar (Figure 2).
Both of the isoelectric points (IEP)25 are located at about pH
2.1 resulting in a negatively charged surface at the bacteria
Figure 2. Zeta-potential, obtained by streaming potential measure-
ments, of sintered ceramics, with and without Cu-doping, sintered at
Tsinter = 900 and 1000 °C, the dashed lines are just to guide the eye.
dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072
Environmental Science & Technology
Figure 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.
Figure 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 of monolith, (e) SEM cross-section of single microbead.
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 are therefore
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, whereas 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 (part b
of Figure 3), the overall Cu release is less than that 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 Fernández-Calviño 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
1068
Article
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, respectively. No significant changes in
CuBs surface morphology were observed after exposure to the
different media (insert in part a of Figure 3 and Figure S.1 of
the Supporting Information) 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. 3D
assembly results in a highly open porous filter structure with
pores in between the beads with average diameters ≥100 μm.
Most bacteria are distinctly smaller than 10 μm.29 Because of
the much larger pore channels, risk of pore clogging during a
filtering process will therefore be moderate.
Part a of Figure 5 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 and 1000 °C in Tris-HCl (part b of
Figure 5). 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
dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072
Environmental Science & Technology Article

Figure 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 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.

Figure 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.

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
1069
larger amounts of CuBs/CuMs are used. To avoid any possibly
detrimental effects, free Cu-ions should therefore be removed
as a precaution after inactivating the bacteria. Parts c and d of
Figure 5 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
dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072
Environmental Science & Technology
Figure 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.
removed and even after 22 h the removal rate of the released
Cu is still 56%.
Presence of B. subtilis slightly reduces Cu-adsorption on
MAds (part d of Figure 5), 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 (Figure S.2 of the Supporting Informa-
tion). 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+, and so
forth, 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 Figure S.3 of the Supporting 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 min31 corresponding to an antibacterial efficiency of
99% and 99.99%, respectively. One gram of CuBs reaches an
antibacterial efficiency of 100% for B. subtilis (part a of Figure 6,
and Table S.2 of the Supporting 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 (part c of Figure 6). 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 gram of CuBs in contact with E. coli and P.
aeruginosa needed incubation times of 3 h to reach an
1070
Article
antibacterial efficiency of 100%, whereas for 2 g only after 15
min living bacteria were observed in the supernatant (parts b
and d of Figure 6). 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 toward 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
Cu,5 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 Cu-ions 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 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 toward other noisome bacteria, that are
reported to be sensitive against Cu like Salmonella, Listeria,5 or
several multidrug-resistant Burkholderia species.40 In contrast to
other antibacterial materials, where organic molecules are
utilized like immobilized proteins41 or enzymes,42 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
cycles, 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.
dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072
Environmental Science & Technology
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 (part a of Figure 7 and Table S.3 of the
Supporting Information). For E. coli, the antibacterial efficiency
decreased from 99.29% at t = 0 min to 92.57% at t = 3 h (part b
of Figure 7). These results have the same tendency as the static
tests, where tested Gram-positive bacteria were slightly more
sensitive toward 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 heterogene-
ities.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 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 (Figure 2). Bacteria are generally negatively charged
at neutral pH values, too.29,45 For E. coli and B. subtilis, for
example, IEPs 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.
■
ASSOCIATED CONTENT
*
S Supporting Information
Supplemental figures and tables as mentioned in the text. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: krezwan@uni-bremen.de, tel: +49 (0)421 218 −
64930, fax: +49 (0)421 218 − 64932.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
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-Rhine-Sieg 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
1071
Article
and Sabine Schulte for proofreading the manuscript. The CT-
image was obtained from GE Measurement & Control,
Wundstorf, Germany.
■
REFERENCES
(1) Kroll, S.; Treccani, L.; Rezwan, K.; Grathwohl, G. Development
and characterisation of functionalised ceramic microtubes for bacteria
filtration. J. Membr. Sci. 2010, 365 (1−2), 447−455.
(2) Martinez, J. L. Environmental pollution by antibiotics and by
antibiotic resistance determinants. Environ. Pollut. 2009, 157 (11),
2893−2902.
(3) Malachová, K.; Praus, P.; Rybková, Z.; Kozák, O. Antibacterial
and antifungal activities of silver, copper and zinc montmorillonites.
Appl. Clay Sci. 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. Appl. Environ. Microbiol. 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. J.
Nanopart. Res. 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.
J. Appl. Microbiol. 2010, 109 (6), 2200−2205.
(9) Li, J.; Dennehy, J. J. Differential bacteriophage mortality on
exposure to copper. Appl. Environ. Microbiol. 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 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), 792−801.
(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. Environ. Sci. Technol. 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. Environ. Sci. Technol. 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), 141−150.
dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072
Environmental Science & Technology Article

(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. J. Appl. Polym. Sci. 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. Environ. Sci. Technol. 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), 907−914.
(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 ed.; 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.
Environ. Sci. Technol. 2011, 46 (2), 1170−1177.
(25) Salmang, H.; Scholze, H. Ceramic (Keramik - German ed.);
Springer Verlag: Heidelberg, 2007; Vol. 7.
(26) Kosmulski, M. Chemical Properties of Material Surfaces; Marcel
Dekker: New York, 2001; Vol. 102.
(27) Fernández-Calviñ o, D.; Bermúdez-Couso, A.; Garrido-
(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. J. Biosci. Bioeng. 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 Surf., B 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 Surf., B 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 spectroscopic and potentiometric study of the
gram-positive bacterium Bacillus subtilis. Environ. Sci. Technol. 2007,
41 (18), 6465−6471.

■
Rodríguez, B.; Peña Rodríguez, S.; Arias-Estévez, M. Copper release
kinetics from a long-term contaminated acid soil using a stirred flow NOTE ADDED AFTER ASAP PUBLICATION
chamber: Effect of ionic strength and pH. J. Colloid Interface Sci. 2012,
367 (1), 422−428. This paper published January 7, 2013 with a missing value in
(28) World Health Organization (WHO), Guidelines for Drinking- Table 1. The corrected version published January 15, 2012.
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. Proc.
Natl. Acad. Sci. U.S.A. 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. Appl. Environ. Microbiol. 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 J. Microbiol. Biotechnol. 1990, 6 (4), 356−365.
(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. Appl. Environ. Microbiol.
2010, 76 (12), 4099−4101.

1072 dx.doi.org/10.1021/es3045828 | Environ. Sci. Technol. 2013, 47, 1065−1072